Us 2018 / 0271910 A1

Home , Albinterferon, Bacteriocin, Oprelvekin, Platelet lysate

US 20180271910A1 ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2018 /0271910 A1 Mata - Fink et al. (43 ) Pub . Date : Sep . 27 , 2018 (54 ) METHODS AND COMPOSITIONS FOR 2014 , provisional application No. 61/ 991, 319 , filed IMMUNOMODULATION on May 9 , 2014 , provisional application No . 61 / 973 , 764, filed on Apr. 1, 2014 , provisional application No. (71 ) Applicant: RUBIUS THERAPEUTICS , INC . , 61/ 973 ,763 , filed on Apr. 1 , 2014 . Cambridge , MA (US ) ( 30 ) Foreign Application Priority Data (72 ) Inventors: Jordi Mata - Fink , Somerville , MA (US ) ; John Round , Cambridge, MA Nov . 12 , 2014 (US ) ...... PCT/ US2014 / 065304 (US ) ; Noubar B . Afeyan , Lexington , MA (US ) ; Avak Kahvejian , Arlington , Publication Classification MA (US ) (51 ) Int. CI. A61K 35 / 28 ( 2015 .01 ) ( 21 ) Appl. No. : 15 /911 , 924 C12N 5 /078 ( 2010 . 01) A61K 48 /00 ( 2006 .01 ) (22 ) Filed : Mar. 5 , 2018 C12N 5 /07 ( 2010 . 01 ) A61K 35 / 12 (2015 . 01) Related U . S. Application Data (52 ) U . S . CI. (63 ) Continuation of application No . 15 / 301, 046 , filed on CPC ...... A61K 35 / 28 ( 2013 .01 ) ; C12N 5 /0644 Sep . 30 , 2016 , filed as application No . PCT/ US2015 / (2013 .01 ) ; C12N 5 / 0641 ( 2013 . 01 ); A61K 020614 on Mar . 13 , 2015 . 2035 / 124 (2013 . 01 ) ; C12N 5 / 06 ( 2013 . 01 ) ; (60 ) Provisional application No . 62/ 059 , 100 , filed on Oct . A61K 2035 / 122 (2013 .01 ); A61K 48 / 00 2 , 2014 , provisional application No . 62/ 025 , 367 , filed ( 2013. 01 ) on Jul. 16 , 2014 , provisional application No. 62 / 006 , 828 , filed on Jun . 2 , 2014 , provisional application No . (57 ) ABSTRACT 62/ 006 ,825 , filed on Jun . 2 , 2014 , provisional appli Provided are cells containing exogenous antigen and uses cation No . 62 /006 ,829 , filed on Jun . 2 , 2014 , provi thereof. sional application No . 62 /006 , 832 , filed on Jun . 2 , Specification includes a Sequence Listing . Patent Application Publication Sep . 27, 2018 Sheet 1 of 27 US 2018 /0271910 A1

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METHODS AND COMPOSITIONS FOR SUMMARY OF THE INVENTION IMMUNOMODULATION [0008 ] The invention , in certain aspects , relates to isolated enucleated hematopoietic cells expressing an antigen . RELATED APPLICATIONS Enucleated hematopoietic cells will be referred to herein as [0001 ] The present application is a continuation of U . S . “ EHCs” ( or in its singular form : “ EHC ” ) . In some embodi Ser. No . 15 /301 , 046 , filed Sep . 30 , 2016 , which is a U . S . ments , the enucleated hematopoietic cells or EHCs lack National Stage Application under 35 U . S . C . § 371 of Inter nuclear material. For example , the EHCs can be are eryth national Application No . PCT /US2015 /020614 , filed Mar. roid cells or thromboid cells . In some embodiments , EHCS 13 , 2015 , which claims priority under 35 U . S . C . § 119 to lacking nuclear material are red blood cells , erythrocytes, U . S . Provisional Application No . 61/ 973 ,764 , filed Apr. 1 , reticulocytes , or platelets. In some embodiments , the enucle 2014 , U . S . Provisional Application No . 61/ 973 , 763, filed ated hematopoietic cells or EHCs are nucleated precursor Apr. 1 , 2014 , U .S . Provisional Application No . 61 /991 , 319 , erythroid cells or precursor thromboid cells that are, e . g . , filed May 9 , 2014 , U . S . Provisional Application No . 62 /006 , induced to lose their nuclear material or are rendered func 825 , filed Jun . 2 , 2014 , U . S . Provisional Application No . tionally enucleated and incapable of replication . In some 62 /006 , 828 , filed Jun . 2 , 2014 , U . S . Provisional Application embodiments , the exogenous antigen - expressing EHC is a No. 62 / 006 ,829 , filed Jun . 2 , 2014 , U . S . Provisional Appli circulating cell , such as a red blood cell . In some embodi cation No . 62 / 006 , 832 , filed Jun . 2 , 2014 , U . S . Provisional ments , the exogenous antigen -expressing EHC is cultured Application No. 62 /025 , 367 , filed Jul. 16 , 2014 , U . S . Pro from a hematopoietic precursor using defined factors . In visional Application No . 62 /059 , 100 , filed Oct . 2 , 2014, and some embodiments, the exogenous antigen -expressing EHC is a thromboid cell , such as a platelet. In some embodiments International Application No . PCT /US2014 / 065304 , filed the thromboid cell is cultured from a hematopoietic precur Nov . 12 , 2014 . The entire contents of each of the foregoing sor using defined factors. In some embodiments , the exog applications are incorporated herein by reference . enous antigen - expressing EHC is a primary cell isolated from a patient, for either autologous or allogeneic use , that SEQUENCE LISTING is contacted with an antigen . [ 0002 ] The instant application contains a Sequence Listing [00091 . Certain aspects of the invention relate to exogenous which has been submitted electronically in ASCII format antigen - expressing EHCs that are capable of inducing and is hereby incorporated by reference in its entirety . Said immune tolerance when administered to a subject, e . g . in ASCII copy , created on Jun . 6 , 2018 , is named R2081 form of a pharmaceutical composition comprising the exog 700320 _ SL . txt and is 66 , 425 bytes in size . enous antigen -expressing EHCs. The exogenous antigen expressed by the EHCs can be tailored to a specific disease , disorder or condition . The exogenous antigen - expressing FIELD OF THE INVENTION EHCs can comprise antigen in multiple ways , such as e . g . [0003 ] The field of the invention is pharmaceutical com surface display, intracellular expression , intracellular load ing , or surface conjugation , of the antigen of interest . The positions for the treatment of diseases and disorders . exogenous antigen -expressing EHCs may manage diseases of aberrant immune activation more effectively and /or with BACKGROUND OF THE INVENTION fewer side effects than existing treatments . For example , [0004 ] Aberrant immune activation is a hallmark ofmany exogenous antigen -expressing EHCsmay selectively modu human diseases and conditions. Autoimmune diseases arise late the immune system while leaving the broader immune when the body ' s immune system improperly senses an system physiology substantially unperturbed . In some autologous antigen as non - self and attacks the body ' s own embodiments , exogenous antigen - expressing EHCs may tissues . Inflammatory diseases and allergies can arise when induce the destruction , deactivation , and / or anergy of anti the body 's immune system is improperly triggered by com gen - specific T and B lymphocytes . Alternatively or in addi mon food -borne or environmental antigens. Polypeptides tion , exogenous antigen - expressing EHCs may induce the and proteins used to treat a range of human diseases are proliferation of antigen - specific regulatory T lymphocytes. often destroyed , neutralized , or otherwise rendered ineffec 0010 ) Certain aspects of the invention relate to exogenous tive by immune cells that respond to them as though they antigen - expressing EHCs that comprise exogenous antigen were foreign antigens. that is recognized by immune cells in autoimmune diseases, such as , e . g . multiple sclerosis , type 1 diabetes, rheumatoid [0005 ] Current treatment of diseases of improper immune arthritis , and membranous nephritis . activation involves immunosuppression with chemical [ 0011 ] Certain aspects of the invention relate to exogenous agents like corticosteroids, or inhibitors of inflammatory antigen - expressing EHCs that comprise exogenous antigen mediators like anti - histamines, antibodies, or cytokines . that is recognized by immune cells in inflammatory diseases , These generalized treatments are associated with significant such as , e . g . Crohn ' s disease , ulcerative colitis , celiac dis morbidities , such as susceptibility to infection , because they ease , or other idiopathic inflammatory bowl diseases . broadly suppress the immune system . [0012 ]. Certain aspects of the invention relate to exogenous [ 0006 ] For some severe allergies , clinical testing is under antigen - expressing EHCs that comprise exogenous antigen way to induce “ tolerance ” to allergens by exposure to slowly that is recognized by immune cells in human leukocyte increasing doses of the offending protein over time. To date antigen (HLA ) mismatch -mediated diseases , such as, e . g . theses treatments lack long- term efficacy and are associated graft - versus - host disease or organ transplant rejection . with a risk of severe anaphylaxis . [0013 ] Certain aspects of the invention relate to exogenous [0007 ] There is a need for novel therapeutics to treat these antigen -expressing EHCs that comprise exogenous antigen diseases . that is recognized by immune cells in allergic diseases, such US 2018 /0271910 A1 Sep . 27 , 2018 as, e . g . asthma, peanut allergy , shellfish allergy, pollen [0025 ] In some embodiments , the concentration of anti allergy, milk protein allergy , insect sting allergy , and latex gen - specific immune cells is decreased by at least about 1 % , allergy . 5 % , 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , [0014 ] Certain aspects of the invention relate to exogenous 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , antigen -expressing EHCs that comprise exogenous antigen 97 % , 98 % , 99 % , 99. 5 % , 99 . 9 % , 99 . 99 % , or greater than that is a therapeutic protein whose efficacy or potency is 99. 99 % during part or the entirety of the treatment period . reduced or impaired by immune cells , such as, e . g ., clotting 10026 ]. In other embodiments , the concentration of anti factor VIII in hemophilia A , clotting factor IX in hemophilia gen - specific immune cells is decreased by at least about 1 % , B , anti -tumor necrosis factor alpha ( TNFa ) antibodies in 5 % , 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , rheumatoid arthritis and other inflammatory diseases, glu 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , cocerebrosidase in Gaucher ' s disease , or asparaginase in 97 % , 98 % , 99 % , 99. 5 % , 99. 9 % , 99 .99 % , or greater than acute lymphoblastic leukemia (ALL ) . 99 .99 % within about 1 , 5 , 10 , 15 , 20 , 30 , 40 , or 50 minutes, [ 0015 ] Certain aspects of the invention relate to exogenous or about 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , antigen - expressing EHCs that comprise exogenous antigen 17, 18 , 19, 20 , 21 , 22 , or 23 hours , or 1 , 2 , 3 , 4 , 5 , or 6 days that comprises full - length , truncations, and chimeric fusions or about 1, 2 , 3 , 4 , 5 , or 6 weeks of the administration . of polypeptides that a ) mediate complement regulation , b ) [0027 ] In some embodiments, the pharmaceutical compo that mediate binding and sequestration of immune com sition is administered a sufficient number of times over a plexes , c ) autoimmune antibodies, or d ) pathogenic par treatment period such that the concentration of antigen ticles. In some embodiments , the exogenous antigen com specific immune cells is substantially decreased for at least prises full - length , truncations , and chimeric fusions of about one week , two weeks , three weeks, four weeks, one polypeptides that are enzymatically active in the conversion month , two months , three months , four months , five months , of one small molecule substrate into another small molecule six months , or greater than six months . product or of one polypetide substrate into a second poly 10028 ] In certain embodiments , the pharmaceutical com peptide product , including , e . g . , cleavage of the polypeptide position is administered a sufficient number of times over a substrate . treatment period such that the concentration of antigen [0016 ] The invention , in certain aspects , also provides specific immune cells is substantially decreased for a period methods of treatment of disease using the exogenous anti of time at least as long as the treatment period. gen - expressing EHCs and pharmaceutical compositions [0029 ] In some embodiments , the pharmaceutical compo thereof provided herein . sition is administered a sufficient number of times over a [ 0017 ] In some aspects , disclosed herein is a method of treatment period such that the concentration of antigen inducing immune tolerance. The method comprises admin specific antibodies in circulation is substantially decreased istering to a human subject suffering from or at risk of during the treatment period . developing an autoimmune disease , disorder or condition , a [0030 ] In some embodiments, the concentration of anti pharmaceutical composition comprising an enucleated gen - specific antibodies in circulation is measured by ELISA . hematopoietic cell expressing an exogenous antigen , 10031 ] In some embodiments , the concentration of anti wherein the pharmaceutical composition is administered in gen - specific circulating antibody is decreased by at least an amount effective to induce immune tolerance in the about 1 % , 5 % , 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , subject to the antigen mediating the autoimmune disease , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , 98 % , 99 % , 99 . 5 % , 99 . 9 % , 99 . 99 % , or disorder or condition . greater than 99 .99 % during part or the entirety of the [0018 ] In some embodiments , the autoimmune disease is treatment period . selected from the group consisting of multiple sclerosis , type [0032 ]. In some embodiments , the concentration of anti 1 diabetes , and those listed in Table F . gen - specific antibody is decreased by at least about 1 % , 5 % , [0019 ] In some embodiments , the method further com 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , prises administering the pharmaceutical composition at least 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , twice over a treatment period such that the autoimmune 98 % , 99 % , 99 . 5 % , 99 . 9 % , 99 . 99 % , or greater than 99 . 99 % disease , disorder or condition is treated , or a symptom within about 1 , 5 , 10 , 15 , 20 , 30 , 40 , or 50 minutes , or about thereof is decreased . 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , [0020 ] In certain embodiments , the method further com 20 , 21 , 22 , or 23 hours, or 1 , 2 , 3 , 4 , 5 , or 6 days or about prises administering the pharmaceutical composition at least 1 , 2 , 3 , 4 , 5 , or 6 weeks of the administration . twice over a treatment period such that the autoimmune [0033 ] In certain embodiments, the pharmaceutical com disease , disorder or condition is prevented . position is administered a sufficient number of times over a [0021 ] In other embodiments, the method further com treatment period such that the concentration of antigen prises administering the pharmaceutical composition a suf specific circulating antibody is substantially decreased for at ficient number of times over a treatment period such that the least about one week , two weeks , three weeks, four weeks, percentage of antigen - specific immune cells is substantially one month , two months , three months , four months, five decreased during the treatment period . months, six months, or greater than six months. [0022 ] In some embodiments , the immune cell is a T cell . [0034 ] In certain embodiments , the pharmaceutical com In some embodiments , the immune cell is a B cell . position is administered a sufficient number of times over a [ 0023 ] In some embodiments, the decrease in concentra treatment period such that the concentration of antigen tion of antigen - specific immune cells is measured by flow specific circulating antibody is substantially decreased for a cytometry from a biological sample taken from the subject . period of time at least as long as the treatment period . [0024 ] In some embodiments , the biological sample is a [0035 ] In some embodiments , the pharmaceutical compo lymph node biopsy, a spleen sample , or peripheral blood . sition is administered a sufficient number of times over a US 2018 /0271910 A1 Sep . 27 , 2018

treatment period such that the percentage of antigen - specific (0047 ] In certain embodiments , the frequency of admin regulatory T cells is substantially increased during the istration is sufficient to effectively increase the concentration treatment period . of antigen - specific , regulatory T cells above a threshold [0036 ] In some embodiments, the decrease in concentra level that is associated with a symptom of autoimmune tion of antigen -specific immune cells is measured by flow disease , disorder or condition . cytometry from a biological sample taken from the subject. [0048 ] In some embodiments , the enucleated hematopoi [ 0037 ] In some embodiments , the biological sample is a etic cell is an erythroid cell, a thromboid cell , or a precursor lymph node biopsy , a spleen sample , or peripheral blood . thereof . In some embodiments , the erythroid cell is an 10038 ] In certain embodiments , the concentration of anti erythrocyte or a reticulocyte . In some embodiments , the gen - specific regulatory T cells is increased by at least about thromboid cell is a platelet. 1 % , 5 % , 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 10049 ] In some embodiments , the enucleated hematopoi etic cell is isolated from a donor. In some embodiments , the 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , enucleated hematopoietic cell is autologously derived from 97 % , 98 % , 99 % , 99. 5 % , 99. 9 % , 99 .99 % , or greater than the subject. In some embodiments , the enucleated 99. 99 % during part or the entirety of the treatment period . hematopoietic cell is allogeneically derived . In some [ 0039 ] In certain embodiments , the concentration of anti embodiments , the enucleated hematopoietic cell is xenoge gen - specific regulatory T cells is increased by at least about neically derived . 1 % , 5 % , 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , [0050 ] In some embodiments, the nucleated hematopoietic 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , cell is derived from a nucleated precursor cell by a culture 97 % , 98 % , 99 % , 99. 5 % , 99. 9 % , 99 .99 % , or greater than based process that induces the expulsion of its nucleus. In 99 .99 % within about 1 , 5 , 10 , 15 , 20 , 30 , 40 , or 50 minutes , some embodiments , the enucleated hematopoietic cell is or about 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , generated from a nucleated precursor cell that is chemically 17 , 18 , 19 , 20 , 21, 22 , or 23 hours , or 1 , 2 , 3 , 4 , 5 , or 6 days or physical manipulated to remove its nucleus . or about 1 , 2 , 3 , 4 , 5 , or 6 weeks of the administration . 10051] In some embodiments , the enucleated hematopoi 10040 ] In some embodiments , the pharmaceutical compo etic cell is generated by irradiation or chemical destruction sition is administered a sufficient number of times over a of the nucleus of a nucleated precursor cell . In some treatment period such that the concentration of antigen embodiments , the chemical destruction is carried out with specific regulatory T cells is substantially increased for at Cytochalasin B . In some embodiments , the irradiation is least about one week , two weeks, three weeks , four weeks , carried out with at least 5 Gy, 7 Gy, 10 Gy, 15 Gy, 25 Gy, one month , two months, three months, four months, five 30 Gy, 40 Gy or at least 50 Gy. months, six months, or greater than six months . [0052 ]. In some embodiments , the exogenous antigen is a 10041 ] In some embodiments , the pharmaceutical compo polypeptide encoded by an exogenous nucleic acid . sition is administered a sufficient number of times over a [ 0053 ] In some embodiments , the exogenous antigen is treatment period such that the concentration of antigen associated with the cell membrane of the enucleated specific regulatory T cells is substantially increased for a hematopoietic cell. period of time at least as long as the treatment period . [ 0054 ] In some embodiments , the exogenous antigen is a [0042 ] In some embodiments , the pharmaceutical compo fusion or a chimera polypeptide. sition is administered a sufficient number of times over a [0055 ] In some embodiments , the fusion or chimera com treatment period such that one or more symptoms of the prises at least one of an S domain , an A domain or a U autoimmune disease , disorder or condition is prevented , domain , wherein the S domain is a surface domain on the decreased or delayed . enucleated hematopoietic cell , wherein the A domain is an [0043 ] In some embodiments , the treatment period is not anchor in or on the cell membrane , wherein the U domain longer than a year , six months, three months, two months, faces the intracellular, unexposed side of the enucleated one month , two weeks, one week , three days , two days, one hematopoietic cell , and wherein the S domain , the A domain , day . and /or the U domain are of different polypeptide origin . [ 00441 In certain embodiments , the time interval between [0056 ] In some embodiments , the S domain and / or the A administrations within the treatment period is no longer than domain comprises at least 5 , 6 , 7 , 8 , 9 , 10 , 15 , 20 , 25 , 30 , 40 , the period in which the number of enucleated hematopoietic 50 , 60 , 70 , 80 , 90 , 100 , 150 , 200 , 250 , or at least 500 amino cells expressing an exogenous antigen is reduced to less than acids. In some embodiments , the S domain and / or A domain about 5 % , 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , comprises at least 500 , 750 , or at least 1 , 000 amino acids . 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , or 95 % [0057 ] In some embodiments , the exogenous antigen is of the number of enucleated hematopoietic cells expressing selected from the group consisting of myelin basic protein , an exogenous antigen present in the administered pharma proteolipid protein , myelin oligodendrocyte glycoprotein , ceutical composition . pancreatic beta cell antigen , insulin , and those listed in Table 0045 ] In some embodiments , the frequency of adminis F , Table 6 , and Table 8 . tration is sufficient to effectively reduce the concentration of 10058 ] In some embodiments, the enucleated hematopoi antigen - specific immune cells below a level that is associ etic cell comprises at least 10 copies , 100 copies , 1 , 000 ated with a symptom of autoimmune disease , disorder or copies , 10 , 000 copies , 25 ,000 copies , 50 , 000 copies , 100 , condition . 000 copies, 500 , 000 copies , 1, 000 , 000 copies, or 2 ,000 , 000 [0046 ] In certain embodiments , the frequency of admin copies of the exogenous antigen . istration is sufficient to effectively reduce the concentration [0059 ] In some embodiments , the pharmaceutical compo of antigen -specific circulating antibody below a level that is sition further comprises a pharmaceutically active agent. associated with a symptom of autoimmune disease, disorder f0060 ] In some embodiments , the method further com or condition . prises the step of administering a pharmaceutically active US 2018 /0271910 A1 Sep . 27 , 2018

agent, wherein the pharmaceutically active agent is admin herein and a medical device for intravenous injection of the istered prior to , after, or concurrent with the pharmaceutical pharmaceutical composition to the subject. composition . 100741 In some aspects , disclosed herein are hematopoi [0061 ] In some embodiments , the pharmaceutical compo etic cells expressing an exogenous antigen of the pharma sition is administered intravenously . ceutical composition administered by any of the methods [0062 ] In some embodiments , the pharmaceutically active described herein . agent is selected from a biological agent, a small molecule [0075 ] In some aspects , disclosed herein is a population of agent, or a nucleic acid agent . hematopoietic cells expressing an exogenous antigen as [0063 ] In some embodiments , the pharmaceutical compo disclosed herein . sition further comprises a pharmaceutically acceptable car [0076 ] In some embodiments , the population of rier. hematopoietic cells expressing an exogenous antigen is [0064 ] In certain embodiments , the method further com formulated as a liquid . prises the step of selecting for treatment a subject suffering [0077 ] In some embodiments , the population of from or at risk of an autoimmune disease , disorder or hematopoietic cells expressing an exogenous antigen is condition selected from the group consisting of: thrombotic frozen . thrombocytopenic purpura , CAPS , APS , myasthenia gravis , [0078 ] In some aspects , disclosed herein is an isolated Goodpasture ' s syndrome , membraneous nephritis , type 1 antigen expressed by the hematopoietic cell population diabetes , rheumatoid arthritis , multiple sclerosis , Crohn ' s described herein . disease , or those listed in Table F and Table G . [0079 ] In some aspects , disclosed herein is an exogenous [0065 ] In some aspects , disclosed herein is a pharmaceu nucleic acid encoding the exogenous antigen described tical composition comprising an enucleated hematopoietic herein . cell expressing an exogenous antigen , wherein administra [0080 ] In some aspects , disclosed herein is an enucleated tion of an effective amount of the pharmaceutical composi hematopoietic cell comprising an exogenous antigen that tion is capable of inducing immune tolerance in a human comprises at least one of an S domain , an A domain or a U subject suffering from or at risk of developing an autoim domain , wherein the S domain is an extracellular surface mune disease , disorder or condition administered by the domain , the A domain is an anchor, and the U domain is method of any one of the preceding claims . intracellularly localized , and wherein the enucleated [0066 ] In some embodiments , the pharmaceutical compo hematopoietic cell is capable of inducing immune tolerance sition further comprises a pharmaceutically acceptable car when administered to a subject . rier . 10081] In some embodiments of the enucleated 100671 In some embodiments , the pharmaceutical compo hematopoietic cell disclosed herein , the exogenous antigen sition comprises a population of hematopoietic cells is a fusion or a chimeric polypeptide . expressing an exogenous antigen . In some embodiments , the [0082 ] In some embodiments of the enucleated pharmaceutical composition comprises at least 1x103 hematopoietic cell disclosed herein , the S domain , the A hematopoietic cells expressing an exogenous antigen . domain , and /or the U domain are of different polypeptide [ 0068 ] In certain embodiments of the pharmaceutical origin . composition , the hematopoietic cells expressing an exog [0083 ] In certain embodiments of the enucleated enous antigen are provided in a volume of about 10 nl, 100 hematopoietic cell disclosed herein , the S domain and /or the nl, 1 ul, 10 ul, 100 ul, 1 ml, 10 ml, 20 ml, or 50 ml. In other A domain comprises at least 5 , 6 , 7 , 8 , 9 , 10 , 15 , 20 , 25 , 30 , embodiments of the pharmaceutical composition , the 40 , 50 , 60 , 70 , 80 , 90 , 100 , 150 , 200 , 250 , or at least 500 hematopoietic cells expressing an exogenous antigen are amino acids . [0084 ] In certain embodiments of the enucleated provided in a volume of about 1 ml, 10 ml, 20 ml, 50 ml, 100 hematopoietic cell disclosed herein , the S domain and/ or A ml, 250 ml, or 500 ml. domain comprises at least 500 , 750 , or at least 1 , 000 amino [0069 ] In some embodiments of the pharmaceutical com acids. position , the composition is formulated for long - term stor [0085 ] In some embodiments of the enucleated age . In some embodiments of the pharmaceutical composi hematopoietic cell disclosed herein , the exogenous antigen tion , the composition is frozen . In some embodiments, the is selected from the group consisting of myelin basic protein , pharmaceutical composition comprises a pharmaceutically proteolipid protein , myelin oligodendrocyte glycoprotein , active agent. pancreatic beta cell antigen , insulin , and those listed in Table [0070 ] In certain embodiments of the pharmaceutical F , Table 6 , and Table 8 . composition , the pharmaceutically active agent is selected [ 0086 ] In some embodiments , the enucleated hematopoi from a biological agent, a small molecule agent, or a nucleic etic cell comprises at least 10 copies, 100 copies, 1 ,000 acid agent. copies, 10 , 000 copies, 25 , 000 copies , 50 , 000 copies, 100 , [0071 ] In some aspects, disclosed herein is a dosage form 000 copies, 500 ,000 copies , 1, 000 ,000 copies, or 2 ,000 ,000 comprising the compositions described herein formulated as copies of the exogenous antigen . a liquid suspension for intravenous injection . [0087 ] In certain embodiments , the enucleated hematopoi [0072 ] In some aspects , disclosed herein is a medical etic cell is an erythroid cell, a thromboid cell , or a precursor device comprising a container holding the pharmaceutical thereof. In some embodiments , the erythroid cell is an compositions described herein and an applicator for intra erythrocyte or a reticulocyte . In some embodiments , the venous injection of the pharmaceutical composition to the thromboid cell is a platelet. subject. [0088 ] In some embodiments , the enucleated hematopoi [0073 ] In some aspects , disclosed herein is a medical kit etic cell is isolated from a donor. In some embodiments , the comprising the pharmaceutical compositions described enucleated hematopoietic cell is autologously derived from US 2018 /0271910 A1 Sep . 27 , 2018

the subject. In some embodiments , the enucleated [0101 ] In some embodiments , the disease is selected from hematopoietic cell is allogeneically derived . In some the group consisting of a self - antibody mediated disease , an embodiments , the enucleated hematopoietic cell is xenoge autoimmune disease , an inflammatory disease , an allergic neically derived . disease , an HLA -mismatch mediated disease , and a disease [0089 ] In certain embodiments , the enucleated hematopoi treatable by an immunogenic therapeutic protein . etic cell is derived from a nucleated precursor cell by a [0102 ] In some aspects , disclosed herein is a method of culture -based process that induces the expulsion of its reducing or alleviating an immune activation in response to nucleus. a therapeutic protein treatment regimen comprising admin (0090 ] In certain embodiments, the enucleated hematopoi istering to a subject in need thereof the pharmaceutical etic cell is generated from a nucleated precursor cell that is compositions described herein in an amount and / or fre chemically or physical manipulated to remove its nucleus . quency sufficient to substantially reduce or alleviate the [ 0091] In certain embodiments , the enucleated hematopoi immune activation . etic cell is generated by irradiation or chemical destruction 10103 ] In some embodiments , the therapeutic protein is of the nucleus of a nucleated precursor cell . In some selected from the group consisting of those listed in Table I , embodiments , the chemical destruction is carried out with Table J , and Table 7 . Cytochalasin B . In some embodiments , the irradiation is [0104 ] In some aspects , disclosed herein is an expression carried out with at least 5 Gy, 7 Gy , 10 Gy, 15 Gy, 25 Gy , vector comprising a nucleic acid sequence encoding an 30 Gy, 40 Gy or at least 50 Gy. endogenous erythroid cell protein fused with one or more 10092 ] In some embodiments of the enucleated exogenous polypeptide antigens selected from the group hematopoietic cell disclosed herein , the exogenous antigen consisting of those listed in Table F , Table G , Table H , Table is a polypeptide encoded by an exogenous nucleic acid . I , Table J , Table 6 , Table 7 , and Table 8 . [0093 ] In some embodiments of the enucleated [0105 ] In some aspects , disclosed herein is a messenger hematopoietic cell disclosed herein , the cell is derived from RNA comprising a nucleic acid sequence encoding an a human source . endogenous erythroid cell protein fused with one or more [0094 ] In some embodiments of the enucleated exogenous polypeptide antigens selected from the group hematopoietic cell disclosed herein , the cell is derived from consisting of those listed in Table F , Table G , Table H , Table a non -human source, selected from the group consisting of I , Table J , Table 6 , Table 7 , and Table 8 . pig , chimpanzee , macaque, a non -human primate , and a [0106 ] In some aspects , disclosed herein is a method of non - primate mammal. inducing immune tolerance comprising administering to a 10095 ] In some embodiments of the enucleated human subject suffering from or at risk of developing an hematopoietic cell disclosed herein , the polypeptide antigen allergen -mediated disease, disorder or condition , a pharma is localized intracellularly . In some embodiments of the ceutical composition comprising an enucleated hematopoi enucleated hematopoietic cell disclosed herein , the polypep etic cell expressing an exogenous antigen , wherein the tide antigen is localized extracellularly on the surface of the pharmaceutical composition is administered in an amount cell . In some embodiments of the enucleated hematopoietic effective to induce immune tolerance in the subject to the cell disclosed herein , the polypeptide antigen is fused to an allergen mediating the disease, disorder or condition . endogenous cell protein . In some embodiments of the 0107 ] In certain embodiments, the exogenous antigen is enucleated hematopoietic cell disclosed herein , the polypep selected from the group consisting of Ara h2 , 2S albumin , tide antigen is fused to an intracellular region of an endog hyalauronidase , and those listed in Table H . enous transmembrane protein . In some embodiments of the [0108 ] In certain embodiments , the allergen -mediated dis enucleated hematopoietic cell disclosed herein , the polypep ease , disorder or condition is selected from the group tide antigen is fused to an extracellular region of an endog consisting of peanut allergy , tree nut allergy , insect venom enous transmembrane protein . In some embodiments of the allergy , and those listed in Table H . enucleated hematopoietic cell disclosed herein , the polypep [0109 ] In some aspects , disclosed herein is a method of tide antigen is fused to a glycosylphosphatidylinisotol (GPI ) inducing immune tolerance comprising administering to a anchored protein . human subject suffering from or at risk of developing a 0096 ] In some aspects , disclosed herein is a tissue culture human leukocyte antigen (HLA ) mismatch -mediated dis batch comprising the enucleated hematopoietic cells ease , disorder or condition , a pharmaceutical composition described herein . comprising an enucleated hematopoietic cell expressing an [ 0097 ] In some aspects , disclosed herein is a population of exogenous antigen , wherein the pharmaceutical composition the enucleated hematopoietic cells described herein . is administered in an amount effective to induce immune [0098 ] In some aspects , disclosed herein is a pharmaceu tolerance in the subject to the HLA mediating the disease , tical composition comprising the cell populations described disorder or condition . herein . [0110 ] In some aspects , disclosed herein is a method of [0099 ] In some aspects , disclosed herein is a method of inducing immune tolerance comprising : administering to a inducing immune tolerance comprising administering to a human subject suffering from or at risk of developing a subject in need thereof the pharmaceutical compositions disease , disorder or condition that can be treated by an described herein in an amount and /or a frequency sufficient immunogenic therapeutic molecule , a pharmaceutical com to induce immune tolerance in the subject. position comprising an enucleated hematopoietic cell [0100 ] In some aspects , disclosed herein is a method of expressing an exogenous antigen , wherein the pharmaceu treating an immune activation disease comprising adminis tical composition is administered in an amount effective to tering to a subject in need thereof the pharmaceutical induce immune tolerance in the subject to the immunogenic compositions described herein in an amount and / or fre - therapeutic molecule used to treat the disease , disorder or quency sufficient to treat the immune activation disease . condition . US 2018 /0271910 A1 Sep . 27 , 2018

[0111 ] In some embodiments , the therapeutic molecule is [0128 ] FIG . 3R — Cytoplasmic expression of adenosine selected from the group consisting of Recombinant ( factor deaminase fused to the intracellular C terminus of glyco VIII) , Benefix ( factor IX ), Humira ( anti- TNFa ) , and those phorin A assessed by anti- HA Western blot. Expected size listed in Table I , Table J , and Table 7 . approximately 55 kDa . [0129 ] FIG . 38 — Cytoplasmic expression of phenylala BRIEF DESCRIPTION OF THE FIGURES nine hydroxylase fused to the intracellular C terminus of glycophorin A assessed by anti -HA Western blot . Expected [0112 ] FIG . 1 is a collection of flow cytometry plots of red size approximately 50 kDa. blood cells contacted with fluorescently labeled IgG encap [0130 ] FIGS. 3 T - AO shows the exogenous expression of sulated within liposomes . Cells are shown that are incubated surface and cytoplasmic proteins on nucleated cultured with no liposomes ( left ), a low dose of liposomes ( center ), erythroid precursor cells . and a high dose of liposomes ( left ) . On the bottom histo [0131 ] FIG . 3T — Expression of glycophorin Awith an HA grams, the percentage of cells that are fluorescent is shown. epitope tag at the cytoplasmic C terminus assessed by [0113 ] FIG . 2 is a plot of cell surface expression levels expression of co - translated GFP . assessed by quantitative flow cytometry . The plot shows of [0132 ] FIG . 30 — Expression of glycophorin A with an various cell surface receptors — glycophorin A ( solid tri HA epitope tag at the N terminus between the leader angles ), CKIT (dashed squares ) , transferrin receptor ( dotted sequence and the body of the gene assessed by anti -HA diamonds) — and an exogenous surface transgene (open staining. circles ) during the course of erythroid cell differentiation . [0133 ] FIG . 3V - Overexpression of complement receptor [0114 ] FIGS. 3 A - C , F , I- M , O - Z , and AA - AU is a col 1 assessed by anti - CR1 staining. lection of flow cytometry plots and Western blots that [0134 ] FIG . 3W - Expression of complement receptor demonstrate the expression of a vast array of exemplary 1 - derived fragment of ~ 70 kDa with an HA epitope tag at the antigens on the surface , in the cytoplasm , as fusions, and as N terminus assessed by anti- HA staining . intact proteins, in three cell types, enucleated erythroid cells, [0135 ] FIG . 3X - Expression of complement receptor nucleated erythroid precursor cells , and erythroleukemic 1 -derived fragment of ~ 210 kDa with an HA epitope tag at cells . the N terminus assessed by anti -HA staining. [0115 ] FIGS. 3 A - C , F , I - M , and O - S shows the exogenous 10136 ] FIG . 3Y — Expression of complement receptor expression of surface and cytoplasmic proteins on enucle 1 -derived fragment of - 230 kDa fused to the N terminus of ated cultured erythroid cells . glycophorin A with an HA epitope tag at the N terminus [0116 ] FIG . 3A - Expression of glycophorin A with an HA assessed by anti- HA staining . epitope tag at the cytoplasmic C terminus assessed by [0137 ] FIG . 3Z — Expression of antibody scFv as N ter expression of co - translated GFP. minal fusion to glycophorin A assessed by anti -HA staining . [0117 ] FIG . 3B — Expression of glycophorin A with an HA [0138 ] FIG . 3AA — Expression of antibody scFv fused to epitope tag at the N terminus between the leader sequence the extracellular C terminus of Kell , assessed by anti - HA and the body of the gene assessed by anti - HA staining. staining . Expected size approximately 108 kDa . [ 0118 ] FIG . 3C — Expression of complement receptor [0139 ] FIG . 3AB — Expression of HA tag fused to the 1 -derived fragmentof ~ 70 kDa with an HA epitope tag at the extracellular C terminus of Kell , assessed by anti -HA stain N terminus assessed by anti- HA staining . ing . [0119 ] FIG . 3F — Expression of antibody scFv as N termi 10140 ] FIG . 3AC — Expression of Kell - derived fragment nal fusion to glycophorin A assessed by anti -HA staining. of 71 amino acids with HA tag at the C ( extracellular ) [0120 ] FIG . 31 — Expression of antibody scFv fused to C terminus assessed by anti -HA staining . terminus of Kell- derived fragment of 71 amino acids [0141 ] FIG . 3AD Expression of Kell - derived fragment assessed by anti- HA staining . of 79 amino acids with HA tag at the C terminus assessed by [0121 ] FIG . 3 ) — Expression of antibody scFv fused to C anti -HA staining . terminus of Kell -derived fragment of 79 amino acids [0142 ] FIG . 3AE — Expression of antibody scFv fused to assessed by anti- HA staining . C terminus of Kell -derived fragment of 71 amino acids [0122 ] FIG . 3K — Expression of CD55 with HA epitope assessed by anti -HA staining tag at the extracellular N terminus after the leader sequence 10143 ] FIG . 3AF - Expression of antibody scFv fused to C assessed by anti -HA staining . terminus of Kell -derived fragment of 79 amino acids [0123 ] FIG . 3L — Expression of CD59 with HA epitope assessed by anti -HA staining . tag at the extracellular N terminus after the leader sequences 10144 ] FIG . 3AG — Expression of CD55 with HA epitope assessed by anti- HA staining . tag at the extracellular N terminus after the leader sequence [0124 ] FIG . 3M - Expression of antibody scFv fused to assessed by anti -HA staining . N -terminus of CD55 -derived fragment of 37 amino acids, [0145 ] FIG . 3AH — Expression of CD59 with HA epitope assessed by anti -HA Western blot. tag at the extracellular N terminus after the leader sequences [0125 ] FIG . 30 — Cytoplasmic expression of adenosine assessed by anti- HA staining . deaminase fused to HA tag assessed by anti -HA Western [0146 ] FIG . 3A1— Expression of antibody scFv fused to blot . Expected size approximately 40 kDa. N -terminus of CD55 - derived fragment of 37 amino acids, [0126 ] FIG . 3P % Cytoplasmic expression of phenylala assessed by anti -HA staining . nine hydroxylase fused to HA tag assessed by anti -HA [0147 ] FIG . 3AJ — Expression of antibody scFv fused to Western blot. Expected size approximately 33 kDa. N - terminus of CD59 assessed by anti- HA staining . [0127 ] FIG . 3Q - Cytoplasmic expression of phenylala [0148 ] FIG . 3AK — Cytoplasmic expression of adenosine nine hydroxylase fused to adenosine deaminase and an HA deaminase fused to HA tag assessed by anti -HA Western tag assessed by anti -HA Western blot. blot . Expected size approximately 40 kDa. US 2018 /0271910 A1 Sep . 27 , 2018

[0149 ] FIG . 3AL - Cytoplasmic expression of phenylala immune complexes ( hashed bars ), complement deficient nine hydroxylase fused to HA tag assessed by anti - HA immune complexes (gray bars ), or no immune complexes Western blot. Expected size approximately 33 kDa. (black bars ) by macrophages ( left set) or macrophages [0150 ] FIG . 3AM Cytoplasmic expression ofphenylala incubated with cultured erythroid cells that overexpress CR1 nine hydroxylase fused to adenosine deaminase and an HA (right set) . tag assessed by flow cytometry for fluorescence from co [0163 ] FIGS. 7A -7D show the activity of an antibody scFv translated GFP . that binds hepatitis B surface antigen ( scFv ) on the surface [0151 ] FIG . 3AN — Cytoplasmic expression of adenosine of a cultured erythroid cell. ( FIG . 7A ) is a flow cytometry deaminase fused to the intracellular C terminus of glyco histogram showing binding of 450 nM antigen (white his phorin A assessed by anti -HA Western blot. Expected size togram ) or no antigen ( gray histogram ). (FIG . 7B ) is a approximately 55 kDa. titration of binding signal assessed by flow cytometry for a [0152 ] FIG . 3A0 _ Cytoplasmic expression of phenylala range of antigen concentrations. ( FIGS . 7C - D ) are flow nine hydroxylase fused to the intracellular C terminus of cytometry plots of blood cells from mice that had been glycophorin A assessed by anti- HA Western blot. Expected injected with fluorescent antigen and cultured erythroid cells size approximately 50 kDa. that (FIG . 7C ) do not or (FIG . 7D ) do express scFv. The [0153 ] FIGS. 3 AP - AU shows the exogenous expression y - axis measures antigen fluorescence. The x - axis measures of surface and cytoplasmic proteins on K562 erythroleuke fluorescence of the cultured cells . mia cells . [ 0164 ] FIGS. 8A -8D show the specific clearance of cir [0154 ] FIG . 3AP - Overexpression of complement recep culating antibodies mediated by exogenous antigen -express tor 1 assessed by anti - CR1 staining . ing EHCs in vivo . (FIG . 8A ) is a set of flow cytometry plots [ 0155 ] FIG . 3AQExpression of antibody scFv as N that show no binding ( top ) and binding (bottom ) of circu terminal fusion to glycophorin A assessed by anti -HA stain lating Dylight650 - labeled mouse anti -HA antibody to ing . CFSE - labeled cultured human erythroid cells isolated from [ 0156 ] FIG . 3AR — Expression of antibody scFv fused to a recipient mouse that either do not ( top ) or do (bottom ) N - terminus of CD55 - derived fragment of 37 amino acids , express HA epitope tag on their surface. The x -axis mea assessed by anti- HA staining . sures CFSE fluorescence . The y - axis measures anti -HA [0157 ] FIG . 3AS — Expression of antibody scFv fused to antibody Dylight650 fluorescence . (FIG . 8B ) is data from an N -terminus of CD59 assessed by anti -HA staining . HA epitope tag substrate ELISA comparing anti -HA anti [0158 ] FIG . 3AT — Cytoplasmic expression of adenosine body levels over time in plasma collected from mice injected deaminase fused to HA tag assessed by anti -HA Western with anti- HA antibody (open circles , solid line ) , anti -HA blot. Expected size approximately 40 kDa. antibody followed by cultured human erythroid cells that do [ 0159 ] FIG . 3AU — Cytoplasmic expression of phenylala not express HA epitope tag (dashed line ), or anti -HA anti nine hydroxylase fused to HA tag assessed by anti - HA body followed by cultured human erythroid cells that do Western blot. Expected size approximately 33 kDa. express HA epitope tag (dotted line ). (FIG . 8C ) is a set of [0160 ] FIGS. 4A -4C are a collection of flow cytometry flow cytometry plots that show no binding ( top ) and binding histograms that measure fluorescence in primary platelets (bottom ) of Dylight650 - labeled mouse anti - biotin antibody that have been transfected with mRNA encoding a fluores to CFSE - labeled primary human erythrocytes that either are cent protein (GFP ) . ( FIG . 4A ) Untransfected platelets . ( FIG . not (top ) or are ( bottom ) conjugated with biotin on their 4B ) Platelets transfected with 3 ug GFP mRNA . ( FIG . 4C ) surface . The x -axis measures CFSE fluorescence . The y - axis Platelets transfected with 6 . 8 ug GFP mRNA . measures anti -biotin antibody Dylight650 fluorescence . [0161 ] FIGS. 5A -5D show protein expression and enzy (FIG . 8D ) is data from a biotin substrate ELISA comparing matic activity of transgenic erythroid cells in culture . ( FIG . anti -biotin antibody levels over time in plasma collected 5A ) is a Western blot of exogenously expressed adenosine from mice injected with anti - biotin antibody (open circles , deaminase detected with an anti -HA antibody over the solid line ) , anti -biotin antibody followed by cultured human course of differentiation , from nucleated precursor cells erythroid cells that are not conjugated to biotin ( dashed line ), ( “ Diff I D5% ) through to enucleated erythroid cells (“ Diff III or anti -biotin antibody followed by cultured human eryth D8 ” ) . ( FIG . 5B ) is a bar chart of inosine produced from roid cells that are conjugated to biotin (dotted line ). adenosine by intact adenosine deaminase - expressing 293T [0165 ) FIGS. 9A -9B show the clearance rate of cultured cells . ( FIG . 5C ) is a Western blot of the exogenously human eyrthroid cells in a mouse . (FIG . 9A ) is a represen expressed phenylalanine hydroxylase detected with an anti tative flow cytometry dot- plot of drawn blood , stained for HA antibody at various time points over the course of human glycophorin A ( y -axis ) and CFSE ( x - axis ), in which differentiation , from nucleated precursor cells (“ Diff I D5 " ) human cultured cells are double -positive . (FIG . 9B ) is a plot through to enucleated erythroid cells (“ Diff III D8” ). (FIG . of the clearance rate over time as a percentage of double 5D ) is a bar chart of tyrosine produced from phenylalanine positive cells remaining after NSG mice were injected with by lysates of cultured phenylalanine hydroxylase -expressing with human red blood cells ( solid circles ) , cultured enucle enucleated erythroid cells . ated erythroid cells (dashed diamonds ) , cultured enucleated [0162 ] FIGS. 6A -6B show immune complex capture and erythroid cells that express an intracellular exogenous pro transfer to macrophages by cultured erythroid cells that tein (dotted squares ) and cultured enucleated erythroid cells overexpress complement receptor 1 (CR1 ) . (FIG . 6A ) is a that express a surface exogenous protein (open triangles) . flow cytometry plot that shows the capture of fluorescent [0166 ] FIGS. 10A - 10D are an assessment of adverse immune complexes ( white histogram ) and complement events following injection of cultured human erythroid cells deficient immune complexes ( shaded histogram ) by cultured into a mouse . ( FIGS . 10A - B ) show levels of ( FIG . 10A ) erythroid cells that overexpress CR1. ( FIG . 6B ) is a bar chart fibrinopeptide A and ( FIG . 10B ) fibrinopeptide B assessed of flow cytometry data assessing the uptake of fluorescent by ELISA in plasma collected from mice 20 minutes (black ) , US 2018 /0271910 A1 Sep . 27 , 2018

6 hours ( gray ) , and 48 hours (white ) after injection with ( 1 ) comprise one or more antigens that comprise or consist of human red blood cells , ( 2 ) cultured human erythroid cells , one or more polypeptides, lipids, and / or carbohydrates , and ( 3 ) cultured human erythroid cells expressing an exogenous any combination thereof. The cells can be circulating cells, cytoplasmic protein , ( 4 ) cultured human erythroid cells such as EHCs . The EHCs can be cultured from hematopoi expressing an exogenous surface transgene , or ( 5 ) recom etic precursors using defined factors such as e . g . stem cell binant protein . (FIGS . 10C - D ) show microscope images of factor , cytokines such as IL - 3 and IL - 6 , insulin , transferrin , histologically stained sections of spleen for mice injected erythropoietin , hydrocortisone , and estrogens . with (G FIG . 10C ) cultured human erythroid cells and ( FIG . 0171 ] Aspects of the invention relate to methods of 10D ) recombinant protein . culturing EHCs to comprise exogenous antigens of interest. [0167 ] FIGS . 11A - 11B track the expression of exogenous The exogenous antigens of interest can be introduced by a protein on cultured erythroid cells in circulation . (FIG . 11A ) number of methods , such as, e . g . intracellular expression , is flow cytometry data of blood drawn from a mouse that cell - surface expression , fusion to an endogenous protein , was injected with cultured human erythroid cells expressing conjugation by chemical or enzymatic means to a cell an exogenous surface protein , showing the the percent of surface protein , or physical loading into the intracellular cultured human erythroid cells that are HA -positive over space . The antigen - comprising cells of the invention may be time. ( FIG . 11B ) is a Western blot of blood drawn from two used as therapeutic agents . mice , wherein one mouse was injected with cultured human [0172 ] Aspects of the invention relate to the use of these erythroid cells expressing an exogenous cytoplasmic pro antigen -comprising cells in the treatment of diseases of tein , and wherein the other mouse was injected with the immune activation by the induction of peripheral tolerance . purified recombinantly -produced exogenous protein in the In some aspects , induction of peripheral tolerance means the absence of any cells , showing the level of HA -containing deletion or inactivation of antigen - specific immune cells , protein in the blood over time. such as, e. g . CD8 T lymphocytes (CD8 T cells ), CD4 T [0168 ] FIGS. 12A - 12C are an assessment of expansion lymphocytes (CD4 T cells ), CD4 T regulatory lymphocytes and differentiation of cultured human erythroid cells . ( FIG . ( Treg ), or B lymphocytes (B cells ). Diseases of immune 12A ) is a plot of expansion rates for distinct cultures of in activation include autoimmune diseases , such as, e . g . mul vitro differentiated erythroid cells that contain transgenes tiple sclerosis , type 1 diabetes , rheumatoid arthritis , and (dashed line and dotted line ) and cells that do not contain a membranous nephritis. Diseases of immune activation also transgene ( solid line ) . ( FIG . 12B ) is a flow cytometry plot of include inflammatory diseases, such as , e . g . Crohn ' s disease , cell surface markers GPA and CKIT for distinct cultures of ulcerative colitis, celiac disease , or other idiopathic inflam cultured human erythroid cells that do not ( left ) or do (right ) matory bowl diseases . Diseases of immune activation also contain a transgene . ( FIG . 12C ) is a flow cytometry plot of include allergic diseases , such as , e . g . asthma, peanut cultured human erythroid cells that do not (left ) or do (right ) allergy , shellfish allergy, pollen allergy , milk protein allergy , contain a transgene , wherein the cells are stained with DNA insect sting allergy , and latex allergy. Diseases of immune stain DRAQ5 ( y - axis ) and anti - glycophorin A ( x - axis ) , activation also include immune activation in response to a which identifies distinct populations of ( 1 ) enucleated cells , therapeutic protein , administered to treat a primary condi ( 2 ) nucleated cells , and ( 3 ) nuclei. tion , that lessens the efficacy of the therapeutic protein , such [ 0169 ] FIG . 13A is a schematic of three ways in which an as, e . g ., clotting factor VIII in hemophilia A , clotting factor antigen may be localized in an exogenous antigen - express IX in hemophilia B , anti- tumor necrosis factor alpha ( TNFa ) ing EHC . FIG . 13B is a schematic of three ways in which an antibodies in rheumatoid arthritis and other inflammatory antigen localized in or on an exogenousan exogenous anti diseases , glucocerebrosidase in Gaucher ' s disease , or gen - expressing EHC may act on a target in circulation . FIG . asparaginase in acute lymphoblastic leukemia (ALL ) . 13C is a schematic of an auto - catalytic fusion of an endog enous polypeptide anchor to an antigen utilizing a SpyTag Biology of Immune Tolerance SpyCatcher mechanism . [0173 ] The body has evolved sophisticated mechanisms for the prevention of aberrant immune activation and auto DETAILED DESCRIPTION OF THE immune disease , collectively termed immune tolerance . INVENTION Central tolerance refers to the antigen - specific deletion of [0170 ] The invention , in certain aspects , provides isolated autoreactive T cells and B cells during development in the cells that are engineered or modified to contain exogenous primary lymphoid organs, e . g . thymus and bone marrow . antigens of interest. In certain aspects , isolated EHCs of the Peripheral tolerance refers to the deletion or inactivation of invention comprise one or more antigens that comprise or mature T and B lymphocytes outside of the primary lym consist of polypeptides . In some embodiments , the antigen phoid organs . Peripheral tolerance includes the suppression is a full - length protein . In some embodiments , the antigen is of autoreactive lymphocytes by regulatory T cells ( Tregs ) or comprised of one or more polypeptides contained within the the induction of anergy or non - responsiveness in antigen full - length protein , of any length greater than approximately specific effector lymphocytes by exposure to continuous low 7 amino acids. The polypeptides comprising the antigen may doses of antigen in the absence of costimulatory “ danger " comprise one or more immunological epitopes which may signals . Both Treg activation and lymphocyte anergy can be be conformational epitopes or may be linear epitopes . The induced by the secretion of inhibitory factors such as, for antigen may be comprised of one or more polypeptides from example , TGF -beta , IL - 10, and IL -4 . one or more different proteins . In certain aspects, EHCs of [0174 ] Immune activation in response to antigen often the invention comprise one or more antigens that comprise requires a secondary " danger ” signal, such as a toll - like or consist of carbohydrates . In certain aspects , EHCs of the receptor ligand , often derived from microbial or viral patho invention comprise one or more antigens that comprise or gens (Matzinger , Annu Rev Immuno 1994 ) . Such danger consist of lipids . In certain aspects , EHCs of the invention signals include double -stranded RNA , single -stranded US 2018 /0271910 A1 Sep . 27 , 2018

DNA , lipopolysaccharide , bacterial lipoproteins, flagellin , Although the exact triggers of erythrocyte clearance remain zymosan , and others. Antigen presenting cells that receive unclear , eryptotic erythrocytes are characterized by phos both antigen and danger signal display costimulatory mol phatidylserine asymmetry , membrane heterogeneity , and ecules on their surface , like CD80 and CD86 , in addition to annexin - V binding, analogous to apoptotic nucleated cells . the antigenic peptides . T cells that recognize both the [0181 ] EHCs are also persistent in the body. Erythrocytes antigenic peptide and the costimulatory molecules become circulate for up to 120 days in the adult human . As such , activated . Those that receive just the antigenic peptide signal EHCs that comprise an antigen of interest may enable the become anergic . persistent exposure of the antigen to the host. As described [0175 ] Therapeutic strategies that take advantage of anti above , though the exact molecular mechanisms are not fully gen presentation in the absence of danger signals to induce understood , it is thought that persistent exposure to antigen immune tolerance have been developed for the experimental can induce peripheral tolerance through antigen presentation treatment ofmany food allergies. The studies take the form in the absence of costimulatory signals, leading to the of prolonged exposure to increasing doses of allergen with expansion of regulatory T cells , the deletion and anergy of the intent to induce tolerance . Thirteen studies since 2007 effector T and B cells , and the secretion of anti - inflammatory have tested a variety of common food allergens like peanut, and pro - tolerogenic cytokines . milk , and egg , in this format. 50 - 100 % of patients become [0182 ] The induction of peripheral tolerance by taking sensitized , that is , able to survive a food challenge without advantage of erythrocytes have been explored experimen anaphylaxis . However, long term tolerance is less success tally as well . In preliminary work , the model antigen oval ful, with only 25 -50 % of patients able to tolerate antigen bumin has been shown to induce antigen - specific CD8 T cell after one month off therapy . See, e .g . Burks et al. , New deletion and antigen - specific Treg induction when non England Journal ofMedicine 2012 . covalently attached to erythrocytes (Kontos et al . , PNAS [ 0176 ] It is thought that allergy is IgE mediated , with 2013 ) or osmotically loaded into erythrocytes (Cremel and activation ofmast cells and basophils . Oral administration of Godfrin , Int J Pharm 2013 ) . the allergen in low dose , such as continuous feeding , induces [ 0183 ] Cultured EHCs of the invention comprising an Tregs via CD11c + dendritic cell presentation of antigen and exogenous antigen of interest may have distinct advantages secretion of TGF -beta , IL - 10 , and IL - 4 . Oral administration over antigen that is non - covalently attached to erythrocytes , at high doses induces antigen - specific T cell deletion and e . g . via a polypeptide binding domain . One advantage may anergy via plasmacytoid dendritic cells . In human studies, be that the bio - distribution of an EHC comprising an exog oral administration of allergen results in a decrease in IgE , enous antigen of interest is more defined than that of a mast cells , and basophils , an increase in IgG4, TGF -beta , polypeptide composition of antigen with targeting domain . IL - 10 , and a temporary uptick in Tregs at the start of therapy . The EHC comprising an exogenous antigen of interest will See, e .g . Herzog , Adv Drug Deliv Rev 2013 . be confined to the vasculature and to places that erythrocytes [0177 ] While not wishing to be limited to any particular typically reside, e . g . spleen . EHCs comprising an exogenous mechanism , it is believed that peripheral immunologial antigen of interest will not be filtered out of the kidney or tolerance can be induced by self antigens from apoptosing exit into peripheral tissue, problems that may arise when cells (Griffith and Ferguson , Immunity 2011 ; Green et al. , polypeptide antigen compositions are administered . The Nat Rev Immunol 2009 ). Though the exact mechanisms are dose of exogenous antigen per EHC may be significantly not fully understood from a molecular perspective , self higher if the cells comprising an exogenous antigen of proteins such as HSP90 and other damage -associated interest are cultured than if a polypeptide antigen is directly molecular patterns facilitate uptake by dendritic cells . Den injected into the bloodstream and distributed across approxi dritic cell receptors like CD205 recognize these signals , mately 10 trillion erythrocytes in the bloodstream . In some cross -present antigen , and induce tolerogenic cytokines and instances , it may be preferable to have the exogenous suppress costimulatory protein expression (Bonifaz , J Exp antigen of interest confined in the intracellular compartment Med 2002 ) . of the EHC . For example, if the antigen is immunogenic , [ 0178 ] Therapeutic strategies that harness the tolerogenic intracellular localization may be advantageous because it potential of apoptosing cells to induce peripheral immune may mask the immunogenic antigen from the immune tolerance are under investigation . These strategies typically system and thus prevent or reduce immune activation . This involve the chemical coupling of antigens of interest to the configuration is not possible with a polypeptide antigen surface of cells . In studies in mice , rat, and guinea pigs, a composition . variety of protein antigens are chemically attached to the [0184 ] Cultured EHCs that express an exogenous antigen surface of splenocytes and leukocytes . See , e . g . , Miller et al. , of interest may have distinct advantages over antigen that is J Exp Med 1979 ; Braley -Mullen et al. , Cell Immunol 1980 ; osmotically loaded into EHCs. The cultured EHCs compris Luo et al. , PNAS 2008 ; Smarr et al . , J Immunol 2011. ing an exogenous antigen of interest will have cell mem 0179 ] In a recent phase I clinical study in humans , a branes and cytoskeleton that are substantially unaltered , in cocktail of peptide antigens associated with multiple scle contrast to the product of an osmotic loading procedure , in rosis were chemically coupled to autologous peripheral which large pores breach the integrity of the cell membrane blood mononuclear cells and reinfused into patients ( Lut and cytoskeleton . The morphology and biophysical charac terotti and Martin , Sci Trans Med 2013 ). The cells were well teristics of EHCs are crucial determinants of the cell ' s tolerated , and there was evidence of a decrease in antigen bio - distribution , circulation , and interaction with the vascu specific T cell responses . lature and immune cells (e . g . Pries et al ., Cardiovasc. Res . [0180 ] EHCs are a prominent source of dying cells . A 1996 ) , and hence maintaining cell integrity may be crucial large number of erythrocytes are cleared after apoptosis - like to retain efficaciousness . Exogenous antigen that is physi programmed cell death , called eryptosis , each day (more cally attached to a cultured EHC , for example by direct than 1 % per day in humans, approximately 1x101 cells ) . fusion to an endogenous cytoplasmic protein or fusion to an US 2018 /0271910 A1 Sep . 27 , 2018 endogenous transmembrane protein , will not leak out of the described herein in the presence of multiple growth factors cell and be exposed to the immune system until the EHC is including , for example , hydrocortisone, stem cell factor, consumed . The problem of leakage may arise if the cell is IL - 3 , and erythropoietin . In the second stage , the cells may contacted with the antigen using an osmotic loading proce optionally be co - cultured , for example , on an adherent dure in which the cell membrane can be damaged . stromal layer in the presence of erythropoietin . In a third [0185 ] Cultured EHCs that express an antigen of interest stage , the cells may be cultured on an adherent stromal layer can be administered directly to a subject in need of the in culture medium in the absence of exogenous factors. The antigen . A separation and purification of the antigen during adherent stromal layer may be murine MS- 5 stromal cells , manufacture of the product is not required . This is in contrast for example . Alternatively , the adherent stromal layer may to an osmotic loading product, in which the antigen must be be mesenchymal stromal cells derived from adult bone synthesized and purified separately and then combined with marrow . The adherent stromal cells may be maintained in the cell , and may provide a significant cost and time advan RPMI supplemented with 10 % fetal calf serum , for example . tage in the manufacture of the product. The cultured EHCs In some embodiments , the erythroid precursor cells and cell that express an antigen of interest can be scaled up by populations derived therefrom are not co - cultured with propagation in culture . Large, industrial- size batches of cells non -EHCs , e . g . , with an adherent stromal layer , i . e . they are may be produced to generate a substantially uniform phar cultured in the absence of non - EHCs. In some embodiments , maceutical composition of EHCs for a given antigen that can EHCs comprising an antigen are cultured in the absence of be used to universally treat many subjects . In contrast , non -EHCs and are differentiated so that greater than 10 % , osmotic loading is generally limited to a one -donor - to -one 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , 60 % , subject scale . 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % or greater than 98 % of EHCs are enucleated and the population of enucleated Acquisition of Cells cells is obtained without an enrichment step , such as gravi tational separation , magnetic or fluorescent sorting , irradia (0186 ] Exogenous antigen - expressing enucleated tion , poisoning of nucleated cells , and the like to select for hematopoietic cells of the invention can be generated by any enucleated cells . method described herein . In some embodiments, the steps [0188 ] In some instances , it may be desirable to expand comprise contacting isolated optionally cultured cells and partially differentiate the CD34 + hematopoietic stem derived from hematopoietic stem cells with an antigen . cells in vitro and to allow terminal differentiation into Hematopoietic stem cells give rise to all of the blood cell mature erythrocytes to occur in vivo (See , e . g ., Neildez types found in mammalian blood including myeloid (mono Nguyen et al . , Nature Biotech . 20 :467 -472 (2002 ) ) . Isolated cytes and macrophages, neutorphils , basophils , eosinophils , CD34 + hematopoietic stem cells may be expanded in vitro erythrocytes, megakaryocytes/ platelets , dendritic cells ) and in the absence of the adherent stromal cell layer in medium lymphoid lineages ( T - cells , B -cells , NK -cells ). Hematopoi containing various factors including, for example , Fit3 etic stem cells may be isolated from the bone marrow of ligand, stem cell factor, thrombopoietin , erythropoietin , and adult bones including, for example , femur, hip , rib , or insulin growth factor. The resulting erythroid precursor cells sternum bones . Cells may be obtained directly from the hip , may be characterized by the surface expression of CD36 and for example , by removal of cells from the bone marrow GPA , and may be transfused into a subject where terminal using aspiration with a needle and syringe . Alternatively, differentiation to mature erythrocytes is allowed to occur. hematopoietic stem cells may be isolated from normal [01891 . In some embodiments, the EHC population com peripheral blood following pre - treatment with cytokines prises a plurality of enucleated EHCs that comprise an such as, for example , granulocyte colony stimulating factor antigen polypeptide that is retained during enucleation . The ( G -CSF ) . G -CSF mobilizes the release of cells from the bone marrow compartment into the peripheral circulation . resulting isolated enucleated EHC comprising an antigen Other sources of hematopoietic stem cells include umbilical polypeptide exhibits substantially the same osmotic mem cord blood and placenta . brane fragility as a corresponding isolated , unmodified , 10187 ] Isolated hematopoietic stem cells may be cultured , uncultured EHC . expanded and differentiated ex vivo to provide a variety of [0190 ] In some embodiments , the EHC population com source material to generate exogenous antigen - expressing prises a plurality of erythrocyte precursor cells in substan EHCs. For example , hematopoietic stem cells isolated from tially the same stage of differentiation and /or cell cycle bone marrow , cytokine - stimulated peripheral blood or stage , wherein the precursor cells comprise a recombinant umbilical cord blood may be expanded and differentiated ex nucleic acid encoding an antigen . The majority of erythro vivo into mature erythrocytes (Giarratana et al . , Nature cyte precursor cells that comprise a recombinant nucleic Biotech . 23 :69 - 74 ( 2005 ) ; U . S . Patent Application 2007 / acid encoding an antigen are capable of differentiating into 0218552 ) . As such , CD34 + cells are isolated from bone mature erythrocytes that retain the antigen without retaining marrow or peripheral or cord blood using , for example , the recombinant nucleic acid . magnetic microbead selection and Mini- MACS columns [0191 ] In some embodiments , the primary cells may be (Miltenyi Biotech ) . In one example , the cells are subse collected through venipuncture , capillary puncture , or arte quently cultured in modified serum - free medium supple rial puncture . From the collected whole blood erythrocytes , mented with 1 % bovine serum albumin (BSA ) , 120 ug /ml platelets or other cells may then be isolated using one , or a iron - saturated human transferrin , 900 ng /ml ferrous sulfate , combination of techniques including plasma depletion , den 90 ng/ ml ferric nitrate and 10 rig /ml insulin and maintained sity gradient, Hetastarch , PrepaCyte - CB , and centrifugation . at 37° C . in 5 % carbon dioxide in air . Expansion and differentiation of the cell culture may occur in multiple Allogeneic / and Autologous Sourcing steps. For example , in the initial growth step following [0192 ] In some embodiments, generating an exogenousan isolation , the cells may be expanded in the medium exogenous antigen - expressing EHC comprises contacting US 2018 /0271910 A1 Sep . 27 , 2018 isolated optionally cultured cells that are autologous and / or galactosaminidase and a - galactosidase enzymatic activities allogeneic to the subject with an antigen . For example , derived from E . meningosepticum bacteria may be used to erythrocytes allogeneic to the subject include one or more of respectively remove the immunodominant A and B antigens blood type specific erythrocytes or one or more universal (Liu et al ., Nat . Biotech . 25 : 454 - 464 (2007 ) ) , if present on donor erythrocytes . In some embodiments , exogenous anti the exogenous antigen -expressing EHCs. In one example , gen - expressing EHCs may be generated through fusion of packed red blood cells isolated as described herein , are erythrocytes , e . g . , between erythrocytes autologous to the incubated in 200 mM glycine (pH 6 . 8 ) and 3 mM NaCl in subject and one or more allogeneic erythrocytes , liposomes, the presence of either a - N -acetylgalactosaminidase and and/ or artificial vesicles . a - galactosidase (about 300 ug /ml packed red blood cells ) for [0193 ] In certain embodiments , autologous transfusion of 60 min at 26° C . After treatment, the red blood cells are exogenous antigen -expressing EHCs includes isolating washed by 3 - 4 rinses in saline with centrifugation and erythrocytes , reticulocytes or hematopoietic stem cells from ABO - typed according to standard blood banking techniques. a subject , generating a suitable exogenous antigen - express [0196 ] In specific embodiments , the exogenous antigen ing EHC by contacting the cell with an antigen by methods expressing EHCs described herein may be generated in the described herein and administering ( e. g ., by transfusion ) the following way . First, erythroid precursor cells are isolated . exogenous antigen - expressing EHC into the same subject. These cells may alternatively be autologous to the patient or [ 01941. In certain embodiments , allogeneic transfusion of from substantially universal donor blood . For example , the exogenous antigen -expressing EHCs includes isolating cells may be ABO type 0 , rhesus factor Rh r/ r , Duffy - / - , erythrocytes, reticulocytes or hematopoietic stem cells from and large Kell antigen K1 negative . In the course of differ a donor , generating a suitable exogenous antigen - expressing entiation from erythroid precursor cell to EHC , a recombi EHC by contacting the cell with an antigen by methods nant nucleic acid encoding the antigen is introduced . The described herein and administering ( e . g . , by transfusion ) the recombinant nucleic acid encoding the antigen can be under exogenous antigen -expressing EHC into a subject that is the control of an erythroid -specific promoter , such as a different from the donor. Where allogeneic cells are used for GATA - 1 promoter ( see e . g . , Repik et al ., Clin Exp Immunol transfusion , care needs to be taken to use a compatible ABO 2005 , 140 : 230 ). The recombinant nucleic acid encoding the blood group to prevent an acute intravascular hemolytic antigen can be introduced in any way known in the art , for transfusion reaction which is characterized by complement example , as plasmid DNA , virus, or mRNA . Nucleic acid activation and lysis of incompatible erythrocytes . The ABO introduction can be achieved by a variety of standard blood types are defined based on the presence or absence of methods, e . g . , transfection , transduction , or electroporation . the blood type antigens A and B , monosaccharide carbohy drate structures that are found at the termini of oligosaccha Platelet Derivation and Maturation ride chains associated with glycoproteins and glycolipids on the surface of the erythrocytes ( reviewed in Liu et al. , Nat. [0197 ] In specific embodiments , the exogenous antigen Biotech . 25 :454 -464 (2007 )) . Group O erythrocytes lack expressing EHCs described herein may be generated by either of these antigenic monosaccharide structures . Sub contacting platelets with an antigen . Each day an adult jects with group A erythrocytes have naturally occurring human produces 2x1011 red blood cells , and about one - half antibodies to group B erythrocytes whereas subjects with as many white cells and platelets . In humans, nearly all group B erythrocytes have antibodies to group A erythro blood cell production occurs in the red bone marrow that cytes . Blood group AB subjects have neither antibody and represents a hierarchical developmental system composed of blood group o individuals have both . Subjects with either hematopoietic stem cells , intermediate level progenitors and anti - A and / or anti - B antibodies cannot receive a transfusion maturing cells committed to each lineage. of blood containing the corresponding antigen . Because [0198 ] Although the morphology of all the major blood group O erythrocytes contain neither A nor B antigens , they cell types is similar through their initial development stages, can be safely transfused into recipients of any ABO blood megakaryocytes , cells committed to platelet production , are group , e . g ., group A , B , AB , or 0 recipients . Group O marked by an obvious structural and functional departure erythrocytes are considered universal and may be used in all beyond the blast cell level of differentiation growing to a blood transfusions. In contrast, group A erythrocytes may be size 10 times the diameter of most other bone marrow and given to group A and AB recipients , group B erythrocytes blood cells , and containing up to 128 times the normal may be given to group B and AB recipients , and group AB chromosomal complement, these cells give rise to blood erythrocytes may only be given to AB recipients . In embodi platelets . After a series of normal cell divisions, the devel ments in which exogenous antigen -expressing EHCs are oping megakaryocyte precursor enters a unique cell cycle generated by contecting erythrocytes or their precursors with characterized by a brief ( about 1 h ) G1 phase, a typical ( 7 h ) an antigen the sourced erythrocytes or their precursors are S phase , a very brief (- 45 min ) G2 phase , followed by the matched for compatibility with the recipient. endomitotic phase ( an aborted M phase ). Once the cell [0195 ] In some instances , it may be beneficial to convert develops a highly polyploid nucleus, it also develops demar an exogenous antigen - expressing EHC comprising a non cation membranes necessary for cytoplasmic fragmentation . group O erythrocyte to a universal blood type . Enzymatic This event is accompanied by expression of glycoprotein removal of the immunodominant monosaccharides on the GPIIbIlla ( platelet fibrinogen receptor; Papayannopoulou et surface of group A and group B erythrocytes may be used to al. , Exp . Hematol. , 24 : 660 - 9 , 1996 ) and GPIb ( von Willi generate a population of group O - like exogenous antigen brand factor receptor ; Kaushansky et al. , Nature, 369 : 568 expressing EHCs (See , e . g . , Liu et al. , Nat. Biotech . 25 : 454 571, 1994 ) , the granules that contain ADP , serotonin , 464 ( 2007 ) ). Group B exogenous antigen - expressing EHCS - thromboglobulin , and other substances critical for mature may be converted using an a - galactosidase derived from platelet function . Finally , highly polyploid megakaryocytes green coffee beans . Alternatively or in addition , a -N - acetyl undergo cytoplasmic partitioning , allowing the release of US 2018 /0271910 A1 Sep . 27 , 2018 thousands of platelets (Choi et al. , Blood , 85 : 402 - 413 , [0207 ] Several physiological substances are involved in 1995 ; Cramer et al. , Blood , 89 : 2336 - 2346 , 1997 ) . the activation of platelets such as collagen , which is exposed [0199 ] Like all blood cell precursors ,megakaryocytes are at the subendothelial surfaces , thrombin , generated by the derived from pluripotent marrow stem cells that retain the coagulation cascade , and thromboxane A2 ( TXA , ) and ADP, capacity to extensively self - renew , or to differentiate into all which are released from activated platelets . Collagen binds of the elements of the blood . Platelet production is in part to several platelet membrane proteins including integrin a2 regulated by signaling mechanisms induced by interaction B1 leading to platelet activation through the release of TXA , between thrombopoietin ( TPO ) and its cellular receptor and ADP ( Shattil , S . J ., et al. , Curr . Opin . Cell Biol. 6 : TPOR /MPUC - MPL . 695 - 704 , 1994 ) . In contrast, thrombin , TXA2, and ADP , [ 0200 ] Thrombopoietin ( TPO ) is a hematopoietic growth activate G -protein coupled receptors directly and induce factor involved in stimulation of megakaryocytopoiesis and platelet aggregation and granule release (Hourani , S . M , and platelet production . TPO is expressed in liver and kidney , Cusack , N . J ., Pharmacol. Rev . 43 : 243 -298 , 1991 ) . The and , in response to platelet demand , its expression may be major events involved in platelet activation are believed to also upregulated in the bone marrow microenvironment be the result of the activation of ß - isoforms of phospholipase (Kato et al. , Stem Cells , 16 : 322 - 328 , 1998 ; McCarty et al . , C (PLC ) leading to the generation of inositol 1 , 4 , 5 triphos Blood , 86 :3668 -3675 , 1995 ). As TPO expression is mostly phate and diacylglycerol. Platelets mainly contain two iso constitutive , the TPO levels are believed to be regulated by forms, PLC - B2 and PLC - B3 . sequestering by platelets ( Fielder et al. , Blood 87: 2154 , [0208 ] Platelet receptors which mediate platelet adhesion 1996 ). and aggregation are located on the two major platelet surface [0201 ] The gene encoding TPO has been cloned and glycoprotein complexes . These complexes are the glycopro characterized (Kuter et al. , Proc . Natl . Acad . Sci. USA , tein Ib - IX complex which facilitates platelet adhesion by 91 : 11104 - 11108 , 1994 ; Bartley et al. , Cell , 77 : 1117 - 1124 , binding von Willebrand factor (vWF ), and the glycoprotein 1994 ; Kaushansky et al. , Nature , 369 : 568 - 571 , 1994 ; Wen IIb - Illa complex which links platelets into aggregates by dling et al. , Nature , 369: 571 - 574 , 1994 , and de Sauvage et binding to fibrinogen . Patients with the Bernard -Soulier al. , Nature , 369 :533 - 538 , 1994 ) . Human TPO (hTPO ) syndrome, a congenital bleeding disorder, show deficient cDNA encodes a 353 amino acid - long polypeptide. The platelet adhesion due to a deficiency in the glycoprotein full - length hTPO secreted from mammalian cells after Ib - IX complex which binds vWF, mild thrombocytopenia , cleavage of the signal peptide consists of 332 amino acids . and large lymphocoid platelets . Although the predicted molecular mass of this protein is 38 [0209 ] Glycoprotein V (GPV ) is a major (~ 12 ,000 mol kDa, the molecular masses reported from measurements of ecules/ platelet) , heavily glycosylated platelet membrane material in serum or in culture fluid from recombinant cells protein (Mr 82 , 000 ). Exposure of platelets to thrombin vary from 18 to 85 kD ( glycosylation , and post - translational liberates a 69 kDa soluble fragment termed GPVf. GPV can proteolytic processing ). interact non - covalently with the GPIB - IX complex a com [0202 ] The cell surface receptor for TPO ( TPOR /MPL / c plex formed by the non -covalent association of GPIb ( con MPL ) is a product of the protooncogene c -mpl , a homologue sisting of GPIba , a 145 kDa protein , disulfide linked to of v -mpl , an envelope protein of the myeloproliferative GPIbß , a 24 kDa protein ) with GPIX (a 22 kDa protein ) . The leukaemia virus (MPLV ) shown to induce a pan -myeloid binding sites for von Willebrand factor and for thrombin on disorder (Wendling , Virol. , 149 :242 - 246 , 1986 ) . The human the GPIB - IX complex have been localized on GPIba . Since c -mpl gene codes for a protein of 635 aa having a predicted thrombin is now known to activate platelets by cleaving the molecular weight of 71 kD (Vigon et al ., Proc . Natl. Acad . thrombin receptor (Vu et . al. , Cell 64 : 1057 - 1068 ( 1990 ) ) , a Sci . USA , 89: 5640 -44 , 1992; Mignotte et al. , Genomics, 20 : G -protein coupled receptor, it is unknown whether thrombin 5 - 12 , 1994 ) . cleaves GPV incidentally as a consequence of thrombin [ 0203] Mice rendered null for the expression of either binding to GPIba , or whether this cleavage has a physi TPO or its receptor ( TPOR /MPL / C -MPL ) manifest a severe ological role . GPIBA , GPIBB , and GPIX contain one or thrombocytopenic phenotype (Gurney et al ., Science , 265 : more homologous 24 amino acid leucine - rich domains . 1445 , 1994 ; Kaushansky et al. , J . Clin . Invest. , 96 : 1683 , These domains are also found in a large family of leucine 1995 ; de Sauvage et al. , J. Exp . Med ., 183 : 651, 1996 ). rich glycoproteins (LRG ) . [0204 ] Multiple cytokines ( e . g . , stem cell factor [ SCF ] , [ 0210 ] GPV is a marker for the megakaryocytic cell lin IL - 1 , IL - 3 , IL - 6 , IL - 11 , leukaemia inhibiting factor [ LIF ], eage . A monoclonal antibody specific for GPV (SW16 ) does G -CSF , GM -CSF , M -CSF , erythropoietin ( EPO ), kit ligand , not bind to red cells , leukocytese endothelial cells , or cell and - interferon ) have been shown to possess thrombocy lines such as HEL or MEG -01 which are known to express topoietic activity . platelet megakaryocyte markers . [0205 ] Platelet Activation [ 0211 ] Mature GPV is composed of 543 amino acids [ 0206 ] The resulting platelets are small disc - shaped cell which contain a single transmembrane domain , a short fragments which undergo a rapid transformation when they cytoplasmic domain ( 16 residues ) and a large extracellular encounter sites of vascular damage . They become more domain with 8 potential N - glycosylation sites . Analysis of spherical and extrude pseudopodia , their fibrinogen recep the extracellular domain revealed the presence of 15 tandem tors are activated leading to aggregation , and they release Leu - rich repeats of 24 amino acids with homology to GPIba , their granule contents and eventually they form a plug which and identified a cleavage site for thrombin near the C -ter is responsible for primary hemostasis (Siess , W ., Physiol. minus with homology to the Au chain of fibrinogen . Rev. 69 : 58 - 178 , 1989 ) . Activation of platelets is also [0212 ] Culturing Conditions implicated in the pathogenesis of unstable angina, myocar [0213 ] Sources for generating exogenous antigen - express dial infarction and stroke (Packham , M . A . , Can J . Physiol ing EHCs described herein include circulating cells such as Pharmacol. 72 : 278 - 284 ) . EHCs . A suitable cell source may be isolated from a subject US 2018 /0271910 A1 Sep . 27 , 2018 13 as described herein from patient- derived hematopoietic or growth factor at 1 -50 ug/ mL . The first step further may erythroid progenitor cells , derived from immortalized EHC optionally comprise contacting the cells in culture with lines , or derived from induced pluripotent stem cells , option transferrin at 0 . 1 - 5 mg/mL . ally cultured and differentiated . Methods for generating [0217 ] The first step may optionally comprise contacting erythrocytes using cell culture techniques are well known in the cells in culture with one or more interleukins ( IL ) or the art, e . g . , Giarratana et al ., Blood 2011 , 118 :5071 , Huang growth factors such as , e .g . , IL - 1, IL -2 , IL - 4 , IL - 5 , IL -6 , et al. , Mol Ther 2013, epub ahead of print September 3 , or IL - 7 , IL - 8 , IL - 9 , IL - 11, IL - 12 , granulocyte colony - stimulat Kurita et al. , PLOS One 2013 , 8 : e59890 . Protocols vary ing factor ( G -CSF ) , macrophage colony -stimulating factor according to growth factors , starting cell lines, culture ( M -CSF ) , granulocyte -macrophage colony -stimulating fac period , and morphological traits by which the resulting cells tor (GM -CSF ), thrombopoietin , fibroblast growth factor are characterized . Culture systems have also been estab ( FGF ), platelet -derived growth factor (PDGF ) , transforming lished for blood production that may substitute for donor growth factor beta ( TGF - B ) , tumor necrosis factor alpha transfusions ( Fibach et al . 1989 Blood 73 : 100 ) . Recently , ( TNF - A ), megakaryocyte growth and development factor CD34 + cells were differentiated to the reticulocyte stage , (MGDF ) , leukemia inhibitory factor (LIF ) , and Flt3 ligand . followed by successful transfusion into a human subject Each interleukin or growth factor may typically be supplied (Giarratana et al. , Blood 2011 , 118 : 5071 ). at a concentration of 0 . 1 - 100 ng/ mL . The first step may also [ 0214 ] Provided herein are culturing methods for EHCS optionally comprise contacting the cells in culture with and exogenous antigen - expressing EHCs derived from serum proteins or non - protein molecules such as , e . g ., fetal EHCs. EHCs can be cultured from hematopoietic progenitor bovine serum ( 1 - 20 % ), human plasma ( 1 - 20 % ) , plasmanate cells , including , for example , CD34 + hematopoietic pro ( 1 - 20 % ) , human serum ( 1 - 20 % ), albumin (0 . 1 - 100 mg/mL ) , genitor cells (Giarratana et al. , Blood 2011 , 118: 5071) , or heparin ( 0 . 1 - 10 U /mL ) . induced pluripotent stem cells ( Kurita et al. , PLOS One [0218 ] The second step may comprise contacting the cells 2013 , 8 : e59890 ) , and embryonic stem cells (Hirose et al. in culture with stem cell factor ( SCF) at 1 - 1000 ng /mL and 2013 Stem Cell Reports 1 : 499 ) . Cocktails of growth and erythropoietin ( EPO ) at 1 - 100 U /mL . The second step may differentiation factors that are suitable to expand and differ also optionally comprise contacting the cells in culture with entiate progenitor cells are known in the art . Examples of an insulin - like molecule , such as e . g . , insulin at 1 - 50 ug /mL , suitable expansion and differentiation factors include , but insulin - like growth factor 1 ( IGF - 1 ) at 1 - 50 ug /mL , insulin are not limited to , stem cell factor (SCF ), an interleukin ( IL ) like growth factor 2 ( IGF - 2 ) at 1- 50 ug /mL , or mechano such as IL - 1 , IL - 2 , IL - 3 , IL - 4 , IL - 5 , IL - 6 , IL - 7 , IL - 8 , IL - 9 , growth factor at 1 - 50 ug /mL . The second step may further IL - 11 , IL - 12, CSF , G - CSF , thrombopoietin ( TPO ) , GM optionally comprise contacting the cells in culture with CSF , erythropoietin (EPO ) , Flt3 , Flt2 , PIXY 321 , and leu transferrin at 0 . 1 - 5 mg/ mL . The second may also optionally kemia inhibitory factor (LIF ) . comprise contacting the cells in culture with serum proteins [ 0215 ] EHCs can be cultured from hematopoietic progeni or non - protein molecules such as, e . g . , fetal bovine serum tors, such as CD34 + cells , by contacting the progenitor cells ( 1 - 20 % ), human plasma ( 1 - 20 % ) , plasmanate ( 1 - 20 % ) , with defined factors in a multi -step culture process . For human serum ( 1- 20 % ) , albumin (0 . 1 - 100 mg/mL ), or hepa example , EHCs can be cultured from hematopoietic pro rin (0 . 1 - 10 U /mL ) . genitors in a three - step process . (0219 ] The third step may comprise contacting the cells in [0216 ] The first step may comprise contacting the cells in culture with erythropoietin (EPO ) at 1 - 100 U /mL . The third culture with stem cell factor ( SCF ) at 1 - 1000 ng /mL , eryth step may optionally comprise contacting the cells in culture ropoietin ( EPO ) at 1 - 100 U / mL , and interleukin - 3 ( IL - 3 ) at with stem cell factor ( SCF ) at 1 - 1000 ng /mL . The third step 0 . 1 - 100 ng /mL . The first step optionally comprises contact may further optionally comprise contacting the cells in ing the cells in culture with a ligand that binds and activates culture with an insulin - like molecule , such as e . g . , insulin at a nuclear hormone receptor, such as e . g . , the glucocorticoid 1 - 50 ug/ mL , insulin - like growth factor 1 ( IGF - 1 ) at 1 - 50 receptor , the estrogen receptor , the progesterone receptor , ug/ mL , insulin - like growth factor 2 ( IGF - 2 ) at 1 - 50 ug/ mL , the androgen receptor, or the pregnane X receptor. The ormechano - growth factor at 1 - 50 ug /mL . The third step may ligands for these receptors include , for example , a corticos also optionally comprise contacting the cells in culture with teroid , such as , e . g . , dexamethasone at 10 nM - 100 uM or transferrin at 0 . 1 - 5 mg/ mL . The third step may also option hydrocortisone at 10 nM - 100 uM ; an estrogen , such as, e. g. , ally comprise contacting the cells in culture with serum beta - estradiol at 10 nM - 100 uM ; a progestogen , such as , proteins or non -protein molecules such as, e . g ., fetal bovine e .g ., progesterone at 10 nM - 100 uM , hydroxyprogesterone serum ( 1 - 20 % ) , human plasma ( 1 - 20 % ) , plasmanate at 10 nM - 100 uM , 5a - dihydroprogesterone at 10 nM - 100 ( 1 - 20 % ), human serum ( 1 - 20 % ), albumin (0 . 1 - 100 mg/ mL ) , uM , 11 - deoxycorticosterone at 10 nM - 100 uM , or a syn or heparin ( 0 . 1 - 10 U /mL ) . thetic progestin , such as , e . g . , chlormadinone acetate at 10 [0220 ] The culture process may optionally comprise con nM - 100 UM ; an androgen , such as , e . g ., testosterone at 10 tacting cells by a method known in the art with a molecule , nM - 100 uM , dihydrotestosterone at 10 nM - 100 uM or e . g ., a DNA molecule , an RNA molecule , a mRNA , an androstenedione at 10 nM - 100 UM ; or a pregnane X receptor siRNA , a microRNA , a lncRNA, a shRNA , a hormone, or a ligand , such as , e . g . , rifampicin at 10 nM - 100 uM , hyper small molecule , that activates or knocks down one or more forin at 10 nM - 100 UM , St. John ' s Wort (hypericin ) at 10 genes. Target genes can include , for example , genes that nM - 100 UM , or vitamin E - like molecules , such as , e . g . , encode a transcription factor, a growth factor, or a growth tocopherol at 10 nM - 100 uM . The first step may also factor receptor, including but not limited to , e . g. , GATA1, optionally comprise contacting the cells in culture with an GATA2, CMyc , hTERT, p53 , EPO , SCF , insulin , EPO - R , insulin - like molecule , such as, e . g . , insulin at 1 -50 ug /mL , SCF - R , transferrin - R , insulin - R . insulin - like growth factor 1 ( IGF - 1 ) at 1 - 50 ug /mL , insulin - (0221 ] In one embodiment, CD34 + cells are placed in a like growth factor 2 ( IGF -2 ) at 1 -50 ug /mL , or mechano culture containing varying amounts of IMDM , FBS , gluta US 2018 /0271910 A1 Sep . 27 , 2018 14 mine , BSA , holotransferrin , insulin , dexamethasone , B - es - (see e . g . Repik et al . , Clin Exp Immunol 2005 , 140 :230 ) . tradiol , IL - 3 , SCF, and erythropoietin , in three separate The nucleic acid encoding the exogenous antigen , can be differentiation stages for a total of 22 days . introduced in any way known in the art , for example , as [0222 ] In one embodiment, CD34 + cells are placed in a plasmid DNA , virus, or mRNA . Nucleic acid introduction culture containing varying amounts of IMDM , FBS , gluta can be achieved by a variety of standard methods , e .g . mine , BSA , holotransferrin , insulin , dexamethasone , B - es transfection , transduction , or electroporation . tradiol, IL -3 , SCF, and thrombopoietin , in three separate [0227 ] Modification of Progenitor Cells . Nucleic acids differentiation stages for a total of 14 days . such as DNA expression vectors or mRNA for producing the 0223 ] In one embodiment, CD34 + cells are placed in a antigen of interest may be introduced into progenitor cells , culture containing varying amounts of IMDM , FBS , gluta which can be isolated from an original source or obtained mine , BSA , holotransferrin , insulin , dexamethasone , B - es from expanded above via routine recombinant technology as tradiol, IL - 3 , SCF , and GCSF, in three separate differentia provided herein . In some instances , the expression vectors tion stages for a total of 15 days . can be designed such that they can incorporate into the [ 0224 ] In certain embodiments , cells that comprise an genome of cells by homologous or non -homologous recom exogenous antigen of interest may be comprised of or bination by methods known in the art . derived from a plurality of circulating cells including, but 10228 ] In some instances , a nucleic acid encoding a poly not limited to , those listed in Table A . In a preferred peptide that can selectively target and cut the genome, for embodiment, the circulating cells of the invention are EHCs, example a CRISPR / Cas9 , transcriptional activator - like such as, e .g . nucleated red blood cells , red blood cell effector nuclease ( TALEN ) , or zinc finger nuclease , is used precursors or enucleated red blood cells . For example , the to direct the insertion of the nucleic acid payload of the EHCs are a cord blood stem cell, a CD34 + cell , a hematopoi expression vector to a particular genomic location , for etic stem cell (HSC ) , a spleen colony forming (CFU - S ) cell, example the CR1 locus ( 1932 . 2 ) , the hemoglobin locus a common myeloid progenitor (CMP ) cell , a blastocyte ( 11p15 .4 ), or another erythroid -associated protein including, colony - forming cell, a burst forming unit -erythroid (BFU but not limited to , those listed in Table C . E ), a megakaryocyte -erythroid progenitor (MEP ) cell , an [0229 ] In some instances , the nucleic acid is an RNA erythroid colony -forming unit (CFU - E ) , a reticulocyte , an molecule , or a DNA molecule that encodes for an RNA erythrocyte , an induced pluripotent stem cell (iPSC ) , a molecule , that silences or represses the expression of a target mesenchymal stem cell MSC( ) , a polychtomratic normo gene . For example , the molecule can be a small interfering blast , an orthochtomratic normoblast , or those listed in Table RNA (siRNA ) , an antisense RNA molecule , or a short A1, or a combination thereof. In some embodiments , the hairpin RNA (shRNA ) molecule . EHCs are immortal or immortalized cells , for example [ 0230 ] Methods for transferring expression vectors into immortalized erythroblast cells generated by retroviral progenitor cells include , but are not limited to , viral medi transduction of CD34 + hematopoietic progenitor cells to ated gene transfer, liposome mediated transfer, transforma express Oct4 , Sox2 , K1f4 , cMyc , and suppress TP53 ( e . g . tion , gene guns , transfection and transduction , e . g . , viral Huang et al. , Mol Ther 2013 , epub ahead of print September mediated gene transfer such as the use of vectors based on 3) . DNA viruses such as adenovirus , adenoassociated virus and [ 0225 ] Erythrocyte compositions are herein provided , herpes virus , as well as retroviral based vectors . Examples of wherein a plurality of erythrocytes express an exogenous modes of gene transfer include e . g . , naked DNA , CaPO4 antigen of interest or a fragment thereof. The cells may be precipitation , DEAE dextran , electroporation , protoplast cultured from patient- derived hematopoietic or erythroid fusion , lipofection , and cell microinjection . progenitor cells , derived from immortalized EHC lines, or [0231 ] Any of the genetically modified progenitor cells derived from induced pluripotent stem cells . Methods for described herein can be cultured under suitable conditions generating erythrocytes in cell culture are known in the art, allowing for differentiation into mature enucleated red blood e .g . Giarratana et al. , Blood 2011 , 118 : 5071, Huang et al ., cells , e . g . , the in vitro culturing process described herein . Mol Ther 2013 , or Kurita et al ., PLOS One 2013 , 8 :e59890 . The resulting enucleated red blood cells display and express Exogenous antigens can be introduced by transfection of proteins associated with mature erythrocytes, e . g . hemoglo single or multiple copies of genes , transduction with a virus, bin , glycophorin A , which can be validated and quantified by or electroporation in the presence of DNA or RNA .Methods standard methods ( e . g . , Western blotting or FACS analysis ) . for expression of exogenous proteins in mammalian cells are well known in the art. For example , expression of exogenous Strategies for Exogenous Antigen Expression factor IX in hematopoietic cells is induced by viral trans [ 0232 ] Provided herein are antigens that are exhibited by duction of CD34 + progenitor cells , see Chang et al. , Nat exogenous antigen -expressing EHCs. In some embodi Biotechnol 2006 , 24 : 1017 . ments , an antigen is capable of interacting with a target , e . g . , [0226 ]. The erythrocyte compositions described herein to associate with or bind to a target. An antigen can comprise may be generated in the following way . First, erythroid or may consist essentially of a polypeptide . In some embodi precursor cells are isolated . These cells may alternatively be ments , the antigen comprises a polypeptide , a carbohydrate , autologous to the patient or from substantially universal a nucleic acid , a lipid , a small molecule , or a combination donor blood . For example , the cells may be ABO type 0 , thereof . In some embodiments antigens do not interact with rhesus factor Rh r/ r , Duffy -/ - , and large Kell antigen K1 a target but act as payloads to be delivered by the exogenous negative . In the course of differentiation from erythroid antigen -expressing EHC to a cell , tissue or other site in the precursor cell to EHC , the nucleic acids encoding the body of a subject. exogenous antigen are introduced . The nucleic acid encod [0233 ] Antigen Polypeptides , Chimeras and Fusions ing the exogenous antigen can be under the control of an [0234 ] In some embodiments , antigens comprise polypep erythroid -specific promoter, such as a GATA -1 promoter tides . Receiver polypeptides may range in size from 6 amino US 2018 /0271910 A1 Sep . 27 , 2018 15

acids to 3000 amino acids and may exceed 6 , 10 , 15 , 20 , 25 , [0237 ] In some embodiments , the antigen polypeptide is 30 , 35 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 150 , 200 , 300 , 400 ormay associated with the membrane of the exogenous antigen exceed 500 amino acids . Receiver polypeptides may range expressing EHC . In other embodiments , the antigen poly in size from about 20 amino acids to about 500 amino acids, peptide is not associated with the membrane of the exog from about 30 amino acids to about 500 amino acids or from enous antigen -expressing EHC . about 40 amino acids to about 500 amino acids. [0238 ] In one embodiment the mass ratio of lipid to [ 0235 ] In some embodiments , the antigen polypeptide antigen in the exogenous antigen - expressing EHC is less comprises a chimeric or fusion protein which may comprise than 1 : 1000 , approximately 1 : 1000 , approximately 1 : 500 , two or more distinct protein domains. These chimeric anti approximately 1: 250 , approximately 1 : 100 , approximately gens are heterologous or exogenous in the sense that the 1 :50 , approximately 1: 25 , approximately 1 : 10 , approxi various domains are derived from different sources , and as mately 1 : 9 , approximately 1 : 8 , approximately 1 : 7 , approxi such , are not found together in nature and can be encoded mately 1 : 6 , approximately 1 : 5 , approximately 1 : 4 , approxi e . g ., by recombinant nucleic acids. Antigen polypeptides can mately 1 : 3 , approximately 1 : 2 , approximately 1 : 1 , be produced by a number of methods , many of which are approximately 2 : 1 , approximately 3 : 1 , approximately 4 : 1 , well known in the art and also described herein . For approximately 5 : 1 , approximately 6 : 1 , approximately 7 : 1 , example , antigen polypeptides can be obtained by extraction approximately 8 : 1 , approximately 9 : 1, approximately 10 : 1, ( e . g . , from isolated cells ) , by expression of a recombinant approximately 25 : 1 , approximately 50 : 1 , approximately nucleic acid encoding the antigen polypeptide , or by chemi 100 : 1 , approximately 250 : 1 , approximately 500 : 1 , approxi cal synthesis . Antigen polypeptides can be produced by, for mately 1000 : 1 , approximately 10 ,000 : 1 , approximately 100 , example , recombinant technology, and expression vectors 000 : 1 , approximately 1 ,000 , 000 : 1 , approximately 10 , 000 , encoding the polypeptide introduced into host cells ( e . g ., by 000 : 1 , approximately 100 , 000 , 000 : 1 , approximately 1 ,000 , transformation or transfection ) for expression of the encoded 000 , 000 : 1 or greater than approximately 1 ,000 , 000 ,000 : 1 . antigen polypeptide . [0239 ] In one embodiment the mass ratio of non -exog enous antigen polypeptide to antigen in the exogenous [ 0236 ] There are a variety of conservative changes that antigen - expressing EHC is less than 1 : 1000 , approximately can generally be made to an amino acid sequence without 1 : 1000 , approximately 1 :500 , approximately 1 : 250 , altering activity . These changes are termed conservative approximately 1 : 100 , approximately 1 :50 , approximately substitutions or mutations; that is , an amino acid belonging 1 :25 , approximately 1 : 10 , approximately 1 : 9 , approximately to a grouping of amino acids having a particular size, charge 1 : 8 , approximately 1 : 7 , approximately 1 : 6 , approximately or other characteristic can be substituted for another amino acid . Substitutions for an amino acid sequence may be 1 : 5 , approximately 1 : 4 , approximately 1 : 3 , approximately selected from other members ofthe class to which the amino 1 : 2 , approximately 1 : 1 , approximately 2 : 1 , approximately acid belongs . For example , the nonpolar (hydrophobic ) 3 :1 , approximately 4 : 1, approximately 5 : 1, approximately amino acids include alanine , leucine, isoleucine , valine , 6 : 1 , approximately 7 : 1 , approximately 8 : 1 , approximately proline , phenylalanine, tryptophan , methionine, and tyro 9 : 1 , approximately 10 : 1 , approximately 25 : 1, approximately sine . The polar neutral amino acids include glycine , serine , 50 : 1 , approximately 100 : 1 , approximately 250 : 1 , approxi threonine, cysteine , tyrosine , asparagine and glutamine. The mately 500 : 1 , approximately 1000 : 1 , approximately 10 , 000 : positively charged (basic ) amino acids include arginine , 1 , approximately 100 ,000 : 1 , approximately 1 , 000 , 000 : 1 , lysine and histidine . The negatively charged ( acidic ) amino approximately 10 ,000 ,000 : 1 , approximately 100 ,000 , 000 : 1 , acids include aspartic acid and glutamic acid . Such altera approximately 1 , 000 , 000 ,000 : 1 or greater than approxi tions are not expected to substantially affect apparent mately 1 ,000 , 000 ,000 : 1 . molecular weight as determined by polyacrylamide gel [0240 ] In certain embodiments , the polypeptide antigen is electrophoresis or isoelectric point . Conservative substitu located on the surface and is exposed to the environment tions also include substituting optical isomers of the around the exogenous antigen -expressing EHC . In some sequences for other optical isomers , specifically D amino embodiments , the polypeptide antigen is located inside and acids for L amino acids for one or more residues of a faces the unexposed side of the exogenous antigen - express sequence . Moreover, all of the amino acids in a sequence ing EHC . may undergo a D to L isomer substitution . Exemplary [ 0241 ] In certain embodiments , the polypeptide antigen conservative substitutions include , but are not limited to , Lys comprises at least one of the following domains , an S for Arg and vice versa to maintain a positive charge ; Glu for domain (surface ) , an A domain (anchor ) , and / or a U domain Asp and vice versa to maintain a negative charge ; Ser for Thr ( unexposed ) , wherein the S domain is a surface domain so that a free ~ OH is maintained ; and Gln for Asn to exposed to the environment around the exogenous antigen maintain a free NH2. Moreover , point mutations, deletions, expressing EHC , wherein the A domain is an anchor, and and insertions of the polypeptide sequences or correspond wherein the U domain is located within and / or faces the ing nucleic acid sequences may in some cases be made unexposed side of the exogenous antigen -expressing EHC . without a loss of function of the polypeptide or nucleic acid [0242 ] Optionally the antigen polypeptide comprises i) fragment. Substitutions may include , e . g . , 1 , 2 , 3 , or more one or more additional S domains , termed S ' domains, or ii ) residues . Any teaching of a specific amino acid sequence or one or more additional U domains, termed U ' domains . a recombinant nucleic acid encoding the polypeptide or [0243 ] In some embodiments , the S domain and the A teaching of the name of the name thereof includes any domain form part of the same polypeptide chain . conservative substitution point mutations, deletions, and [ 0244 ] In some embodiments , the A domain and the U insertions of those polypeptide sequences or corresponding domain form part of the same polypeptide chain . nucleic acid sequences and any sequence deposited for the [0245 ] In some embodiments , any one or more of the S , A , protein or gene in a database that can bemade without a loss U domain is added to the exogenous antigen - expressing of function of the polypeptide or nucleic acid fragment. EHC externally. US 2018 /0271910 A1 Sep . 27 , 2018

[0246 ] In some embodiments , any one or more of the S , A , LGALS3 protein , Urea transporter, Rh blood CE group U domain is produced within the exogenous antigen - ex antigen poypeptide , Rh -associated glycoprotein , Dematin , pressing EHC . ABO blood groups , Aquaporin 3 , Aubergers , Band 3 , Basi [ 0247 ] In some embodiments , any one or more of the S , A , gin , C41, CD44 , Cis AB , Colton antigen , Complement U domain is a polypeptide. Component 4 , CR1, DAF, Diego , Duffy , Hh/ Bombay anti [ 0248 ] In some embodiments , any one or more of the S , A , gen , ii antigen , Indian blood group , Kell , Kidd , Lewis U domain is not a polypeptide . antigen , Lutheran antigen ,MNS antigen system , Cost group , [0249 ] Schematics of exemplary conformations of anti Er group , Dematin , Stomatin , Tropomyosin , Glucose trans gens within or on exogenous antigen - expressing EHCs are porter, Adducin , Rabphilin , C1 tetrahydrofolate synthase , shown in FIGS. 13A , 13B , and 13C . Vel group , Lan antigen , At antigen , Jr antigen , AnWj anti [0250 ) The A Domain gen , Sd antigen , Batty , Bilkes, Box , Christiansen , HJK , [0251 ] In certain embodiments , the A domain is a mem HOFM , JFV , JONEs , Jensen , Katagiri, Livesay, Milne , brane polypeptide . The A domain can be, e .g ., an integral Oldeide , Peters , Rasmussen , Reid , REIT , SARA , Rhesus membrane polypeptide or a membrane associated polypep blood D group , Aldolase , Tropomodulin , Arginase , Creatine tide . kinase , B - Cam protein , Rapl? , Bennett -Goodspeed , P anti [0252 ] The A domain may be selected from one of the gen system , Rh blood groupXg antigen system , XK protein , following classes, including but not limited to , for example , Yt/ Cartwright antigen system , CD58 , Rh, Scianna , Radin , alpha -helical bitopic , alpha -helical polytopic , beta -barrel DARC (Duffy ) , CR1 Knops -McCoy , DAF Cromer, Gerbich transmembrane , all alpha monotopic /peripheral , all beta (GYPC ) , CD47 , Glycophorin A , Band 3 ( AE3) , GYPB Ss, monotopic /peripheral , alpha/ beta monotopic /peripheral , C4A , C4B Chido , Rodgers C4 component of complement, alpha + beta monotopic /peripheral , alpha helical peptides , HLA Bg HLA class I , RHAG Rh - associated Ammonium beta -hairpin peptides , beta -helical peptides , type 1 trans transport, Glycoprotein , Colton ( Co ) Water channel protein , membrane protein ( N - terminus extracellular ) , type 2 trans ACHE Cartwright ( Yt ) Acetylcholinesterase , Glutathione membrane protein ( N - terminus intracellular) , type 3 trans transferase , Glycophorin C , Aquaporin , Erythroblast asso membrane protein , type 4A transmembrane protein , type 4B ciated membrane protein , CD44 , Synaptobrevin 2 , Ribonu transmembrane protein , lipid - anchored protein , glycosyl clease , Duodenal cytochrome B , ABO glycosyl transferases , CD59 , CD44 Indian ( In ) , AnWj Adhesion receptor , MER2, phosphatidylinositol (GPI ) anchored protein , prenyl chain DOK Dombrock ADP -ribosyltransferase , SEMAZA JMH anchored protein , or peptides of nonregular structure . Putative adhesion receptor, UMOD Sda Tamm - Horsfall pro [ 0253] In certain embodiments , the A domain is endog tein (uromodulin ) , Diego (Di ) , Wright ( Wr) Anion channel enous, e . g . , endogenous to an EHC , a platelet , or a protein ( band 3 , AE1 ) , Kidd ( Jk ) Urea transporter , FUT3 hematopoietic cell. In some embodiments , the A domain is Lewis (Le ) alpha ( 1 , 3 ) fucosyltransferase , OK Oka Neu endogenous to a mammalian cell . rothelin , putative adhesion molecule , LW Adhesion receptor, [ 0254 ) In certain embodiments , the A domain is exog FUT2 Secretor (Se ) alpha ( 1 , 2 ) fucosyltransferase, FUT1 Hh enous, e . g . , exogenous to an EHC , a platelet , or a hematopoi alpha ( 1, 2 ) fucosyltransferase, LU Lutheran (Lu ) Adhesion etic cell . In some embodiments , the A domain is exogenous receptor , P1 Glycosyltransferase , XK Kx Putative neu to a mammalian cell . rotransmitter transporter , XG Xg formerly called PBDX , [0255 ) The A domain may be selected from the the fol MIC2, Hemoglobin , Ankyrin , Spectrin , KEL Kell ( forms lowing molecules or fragments thereof, including but not K , K ,Kp , Js ) Metalloproteinase , Torkildsen antigen , coen limited to , CD1, CD2, CD3 , CD4, CD5 , CD , CD7, CD8, zyme Q10 , Rab 35 , Ral A binding protein , Zona pellucida CD9, CD10 , CD11a , CD11b , CD11c, CD12w , CD13 , CD14 , binding protein , Lyn B protein , Klaa1741 protein , DC38 , CD15 , CD16 , CDw17 , CD18 , CD19 , CD20 , CD21, CD22 , Calcium transporting ATPase , GPIX , GPIba, GPIbb , GPV , CD23 , CD24 , CD25 , CD26 , CD27 , CD28 , CD29 , CD30 , GPIB - IX - V , GPVI, GPIa / IIa , GPIIB / IIIa , GPV / IIa . CD31, CD32 , CD33 , CD34 , CD35 , CD36 , CD37 , CD38 , CD39 , CD40 , CD41 , CD42 , CD43 , CD44 , CD45 , CD46 , [ 0256 ] The S Domain CD47 , CD48 , CD49a, CD49b , CD49c , CD49d , CD49e , [0257 ] In some embodiments , the S domain is a protein or CD49f , CD53 , CD54 , CD55 , CD56 , CD57 , CD58 , CD59 , a polypeptide . In other embodiments , the S domain is a CD61 . CD62E . CD62L . CD62P . CD63 . CD68 . CD69 . nucleic acid . In some embodiments , the S domain is a CD71 , CD72 , CD73, CD74 , CD80 , CD81, CD82 , CD83 , chemical. In certain embodiment the S domain is a small CD86 , CD87 , CD88 , CD89 , CD90 , CD91, CD95 , CD96 , molecule . CD100 , CD103 , CD105, CD106 , CD107, CD107a, [ 0258 ] In some embodiments , the S domain is a polypep CD107b , CD109 , CD117 , CD120 , CD122 , CD123 , CD127 , tide selected from or derived from one or more of the CD132 , CD133, CD134 , CD135 , CD138 , CD141, CD142 , following classes, including but not limited to , a flexible CD143 , CD144 , CD147 , CD151, CD152, CD154, CD155 , linker , an epitope tag , an enzyme, a protease , a nuclease, an CD156 , CD158 , CD163 , CD165 , CD166 , CD168 , CD184 , antigen , an antibody - like molecule , a ligand of an antibody , CDw186 , CD195 , CD197 , CDw199 , CD209 , CD202a , a growth factor, a cytokine , a chemokine , a growth factor CD220 , CD221 , CD235a , CD271, CD279 , CD303 , CD304 , receptor, a cytokine receptor, a chemokine receptor, an CD309, CD326 , Ras- Related protein 1A , semaporin 7A enzymatic recognition sequence , a transpeptidase recogni precursor, Calcium and integrin -binding protein 1 , 55 kDa tion sequence , a protease recognition sequence , a cleavable erythrocyte membrane protein , Flotillin - 1 , Flotillin - 2 , domain , an intein , a DNA binding protein , and RNA binding Erythroid membrane -associated protein , eukaryotic transla protein , a complement regulatory molecule , a complement tion initiation factor 2C 2 , cytochrome b5 reductase , cell cascade molecule , a clotting cascade molecule , a chelator, a division control protein 42 homolog , KIAA1363 protein , complement regulatory domain , an SCR domain , a CCP band3 , annexin VII , aquaporin , Ecto - ADP - ribosyltrans - domain , an immunoglobulin or immunogloblulin - like ferase 4 , Kell, LFA - 3 , soulute carrier family 2 member 1 , domain , an armadillo repeat, a leucine zipper, a dealth US 2018 /0271910 A1 Sep . 27 , 2018 17 effector domain , a cadherein repeat, an EF hand , a phos tag , and the body sequence of glycophorin A ; the polypep photyrosine binding domain , a pleckstrin homology domain , tide antigen comprises complement receptor 1 (CR1 ); the an SCR homology 2 domain , a zinc finger domain , a cyclic polypeptide antigen comprises the leader sequence of CR1 , peptide, a cell- penetrating peptide . HA epitope tag , the body sequence of CR1; the polypeptide [0259 ] In some embodiments , the S domain is a non antigen comprises the leader sequence of CR1, HA epitope polypeptide molecule , for example a nucleic acid , a carbo tag , six SCR domains of LHR - A and LHR - B of CR1, the hydrate , or a small molecule . In some embodiments , the S membrane proximal two SCR domains of CR1, the trans domain is a nucleic acid selected from one or more of the membrane region of CR1, and the intracellular region of following classes , including but not limited to , a DNA CR1; the polypeptide antigen comprises the leader sequence aptamer , an RNA aptamer, an siRNA , a shRNA , a single of CR1, HA epitope tag , nine SCR domains of LHR - A and strand RNA probe , a single strand DNA probe , an mRNA, LHR - B and LHR - C of CR1, the membrane proximal two a chemically modified oligonucleotide . In some embodi SCR domains of CR1, the transmembrane region of CR1, ments, the S domain is a small molecule selected from one and the intracellular region of CR1; the polypeptide antigen or more of the following classes , including but not limited comprises the leader sequence of CR1, LHR - A of CR1, to , a chelator, DOTA , a radionuclide, an isotope, an imaging LHR - B of CR1, LHR - C of CR1, the membrane proximal agent, a fluorescentmolecule , a chemiluminescentmolecule , two SCR domains of CR1, the transmembrane region of a gas . CR1, and the intracellular region of CR1 ; the polypeptide [0260 ] The U Domain antigen comprises leader sequence of CR1, LHR - A of CR1 , [0261 ] In some embodiments , the U domain is a protein or LHR - B of CR1, LHR - C of CR1, the membrane proximal a polypeptide . In other embodiments , the U domain is a two SCR domains of CR1, the transmembrane region and nucleic acid . In some embodiments , the U domain is a intracellular region of glycophorin A ; the polypeptide anti chemical . In certain embodiment the U domain is a small gen comprises the leader sequence of glycophorin A , an molecule . antibody scFv against hepatitis B surface antigen ( scFv ) , a [ 0262] In some embodiments , the U domain is a polypep (Gly3Ser ) 2 flexible linker (Seq . ID No . 23 ), HA epitope tag, tide selected from or derived from one or more of the and the body of glycophorin A ; the polypeptide antigen following classes , including but not limited to , a flexible comprises Kell , a (Gly3Ser ) 2 flexible linker (Seq . ID No . linker, an epitope tag , an enzyme, a protease , a nuclease , an 23 ) , HA epitope tag , and scFv ; the polypeptide antigen antigen , an antibody - like molecule, a ligand of an antibody, comprises Kell and HA epitope tag ; the polypeptide antigen a growth factor, a cytokine, a chemokine, a growth factor comprises a 71 -amino acid N -terminal fragment of Kell and receptor, a cytokine receptor , a chemokine receptor, an an HA epitope tag ; the polypeptide antigen comprises a enzymatic recognition sequence, a transpeptidase recogni 71 - amino acid N - terminal fragment of Kell , a (Gly3Ser ) 2 tion sequence , a protease recognition sequence , a cleavable flexible linker ( Seq . ID No. 23 ) , and an HA epitope tag; the domain , an intein , a DNA binding protein , and RNA binding polypeptide antigen comprises a 79 -amino acid N -terminal protein , a complement regulatory molecule , a complement fragment of Kell and an HA epitope tag ; the polypeptide cascade molecule , a clotting cascade molecule , a chelator, a antigen comprises a 79 -amino acid N - terminal fragment of complement regulatory domain , an SCR domain , a CCP Kell, a (Gly3Ser ) 2 flexible linker (Seq . ID No . 23 ) , and an domain , an immunoglobulin or immunogloblulin - like HA epitope tag ; the polypeptide antigen comprises a domain , an armadillo repeat, a leucine zipper, a dealth 71 -amino acid N - terminal fragment of Kell , a (Gly3Ser ) 2 effector domain , a cadherein repeat , an EF hand , a phos flexible linker (Seq . ID No . 23 ), scFv, and an HA epitope photyrosine binding domain , a pleckstrin homology domain , tag ; the polypeptide antigen comprises a 79 - amino acid an SCR homology 2 domain , a zinc finger domain , a cyclic N - terminal fragment of Kell , a (Gly3Ser ) 2 flexible linker peptide , a cell -penetrating peptide, a kinase domain , aphos (Seq . ID No. 23 ) , scFv, and an HA epitope tag; the poly phatase domain , a cytoskeletal protein , a protein that inter peptide antigen comprises the leader sequence of CD55 , acts with the cytoskeletal protein , a G -protein coupled scFv , an HA epitope tag , and the terminal 37 amino acids of receptor, a tyrosine kinase , an ITIM domain , an ITAM CD55 ; the polypeptide antigen comprises the leader domain . sequence of CD55 , an HA epitope tag , and the body of [0263 ] In some embodiments, the U domain is a non CD55 . In one embodiment, the polypeptide antigen com polypeptide molecule , for example a nucleic acid , a carbo prises the leader sequence ofCD59 , scFv , an HA epitope tag , hydrate , or a small molecule . In some embodiments , the U and the body of CD59 ; the polypeptide antigen comprises domain is a nucleic acid selected from one or more of the the leader sequence of CD59 , and HA epitope tag , and the following classes, including but not limited to , a DNA body of CD59 ; the polypeptide antigen comprises adenosine aptamer , an RNA aptamer , an siRNA , a shRNA , a single deaminase and an HA epitope tag; the polypeptide antigen strand RNA probe , a single strand DNA probe, an mRNA , comprises phenylalanine hydroxylase and an HA epitope a chemically modified oligonucleotide . In some embodi tag ; the polypeptide antigen comprises adenosine deami ments , the U domain is a small molecule selected from one nase, a (Gly3Ser )2 flexible linker (Seq . ID No. 23 ), phenyl or more of the following classes , including but not limited alanine hydroxylase , and an HA epitope tag ; the polypeptide to , a chelator, DOTA , a radionuclide , an isotope , an imaging antigen comprises glycophorin A , adenosine deaminase at agent, a fluorescent molecule , a chemiluminescent molecule , the cytoplasmic C terminus, and an HA epitope tag ; the a gas . polypeptide antigen comprises glycophorin A , phenylala [0264 ] Examples of Antigen Polypeptides nine hydroxylase at the cytoplasmic C terminus, and an HA [ 0265 ] Examples of antigen polypeptides include: the epitope tag. polypeptide antigen comprises glycophorin A with HA [0266 ] In certain embodiments , the antigen is capable or epitope tag at the N terminus; the polypeptide antigen interacting with a macrophage . The antigen polypeptide may comprises the leader sequence of glycophorin A , HA epitope comprise one or more of: the complement receptor (Rieu et US 2018 /0271910 A1 Sep . 27 , 2018

al ., J. Cell Biol. 127 : 2081 - 2091 ( 1994 ) ) , the scavenger In another embodiment, the CR1 antigen polypeptide may receptor (Brasseur et al. , Photochem . Photobiol. 69 :345 -352 comprise one or more than one extracellular domains of ( 1999 ) ) , the transferrin receptor (Dreier et al. , Bioconjug . CR1 fused to another cell membrane protein , e . g ., glyco Chem . 9 : 482 - 489 ( 1998 ) ; Hamblin et al . , J . Photochem . phorin A , glycophorin B , glycophorin C , glycophorin D , Photobiol. 26 : 4556 ( 1994 ) ) ; the Fc receptor (Rojanasakul et kell, band 3 , aquaporin 1 , glut 1, kidd antigen protein , rhesus al ., Pharm . Res. 11: 1731- 1733 ( 1994 ) ); and the mannose antigen , including, but not limited to the cell surface moi receptor ( Frankel et al. , Carbohydr. Res. 300 :251 -258 eties listed in table 1 and table 6 . ( 1997) ; Chakrabarty et al ., J. Protozool. 37 :358 - 364 ( 1990 ) ) . 0270 ]. In some embodiments, an EHC contains a recom [0267 ] Other antigens capable or interacting with a mac binant nucleic acid encoding a complement receptor antigen rophages include: low density lipoproteins (Mankertz et al. , polypeptide, or alternatively or in combination , a comple Biochem . Biophys. Res . Commun . 240 : 112 - 115 ( 1997 ) ; von ment receptor agonist antigen polypeptide or complement Baeyer et al. , Int . J . Clin . Pharmacol. Ther. Toxicol. 31 : 382 associated antigen polypeptide including but not limited to , 386 (1993 ) ) , very low density lipoproteins ( Tabas et al. , J . the polypeptides, and agonists to the polypeptides, listed in Cell Biol. 115 : 1547 - 1560 ( 1991 )) , mannose residues and table 8 . In some embodiments , the EHCs further contain an other carbohydrate moieties ( Pittet et al. , Nucl . Med. Biol. exogenous decay - accelerating factor ( CD59, GenBank : 22 : 355 - 365 (1995 ) ) , poly -cationic molecules, such as poly CAG46523 . 1 ) polypeptide , or an exogenous membrane L -lysine (Hamblin et al. , J . Photochem . Photobiol. 26 :45 -56 cofactor ( CD46 , GenBank : BAA12224 . 1 ) polypeptide, or a ( 1994 ) ) , liposomes (Bakker - Woudenberg et al. , J . Drug variant or functional fragment thereof, or a combination Target . 2 : 363 -371 (1994 ); Betageri et al ., J . Pharm . Phar thereof . macol. 45 :48 -53 ( 1993 ) ) and 2 -macroglobulin (Chu et al . , J . 10271 ] CR1 activities include binding to C3b -containing Immunol. 152: 1538 - 1545 ( 1994 ) ) . immune complexes and shuttling of these immune com [ 0268] Provided herein are compositions containing EHCs plexes from circulation to liver and spleen macrophages of comprising an antigen having functional activities that are the reticuloendothelial system . Upon encounter with cells of either i ) not present in native EHCs of the same lineage , or the reticuloendothelial system , the immune complex is ii ) present in native EHCs of the same lineage in reduced endocytosed by the phyagocytic cell but the red blood cell levels or reduced activity levels as compared to the EHCS is spared to continue its circulation . The removal of the comprising the antigen . Such functional activities include immune complex sometimes results in proteolytic cleavage complement inhibition , immune complex clearance , artifi of CR1 from the surface of the red blood cell . To measure cial antigen presentation , modulation of the coagulation binding activity , one can perform an in vitro binding assay cascade , oxygen transfer, drug delivery , cytotoxin adsorp between EHCs and immune complexes. To measure sparing tion , avoidance of phagocytosis , and extension of circulation of the EHC , one can perform an in vitro phagocytosis assay time. with phagocytic cells and immune complex - loaded EHCS. [ 0269 ] In some embodiments , EHCs have higher levels of To measure in vivo clearance of circulating immune com a complement receptor polypeptide , such as CR1, than plexes to the liver, one can perform a clearance and biodis native EHCs of the same lineage by virtue of comprising a tribution assay using radiolabeled immune complexes . CR - 1 antigen . In an alternative embodiment, the EHCS [0272 ] Provided are compositions containing EHCs con comprising an antigen have higher levels of a complement taining an antigen comprising a native polypeptide at a level receptor agonist polypeptide or complement associated greater than that of a hematopoietic cell of the same lineage polypeptide than native EHCs of the same lineage , including not comprising the antigen polypeptide . For example , popu but not limited to , the polypeptides listed in table 6 and table lations of EHCs contain antigens, such as complement 8 . The complement receptor antigen polypeptide comprises receptor 1 levels at least about 1 . 1 , e . g ., 1 . 2 , 1 .3 , 1 . 4 , 1 .5 , 1 . 6 , a human Complement Receptor - 1 (CRI ) polypeptide, vari 1 . 7 , 1 . 8 , 1 . 9 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 15 , 20 , 25 , 30 , 35 , 40 , ant, or functional fragment thereof . The CR1 antigen poly 45 , 50 , 60 , 70 , 80 , 90 , 100 , 150 , 200 , 250 , 300 , 350 , 400 , peptide may be derived from one or more than one of the 450 , 500 , 600 , 700 , 800 , 900 , 1000 , 2000 , 3000 , 4000 , 5000 , native alleles of CR1, e . g . , the A allele ( also termed the F 6000 , 7000 , 8000 , 9000 , 10000 , or more than 10000 times allele or CR1 * 1 allele ) , the B allele ( also termed the S allele greater than corresponding hematopoietic cells of the same or CR1 * 2 allele ) , the C allele ( also termed the F ' allele or lineage that lack the CR1 antigen polypeptide . CR1 levels on CR1 * 3 allele ), or the D allele ( also termed the CR1 * 4 reticulocytes and erythrocytes are typically between allele ) . The sequences and database accession numbers for 50 - 2000 molecules per cell (Lach - Trifilieff , J Immunol these native forms are provided in table 3 . In some embodi 1999 , 162 :7549 ). Provided are compositions that contain ments , the CR1 antigen polypeptide contains a domain of a populations of EHCs with CR1 levels of at least about 2500 , CR1 polypeptide . For example , the CR1 polypeptide may 5000 , 6000 , 7000 , 8000 , 9000 , 10000 , 15000 , 20000 , 25000 , comprise one or more short consensus repeat ( SCR ) 30000 , 40000 , 50000 , 100000 , 200000 , 300000 , 400000 , domains , also termed complement control protein ( CCP ) 500000 , 600000 , 700000 , 800000 , 900000 , 1000000 , or modules or Sushi domains , e . g ., Genbank accession number more than 1000000 molecules per cell. CR1 levels in AAV65577 . 1 . In one embodiment , the CR1 antigen poly wild - type and exogenous antigen - expressing EHCs can be peptide comprises one or more Short Consensus Repeats measured and quantified by , for example , flow cytometry ( SCRs ) , e . g . , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11, 12 , 13 , 14 , 15 , with antibodies specific for CR1. 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 10273 ] Provided herein , in some embodiments , are EHCS 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41, 42 , 43 , 44 or greater comprising an antigen , populations of EHCs comprising an than 44 SCRs. In another embodiment, the CR1 antigen antigen , and compositions of EHCs comprising an antigen . polypeptide comprises one or more long homologous repeat In some embodiments , the antigen interacts with a circulat ( LHR ) units of CR1, e . g ., LHR - A , LHR - B , LHR - C , or ing pathogen , such as a virus or a bacterium . In some LHR - D , e . g . , 1 , 2 , 3 , 4 , 5 , 6 or greater than 6 LHR domains . embodiments , the EHC expresses a recombinant gene US 2018 /0271910 A1 Sep . 27 , 2018 19 encoding an antibody, scFv, or nanobody specific for the specific ; b ) unable to initiate intracellular signals , and c ) circulating pathogen . The antibody, scFv, or nanobody may chemokines bound to erythrocyte surface are believed to be be expressed as a fusion protein . In other embodiments , the inaccessible to their normal target inflammatory cells (Ne antibody , scFv, or nanobody antigen or another antigen with ote , J Biol Chem , 1993 ) . Erythrocytes may play a role in the affinity to circulating pathogens is loaded into or onto the regulation of inflammatory processes through the presence EHC . The antibody , scFv , or nanobody antigen or the other antigen with affinity to circulating pathogens may be local of DARC ized intracellularly or extracellularly . In some embodiments , [0279 ] Inflammatory signaling molecules, such as cytok the antigen is specific for a viral or bacterial antigen , such as ines, can trigger local and systemic tissue damage when a surface , envelope or capsid antigen . present in high concentrations . Bursts of cytokines are [ 0274 ] Provided herein , in certain embodiments , are EHCs implicated in the pathogenesis of bacterial sepsis , rheuma comprising an antigen , populations of EHCs comprising an toid arthritis , and several other inflammatory diseases . antigen , and compositions of EHCs comprising an antigen . Modified EHCs that exogenously express natural cytokine In some embodiments , the antigen interacts with a toxin , receptors or synthetic antibody - like receptor mimics can preferably a foreign toxin , such as derived from a pathogen sequester the inflammatory cytokines . An exemplary or otherwise from the environment. In some embodiments , chemokine receptor is DARC . Provided herein are EHCS the EHC expresses a recombinant gene encoding an antigen comprising an antigen that is a cytokine receptor or comprising an amino acid sequence derived from lipopoly chemokine receptor , including , but not limited to DARC . saccharide -binding protein (LBP ) , bactericidal/ permeabil For example , EHCs expressing DARC antigen ( thereby ity - increasing protein (BPI ) , amyloid P component, or a increasing the amount present on native erythrocytes ) may cationic protein . Toxin -binding antigens may be expressed be used to modulate chemokine levels in circulation and / or as a fusion protein . In other embodiments, toxin - binding within the body ' s peripheral tissues . The EHCs comprising antigensmay be loaded into or onto the EHC . Toxin -binding a DARC antigen can either be marked for destruction or can antigens may be localized intracellularly or extracellularly . slowly release the inflammatory mediators back into circu In some embodiments , the toxin binding antigen is specific lation , but at a low and diffuse concentration . The EHC for a bacterial toxin such as botulinum or anthrax . comprising an antigen that comprises a chemokine or [ 0275 ] Further , exogenous antigen -expressing EHCs may cytokine receptormay act as a reservoir for signal transduc express an antigen capable of enhancing its ability to seques tion peptides. ter a target . Potential sequestration enhancement antigens [0280 ] In one embodiment, the antigen comprises a poly include the polypeptide transporters including , but not lim peptide that comprises an amino acid sequence derived from ited to , those in table 1 . an antibody . In one embodiment, the EHC expresses a [ 02761. In one embodiment, the antigen comprises a poly recombinant gene encoding an amino acid sequence derived peptide that comprises an amino acid sequence derived from from an antibody. The antibody antigen may be expressed as Duffy Antigen Receptor for Chemokines (DARC ) . In one a full -length protein or a fragment thereof. The antibody embodiment, the EHC expresses a recombinant gene encod may be expressed as a fusion protein . In other embodiments , ing an amino acid sequence derived from Duffy Antigen the antibody protein is loaded into or onto the EHC . In some Receptor for Chemokines (DARC ) . The DARC antigen may embodiments , the loaded antibody is additionally function be expressed as a full - length protein or a fragment thereof. alized or otherwise modified . The antibody antigen may be DARC may be expressed as a fusion protein . In other localized intracellularly or extracellularly . In one embodi embodiments , DARC protein is loaded into or onto the ment, the antigen comprises an antibody amino acid EHC . In some embodiments , the loaded DARC is addition sequence that is specific for a desired target . In some ally functionalized or otherwise modified . The DARC anti embodiments , the antibody is a scFv . In other embodiments , gen molecule may be localized intracellularly or extracel lularly . the antibody is a nanobody . [0277 ] DARC was identified as a potent multi - ligand [ 0281 ] In certain embodiments , the EHCs comprise an chemokine receptor . DARC belongs to the family of rho antigen that comprises an antibody or fragment thereof that dopsin - like seven -helix transmembrane proteins. Besides is specific for a target and is located on the cell surface . For erythrocytes DARC is expressed in post capillary venular example , a variable fragment ( Fv ) of an antibody specific for endothelial cells , which are the primary site of leukocyte botulinum toxin binding is expressed on the surface of the transmigration in most tissues . DARC provides a highly EHC . Botulinum toxin binding antibodies are known in the specific binding site for both CC and CXC chemokines . art ( Amersdorfer , Inf and Immunity , 1997 ) , as is the expres DARC is thought to possess a higher affinity for ELR motif sion of the Fv portion of an antibody (Hoedemaeker , Journ CXC chemokines . CXC chemokines are neutrophil che of Bio Chemistry , 1997 ) . Upon binding , the toxin is retained moattractants and may potentially be pro -angiogenic . by the EHC through the Fv region , sequestered and shuttled [0278 ] Interaction between DARC and CXCL8 has dem via the circulatory system to the liver for clearance from the onstrated a dissociation constant (KJ ) of 5 nmol/ L and body. receptor binding sites estimated at 1000 - 9000 per erythro [0282 ] In one embodiment, the antigen comprises a poly cyte (Hadley , Blood , 1997 ) Unlike other seven - transmem peptide that comprises an amino acid sequence derived from brane chemokine receptors , DARC lacks the highly con a scFv antibody. In one embodiment, the EHC expresses a served G protein coupling motif located in the second recombinant gene encoding an amino acid sequence derived cytoplasmic loop (Meny , Immunohematology , 2010 ) . from a scFv antibody. The scFv antibody antigen may be DARC is not G -protein coupled and has no known alterna expressed as a full -length protein or a fragment thereof. The tive signaling mechanism . The biological role of DARC is scFv antibody may be expressed as a fusion protein . In other not fully understood . DARC is thought to be a ) multi embodiments , the scFv protein is loaded into or onto the US 2018 /0271910 A1 Sep . 27 , 2018 20

EHC . Suitable scFv antigen polypeptides that may be expresses a recombinant gene encoding an amino acid expressed by EHCs include , but are not limited to , those sequence derived from one of proteases , nucleases, amylase , listed in table 6 . lyase (sucrase ) or hydrolase (DNase , lipase ). Antigen pro [ 0283 ] scFv antibodies have been constructed mainly from teases, nucleases , amylases , lyases and hydrolases may be hybridoma, spleen cells from immunized mice , and B lym expressed as a full - length protein or a fragment thereof. phocytes from human . The variable region of an antibody is Antigen proteases , nucleases, amylases , lyases and hydro formed by the noncovalent heterodimer of the variable lases may be expressed as a fusion protein . In other embodi domains of the V ( H ) and V ( L ) domains, which can then be ments , antigen proteases , nucleases , amylases , lyases or used in the construction of a recombinant scFv antibody . hydrolases are loaded into or onto the EHC . In some [0284 ] The production of scFvs is known in the art and embodiments , the loaded antigen proteases, nucleases, amy require mRNA to first be isolated from hybridoma ( or also lases, lyases or hydrolases are additionally functionalized or from the spleen , lymph cells , and bone morrow ) followed by otherwise modified . The antigen protease, nuclease , amy reverse transcription into cDNA to serve as a template for lase , lyase or hydrolase antigen molecule may be localized antibody gene amplification (PCR ) . With this method , large intracellularly or extracellularly . libraries with a diverse set of antibody - derived scFvs ( a set [0290 ] In certain embodiments , EHCs comprise an antigen comparable to that of the original antibodies from which the comprising a protease , a nuclease , an amylase , a lyase or a scFvs are modeled ) can be created . hydrolase . The EHC comprising a protease , a nuclease , an [0285 ] The scFv antigen may be made specific to any amylase , a lyase or a hydrolase antigen is capable of target molecule including, but not limited to , those in table degrading a target on the EHC independent of circulatory clearance , e . g . , by macrophages in the liver. In certain ( 0286 ] In one example , a scFv antigen specific for anthrax embodiments , EHCs comprising an antigen comprising a toxin may be expressed on a EHC . Upon administration to protease , a nuclease , an amylase , a lyase or a hydrolase may a subject in need thereof an effective dose of a population of be administered to a subject in need thereof to treat a cancer EHC comprising an antigen molecule specific for anthrax by selectively degrading metabolites that are essential for toxin can be used to capture and sequester the anthrax toxin . cancer cell growth . For example , asparaginase is used to The EHC migrates to the liver where clearance occurs . decrease local asparagine levels to treat acute lymphoblastic [ 0287 ] In certain embodiments , erythrocytes comprise an leukemia and acute myeloid leukemia . Suitable antigens antigen comprising a camelid - derived nanobody expressed may, e. g ., comprise one or both of the two major classes of on the surface of the cell. Nanobodies are usually 12 - 15 kDa. enzymes capable of degrading target molecules , lyases and They are considerably smaller than antibodies and scFv. hydrolases . In certain embodiments , EHCs are provided Nanobodies may thus be easier to transfect, and the nano comprising an antigen comprising a molecule including but body antigen will be more easily expressed , translated and not limited to those listed in table 6 . or transported to the cell surface in an EHC . In certain [0291 ] In certain embodiments , erythrocytes comprise an embodiments , nanobody antigens are employed to minimize antigen comprising a lyase. In one embodiment, the lyase is immunogenic effects caused by a specific antigen . Nano valine decarboxylase . Valine decarboxylase antigen may be bodies because of their small size will offer reduced immu expressed within the intracellular space of the EHC . EHCs nogenic potential. In certain embodiments , antigen nano comprising a valine decarboxylase antigen may be admin bodies are employed because they limit changes in the istered to a subject in need thereof to modulate valine levels mechanical and morphological behavior of the plasma mem within the blood . EHCs comprising a valine decarboxylase brane of the EHC . This may allow the EHC to exhibit antigen are suitable to treat valinemia , an inherited disorder normal circulatory red blood cell behavior. In certain that increases levels of the amino acid valine in the blood . embodiments , antigen nanobodies are employed because Affected individuals typically develop vomiting , failure to they have an increased ability to recognize hidden or uncom thrive , intellectual disability , and fatigue . Valinemia is mon epitopes compared to standard antibodies . For example , caused by a deficiency of the valine transaminase enzyme they can bind to small enzymatic cavities of a target and and has an autosomal recessive pattern of inheritance . modulate the molecular behavior of the target . [0292 ] In certain embodiments , erythrocytes comprise an [ 0288 ] In certain embodiments , EHCs comprise antigen antigen comprising a hydrolase . In one embodiment, the nanobodies with specificity to target epitopes of molecules hydrolase is deoxyribonuclease I (DNase I) . DNase I antigen in the human complement system . Such EHCs may be may be expressed on the surface of the EHC . EHCs com administered to a subject in need thereof to selectively prising a DNase I antigen may be administered to a subject deplete one or more over -active factors of the complement in need thereof to preferentially cleave circulating DNA at system . For example , C5 may be targeted by EHCs com phosphodiester linkages adjacent to a pyrimidine nucleotide , prising antigen nanobodies with specificity to target epitopes yielding 5 '- phosphate -terminated polynucleotides with a of C5 and cleared from the system by the EHCs upon free hydroxyl group on position 3 '. On average tetra - nucleo administration of the cells into a subject . This approach is tides are produced . EHCs comprising a DNase I antigen are suitable to provide a therapeutic effect, e . g . , for a comple suitable to treat conditions exacerbated by high levels of ment disorder , such as paroxysmal nocturnal hemoglobinu immunogenic DNA in circulation , such as systemic lupus ria . In certain embodiments , EHCs comprise antigen nano erythematosus ( SLE ). bodies with specificity to target epitopes of molecules 10293 ]. In certain embodiments the antigen is capable of including , but not limited to , those listed in table 4 . responding to an external stimulus , e . g . , upon binding to a [ 0289 ] In some embodiments , the antigen comprises a ligand or contacting the stimulus , wherein responding polypeptide that comprises an amino acid sequence derived entails , for example , moving, re - folding, changing confor from one of proteases , nucleases , amylase , lyase ( sucrase ) or mation , forming a dimer, forming a homodimer , forming a hydrolase (DNase , lipase ). In one embodiment, the EHC heterodimer, forming a multimer, transducing a signal, emit US 2018 /0271910 A1 Sep . 27 , 2018 21

ting energy in a detectable form ( e . g . , fluorescence ), func eria monocytogenes; Mycoplasma spp . ; Pseudomonas fluo tionally interacting with another antigen , or functionally rescens ; Vibrio cholerae ; Haemophilus influenzae ; Bacillus interacting with a non -exogenous antigen polypeptide. anthracis ; Treponema pallidum ; Leptospira ; Borrelia ; Corynebacterium diphtheriae ; Francisella ; Brucella Targets melitensis ; Campylobacter jejuni ; Enterobacter; Proteus [0294 ] Provided herein are exogenous antigen -expressing mirabilis ; Proteus ; and Klebsiella pneumoniae . EHCs comprising an exogenous antigen polypeptide capable of interacting with a target . Further provided herein [0300 ] In some embodiments , the target is a virus, includ are exogenous antigen - expressing EHCs comprising a non ing but limited to, those whose infection involves injection polypeptide exogenous antigen capable of interacting with a of genetic materials into host cells upon binding to cell target. The exogenous antigen - expressing EHCs may be surface receptors, viruses whose infection is mediated by administered to a subject in need thereof to modulate the cell surface receptors. Non - limiting examples of these amount or concentration of a target residing in the circula viruses can be selected from Paramyxoviridae ( e . g . , pneu tory system of the subject. A suitable exogenous antigen may movirus , morbillivirus , metapneumovirus , respirovirus or be chosen to interact with a specific target. Suitable targets rubulavirus ), Adenoviridae ( e . g . , adenovirus ) , Arenaviridae include entities that are associated with a specific disease , ( e . g ., arenavirus such as lymphocytic choriomeningitis disorder, or condition . However, targets may also be chosen virus ), Arteriviridae ( e . g . , porcine respiratory and reproduc independent of a specific disease , disorder , or condition . tive syndrome virus or equine arteritis virus ) , Bunyaviridae [0295 ] In some embodiments , the target is an antibody or ( e . g ., phlebovirus or hantavirus ) , Caliciviridae ( e . g . , Nor antibody - like molecule , for example an autoimmune or a walk virus ) , Coronaviridae ( e . g ., coronavirus or torovirus) , self- antibody , or a foreign antibody, or a therapeutic anti Filoviridae (e . g ., Ebola - like viruses ) , Flaviviridae ( e. g . , body, including but not limited to , e . g . , an antibody against hepacivirus or flavivirus ) , Herpesviridae ( e . g ., simplexvirus, beta - 2 glycoprotein 1 , an antibody against I/ i antigen , an varicellovirus , cytomegalovirus, roseolovirus, or lymphoc antibody against the NC1 domain of collagen a3 (IV ) , an ryptovirus ) , Orthomyxoviridae ( e . g . , influenza virus or antibody against platelet glycoprotein , an antibody against thogotovirus ), Parvoviridae (e .g . , parvovirus) , Picomaviri phospholipase A2 receptor, an antibody against erythrocyte dae ( e . g . , enterovirus or hepatovirus) , Poxviridae ( e . g . , glycophorin A , B , or C , or an antibody against erythrocyte orthopoxvirus , avipoxvirus, or leporipoxvirus ), Retroviridae Rh antigen . ( e . g . , lentivirus or spumavirus ) , Reoviridae ( e . g ., rotavirus ) , [ 0296 ] In some embodiments , the target is a molecule of Rhabdoviridae ( e . g . , lyssavirus , novirhabdovirus, or vesicu the complement cascade , for example C1, C1r, Cis , Cig , lovirus ), and Togaviridae ( e . g ., alphavirus or rubivirus) . C2 , C2a , C2 , C3 , C3a , C3b , C4 , C4 , C4a , C3bBb , Specific examples of these viruses include human respira C3bBb3b , C4b2b , C4b2b3b , C5, C5a , C5b , C6 , C7 , C8, C9 , tory coronavirus, influenza viruses A - C , hepatitis viruses A poly -C9 , membrane attack complex . Factor B , Factor D , to G , and herpes simplex viruses 1 - 9 . Properdin , C3, C3a, C3b , iC3b , C3c, C3dg , C3dk , C3e , Bb , [0301 ] In some embodiments , the target is a parasite , Factor I , C1q , C1r, Cis , C4 , C4a , C4b , C2 , C4 bp , Mannose including but not limited to , for example , intestinal or Binding Lectin (MBL ) , MBL - Associated Serine Protease 1 blood -borne parasites, protozoa , trypanosomes ; haemopro (MASP1 ) , MBL - Associated Serine Protease 2 (MASP2 ) , tozoa and parasites capable of causing malaria ; enteric and C5, C5a , C6 , C7 , C8 , C9 , CR1, CR2, CR3, CR4, C3AR , systemic cestodes including taeniid cestodes ; enteric coc C3eR , Decay - accelerating factor (DAF ) , Membrane cofac cidians ; enteric flagellate protozoa ; filarial nematodes ; gas tor protein (MCP ), CD59 , C3 Beta chain Receptor, C1 trointestinal and systemic nematodes and hookworms. inhibitor , C4 binding protein , Factor 1 , Factor H . [0297 ] In some embodiments , the target is an immune [ 0302 ] In some embodiments , the target is a fungus , complex , for example an IgG immune complex , an IgA including but not limited to , for example , Candida albicans , immune complex , an IgM immune complex . Candida glabrata, Aspergillus, T. glabrata , Candida tropi [ 0298 ] In some embodiments , the target is an amyloid calis , C . krusei , and C . parapsilosis . placque , for example a placque comprised of beta amyloid , [0303 ] In some embodiments , the target is a bacterial IAPP ( Amylin ), alpha - synuclein , PrPSc , huntingtin , calci toxin , including but not limited to , for example , AB toxin , tonin , atrial natriuretic factor, apolipoprotein Al, serum alpha toxin , anthrax toxin , bacteriocin , botunlinum toxin , amyloid A , medin , prolactin , transthyretin , lysozyme, beta 2 cholesterol- dependent cytolysin , Clostridium botulinum C3 microglobulin , gelsolin , keratoepithelin , cystatin , immuno toxin , Clostridium difficile toxin A , Clostridium difficile globulin light chain AL , S - IBM . toxin B , Clostridium enterotoxin , Clostridium perfringens [0299 ] In some embodiments , the target is a bacterium , for alpha toxin , Clostridium perfringens beta toxin , Cord factor , example Enterococcus, Streptococcus, or Mycobacteria , CrylAc , Cryptophycin , Delta endotoxin , Diphtheria toxin , Rickettsia , Mycoplasma, Neisseria meningitides , Neisseria Enterotoxin type B , erythrogenic toxin , exfoliatin , haemoly gonorrheoeae, Legionella , Vibrio cholerae, Streptococci, sin E , heat- labile enterotoxin , heat- stable enterotoxin , Staphylococcus aureus, Staphylococcus epidermidis, hemolysin , leukocidin , lipopolysaccharide , Listeriolysin 0 , Pseudomonas aeruginosa , Corynobacteria diphtheriae, microcin , Panton - Valentine leucocidin , pathogenicity island , Clostridium spp ., enterotoxigenic Eschericia coli, and Bacil phenol- soluble modulin , pneumolysin , pore - forming toxin , lus anthracis . Other pathogens for which bacteremia has Pseudomonas exotoxin , RTX toxin , sakacin , Staphylococ been reported at some level include the following : Rickett cus aureus alpha toxin , Staphylococcus aureus beta toxin , sia , Bartonella henselae, Bartonella quintana , Coxiella bur - Staphylococcus aureus delta toxin , Streptolysin , Symploc netii , chlamydia , Mycobacterium leprae , Salmonella ; shi amide A , tabtoxin , tetanolysin , tetanospasmin , thiol- acti gella ; Yersinia enterocolitica ; Yersinia pseudotuberculosis ; vated cytolysin , tolaasin , toxic shock syndrome toxin , toxo Legionella pneumophila ; Mycobacterium tuberculosis ; List flavin , trehalose dimycolate , verocytotoxin , and vibriocin . US 2018 /0271910 A1 Sep . 27 , 2018

[0304 ] In some embodiments , the target is a prion protein , f0308 ] In some embodiments , the target is a mammalian including but not limited to , for example, PRP , PRPC, cell , including but not limited to , for example, a human cell , PRPsc , PRPres. a circulating cell , an immune cell , a neutrophil , an eosino [ 0305 ] In some embodiments, the target is a cytokine or a phil, a basophil , a lymphocyte , a monocyte , a B cell, a T cell , chemokine or a growth factor, including but not limited to , a CD4 + T cell , a CD8 + T cell, a gamma - delta T cell , a for example , acylation stimulating protein , adipokine , regulatory T cell , a natural killer cell , a natural killer T cell , albinterferon , CCL1, CCL11, CCL12 , CCL13 , CCL14 , a macrophage , a Kupffer cell , a dendritic cell, a cancer cell , CCL15 , CCL16 , CCL17 , CCL18 , CCL19 , CCL2, CCL20 , a cancer stem cell , a circulating tumor cell , a cancer cell CCL21, CCL22 , CCL23 , CCL24 , CCL25 , CCL26 , CCL27 , from one of the following cancers including , but not limited CCL28 , CCL3 , CCL5, CCL6 , CCL7 , CCL8 , CCL9, colony to , stimulating factor, CX3CL1, CX3CR1 , CXCL1, CXCL10 , [0309 ) ACUTE lymphoblastic leukaemia ( ALL ) , ACUTE CXCL11 , CXCL13 , CXCL14 , CXCL15 , CXCL16 , myeloid leukaemia (AML ) , anal cancer , bile duct cancer, CXCL17 , CXCL2 , CXCL3, CXCL5 , CXCL6 , CXCL7 , bladder cancer , bone cancer, bowel cancer, brain tumours , CXCL9, erythropoietin , GC- MAF , granulocyte colony breast cancer, cancer of unknown primary , cancer spread to stimulating factor, granulocyte macrophage colony - stimu bone , cancer spread to brain , cancer spread to liver , cancer lating factor, hepatocyte growth factor, IL 10 family , IL 17 spread to lung , carcinoid , cervical cancer , choriocarcinoma , family , IL1A , ILIB , interferon , interferon beta la , interferon chronic lymphocytic leukaemia (CLL ) , chronic myeloid beta 1b , interferon gamma, interferon type I , interferon type leukaemia (CML ) , colon cancer , colorectal cancer , endome II , interferon type III, interleukin , interleukin 1 family, trial cancer, eye cancer, gallbladder cancer , gastric cancer, interleukin 1 receptor antagonist , interleukin 10 , interleukin gestational trophoblastic tumours (GTT ), hairy cell leukae 12 , interleukin 12 subunit beta, interleukin 13 , interleukin mia , head and neck cancer, hodgkin lymphoma, kidney 16 , interleukin 2 , interleukin 23 , interleukin 23 subunit cancer, laryngeal cancer, leukaemia , liver cancer, lung can alpha , interleukin 34 , interleukin 35 , interleukin 6 , interleu cer, lymphoma, melanoma skin cancer , mesothelioma , kin 7 , interleukin 8 , interleukin - 36 , leukemia inhibitory men ' s cancer, molar pregnancy , mouth and oropharyngeal factor, leukocyte - promoting factor, lymphokine, lympho cancer, myeloma , nasal and sinus cancers , nasopharyngeal toxin , lymphotoxin alpha , lymphotoxin beta , macrophage cancer, non hodgkin lymphoma (NHL ) , oesophageal cancer , colony -stimulating factor, macrophage inflammatory pro ovarian cancer, pancreatic cancer, penile cancer, prostate tein , macrophage -activating factor, monokine , myokine , cancer, rare cancers, rectal cancer, salivary gland cancer, myonectin , nicotinamide phosphoribosyltransferase , secondary cancers , skin cancer (non melanoma) , soft tissue oncostatin M , oprelvekin , platelet factor 4 , proinflammatory sarcoma, stomach cancer, testicular cancer, thyroid cancer, cytokine , promegapoietin , RANKL , stromal cell -derived unknown primary cancer, uterine cancer, vaginal cancer, and factor 1, talimogene laherparepvec , tumor necrosis factor vulval cancer. alpha , tumor necrosis factors , XCL1 , XCL2, XCR1, angiopoietin , basic fibroblast growth factor, betacellulin , Antigen Expression , Conjugation , Loading bone morphogenetic protein , brain - derived neurotrophic [0310 ] In certain embodiments , the polypeptide antigen is factor, CCN intercellular signaling protein , CTGF, darbepo expressed within the exogenous antigen -expressing EHC . etin alfa , endoglin , epidermal growth factor, epoetin alfa , The polypeptide antigen may be exhibited on the surface of epoetin beta , erythropoietin , FGF15 , FGF15 /19 , fibroblast the exogenous antigen - expressing EHC or may reside within growth factor , fibroblast growth factor 23 , filgrastim , GLIA the exogenous antigen -expressing EHC . maturation factor, granulocyte colony - stimulating factor, [0311 ] In certain embodiments , the polypeptide antigen is granulocyte macrophage colony - stimulating factor, growth conjugated to the exogenous antigen - expressing EHC . The differentiation factor - 9 , heberprot- P , hemopoietic growth polypeptide antigen usually is conjugated to the surface of factors , heparin -binding EGF - like growth factor, hepatocyte the exogenous antigen -expressing EHC . Conjugation may growth factor, insulin - like growth factor, insulin - like growth be achieved chemically or enzymatically , by methods factor 1 , insulin -like growth factor 2 , keratinocyte growth known in the art and described herein . Non -polypeptide factor, myostatin , nerve growth factor, neurotrophin - 3 , neu antigens may also be conjugated to an exogenous antigen rotrophin - 4 , oncomodulin , osteopromotive , palifermin , expressing EHC . In some embodiments , the antigen is not PDGFB , placental growth factor , platelet alpha - granule , conjugated to the exogenous antigen -expressing EHC . platelet- derived growth factor, platelet - derived growth fac [ 0312 ] In certain embodiments , the polypeptide antigen is tor receptor, proliferative index , thrombopoietin , transform loaded into the exogenous antigen - expressing EHC . Non ing growth factor, vascular endothelial growth factor. polypeptide antigens may also be loaded within an exog [0306 ] In some embodiments , the target is a small mol enous antigen -expressing EHC . In some embodiments , the ecule , for example a chemical , an amino acid , an atom , an antigen is not loaded into or onto the exogenous antigen element, an organic acid , < 2000 Da, < 1000 Da , < 500 Da , expressing EHC . including but not limited to , for example, iron , copper , [0313 ] In some embodiments , the exogenous antigen calcium , potassium , ethanol, methanol, glycine , alanine , expressing EHC comprises an antigen that is optionally valine , leucine , isoleucine, serine , cysteine, selenocysteine , expressed from a recombinant nucleic acid , conjugated to threonine, methionine , proline , phenylalanine , tyrosine , the EHC , loaded into or onto the EHC , and any combination tryptophan , histidine, lysine , arginine , aspartate , glutamate , thereof . Optionally , the exogenous antigen - expressing EHC asparagine , glutamine. comprises a therapeutic agent or other payload . [0307 ] In some embodiments , the target is a lipid , lipid [ 0314 ] In some embodiments , the exogenous antigen complex , proteolipid complex , or cholesterol, including but expressing EHC is generated by contacting a suitable iso not limited to for example , LDL , VLDL , HDL , HDL2B , lated cell , e . g . , an EHC , a reticulaocyte , an EHC precursor , triglycerides, LP ( a ), cholesterol. a platelet , or a platelet precursor, with a recombinant nucleic US 2018 /0271910 A1 Sep . 27 , 2018

acid encoding an antigen polypeptide . In some embodi e . g . Band 3 , glycophorin A , or Kell ; or fusion to the ments , the antigen polypeptide is encoded by a DNA , which GPI- linker acceptor peptide of an endogenous erythroid is contacted with a nucleated erythroid precursor cell or a GPI- linked cell surface protein such as, e . g . acetylcholin nucleated platelet precursor cell . In some embodiments , the esterase , CD55 , CD58 or CD59 ( see , e .g . Kooyman et al. , antigen polypeptide is encoded by an RNA , which is con Science 1995 ) . tacted with a platelet, a nucleate EHC , a nucleated platelet [0320 ] The antigen of interest can be conjugated to the precursor cell , or a reticulocyte . In some embodiments , the surface of a cultured EHC by various chemical and enzy antigen is a polypeptide , which is contacted with a primary matic means, including but not limited to those listed in platelet , a nucleated EHC , a nucleated platelet precursor cell , Table D , Table Di, and Table E . These methods include a reticulocyte , or an erythrocyte. chemical conjugation with bifunctional cross - linking agents [ 0315 ] A antigen polypeptide may be expressed from a such as , e . g . an NHS ester -maleimide heterobifunctional transgene introduced into an EHC by electroporation , crosslinker to connect a primary amine group with a reduced chemical or polymeric transfection , viral transduction , thiol group . These methods also include enzymatic strate mechanical membrane disruption , or other method ; an anti gies such as , e . g. transpeptidase reaction mediated by a gen polypeptide that is expressed from mRNA that is sortase enzyme to connect one polypeptide containing the introduced into a cell by electroporation , chemical or poly acceptor sequence LPXTG (Seq . ID No. 24 ) or LPXTA meric transfection , viral transduction , mechanical mem (Seq . ID No. 25 ) with a polypeptide containing the N -ter brane disruption , or other method ; an antigen polypeptide minal donor sequence GGG , see e . g . Swee et al. , PNAS that is over - expressed from the native locus by the intro 2013 . The methods also include combination methods, such duction of an external factor , e . g . , a transcriptional activator, as e . g . sortase- mediated conjugation of Click Chemistry transcriptional repressor, or secretory pathway enhancer ; handles (an azide and an alkyne ) on the antigen and the cell , and / or an antigen polypeptide that is synthesized , extracted , respectively , followed by a cycloaddition reaction to chemi or produced from a production cell or other external system cally bond the antigen to the cell , see e . g . Neves et al. , and incorporated into the EHC . Bioconjugate Chemistry , 2013 . [0316 ] In some embodiments , the antigen is a full- length [0321 ] If desired , a catalytic bond - forming polypeptide protein . In some embodiments , the antigen is comprised of domain can be expressed on or in an EHC , either intracel one or more polypeptides contained within the full- length lularly or extracellularly . Many catalytic bond - forming poly protein , of any length greater than approximately 7 amino peptides exist , including transpeptidases , sortases , and iso acids. For example , the polypeptides can be 7 , 8, 9, 10 , 11, peptidases, including those derived from Spy0128 , a protein 12 , 13 , 14 , 15 , 16 , 17 , 18, 19 , 20 , or more than 20 amino isolated from Streptococcus pyogenes . acids , e . g 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , or more than 100 [0322 ] It has been demonstrated that splitting the auto amino acids. The polypeptides comprising the antigen may catalytic isopeptide bond - forming subunit (CnaB2 domain ) comprise one or more immunological epitopes which may of Spy0128 results in two distinct polypeptides that retain be conformational epitopes or may be linear epitopes . The catalytic activity with specificity for each other . The poly antigen may be comprised of one or more polypeptides from peptides in this system are termed SpyTag and SpyCatcher. one or more different proteins. Upon mixing , SpyTag and SpyCatcher undergo isopeptide [0317 ] The antigen of interest can be expressed in the bond formation between Asp117 on SpyTag and Lys31 on circulating cell by fusion to an endogenous cell protein , SpyCatcher ( Zakeri and Howarth , JACS 2010 , 132 :4526 ) . including but not limited to those listed in Table B and Table The reaction is compatible with the cellular environment and C . Fusion to an endogenous protein may be necessary highly specific for protein / peptide conjugation ( Zakeri, B .; because it is thought that during the natural process of Fierer, J . O . ; Celik , E .; Chittock , E . C . ; Schwarz - Linek , U .; differentiation and enucleation , the EHC sheds and discards Moy , V . T. ; Howarth , M . Proc . Natl . Acad . Sci. U . S . A . 2012 , many of the endogenous proteins required for erythropoiesis 109, E690 - E697 ) . SpyTag and SpyCatcher has been shown but not for mature erythrocyte function such as, e . g . c - Kit to direct post - translational topologicalmodification in elas ( SCF receptor ) and transferrin . See e . g . Keerthivasan et al. , tin - like protein . For example , placement of SpyTag at the Stem Cells International 2011 ; Migliaccio , Haematologica N -terminus and SpyCatcher at the C - terminus directs for 2010 . Proteins that are retained include certain membrane mation of circular elastin - like proteins ( Zhang et al, Journal proteins such as, e .g . glycophorin A , band 3 , and aquaporin ; of the American Chemical Society , 2013 ) . certain cytoplasmic proteins such as, e . g . hemoglobin alpha , [0323 ] The components Spy Tag and SpyCatcher can be hemoglobin beta , and adenosine deaminase ; and cytoskel interchanged such that a system in which molecule A is etal proteins. fused to SpyTag and molecule B is fused to SpyCatcher is [0318 ] The antigen of interest can be expressed in the functionally equivalent to a system in which molecule A is intracellular space of the EHC by a number of methods , fused to SpyCatcher and molecule B is fused to SpyTag . For including direct expression of the transgene, fusion to an the purposes of this document, when SpyTag and Spy endogenous intracellular protein such as , e . g . , hemoglobin , Catcher are used , it is to be understood that the complemen fusion to the intracellular domain of endogenous cell surface tary molecule could be substituted in its place . proteins such as , e . g . Band 3 , glycophorin A , Kell , or fusion [0324 ] A catalytic bond - forming polypeptide, such as a to a structural component of the erythroid cytoskeleton . SpyTag / SpyCatcher system , can be used to attach an exog [ 0319] The antigen of interest can be expressed on the enous antigen of interest to the surface of an EHC . The extracellular surface of the EHC by a number ofmethods , SpyTag polypeptide sequence can be expressed on the including direct expression of the transgene if it contains a extracellular surface of the EHC . The SpyTag polypeptide transmembrane domain or other membrane attachment can be, for example , fused to the N terminus of a type - 1 or domain , fusion to an endogenous erythroid membrane pro type - 3 transmembrane protein , e . g . glycophorin A , fused to tein or to the transmembrane domain of said protein such as , the C terminus of a type - 2 transmembrane protein , e . g . Kell, US 2018 /0271910 A1 Sep . 27 , 2018 24.

inserted in - frame at the extracellular terminus or in an hemoglobin promoter , beta globin gene and a Spy Tag extracellular loop of a multi -pass transmembrane protein , sequence such that upon translation , intracellular beta globin e . g . Band 3 , fused to a GPI - acceptor polypeptide, e . g . CD55 is fused to Spy Tag at is C terminus. In addition , the EHC or CD59 , fused to a lipid -chain -anchored polypeptide , or expresses a Gatal promoter -led gene that codes for Spy fused to a peripheral membrane protein . The nucleic acid Catcher driving phenylalanine hydroxylase ( PAH ) expres sequence encoding the Spy Tag fusion can be expressed sion such that upon translation , intracellular PAH is fused to within an EHC . An exogenous antigen of interest can be SpyCatcher at its N terminus. Upon expression of both fused to SpyCatcher. The nucleic acid sequence encoding fusion proteins the Spy Tag bound beta globin is linked the SpyCatcher fusion can be expressed and secreted from through an isopeptide bond to the SpyCatcher bound PAH in the same EHC that expresses the SpyTag fusion . Alterna the intracellular space , allowing PAH to be anchored to beta tively , the nucleic acid sequence encoding the SpyCatcher globin and retained during maturation . fusion can be produced exogenously , for example in a [0329 ] In another embodiment, the SpyTag polypeptide bacterial, fungal, insect , mammalian , or cell- free production can be expressed as a fusion to the exogenous antigen of system . Upon reaction of the SpyTag and SpyCatcher poly interest within an EHC . The SpyCatcher polypeptide can be peptides , a covalent bond will be formed that attaches the expressed as a fusion to the C terminus ( intracellular ) of exogenous antigen of interest to the surface of the EHC . glycophorin A within the same EHC . Upon expression of [ 0325 ] In one embodiment, the SpyTag polypeptide may both fusion polypeptides, an isopeptide bond will be formed be expressed as a fusion to the N terminus of glycophorin A between the Spy Tag and SpyCatcher polypeptides , forming under the control of the Gatal promoter in an EHC . An a covalent bond between the membrane -anchored endog exogenous antigen of interest, for example the exogenous enous erythroid polypeptide and the exogenous antigen of antigens listed in Table F, Table G , Table H , Table I and Table J , fused to the SpyCatcher polypeptide sequence can interest. be expressed under the control of the Gatal promoter in the [0330 ] In another example , the exogenous antigen of same EHC . Upon expression of both fusion polypeptides , an interest may be physically loaded into a cultured EHC (as isopeptide bond will be formed between the SpyTag and opposed to expressed ) by a number of methods, including SpyCatcher polypeptides , forming a covalent bond between osmotic loading or hypotonic -hypertonic cycling in which the EHC surface and the exogenous antigen of interest. exogenous antigen diffuses through pores introduced into [ 0326 ] In another embodiment , the Spy Tag polypeptide the EHC membrane ( see e . g . Cremel and Godfrin , Int J may be expressed as a fusion to the N terminus of glyco Pharm 2013 ) and fusion to a cell penetrating peptide, such phorin A under the control of the Gatal promoter in an EHC . as one derived from a bacterial toxin , see e . g . Kwon et al. , An exogenous antigen of interest, for example the exog J Contr Rel 2009. enous antigens listed in Table F , Table G , Table H , Table I [0331 ] The exogenous antigen of interest may be and Table J , fused to the SpyCatcher polypeptide sequence expressed from a transgene introduced into an EHC by can be expressed in a suitable mammalian cell expression electroporation , chemical or polymeric transfection , viral system , for example HEK293 cells . Upon expression of the transduction , mechanical membrane disruption , or other SpyTag fusion polypeptide on the EHC , the SpyCatcher method ; an exogenous antigen that is expressed from mRNA fusion polypeptide can be brought in contact with the cell . that is introduced into a cell by electroporation , chemical or Under suitable reaction conditions, an isopeptide bond will polymeric transfection , viral transduction , mechanicalmem be formed between the SpyTag and SpyCatcher polypep brane disruption , or other method ; an exogenous antigen tides , forming a covalent bond between the EHC surface and polypeptide that is over -expressed from the native locus by the exogenous antigen of interest . the introduction of an external factor , e . g . a transcriptional [ 0327 ] A catalytic bond - forming polypeptide, such as a activator , transcriptional repressor, or secretory pathway SpyTag /SpyCatcher system , can be used to anchor the enhancer ; an exogenous antigen that is synthesized , exogenous antigen of interest to the intracellular space of an extracted , or produced from a production cell or other EHC . The SpyTag polypeptide sequence can be expressed in external system and incorporated into the EHC . the intracellular space of the EHC by a number of methods , (0332 ] EHCs of the invention may optionally be loaded including direct expression of the transgene, fusion to an with materials (payload ) such as peptides , proteins , DNA , endogenous intracellular protein such as , e . g . , hemoglobin , RNA , siRNA , and other macromolecules by applying con fusion to the intracellular domain of endogenous cell surface trolled injury to the cell for a predetermined amount of time proteins such as, e . g . Band 3 , glycophorin A , Kell, or fusion in order to cause perturbations in the cell membrane such to a structural component of the erythroid cytoskeleton . The that the materials can be delivered to the inside of the cell SpyTag sequence is not limited to a polypeptide terminus ( e . g . cytoplasm ). and may be integrated within the interior sequence of an [0333 ] In preferred embodiments , the EHC is a reticulo endogenous polypeptide such that polypeptide translation cyte . For example , reticulocytes may be loaded with an and localization is not perturbed . An exogenous antigen of mRNA encoding an exogenous antigen by controlled cell interest can be fused to SpyCatcher. The nucleic acid injury . The mRNA may be naked or modified , as desired . sequence encoding the SpyCatcher fusion can be expressed mRNA modification that improve mRNA stability and /or within the same EHC that expresses the Spy Tag fusion . decrease immunogenicity include , e . g . ARCA : anti -reverse Upon reaction of the SpyTag and SpyCatcher polypeptides , cap analog ( m - 3 - GP , G ) , GP G (Unmethylated Cap Ana a covalent bond will be formed that acts to anchor the log ), m 'GP G (Monomethylated Cap Analog ), m ; 2 .2 . 7GP2G antigen of interest in the intracellular space of the EHC . ( Trimethylated Cap Analog ) , m5CTP ( 5 -methyl - cytidine [ 0328 ] In one embodiment, an EHC may express SpyTag triphosphate ), m6ATP (N6 -methyl - adenosine - 5 - triphos fused to hemoglobin beta intracellularly . The EHC may be phate ) , S2UTP ( 2 - thio -uridine triphosphate ), and V genetically modified with a gene sequence that includes a (pseudouridine triphosphate ) . US 2018 /0271910 A1 Sep . 27 , 2018 25

[0334 ] In another preferred embodiment, the EHC is an [0339 ] Controlled cell injury as used herein includes: i ) erythrocyte . For example, erythrocytes may be loaded with virus -mediated transfection ( e . g . Herpes simplex virus , an exogenous antigen by controlled cell injury . The cell Adeno virus, Adeno - associated virus, Vaccinia virus , or injury can be caused by , for example , pressure induced by Sindbis virus ), ii ) chemically -mediated transfection , e . g . mechanical strain or shear forces, subjecting the cell to cationic polymer, calcium phosphate , cationic lipid , poly deformation , constriction , rapid stretching , rapid compres mers , and nanoparticles , such as cyclodextrin , liposomes , sion , or pulse of high shear rate . The controlled cell injury cationic liposomes, DEAE -dextran , polyethyleneimine , den leads to uptake of material ( payload ) into the cytoplasm of drimer , polybrene , calcium phosphate , lipofectin , DOTAP, the cell from the surrounding cell medium . lipofectamine , CTAB /DOPE , DOTMA ; and iii ) physically [ 0335 ] Using controlled cell injury based on controlled mediated transfection , including direct injection , biolistic cell deformation ( e . g .mechanical deformation of the cell as particle delivery , electroporation , laser- irradiation , sonopo it passes through the constriction ) leads to uptake of material ration, magnetic nanoparticles , and controlled deformation (payload ) by diffusion rather than endocytosis . The material ( e . g . cell squeezing ) , as exemplified by micro - needle , nano ( payload ) is present in the cytoplasm rather than in endo needle , femtosyringe , atomic - force microscopy (AFM ) tip , somes following cellular uptake upon the controlled injury gene gun ( e . g . gold nanoparticles ), Amaxa Nucleofector, thereby making the material readily available to the cell. phototransfection (multi -photon laser ), impalefection , and Controlled cell injury, e . g . by controlled deformation , pre magnetofection , and other suitable methods known in the serves cell viability ( e . g . greater than 50 % , 70 % , or greater art . Any suitable method may be used to obtain the EHCS than 90 % ) . In certain embodiments , controlled cell injury , described herein comprising one or more desired DNA , e . g . by controlled deformation , preserves the state of cellular RNA ( e . g . mRNA ) , or polypeptides comprising antigen . differentiation and activity . If desired, a combination treat [0340 ] Exogenous antigen of interest can be detected on ment is used , e . g . , controlled injury by deformation followed the EHC of the invention . The presence of the exogenous by or preceded by , e . g . , electroporation or another cell antigen can be validated and quantified using standard molecular biology methods , e . g . Western blotting or FACS membrane permeability increasing method . Optionally , sur analysis . Exogenous antigens present in the intracellular factants may be used . environment may be quantified upon cell lysis or using [ 0336 ] Mechanical deformation methods are particularly fluorescent detection . suitable for cells that do not tolerate other membrane per meability increasing methods well , e .g . show decreased Manufacturing viability or a different state of differentiation after perform ing such methods . Mechanical deformation methods are also [0341 ] In some embodiments , the EHC is generated using suitable for material ( payload ) that does not tolerate other a precursor hematopoietic cell, e . g ., a CD34 + cell, an membrane permeability increasing methods well . Alterna erythrocyte , a platelet, a megakaryocyte , or a neutrophil as tively or in addition , the payload may not be sufficiently a source . In some embodiments, the precursor hematopoietic introduced into the cell using alternative methods, e. g . cell is isolated from a human donor by a GMP - compliant because of e . g . charge , hydrophobicity , or size of the pay process . In some embodiments, the starting cells are sourced load . from an autologous donor. In some embodiments , the start ing cells are sourced from an allogeneic donor . The donor [0337 ] One exemplar method of controlled injury by may be typed for blood cell antigen polymorphisms and /or deformation and devices suitable for such methods is the donor is genotyped for blood cell antigens . The donor described , e . g . in PCT Publication No. WO2013059343 can be a universal blood donor. In some embodiments , the INTRACELLULAR DELIVERY, incorporated herein by donor has the Bombay phenotype , . ie . does not express the reference . H antigen . In some embodiments , the donor has ABO blood [ 0338 ] In a specific embodiment, a population of reticu type O and is Rh -negative . locytes is provided that has been subjected to controlled cell [0342 ] In some embodiments, the EHC is generated using injury by controlled deformation . The cells can , e . g . , be CD34 + hematopoietic progenitor cells , mobilized peripheral compressed and deformed by passage through a micro CD34 + cells , or bone marrow - derived CD34 + cells as a channel having a diameter less than that of an individual source for the starting material. In some embodiments , the reticulocyte , thereby causing perturbations in the cell mem starting cells are derived from umbilical cord blood , are brane such that the membrane becomes porous . Cells are induced pluripotent stem cells or are embryonic stem cells . moved , e . g . , pushed , through the channels or conduits by [0343 ] The exogenous antigen - expressing EHC may be application of pressure . The compression and deformation cultured . Cultured EHCs can be scaled up from bench - top occurs in a delivery medium comprising a payload , e . g . an scale to bioreactor scale . For example , the EHCs are cul exogenous polypeptide or oligonucleotide (e . g . DNA , RNA , tured until they reach saturation density , e . g ., 1x10 % , 1x10 " , such as mRNA ). For example , the delivery medium may 1x10 " , or greater than 1x10 ' EHCs per ml. Optionally , upon comprise an exogenous antigen listed in Table F , Table G , reaching saturation density , the EHCs can be transferred to Table H , Table I and Table J or coding mRNA thereof. Upon a larger volume of fresh medium . The exogenous antigen deformation the reticulocyte takes up and retains the exog expressing EHCs may be cultured in a bioreactor, such as , enous material. Following controlled injury to the cell by e . g ., a Wave -type bioreactor , a stirred - tank bioreactor. Vari constriction , stretching , and/ or a pulse of high shear rate , the ous configurations of bioreactors are known in the art and a cells are optionally incubated in a delivery medium that suitable configuration may be chosen as desired . Configu contains the material ( payload ) . The cells may be maintained rations suitable for culturing and / or expanding populations in the delivery medium for a few minutes to recover, e . g . to of exogenous antigen - expressing EHCs can easily be deter close the injury caused by passing through the constriction . mined by one of skill in the art without undue experimen This may occur at room temperature . tation . The bioreactor can be oxygenated . The bioreactor US 2018 /0271910 A1 Sep . 27 , 2018 26 may optionally contain one or more impellers , a recycle ments , the identity of the exogenous antigen - expressing stream , a media inlet stream , and control components to EHCs is assessed by analysis of the exogenous antigen regulate the influx of media and nutrients or to regulate the content of the EHCs, e . g ., by flow cytometry , Western blot, outflux of media , nutrients, and waste products . immunoprecipitation , fluorescence spectroscopy, chemilu [0344 ] In some embodiments , the bioreactor may contain minescence , mass spectrometry , or absorbance spectros a population of human EHCs comprising an exogenous copy . For example , the identity of the exogenous antigen antigen that shed their intracellular DNA over the course of expressing EHCs can be assessed by the mRNA content of the culture process . For example , the bioreactor may contain the EHCs, e . g ., by RT- PCR , flow cytometry , or northern blot . a population of human EHCs , enucleated EHCs, and pyre - The identity of the exogenous antigen -expressing EHCs can nocytes after culture . In a specific embodiment, the human be assessed by nuclear material content, e .g ., by flow EHCs and enucleated EHCs comprise an exogenous antigen cytometry, microscopy , or southern blot, using , e . g. , a and the exogenous antigen is retained by the enucleated nuclear stain or a nucleic acid probe . Alternatively or in EHC , whereas the recombinant nucleic acid encoding the addition , the identity of the exogenous antigen - expressing exogenous antigen is not retained by the enucleated cell. In EHCs is assessed by lipid content of the EHCs, e .g by flow certain embodiments , the enucleated EHC comprising the cytometry, liquid chromatography, or by mass spectrometry. exogenous antigen exhibits substantially the same osmotic [0348 ] In some embodiments , the identity of the exog membrane fragility as a corresponding isolated unmodified , enous antigen - expressing EHCs is assessed by metabolic uncultured EHC . activity of the EHCs, e . g by mass spectrometry , chemilu [0345 ] In one embodiment. The population of exogenous minescence , fluorescence spectroscopy , absorbance spec antigen -expressing EHCs generated from EHCs or EHC troscopy . Metabolic activity can be assessed by ATP con precursors in the bioreactor undergo a total expansion of sumption rate and / or the metabolic activity is assessed greater than 20 ,000 - fold in 14 days or greater. In some measuring 2 ,3 - diphosphoglycerate ( 2, 3 -DPG ) level in the embodiments , the exogenous antigen is introduced into a exogenous antigen -expressing EHC . The metabolic activity cultured or freshly isolated EHC precursor and after intro can be assessed as the rate of metabolism of one of the duction of a recombinant nucleic acid encoding the exog following , including but not limited to , Acetylsalicylic acid , enous antigen the population of exogenous antigen - express N -Acetylcystein , 4 - Aminophenol, Azathioprine, Bunolol, ing EHCs generated from the EHC precursors in the Captopril, Chlorpromazine, Dapsone , Daunorubicin , Dehy bioreactor expands in the bioreactor from the precursor cells droepiandrosterone , Didanosin , Dopamine , Epinephrine, by more than 20 ,000 - fold . Esmolol , Estradiol, Estrone , Etoposide, Haloperidol, [0346 ] In some embodiments , the bioreactor is a Wave Heroin , Insulin , Isoproterenol, Isosorbide dinitrate , LY bioreactor or a impeller- driven agitator. The bioreactor may 217896 , 6 -mercaptopurine , Misonidazole , Nitroglycerin , be aerated by means of a sparger. In one embodiment, the Norepinephrine , Para - aminobenzoic acid . In some embodi bioreactor is disposable. In one embodiment, the bioreactor ments , the identity of the exogenous antigen -expressing is CIP ( cleaned in place ) . The final number of exogenous EHCs is assessed by partitioning of a substrate by the EHCs, antigen -expressing EHCs that may be obtained in a biore e .g by mass spectrometry , chemiluminescence, fluorescence actor setting as described herein can be greater than 10°, spectroscopy, or absorbance spectroscopy . The substrate can 1010 , 1011 , 1012 , 1013 or greater than 1013 EHCs. The density be one of the following , including but not limited to , of exogenous antigen - expressing EHCs may be monitored Acetazolamide, Arbutine , Bumetamide , Creatinine , during culture by measuring cell density by hemacytometer Darstine , Desethyldorzolamide , Digoxigenin digitoxoside , counting or by optical density reading at 600 nm . Optionally , Digoxin - 16 - glucuronide, Epinephrine , Gentamycin , Hippu the culture process is monitored for pH levels , oxygenation , ric acid ,Metformin , Norepinephrine , p - Aminohippuric acid , agitation rate , and / or recycle rate . Papaverine, Penicillin G , Phenol red , Serotonin , Sulfosali cylic acid , Tacrolimus, Tetracycline , Tucaresol, and Vanco Processes and Properties mycin . [0347 ] The identity of the exogenous antigen -expressing [0349 ] In one embodiment , the population of exogenous EHCs can be assessed by in vitro assays. For example , the antigen -expressing EHCs is differentiated from a precursor identity of the exogenous antigen -expressing EHCs is cell. In this embodiment, the differentiation state of the assessed by counting the number of EHCs in a population , population of exogenous antigen - expressing EHCs is e . g . , by microscopy, by flow cytometry , or by hemacytom assessed by an in vitro assay . The in vitro assays include etry . Alternatively or in addition , the identity of the exog those described herein for assessing the identity of the enous antigen - expressing EHCs is assessed by analysis of EHCs, including but not limited to expansion rate , number, protein content of the EHCs, e .g . , by flow cytometry, West protein content or expression level, mRNA content or ern blot , immunoprecipitation , fluorescence spectroscopy, expression level, lipid content, partition of a substrate , chemiluminescence , mass spectrometry , or absorbance spec catalytic activity , or metabolic activity . troscopy. In one embodiment, the protein content assayed is [0350 ] In some embodiments , the exogenous antigen a non - surface protein , e . g ., an integral membrane protein , expressing EHCs are cultured and the differentiation state of hemoglobin , adult hemoglobin , fetal hemoglobin , embry the EHCs is assessed at multiple time points over the course onic hemoglobin , a cytoskeletal protein . In one embodiment, of the culture process . the protein content assayed is a surface protein , e . g . , a [ 0351 ] Exogenous antigen - expressing EHCsmay be gen differentiation marker , a receptor, a co - receptor, a trans erated using reticulocytes as a source for starting material. porter , a glycoprotein . In one embodiment, the surface The purity of isolated reticulocytes may be assessed using protein is selected from the list including , but not limited to , microscopy in that reticulocytes are characterized by a glycophorin A , CKIT , transferrin receptor , Band3 , Kell , reticular (mesh - like ) network of ribosomal RNA that CD45, CD46 , CD47 , CD55 , CD59 , CR1 . In some embodi becomes visible under a microscope with certain stains such US 2018 /0271910 A1 Sep . 27 , 2018 27 as new methylene blue or brilliant cresyl blue. Surface ment the volume of the mean corpuscular volume of the expression of transferrin receptor (CD71 ) is also higher on EHCs is greater than 10 fL , 20 fL , 30 fL , 40 fL , 50 fL , 60 reticulocytes and decreases and they mature to erythrocytes, fL , 70 fL , 80 fL , 90 fL , 100 fL , 110 fL , 120 fL , 130 fL , 140 allowing for enrichment and analysis of reticulocyte popu - fL , 150 fL , or greater than 150 fL . In one embodiment the lations using anti- CD71 antibodies (See , e . g. , Miltenyi mean corpuscular volume of the EHCs is less than 30 fL , 40 CD71 microbeads product insert No . 130 - 046 - 201) . Alter fL , 50 fL , 60 fL , 70 fL , 80 fL , 90 fL , 100 fL , 110 fL , 120 natively , analysis of creatine and hemoglobin A1C content fL , 130 fL , 140 fL , 150 fL , 160 fL , 170 fL , 180 fL , 190 fL , and pyruvate kinase , aspartate aminotransferase , and por 200 fL , or less than 200 fL . In one embodiment the mean phobilinogen deaminase enzyme activity may be used to corpuscular volume of the EHCs is between 80 - 100 femto assess properties of the isolated reticulocytes relative to liters ( fL ) . mature erythrocytes (See , e. g . , Brun et al. , Blood 76 : 2397 [0356 ]. In one embodiment the average buoyant mass of 2403 ( 1990 ) ) . For example , the activity of porphobilinogen the exogenous antigen - expressing EHCs (pg /cell ) is mea deaminase is nearly 9 fold higher whereas the hemoglobin sured using a suspended microchannel resonatory or a A1C content is nearly 10 fold less in reticulocytes relative to double suspended microchannel resonatory ( see e . g . , Byun mature erythrocytes . et al PNAS 2013 110 ( 19 ) : 7580 and Bryan et al. Lab Chip [ 0352] In some embodiments , cells suitable for generating 2014 14 (3 ): 569 ). exogenous antigen -expressing EHCs are differentiated ex [0357 ] In one embodiment the dry density of the exog vivo and / or in vivo from one or more stem cells . In one enous antigen - expressing EHCs is measured by buoyant embodiment, the one or more stem cells are one or more mass in an H2O -D20 exchange assay ( see e. g ., Feijo hematopoietic stem cells. Various assays may be performed Delgado et al ., PLOS One 2013 8 (7 ): e67590 ). to confirm the ex vivo differentiation of cultured hematopoi etic stem cells into reticulocytes and erythrocytes, including , [ 0358 ] In some embodiments , the exogenous antigen for example , microscopy, hematology , flow cytometry, expressing EHCs have an average membrane deformability deformability measurements , enzyme activities, and hemo fluctuation of standard deviation greater than 10 , 20 , 30 , 40 , globin analysis and functional properties (Giarratana et al. , 50 , 60 , 70 , 80 , 90 , 100 or greater than 100 mrad as measured Nature Biotech . 23 :69 -74 ( 2005 ) ) . The phenotype of cul by spatial light interference microscopy (SLIM ) ( see e . g . , tured hematopoietic stem cells may be assessed using Bhaduri et al. , Sci Reports 2014 , 4 :6211 ) . microscopy of cells stained , for example , with Cresyl Bril [0359 ] In one embodiment , the average membrane viscos liant blue . Reticulocytes , for example , exhibit a reticular ity of a population of exogenous antigen -expressing EHCs is network of ribosomal RNA under these staining conditions measured by detecting the average fluorescence upon incu whereas erythrocytes are devoid of staining . Enucleated bation with viscosity -dependent quantum yield fluorophores cells may also be monitored for standard hematological ( see e . g ., Haidekker et al. Chem & Biol 2001 8 ( 2 ): 123 ). variables including mean corpuscular volume (MCV ; fem [ 0360 ] In one embodiment , the membrane fluidity of the toliters ( fL ) ) , mean corpuscular hemoglobin concentration exogenous antigen - expressing EHCs is measured by fluo (MCHC ; % ) and mean corpuscular hemoglobin (MCH ; rescence polarization , e. g ., with BMG Labtech POLARstar pg /cell ) using , for example , an XE2100 automat (Sysmex , Omega microplate reader . Roche Diagnostics ). [ 0361 ] For example , to measure deformability reticulo [ 0353] In some embodiments , the exogenous antigen cytes may be separated from nucleated cells on day 15 of expressing EHCs are assessed for their basic physical prop culture , for example , by passage through a deleukocyting erties , e . g . , size , mass, volume, diameter, buoyancy , density , filter ( e . g ., Leucolab LCG2, Macopharma ) and subsequently and membrane properties , e. g ., viscosity , deformability fluc assayed using ektacytometry . The enucleated cells are sus tuation , and fluidity. pended in 4 % polyvinylpyrrolidone solution and then [ 0354 ] In one embodiment, the diameter of the exogenous exposed to an increasing osmotic gradient from 60 to 450 antigen - expressing EHCs is measured by microscopy or by mosM . Changes in the laser diffraction pattern ( deformabil automated instrumentation , e . g ., a hematological analysis ity index ) of the cells are recorded as a function of osmo instrument. In one embodiment the diameter of the exog larity , to assess the dynamic deformability of the cell mem enous antigen -expressing EHCs is between about 1 - 20 brane. The maximum deformability index achieved at a microns . In one embodiment, the diameter of the exogenous physiologically relevant osmolarity is related to the mean antigen - expressing EHCs is at least in one dimension surface area of erythrocytes . between about 1 - 20 microns . In one embodiment , the diam [0362 ] In some embodiments , the exogenous antigen eter of the exogenous antigen - expressing EHCs is less than expressing EHCs are analyzed for hemoglobin contents . about 1 micron . In one embodiment , the diameter of the Assays of hemoglobin may be used to assess the phenotype EHCs in one dimension is larger than about 20 microns. In of differentiated cells (Giarratana et al. , Nature Biotech . one embodiment, the diameter of the exogenous antigen 23 :69 -74 (2005 ) ) . For example , high performance liquid expressing EHCs is between about 1 micron and about 20 chromatography (HPLC ) using a Bio -Rad Variant II Hb microns , between about 2 microns and about 20 microns analyzer ( Bio -Rad Laboratories ) may be used to assess the between about 3 microns and about 20 microns between percentage of various hemoglobin fractions. Oxygen equi about 4 microns and about 20 microns between about 5 librium may be measured using a continuous method with a microns and about 20 microns between about 6 microns and double -wavelength spectrophotometer ( e . g . , Hemox ana about 20 microns, between about 5 microns and about 15 lyzer, TCS ) . The binding properties of hemoglobin may be microns or between about 10 microns and about 30 microns . assessed using flash photolysis . In this method , the rebinding [0355 ] In one embodiment, the mean corpuscular volume of CO to intracellular hemoglobin tetramers are analyzed at of the exogenous antigen - expressing EHCs is measured 436 nm after photolysis with a 10 nanosecond pulse at 532 using a hematological analysis instrument. In one embodi nm . US 2018 /0271910 A1 Sep . 27 , 2018 20

[0363 ] The exogenous antigen - expressing EHCS Radiations that cause dense ionization along their track described herein can be purified following manufacture if ( such as neutrons ) are called high - linear- energy - transfer desired . Many suitable methods of purification are known in (high -LET ) radiation , a physical parameter to describe aver the art. For example , the exogenous antigen - expressing age energy released per unit length of the track . (See the EHCs are purified by centrifugation , magnetophoresis , irra accompanying figure . ) Low -LET radiations produce ioniza diation , acoustophoresis , and chemical or physical enucle tions only sparsely along their track and , hence, almost ation . In one embodiment exogenous antigen -expressing homogeneously within a cell . Radiation dose is the amount EHCs are purified by ex vivo maturation with , e . g ., a stromal of energy per unit of biological material ( e . g . , number of cell co - culture. In one embodiment, exogenous antigen ionizations per cell ) . Thus , high -LET radiations are more expressing EHCs are purified by chemical or enzymatic destructive to biologicalmaterial than low - LET radiations treatment of EHCs, e . g by treatment with a deglycosylation such as X and gamma rays — because at the same dose , the enzyme. low -LET radiations induce the same number of radicals [ 0364 ] In one embodiment the exogenous antigen -ex more sparsely within a cell , whereas the high -LET radia pressing EHCs are purified by disabling any residual repli tions , such as neutrons and alpha particles — transfer most cative potential of the exogenous antigen -expressing EHCs. of their energy to a small region of the cell. The localized In one embodiment the exogenous antigen - expressing EHCS DNA damage caused by dense ionizations from high -LET are subjected to radiation , e . g . , X rays, gamma rays , beta radiations is more difficult to repair than the diffuse DNA particles, alpha particles, neutrons, protons, elemental damage caused by the sparse ionizations from low -LET nuclei, UV rays in order to damage residual replication radiations . competent nucleic acids. [0369 ] In one embodiment, a population of exogenous [ 0365 ] Ionizing radiation is energy transmitted via X rays , antigen - expressing EHCs are subjected to gamma irradia gamma rays, beta particles ( high - speed electrons ), alpha tion using an irradiation dose of more than 1 , 5 , 10 , 15 , 20 , particles (the nucleus of the helium atom ) , neutrons , protons, 25 , 30 , 35, 40 , 50 , 60 , 70 , 80 , 90 , 100 , ormore than 100 kGy. and other heavy ions such as the nuclei of argon , nitrogen , carbon , and other elements . X rays and gamma rays are [ 0370 ] In one embodiment, a population of exogenous electromagnetic waves like light, but their energy is much antigen - expressing EHCs are subjected to X -ray irradiation higher than that of light ( their wavelengths are much using an irradiation dose of more than 0 .1 , 0 . 5 , 1 , 5 , 10 , 15 , shorter) . Ultraviolet (UV ) light is a radiation of intermediate 20 , 25 , 30 , 35 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 200 , 300 , 400 , energy that can damage cells but UV light differs from the 500 , 600 , 700 , 800 , 900 , 1000 , 2000 , 3000 , 4000 , 5000 , forms of electromagnetic radiation mentioned above in that 6000 , 7000 , 8000 , 9000 , 10000 , or greater than 10000 mSv . it does not cause ionization ( loss of an electron ) in atoms or [ 0371 ] The purity of a population of exogenous antigen molecules , but rather excitation ( change in energy level of expressing EHCs can be assessed by measuring the homo an electron ) . The other forms of radiation - particles are geneity of the population . In one embodiment, the average either negatively charged ( electrons ), positively charged distribution width of the exogenous antigen - expressing (protons , alpha rays , and other heavy ions ) , or electrically EHCs is measured by a hematological analysis instrument. neutral (neutrons ). In one embodiment, the population of exogenous antigen 10366 ] Radiation - induced ionizations may act directly on expressing EHCs has a reticulocyte to non - reticulocyte ratio the cellular component molecules or indirectly on water greater than 10 , 100 , 1000 , 104, 10 % , 10 % , or greater than 10 % . molecules, causing water -derived radicals . Radicals react The homogeneity of the population of exogenous antigen with nearby molecules in a very short time, resulting in expressing EHCsmay be assessed by measuring the stromal breakage of chemical bonds or oxidation (addition of oxy cell content of the population . In one embodiment, the gen atoms) of the affected molecules . The major effect in population of exogenous antigen - expressing EHCs has less cells is DNA breaks . Since DNA consists of a pair of than 1 ppb of stromal cells . Alternatively or in addition , the complementary double strands, breaks of either a single homogeneity of the population of exogenous antigen - ex strand or both strands can occur . However , the latter is pressing EHCs is assessed by measuring the viral titer and /or believed to be much more important biologically . Most a bacterial colony forming potential of the population . single - strand breaks can be repaired normally thanks to the [0372 ] In one embodiment the homogeneity of a popula double -stranded nature of the DNA molecule (the two tion of exogenous antigen - expressing EHCs is assessed by strands complement each other, so that an intact strand can an in vitro assay . The in vitro assays include those described serve as a template for repair of its damaged , opposite herein for assessing the identity of the EHCs, including but strand ) . In the case of double - strand breaks , however , repair not limited to expansion rate , number, protein content or is more difficult and erroneous rejoining of broken ends may expression level , mRNA content or expression level, lipid occur. These so - called misrepairs result in induction of content, partition of a substrate , catalytic activity , or meta mutations, chromosome aberrations, or cell death . bolic activity . [ 0367 ] Deletion of DNA segments is the predominant [0373 ] Mature erythrocytes for use in generating the exog form of radiation damage in cells that survive irradiation . It enous antigen - expressing EHCs may be isolated using vari may be caused by ( 1 ) misrepair of two separate double ous methods such as , for example , a cell washer, a continu strand breaks in a DNA molecule with joining of the two ous flow cell separator , density gradient separation , outer ends and loss of the fragmentbetween the breaks or ( 2 ) fluorescence -activated cell sorting (FACS ), Miltenyi immu the process of cleaning ( enzyme digestion of nucleotides nomagnetic depletion (MACS ) , or a combination of these the component molecules of DNA ) of the broken ends methods ( See , e . g . , van der Berg et al. , Clin . Chem . 33 : 1081 before rejoining to repair one double - strand break . 1082 ( 1987 ) ; Bar - Zvi et al . , J . Biol. Chem . 262: 17719 - 17723 [0368 ] Radiations differ not only by their constituents (1987 ) ; Goodman et al. , Exp . Biol. Med . 232 : 1470 - 1476 ( electrons, protons, neutrons, etc .) but also by their energy. (2007 ) ). US 2018 /0271910 A1 Sep . 27 , 2018

[0374 ] Erythrocytes may be isolated from whole blood by as, for example, CD71 and CD36 . The antibodies may be simple centrifugation (See , e . g ., van der Berg et al. , Clin . directly attached to magnetic beads or conjugated to PE , for Chem . 33 :1081 - 1082 (1987 )) . For example , EDTA -antico example , to which magnetic beads with anti -PE antibody agulated whole blood may be centrifuged at 800xg for 10 will react. The antibody -magnetic bead complex is able to min at 4° C . The platelet- rich plasma and buffy coat are selectively extract residual reticulocytes, for example , from removed and the red blood cells are washed three times with the erythrocyte population . isotonic saline solution (NaCl , 9 g / L ) . [0379 ] Erythrocytes may also be isolated using apheresis . [ 0375 ] Alternatively , erythrocytes may be isolated using The process of apheresis involves removal of whole blood density gradient centrifugation with various separation from a patient or donor, separation of blood components mediums such as , for example , Ficoll , Hypaque , His using centrifugation or cell sorting, withdrawal of one or topaque , Percoll , Sigmacell, or combinations thereof. For more of the separated portions , and transfusion of remaining example , a volume of Histopaque - 1077 is layered on top of components back into the patient or donor. A number of an equal volume of Histopaque - 1119 . EDTA -anticoagulated instruments are currently in use for this purpose such as for whole blood diluted 1 : 1 in an equal volumeof isotonic saline example the Amicus and Alyx instruments from Baxter solution (NaCl , 9 g / L ) is layered on top of the Histopaque ( Deerfield , ill ., USA ), the Trima Accel instrument from and the sample is centrifuged at 700xg for 30 min at room Gambro BCT ( Lakewood , Colo . , USA ) , and the MCS + 9000 temperature . Under these conditions, granulocytes migrate instrument from Haemonetics (Braintree , Mass. , USA ). to the 1077 / 1119 interface , lymphocytes, other mononuclear Additional purification methods may be necessary to cells and platelets remain at the plasma/ 1077 interface , and achieve the appropriate degree of cell purity . the red blood cells are pelleted . The red blood cells are 10380 ] In some embodiments, the exogenous antigen washed twice with isotonic saline solution . expressing EHCs are differentiated ex vivo and / or in vivo [0376 ] Alternatively , erythrocytes may be isolated by cen from one or more reticulocytes. Reticulocytes may be used trifugation using a Percoll step gradient (See , e . g . , Bar- Zvi to generate exogenous antigen - expressing EHCs . Reticulo et al ., J. Biol. Chem . 262 : 17719 - 17723 ( 1987 ) ) . For cytes are immature red blood cells and compose approxi example , fresh blood is mixed with an anticoagulant solu mately 1 % of the red blood cells in the human body . tion containing 75 mM sodium citrate and 38 mM citric acid Reticulocytes develop and mature in the bone marrow . Once and the cells washed briefly in Hepes - buffered saline . Leu released into circulation , reticulocytes rapidly undergo ter kocytes and platelets are removed by adsorption with a minal differentiation to mature erythrocytes . Like mature mixture of a -cellulose and Sigmacell (1 : 1) . The erythrocytes erythrocytes , reticulocytes do not have a cell nucleus. Unlike are further isolated from reticulocytes and residual white mature erythrocytes , reticulocytes maintain the ability to blood cells by centrifugation through a 45 / 75 % Percoll step perform protein synthesis . In some embodiments , recombi gradient for 10 min at 2500 rpm in a Sorvall SS34 rotor. The nant nucleic acid ( such as mRNA ) encoding an exogenous erythrocytes are recovered in the pellet while reticulocytes antigen is introduced into reticulocytes to generate exog band at the 45 /75 % interface and the remaining white blood enous antigen -expressing EHCs. cells band at the 0 / 45 % interface . The Percoll is removed [0381 ] Reticulocytes of varying age may be isolated from from the erythrocytes by several washes in Hepes - buffered peripheral blood based on the differences in cell density as saline. Other materials that may be used to generate density the reticulocytes mature . Reticulocytes may be isolated from gradients for isolation of erythrocytes include OptiPrepTM , a peripheral blood using differential centrifugation through 60 % solution of iodixanol in water ( from Axis - Shield , various density gradients . For example , Percoll gradients Dundee , Scotland ) . may be used to isolate reticulocytes (See , e . g . , Noble el al . , [ 0377 ] Erythrocytes may be separated from reticulocytes , Blood 74 : 475 - 481 ( 1989 ) ) . Sterile isotonic Percoll solutions for example , using flow cytometry ( See , e . g ., Goodman el ofdensity 1 .096 and 1 . 058 g /ml are made by diluting Percoll al ., Exp . Biol. Med . 232 : 1470 - 1476 ( 2007 ) ) . In this instance , ( Sigma - Aldrich , Saint Louis , Mo. , USA ) to a final concen whole blood is centrifuged ( 550xg , 20 min , 25° C . ) to tration of 10 mM triethanolamine , 117 mM NaCl, 5 mM separate cells from plasma. The cell pellet is resuspended in glucose , and 1 . 5 mg/ ml bovine serum albumin (BSA ) . These phosphate buffered saline solution and further fractionated solutions have an osmolarity between 295 and 310 mOsm . on Ficoll -Paque ( 1 .077 density ) , for example, by centrifu Five milliliters , for example , of the first Percoll solution gation (400xg , 30 min , 25° C . ) to separate the erythrocytes ( density 1 .096 ) is added to a sterile 15 ml conical centrifuge from the white blood cells . The resulting cell pellet is tube. Two milliliters , for example , of the second Percoll resuspended in RPMI supplemented with 10 % fetal bovine solution (density 1 . 058 ) is layered over the higher density serum and sorted on a FACS instrument such as, for first Percoll solution . Two to four milliliters of whole blood example , a Becton Dickinson FACSCalibur (BD Biosci are layered on top of the tube . The tube is centrifuged at ences , Franklin Lakes , N . J. , USA ) based on size and granu 250xg for 30 min in a refrigerated centrifuge with swing - out larity . tube holders . Reticulocytes and somewhite cells migrate to [ 0378 ] Erythrocytes may be isolated by immunomagnetic the interface between the two Percoll layers . The cells at the depletion ( See , e . g . , Goodman , el al. , ( 2007 ) Exp . Biol. Med . interface are transferred to a new tube and washed twice 232: 1470 - 1476 ) . In this instance , magnetic beads with cell with phosphate buffered saline (PBS ) with 5 mM glucose , type specific antibodies are used to eliminate non - erythro 0 . 03 mM sodium azide and 1 mg/ ml BSA . Residual white cytes . For example , erythrocytes are isolated from the blood cells are removed by chromatography in PBS over a majority of other blood components using a density gradient size exclusion column . as described herein followed by immunomagnetic depletion [ 0382 ] Alternatively , reticulocytes may be isolated by of any residual reticulocytes. The cells are pre - treated with positive selection using an immunomagnetic separation human antibody serum for 20 min at 25° C . and then treated approach (See , e. g ., Brun et al . , Blood 76 :2397 - 2403 with antibodies against reticulocyte specific antigens such ( 1990 )) . This approach takes advantage of the large number US 2018 /0271910 A1 Sep . 27 , 2018 30 of transferrin receptors that are expressed on the surface of 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 99 % , 99. 5 % , reticulocytes relative to erythrocytes prior to maturation . 99 . 9 % , or greater than 99 . 9 % enucleated . Magnetic beads coated with an antibody to the transferrin 0387 ] In some embodiments , the exogenous antigen receptor may be used to selectively isolate reticulocytes expressing EHCs are not co - cultured with support cells , e . g . , from a mixed blood cell population . Antibodies to the with an adherent stromal layer . In some embodiments , the transferrin receptor of a variety of mammalian species , population of exogenous antigen - expressing EHCs is gen including human , are available from commercial sources erated by contacting EHCs with an exogenous antigen and ( e . g . , Affinity BioReagents , Golden , Colo . , USA ; Sigma differentiating the EHCs to obtain a population of enucleated Aldrich , Saint Louis , Mo. , USA ) . The transferrin antibody cells comprising the exogenous antigen . The population of may be directly linked to the magnetic beads. Alternatively , exogenous antigen -expressing EHCs is obtained without an the transferrin antibody may be indirectly linked to the enrichment step , such as gravitational separation , magnetic magnetic beads via a secondary antibody. For example , or fluorescent sorting, irradiation , poisoning of nucleated mouse monoclonal antibody 10D2 (Affinity BioReagents , cells , and the like to select for enucleated cells . Golden , Colo . , USA ) against human transferrin may be mixed with immunomagnetic beads coated with a sheep [ 0388 ] In some embodiments , the population of exog anti -mouse immunoglobulin G (Dynal / Invitrogen , Carlsbad , enous antigen -expressing EHCs is comprised of greater than Calif ., USA ) . The immunomagnetic beads are then incu 5 % , 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , bated with a leukocyte -depleted red blood cell fraction . The 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 99 % , beads and red blood cells are incubated at 22° C . with gentle 99 . 5 % , 99 . 9 % , or greater than 99 . 9 % of exogenous antigen mixing for 60 - 90 min followed by isolation of the beads with expressing EHCs that lack nuclear material as assessed by attached reticulocytes using a magnetic field . The isolated an assay to detect nuclear material such as those described reticulocytes may be removed from the magnetic beads herein . using , for example , DETACHABEAD® solution ( from [ 0389 ] In some embodiments , the presence, biological Invitrogen , Carlsbad , Calif. , USA ) . Alternatively , reticulo activity and /or function of an exogenous antigen , such as an cytes may be isolated from in vitro growth and maturation exogenous antigen polypeptide exhibited by exogenous anti of CD34 + hematopoietic stem cells using the methods gen -expressing EHCs is assessed . Many suitable assays are described herein . available and known in the art. [ 0383] Terminally - differentiated , enucleated erythrocytes [0390 ] In one embodiment, the exogenous antigen is a can be separated from other cells based on their DNA polypeptide on the surface of the exogenous antigen - ex content. In a non - limiting example , cells are first labeled pressing EHC . The presence of the exogenous antigen can with a vital DNA dye , such as Hoechst 33342 ( Invitrogen be assessed by assays including but not limited to flow Corp . ) . Hoechst 33342 is a cell -permeant nuclear counter cytometry , western blotting , RT- PCR , Northern blotting , stain that emits blue fluorescence when bound to double Coombs rosetting , mass spectrometry . In one embodiment, stranded DNA . Undifferentiated precursor cells , macro the exogenous antigen is a polypeptide in the interior of the phages or other nucleated cells in the culture are stained by exogenous antigen -expressing EHC . The presence of the Hoechst 33342 , while enucleated erythrocytes are Hoechst exogenous antigen can be assessed by assays including but negative . The Hoechst- positive cells can be separated from not limited to Western blotting , RT- PCR , Norther blotting , enucleated erythrocytes by using fluorescence activated cell PCR , Southern blotting , mass spectrometry . sorters or other cell sorting techniques. The Hoechst dye can [0391 ] In one embodiment, the exogenous antigen is a be removed from the isolated erythrocytes by dialysis or nucleic acid on the surface of the exogenous antigen other suitable methods . expressing EHC . The presence of the exogenous antigen can [0384 ] A population of exogenous antigen - expressing be assessed by assays including but not limited to flow EHCs can be purified by reducing the nuclear material cytometry, flow cytometry with a homologous fluorescent content of the population of EHCs. For example , the enucle probe, southern blotting, northern blotting , PCR . In one ation rate of the population of EHCs is increased , and / or the embodiment , the exogenous antigen is a nucleic acid in the number of enucleated exogenous antigen - expressing EHCs interior of the exogenous antigen -expressing EHC . The is increased or enriched . presence of the exogenous antigen can be assessed by assays [ 0385 ) Populations of exogenous antigen - expressing including but not limited to southern blotting , northern EHCs can be incubated with a small molecule , e . g ., an actin blotting , PCR . inhibitor, e . g . , cytochalasin A , B , C , D , E , F , H , J , and then [0392 ] In one embodiment, the exogenous antigen is a centrifuged to remove nuclear material. Alternatively or in small molecule on the surface of the exogenous antigen addition , a population of exogenous antigen -expressing expressing EHC . The presence ofthe exogenous antigen can EHCs can be mechanically manipulated by passing through be assessed by assays including but not limited to flow progressively smaller size -restrictive filters to remove cytometry , mass spectrometry . In one embodiment, the nuclear material . The population of exogenous antigen exogenous antigen is a small molecule in the interior of the expressing EHCs may also be incubated on a fibronectin exogenous antigen - expressing EHC . The presence of the coated plastic surface to increase the removal of nuclear exogenous antigen can be assessed by assays including but material. In one embodiment, the population of exogenous not limited to nass spectrometry , fluorescence spectroscopy . antigen - expressing EHCs is incubated in co - culture with [0393 ] In one embodiment, the exogenous antigen is a stromal cells , e . g ., macrophages, to increase the removal of lipid in the membrane of the exogenous antigen - expressing nuclear material. EHC . The presence of the exogenous antigen can be [ 0386 ] In some embodiments , the population of exog assessed by assays including but not limited to mass spec enous antigen - expressing EHCs is greater than 5 % , 10 % , trometry , flow cytometry , membrane solubility , fluorescence 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , 60 % , polarization , spatial light interferences microscopy . US 2018 /0271910 A1 Sep . 27 , 2018 31

[0394 ] In one embodiment, the exogenous antigen is fluo - ing EHC , the overall biological activity of the EHC , and the rescent or is fused to a fluoresecent molecule or is co overall activity of a population of EHCs can be assessed by expressed from a recombinant nucleic acid ( e . g . , in a vector ) in vitro assays . with a fluorescent reporter protein like GFP . The presence of f0401 ] In some embodiments, the activity of the exog the exogenous antigen in or on the exogenous antigen enous antigen - expressing EHC is rapidly iterated using a expressing EHC can be assessed by assays including but not model cell line . For example , a library of suitable exogenous limited to flow cytometry , fluorescence spectroscopy, absor antigens is expressed in a model cell line, e . g ., HEK293T or bance spectroscopy. K562 , and the activity is assessed via a suitable assay ; then [ 0395 ] In one embodiment, the exogenous antigen is a the best exogenous antigen candidate , e .g ., the one that is gaseous molecule . The presence of the exogenous antigen in expressed at the highest level or one that demonstrates the or on the exogenous antigen - expressing EHC can be highest activity in the suitable assay, is expressed , e . g . , in assessed by assays including but not limited to chemilumi cultured EHCs to generate exogenous antigen - expressing nescence assays, mass spectroscopy . EHCs. [0402 ] In one embodiment, the activity of the exogenous [ 0396 ] The presence of the exogenous antigen in or on the antigen - expressing EHC is rapidly iterated using a cultured exogenous antigen - expressing EHC can be assessed by flow mouse model . For example , a library of suitable exogenous cytometry in a quantitative fashion using calibration beads antigens is expressed in cultured mouse EHCs; activity is such as commercially available cytometry calibration beads assessed in a suitable mouse model of disease or a suitable to quantify the number of exogenous antigens on an indi mouse model system for assessing activity ; the best exog vidual EHC . Alternatively or in addition , the presence of the enous antigen candidate , e . g ., the one that is expressed at the exogenous antigen in or on the exogenous antigen - express highest level or the one that demonstrates the highest ing EHC can be assessed by Western blot in a quantitative activity in the suitable assay, is then expressed , e . g ., in fashion using a standard of known concentration that is cultured EHCs to generate exogenous antigen -expressing detectable using the same detection reagents as the exog EHC . enous antigen , and in this way the number of exogenous 10403 ]. In some instances , the exogenous antigen is an antigens on an individual EHC can be quantified . enzyme and the activity of the exogenous antigen can be [ 0397] In some embodiments , the presence of two or more assessed by an enzymatic assay in which the disappearance different exogenous antigens can be assessed by the same or of a specific substrate molecule is detected or the appearance different methods , either simultaneously , in sequential fash of a specific product molecule is detected . Such assays ion , or in parallel. For example , in one embodiment an include but are not limited to , colorimetric assays , mass exogenous antigen on the surface can be assessed by flow spectrometry, HPLC , fluorescent assays . cytometry using an antibody specific to the exogenous [ 0404 ] For example , a ) the exogenous antigen is adenosine antigen and a different exogenous antigen not on the surface deaminase (ADA ) and the enzymatic assay detects the that is fluoresecent can be assessed by fluorescent signal conversion of adenosine to inosine ; b ) the exogenous anti using a different channel in flow cytometry . In a different gen is phenylalanine hydroxylase ( PAH ) and the assay example , an exogenous antigen on the surface can be detects the conversion of phenylalanine to tyrosine ; c ) the assessed by flow cytometry and a different exogenous anti exogenous antigen is phenylalanine ammonia lyase (PAL ) gen not on the surface can be assessed by Western blot. and the assay detects the conversion of phenylalanine to [ 0398 ] In a specific embodiment , the exogenous antigen is trans -cinnamic acid ; d ) the exogenous antigen is thymidine retained on the exogenous antigen - expressing EHC follow phosphorylase ( TP ) and the assay detects the conversion of ing terminal differentiation of the cell source . For example , thymidine to thymine and 2 -deoxy - ribose; e ) the exogenous the exogenous antigen - expressing EHC is generated from a antigen is Purine nucleoside phosphorylase (PNP ) and the cultured EHC and the expression or presence of the exog assay detects the conversion of inosine to hypoxanthine , enous antigen is assessed following terminal differentiation adenosine into adenine, and guanosine into guanine ; f ) the of the cell by a suitable method , e . g . , by flow cytometry, exogenous antigen is homogentisate 1, 2 - dioxygenase (HDG ) and the assay detects the conversion of homogenti Western blot , immunoprecipitation , fluorescence spectros sate to maleylacetoacetate ; g ) the exogenous antigen is copy, chemiluminescence , Southern blot, Northern blot, or cystathionine beta synthase and the assay detects the con absorbance spectroscopy . version of serine and homocysteine to cystathionine ; h ) the [ 0399] In a specific embodiment, the exogenous antigen is exogenous antigen is oxalate oxidase and the assay detects retained on the exogenous antigen - expressing EHC follow the oxidation of oxalate . ing circulation in vivo after administration of the exogenous [ 0405 ] In some embodiments , activity of the exogenous antigen - expressing EHC to a subject. The exogenous anti antigen - expressing EHC is assessed in an animal model, for gen - expressing EHC can be injected into a laboratory animal example a mouse model, and immunodeficientmouse , or an or animal model, such as a mouse intravenously , e . g ., via the NSG mouse , of a disease , for example a metabolic disease tail vein , or is injected into a human intravenously . Then or an enzyme deficiency , or that can demonstrate the effect blood is drawn and the presence of the exogenous antigen on of the exogenous antigen - expressing EHC , for example a the exogenous antigen -expressing EHC is assessed by suit mouse into which a substrate is injected and the product of able assay , e . g . , by flow cytometry , Western blot, immuno the exogenous antigen -mediated conversion measured . precipitation , fluorescence spectroscopy , chemilumines [0406 ] In one embodiment, the exogenous antigen is cence, Southern blot, Northern blot, or absorbance complement receptor 1 (CR1 ) polypeptide, a derivative or spectroscopy . functional fragment thereof. The activity of the CR1 exog [0400 ] In some embodiments , the biological activity of the enous antigen can be assessed in several ways including , for exogenous antigen in or on the exogenous antigen -express example , the specific capture of immune complexes by the US 2018 /0271910 A1 Sep . 27 , 2018 32

CR1 exogenous antigen , the efficient transfer of the immune disease , such as lupus. Mouse models include NZBWF1/ J, complexes to macrophages , or the in vivo clearance of MRL /MPJ , MRL /MpJ - Fas ( lpr ) , Smn . C3 - Fasl/ J, NZM2410 / immune complexes from a mouse . Aeg, 12984 - Cd48 , Cg - Slel , NZM -Slel Sle2 Sle3 /Lmod , [0407 ] Functionality of EHCs overexpressing CR1 exog and BXSB . 129P2 . Alternatively or in addition , a disease enous antigen may be assessed by one or more processes : phenotype may be introduced into a mouse , e . g ., by injection capture of immune complexes on the EHC surface compris of immune complexes . The exogenous CR1 antigen - ex ing CR1 exogenous antigen , release of the immune com pressing EHCs may be injected into any suitable mouse (or plexes to macrophages while sparing the EHC comprising other animal model) to test one or more biological effects of CR1 exogenous antigen , and proper circulation of the EHCS the EHC , e . g . , the clearance of the injected immune com comprising CR1 exogenous antigen . These three parameters plexes by the exogenous CR1 antigen - expressing EHC . can be assayed in vitro . Immune complex capture assays are [0411 ] In one embodiment, the exogenous antigen is a described in the art , e. g . , Oudin et al. , J Immunol 2000 and complement regulatory molecule or has complement regu Schifferli et al. , J Immunol 1991 . For example , labeled latory activity . This activity of the exogenous antigen can be immune complexes are incubated with EHCs expressing assessed by both in vitro and in vivo assays . For instance , the native CR1 or CR1 exogenous antigen polypeptide or a activity of the exogenous antigen can be assessed by mea fragment thereof and the number of immune complexes suring the reduction in an in vitro complement activation captured by the EHCs is assayed by flow cytometry. Mac assay, e. g. , CH50 assay that measures complement- mediated rophage transfer assays are described in the art , e . g . , Kuhn lysis of sensitized sheep erythroctyes , or AH50 assay that et al. , J Immunol 1998 . For example , labeled immune measured alternate pathway complement -mediated lysis of complexes loaded onto erythrocytes expressing native CR1 non - sensitized rabbit erythrocytes. Alternatively , the activity or CR1 exogenous antigen polypeptide or a fragment thereof of the exogenous antigen can be assessed by detecting the are incubated with macrophages . The transfer of immune cleavage or absence of cleavage , which may or may not complex from erythrocyte surface to macrophage , and the expose a neoepitope , of a recombinant complement compo consumption or sparing of erythrocytes by macrophages , nent that has been incubated with the exogenous antigen , can be measured by flow cytometry . Proper circulation can including but not limited to e . g . , the cleavage of recombinant be predicted by analyzing EHC morphology and deform C2 into C2a and C2b , the cleavage of factor B into factor Ba ability . Morphology of EHCs expressing native CR1 or CR1 and factor Bb , the cleavage of factor C3b into iC3bH and exogenous antigen polypeptide or a fragment thereof can be iC3bL , the cleavage of C3bBb into C3b and Bb , the cleavage assessed by eye using standard microscopy techniques , as of C4bBb into C4b and Bb , or the cleavage of factor C4b described e . g ., by Giarratana et al ., Blood 2011 and Repik et into iC4bH and iC4bL . The cleavage or absence of cleavage al ., Clin Exp Immunol 2005 . Deformability of EHCS of a suitable recombinant complement component can be expressing native CR1 or CR1 exogenous antigen polypep assessed by protein analysis methods known in the art tide or a fragment thereof can be assessed by ektacytometry, including , but not limited to , e . g . , chromatography, gel also known as laser- assisted optical rotational cell analysis electrophoresis , ELISA , and western blotting . Suitable in (LORCA ), as described e. g. , Giarratana et al ., Blood 2011. vivo assays for exogenous antigen activity include injection [0408 ] For example , an exogenous CR1 antigen - express of the exogenous antigen -expressing EHC into animal, for ing EHC (the EHC comprises a CR1 polypeptide exogenous example a mouse , and examining the deposition of comple antigen ) is incubated with immune complexes , such as in ment factors , for example membrane attack complex , by vivtro generated immune complexes or patient- derived histological staining . immune complexes . The capture of the immune complexes [0412 ] In one embodiment , the exogenous antigen is by the CR1 exogenous antigen is assessed by , for example , capable of binding or capturing a target and the activity of flow cytometry using a fluorescent marker in the immune the exogenous antigen can be assessed by detecting the complex or by flow cytometry using a secondary detection captured target on the exogenous antigen in vitro or in vivo . agent against an element of the immune complex . [0413 ]. In one embodiment , the exogenous antigen - ex [0409 ] In one embodiment, the exogenous CR1 antigen pressing EHC is incubated with the target in vitro , and the expressing EHC is first incubated with immune complexes capture of the target by the exogenous antigen is detected and then incubated with macrophages , such as primary using an in vitro assay including but not limited to , for macrophages, primary monocytes, cultured macrophages , example , flow cytometry , immunohistochemistry , magnetic cultured monocytpes , U937 cells, PMA - activated U937 separation , radiography, colony - forming assays, micros cells , AA9 cells , RAW 264 . 7 cells , J774 Cells , THP1 cells , copy. KG - 1 cells , NR8383 cells , MV - 4 - 11 cells , 3D4 /31 cells , MD [0414 ] In one embodiment, the exogenous antigen -ex cells , Fcwf- 4 cells , DH82 cells . The macrophages are pressing EHC is incubated with the target in vitro , and the assayed by, for example , flow cytometry or radiography , for capture of the target by the exogenous antigen is detected the presence of immune complexes transferred by the exog using an in vitro co - culture assay including but not limited enous CR1 antigen - expressing EHC . The transfer of cap to for example a macrophage consumption assay of tured immune complexes from cultured EHCs to macro opsonized exogenous antigen - expressing EHC , a T cell phages is a standard assay in the art, see for example : Repik activation assay, a B cell stimulation assay , a mast cell et al. 2005 Clin Exp Immunol. 140 : 230 ; Li et al . 2010 degranulation assay , an infectious potential assay . Infection Immunity 78 ( 7 ) :3129 . [ 0415 ]. In an embodiment , the exogenous antigen - express [0410 ] In one embodiment, activity of the exogenous CR1 ing EHC is incubated with the target in vitro , and the release antigen - expressing EHC is assessed in an animal model. For or off -rate of the captured target is measured using an in vitro example , a suitable mouse model may be used , such as an assay including but not limited to , for example , flow cytom immunodeficient mouse , or an NSG mouse . The mouse etry , immunohistochemistry , magnetic separation , radiogra disease model can be for example an immune complex phy, colony - forming assays, microscopy. US 2018 /0271910 A1 Sep . 27 , 2018 33

[ 0416 ] The capture of the target by the exogenous antigen - antigen - expressing EHC is assayed either by examining the expressing EHC can be assayed in an in vivo assay, for animal, e . g the plasma, the tissue , for reduction of the target , example in an animal, including a mouse model of diseases or by isolating or collecting the exogenous antigen - express in which the target is naturally present in themouse . Suitable ing EHC from the animal, e . g . , from the blood , from the diseases include bacterial infections , viral infections, fungal plasma , from a tissue , and assaying the presence of the target infections, immune complex diseases, self- antibody dis on the exogenous antigen using an in vitro assay including , eases , hyperlipidemia , hyperglycemia . In other mouse mod but not limited to , for example , flow cytometry , immuno els , the target is administered to the mouse externally , e . g . , histochemistry, magnetic separation , radiography, colony by injection or by feeding . In these assays, the capture of the forming assays, microscopy . target by the exogenous antigen - expressing EHC is assayed [0421 ] In some embodiments , the exogenous antigen either by examining the animal, e . g the plasma, the tissue , expressing EHC is capable of delivering a suitable exog for reduction or retention of the target , or by isolating or enous antigen to a specific subcellular compartment, for collecting the exogenous antigen - expressing EHC from the example a lysosome . animal, e . g ., from the blood , from the plasma, from a tissue , and assaying the presence of the target on the exogenous [ 0422 ] For example , an exogenous antigen may be deliv antigen using an in vitro assay including , but not limited to , ered to the lysosomal compartment of a target cell, e . g . , a for example , flow cytometry, immunohistochemistry , mag macrophage . The successful delivery of the exogenous anti netic separation , radiography , colony - forming assays , gen to the lysosomal compartment of a target cell is assessed microscopy . by microscopy and the detection of punctate spots corre [ 0417 ]. In some embodiments , the exogenous antigen is sponding to a fluorescent exogenous antigen or fluorescent capable of binding or capturing a target and substantially exogenous antigen detection agent. Alternatively or in addi increasing the clearance of the target in vivo , or substantially tion , the successful delivery of the exogenous antigen to the reducing the concentration of the target in circulation . The lysosomal compartment of a target cell is assessed by activity of the exogenous antigen on the exogenous antigen microscopy and the colocalization of a fluorescent exog expressing EHC can be assessed by detecting the enhanced enous antigen detection agent with a fluorescent detection clearance of the target in vitro or in vivo . agent for a known lysosomal marker , e . g ., lysotracker, [ 0418 ] In one embodiment , the exogenous antigen - ex LAMP - 1 . pressing EHC is incubated with the target in vitro , and the [0423 ] In some embodiments , the exogenous antigen is an capture of the target by the exogenous antigen is detected enzyme that can degrade toxic components that have built using an in vitro assay including but not limited to , for up in the lysosome of a cell exhibiting the genotype or example , flow cytometry, immunohistochemistry , magnetic phenotype of, or derived from a patient with , a lysosomal separation , radiography , colony - forming assays , micros storage disease . For example , the exogenous antigen is copy . Subsequently , the exogenous antigen -expressing EHC capable of degrading the toxic material built up in the cell is incubated in a co - culture assay with a cell known to and rescue the cell phenotype , e. g ., preventing cell death . promote clearance , for example a macrophage or a mono [0424 ] The population of exogenous antigen - expressing cyte, and the clearance of the target and exogenous antigen EHCs can be assessed for the inability of the EHCs to expressing EHC is assessed by , for example , flow cytometry, replicate , the ability of the EHCs to circulate safely through immunohistochemistry , magnetic separation , radiography, the vasculature, and the lack of immunogenicity of the colony - forming assays , microscopy . EHCs. [ 0419 ]. In one embodiment, the exogenous antigen - ex [0425 ] In some embodiments , the safety of the population pressing EHC is incubated with the target in vitro , and the of exogenous antigen - expressing EHCs is assessed by mea capture of the target by the exogenous antigen is detected suring the replication potential of the population of EHCs using an in vitro assay including but not limited to , for using a suitable in vitro or in vivo assay. For example , tests example , flow cytometry , immunohistochemistry , magnetic for a substantial inability of the exogenous antigen -express separation , radiography , colony - forming assays, micros ing EHCs to self - replicate include : a ) a susbstanital inability copy . Subsequently , the exogenous antigen -expressing EHC to form a tumor when injected into an immunocompromised is incubated in a physical system that mimics the clearance mouse ; b ) a substantial inability to form a colony when mechanism of the EHC in vivo , for example an artificial cultured in soft agar; c ) a substantial inability to incorporate spleen , a microchannel, a packed column , a resin , a tissue thymidine in a thymidine incorporation assay ; d ) a substan explant, a centrifuge , and the clearance of the target and tial lack of positive signal upon transfection with DNA exogenous antigen - expressing EHC is assessed by, for encoding a fluorescent reporter, e . g . , less than 10 % , 1 % , example , flow cytometry , immunohistochemistry , magnetic 0 . 1 % , 0 .01 % , 0 .001 % , 1 ppm , 100 ppb , 10 ppb , 1 ppb , 100 separation , radiography , colony - forming assays, micros ppt , 10 ppt, 1 ppt, or less than 1 ppt positive signal; e ) a copy. substantial lack of positive signal upon staining with a [ 0420 ] In one embodiment, the clearance of the target by nuclear dye, e . g ., less than 10 % , 1 % , 0 . 1 % , 0 .01 % ; and the exogenous antigen - expressing EHC is assayed in an in 0 .001 % , 1 ppm , 100 ppb , 10 ppb , 1 ppb , 100 ppt, 10 ppt, 1 vivo assay , for example in an animal, including , for example , ppt, or less than 1 ppt positive signal; f ) a substantial lack of a mouse model of diseases in which the target is naturally positive signal upon staining with cell markers of hemato present in the mouse , for example bacterial infection , viral logical malignancy , e . g ., CKIT , CD34 , EpCam , e . g . , less infection , fungal infection , immune complex disease , self than 10 % , 1 % , 0 . 1 % , 0 .01 % , 0 .001 % , 1 ppm , 100 ppb , 10 antibody disease , hyperlipidemia , hyperglycemia , or for ppb , 1 ppb , 100 ppt, 10 ppt, 1 ppt , or less than 1 ppt positive example , a mouse model in which the target is administered signal. In certain embodiments , exogenous antigen - express to the mouse externally , e . g ., by injection or by feeding . In ing EHCs are provided that do not contain a substantial these assays, the clearance of the target by the exogenous amount of a replicating nucleic acid . US 2018 /0271910 A1 Sep . 27 , 2018 34

[0426 ] In some embodiments , the safety of the population (0431 ] In one embodiment, any adverse events induced by of exogenous antigen - expressing EHCs is assessed by mea the exogenous antigen - expressing EHCs are assessed by suring the ability of an administered EHC to circulate in vivo injecting the EHCs into an animal intravenously and assess in the circulatory system of a subject ) without causing ing the status of tissues, e . g . , liver , spleen , heart, lungs , substantial vascular occlusion or induction of the clotting brain , skin , kidneys . The status of tissue can be assessed by cascade . Optionally , the circulation pharmacokinetics of the gross necropsy , dissection of the tissue , histological staining , exogenous antigen - expressing EHCs may be assessed . and imaging by microscopy . [0427 ] In one embodiment, the circulation pharmacoki [ 0432 ] In one embodiment, the ability of the exogenous netics of the exogenous antigen - expressing EHCs is antigen - expressing EHC to circulate in vivo without causing assessed by injecting the EHC into an animal intravenously , substantial vascular occlusion or activation of the clotting such as a mouse . The mouse can be an NSG (nod - SCID cascade is assessed by measuring the deformability of the gamma ) immunodeficient mouse . The mouse is depleted of EHCs. The deformability of the exogenous antigen - express macrophages prior to injection with the EHC , e . g ., by ing EHC is assessed using an in vitro assay . For example , the intraperitoneal injection of human red blood cells , or by assay is an osmotic fragility assay. Mechanical fragility of intravenous injection with clodronate liposomes . The exog the exogenous antigen - expressing EHC can be assessed by enous antigen - expressing EHCs can be labeled with a fluo measuring the structural integrity in response to shear stress resecent dye , e . g . , CFSE . After injection of the EHCs, blood in a Couett -type shearing system . In one embodiment, the is drawn and the number of exogenous antigen - expressing deformability of the exogenous antigen - expressing EHC is EHCs remaining is assessed by , e . g . , flow cytometry , west assessed using an Ektacytometer . In one embodiment, the ern blot, and the clearance rate of the exogenous antigen deformability of the exogenous antigen - expressing EHC is expressing EHCs is deduced from these data . Human red assessed by measuring the elongation index at a defined blood cells can be injected into the same animal model as the pressure by laser diffraction using a laser -assisted optical exogenous antigen - expressing EHCs and the clearance rates rotational cell analyzer (LORCA ) instrument. In one of the EHCs and human red blood cells are compared . embodiment, the deformability of the exogenous antigen expressing EHC is assessed by measuring the transit time [0428 ] In one embodiment, the risk of activation of the through a series of micron -scale constrictions of defined clotting cascade by the exogenous antigen -expressing EHC dimensions at a fixed pressure in a microfluidic device . In is assessed with an in vitro assay . In one embodiment, the one embodiment , the deformability of the exogenous anti exogenous antigen -expressing EHC is incubated with gen - expressing EHC is assessed by measuring the survival human blood and clotting cascade activation is assessed by rate through a series of micron - scale constrictions of defined measuring the time required for coagulation in the presence dimensions at a fixed pressure in a microfluidic device. The of kaolin , negatively - charged phospholipids , and calcium microfluidic device can be selected from one of the follow (activated partial thromboplastn time ( aPTT ) test) , see e . g ., ing , including but not limited to , a poly dimethyl siloxane Exner and Rickard , Biomedicine 1977 27 ( 2 ) : 62 , or by ( PDMS ) chip with micron - scale constrictions ( e . g ., Hoelzle measuring the time required for coagulation in the presence et al. J Vis Exp 2014 91 :e51474 ) ; a chip with funnel - shaped of thromboplastin and calcium ( prothrombin time (PT ) test ) , constrictions ( e . g ., Guo et al . Lab Chip 2012 12 : 1143 ) ; a see e .g ., Jaques and Dunlop 1945 , Am J Physiol 145 :67 . The PDMS chip with pillars ( e . g . , Zhang et al. PNAS 2012 normal range for the aPTT test is approximately 25 - 38 109 ( 46 ) : 18707 ) ; or an in vitro artificial spleen microbead seconds. The normal range for the PT test is approximately packed column (Guillaume DePlaine et al. , Blood 2011 , 10 - 12 seconds. 117 (8 )) . [ 0429 ] In one embodiment, any adverse events induced by [0433 ] In one embodiment, the ability of the exogenous the exogenous antigen - expressing EHCs are assessed by antigen - expressing EHC to circulate in vivo without causing injecting the EHCs into an animal intravenously and assess substantial vascular occlusion or activation of the clotting ing the activation of the clotting cascade . The level of cascade is assessed by measuring the vascular occlusion of clotting cascade induction is assessed by drawing blood and the EHCs. Vascular occlusion of the exogenous antigen assessing the levels of clotting cascade components in the expressing EHC can be assessed using an in vitro assay. For plasma by, e . g . , Western Blot or ELISA . The clotting cas example , the vascular occlusion of the exogenous antigen cade components are typically fibrinogen breakdown prod expressing EHC is assessed using an ex vivo assay. The ucts , e . g . , fibrinopeptide A and fibrinopeptide B . For exogenous antigen -expressing EHC is incubated at a 1: 1 example , the level of clotting cascade induction is assessed ratio with reference human red blood cells and induction of by drawing blood and assessing the levels of clotting activity multi - cell rosettes are assessed by light microscopy in in the plasma by platelet activation assay, e . g . , incubating the comparison to a reference assay with Rh -mismatched blood . plasma with platelets and assessing the activation of the The vascular occlusion of the exogenous antigen - expressing platelets by flow cytometry , e . g ., by staining for markers of EHC is assessed by measuring the adhesion of the EHCs to activation , e . g ., by staining for PAC - 1 , CD62p, or CD40L . human vascular endothelial cells under flow conditions, see [ 0430 ] In one embodiment, any adverse events induced by e .g ., Kaul D K , Finnegan E , and Barabino G A ( 2009 ) the exogenous antigen - expressing EHCs are assessed by Microcirculation 16 ( 1 ) : 97 - 111 . Alternatively or in addition , injecting the EHCs into an animal intravenously and assess vascular occlusion is assessed by measuring the peripheral ing the activation of inflammatory pathways. The level of resistance unit (PRU ) increase in an ex vivo perfusion assay inflammation can be assessed by drawing blood and assess of rat vascular endothelium , see e . g. , Kaul , Fabry and Nagel, ing the levels of inflammatory cytokines in the plasma by , PNAS 1989, 86 : 3356 . Further , vascular occlusion is e . g ., Western Blot or ELISA . In one embodiment, the assessed by intravital microscopy , see e . g . , Zennadi et al. inflammatory citokines are interferon gamma, tumor necro 2007 Blood 110 ( 7 ) : 2708 . Vascular occlusion may also be sis factor alpha , or IL - 12 fragment p70 . assessed by measuring flow rates and adhesion of the EHCs US 2018 /0271910 A1 Sep . 27 , 2018 35 in vitro graduated height flow chambers , see e. g ., Zennadi et [0443 ] Alternatively , the cultured EHCs of the invention al 2004 , Blood 104 ( 12 ) : 3774 . can stored in a glycerol cryoprotective solution . The com [0434 ] In some embodiments , the safety of the population positionsmay be frozen and stored for up to 10 years. Frozen of exogenous antigen - expressing EHCs is assessed by mea cells may be thawed and deglycerolized by successive suring the immunogenicity of the population of EHCs using washing steps , for example with 0 . 9 % sodium chloride a suitable in vitro or in vivo assay . before use . [ 0435 ] For example , the population of exogenous antigen [0444 ] Considering the life span of human and murine expressing EHCs a ) does not induce agglutination in a erythrocytes ( 120 and 50 days , respectively (see , e . g . Khan Coombs test using serum from the intended recipient subject delwal et al ., Transfusion 2007 ), it may be advantageous to or b ) does not induce agglutination in a Coombs test using stimulate artificial erythrocyte aging to increase phagocyto pooled human serum . sis by antigen presenting cells . The most important and [ 0436 ] In one embodiment , the population of exogenous physiological mechanism of erythrocyte removal from the antigen - expressing EHCs is derived from a progenitor cell circulation is immune -mediated (Singer et al. , PNAS 1986 ) that has been genotyped for the predominant blood group after exposure of new antigenic sites on RBC cell surface antigens and matched to the blood group antigen genotype such as phosphatidylserine externalization (Schroit et al. , J of the recipient. Biol Chem 1985 ) or Band 3 protein clustering (Kay , PNAS [0437 ] In one embodiment, the population of exogenous 1975 ; Turrini et al. , J Biol Chem 1991 ) induced artificially antigen - expressing EHCS comprises an exogenous antigen with calcium ionophore or BS3 chemical treatments , respec or other exogenous protein that has less than 10 % , 1 % , tively . 0 . 1 % , 0 .01 % , 0 . 001 % , or less than 0 .001 % predicted T cell [0445 ] The cultured EHCs of the invention may be treated reactivity by an in silico T cell epitope prediction algorithm . with a phagocytosis - inducing agent such as , e . g . calcium [ 0438 ] In one embodiment, the population of exogenous ionophore or BS3 . The plurality of cultured EHCs of the antigen - expressing EHCS comprises an exogenous antigen invention may comprise EHCs that have been treated with a or other exogenous protein that has less than 10 % , 1 % , phagocytosis - inducing agent, such as, e . g . calcium iono 0 .1 % , 0 .01 % , 0 .001 % , or less than 0 .001 % reactivity in an phore or BS3 , for differing lengths of time, such that upon in vitro T cell activation assay, e. g. , Antitope Inc. EpiScreen administration to a subject , the plurality of cultured EHCs of assay . the invention are phagocytosed at different rates, e . g . con [0439 ] For example , exogenous antigen -expressing EHCs tinuously over the course of one day or several days rather derived from erythrocytes can be centrifuged and resus than as a bolus. pended in appropriate solution ( e . g . , standard AS - 3 solution ) for infusion into subjects in need thereof. In some embodi [0446 ] Provided herein are compositions and pharmaceu ments , the exogenous antigen - expressing EHCs to be tical compositions comprising a plurality of cultured EHCs infused have the same ABO type as that of the recipient to that comprise an exogenous antigen of interest . The com minimize the risk of infusion - associated immune reactions . positions and pharmaceutical compositions may comprise a The exogenous antigen - expressing EHCs can also be pre solution of appropriate storage buffer such as, e . g . antico agulant citrate - dextrose A . The compositions and pharma treated to remove blood type - specific antigens or otherwise ceutical compositions comprising the plurality of cultured reduce antigenicities. Methods suitable for this purpose EHCs that comprise an exogenous antigen of interest may include , but are not limited to , those described in U . S . Patent additionally comprise an approved additive such as , e . g . Application Publication Nos . 20010006772 and Adsol. The compositions and pharmaceutical compositions 20030207247 . comprising the plurality of cultured EHCs that comprise an exogenous antigen of interest may additionally comprise a Therapeutic Compositions glycerol cryoprotective solution for frozen storage . [ 0440 ] Provided are compositions containing EHCs hav [0447 ] Provided herein are EHCs comprising an exog ing effective levels of exogenous antigen of interest. Such enous antigen of interest selected from one or more of the compositions contain a plurality of EHCs, e . g . , 1x10 cells , antigens of Table F , Table G , Table H , Table I and Table J, or 1x104, 1x109, 1x10 " , 1x107, 1x108, 1x10°, 1x1010 , such as e . g . myelin basic protein , proteolipid protein , myelin 1x1011 , 1x1012 , or greater than 1x1012 cells . EHCs of the oligodendrocyte glycoprotein , pancreatic beta cell antigen , invention can , for example , be administered as packed red insulin , flagellin , gluten , 2S albumin , hyalauronidase , factor blood cells in a saline solution at a concentration of 10 % , VIII, factor IX , and anti- TNFa , adenosine deaminase , L -as 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , or greater than paraginase , rasburicase, antithymocyte globulin , L - arginase , 90 % mass to volume ratio ( % m / v ) . The time of adminis L -methionase , preproinsulin , proinsulin , insulin , GAD65 , tration to a patientmay range from 10 minutes to four hours , GAD67, IA - 2 , IA - 2B , thyroglobulin , thyroid peroxidase , or more . thyrotropin receptor, myelin oligodendrocyte glycoprotein , [ 0441 ] The cultured EHCs of the invention can be stored proteolipid protein , collagen II , collagen IV , acetylcholine in an appropriate buffer, e . g . an FDA - approved anticoagu receptor, matrix metalloprotein 1 and 3 , molecular chaper lant preservative solution such as anticoagulant citrate one heat- shock protein 47 , fibrillin - 1 , PDGF receptor a , dextrose A ( ACD - A ), citrate -phosphate dextrose (CPD ), PDGF receptor B , nuclear protein SS - A , conarachin (Ara h Citratephosphate - dextrose - dextrose (CP2D ) , or citrate 1 ) , allergen II ( Ara h 2 ) , arachis agglutinin ( Ara h 6 ) , phosphate - dextrose -adenine ( CPDA - 1 ) . The compositions a - lactalbumin (ALA ) , lactotransferrin , glutein , low molecu may be stored for up to 21 days . lar weight glutein , a - gliadin , y - gliadin , hordein , secalin , [0442 ] Alternatively , the cultured EHCs of the invention avenin and combinations thereof. A plurality of EHCs com can be stored in an approved additive solution , e . g . AS - 1 prising an exogenous antigen of interestmay be provided as ( Adsol) , AS - 3 (Nutricel ) , AS - 5 (Optisol ) , or AS - 7 (SOLX ) . a composition or pharmaceutical composition . US 2018 /0271910 A1 Sep . 27 , 2018 36

[0448 ] Provided herein are expression vectors encoding ide . The parenteral preparation can be enclosed in ampoules, one or more antigens of Table F , Table G , Table H , Table I disposable syringes or multiple dose vials made of glass or and Table J , such as e . g . myelin basic protein , proteolipid plastic . protein , myelin oligodendrocyte glycoprotein , pancreatic 10453 ] Pharmaceutical compositions suitable for inject beta cell antigen , insulin , flagellin , gluten , Ara h2, 2S able use include sterile aqueous solutions (where water albumin , hyalauronidase , factor VIII , factor IX , and anti soluble ) or dispersions and sterile powders for the extem TNFa, optionally fused to one of the endogenous erythroid poraneous preparation of sterile injectable solutions or dis proteins of Table C . persion . For intravenous administration , suitable carriers [0449 ] Provided herein are pharmaceutical compositions include physiological saline , bacteriostatic water, Cremo comprising exogenous antigen - expressing EHCs that are phor ELTM (BASF , Parsippany, N . J. ) or phosphate buffered suitable for administration to a subject . The pharmaceutical saline (PBS ). In all cases, the composition must be sterile compositions generally comprise a population of exogenous and should be fluid to the extent that easy syringeability antigen -expressing EHCs and a pharmaceutically -accept exists . It must be stable under the conditions of manufacture able carrier in a form suitable for administration to a subject. and storage and must be preserved against the contaminating Pharmaceutically - acceptable carriers are determined in part action of microorganisms such as bacteria and fungi . The by the particular composition being administered , as well as carrier can be a solvent or dispersion medium containing, by the particular method used to administer the composition . e . g . , water , ethanol, polyol ( e . g . , glycerol, propylene glycol, Accordingly , there is a wide variety of suitable formulations and liquid polyethylene glycol, and the like ) , and suitable of pharmaceutical compositions comprising a population of mixtures thereof. The proper fluidity can be maintained , e . g . , exogenous antigen -expressing EHCs . (See , e . g . , Reming by the use of a coating such as lecithin , by the maintenance ton ' s Pharmaceutical Sciences , Mack Publishing Co ., Eas of the required particle size in the case of dispersion and by ton , Pa . 18th ed . (1990 )) . The pharmaceutical compositions the use of surfactants . Prevention of the action of microor are generally formulated as sterile , substantially isotonic and ganisms can be achieved by various antibacterial and anti in full compliance with all Good Manufacturing Practice fungal compounds, e . g . , parabens , chlorobutanol, phenol, (GMP ) regulations of the U . S . Food and Drug Administra ascorbic acid , thimerosal, and the like . In many cases , it will tion . be preferable to include isotonic compounds, e . g . , sugars , [ 0450 ] Pharmaceutically - acceptable excipients include polyalcohols such as manitol, sorbitol, sodium chloride in excipients that are generally safe , non - toxic, and desirable , the composition . Prolonged absorption of the injectable including excipients that are acceptable for veterinary use as compositions can be brought about by including in the well as for human pharmaceutical use . Such excipients can composition a compound which delays absorption , e . g ., be solid , liquid , semisolid , or, in the case of an aerosol aluminum monostearate and gelatin . composition , gaseous . [ 0454 ] Sterile injectable solutions can be prepared by [0451 ] Examples of carriers or diluents include , but are incorporating the exogenous antigen -expressing EHCs in an not limited to , water, saline , Ringer ' s solutions, dextrose effective amount and in an appropriate solvent with one or solution , and 5 % human serum albumin . The use of such a combination of ingredients enumerated herein , as desired . media and compounds for pharmaceutically active sub Generally , dispersions are prepared by incorporating the stances is well known in the art. Except insofar as any exogenous antigen - expressing EHCs into a sterile vehicle conventional media or compound is incompatible with the that contains a basic dispersion medium and any desired exogenous antigen - expressing EHCs described herein , use other ingredients . In the case of sterile powders for the thereof in the compositions is contemplated . Supplementary preparation of sterile injectable solutions , methods of prepa therapeutic agents may also be incorporated into the com ration are vacuum drying and freeze -drying that yields a positions. Typically , a pharmaceutical composition is for powder of the active ingredient plus any additional desired mulated to be compatible with its intended route of admin ingredient from a previously sterile - filtered solution thereof. istration . The exogenous antigen - expressing EHCs can be The exogenous antigen - expressing EHCs can be adminis administered by parenteral, topical , intravenous, oral, sub tered in the form of a depot injection or implant preparation cutaneous , intraarterial, intradermal, transdermal, rectal, which can be formulated in such a manner to permit a intracranial, intraperitoneal, intranasal; intramuscular route sustained or pulsatile release of the exogenous antigen or as inhalants . The exogenous antigen - expressing EHCS expressing EHCs, their exogenous antigen (s ) and /or their can optionally be administered in combination with other optional payload ( s ) . therapeutic agents that are at least partly effective in treating [ 0455 ] Oral compositions generally include an inert the disease , disorder or condition for which the exogenous diluent or an edible carrier . They can be enclosed in gelatin antigen - expressing EHCs are intended . capsules or compressed into tablets . For the purpose of oral [0452 ] Solutions or suspensions used for parenteral, intra therapeutic administration , the exogenous antigen -express dermal, or subcutaneous application can include the follow ing EHCs can be incorporated with excipients and used in ing components : a sterile diluent such as water for injection , the form of tablets, troches , or capsules. Oral compositions saline solution , fixed oils , polyethylene glycols , glycerine , can also be prepared using a fluid carrier for use as a propylene glycol or other synthetic solvents ; antibacterial mouthwash , wherein the compound in the fluid carrier is compounds such as benzyl alcohol or methyl parabens ; applied orally and swished and expectorated or swallowed . antioxidants such as ascorbic acid or sodium bisulfite ; Pharmaceutically compatible binding compounds, and /or chelating compounds such as ethylenediaminetetraacetic adjuvant materials can be included as part of the composi acid ( EDTA ) ; buffers such as acetates , citrates or phos tion . The tablets , pills, capsules, troches and the like can phates , and compounds for the adjustment of tonicity such contain any of the following ingredients , or compounds of a as sodium chloride or dextrose . The pH can be adjusted with similar nature : a binder such as microcrystalline cellulose , acids or bases, such as hydrochloric acid or sodium hydrox gum tragacanth or gelatin ; an excipient such as starch or US 2018 /0271910 A1 Sep . 27 , 2018 37 lactose , a disintegrating compound such as alginic acid , supplementation of metabolic precursors , manipulation of Primogel, or corn starch ; a lubricant such as magnesium osmotic balance , increasing of the volume of the suspending stearate or Sterotes ; a glidant such as colloidal silicon medium , and /or reduction of oxidative stress by adding dioxide; a sweetening compound such as sucrose or saccha protectivemolecules can be used to maintain the viability of rin ; or a flavoring compound such as peppermint, methyl the exogenous antigen -expressing EHCs . Several studies salicylate , or orange flavoring . employing a combination of these strategies have reported [ 0456 ] For administration by inhalation , the exogenous maintenance of viability of erythrocytes allowing an exten antigen -expressing EHCs are delivered in the form of an sion of storage beyond 6 weeks (see e . g ., Yoshida and aerosol spray from pressured container or dispenser which Shevkoplyas, Blood Transfus 2010 8 : 220 ). contains a suitable propellant, e . g . , a gas such as carbon [ 0463 ] Pharmaceutically acceptable carriers or excipients dioxide , or a nebulizer. may be used to deliver the exogenous antigen -expressing [0457 ] Systemic administration of compositions compris EHCS described herein . Excipient refers to an inert sub ing exogenous antigen - expressing EHCs can also be by stance used as a diluent or vehicle . Pharmaceutically accept transmucosal or transdermal means . For transmucosal or able carriers are used , in general, with a compound so as to transdermal administration , penetrants appropriate to the make the compound useful for a therapy or as a product. In barrier to be permeated are used in the formulation . Such general, for any substance , a pharmaceutically acceptable penetrants are generally known in the art, and include , e . g ., carrier is a material that is combined with the substance for for transmucosal administration , detergents , bile salts , and delivery to a subject . Conventional pharmaceutical carriers , fusidic acid derivatives . Transmucosal administration can be aqueous , powder or oily bases , thickeners and the like may accomplished through the use of nasal sprays or supposito be necessary or desirable . In some cases the carrier is ries . For transdermal administration , the modified red blood essential for delivery , e . g . , to solubilize an insoluble com cells are formulated into ointments , salves , gels, or creams pound for liquid delivery ; a buffer for control of the pH of as generally known in the art . the substance to preserve its activity ; or a diluent to prevent [0458 ] The exogenous antigen - expressing EHCs can also loss of the substance in the storage vessel . In other cases , be prepared as pharmaceutical compositions in the form of however , the carrier is for convenience , e . g . , a liquid for suppositories ( e . g . , with conventional suppository bases more convenient administration . Pharmaceutically accept such as cocoa butter and other glycerides ) or retention able salts of the compounds described herein may be syn enemas for rectal delivery . thesized according to methods known to those skilled in the [ 0459 ] In some embodiments , the exogenous antigen arts . expressing EHCs are prepared with carriers that will [0464 ] Typically , pharmaceutically acceptable composi decrease the rate with which exogenous antigen - expressing tions are highly purified to be free of contaminants , are EHCs are eliminated from the body of a subject. For biocompatible and not toxic , and are suited to administration example , controlled release formulation are suitable, includ ing implants and microencapsulated delivery systems. Bio to a subject . If water is a constituent of the carrier, the water degradable , biocompatible polymers can be used , such as is highly purified and processed to be free of contaminants , ethylene vinyl acetate , polyanhydrides , polyglycolic acid , e . g . , endotoxins . collagen , polyorthoesters , and polylactic acid . Methods for [0465 ] The pharmaceutically acceptable carrier may be preparation of such formulations will be apparent to those lactose , dextrose , sucrose , sorbitol, mannitol, starch , gum skilled in the art . The materials can also be obtained com acacia , calcium phosphate , alginates , gelatin , calcium sili mercially from Alza Corporation and Nova Pharmaceuticals , cate , micro - crystalline cellulose , polyvinylpyrrolidone, cel Inc . lulose , water, syrup , methyl cellulose , methylhydroxy ben [0460 ] In one embodiment the pharmaceutical composi zoate , propylhydroxy benzoate , talc , magnesium stearate , tion comprising exogenous antigen - expressing EHCs is and /or mineral oil, but is not limited thereto . The pharma administered intravenously into a subject that would benefit ceutical composition may further include a lubricant, a from the pharmaceutical composition . In other embodi wetting agent, a sweetener, a flavor enhancer, an emulsifying ments , the composition is administered to the lymphatic agent , a suspension agent , and / or a preservative . system , e . g ., by intralymphatic injection or by intranodal [046 ] Provided are pharmaceutical compositions con injection ( see e . g ., Senti et al ., 2008 PNAS 105 ( 46 ) : 17908 ) , taining exogenous antigen - expressing EHCs having effec or by intramuscular injection , by subcutaneous administra tive levels of exogenous antigens . Such compositions con tion , by direct injection into the thymus, or into the liver. tain a plurality of exogenous antigen - expressing EHCs , e . g . , [ 0461 ] In one embodiment , the pharmaceutical composi 1x10 EHCs, or 1x104 , 1x105 , 1x10 " , 1x107, 1x108, 1x10° . tion comprising exogenous antigen - expressing EHCs is 1x1010 , 1x1011 , 1x1012, or greater than 1x1012 EHCs . In administered as a liquid suspension . In one embodiment the specific examples , exogenous antigen -expressing EHCs pharmaceutical composition is administered as a coagulated generated from EHCs may be administered as packed red formulation that is capable of forming a depot following blood cells in a saline solution at a concentration of 10 % , administration , and in a preferred embodiment slowly 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , or greater than release exogenous antigen - expressing EHCs into circula 90 % mass to volume ratio ( % m / v ) . The time of adminis tion , or in a preferred embodiment remain in depot form . tration to a patientmay range from 10 minutes to four hours , [0462 ] In one embodiment, the pharmaceutical composi or more . tion comprising exogenous antigen - expressing EHCs is [0467 ] In specific examples, exogenous antigen -express stored using methods and buffer compositions that are ing EHCs generated from EHCs can be stored in an appro capable of maintaining viability of the exogenous antigen priate buffer, e . g ., an FDA -approved anticoagulant preser expressing EHCs . For example , deoxygenation prior to vative solution such as anticoagulant citrate - dextrose A storage to maintain an anaerobic state , manipulation of pH , ( ACD - A ) , citrate - phosphate dextrose (CPD ), Citratephos US 2018 /0271910 A1 Sep . 27 , 2018 38

phate -dextrose - dextrose (CP2D ), or citrate -phosphate -dex 1x1011, 2x1011 , 3x1011 , 4x1011 , 5x1011 , 6x1011 , 7x1011, trose - adenine (CPDA - 1 ). The compositions may be stored 8x1011 , 9x1011 , 1x1012 , or more exogenous antigen -ex for up to 21 days . pressing EHCs . Each dose of exogenous antigen -expressing [ 0468 ] Alternatively , exogenous antigen - expressing EHCs EHCs can be administered at intervals such as once daily , generated from EHCs can be stored in an approved additive once weekly , twice weekly , once monthly, or twice monthly . solution , e . g. , AS - 1 (Adsol ) , AS - 3 (Nutricel ) , AS - 5 (Opti Each EHC may express a range of antigen molecules, for sol) , or AS - 7 (SOLX ) . example , from about 100 to 10 ^ 7 , or from about 10 ^ 3 to [ 0469) Alternatively , exogenous antigen - expressing EHCS 10 ^ 6 . Specific examples include about 1000 , 3000 , 5000 , generated from EHCs can stored in a glycerol cryoprotective 1x10 ^ 4 , 3x10 ^ 4 , 5x10 ^ 4 , 1x10 ^ 5 , 3x10 ^ 5 , 5x10 ^ 5 , 1x10 ^ 6 , solution . The compositions may be frozen and stored for up 3x10 ^ 6 , 5x10 ^6 , 1x10 ^ 7 , or more exogenous antigen mol to 10 years . Frozen cells may be thawed and deglycerolized ecules per EHC . by successive washing steps, for example with 0 .9 % sodium [0475 ] “ EHC -based proportional dosage” is the number of chloride before use . exogenous antigen - expressing EHCs administered as a dose [0470 ] Provided herein are compositions and pharmaceu relative to a naturally occurring quantity of circulating tical compositions comprising a plurality of cultured EHCS entities . The circulating entities may be cells , e . g ., erythro that comprise an exogenous antigen . The compositions and cytes, reticulocytes , or lymphocytes, or targets , e. g ., anti pharmaceutical compositions may comprise a solution of gens, antibodies , viruses, toxins, cytokines , etc . The units appropriate storage buffer such as, e . g . , anticoagulant cit are defined as exogenous antigen - expressing EHC per cir rate -dextrose A . The compositions and pharmaceutical com culating entity , ie SCMRC / CE . This dosage unit may positions comprising the plurality of cultured EHCs that include 10 - 7 , 10 - 6 , 10 - 5 , 10 - 4 , 10 - 3 , 10 - 2 , 10 - 1 , 1 , 10 , 102 , comprise an exogenous antigen may additionally comprise 103 , 104, 105 , 106, 107, 108 , 10 % . an approved additive such as, e . g . , Adsol. The compositions [0476 ] The pharmaceutical compositions described herein and pharmaceutical compositions comprising the plurality comprise an exogenous antigen -expressing EHC and option of cultured EHCs that comprise exogenous antigen may ally a pharmaceutically active or therapeutic agent. The additionally comprise a glycerol cryoprotective solution for therapeutic agent can be a biological agent, a small molecule frozen storage. agent, or a nucleic acid agent. [0471 ] In one embodiment, the exogenous antigen - ex [0477 ] Dosage forms are provided that comprise a phar pressing EHC is able to form a multi - complex aggregate , maceutical composition comprising an exogenous antigen e . g . , a dimer, a trimer , a multimer, with another exogenous expressing EHC described herein . In some embodiments , antigen - expressing EHC . the dosage form is formulated as a liquid suspension for [ 0472 ] In one embodiment the exogenous antigen - ex intravenous injection . pressing EHC is able to form a multi - complex aggregate , [0478 ] Medical devices are provided that comprise a con e .g ., a dimer, a trimer, a multimer, with component of the tainer holding a pharmaceutical composition comprising an circulatory system , e . g an erythrocyte , a reticulocyte , a exogenous antigen -expressing EHC described herein and an platelet , a macrophage , a lymphocyte , a T cell , a B cell , a applicator for intravenous injection of the pharmaceutical mast cell . composition to a subject. [0473 ] The dosage and frequency of the administration of [0479 ] Medical kits are provided that comprise a pharma the exogenous antigen - expressing EHCs and pharmaceutical ceutical composition comprising an exogenous antigen compositions thereof can be determined by the attending expressing EHC described herein and a medical device for physician based on various factors such as the severity of intravenous injection of the pharmaceutical composition to disease , the patient' s age , sex and diet, the severity of any a subject. inflammation , time of administration , and other clinical [0480 ] A pharmaceutically acceptable suspension of exog factors . In one example , an intravenous administration is enous antigen -expressing EHCs is preferably packaged in a initiated at a dose which is minimally effective, and the dose volume of approximately 10 to approximately 250 ml. The is increased over a pre - selected time course until a positive packaging can be a syringe or an IV bag suitable for effect is observed . Subsequently , incremental increases in transfusions . Administration of the suspension is carried out , dosage are made limiting to levels that produce a corre e . g ., by intravenous or intra - arterial injection , optionally sponding increase in effect while taking into account any using a drip from an IV bag or the like . The administration adverse affects that may appear. is typically carried out intravenously in the arm or via a [0474 ] Non - limited examples of suitable dosages can central catheter. For administrations exceeding 50 ml use of range , for example , from 1x103 to 1x1014 , from 1x103 to a drip is preferred . 1x107, from 1x105 to 1x10 " , from 1x107 to 1x1011 , from 1x0 to 1x10° , from 1x1010 to 1x1014, from 1x1011 to Treatment of Diseases 1x1013 , or from 5x1011 to 5x1012 exogenous antigen -ex [0481 ] Provided are methods of inducing immune toler pressing EHCs. Specific examples include about 1x10°, ance . The methods include administering to a subject in need 2x103 , 3x103, 4x103, 5x103, 6x103, 7x10°, 8x109, 9x103 , of induction of immune tolerance a pharmaceutical compo 1x104, 2x104, 3x104, 4x104, 5x104, 6x104, 7x104, 8x104, sition of the erythrocyte cells that comprise an exogenous 9x104 , 1x105 , 2x105, 3x105 , 4x105, 5x105 , 6x105 , 7x105 . antigen of interest provided herein in an amount and/ or a 8x10 % , 9x109, 1x10 " , 2x106, 3x10 " , 4x106 , 5x10 “ , 6x10 “ , dosing frequency sufficient to induce immune tolerance in 7x106, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107, the subject . 6x107, 7x107, 8x107, 9x107, 1x108, 2x108 , 3x108, 4x108, [0482 ] The pharmaceutical compositions of the invention 5x108 , 6x108, 7x108, 8x108, 9x109, 1x10°, 2x10°, 3x10° , provide erythrocyte cells that comprise an exogenous anti 4x109 , 5x10°, 6x109, 7x10° , 8x109, 9x10°, 1x1010 , 2x1010 , gen of interest that are useful to promote or enhance immune 3x1010 , 4x1010 , 5x1010 , 6x1010, 7x1010 , 8x1010, 9x1010 , tolerance . Immune tolerance may be used to treat, prevent, US 2018 /0271910 A1 Sep . 27 , 2018 39 or reduce the severity of a disease , disorder , or condition ance to ADAMTS13 , thus reducing the quantity of inhibi associated with immune activation . tory anti - ADAMTS13 self- antibodies in circulation and [0483 ] Diseases of immune activation include autoim restoring the ability of the body to cleave von Willebrand mune diseases , such as , e . g . multiple sclerosis , type 1 Factor thus reducing the symptoms of the disease . In a diabetes , rheumatoid arthritis , and membranous nephritis , preferred embodiment, only the antigenic fragment of and those listed in Table F . Diseases of immune activation ADAMTS13 is expressed on the enucleated hematopoietic also include inflammatory diseases, such as, e . g . Crohn ' s cell. In a preferred embodiment, the full - length ADAMTS13 disease, ulcerative colitis , celiac disease , or other idiopathic is expressed on the enucleated hematopoietic cell. In a inflammatory bowl diseases, and those listed in Table G . preferred embodiment, the full - length ADAMTS13 is Diseases of immune activation also include allergic dis expressed on the enucleated hematopoietic cell and is enzy eases , such as , e . g . asthma, peanut allergy , shellfish allergy, matically active , such that the administered cell product is pollen allergy, milk protein allergy , insect sting allergy , and able to both induce tolerance and also therapeutically cleave latex allergy, and those listed in Table H . Diseases of von Willebrand Factor. immune activation also include immune activation in [ 0487] In another example , a patient suffering from atypi response to a therapeutic protein , administered to treat a cal hemolytic anemic syndrome (aHUS ) has an aberrant primary condition , that lessens the efficacy of the therapeutic self - antibody response to the endogenous protein Comple protein , such as, e . g . , clotting factor VIII in hemophilia A , ment Factor H ( CFH ) , preventing CFH from performing it ' s clotting factor IX in hemophilia B , anti- tumor necrosis complement regulatory function . As a result, complement factor alpha ( TNFa ) antibodies in rheumatoid arthritis and overactivation occurs in the vasculature leading to intravas other inflammatory diseases , glucocerebrosidase in Gauch cular hemolysis . In this embodiment, the CFH antigen is er ' s disease , or asparaginase in acute lymphoblastic leuke expressed on an enucleated hematopoietic cell and admin mia ( ALL ) , and those listed in Table 1 , Table J , Table 5 , and istered to a patient suffering from aHUS in an amount Table 7 . effective to induce tolerance to CFH , thus reducing the [0484 ] Further provided are methods for treating an quantity of inhibitory anti -CFH self -antibodies in circulation immune activation disease . The methods include adminis and restoring the ability of the body to inhibit complement tering to a subject in need of induction of treatment a thus reducing the symptoms of the disease . In a preferred pharmaceutical composition of the erythrocyte cells that embodiment, only the antigenic fragment of CFH is comprise an exogenous antigen of interest provided herein expressed on the enucleated hematopoietic cell. In a pre in an amount sufficient to treat the immune activation ferred embodiment, the full - length CFH is expressed on the disease . For example , a subject that has or is suspected of enucleated hematopoietic cell . In a preferred embodiment, having an immune activation disease such as autoimmune the full - length CFH is expressed on the enucleated disease , inflammatory disease or allergic disease would hematopoietic cell and is therapeutically active, such that the benefit from the treatment methods provided . administered cell product is able to both induce tolerance [0485 ] In some embodiments a patient is suffering from an and also therapeutically promote complement regulation . autoimmune disease or condition or a self- antibody medi [0488 ] In another example , a patient suffering from mul ated disease or condition , in which the patient' s immune tiple sclerosis (MS ) has an autoimmune response to the system is active against an endogenous molecule , for polypeptide myelin that sheathes neurons. As a result , T cells example a protein antigen , such that the immune system attack the myelin and the resultant inflammation causes attacks the endogenous molecule, induces inflammation , demyelination of the nerve fibers and impairs the ability for damages tissue , and otherwise causes the symptoms of the electrical signals to be sent along the nerves leading to the autoimmune or self - antibody disease or conditions . The symptoms of multiple sclerosis. In this embodiment, the immune response might be driven by antibodies that bind to myelin antigen is expressed on an enucleated hematopoietic the endogenous molecule , or it may be driven by overactive cell and administered to a patient suffering from MS in an T cells that attack cells expressing the endogenous molecule , amount effective to induce tolerance to myelin antigen , thus or it may be driven by other immune cells such as regulatory reducing the anti -myelin immune response and restoring the T cells , NK cells , NKT cells , or B cells . In these embodi ability of the body to send electrical impulses down myeli ments , the antigenic protein or a fragment thereof may be nated nerve fibers , thus reducing the symptoms of the expressed on an enucleated hematopoietic cell of the inven disease . In a preferred embodiment , only one or more tion . A population of these cells , when administered once or antigenic fragments ofmyelin are expressed on the enucle more to the patient suffering from the disease or condition , ated hematopoietic cell . In a preferred embodiment, the would be sufficient to induce tolerance to the antigenic full -length myelin protein is expressed on the enucleated protein such that it no longer induced activation of the hematopoietic cell . immune system , and thus would treat or ameliorate the [0489 ] In another example , a patient suffering from type 1 symptoms of the underlying disease or condition . diabetes ( T1D ) has an autoimmune response to the beta islet [0486 ] For example , a patient suffering from acquired cells of the pancreas. As a result , T cells kill the beta islet thrombotic thrombocytopenic purpura ( TTP ) has an aberrant cells reducing or eliminating the pancreas ' ability to produce self -antibody mediated disease in which antibodies are gen and secrete insulin , which leads to the symptoms and erated against endogenous ADAMTS13 protein rendering it pathology of T1D . In this embodiment, the beta cell antigen ineffective at performing its von Willebrand Factor -cleaving is expressed on an enucleated hematopoietic cell and admin activities, which results in microthrombi forming throughout istered to a patient suffering from T1D in an amount the vasculature and consequent thrombocytopenia . In this effective to induce tolerance to beta cell antigen , thus embodiment, the ADAMTS13 antigen is expressed on an reducing the anti -beta cell immune response and restoring eucleated hematopoietic cell and administered to a patient the ability of the pancreas to produce and secrete insulin , suffering from TTP in an amount effective to induce toler thus reducing the symptoms of the disease . In a preferred US 2018 /0271910 A1 Sep . 27 , 2018 40 embodiment, only one or more antigenic fragments of beta a level necessary to ameliorate the symptoms and prevent cell antigen are expressed on the enucleated hematopoietic severe uncontrolled bleeding, and such that ( 2 ) tolerance is cell. In a preferred embodiment, the full - length beta cell induced to FVIII . protein is expressed on the enucleated hematopoietic cell. [0493 ] In another example , a patient suffering from rheu [0490 ] Further provided are methods of reducing or alle matoid arthritis requires injections of anti - TNFa antibody to viating an immune activation in response to a therapeutic reduce the inflammation associated with that disease . How protein treatment regimen . The methods include adminis ever may patients develop neutralizing antibodies against tering to a subject in need of reducing or alleviating an the anti - TNFa antibody that render the therapeutic antibody immune activation in response to a therapeutic protein ineffective . In this instance , the patient typically suffers a treatment regimen a pharmaceutical composition of the worsening of symptoms and either has to increase the dose erythrocyte cells that comprise an exogenous antigen of of the anti - TNFa antibody or switch to a different anti - TNFa interest provided herein in an amount sufficient to reduce or antibody with a different coding sequence of amino acids . In alleviate the immune activation in response to a therapeutic this embodiment, an enucleated hematopoietic cell express protein treatment regimen . ing an antigenic fragment of anti - TNFa is administered to a [0491 ] In some embodiments a patient is suffering from a patient with rheumatoid arthritis who has developed neu disease or condition for which a therapeutic protein can be tralizing antibodies against the anti - TNFa antibody . The administered to treat or ameliorate the symptoms of the composition is administered at a dose sufficient to induce disease or condition , but the therapeutic protein is immu tolerance to the anti - TNFa antibody, allowing effective nogenic such that the patient elicits an immune response administration of anti - TNFa to reduce the circulating TNFa against the therapeutic protein such that it is no longer levels and thus reduce the symptoms of rheumatoid arthritis effective at treating or ameliorating the original disease . For in the patient. example , the immunogenic therapeutic protein might be [ 0494 ] In another example , a patient suffering from phe derived from a non -human source , e . g . bovine , porcine , or nylketonuria ( PKU ) is treated with pegylated phenylalanine non -human primate , or from a non -mammalian source , e . g . ammonia lyase (PAL ) , a non -human enzyme. The patient bacterial , fungal, or plat- derived , or the immunogenic thera develops opsonizing and neutralizing antibodies against peutic protein may be derived from a human source but the PAL that also elicit an allergic reaction upon administration repetitive exposure and dosing might be sufficient to induce of the therapeutic protein . This immune response not only immunogenicity . The immune response might be driven by renders the PAL ineffective , it also threatens the health of the antibodies that bind to the immunogenic therapeutic protein patient. In one embodiment, enucleated hematopoietic cells and inhibit its function (neutralizing antibodies ) or that bind expressing exogenous PAL are administered to a patient to the immunogenic therapeutic protein and trigger its suffering from PKU in an amount sufficient to induce clearance by other immune cells (opsonizing antibodies ) . In tolerance to PAL . In a preferred embodiment, the cell these embodiments , the immunogenic therapeutic protein or expressed PAL is active on the cell , and the composition is an antigenic fragment thereof may be expressed on an able to reduce the circulating levels of phenylalanine and enucleated hematopoietic cell of the invention . A population treat or ameliorate the symptoms of phenylketonuria in of these cells, when administered once or more to the patient addition to preventing a dangerous immune reaction against suffering from the disease , would be sufficient to induce PAL . In another preferred embodiment, an antigenic frag tolerance to the immunogenic therapeutic protein such that ment of PAL is expressed on the enucleated hematopoietic it was no longer neutralized or opsonized by the immune cell , and this cell - expressed fragment is not therapeutically system . In one preferred embodiment , the immunogenic active , so a separate formulation of PAL is administered to therapeutic protein expressed on the surface of the enucle the patient to treat or ameliorate the symptoms of phenylke ated hematopoietic cell of the invention is therapeutically tonuria . The tolerance - inducing cell composition can be active on the cell in circulation , such that the composition of administered prior to administering the therapeutic formu cell and protein is able to both induce tolerance and treat or lation of PAL , or the tolerance - inducing cell composition ameliorate the symptoms of the underlying disease or con can be administered concurrent to the administration of the dition when administered to the patient. In another preferred therapeutic formulation of PAL . embodiment the antigenic fragment of the immunogenic [ 0495 ] In some embodiments a patient is suffering from an therapeutic protein expressed on the surface of the enucle allergic disease , for example an allergy to animal dander , ated hematopoietic cell of the invention is not therapeuti black walnut, brazil nut, cashew nut, chestnut, dust mites, cally active on the cell in circulation , such that the compo egg , english walnut, fish , hazelnut, insect Venom , latex , sition of cell and protein is able to induce tolerance when milk , mold , peanuts , pollen , grass, shellfish , soy , tree nuts , or administered to the patient but a separate formulation of wheat. A patient suffering from an allergy may mount an immunogenic therapeutic protein is administered to treat or immune response upon contact with the antigenic fragment ameliorate the symptoms of the underlying disease or con of the allergen , for example through diet, skin contact, dition . injection , or environmental exposure . The immune response [ 0492 ] For example , a patient suffering from hemophilia A may involve IgE antibody, sensitized mast cells , degranu requires infusions of clotting factor VIII (FVIII ) to restore lation , histamine release , and anaphylaxis , as well as canoni proper coagulation . However many patients develop neu cal immune cells like T cells , B cells , dendritic cells , T tralizing antibodies to FVIII despite it being human derived , regulatory cells , NK cells , neutrophils , and NKT cells . The which render the therapeutic ineffective and lead to a life allergic reaction may cause discomfort or it may be severe threatening risk of bleeding . In one embodiment, an exog enough to cause death , and thus requires constant vigilance enous FVIII expressing enucleated hematopoietic cell is on the part of the sufferer as well as his or her family and administered to the patient suffering from hemophilia A such caretakers. In these embodiments, the antigenic protein or a that ( 1 ) the levels of circulating active FVIII are restored to fragment thereof may be expressed on an enucleated US 2018 /0271910 A1 Sep . 27 , 2018 hematopoietic cell of the invention . A population of these [ 0500 ] The pharmaceutical compositions may be admin cells , when administered once or more to the patient suf istered to the subject for example by intravenous transfusion fering from the allergic disease or condition , would be or intramuscular injection . sufficient to induce tolerance to the antigenic protein such that it no longer induced activation of the immune system OTHER EMBODIMENTS upon exposure , and thus would treat or ameliorate the [0501 ] It is to be understood that this invention is not symptoms of the underlying allergic disease or condition . limited to particular methods, reagents , compounds, com positions or biological systems, which can , of course , vary . [ 0496 ] In one example a patient suffering from peanut It is also to be understood that the terminology used herein allergy has an immune response following exposure to is for the purpose of describing particular aspects only , and peanut antigen AraH1. In this embodiment, AraHl is is not intended to be limiting. expressed on an enucleated hematopoietic cell and admin [0502 ] All of the features disclosed in this specification istered to a patient suffering from peanut allergy in an may be combined in any combination . Each feature dis amount effective to induce tolerance to AraH1 antigen , thus closed in this specification may be replaced by an alternative reducing the allergic immune response and restoring the feature serving the same, equivalent , or similar purpose . ability of the individual to safely consume peanuts , thus Thus, unless expressly stated otherwise , each feature dis reducing the symptoms of the disease. In a preferred closed is only an example of a generic series of equivalent embodiment, only one or more antigenic fragments of or similar features. AraH1 are expressed on the enucleated hematopoietic cell. [ 0503] In some embodiments , the exogenous antigen In a preferred embodiment, the full- length AraH1 protein is expressing EHC comprising a CR1 exogenous antigen is not expressed on the enucleated hematopoietic cell . generated in a mouse and/ or is not generated from mouse [0497 ] Certain aspects of the invention relate to EHCs that erythroid cells . In some embodiments , the exogenous anti comprise antigen that is recognized by immune cells in gen - expressing EHC comprising a CR1 exogenous antigen human leukocyte antigen ( HLA ) mismatch -mediated dis is not generated in a laboratory animal and /or is not gener eases , such as , e . g . graft - versus -host disease or organ trans ated from an erythroid cells derived from a laboratory plant rejection . animal. In some embodiments , the exogenous antigen - ex pressing EHC is generated from megakaryocytes or plate [0498 ] In some embodiments , a patient is suffering from a lets . In some embodiments , the exogenous antigen - express disease or condition of human leukocyte antigen (HLA ) ing EHC is generated from an erythroid cell , such as, e . g . an mismatch in which immune cells are activated against HLA erythrocyte or a reticulocyte . In some embodiments , the antigens on a tissue and attack the tissue . This commonly exogenous antigen - expressing EHC is not generated from a occurs following allogeneic transplantation of an organ or neutrophil, an eosinophil , or a basophil . In some embodi tissue from a donor who is not a perfect match and leads to ments , the exogenous antigen - expressing EHC is not gen the medical condition of transplant rejection , in which the erated from a monocyte or a macrophage . In some embodi patient' s immune system attacks the foreign tissue or organ ments , the exogenous antigen -expressing EHC is not and causes the transplanted organ or tissue to die . Another generated from a CD34 + Thy - 17 hematopoietic stem cell or common HLA -mismatch condition is Graft - versus -Host cell populations enriched in CD34 + Lin ' or CD34 + Thy - 1 * Disease (GVHD ) in which a patient has received an allo Lin " cells . In some embodiments, the exogenous antigen geneic bone marrow transplantation from a donor who is not expressing EHC does not comprise an exogenous antigen a perfect match and in which the transplanted immune cells comprising an extracellular domain of an HIV coreceptor. In ( graft ) become activated and attack the recipients organs some embodiments , the exogenous antigen - expressing EHC ( host ), which are recognized as foreign , causing the damage does not comprise an exogenous antigen capable of binding of host tissues and organs and leading to severe conse to a virus. In some embodiments , the exogenous antigen quences including death . HLA -mismatch immune activation expressing EHC does not comprise an exogenous antigen is typically mediated by T cells , but can also involve T comprising CD4 . In some embodiments , the exogenous regulatory cells , NK cells , NKT cells , B cells , antibodies , antigen - expressing EHC does not comprise an exogenous dendritic cells , monocytes, macrophages , and neutrophils . In antigen comprising an HIV coreceptor . In some embodi these embodiments , the antigenic HLA molecule or a frag ments , the exogenous antigen - expressing EHC does not ment thereof may be expressed on an enucleated hematopoi comprise an exogenous antigen comprising CXCR4 , CCR5 , etic cell of the invention . A population of these cells , when CCR1, CCR2 , CCR3 , CCR4 , CCR8 , CXCR1, CXCR2, administered once or more to the patient suffering from the CXCR3 , CXCR6 , GPR15 , APJ, CMKLR1, or CX3CR1 or HLA -mismatch disease or condition , would be sufficient to any combination thereof . In some embodiments , the exog induce tolerance to the antigenic HLA molecule such that it enous antigen - expressing EHC does not contain an exog no longer induced activation of the immune system and thus enous nucleic acid encoding an adenosine deaminase anti would treat or ameliorate the symptoms of the underlying gen . In some embodiments , the exogenous antigen HLA -mismatch disease or condition , for example the sur expressing EHC does not comprise an exogenous antigen vival of the transplanted organ or the survival of the patient. comprising adenosine deaminase (ADA ) . In some embodi In a preferred embodiment, the HLA molecule expressed on ments , the exogenous antigen - expressing EHC does not the surface of the cell also contains a peptide loaded into the comprise an exogenous nucleic acid encoding an oncogene . HLA molecule . In some embodiments, the exogenous antigen -expressing [ 0499 ] The erythrocyte cells that comprise an exogenous EHC does not comprise an exogenous antigen comprising antigen that are used for the methods described herein can be oncogene . In some embodiments, the exogenous antigen derived autologously , i. e . from the same subject , or may be expressing EHC does not contain an exogenous nucleic acid allogeneically derived , i . e . from a different cell donor. encoding cdx1, cdx2 , or cdx4 . In some embodiments , the US 2018 /0271910 A1 Sep . 27 , 2018 exogenous antigen - expressing EHC does not comprise an ing EHC is not a differentiated , mature human blood cell. In exogenous antigen comprising cdx1 , cdx2 , or cdx4, or any some embodiments , the exogenous antigen -expressing EHC combination thereof. In some embodiments , the exogenous is not generated from a blood cell derived from a universal antigen - expressing EHC does not comprise an exogenous donor, e .g . blood type O , Rh factor negative. In some antigen comprising a chimeric polypeptide comprising a embodiments , an exogenous ADA polypeptide antigen - ex ligand binding domain . In some embodiments , the exog pressing EHC is not used to treat severe combined immune enous antigen - expressing EHC does not comprise an exog deficiency (ADA - SCID ) . In some embodiments, methods of expansion and differentiation of the exogenous antigen enous antigen comprising an S domain that is capable of expressing EHCs do not include culturing the exogenous binding a ligand . In some embodiments , the exogenous antigen - expressing EHCs in a medium comprising a myelo antigen - expressing EHC does not comprise an exogenous proliferative receptor (mpl ) ligand . In some embodiments , antigen comprising CD3C , CD3n , an IL - 2 receptor , an IL - 3 the exogenous antigen - expressing EHC does not comprise a receptor, an IL - 4 receptor, an IL - 7 receptor, an IL - 11 recep payload comprising a synthetic triphosphorylated nucleo tor, an IL - 13 receptor, a GM -CSF receptor, a LIF receptor, side analog . In some embodiments, the exogenous antigen a CNTF receptor, an oncostatin M receptor, a TGF - B recep expressing EHC does not comprise a payload comprising tor, an EGF receptor, ATR2/ neu , a HER2 /neu , a HER3/ c 2 ' , 3 ' - dideoxycytidine - 5 ' -triphosphate (ddCTP ) and / or erbB - 3 , Xmrk , an insulin receptor, an IGF - 1 receptor, IRR , 3 '- azido - 3' - deoxythymidine - 5 - triphosphate (AZT - TP ). In PDGF receptor, a CSF - 1 receptor , c -kit , STK - 1 / flk - 2 , an some embodiments , the exogenous antigen - expressing EHC FGF receptor , fig , bek , an NGF receptor, Rorl and Ror2 or does not comprise a payload comprising a bisphosphonate . any combination thereof . In some embodiments , the exog In some embodiments, the exogenous antigen - expressing enous antigen - expressing EHC does not comprise an exog EHC is generated by contacting an erythroid cell with an enous antigen comprising E6 or E7 genes of human papil exogenous antigen and optionally a payload without lysing lomavirus . In some embodiments , the exogenous antigen and resealing the cells to incorporate the exogenous antigen expressing EHC does not comprise an exogenous antigen and /or payload . In some embodiments , the exogenous anti comprising a tumor antigen . In some embodiments , the gen -expressing EHC is generated by contacting an erythroid exogenous antigen - expressing EHC does not comprise an cell with an exogenous antigen and optionally a payload , exogenous antigen comprising glucocerebrosidase . In some wherein contacting does not comprise hypotonic dialysis . In embodiments , the exogenous antigen - expressing EHC does some embodiments , the exogenous antigen - expressing EHC not comprise an exogenous antigen comprising asparagi is generated by contacting an erythroid cell with an exog nase . In some embodiments , the exogenous antigen - express enous antigen and optionally a payload , wherein contacting ing EHC does not comprise an exogenous antigen compris does not include loading the exogenous antigen and /or ing arginine deiminase . In some embodiments , the payload into or onto the erythroid cell . In some embodi exogenous antigen - expressing EHC does not comprise a ments , the exogenous antigen is generated in an entity that fusion molecule capable of promoting fusion of the exog is not the erythroid cell to be contacted and / or the exogenous enous antigen - expressing EHC to a target cell that is i ) antigen is isolated from a sample that does not comprise the different from and / or ii ) acts independent of the exogenous erythroid cell to be contacted . For example , for a polypep antigen , wherein the exogenous antigen is capable of inter acting with a target. In some embodiments , the exogenous tide exogenous antigen suitable entities include a cell line , antigen -expressing EHC does not comprise an exogenous an in vitro expression system , a bacterial expression system , antigen comprising Syncytin - 1. In some embodiments , the etc . exogenous antigen -expressing EHC does not comprise a [0504 ] In some embodiments , the exogenous antigen poly photosensitive synthetic compound , such as, e . g. a com peptide expressed by the EHC is present on the surface of pound that can be activated by photons or quenchable the EHC but is not non - covalently bound to the surface of compounds. In some embodiments , the exogenous antigen the EHC . In some embodiments , the non - covalent attach expressing EHC does not comprise an activatable molecular ment of the antigen to the surface of the EHC is not mediated detection agent capable of producing a detectable response . by an antibody , an antibody - fragment, an antibody - like In some embodiments , the exogenous antigen - expressing polypeptide , or a non - antibody polypeptide binding scaffold . EHC does not comprise a diagnostic compound . In some In some embodiments , the non - covalent attachment of the embodiments , the exogenous antigen - expressing EHC does exogenous antigen to the surface of the EHC is not directed not comprise a virus or bacterium . In some embodiments , against an erythroid cell antigen such as Band 3 (CD233 ) , the exogenous antigen - expressing EHC is not generated aquaporin - 1 , Glut- 1 , Kidd antigen , RhAg /Rh50 (CD241 ) , from or does not comprise an autologous CD34 + cell . In Rh ( CD240 ), Rh30 CE ( CD240CE ) , Rh30D (CD240D ) , Kx , some embodiments , methods of treatment and prevention glycophorin B (CD235b ), glycophorin C (CD235c ), glyco using exogenous antigen -expressing EHCs generated from phorin D (CD235d ) , Kell (CD238 ) , Duffy /DARCi (CD234 ) , erythroid cells described herein do not comprise the step of CR1 (CD35 ) , DAF (CD55 ) , globoside , CD44 , ICAM - 4 detecting the erythroid cell in vivo , e . g ., through a detection (CD242 ) , Lu/ B -CAM (CD239 ) , XG1/ XG2 ( CD99 ) , EMM agent that is associated with the erythroid cell . In some PRIN /neur . thelin (CD147 ) , JMH , glycosyltransferase , Cart embodiments , the exogenous antigen - expressing EHC is not wright , Dombrock , C4A /CAB , Scianna , MER2 , s tomatin . generated from a human donor pluripotent hematopoietic BA - 1 (CD24 ), GPIV (CD36 ) , CD108 , CD139 , and H anti stem cell . In some embodiments , a population of exogenous gen (CD173 ) , or another erythrocyte -binding moiety . antigen - expressing EHCs is not expanded in a bioreactor. In [ 0505 ] In some embodiments , the exogenous antigen poly some embodiments , the population of exogenous antigen peptide is not generated apart from the EHC and then expressing EHCs after expansion and / or differentiation does conjugated to the EHC . In some embodiments , the exog not comprise a single species of differentiated human blood enous antigen polypeptide is not enzymatically conjugated , cells . In some embodiments , the exogenous antigen - express e . g . through an autocatalytic isopeptide bond - forming reac US 2018 /0271910 A1 Sep . 27 , 2018 43 tion such as carried out, e. g . by a transpeptidase , a sortase , indicated to be incorporated by reference for all purposes . and /or isopeptidase . In one embodiment, the exogenous The publications discussed herein are provided solely for antigen polypeptide is not enzymatically conjugated using a their disclosure prior to the filing date of the present appli sortase . cation . Nothing herein is to be construed as an admission [0506 ] In some embodiments , the exogenous antigen poly that the inventors described herein are not entitled to ante peptide is not chemically conjugated , e . g . through a cross date such disclosure by virtue of prior invention or for any linking agent such as a carbodiimide ( including sortase other reason . 1 -Ethyl - 3 - ( 3 -dimethylaminopropyl ) carbodiimide ( EDC ) ) . [0507 ] In one embodiment, the exogenous antigen is not Definitions generated apart from the EHC and then encapsulated by the [ 0517] “ Administration ,” “ administering ” and variants EHC . In one embodiment, the encapsulation of the exog thereof means introducing a composition , such as an exog enous antigen is not mediated by hypotonic dialysis of the enous antigen -expressing EHC , or agent into a subject and EHC in the presence of exogenous antigen . includes concurrent and sequential introduction of a com 0508 ] Many modifications and other embodiments of the position or agent. The introduction of a composition or agent inventions set forth herein will easily come to mind to one into a subject is by any suitable route , including orally , skilled in the art to which these inventions pertain having the pulmonarily , intranasally , parenterally ( intravenously , intra benefit of the teachings presented in the foregoing descrip muscularly, intraperitoneally, or subcutaneously ) , rectally , tions and the associated drawings . Therefore , it is to be intralymphatically , or topically . Administration includes understood that the inventions are not to be limited to the self - administration and the administration by another. A specific embodiments disclosed and that modifications and suitable route of administration allows the composition or other embodiments are intended to be included within the the agent to perform its intended function . For example , if a scope of the appended claims. Although specific terms are suitable route is intravenous , the composition is adminis employed herein , they are used in a generic and descriptive tered by introducing the composition or agent into a vein of sense only and not for purposes of limitation . the subject . Administration can be carried out by any suit [0509 ] As used in this specification and the appended able route , claims, the singular forms “ a ” , “ an ” and “ the ” include plural [0518 ] “ Anchor” or “ anchor domain ” or “ A domain ” is references unless the content clearly dictates otherwise . used to refer to the portion of an exogenous antigen poly [0510 ] The use of the alternative (e .g ., " or” ) should be peptide , including a fusion or chimeric exogenous antigen understood to mean either one, both , or any combination polypeptide that is in contact with the cell membrane of an thereof of the alternatives . EHC . The exogenous antigen polypeptide may interact with [0511 ] The term “ about” as used herein when referring to the lipid cell membrane layer via a phospholipid tail inser a measurable value such as an amount, a temporal duration , tion , covalent binding to a lipid layer constituent, an ionic and the like , is meant to encompass variations of + 20 % or bond , hydrogen bond , or via a single or multi- pass trans + 10 % , more preferably + 5 % , even more preferably + 1 % , membrane polypeptide domain that cross one or more of the and still more preferably + 0 . 1 % from the specified value , as lipid cell membrane layers . such variations are appropriate to perform the disclosed [ 0519 ] As used herein , the term “ antibody " encompasses methods . an immunoglobulin whether natural or partly or wholly [ 0512] As used herein , any concentration range , percent synthetically produced , and fragments thereof. The term also age range , ratio range , or integer range is to be understood covers any protein having a binding domain which is to include the value of any integer within the recited range homologous to an immunoglobulin binding domain . These and , when appropriate , fractions thereof (such as one tenth proteins can be derived from natural sources, or partly or and one hundredth of an integer ) , unless otherwise indicated . wholly synthetically produced . “ Antibody ” further includes [0513 ] “ Comprise, " " comprising , ” and “ comprises” and a polypeptide comprising a framework region from an " comprised of" as used herein are synonymous with immunoglobulin gene or fragments thereof that specifically “ include ” , “ including ” , “ includes " or " contain " , " contain binds and recognizes an antigen . Use of the term antibody is ing " , " contains” and are inclusive or open -ended terms that meant to include whole antibodies , polyclonal, monoclonal specifies the presence of what follows e . g . component and and recombinant antibodies, fragments thereof, and further do not exclude or preclude the presence of additional, includes single - chain antibodies, humanized antibodies ; non - recited components, features , element, members , steps , murine antibodies; chimeric , mouse -human , mouse - primate , known in the art or disclosed therein . primate -human monoclonal antibodies , anti -idiotype anti [0514 ] As used herein , the terms “ such as” , “ for example " bodies, antibody fragments , such as, e . g ., scFv, ( scFv ) 2 , Fab , and the like are intended to refer to exemplary embodiments Fab ', and F ( ab ') 2 , F ( ab1 ) 2 , Fv, dAb , and Fd fragments , and not to limit the scope of the present disclosure. diabodies, and antibody - related polypeptides. Antibody 0515 ]. Unless defined otherwise , all technical and scien includes bispecific antibodies and multispecific antibodies tific terms used herein have the samemeaning as commonly so long as they exhibit the desired biological activity or understood by one of ordinary skill in the art to which the function . invention pertains . Although any methods and materials [0520 ] The term “ antigen binding fragment” used herein similar or equivalent to those described herein can be used refers to fragments of an intact immunoglobulin , and any in the practice for testing of the present invention , preferred part of a polypeptide including antigen binding regions materials and methods are described herein . having the ability to specifically bind to the antigen . For [0516 ] All publications and patent applications cited in example , the antigen binding fragment may be a F ( ab ') 2 this specification are herein incorporated by reference in fragment, a Fab ' fragment, a Fab fragment, a Fv fragment, or their entirety for all purposes as if each individual publica a scFv fragment, but is not limited thereto . A Fab fragment tion or patent application were specifically and individually has one antigen binding site and contains the variable US 2018 /0271910 A1 Sep . 27 , 2018 44 regions of a light chain and a heavy chain , the constant interactions, hydrogen bonding, London forces, van der region of the light chain , and the first constant region CH1 Waals forces , hydrophobic interaction , lipophilic interac of the heavy chain . A Fab ' fragment differs from a Fab tions , and similar. fragment in that the Fab ' fragment additionally includes the [ 0526 ] The “ biological activity of a polypeptide” refers to hinge region of the heavy chain , including at least one any molecular activity or phenotype ( such as, e. g ., binding , cysteine residue at the C - terminal of the heavy chain CH1 signal transduction , catalytic , etc . ) that is caused by the region . The F ( ab' ) 2 fragment is produced whereby cysteine polypeptide , such as an exogenous antigen polypeptide . residues of the Fab ' fragment are joined by a disulfide bond f0527 ] As used herein , the term “ biological sample ” refers at the hinge region . A Fv fragment is the minimal antibody to any type ofmaterial of biological origin isolated from a fragment having only heavy chain variable regions and light subject, including, for example , DNA , RNA , lipids , carbo chain variable regions , and a recombinant technique for hydrates, and protein . The term “ biological sample” includes producing the Fv fragment is well known in the art . Two tissues , cells and biological fluids isolated from a subject . chain Fv fragments may have a structure in which heavy Biological samples include , e. g ., but are not limited to , chain variable regions are linked to light chain variable whole blood , plasma, serum , semen , saliva , tears , urine , regions by a non -covalent bond . Single -chain Fv (scFv ) fecal material, sweat, buccal, skin , cerebrospinal fluid , bone fragments generally may have a dimer structure as in the marrow , bile, hair, muscle biopsy , organ tissue or other two - chain Fv fragments in which heavy chain variable material of biological origin known by those of ordinary regions are covalently bound to light chain variable regions skill in the art . Biological samples can be obtained from , via a peptide linker or heavy and light chain variable regions e . g ., biopsies of internal organs or from cancers . Biological are directly linked to each other at the C - terminal thereof. samples can be obtained from subjects for diagnosis or The antigen binding fragment may be obtained using a research or can be obtained from healthy subjects , as con protease (for example , a whole antibody is digested with trols or for basic research . papain to obtain Fab fragments , and is digested with pepsin [0528 ] The “ clearance rate ” as used herein is calculated by to obtain F (ab ') 2 fragments ), and may be prepared by a measuring the amount or concentration of, e . g . , exogenous genetic recombinant technique . A dAb fragment consists of antigen , target -exogenous antigen , or exogenous antigen a VH domain . Single -chain antibody molecules may com expressing EHCs remaining in the circulatory system of a prise a polymer with a number of individual molecules, for subject over time. For example, 1 % , 2 % , 3 % , 4 % , 5 % , 10 % , example , dimmer, trimer or other polymers . 15 % , 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , 95 % , or [ 0521 ] " Applicator ” refers to any device used to connect 99 % of target detected in a first sample may still be detected to a subject. This includes, e . g ., needles , cannulae , catheters , in a second sample that is taken 1 hour, 5 hours , 10 hours, and tubing . 24 hours , 2 days , 3 days , 4 days, 5 days , 6 days, 7 days , 2 weeks , 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 [0522 ] “ Associated with ” when used to describe the rela months , 6 months, 7 months , 8 months, 9 months , 10 tionships among multiple compounds or molecules encom months, 11 months, 12 months , 2 years , 3 years, 4 years, or passes such as, e . g ., any interaction between an exogenous 5 years later. The clearance rate may alternatively be antigen and a target or between an exogenous antigen expressed as : number of entities ( e . g . , target / exogenous expressing EHC and a target. This includes enzymatic antigen ) per unit of time ( e . g ., per day ) . An increase in interaction , ionic binding , covalent binding , non - covalent clearance rate is a rate greater than that exhibited in an binding , hydrogen bonding , London forces , van der Waals untreated or healthy suitable control subject . A decrease in forces , hydrophobic interaction , lipophilic interactions, clearance rate is a rate less than that exhibited in an untreated magnetic interactions , electrostatic interactions , and the like . or healthy suitable control subject. The increase or decrease [0523 ] “ Associated with ” when used to describe the rela may be 1 % , 2 % , 3 % , 4 % , 5 % , 10 % , 15 % , 20 % , 30 % , 40 % , tionships among a target , entity , compound , agent, or mol 50 % , 60 % , 70 % , 80 % , 90 % , 100 % , 150 % , 200 % , 500 % , ecule and a disease , disorder , condition , symptom or phe 1000 % or may be 1 . 1 - fold, 1 . 2 - fold , 1 . 3 fold , 1 . 4 - fold , notype is any link that may reasonably be made between 1 . 5 - fold , 2 - fold , 3 - fold , 4 - fold , 5 - fold , 10 - fold , 20 - fold , them , including a causal link , or a statistical significant link , 50 - fold , 100 -fold , 500 - fold , or 1000 - fold . an empirically established link , a suggested link , whether or [ 0529 ] " Cleaving ” as used herein is a process that disrupts not causative of the disease , disorder, condition , symptom or a bonding interaction present in a target, such as a polypep phenotype. tide or nucleic e. g ., to produce two or more entities that after cleaving can be separated from one another. The separation [0524 ] “ Autoimmune disorders ” generally are conditions can involve , e . g ., disrupt an ionic bond , a covalent bond , a in which a subject ' s immune system attacks the body 's own polar covalent bond , a non - polar covalent bond , or a metallic cells , causing tissue destruction . Autoimmune disorders may bond . As cleaving applies to polypeptide targets , cleavage be diagnosed using blood tests , cerebrospinal fluid analysis , can involve breaking one or more peptide bonds. As cleav electromyogram (measures muscle function ) , and magnetic ing applies to small molecule targets , cleavage can involve resonance imaging of the brain , but antibody testing in the breaking one or more carbon or sulfide bonds. As cleaving blood , for self -antibodies (or auto -antibodies ) is particularly applies to nucleotide sequences, cleavage can involve break useful. Usually , IgG class antibodies are associated with ing one or more phosphodiester bonds . As cleaving applies autoimmune diseases. to microbes such as bacteria , fungi , or viruses, cleavage can [0525 ] “ Binding “ describes an interaction among com involve lysis of a membrane or capsid structure . Cleaving pounds or molecules , e . g . , between an exogenous antigen can be carried out by an enzyme, e . g ., a catalytically active and a target or between an exogenous antigen - expressing exogenous antigen polypeptide . Exogenous antigens can EHC and a target, that comes about by covalent binding or comprise, e . g ., exonuclease, endonuclease , or protease non -covalent binding , including ionic binding, electrostatic activity . US 2018 /0271910 A1 Sep . 27 , 2018 45

[ 0530 ] The " circulatory system of a subject, ” as used example , for self -antibody mediated diseases, the decrease herein , encompasses the space occupied by whole blood and may be quantified as one , or a combination of the following optionally the lymphatic system in a human , inclusive of parameters : reduced inflammation , reduced flare - ups , plasma and all circulating cells and molecules , and distrib reduced fatigue , reduced blood clotting , reduced swelling , uted throughout arteries, veins , capillaries, and lymphatic increased energy , or increased hair growth , etc . The param vessels of all tissues. The " circulatory concentration ” is the eters that may be quantified are those appropriate for assess concentration of a target, e . g ., a cell , polypeptide (such as an ing the specific disease , disorder or condition that is being antibody , pathogenic antigen , etc . ) , therapeutic agent, small treated . Delay, in the context of symptoms of a treated molecule, metabolite or other entity , an exogenous antigen disease , disorder or condition , refers to the significant exten or an exogenous antigen - expressing EHC in the space sion of a manageable health condition that would otherwise defined as the circulatory system . In certain embodiments , become exacerbated , using a treatment. the concentration may be defined as the number of free f0535 ] “ Degrading ” is defined as the process in which a ( unbound ) entities in a given volume. In other embodiments , target is either directly , or indirectly , reduced , inactivated , the concentration may be defined as the total number of decomposed , deconstructed , lysed , dissolved , broken , less entities in a given volume. ened , impaired , weakened , deteriorated , diminished , or par [ 0531] The term “ complementarity determining region titioned . ( CDR ) ” used herein refers to an amino acid sequence found [0536 ] “ Different polypeptide origin ” refers to the organ in the variable region of a heavy chain or a light chain of an ism or species from which a genetic sequence encoding the immunoglobulin . The CDRs determine the specificity of an polypeptide , the polypeptide , or portion thereof, is sourced . antibody and may provide a contact residue for binding to a In certain embodiments , a fusion comprising polypeptides of specific epitope of an antigen . The heavy chain and the light different polypeptide origin may include an exogenous anti chain may respectively include three CDRs (CDRHI , gen polypeptide that is encoded by the genetic sequence for CDRH2, and CDRH3, and CDRL1 , CDRL2, and CDRL3) . human adenosine deaminase and the genetic sequence for Four framework regions, which havemore highly conserved phenylalanine hydroxylase from chromobacterium viola amino acid sequences than the CDRs, separate the CDR ceum . regions in the VH or VL . [0537 ] A " domain ” is a part of a polypeptide, such as an [ 0532 ] A “ complex ” as used herein comprises an associa exogenous antigen polypeptide that is generally having a tion of two or more entities . A complex may comprise one 3 -dimensional structure and may exhibit a distinct activity , or more polypeptides , nucleic acid , lipids, carbohydrates, function , such as, e . g . , a catalytic , an enzymatic , a structural inorganic compounds, organic compounds, and the like . A role , or a binding function . complex can be functional (multiunit polypeptides ) or non [0538 ] By an “ enriched population of cells ” it is meant a functional ( e . g ., aggregates or precipitates ) and may have population of cells that is substantially comprised of a beneficial or detrimental properties ( e . g ., immune com particular cell of interest . In an enriched population , 50 % or plexes) . Complexes may be naturally occurring or may be more of the cells in the population are the cells of interest , man -made or synthetic . Synthetic complexes include higher e . g ., 50 % , 60 % , 70 % , usually 80 % , 85 % , 90 % , more usually order entities, e . g ., subcellular structures and cells if they 92 % , 95 % , 96 % , 97 % , 98 % , or 99 % , sometimes as much as comprise a synthetic compound or molecule . 100 % of the cells in the population . The separation of cells [0533 ] As to amino acid sequences, one of skill will of interest from a complex mixture or heterogeneous culture recognize that individual substitutions , deletions or addi of cells may be performed by any convenient means known tions to a nucleic acid , peptide, polypeptide, or protein in the art , for example , by affinity separation techniques such sequence which alters, adds or deletes a single amino acid or as magnetic separation using magnetic beads coated with an a small percentage of amino acids in the encoded sequence affinity reagent, affinity chromatography , or “ panning” with is a “ conservatively modified variant” where the alteration an affinity reagent attached to a solid matrix , e .g ., plate , or results in the substitution of an amino acid with a chemically other convenient technique . Other techniques providing similar amino acid . As used herein the term “ conservative accurate separation include fluorescence activated cell sort amino acid substitution ” is illustrated by a substitution ers , which can have varying degrees of sophistication , such among amino acids within each of the following groups: ( 1 ) as multiple color channels , low angle and obtuse light glycine , alanine , valine , leucine , and isoleucine , ( 2 ) phenyl scattering detecting channels, impedance channels , etc . The alanine , tyrosine , and tryptophan , ( 3 ) serine and threonine , cells may be selected against dead cells by employing dyes ( 4 ) aspartate and glutamate , ( 5 ) glutamine and asparagine, associated with dead cells . Any technique may be employed and (6 ) lysine , arginine and histidine . which is not unduly detrimental to the viability of the desired [ 0534 ] “ Decrease , ” in the context of a symptom of a cells. treated disease , disorder or condition , refers to a reduction in [0539 ] “ Enucleation ” is the rendering of a cell to a non measurable or conveyable parameters associated with the replicative state , either through inactivation or removal of disease or condition that manifest as symptoms. Examples the nucleus . of measurable parameters are a reduction in the subject' s [0540 ] An “ epitope ” includes any segment on an antigen body temperature , a reduction in the concentration of targets to which an antibody or other ligand or binding molecule in a sample taken from the subject, reduction in the intensity binds . An epitope may consist of chemically active surface of inflammation or size of an inflamed area, reduction in the groupings of molecules such as amino acids or sugar side number of infiltrating cells , reduction in the number of chains and usually have specific three dimensional structural episodes associated with the disease , disorder or condition , characteristics , as well as specific charge characteristics. In increase /decrease in organ size , weight gain / loss , etc . some embodiments , exogenous antigens comprise specific Examples of conveyable parameters are, e .g ., the subject' s epitopes . In some embodiments , targets comprise specific own assessment of well being and quality of life . For epitopes . US 2018 /0271910 A1 Sep . 27 , 2018 46

[0541 ] “ Erythroid cells ” as used herein , include nucleated antigen polypeptide occurs , e .g ., in embodiments in which red blood cells , red blood cell precursors , and enucleated red the exogenous antigen is overexpressed as compared to the blood cells and those listed in Table Al. For example , the expression of a naturally occurring polypeptide in an erythroid cells are a cord blood stem cell , a CD34 + cell , a unmodified cell. In some embodiments , the polypeptide hematopoietic stem cell (HSC ) , a spleen colony forming exogenous antigen is expressed from an exogenous nucleic ( CFU - S ) cell , a common myeloid progenitor (CMP ) cell , a acid . In some embodiments , the exogenous antigen is iso blastocyte colony - forming cell, a burst forming unit - eryth lated from a source and loaded into or conjugated to an roid (BFU - E ) , a megakaryocyte - erythroid progenitor (MEP ) exogenous antigen - expressing EHC . The term " exogenous " cell , an erythroid colony -forming unit (CFU - E ) , a reticulo when used in the context of nucleic acid includes a transgene cyte , an erythrocyte , an induced pluripotent stem cell and recombinant nucleic acids. ( iPSC ) , a mesenchymal stem cell (MSC ) , a polychromatic normoblast , an orthochromatic normoblast , or a combina [ 0546 ] As used herein , the term " expression ” or “ express tion thereof. In some embodiments , the erythroid cells are ing ” refers to the process to produce a polypeptide , such as immortal or immortalized cells . For example , immortalized an exogenous antigen polypeptide including transcription erythroblast cells can be generated by retroviral transduction and translation . Expression may be, e . g . , increased by a of CD34 + hematopoietic progenitor cells to express Oct4 , number of approaches , including : increasing the number of Sox2 , K1f4 , Myc , and suppress TP53 ( e . g . , as described in genes encoding the polypeptide , increasing the transcription Huang et al . , Mol Ther 2013 , epub ahead of print September of the gene ( such as by placing the gene under the control of 3 ) . In addition , the cells may be intended for autologous use a constitutive promoter ), increasing the translation of the or provide a source for allogeneic transfusion . Erythroid gene , knocking out of a competitive gene , or a combination cells can be contacted with an exogenous antigen to generate of these and / or other approaches. The term “ expression " or an exogenous antigen - expressing EHC . Erythroid cells com “ expressing ” also include EHCs that comprise an exogenous prising an exogenous antigen are one example of an exog polypeptide that was at one time actively expressed by the EHC but active expression (defined as transcription and enous antigen -expressing EHC . In some embodiments, translation ) since has ceased . For example , the exogenous erythroid cells are cultured . In some embodiments , erythroid antigen polypeptide was actively expressed ( i . e . transcribed progenitor cells are contacted with an exogenous antigen to and translated ) by an EHC prior to the enucleation event and generate an exogenous antigen -expressing EHC . the antigen polypeptide is retained by the EHC after enucle [ 0542 ] As used herein , the term “ thromboid cell” refers to ation but no longer actively expressed , e . g . for lack of a cell of the stem cell- megakaryocyte - platelet lineage , encoding nucleic acid . For example , the EHC may comprise including for example megakayrocytes and platelets, or cells an exogenous antigen polypeptide encoded by an exogenous that are induced to differentiate by thrombopoietin , or cells nucleic acid . During enucleation the exogenous antigen that express surface markers associated with this lineage , for polypeptide is retained by the EHC whereas the exogenous example CD41 (GP IIb / IIIa ), CD42a (GPIX ) , CD42b nucleic acid is not retained , such EHC is said to be “ antigen (GPIb ), and CD61 (avb3 , vitronectin receptor ), PAC - 1 (acti expressing ” or “ expressing antigen ” even in the event that vated IIb / IIIa ) , CD62P ( P - selectin ) , CD31 (PECAM ) and the active expression ( transcription and translation ) of the CD63 . antigen polypeptide is effectively terminated and /or the EHC [0543 ] As used herein , the term “ enucleated hematopoietic does not contain a substantial amount of a replicating cell” ( EHC ) refers to a hematopoietic cell , human or non nucleic acid . human , that is or has been rendered enucleated as defined herein . This definition encompasses the both " erythroid [0547 ] A " functional” exogenous antigen or exogenous cells ” and “ thromboid cells ” as defined herein . antigen - expressing EHC exhibits a desired or specified [0544 ] As used herein , the term “ excipient” refers to an activity or characteristic , including enzymatic , catalytic or inert substance added to a pharmaceutical composition to metabolic activity , structural integrity , immunogenic further facilitate administration of a compound . Examples of complementarity, target binding, and correct localization or excipients include , but are not limited to , calcium carbonate , is capable of promoting a desired or specified effect or calcium phosphate , various sugars and types of starch , phenotype . cellulose derivatives , gelatin , vegetable oils , anticoagu [0548 ] “ Fusion or chimera ” is a polypeptide sequence, or lants , and polyethylene glycols. corresponding encoding nucleotide sequence , that is derived 0545 . The term " exogenous ” as used herein means a from the combination of two or more sequences that are not cellular component or function that is generated by a car found together in nature . This may be a combination of bohydrate , polysaccharide, lipid , oligonucleotide or poly separate sequences derived from separate genes within the peptide not found naturally within a cell or the enhancement same genome, or from heterologous genes derived from or manipulation of a cellular component or function that is distinctly different species' genomes . endogenous to a cell, including , e . g ., a fusion protein com prising an exogenous polypeptide antigen and an endog [0549 ] “ Genetic material” refers to nucleic acid molecules enous protein or a functional fragment thereof. The exog having nucleotide sequences of adenosine , thymine , uracil , enous antigen , including an exogenous antigen polypeptide cytosine, and guanine capable of encoding a gene . is " exogenous” or “ heterologous ” , thus it may either not [0550 ] The term “ heavy chain ” used herein is understood naturally exist, such as a fusion or chimera comprising to include a full -length heavy chain including a variable domains of different polypeptide or species origin , it may region ( VH ) having amino acid sequences that determine not naturally occur in a naturally occurring cell , such as an specificity for antigens and a constant region having three unmodified erythrocyte or platelet, it may not function in the constant domains (CH1 , CH2, and CH3) , and fragments same way as a naturally occurring polypeptide would , or it thereof . In addition , the term “ light chain ” used herein is may not naturally occur in the quantity that the exogenous understood to include a full- length light chain including a US 2018 /0271910 A1 Sep . 27 , 2018 47

variable region (VL ) having amino acid sequences that [0559 ] As used herein , the term “ modulate ," “modulat determine specificity for antigens and a constant region ing ” , “ modify , ” and / or “ modulator” generally refers to the ( CL ) , and fragments thereof. ability to alter , by increase or decrease , e . g ., directly or [0551 ] The term “ homolog ” indicates polypeptides , indirectly promoting / stimulating /upregulating or interfering including exogenous antigen polypeptide that have the same with /inhibiting / downregulating a specific concentration , or conserved residues at a corresponding position in their level, expression , function or behavior, such as, e . g ., to act primary , secondary or tertiary structure . Functional as an antagonist or agonist. In some instances a modulator homologs include exogenous antigens and other polypep may increase and /or decrease a certain concentration , level , tides that exhibit similar function and / or specificity ( e. g. , for activity or function relative to a control, or relative to the a particular target) . average level of activity that would generally be expected or [0552 ] A naturally occurring intact antibody, or immuno relative to a control level of activity . globulin , includes four polypeptides : two full- length light [0560 ] “Membrane ” as used herein is a boundary layer chains and two full - length heavy chains , in which each light that separates an interior space from an exterior space chain is linked to a heavy chain by disulfide bonds . Each comprising one or more biological compounds, typically heavy chain has a constant region and a variable region . lipids, and optionally polypeptides . Membranes can be lipid Similarly , each light chain has a constant region and a bilayers . In certain embodiments , membranes comprise one variable region . There are five heavy chain classes ( iso or more of phosphatidylcholine, sphingomyelin , lysophos types ) : gamma ( V ) , mu ( u ) , alpha ( a ) , delta ( d ) , or epsilon phatidylcholine , phosphatidylethanolamine , phosphatidyl ( c ) , and additionally several subclasses gamma 1 (y1 ) , serine, phosphatidylinositol, or phosphatidic acid . In some gamma 2 ( y2 ) , gamma 3 ( Y3 ) , gamma 4 (14 ) , alpha 1 ( al ) , and embodiments , membranes comprise one or more polypep alpha 2 (a2 ) . The light chain constant region can be either tides such as ankyrin and coenzyme Q10 . Included in the kappa ( K ) or lambda ( a ) type . The variable regions differ in definition ofmembrane are cell membranes comprising, e .g ., sequence among antibodies and are used in the binding and a phospholipid bilayer and cell membrane associated poly specificity of a given antibody to its particular antigen . peptides . [0561 ] The phrase “ nucleic acid molecule” refers to a 10553 ]. As used herein , the term " increase ," " enhance, " single or double - stranded polymer of deoxyribonucleotide " stimulate , " and /or " induce ” (and like terms) generally or ribonucleotide bases . It includes chromosomal DNA and refers to the act of improving or increasing , either directly or self- replicating plasmids, vectors , mRNA , TRNA, siRNA , indirectly, a concentration , level, function , activity , or etc . which may be recombinant and from which exogenous behavior relative to the natural, expected , or average , or polypeptides may be expressed when the nucleic acid is relative to a control condition . introduced into a cell. [0554 ] As used herein , the term “ inhibit, ” “ suppress, " [0562 ] Orthologs are defined as genes in different species " decrease, ” “ interfere ,” and /or " reduce” ( and like terms) that evolved from a common ancestral gene by speciation . generally refers to the act of reducing , either directly or [ 0563 ] The term “ pharmaceutically - acceptable ” and indirectly , a concentration , level, function , activity , or grammatical variations thereof, refers to compositions , car behavior relative to the natural, expected , or average , or riers, diluents and reagents capable of administration to or relative to a control condition . upon a subject without the production of undesirable physi [ 0555 ] A “ library ” as used herein includes a collection of ological effects to a degree that would prohibit administra nucleic acid molecules ( e. g ., DNA , RNA ) having diverse tion of the composition . For example , " pharmaceutically nucleic acid sequences , a genetically diverse collection of acceptable excipient" includes an excipient that is useful in clones , a collection of diverse polypeptides, a diverse col preparing a pharmaceutical composition that is generally lection of cells , such as EHCs , etc . safe, non - toxic , and desirable , and includes excipients that [0556 ] As used herein , “ a mammalian subject includes all are acceptable for veterinary use as well as for human mammals , including without limitation , humans, domestic pharmaceutical use. Such excipients can be solid , liquid , animals ( e. g ., dogs, cats and the like ), farm animals ( e .g ., semisolid , or, in the case of an aerosol composition , gaseous . cows, sheep , pigs , horses and the like ) and laboratory 10564 ]. As used herein , the term “ pharmaceutically accept animals ( e . g . , monkey , rats , mice , rabbits, guinea pigs and able carrier ” includes any of the standard pharmaceutical the like ). The terms " individual, ” “ subject, " " host, " and carriers , such as a phosphate buffered saline solution , water, “ patient, ” are used interchangeably herein and refer to any emulsions such as an oil/ water or water /oil , and various mammalian subject for whom diagnosis , treatment, or types of wetting agents . The term also encompasses any of therapy is desired , particularly humans . The methods the agents approved by a regulatory agency of the US described herein are applicable to both human therapy and Federal government or listed in the US Pharmacopeia for veterinary applications . In some embodiments , the subject is use in animals , including humans, as well as any carrier or a mammal, and in other embodiments the subject is a human . diluent that does not cause significant irritation to a subject [0557 ] “ Medical device” refers to any device , apparatus or and does not abrogate the biological activity and properties machine used to deliver a dose of an exogenous antigen of the administered compound . expressing EHC and / or a therapeutic agent. This includes [0565 ] Some agents may be administered as “ pharmaceu containers , bottles , vials , syringes, bags , cartridges, cas tically acceptable salt ” , e . g . , prepared from inorganic and settes, magazines, cylinders, or canisters . organic acids . Salts derived from inorganic acids include [ 0558 ) “Medical kit” refers to a packaged unit that hydrochloric acid , hydrobromic acid , sulfuric acid , nitric includes a medical device or applicator , an appropriate acid , phosphoric acid , and the like. Salts derived from dosage of exogenous antigen -expressing EHC , optionally organic acids include acetic acid , propionic acid , glycolic including a therapeutic agent , and relevant labeling and acid , pyruvic acid , oxalic acid , malic acid , malonic acid , instructions. succinic acid , maleic acid , fumaric acid , tartaric acid , citric US 2018 /0271910 A1 Sep . 27 , 2018 48 acid , benzoic acid , cinnamic acid , mandelic acid , methane [ 0570 ] A “ exogenous antigen ,” as used herein , is an entity sulfonic acid , ethanesulfonic acid , p - toluene -sulfonic acid , capable of interacting with a target, e . g ., to associate with or salicylic acid , and the like . Salts can also be prepared from bind to a target . An exogenous antigen can comprise or can inorganic and organic bases. Salts derived from inorganic consist essentially of a polypeptide . In some embodiments , bases , include by way of example only , sodium , potassium , the exogenous antigen comprises a polypeptide , a carbohy lithium , ammonium , calcium and magnesium salts . Salts drate , a nucleic acid , a lipid , a small molecule , or a combi derived from organic bases include , but are not limited to , nation thereof. In embodiments in which an exogenous salts of primary , secondary and tertiary amines . Any ordi antigen is a naturally occurring compound or molecule , the nary skilled person in the art will know how to select a antigen is “ exogenous ” in the sense that it is an exogenous proper pharmaceutically acceptable carrier , a pharmaceuti or heterologous compound or molecule with regard to its cally acceptable salt thereof for implementing this invention presence in the EHC . In other embodiments the antigen is without undue experimentation . “ exogenous ” in the sense that it is a man -made compound or [0566 ] As used herein , the term “ pharmaceutical compo molecule , such as a fusion or chimera , a non -naturally sition ” refers to one or more of the compounds described occurring polypeptide , carbohydrate , nucleic acid , lipid , or herein , such as, e .g ., an exogenous antigen - expressing EHC combination thereof, or a man -made small molecule or other mixed or intermingled with , or suspended in one or more therapeutic agent. For example , the exogenous antigen may other chemical components , such as physiologically accept comprise a fusion or chimera comprising one or more of an able carriers and excipients . One purpose of a pharmaceu S domain , an A domain and a U domain . The S domain is a tical composition is to facilitate administration of a com surface domain exposed to the environment around the pound to a subject . EHC , such as the circulatory system of a subject. The A [ 0567] Certain embodiments provide various polypeptide domain is an anchor domain that attaches the S domain to molecules having sequences associated with a desired func the cell membrane of the EHC . The U domain faces the tion or activity, such as exogenous antigen polypeptides. A unexposed side of or is located within ( i . e . in the intracel polypeptide is a term that refers to a chain of amino acid lular space of ) the EHC . Irrespective of any domains , an residues , regardless of post- translational modification ( e . g ., exogenous antigen may be located on the surface of the phosphorylation or glycosylation ) and /or complexation with exogenous antigen - expressing EHC or may be located additional polypeptides , synthesis into multisubunit com within the EHC . The exogenous antigen may be associated plexes , with nucleic acids and/ or carbohydrates, or other with the membrane of the exogenous antigen -expressing molecules . Proteoglycans therefore also are referred to EHC , e . g . , the exogenous antigen is anchored in , conjugated herein as polypeptides . In certain embodiments, the exog to or otherwise bound to the membrane. In some embodi enous antigen -expressing EHC comprises a polypeptide ments, the exogenous antigen may be conjugated to the exogenous antigen . In certain embodiments , the exogenous membrane of the exogenous antigen - expressing EHC by antigen - expressing EHC comprises one or more non - exog chemical or enzymatic conjugation . In other embodiments , enous antigen polypeptides that are optionally membrane the exogenous antigen is not conjugated to the membrane . In associated . some embodiments , the exogenous antigen is not associated [ 0568 ] The term “ pharmaceutically active agent” or “ phar with the membrane of the exogenous antigen -expressing maceutical agent” is defined as any compound , e. g ., a small EHC and is located within the membrane - encapsulated molecule drug, or a biologic ( e . g . , a polypeptide drug or a intracellular space of the EHC . In some embodiments , an nucleic acid drug ) that when administered to a subject has a exogenous antigen located within the intracellular space of measurable or conveyable effect on the subject , e . g ., it the EHC does not substantially diffuse out of the EHC and /or alleviates or decreases a symptom of a disease , disorder or may not permeate the membrane . In other embodiments, the condition . In some embodiments , the pharmaceutical agent exogenous antigen may substantially diffuse out of the EHC may be administered prior to , in combination with , or and / or may permeate the membrane . In some embodiments, following the delivery of an exogenous antigen - expressing the exogenous antigen is loaded , e . g . , introduced into or put EHC . In some embodiments , the pharmaceutically active onto the EHC . An exogenous antigen that is loaded is not agent exerts a synergistic treatment effect with the exog biologically synthesized by the exogenous antigen - express enous antigen -expressing EHC . In some embodiments , the ing EHC . An exogenous antigen suitable for loading may be pharmaceutically active agent exerts an additive treatment e . g ., produced in a cell -based expression system , isolated effect with the exogenous antigen -expressing EHC . from a biological sample , chemically or enzymatically syn [ 0569 ] A “ promoter ” is defined as an array of nucleic acid thesized , and then loaded into or onto the EHC . In some control sequences that direct transcription of an operably embodiments , the exogenous antigen may be further modi linked nucleic acid . Promoters include necessary nucleic fied by the exogenous antigen - expressing EHC after load acid sequences near the start site of transcription . A promoter ing . In other embodiments , the exogenous antigen is not also optionally includes distal enhancer or repressor ele modified after loading . In some embodiments , the exog ments . A “ constitutive” promoter is a promoter that is active enous antigen polypeptide is not loaded onto or into the under most environmental and developmental conditions . EHC . In some embodiments , the exogenous antigen is made, An “ inducible " promoter is a promoter that is active under e . g . , biologically synthesized by the exogenous antigen environmental or developmental regulation . The term “ oper expressing EHC . Typically an exogenous antigen polypep ably linked ” refers to a functional linkage between a nucleic tide is expressed by the exogenous antigen - expressing EHC acid expression control sequence (such as a promoter , or from an exogenous nucleic acid molecule (e . g ., a DNA or array of transcription factor binding sites ) and a second mRNA ) that was introduced into the EHC . The exogenous nucleic acid sequence , wherein the expression control antigen may have a biological function that is retained when sequence directs transcription of the nucleic acid corre the antigen is expressed on the EHC . The exogenous antigen sponding to the second sequence . may bind to and / or sequester a target . Alternatively or in US 2018 /0271910 A1 Sep . 27 , 2018 49 addition the exogenous antigen may exhibit a catalytic present in a particular space if it is barely detectable but only activity toward the target, e . g. , the exogenous antigen may in non - functional quantities or minute quantities that do not convert or modify the target, or may degrade the target. A cause or change a phenotype . In other embodiments , an product may then optionally be released from the exogenous entity may not be substantially present in a particular antigen . population if it is present and can be detected only in a small [ 0571 ] “ Residency ” of an exogenous antigen - expressing number of constituents making up the population , e . g . , less EHC refers to the period of time it spends in a physiological than 10 % , 9 % , 8 % , 7 % , 6 % , 5 % , 4 % , 3 % 2 % or less than location . The specific location of the exogenous antigen 1 % , 0 . 5 % , 0 . 1 % of constituents of the population . For expressing EHC may change during its lifetime and “ resi example , an exogenous nucleic acid may not be retained dency ” applies to the period of time spent in various upon enucleation , the cell is rendered non -replicative , and environments , including vascular circulation , peripheral tis the enucleated cell is incapable of continued expression of sues, capillaries, digestive system , pulmonary system , nasal the exogenous antigen polypeptide encoded by the exog tissues , epidermal surface , and interstitial tissue . In specific enous nucleic acid . The loss of the ability of the cell to embodiments , the exogenous antigen - expressing EHC continue to significantly translate the exogenous polypeptide resides in the circulatory system of a subject. “ effectively terminates ” protein expression . In certain [0572 ] “ Replicating nucleic acid ” refers to deoxyribo embodiments , the exogenous antigen - expressing EHC is nucleic acid (DNA ) that is capable of being copied by substantially incapable of self - replication , e . g ., the replica enzymes dedicated to the increasing the number of copies of tion of nucleic acids. For example , the exogenous antigen the DNA . Usually , DNA replication leads to the production expressing EHC does not substantially incorporate a nucleo of two identical replicas from one original DNA molecule . side if contacted with labeled nucleoside, such as thymidine, DNA replication comprises the incorporation of nucleotides in an incorporation assay. In some embodiments, the exog into a growing DNA strand by DNA polymerase matched to enous antigen -expressing EHC does not contain a substan the template strand one at a time via the creation of phos tial amount of self - replicating nucleic acids. The term “ sub phodiester bonds. stantial identity ” of polynucleotide or nucleic acid sequences [ 0573] “ Sequestering” is defined as cloistering, occluding , means that a polynucleotide comprises a sequence that has separating , segregating , hiding , insulating , or isolating of a at least 25 % sequence identity . Alternatively , percent iden target and preventing it from freely interacting with its tity can be any integer from 25 % to 100 % . More preferred environment. embodiments include at least : 25 % , 30 % , 35 % , 40 % , 45 % , [0574 ] “ Specifically binding” or “ specifically interacting ” , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , or as used herein , describes any interaction between two enti 99 % compared to a reference sequence using the programs ties ( e . g . , a target with an exogenous antigen , such as an described herein ; preferably BLAST using standard param antibody with an antigen , a receptor with a ligand , an eters. enzyme with a substrate , biotin with avidin , etc . ) that is [0576 ] “ Synthetic ” refers to a compound ormolecule that saturable , often reversible and so competitive , as these terms is either man - made and non -naturally occurring , or if it is are understood by those of ordinary skill in the chemical and naturally occurring is placed in a context or location that it biochemical arts . e . g . , Specific binding involving biological would not naturally exist, or if it naturally exists in the molecules such as , e . g . , proteins, peptides and nucleic acid context or location is in a state of purity , or is present in an occurs when one member of the binding pair has a site with amount, concentration or number that it would not naturally a shape and distribution of charged , polar , or hydrophobic be present in the context or location . Synthetic entities can moieties such that the interaction of the cognate ligand with be isolated or purified compounds that are optionally chemi that site is characterized by favorable energetics ( i . e ., a cally or enzymatically modified from their natural state , negative free energy of binding ). The specificity of the exogenous nucleic acids , exogenous (heterologous ) exog interaction may be measured or expressed as a binding enous antigens, and the like . The presence of a synthetic constant (Kd ) . The Kd may range from a mM range to a fM compound or molecule , as defined herein , in any entity range , including pM ranges , uM ranges and nM ranges . renders the entire entity " synthetic ” . For example , a cell Typical Kd values are below about 10 - 6 M , below about comprising an exogenous antigen is a synthetic cell . 10 - 7 M , below about 10 - 8 M , and in some embodiments [0577 ] A “ target ," as used herein , is an entity capable of below about 10 - 9 M . interacting with an exogenous antigen , e . g . , to associate with [0575 ] As used herein , the term “ substantially ” or “ sub or bind to an exogenous antigen . A “ target " includes , but is stantial” refers, e . g ., to the presence , level, or concentration not limited to a polypeptide ( e . g . , an antibody or antibody of an entity in a particular space, the effect of one entity on related polypeptide , a complement constituent, an amyloid another entity , or the effect of a treatment . For example , an protein , a pathogen , a toxin , a prion ), a molecule ( e . g . , a activity, level or concentration of an entity is substantially metabolite , a steroid , a hormone , a carbohydrate ; an oli increased if the increase is 2 - fold , 3 - fold , 4 - fold , 5 - fold , gosaccharide ; a chemical ; a polysaccharide, a DNA ; an 10 - fold , 50 - fold , 100 - fold , or 1000 - fold relative to a base RNA ; a lipid , an amino acid , an element, a toxin or patho line . An activity , level or concentration of an entity is also gen ) , a complex ( e . g . , an immune complex ) , or a cell ( e . g . , substantially increased if the increase is 5 % , 10 % , 20 % , a cancer cell , a macrophage , a bacterium , a fungus, a virus , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , 100 % , 200 % , or or a parasite ) . A target is intended to be detected , diagnosed , 500 % relative to a baseline. An entity may be substantially impaired , destroyed or altered ( e . g . , functionally comple present in a particular space if it can be detected by methods mented ) by the methods provided herein . The specific target known in the art . An entity may not be substantially present may occur free or is associated with other entities in the in a particular space if it is present at levels below the limit circulatory system of a subject . of detection for assays and methods known in the art. In [0578 ] A “ target self- antibody, " as used herein , is a self some embodiments, an entity may not be substantially antibody associated with an autoimmune disease . Such US 2018 /0271910 A1 Sep . 27 , 2018 50 self- antibodies may be detected and analyzed using antibody sensitivity . The skilled artisan will be able to determine binding tests involving contacting the subject' s antibodies to appropriate dosages depending on these and other factors . samples of the subject' s own tissue, usually thyroid , stom The compositions can also be administered in combination ach , liver , and kidney tissue . Antibodies binding to the " self " with one or more additional therapeutic compounds. A tissue (comprising self -antigens ) indicate an autoimmune desirable dosage of the pharmaceutical composition may be disorder. in the range of about 0 .001 to 100 mg/ kg for an adult. In one [0579 ] “ Transgene” or “ exogenous nucleic acid ” refers to example , an intravenous administration is initiated at a dose a foreign or native nucleotide sequence that is introduced which is minimally effective , and the dose is increased over into an EHC . Transgene and exogenous nucleic acid are used a pre - selected time course until a positive effect is observed . interchangeably herein and encompass recombinant nucleic Subsequently , incremental increases in dosage are made acids . limiting to levels that produce a corresponding increase in [0580 ] As used herein , “ treat, ” “ treating, " and / or " treat effect while taking into account any adverse affects that may ment” are an approach for obtaining beneficial or desired appear . Non - limited examples of suitable dosages can range , clinical results , pharmacologic and/ or physiologic effect, for example , from 1x1010 to 1x1014, from 1x1011 to 1x1013, e . g ., alleviation of the symptoms, preventing or eliminating or from 5x1011 to 5x1012 exogenous antigen - expressing said symptoms, and refer to both therapeutic treatment and EHCs of the present invention . Specific examples include prophylactic or preventative treatment of the specific dis about 5x1010 , 6x1010 , 7x1010 . 8x1010 . 9x1010 . 1x1011 , ease , disorder or condition . Beneficial or desired clinical 2x1011 , 3x1011, 4x1011, 5x1011 , 6x1011, 7x1011, 8x1011 , results , pharmacologic and /or physiologic effect include , but 9x1011, 1x101? , or more exogenous antigen -expressing are not limited to , preventing the disease, disorder or con EHCs of the present invention . Each dose of exogenous dition from occurring in a subject that may be predisposed antigen - expressing EHCs can be administered at intervals to the disease , disorder or condition but does not yet such as once daily , once weekly , twice weekly , once experience or exhibit symptoms of the disease (prophylactic monthly , or twice monthly . treatment) , alleviation of symptoms of the disease , disorder [0583 ] “ Unbound ” refers to the state of a target with which or condition, diminishmentof extent of the disease , disorder the exogenous antigen is capable of interacting . An unbound or condition , stabilization ( i. e . , not worsening ) of the dis target is not associated with another entity or an exogenous ease , disorder or condition , preventing spread of the disease , antigen . An unbound exogenous antigen is not associated disorder or condition , delaying or slowing of the disease , with another entity or a target. A target is considered disorder or condition progression , amelioration or palliation “ bound ” once it is associated with the exogenous antigen or of the disease , disorder or condition , and combinations another entity . Unbound targets include soluble forms of the thereof, as well as prolonging survival as compared to target in circulation . Bound targets include targets that are expected survival if not receiving treatment . embedded , associated with , linked to , or otherwise interact [ 0581 ] A " therapeutic agent” or “ therapeutic molecule ” ing with entities in circulation or peripheral tissue . Entities includes a compound or molecule that , when present in an with which a target may interact include circulating cells , effective amount, produces a desired therapeutic effect, peripheral endothelial tissue, immune complexes , glycolip pharmacologic and / or physiologic effect on a subject in need ids , microbes, immunoglobulins , serum albumin , clotting thereof. factors , lipoproteins, and electrolytes. [ 0582 ] The term " therapeutically effective amount" or [ 0584 ] A “ variant” is a polypeptide which differs from the " effective amount" is an amount of an agent being admin original protein by one or more amino acid substitutions , istered to a subject sufficient to effect beneficial or desired deletions , insertions , or other modifications . These modifi clinical results , pharmacologic and /or physiologic effects . cations do not significantly change the biological activity of An effective amount can be administered in one or more the original protein . In many cases , a variant retains at least administrations . An effective amount is typically sufficient 10 % , 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , 95 % , or to palliate , ameliorate , stabilize , reverse , slow or delay the 100 % of the biological activity of original protein . The progression of the disease state. The effective amount thus biological activity of a variant can also be higher than that refers to a quantity of an agent or frequency of administra of the original protein . A variant can be naturally occurring , tion of a specific quantity of an agent sufficient to reasonably such as by allelic variation or polymorphism , or be delib achieve a desired therapeutic and /or prophylactic effect . For erately engineered . example , it may include an amount that results in the [0585 ] The amino acid sequence of a variant is substan prevention of, treatment of, or a decrease in , the symptoms tially identical to that of the original protein . In many associated with a disease or condition that is being treated , embodiments , a variant shares at least 50 % , 60 % , 70 % , e . g ., the disease or medical conditions associated with 80 % , 85 % , 90 % , 95 % , 99 % , or more global sequence autoimmune response , overactive immune activation , or identity or similarity with the original protein . Sequence inhibitory antibody generation for which immune tolerance identity or similarity can be determined using various meth is desired , or the diseases or medical conditions associated ods known in the art, such as Basic Local Alignment Tool with a target polypeptide . The amount of a therapeutic (BLAST ) , dot matrix analysis , or the dynamic programming composition administered to the subject will depend on the method . In one example , the sequence identity or similarity type and severity of the disease and on the characteristics of is determined by using the Genetics Computer Group (GCG ) the individual, such as generalhealth , pathologic conditions, programs GAP (Needleman -Wunsch algorithm ) . The amino diets , age, sex , body weight and tolerance to drugs . It will acid sequences of a variant and the original protein can be also depend on the degree , severity and type of disease . substantially identical in one or more regions, but divergent Further, the effective amount will depend on the methods of in other regions . formulation and administration used , e .g ., administration (0586 ] As used herein , the term “ vector” is a nucleic acid time, administration route , excretion speed , and reaction molecule , preferably self -replicating , which transfers and / or US 2018 /0271910 A1 Sep . 27 , 2018 51 replicates an inserted nucleic acid molecule , such as a reducing agent is removed by size exclusion chromatogra transgene or exogenous nucleic acid into and / or between phy prior to conjugation . Cells are incubated with maleim host cells . It includes a plasmid or viral chromosome into ide -PEG - NHS crosslinker, e . g . SM (PEG ) . ( Thermo Scien whose genome a fragment of recombinant DNA is inserted tific ) for 30 minutes . The NHS group reacts with free amine and used to introduce recombinant DNA, or a transgene, into groups on cell surface antigens . Reacted cells are washed in an EHC . PBS to remove excess crosslinker. Antigen - SH solution is introduced to the cells - plus - crosslinker solution , and the EXAMPLES maleimide - SH reaction is allowed to proceed for 30 min [0587 ] The following examples are offered by way of utes . Cells are pelleted and washed to remove unbound illustration and not by way of limitation . antigen . Example 1 : Culture of Erythroid Cells with Example 4 : Cell Surface Labeling with Sortase Heterologous Gene Expression [0592 ] Cells expressing surface proteinst that contain the [0588 ] CD34 cells are isolated from peripheral blood by Sortase A acceptor sequence , LPXTG ( Seq . ID No. 24 ) , are supermagnetic microbead selection by the use of Mini incubated with 100 uM sortase A in a reaction buffer MACS columns (Miltenyi Biotec ; 94 % + / - 3 % purity ) . The consisting of 50 mM Tris - C1 , pH 7 . 5 , 150 mM NaCl, 1 mM cells are cultured in erythroid differentiation medium (EDM ) CaCl2 , for 4 h at 37° C . 10x molar excess of nucleophile , on the basis of IMDM supplemented with stabilized gluta GGG - antigen , is introduced and the reaction is allowed to mine , 330 ug /mL holo - human transferrin , 10 ug /mL recom proceed at room temperature overnight. Following the reac binant human insulin , 2 IU /mL heparin , and 5 % solvent / tion , excess sortase and unreacted nucleophile is removed by detergent virus - inactivated plasma . The expansion pelleting and washing the cells . procedure comprises 3 steps. In the first step (day 0 to day 7 ) , 10 ^ 4 /mL CD34 cells are cultured in EDM in the presence Example 5 : Cell Surface Labeling with Click of 1 uM hydrocortisone , 100 ng /mL SCF , 5 ng /mL IL - 3 , and Chemistry 3 IU /mL Epo . On day 4 , 1 volume of cell culture is diluted [0593 ] Cells are reacted with Alkyne- NHS Ester to con in 4 volumes of fresh medium containing SCF , IL - 3 , Epo , jugate the alkyne group to exposed primary amines on the and hydrocortisone . In the second step (day 7 to day 11) , the cell surface , per manufacturer' s instructions ( e . g . Glen cells are resuspended at 10 ^ 5 /mL in EDM supplemented Research ) . Excess alkyne -NHS ester is removed by pelleting with SCF and Epo . In the third step (day 11 to day 18 ) , the and washing the cells . Antigen is reacted with Azide- NHS cells are cultured in EDM supplemented with Epo alone . ester to conjugate the azide group to exposed primary Cell counts are adjusted to 7 .5x10 ^ 5 to 1x10 ^ 6 and 5 - 10x amines on the antigen , per manufacturer ' s instructions ( e . g . 10 ^ 6 cells /mL on days 11 and 15 , respectively . Beyond day Thermo Scientific ) . Excess azide -NHS ester is removed by 18 , the culture medium containing Epo is renewed twice a size exclusion chromatography. Alkyne -cells and azide week . The cultures are maintained at 37° C . in 5 % CO2 in antigen are reacted by copper- catalyzed cycloaddition at air. room temperature . [0589 ] The coding sequence of the antigen of interest is placed under the control of an erythroid - specific promoter , Example 6 : Measurement of T Cell Proliferation in e . g . GATA - 1 , and terminated with a poly - A tail , see e . g . a Mouse Repik et al ., Clin Exp Immunol 2005 , 140 : 230 . This [0594 ] CD8 + T cells from OTI (CD45 . 2 + ) mouse spleens sequence is encoded in a lentiviral vector ( e . g . EFI, System are isolated using a CD8 magnetic bead negative selection Biosciences , Inc .) . The vector is produced by standard kit (Miltenyi Biotec ) as per the manufacturer 's instructions . methods from 293T cells . The lentiviral vector is transduced Freshly isolated CD8 + OTI cells are resuspended in PBS and into human hematopoietic progenitor cell , e . g . CD34 + cell labeled with 1 uM CFSE ( Invitrogen ) for 6 min at room or immortalized erythroblast or iPS cell , for example as temperature, and the reaction is quenched for 1 min with an described by Chang et al ., Nat Biotechnol 2006 , 24 : 1017 , equal volume of Iscove ' s modified Dulbecco ' s medium during days 1 -4 of culturing . Subsequent expansion and ( IMDM ) with 10 % ( vol/ vol ) FBS (Gibco ) . Cells are washed , differentiation stages are performed as described above . counted , and resuspended in pure IMDM before injection . A total of 3x10 CFSE - labeled CD8 + OTI cells are injected i. v . Example 2 : Loading of Protein into Erythroid Cell into the tail vein of recipient CD45 . 1 + mice . For short -term by Hypotonic /Hypertonic Cycling proliferation studies , OVA -comprising erythroid cells are [0590 ] Antigen , in this case OVA , is added to a RBC injected 24 h following adoptive transfer. Splenocytes are suspension at a final concentration of 0 . 5 or 5 mg/ ml with a harvested 5 d following antigen administration and stained hematocrit (Hct ) of 70 % . After a hypotonic dialysis process for analysis by flow cytometry . (50 mOsmol/ kg ), RBC are resealed by adding a hypertonic solution ( 1900 mOsmol/ kg ) at 37 C for 30 min . OVA - loaded Example 7 : OVA Antigen Challenge Model in a RBC are washed four times with a 0 . 9 % NaC1 + 0 . 2 % glucose Mouse solution , centrifuged at 1000xg for 10 min at 4 C and [ 0595 ] A total of 3x10 - CFSE - labeled OTI CD8+ T cells adjusted to a Hot of 50 % with plasma or buffer. are injected into CD45 . 1 + recipient mice as described above . At 1 and 6 d following adoptive transfer , mice are i . v . Example 3 : Cell Surface Labeling with administered 100 uL of OVA - comprising erythroid cells into Heterobifunctional Crosslinker the tail vein . At 15 d following adoptive transfer , mice are [0591 ] Antigen with exposed , reduced thiol group (anti challenged with 5 ug of OVA and 25 ng of ultrapure E . coli gen -SH ) is prepared by incubation with 5 mM TCEP . The LPS ( InvivoGen ) in 25 uL of saline injected intradermally US 2018 /0271910 A1 Sep . 27 , 2018 into each rear leg pad (Hock method : total dose of 10 ug of [ 0600 ] 1 . Single Gene Cloning (CR1 ) OVA and 50 ng of LPS ) . Quantification of antigen specific [0601 ] Genes were assembled into expression vectors by B and T cells and serum antibody titer is described below . standard molecular biology methods known in the art . The gene for complement receptor 1 (CR1 ) was synthesized by Example 8 : Quantification of Antigen Specific B a commercial vendor (DNA2 . 0 ) and supplied in a standard and T Cells in a Mouse cloning vector ( PJ series ) . The gene was amplified out of the [0596 ] Mice are killed 4 d following OVA challenge , pJ vector by polymerase chain reaction (PCR ) using oligos described above, and spleen and draining lymph node cells with non -homologous terminal sequences to prepare for are isolated for restimulation . For flow cytometry analysis of insertion into the mammalian expression vector (System intracellular cytokines , cells are restimulated in the presence Biosciences , PM series ) : the upstream oligo consisted of 25 of 1 mg/mL OVA or 1 rig /mL SIINFEKL peptide ( Seq . ID nt homologous to the upstream PM insertion site and 25 nt No . 26 ) (Genscript ) for 3 h . Brefeldin - A ( 5 ug /mL ; Sigma ) homologous to the start of CR1; the downstream oligo is added , and restimulation is resumed for an additional 3 h consisted of 25 nt homologous to the downstream PM before staining and flow cytometry analysis . For ELISA insertion site and 25 nt homologous to the end of CR1. The measurements of secreted factors , cells are restimulated in amplified product was purified by gel electrophoresis ( Qia the presence of 100 ug /mL OVA or 1 ug /mL SIINFEKL gen ). The pM vector was linearized by PCR with tail- to - tail peptide (Seq . ID No . 26 ) for 4 d . Cells are spun , and the oligos homologous to the upstream and downstream inser tion sites and purified by PCR purification (Qiagen ) . The media are collected for ELISA analysis using IFN - y and CR1 amplicon was ligated into the linearized pM vector by IL - 10 Ready - Set -Go kits ( eBioscience ) as per the manufac Gibson assembly, described in detail in Gibson 2011 , Meth turer 's instructions . ods Enzymology Vol 498 , p . 394 . Sequences were confirmed Example 9 : Quantification of Circulating Antibody by Sanger sequencing . Titer [0602 ] 2 . Fusion of Two Genes (Membrane Kell - scFv ) [0603 ] The gene for Kell was purchased as cDNA and [ 0597] OVA - specific serum IgG is detected by incubating supplied in a standard cloning vector (PJ series) . The gene mouse serum at varying dilutions on OVA - coated plates, for an antibody scFv specific to hepatitis B surface antigen followed by a final incubation with goat anti -mouse IgG ( scFv , described in Bose 2003, Molecular Immunology HRP ( Southern Biotech ) . 40 :617 ) was synthesized by a commercial vendor (DNA2 . 0 ) and supplied in a standard cloning vector (PJ series ) . The Example 10 : Extracellular SpyTag - SpyCatcher genes was amplified out of the pJ vectors by polymerase Tolerance Induction chain reaction (PCR ) using oligos with non -homologous [ 0598 ] An expression cassette containing the coding terminal sequences to prepare for insertion into the mam sequence of Kell and Spy Tag is synthesized and inserted in malian expression vector (System Biosciences , PM series ) . the lentiviral vector EF1. A population of CD34 + cells is Kell was amplified with an upstream oligo consisting of 25 transformed with the vector. The expression of Kell - SpyTag nt homologous to the upstream pM insertion site and 25 nt fusion protein is quantified by FACS . Cells that express homologous to the 5 ' terminus of Kell , and a downstream Kell -Spy Tag extracellularly are then placed in a solution of oligo consisting of 25 nt homologous to the 5 ' terminus of Ara h ( 1 -6 ) peptides fused to the SpyCatcher sequence and a scFv and 25 nt homologous to the 3 ' terminus of Kell. scFv cMyc tag . Following incubation , the cells are sorted using was amplified with an upstream oligo consisting of 25 nt FACs to quantify covalent isopeptide conjugation of Kell homologous to the 3 ' terminus of Kell insertion site and 25 SpyTag to SpyCatcher - ArahX -cMyc . The cells are also nt homologous to the 5 ' terminus of scFv , and a downstream hypotonically lysed and the presence of Kell - SpyTag - Spy oligo consisting of 25 nt homologous to the downstream PM Catcher- ArahX - Myc is quantified by Western blot. insertion site and 25 nt homologous to the 3 ' terminus of scFv. The amplified products were purified by gel electro Example 11 : Gene Assembly phoresis ( Qiagen ). The pM vector was linearized by PCR [ 0599] DNA encoding the following genes - glycophorin with tail - to -tail oligos homologous to the upstream and A (Uniprot ID P02724 ) , Kell (Uniprot ID P23276 ) , antibody downstream insertion sites and purified by PCR purification scFv against hepatitis B surface antigen ( Bose et al. 2003 ( Qiagen ) . The Kell and scFv amplicons were ligated into the Mol Immunol 40 ( 9 ) :617 , GenBank ID AJ549501 . 1 ) , linearized pM vector by one - pot Gibson assembly , described adenosine deaminase (Uniprot ID P00813 ) , phenylalanine in detail in Gibson 2011 , Methods Enzymology Vol 498 , p . hydroxylase from Chromobacterium violaceum (GenBank 394 . Sequences were confirmed by Sanger sequencing . ID AF146711 . 1 ) , complement receptor 1 (Uniprot ID [0604 ] 3. Linker - Assembly Between Genes (Kell - scfv ) P17927 ) , CD46 (GenBank : BAA12224 . 1 ), CD55 ( Uniprot [0605 ] The gene for Kell was purchased as cDNA and ID P08174 ), CD59 (Uniprot ID P13987 ), green fluorescent supplied in a standard cloning vector (pJ series ) . The gene protein (Uniprot ID P42212 ), thymidine phosphorylase for an antibody scFv specific to hepatitis B surface antigen (Uniprot ID P19971) , glucocerebrosidase (Uniprot ID ( scFv , described in Bose 2003 , Molecular Immunology P04062) , beta2 glycoprotein 1 (Uniprot ID P02749 ) , phos 40 :617 ) was synthesized by a commercial vendor (DNA2 . 0 ) pholipase a2 receptor (Uniprot ID Q13018 ), collagen alpha and supplied in a standard cloning vector (PJ series ) . The 3 ( IV ) ( Uniprot ID Q01955 ) , serum amyloid P (Uniprot ID genes was amplified out of the pJ vectors by polymerase P02743 ) , lipoprotein lipase (Uniprot ID P06858 ), asparagi chain reaction (PCR ) using oligos with non - homologous nase (Uniprot ID P00805 ) , factor IX (Uniprot ID F2RM35 ) , terminal sequences to prepare for insertion into the mam ADAMTS13 (Uniprot ID Q76LX8 ) — were purchased as malian expression vector (System Biosciences , PM series ) . cDNA from Dharmacon (GE Life Sciences ) or synthesized Kell was amplified with an upstream oligo consisting of 25 de novo by DNA2 . 0 and Genscript. nt homologous to the upstream pM insertion site and 25 nt US 2018 /0271910 A1 Sep . 27 , 2018 53 homologous to the 5' terminus of Kell ; and a downstream the upstream PM insertion site and 25 nt homologous to the oligo consisting of 25 nt homologous to the 5 ' terminus of start of CR1; the downstream oligo consisted of 54 nt scFv , 24 nt encoding a (GlyGlyGlySer ) x2 spacer (Seq . ID homologous to the viral- derived T2A sequence gagggcagag No . 23 ) , and 25 nt homologous to the 3 ' terminus of Kell . gaagtcttctaacatgcggtgacgtggaggsgsstcccggccct (Seq . ID No. scFv was amplified with an upstream oligo consisting of 25 7 ) . The GFP gene was amplified out of the pJ vector by nt homologous to the 3 ' terminus of Kell insertion site , 24 nt polymerase chain reaction (PCR ) using oligos with non encoding a (GlyGlyGlySer ) x2 spacer (Seq . ID No . 23 ), and homologous terminal sequences to prepare for insertion into 25 nt homologous to the 5 ' terminus of scFv ; and a down the mammalian expression vector (System Biosciences, PM stream oligo consisting of 25 nt homologous to the down series ): the upstream oligo consisted of 54 nt homologous to stream PM insertion site and 25 nt homologous to the 3 ' the viral- derived T2A sequence gagggcagaggaagtcttctaacat terminus of scFv . The amplified products were purified by gcggtgacgtggaggsgsstcccggccct ( Seq . ID No. 7 ) and 25 nt gel electrophoresis ( Qiagen ) . The pM vector was linearized homologous to the start of GFP ; the downstream oligo by PCR with tail -to -tail oligos homologous to the upstream consisted of 25 nt homologous to the downstream PM and downstream insertion sites and purified by PCR purifi insertion site and 25 nt homologous to the end of GFP . The cation ( Qiagen ) . The Kell and scFv amplicons were ligated amplified products were purified by gel electrophoresis into the linearized PM vector by one- pot Gibson assembly, ( Qiagen ) . The pM vector was linearized by PCR with described in detail in Gibson 2011 , Methods Enzymology tail- to - tail oligos homologous to the upstream and down Vol 498 , p . 394 . Sequences were confirmed by Sanger stream insertion sites and purified by PCR purification sequencing. ( Qiagen ) . The CR1 and GFP amplicons were ligated [0606 ] 4 . Epitope Tag Attachment (Kell - scFv ) together and into the linearized pM vector by Gibson [ 0607 ] The gene for Kell was purchased as cDNA and assembly , described in detail in Gibson 2011, Methods supplied in a standard cloning vector (PJ series ) . The gene Enzymology Vol 498 , p . 394 . Sequences were confirmed by for an antibody scFv specific to hepatitis B surface antigen Sanger sequencing. ( scFv , described in Bose 2003 , Molecular Immunology 40 :617 ) was synthesized by a commercial vendor ( DNA2 . 0 ) Example 12 : mRNA Assembly and supplied in a standard cloning vector (PJ series ) . The [0608 ] gene of interest is cloned into the multiple genes was amplified out of the pJ vectors by polymerase cloning site of the PSP64 vector ( Promega ) using standard chain reaction ( PCR ) using oligos with non - homologous molecular biology methods . The vector is digested with terminal sequences to prepare for insertion into the mam EcoRI (NEB ) to generate a linearized dsDNA vector con malian expression vector ( System Biosciences, PM series) . taining the SP6 promoter, gene of interest , and 30 nucleotide Kell was amplified with an upstream oligo consisting of 25 long poly - A tail. mRNA is synthesized by reaction with SP6 nt homologous to the upstream pM insertion site and 25 nt RNA polymerase (Promega ) according to manufacturer ' s homologous to the 5 ' terminus of Kell; and a downstream instructions, including recommended concentrations of 5' oligo consisting of 25 nt homologous to the 5 ' terminus of cap analog (ARCA ) to synthesize capped mRNA transcript. scFv, 24 nt encoding a (GlyGlyGlySer )x2 spacer (Seq . ID The reaction mixture is then treated with DNAse to digest No . 23 ) , and 25 nt homologous to the 3 ' terminus of Kell . the template vector (Riboprobe from Promega ) and the scFv was amplified with an upstream oligo consisting of 25 mRNA is purified using the EZNA MicroElute RNA Clean nthomologous to the 3 ' terminus of Kell insertion site , 24 nt Up kit (Omega ). encoding a (GlyGlyGlySer ) x2 spacer (Seq . ID No. 23) , and 25 nt homologous to the 5 ' terminus of scFv ; and a down Example 13: Cell Culture stream oligo consisting of 25 nt homologous to the down stream PM insertion site , the 27 nt sequence tacccctatgacgt [ 0609] 1 . Human Red Blood Cells (RBCs ) gcccgactatgcc (Seq . ID No . 8 ) encoding an HA epitope tag , [ 0610 ] CD34 cells are isolated from peripheral blood by and 25 nt homologous to the 3 ' terminus of scFv . The supermagnetic microbead selection by the use of Mini amplified products were purified by gel electrophoresis MACS columns (Miltenyi Biotec ; 94 % + / - 3 % purity ) . The ( Qiagen ). The pM vector was linearized by PCR with cells are cultured in erythroid differentiation medium ( EDM ) tail - to - tail oligos homologous to the upstream and down on the basis of IMDM supplemented with stabilized gluta stream insertion sites . The downstream primer additionally mine, 330 ug /mL holo -human transferrin , 10 ug /mL recom contained the 27 nt sequence tacccctatgacgtgcccgactatgcc binant human insulin , 2 IU /mL heparin , and 5 % solvent / (Seq . ID No. 8 ) encoding an HA epitope tag . The linearized detergent virus - inactivated plasma. The expansion vector was purified by PCR purification ( Qiagen ) . The Kell procedure comprises 3 steps. In the first step ( day 0 to day and scFv amplicons were ligated into the linearized PM 7 ) , 10 ^ 4 /mL CD34 + cells are cultured in EDM in the vector by one- pot Gibson assembly , described in detail in presence of 1 uM hydrocortisone , 100 ng /mL SCF, 5 ng /mL Gibson 2011, Methods Enzymology Vol 498 , p . 394 . IL - 3 , and 3 IU /mL EPO . On day 4 , 1 volume of cell culture Sequences were confirmed by Sanger sequencing . 5 . Fusion is diluted in 4 volumes of fresh medium containing SCF , of Two Genes (Reporter Assembly ) (GPA - HA ) The genes IL - 3 , EPO , and hydrocortisone. In the second step (day 7 to for complement receptor 1 (CR1 ) and green fluorescent day 11 ) , the cells are resuspended at 10 ^ 5 /mL in EDM protein (GFP ) were synthesized by a commercial vendor supplemented with SCF and EPO . In the third step ( day 11 (DNA2 . 0 ) and supplied in standard cloning vectors (PJ to day 18 ) , the cells are cultured in EDM supplemented with series ). The CR1 gene was amplified out of the PJ vector by EPO alone . Cell counts are adjusted to 7 .5x10 ^ 5 to 1x10 ^ 6 polymerase chain reaction (PCR ) using oligos with non and 5 - 10x10 ^ 6 cells /mL on days 11 and 15 , respectively . homologous terminal sequences to prepare for insertion into Beyond day 18 , the culture medium containing EPO is the mammalian expression vector (System Biosciences, PM renewed twice a week . The cultures are maintained at 37° C . series ): the upstream oligo consisted of 25 nt homologous to in 5 % CO2 in air. US 2018 /0271910 A1 Sep . 27 , 2018 54

[0611 ] 2 . Mouse Red Blood Cells for colony - forming unit -megakaryocyte (CFU -Mk ) , accord [ 0612 ] Methods of culturing mouse erythroid cells from ing to manufacturer ' s instructions (StemCell Technologies, mouse fetal liver erythroid progenitors are known in the art , Vancouver, BC , Canada ) . To assess differentiation , cells are see e . g ., Shi et al . 2014 , PNAS 2014 111( 28 ) : 10131 . stained with antibodies against CD61m CD42b , CD41 , [ 0613] Mouse erythroid progenitors are isolated from fetal CD61, and CD49b by flow cytometry using a FACS -Calibur livers. Fetal livers are purchased from Charles River Labs . (Becton Dickinson ). For cell cycle analysis , cells are rinsed Livers are put in 1 ml PBS on ice . Pipette up and down to with phosphate -buffered saline ( PBS ) , fixed with formalde get a single - cell suspension solution and pass by a 70 um hyde 2 % (Sigma , St Louis , Mo . , USA ) for 5 min and strainer (BD Falcon 35 - 2235 ) . Rinse the mesh with 1 ml permeabilized with 0 . 1 % of Triton X - 100 (Bio - Rad , Hercu PBS . Combine the flow through ( 1 ml per embryo ). Pellet les , Calif ., USA ) . Cells are then marked with mAb -Ki - 67 the cells at 1 . 5 k RPM for 5 min , re - suspend with red cell FITC (BD Bioscience, San Jose , Calif. , USA ), washed and lysis buffer (Ammonium Chloride Solution from Stemcell ) , resuspended in 0 . 5 mL PBS - 1 % fetal bovine serum ( FBS ) and incubate on ice for 10 mins. Pellet the cells at 1 . 5 k RPM 0 . 01 % azide 7 - amino - actinomycin D ( 7 -AAD ) following the for 5 min , remove the lysis buffer, and re - suspend with 10 ml manufacturer 's instructions ( BD Biosciences ) . PBS - 2 % FBS . Add chromPure Rat IgG ( Jackson Immu noResearch , # 012 - 000 - 003 ) at 50 ul/ mouse and incubate at Example 14 : Cell Isolation 4 C for 5 min . Add Biotinylated anti -mouse TER119 (BD [ 0618 ] 1. Primary RBCs Pharmingen , # 553672 ) at (at 1 ul/ 1 * 10 ^ 6 cells ) and incubate [0619 ] Whole blood is collected using aseptic techniques at 4 C for 15 min . Add Ms Lineage Panel ( Fisher Scientific in tubes containing low molecular weight heparin , dalteparin ( Thermo Fisher Scientific ) # BDB559971 ) to the cells at ( 2 sodium ( 9 units /mL blood ) . Blood is centrifuged at 5000xg ul/ 1 * 10 ^ 6 cells ) and incubate at 4 C for 15 min . Washing for 5 minutes and after removal of plasma and buffy coat once with 10x volume of PBS / and Spin the cells with 1 . 5 (both can be retained for later use ) , the erythrocytes are k RPM for 5 min at 4 degree. Add Streptavidin Particles washed twice in cold ( 4 C ) phosphate buffered saline (PBS ) Plus - DM (magnetic beads ) (BD Pharmigen , # 557812 ) (5 with centrifugation . The resultant red blood cell population ul/ 1 * 10 ^ 6 cells ) and incubate at 4 C for 30 min . Prepare 2 - 4 is stored at 4 C in CPDA - 1 anticoagulant or a glycerol FACS tubes on a magnetic holder. Aliquot 2 ml cells into solution for long - term preservation . each tube ( 4 ml in total) , and carefully take the cells out of [0620 ] 2 . Primary Platelets the tube and put into the other tube on the other side avoiding [0621 ] Whole blood (40 ml) is collected in 3 .8 % sodium the disruption of the magnetic stick beads. Repeat the same citrate ( 1 : 9 citrate to blood vol/ vol) from healthy individuals procedure and take the Ter119 negative and linkage negative under an appropriate IRB protocol. Blood is centrifuged at cells to a new tube . Concentrate the cells , and resuspend the 200 g for 15 minutes to isolate platelet- rich plasma (PRP ) . cells with 50 - 100 ul PBS ( 2 % FBS ) . Platelets are then washed in modified Tyrode 's buffer ( con [ 0614 ] Purified erythroid progenitors are cultured in dif taining 138 mM NaCl, 5 . 5 mM dextrose , 12 mM NaHCO3, ferentiation medium comprising ( for 40 mL ): IMDM : 29 ml, 0 . 8 mM CaC12 , 0 . 4 mM MgC12 , 2 . 9 mM KC12 , 0 . 36 mM FBS (Stem Cell ) : 6 ml ( Final 15 % ) , 10 % BSA in IMDM Na2HPO4 and 20 mM Hepes, pH 7 .4 ) in presence of 1 uM ( Stem Cell) : 4 ml ( Final 1 % ) , 10 mg/ml Holo - transferrin : prostaglandin 12 , and resuspended in the same buffer. 2000 ul ( Final: 500 ug /ml ) , 100 * L -Glutamine : 400 ul, 100 * penicillin streptomycin : 400 ul, 10U /ul Epo : 2 ul (Final : 0 .5 Example 15 : Irradiation of Primary or Cultured U /ml ), 10 mg/ ml Insulin : 40 ul ( Final: 10 ug/ ml ) . Culture Cells 2 * 10 ^ 5 cells /ml in the differentiation medium in 24 wells plate at 37 C . After a total culture of 44 -48 hours, analyses 10622 ] Irradiation of a population of exogenous antigen are performed , for example by flow cytometry as performed expressing EHCs can be performed to ensure that they are herein . Enucleated red blood cells are gated out using incapable of replication . Such protocols are similar to those (Hoechst stain ) for differentiation profile analysis . A suc known in the art for irradiating cells , e . g ., primary red blood cessful culture will yield 16 fold increase . cells . Briefly , one unit (350 ml) of whole blood is taken and [0615 ] 3 . Platelets divided into two aliquots of 175 ml each , 10 such units are [0616 ] Donated CD34 + cells are acquired from the Fred thus divided into 20 aliquots . One aliquot ( 175 ml) from Hutchinson Cancer Research Center. The CD34 + enriched each unit of blood is subjected to gamma irradiation of 25 cells are plated in a serum - free medium at 2 -4x10 ^ 4 cells / Gy, and not exceeding 50 Gy, by a self -contained gamma mL and medium refreshment is done on day 4 by adding an cell irradiator (GammaCell 1000 , Theratronics ) . The blood equal volume of media . On day 6 , cells are counted and is then stored at 4 C under conventional blood banking analyzed : 1 . 5x10 ^ 5 cells are washed and placed in 1 mL of conditions. Sampling is done from these 10 irradiated and 10 the same medium supplemented with a cytokine cocktail non - irradiated blood bags on days 0 , 7 , 14 , and 21 with the consisting of TPO 30 ng/ mL , SCF 1 ng /mL , interleukin help of sampling site coupler ( Fenwal, USA ). Tests for cell ( IL ) - 6 7 . 5 ng /mL and IL - 9 13 . 5 ng /mL ] to induce mega proliferation are conducted , including a thymidine incorpo karyocyte differentiation . At day 10 , 1 /2 - 1/ 4 of the suspension ration assay to quantify any mitotic potential. Supernatant is culture is replaced with fresh medium . All cytokines are assayed for free hemoglobin by absorbance spectroscopy, purchased from Peprotech . The cultures are incubated in a and for free lactate dehydrogenase by colorimetric assay humidified atmosphere ( 10 % CO2 ) at 39° C . for the first 6 (Pierce ) to evaluate levels of cell lysis . days of culture and 37° C . for the last 8 days . Viable nucleated cells are counted with a hemocytometer ( 0 . 4 % Example 16 : Enucleation of Erythroid Cells trypan blue ; Invitrogen , Burlington , ON , Canada ) . [0623 ] Erythroid cells are grown to semiconfluence ( 1 to [ 0617] Clonogenic progenitor cells (CPC ) are assayed 4x10 ^ 4 cells per cm2) on 12 -mm diameter coverslips coated using MethoCult H4436 for myeloid CPC , and MegaCult - C with collagen in IMDM medium supplemented with 100 US 2018 /0271910 A1 Sep . 27 , 2018 55

units /mi of penicillin and 100 units /ml of streptomycin . The medium is changed to DMEM / 10 % FBS . The virus super collagen is necessary to prevent all the cells from falling off natant is collected 48 hours post- transfection by centrifuga the coverslip during centrifugation . Cells are grown to tion at 1 , 500 rpm for 5 minutes . The supernatant is collected monolayers (5x104 cells per cm2 ) on coverslips either in the and frozen in 1 ml aliquots at - 80° C . same medium or in Dulbecco ' s modified Eagle ' s medium [0629 ] Target cells are transduced at day 3 - 7 of the culture with 10 % calf serum . It is not necessary to coat the cell process described herein . 5x10 ^ 5 cultured cells are plated in coverslips with collagen . In order to enucleate the cells , the 500 uL of medium containing 20 ug /mL polybrene in a coverslips are inverted (cell side down ) and placed into the 24 -well plate . For each virus, cells are transduced in tripli bottom of 15 -ml Corex centrifuge tubes containing 2 - 5 ml of cate wells . Virus supernatant is added in another 500 uL of medium with 10 g of cytochalasin B per ml. The centrifuge medium and the sample is mixed by pipetting . Infection is tubes with the coverslips are placed immediately into a achieved by spinoculation , spinning the plate at 2000 rpm Sorvall RC - 2 centrifuge that has been warmed to 37 C by for 90 minutes at room temperature. After spinoculation , the spinning the ( SS 34 ) rotor with the head in place for about cells are incubated at 37 C overnight, and the next day 1 mL 1 hr at 10 ,000 rpm (with the temperature regulator set at of fresh IMDM medium with appropriate cytokines is 37 -399 ) . The length of time and speed of centrifugation are added . crucial factors for successful enucleation . Cells are spun at [0630 ] 3 . Nucleic Acid - Cationic Polymer 9000 rpm for 1 hr at 37 + 20 and cells are spun at 6500 rpm [ 0631] An mRNA ecoding the transgene of interest , and for 50 min at 37 + - 20 . After centrifugation , the coverslips are including an upstream promoter sequence and a downstream removed from the centrifuge and placed cell side up into poly A tail , can be purchased from multiple commercial 35 -mm (Falcon ) tissue culture dishes (Biolquest ) containing vendors ( e . g . , IDT- DNA, Coralville Iowa) . RNA transfec 3 ml of medium without cytochalasin B . Within 30 -60 min tions are carried out using RNAIMax ( Invitrogen , Carlsbad , at 370 , the cells are morphologically normal and 90 - 99 % Calif .) or TRANSIT -mRNA (Mims Bio , Madison , Wis .) lacked nuclei. Enucleated cells are removed from the cov cationic lipid delivery vehicles. RNA and reagent are first erslips by treatment with trypsin - EDTA (Grand Island Bio diluted in Opti -MEM basal media ( Invitrogen , Carlsbad , logical Co . ) and the cells are suspended in normal medium . Calif .) . 100 ng /uL RNA is diluted 5x and 5 uL , of RNAIMax The enucleated cells are then replated in small drops on per ug of RNA is diluted 10x . The diluted components are 22 -mm2 coverslips kept in 35 - mm tissue culture dishes and pooled and incubated 15 minutes at room temperature before placed in an incubator . At time intervals after replating , the they are dispensed to culture media . For TRANSIT -mRNA coverslips are mounted on slides ( 12 ) and observations on transfections, 100 ng /uL RNA is diluted 10x in Opti- MEM the enucleates are made with Zeiss phase contrast , polarized and BOOST reagent is added (at a concentration of 2 uL , per light, and Nomarski optics. ug of RNA ) , TRANSIT -mRNA is added ( at a concentration of 2 uL , per ug of RNA ), and then the RNA - lipid complexes Example 17: Contacting of Cells are delivered to the culture media after a 2 -minute incuba [0624 ] 1 . Nucleic Acid - Transfection tion at room temperature . RNA transfections are performed [ 0625 ] The nucleic acid of interest is scaled up to provide in Nutristem xenofree hES media (STEMGENT® , Cam approximately 5 ug nucleic acid per 10 ^ 5 EHCs to be loaded , bridge , Mass .) or Opti -MEM plus 2 % FBS. Successful e . g ., a cell, such as an erythroid cell, a platelet, or a introduction of the mRNA transcript into host cells can be hematopoietic precursor cell. The nucleic acid is diluted in monitored using various known methods , such as a fluores Opti- MEM Medium (Life Technologies ) at a ratio of 1 ug to cent label or reporter protein , such as Green Fluorescent 50 uL medium . The diluted nucleic is then combined with a Protein (GFP ) . Successful transfection of a modified mRNA transfection reagent ( Trans- IT for DNA , Trans- IT mRNA for can also be determined by measuring the protein expression mRNA , Trans - IT siRNA for siRNA , Mirus Bio ) at a 1 : 1 level of the target polypeptide by e . g . , Western Blotting or volume ratio and allowed to form complexes for 5 minutes immunocytochemistry . Similar methods may be followed at room temperature . The nucleic acid complex is added to for large volume scale - up to multi- liter ( 5 - 10 , 000 L ) culture cells for 12 - 24 hours . Optionally , after this period of time, format following similar RNA - lipid complex ratios . the media can be exchanged with fresh media such that the [ 0632] 4 . Nucleic Acid - Electroporation transfection reagents are no longer present. [ 0633] mRNA ecoding the transgene of interest, and [ 0626 ] 2 . Nucleic Acid — Viral Transduction including an upstream promoter sequence and a downstream [0627 ] The gene of interest is cloned into the multiple poly A tail , can be purchased from multiple commercial cloning site of lentivirus vector pCDH with the MSCV vendors ( e . g ., IDT- DNA , Coralville Iowa ). Electroporation promoter sequence from System Biosciences . parameters are optimized by transfecting erythroid lineage [ 0628 ] Lentivirus is produced in 293T cells by transfecting cells with mRNA transcripts and measuring transfection the cells with lipofectamine . 5x10 ^ 6 293T cells ( Lenti - X efficiency by quantitative RT- PCR with primers designed to 293T Cell Line , Clontech catalog # 632180 ) are plated in a specifically detect the exogenous transcripts . For certain P10 petri dish the day before transfection . Cell confluency cells preparations , discharging a 150 uF capacitor into should be around 70 % . One plate is transfected per con 2 .5x10 ^ 6 cells suspended in 50 ul of Opti -MEM ( Invitrogen , struct. 20 ul ( 10 ug ) PPACKH1 (System Biosciences) plas Carlsbad , Calif . ) in a standard electroporation cuvette with mid mix + 2 ug lenti construct + 20 ul Plus reagent ( LifeTech - a 2 mm gap is sufficient for repeated delivery in excess of nologies , Catalog # 11514 - 015 ) are combined in 400 ul 10 , 000 copies of modified mRNA transcripts per cell , as Optimem and incubated 15 min at RT. 30 ul of LF2000 determined using the standard curve method , while main (Life Technologies , Catalog # 11668 -019 ) is diluted into 400 taining high viability ( > 70 % ) . Cell density may vary from ul Optimem , added dropwise to DNA mix , and incubated for 1x10 ^ 6 cell/ 50 ul to a density of 2 .5x10 ^ 6 cells / 500 and 15 min RT. DNA mix is added to cells ( cells are in 9 ml of require from 110V to 145V to transfect cells with similar Optimem ) . Cells are incubated for 6 hours and then the efficiencies measured in transcript copies per cell. Large US 2018 /0271910 A1 Sep . 27 , 2018 56 multi- liter (5 - 10 ,000 L ) electroporation may be performed the desired delivery material, and placed in the device ' s inlet similar to large volume flow electroporation strategies simi reservoir . This reservoir is connected to a compressed air lar to methods described with the above described con line controlled by a regulator , and the selected pressure ( 0 - 70 straints (Li et al. , 2002; Geng et al ., 2010 ) . psi) is used to drive the fluid through the device . Treated [0634 ] 5 . Polypeptide — Liposome cells are then collected from the outlet reservoir. Cells are [ 0635 ] Cells , including primary terminally - differentiated incubated at room temperature in the delivery solution for cells e . g. , erythrocytes , can be loaded with exogenous pro 5 - 20 min after treatment to ensure hole closure before being tein on their surface and in their cytoplasm . The loading of subjected to any further treatment . To deliver fluorescently proteins can be performed using liposomes . labeled phenylalanine ammonia hydroxylase ( PAH ), the [ 0636 ] Lipids ( Pro - Ject reagent, Pierce ) in organic solvent experiments are conducted as described above such that the were dried under nitrogen into a thin film in glass scintil delivery buffer contained 0 . 1 - 0 . 3 mg/ mL PAH . GFP knock lation vial. Approximately 2 uL lipids were used per 1095 down is measured as the percentage reduction in a cell cells . Polyclonal mouse IgG (Abcam ) was labeled with population ' s average fluorescence intensity relative to Dylight -650 ( Pierce ) per manufacturer ' s instructions . Pro untreated controls . tein solution at 0 . 1 mg/ mL in PBS was added to the dried [0641 ] 7 . Polypeptide Surface Conjugation lipid mixture . The solution was pipetted several times , [0642 ] The cell surface is treated with Traut' s reagent incubated for 5 minutes at room temperature , then vortexed ( 2 - iminothiolane HCI, Pierce ) to thiolate primary amines. vigorously to generate encapsulating liposomes . Serum - free Traut' s reagent is dissolved in Tris buffer pH 8 with EDTA medium was added to bring the total volume to 500 UL per to prevent oxidation of sulfhydryls . Approximately 1 umol 10 ^ 5 cells . The liposomal mixture was then incubated with Traut’ s reagent is used to treat 10 ^6 cells . Incubate Traut' s the cells for 3 - 4 hours at 37 C . reagent with cells for 1 hour at room temperature . Remove [ 0637] FIG . 1 shows the loading of an exogenous protein , excess or unreacted reagent by centrifugation and washing in this case fluorescently - labeled IgG , into primary erythro the cells . The number of available sulfhydryl groups can be cytes with liposomes . The loading is measured by flow measured using Ellman ' s Reagent. In the meantime, treat cytometry . The loading is dose -dependent , as 0 . 06 % of cells suitable exogenous antigen polypeptide with amine- to - sulf are fluorescent without liposomes , - 60 % of cells are fluo hydryl crosslinker , such as SMCC ( Pierce ) according to rescent at a low liposome dose , and - 85 % of cells are manufacturer ' s instructions. Excess crosslinking reagent is fluorescent at a high liposome dose . The data in FIG . 1 is removed by desalting . Themaleimide - functionalized protein strong proof that exogenous proteins can be loaded into is then incubated with the thiolated cells for several hours . erythroid cells with liposomes . Unreacted protein is separated from the conjugated cells by [ 0638 ] 6 . Polypeptide Mechanical Disruption centrifugation and washing . [ 0639] Cells may be loaded using a microfluidic device [ 0643] 8 . Polypeptide - Non - Covalent Surface Attach containing 1 um , 2 um , 3 um , 4 um , 5 um , 10 um wide ment channels that transiently porate the cells , allowing a payload [0644 The gene for an antibody scFv against hepatitis B to enter when the cells are pressured through the system . surface antigen ( scFv, described in Bose 2003 , Molecular 10640 ] The silicon -based devices are fabricated at the Immunology 40 :617 ) is fused to a 6 - histidine affinity tag Massachusetts Institute of Technology microfabrication (Seq . ID No. 27) and to the gene encoding the polypeptide facility using photolithography and deep reactive ion etching sequence that binds mouse glycophorin A , HWMVLP techniques . In this process , 6 " silicon wafers with a 450 - um WLPGTLDGGSGCRG (Seq . ID No . 28 ) , in a mammalian thickness are treated with hexamethyldisilazane , spin coated expression vector (Genlantis ) . The full fusion protein is with photoresist (OCG934 ; FujiFilm ) for 60 s at 3 , 000 rpm , produced by transient transfection of HEK - 293T cells using exposed to UV light ( EV1; EVG ) through a chrome mask standard methods and purified on a Ni- NTA affinity resin with theconstriction channel design , and developed in ( Pierce ) according to manufacturer ' s instructions . The puri AZ405 (AZ Electronic Materials ) solution for 100 s . After fied fusion protein is incubated with mouse erythrocytes at 20 min of baking at 90° C . , the wafer is etched by deep > 100 nM concentration to allow for rapid equilibration and reactive ion etching (SPTS Technologies ) to the desired binding of the peptide to glycophorin A . depth (typically 15 um ) . The process is repeated on the [ 0645 ] 9 . Polypeptide - Lipid Insertion into Membrane opposite side of the wafer ( i. e ., the one not containing the [0646 ] Traut ' s reagent ( Thermo Fisher ) is used to generate etched channels ) using a different mask , which contains the sulfhydryl groups on an amine - containing suitable exog access hole patterns, and using a thicker photoresist AZ9260 enous antigen polypeptide molecule following manufactur (AZ Electronic Materials ) . Wet oxidation is then used to er' s protocol. The reaction mixture is incubated for 1 h at grow 100 - 200 nm of silicon oxide before the wafer is room temperature (RT ) on a shaker and washed through a anodically bonded to a Pyrex wafer and diced into individual spin desalting column ( Zeba , MWCO 7K , Thermo Scien devices . Before each experiment, devices are visually tific ) following the manufacturer ' s instructions to remove inspected and mounted onto a holder with inlet and outlet the unreacted Traut' s reagent. The generation of sulfhydryl reservoirs (all designed in - house and produced by Firstcut ) . groups on the modified polypeptide is quantified using These reservoirs interface with the device using Buna - N Ellman ' s Reagent (Pierce ) based on the manufacturer ' s O - rings (McMaster - Carr ) to provide proper sealing . The protocol . inlet reservoir is connected to a home- made pressure regu [0647 ] DSPE - PEG 3400 -mal ( 1x10 ^ - 3M in PBS , 4 ul , lator system using Teflon tubing to provide the necessary molar ratio lipid :Polypeptide = 1 :1 ) (all lipids purchased from driving force to push material through the device . A popu Avanti Polar Lipids and stored as chloroform solution under lation of erythroid cells is first suspended in the desired argon at - 20 C ) are added to the desalted polypeptide delivery buffer [ growth medium , PBS , or PBS supplemented solution and incubated at RT on a shaker. After 1 h , the with 3 % FBS and 1 % F -68 Pluronics (Sigma ) ] , mixed with sample solution is filtered using a centrifugal filter device US 2018 /0271910 A1 Sep . 27 , 2018 57

(Microcon , Millipore Co . ) at 14 000 g for 15 min at 4° C . to solution . Afterwards, the suitable exogenous antigen poly remove the small molecules and suspended in 600 uL PBS peptide was added in Ringer ' s solution ( 1 to 4 uM ) to a final ( 1 mg/mL polypeptide ). volume of 400 ul and incubated for 30 min at room tem [ 0648 ] 200 uL of whole blood is suspended in 1000 uL perature under mild agitation . After incubation , cells were PBS and spun at 1500 g for 30 s , repeated four times. washed twice , resuspended in Ringer and collected for Finally , the RBCs are suspended in 800 uL PBS . The analysis . conjugation of RBC /DSPE -PEG - Polypeptide is prepared by [0657 ] 13 . PolypeptideEnzymatic Conjugation mixing the above RBCs suspensions and various amounts of 10658 ] Cell surface enzymatic conjugations with sortase DSPE - PEG -Polypeptide solution ( 1 mg per mL ) followed are known in the art , see e . g ., Shi et al PNAS 2014 by incubation for 15 - 30 min at 37° C . The mixture is kept 111( 28 ) : 10131 . To label the GPA N terminus with polypep for 5 min at room temperature , then washed three times in tide , 30 uL of 500 M S aureus sortase and 1 mM polypep PBS and resuspended to a final RBC concentration of tide with LPETGG (Seq . ID No . 29 ) at the C terminus is 5x10 ^ 8 per mL . An automated cell counter ( Countess, preincubated in 50 mM Tris pH 7 . 5 , 150 mM NaCl, on ice Invitrogen ) is used to measure the cell concentration . for 15 minutes and added to 5x10 ^7 RBCs in DMEM . The [0649 ] 10 . Polypeptide — Hypotonic Loading sortase and cell mixture is incubated on ice for 30 min with [ 0650 ) A suitable exogenous antigen polypeptide , in this occasional gentle mixing , then spun at 500xg for 2 min at 4 instance mouse IgG , was purchased from Abcam and was C to remove buffer/ DMEM , then washed three times with 1 added at 0 .25 mg/ mL to a RBC suspension in isotonic mL of ice -cold PBS . solution at a hematocrit (Hct ) of 70 % . The suspension was dialyzed in 250 mL of a hypotonic solution containing 10 Example 18 : Assessment of Polypeptide Presence mM sodium phosphate pH 7 . 4 , 10 mM sodium bicarbonate , 10659 ] 1 . Fluorescent Transgene and 20 mM glucose , stirred at 15 rpm for 1 hour at 4 C . The [0660 ] Erythroid cells were cultured as described herein . cells were then isotonically resealed by adding 1 / 10 volume A transgene encoding glycophorin A with an HA tag on the of resealing solution comprising 5 mM adenine, 100 mm C -terminus fused to GFP with an intervening viral T2A inosine, 100 mM sodium pyruvate , 100 mM sodium phos peptide was constructed by Gibson assembly as described phate , 100 mM glucose , 12 % ( w / v ) NaCl at pH 7 . 4 . Cells herein . The transgene was introduced into the erythroid cells were then incubated at 37 C for 30 minutes. by lentiviral transduction as described herein . Two days after [0651 ] 11. PolypeptideCell -Penetrating Peptide transduction , cells were collected , washed in PBS buffer. [ 0652] The manufacture of protamine- conjugated poly and analyzed on a flow cytometer (Attune , Life Technolo peptide is known in the art , see e . g ., Kwon et al . 2009 J gies ) . Transduction efficiency was assessed as the percent Contr Rel 139 ( 3 ) : 182 . 5 mg/ ml of Low Molecular Weight age of GFP -positive cells in the population . Protamine (LMWP ) in 50 mM HEPES buffer ( pH 8 ) is [0661 ] 2 . Cell Surface Proteins mixed with the heterobifunctional cross - linker 3 - ( 2 - pyridyl 10662 ) For cell surface proteins , the level of protein dithio ) propionic acid N -hydroxysuccinimide (SPDP , Sigma expression can be detected as early as 2 days after transfec Aldrich ) at a 1 : 10 molar ratio in DMSO and shaken for 1 h tion by flow cytometry with antibodies specific for the at room temperature . The reaction mixture is then treated protein or for a co -expressed epitope tag . Erythroid cells with 50 mM dithiothreitol (DTT , Sigma - Aldrich ) and the were cultured as described herein . A transgene encoding thiolated LMWP is purified by HPLC on a heparin affinity glycophorin A with an HA tag at the N -terminus was column . The product is collected by ultrafiltration , constructed by Gibson assembly as described herein . The lyophilized, and stored at - 20° C . until further use . transgene was introduced into the erythroid cells by lenti [ 0653] For conjugation , 5 mg/ ml suitable exogenous anti viral transduction as described herein . Two days after trans gen polypeptide is mixed with SPDP ( 40 ul of 0 . 1M SPDP duction , cells were collected , washed in PBS buffer , and in ethanol to 1 ml protein solution ) in phosphate buffer, and stained with 1 : 50 dilution ofmouse anti -HA antibody (Ab stirred at room temperature for 1 h . Unreacted SPDP is cam ) for 1 hr. Cells were washed and then stained with a removed by rapid desalting and buffer exchange by FPLC 1 : 100 dilution of alexa 488 - labeled goat anti -mouse second with 0 . 1M phosphate buffer (pH 7 . 4 ). Activated polypeptide ary antibody (Life Technologies ) for 30 minutes on ice . is then conjugated with a 10 - fold molar excess of the Cells were washed and analyzed on a flow cytometer above- prepared LMWP- SH for 24 h at 4° C . The LMWP ( Attune, Life Technologies ) . Transduction efficiency was polypeptide conjugates are isolated by ion - exchange chro assessed as the percentage of alexa 488 - positive cells in the matography using a heparin affinity column followed by five population . rounds of centrifugal filtration (molecular weight cut- off : [0663 ] 3 . Intracellular Proteins 5 , 000 Da ). Pooled LMWP - polypeptide conjugates are con 10664 ] For intracellular proteins, the level of protein centrated , and the degree of conjugation determined by expression can be detected as early as 8 - 12 hours after MALDI- TOF mass spectroscopy. transfection by Western Blot. Erythroid cells were cultured [ 0654 ] For uptake experiments , fresh sheep erythrocytes as described herein . A transgene encoding adenosine deami (MP Biomedicals , Solon , Ohio ) are suspended in Hank ' s nase with an HA tag at the C -terminus was constructed by balanced salt solution (HBSS ) at a density of 5x10 ^ 8 cells / Gibson assembly as described herein . The transgene was ml, and are then incubated with a 0 . 5 mg/ ml solution of the introduced into the erythroid cells by lentiviral transduction LMWP- polypeptide conjugates for 30 min at room tempera as described herein . Two days after transduction , cells were ture under gentle shaking . RBCs are then washed with collected , washed in PBS buffer , and lysed in RIPA cell lysis HBSS and stored at 2 -8 C . buffer ( Pierce ) . Cell lysate was denatured by boiling in 100 [ 0655 ] 12 . Polypeptide _ Chemical Permeability mM DTT, then loaded onto a NuPage SDS -PAGE pre -cast [0656 ] 3x10 ^8 RBCs were preincubated for 30 min with gel. After electrophoresis and transfer to nitrocellulose chlorpromazine (Sigma Aldrich ) at 200 uM in Ringer' s membrane , protein bands were developed by staining with US 2018 /0271910 A1 Sep . 27 , 2018 58

1 :5000 dilution of mouse anti -HA antibody (Abcam ) fol fied on an HA affinity column (Pierce ) according to manu lowed by 1 : 5000 dilution of goat anti -mouse HRP (Pierce ) , facturer' s instructions . Protein concentration was assessed and subsequent treatment with HRP substrate (SuperSignal , by absorbance at 280 nm . Pierce ). Images were captured using an Amersham imager [ 0672] Western blotting was performed as described (GE healthcare ). herein . In addition to the cell lysate samples , known amounts of the recombinant adenosine deaminase were run Example 19: Contacting Cells with Chemical on the same gel. Following image collection , the intensity of Modifying Agent the recombinant bands were used to generate a standard curve to quantify the amount of protein present in the cell [ 0665 ] To increase antigen -presenting cell phagocytosis samples. and to promote liver targeting , the cell compositions [ 0673] The robust expression of transgenes at high levels described herein are treated for 30 min with 0 . 15 microM of has important implications for the therapeutic capacity of the calcium ionophore A23187 (Sigma Aldrich , Saint Quentin final cell population . FIG . 2 quantifies the expression of Fallavier , France ) at 37 C or with 5 mM of BS3 ( Fischer three surface proteins indicative of differentiation and one Bioblock Scientific , Illkirch , France ) at room temperature exogenous transgene by quantitative flow cytometry , and (RT ) . After processing, the final products are stored at 2 - 8 C demonstrates that the transgene is robustly expressed at a in suitable buffer . high level. Example 20 : Assessment of Expression and [ 0674 ] Erythroid cells in culture were collected at seven Activity time points during a four - stage in vitro differentiation pro cess. At the first time point (“ Expand D6 ” ) the cells are [0666 ] The expression of exogenous proteins in and on nucleated hematopoietic precursors . By the final time point cultured cells can be assessed quantitatively by flow cytom (“ Diff 3 D8” ) the cells are predominantly enucleated eryth etry ( if the protein is expressed on the surface ) or by Western roid cells . GPA (solid triangles ), a canonical marker of blot ( for proteins expressed in the cytoplasm ). erythroid cells , starts low in the precursor cells and rapidly [ 0667] 1 . Quantitative Flow Cytometry reaches > 1x10 ^ 6 copies per cell. CKIT (dashed squares) , a [ 0668 ] Anti -mouse Fc- binding quantitative flow cytom receptor for stem cell factor, starts high then decreases to etry beads (Simply Cellular Calibration ) were purchased < 1x10 ^ 4 copies per cell as differentiation ensues. TR ( dotted from Bangs Labs . Fluorescently labeled mouse antibodies diamonds) , necessary for the transport of iron into erythroid against relevant cell surface receptors glycophorin A , Ckit, cells , increases initially then gradually declines to < 1x10 ^ 5 and transferrin receptor — were purchased from BioLegend . copies per cell . The transgene (open circles ) is introduced at Fluorescently labeled mouse antibody against the HA the end of the second differentiation stage (“ Diff 1 ” ) and is epitope tag was purchased from Life Technologies . Eryth steadily expressed at approximately 1x10 ^ 5 copies per cell roid cells were cultured as described herein . A transgene throughout differentiation . The above data demonstrate that encoding glycophorin A with an HA tag at the N - terminus transgenes are robustly expressed in cultured cells . was constructed by Gibson assembly as described herein . 10675 ]. The expression of exogenous proteins in and on The transgene was introduced into the erythroid cells by cultured cells can be assessed by flow cytometry ( if the lentiviral transduction as described herein . At least two days protein is expressed on the surface ) as described herein , or after transduction , 2x10 ^ 5 cells were collected , washed in by Western blot ( for proteins expressed in the cytoplasm ) as PBS buffer, and stained with 1 : 100 dilution of one of the described herein . In instances where an exogenous gene is in above - listed antibodies for 1 hr . Cells were washed and a single - transcript construct that contains a downstream analyzed on a flow cytometer ( Attune, Life Technologies ). fluorescent reporter protein , the fluorescence of the reporter The protocol was repeated for each of the four antibodies protein can be used as a proxy for expression of the upstream listed above . Quantification was performed according to gene , and assessed by flow cytometry as described herein . manufacturer ' s instructions. Briefly , one drop of each of the [0676 ] FIGS . 3 A - S shows the exogenous expression of five bead samples was incubated with 1 : 100 dilution of an surface and cytoplasmic proteins on enucleated cultured above -listed antibody . The beads were incubated for 1 hr, erythroid cells . The above data conclusively demonstrate washed in PBS , and analyzed on a flow cytometer (Attune , that multiple protein classes including cytoplasmic , sur Life Technologies ). The protocol was repeated for each of face , intact, fusions to type I membrane proteins , fusions to the four antibodies listed above . Calibration curves were fit type II membrane proteins, fusions to GPI- linked membrane using the manufacturer ' s provided excel spreadsheets , from proteins , intracellular fusions , overexpressed , and de novo which quantification of fluorescence intensity for the cell expressed can be expressed on multiple cell types includ based signals was derived . ing cultured enucleated erythroid cells , cultured nucleated [ 0669 ] 2 . Quantitative Western Blot erthyroid precursor cells , and K562 erythroleukemia cells . [0670 ] Erythroid cells were cultured as described herein . 10677 ] FIGS . 3 B and 3 F demonstrate the simultaneous A transgene encoding adenosine deaminase with an HA tag expression of two exogenous proteins in an enucleated at the C - terminus was constructed by Gibson assembly as cultured cell . described herein . The transgene was introduced into the [0678 ] In FIG . 3 B , Erythroid cells were cultured as erythroid cells by lentiviral transduction as described herein . described herein . A transgene construct encoding glyco Two days after transduction , cells were collected , washed in phorin A signal sequence , an HA epitope tag , glycophorin A PBS buffer, and lysed in RIPA cell lysis buffer (Pierce ) . coding sequence , viral T2A cleavable sequence and GFP [0671 ] The transgene was introduced into HEK293T cells was assembled by Gibson assembly as described herein . The by transient transfection using lipofectamine 2000 (Life transgene was introduced into the erythroid cells by lenti technologies ) . Cells were cultured for one week and the viral transduction as described herein . The cells were cul supernatant was harvested . Recombinant protein was puri- tured to terminal differentiation as described herein . Cells US 2018 /0271910 A1 Sep . 27 , 2018 59 were analyzed by flow cytometry as described herein , using of a protein over the course of differentiation . Biological a fluorescent anti -HA antibody and GFP fluorescence to activity of cytoplasmic enzymes was assessed by in vitro detect expression of both transgenes . enzymatic activity assay . [ 0679 ] In FIG . 3 F , Erythroid cells were cultured as [ 0685 ] FIGS . 5A -5D show the activity of two different described herein . A transgene construct encoding glyco intracellular enzymes expressed in cultured erythroid cells . phorin A signal sequence, antibody scFv specific to hepatitis [0686 ] 1. Adenosine Deaminase . B surface antigen , HÀ epitope tag , glycophorin A coding [0687 ] A transgene encoding adenosine deaminase with an sequence, viral T2A cleavable sequence and GFP was HA tag at the C -terminus was constructed by Gibson assem assembled by Gibson assembly as described herein . The bly as described herein . The transgene was introduced into transgene was introduced into the erythroid cells by lenti HEK - 293T cells by lipofectamine transfection (Life Tech viral transduction as described herein . The cells were cul nologies ) as described herein . Enzymatic activity is assayed tured to terminal differentiation as described herein . Cells using a protocol derived from Helenius 2012 , Biochim were analyzed by flow cytometry as described herein , using Biophys Acta 1823 ( 10 ) : 1967, in which a specific mixture of a fluorescent anti -HA antibody and GFP fluorescence to enzymes convert purines into uric acid and H202 followed detect expression of both transgenes . by fluorometric detection of the generated H202. In brief , two days after transfection , cells were collected , media Example 21: Expression of Protein from mRNA in aspirated , and Krebs Ringer phosphate glucose (KRPG ; Platelets comprising: 145 mM NaCl, 5 .7 mM sodium phosphate , 4 .86 mM KC1, 0 .54 mM CaC12 , 1 . 22 mM MgSO4 , and 5 . 5 mM [ 0680 ] The expression in platelets of exogenous proteins glucose ; pH 7 .35 ) added to the cells at 2x10 ^5 cells/ mL . translated from exogenous transfected mRNA was measured Adenosine was added at 50 uM . After reaction for 6 hours , by flow cytometry . In brief, platelet - enriched serum was supernatant was collected and heat inactivated for 5 minutes centrifuged at 190 g for 15 minutes to remove erythrocytes at 60 C . Aliquots of supernatant were transferred to wells in and leukocytes . The supernatant was then spun for an a white 96 -well microplate containing 0 .25 U /ml bacterial additional 5 minutes at 2500 g to pellet platelets . Platelets purine nucleoside phosphorylase (PNP ) and 0 . 15 U /ml were resuspended in 5 mL of Tyrode ' s buffer with 1 uM microbial xanthine oxidase (XO ) , both from Sigma . After 20 prostaglandin , washed , and resuspended in 750 L of min incubation at RT, 30 ul of H2O2 -detecting mixture Tyrode ' s buffer with 1 uM prostaglandin . mRNA encoding containing HRP ( final concentration 1 U /ml , Sigma ) and the gene of interest , in this example GFP , was mixed with Amplex Red reagent (60 UM , Invitrogen , Molecular Probes ) lipofectamine at a 1 : 1 mg/mL ratio . The mixture was incu was added to the microwells , followed by measurement of bated for 5 minutes , then added to the washed platelet the fluorescence intensity at the emission and excitation population . The combination was incubated for 24 hours at wavelengths of 545 and 590 nm , respectively ( Tecan Infinite room temperature with slow rocking . Platelet expression of M200 ) . the transgene was assayed by flow cytometry measuring [0688 ] 2 . Phenylalanine Hydroxylase GFP fluorescence . Surface proteins can also be assayed by [0689 ] Erythroid cells were cultured as described herein . flow cytometry . Cytoplasmic or other intracellularly -ex A transgene encoding phenylalanine hydroxylase with an pressed proteins can also be assayed by Western blot. HA tag at the C -terminus was constructed by Gibson assem [ 0681 ] There is therapeutic relevance to introducing exog bly as described herein . The transgene was introduced into enous proteins into and onto platelets . Since platelets do not the erythroid cells by lentiviral transduction as described possess a nucleus or RNA transcription machinery , DNA herein . Two days after transduction , cells were collected , transfection is not a viable means of inducing exogenous washed in PBS buffer , and lysed in RIPA cell lysis buffer protein expression in platelets . However , mRNA transfec ( Pierce ). Cell lysates (64 ug total protein ) were added to 1 tion and translation is a way of introducing exogenous mL reaction buffer containing 100 mM Tris -HC1 , PH 7 .5 , 4 proteins into cells . It is thought that platelets contain mRNA mM DTT, 4 mM Phenylalanine, 33 ug catalase , and 0 . 4 mM translation machinery , but until now it was not known DMPH4 (all from Sigma ) . Reactions were run overnight at whether they are able to accept and translate exogenous 37 C . After incubation , samples were de -proteinized by mRNA into protein . centrifugal filtration in an Amicon Ultra - 4 Centrifugal Filter [ 0682 ] FIGS. 4A -4C are a collection of flow cytometry 10 KD (Millipore UFC801024 ) spinning at 3700 rpm for 10 plots that demonstrate the translation of exogenous mRNA min . Samples were collected and assayed for tyrosine con encoding a transgene , in this case GFP , by platelets . The centration by absorbance at 540 nm . GFP is detected by fluorescence in the FL1 channel after [ 0690 ] Both of these exogenous proteins were retained excitation with a 488 nm laser . ( 4A ) Untransfected platelets through the end of terminal differentiation , a non - trivial feat ( 1 . 7 % GFP + ) . ( 4B ) Platelets transfected with 3 ug GFP given that it is well -known in the field that erythroid cells mRNA ( 8 .6 % GFP + ) . (4C ) Platelets transfected with 6 . 8 ug undergo a rigorous program of elimination of proteins GFP mRNA ( 3 . 3 % GFP + ) . unnecessary for basic function (Liu J et al. (2010 ) Blood [ 0683 ] The data conclusively demonstrate , for the first 115 ( 10 ): 2021- 2027 , Lodish H F et al. ( 1975 ) Developmental time, the translation of exogenous mRNA into exogenous Biology 47 (1 ): 59 ). In FIG . 5A , the exogenously over -ex protein by platelets . pressed protein adenosine deaminase is detected by anti -HA Western blot at various time points over the course of Example 22 : Activity of Enzymes differentiation , from nucleated precursor cells ( “ Diff I D5 % ) through to enucleated erythroid cells (“ Diff III D8” ) . In FIG . [ 0684 ] FIGS. 5A -5D demonstrate the activity for enzymes 5C , the exogenously expressed microbial protein phenylala contained on erythroid cells . Biochemical activity of cyto - nine hydroxylase is detected by anti- HA Western blot at plasmic enzymes was assessed by Western blot for retention various time points over the course of differentiation , from US 2018 /0271910 A1 Sep . 27 , 2018 60 nucleated precursor cells (“ DiffI D5 % ) through to enucleated binding to complexes of BSA and IgG that lack human erythroid cells ( “ Diff III D8” ) . complement, demonstrating that the binding event is CR1 [0691 ] Additionally , both of these enzymes maintained mediated . their ability to enzymatically convert substrate into product. [0702 ] FIG . 6B shows the biological activity of CR1, FIG . 5B shows the enzymatic conversion of adenosine to defined as the transfer of captured immune complexes from inosine by intact adenosine deaminase - expressing 293T cultured erythroid cells to macrophages . This is a standard cells . FIG . 5D shows the enzymatic conversion of phenyl assay in the field , see : Repik et al. 2005 Clin Exp Immunol. alanine to tyrosine by lysates of cultured phenylalanine 140 :230 ; Li et al . 2010 Infection Immunity 78 (7 ): 3129 . hydroxylase - expressing enucleated erythroid cells . Transfer is assessed by flow cytometry and measured as the [0692 ] These data conclusively demonstrate that exog intensity of labeled immune complex -derived fluorescence enous enzymes are retained on erythroid cells throughout the on macrophages . In this assay , macrophages that are incu culture process and that they are enzymatically active in bated with no immune complexes (black bars ) do not erythroid cells , which has profound therapeutic implica become fluorescent. Macrophages that are incubated with tions . complexes of BSA and IgG that lack complement ( and therefore do not bind CR1) take up only a small amount of Example 23: Activity of CR1 immune complex solid gray bars ) , independent of the presence of cultured CR1 - overexpressing erythroid cells . [ 0693] FIGS. 6A -6B show both biochemical and biologi This uptake is likely due to Fc- gamma receptors on the U937 cal activity for complement receptor 1 (CR1 ) overexpressed cells interacting with the Fc regions of the IgG molecules . on the surface of cultured erythroid cells . Biochemical Macrophages that are incubated with immune complexes activity of CR1 was assessed by flow cytometry for binding (BSA + IgG + complement) in the absence of CR1- overex to an immune complex . Biological activity of CR1 was pressing cells (hashed bar , left ) take up the same amount of assessed by transfer of immune complexes to macrophages immune complex as in the absence of complement, likely by in a co - culture assay . the same Fc - gamma mediated method . However, the mac [0694 ] 1 . Immune Complex Binding of CR1- Expressing rophages that are incubated with immune complexes in the Cells . presence of CR1- overexpressing cells (hashed bar, right ) [ 0695 ] Erythroid cells were cultured as described herein . take up nearly double the number of immune complexes as A transgene construct encoding complement receptor 1 measured by fluorescence . (CRI ) was constructed by Gibson assembly as described [0703 ] These data conclusively demonstrate that CR1 herein . The transgene was introduced into the erythroid cells overexpression on cultured erythroid cells enables the cap by lentiviral transduction as described herein . Transgene ture of immune complexes on said erythroid cells , facilitates expression levels were assessed by flow cytometry as the transfer of immune complexes from erythoroid cells to described herein using an anti -CR1 antibody ( Abcam ) . The macrophages , and significantly increases the rate and num cells were cultured to terminal differentiation as described ber of immune complexes taken up by macrophages . herein . [ 0696 ] Dylight 650 - labeled bovine serum albumin (BSA Example 24 : Activity of scFv 650 ) was incubated with polyclonal rabbit anti - BSA ( Ab [0704 ] FIGS . 7A -7D show the biochemical and biological cam ) in an excess of antibody for 30 minutes at room temp . activity of antibody scFv exogenously expressed on the The complexes were then mixed with human serum at a 1 : 1 surface of cultured erythroid cells as a fusion to the trans volume ratio for 30 minutes at 37 C . Control complexes membrane protein GPA . were either not mixed with human serum or mixed with [0705 ] Erythroid cells were cultured as described herein . heat- inactivated human serum . A transgene construct encoding the leader sequence of [ 0697 ] Complexes were incubated with the CR1- express glycophorin A , an antibody scFv specific to hepatitis B ing cells for 30 minutes at 37 C . Cells were washed and surface antigen (scFv , described in Bose 2003, Molecular analyzed by flow cytometry for capture of immune com Immunology 40 :617 ) , an HA epitope tag , a [Gly - 3 -Ser ] 2 plexes by detecting Dylight 650 fluorescence . flexible linker (Seq . ID No . 23 ) , and the body of glycophorin [ 0698 ] 2 . Immune Complex Transfer to Macrophages A was assembled by Gibson assembly as described herein . [0699 ] Cultured U937 monocytes were activated by incu The transgene was introduced into the erythroid cells by bation with 100 nM phorbol myristate acetate (PMA ) for 24 lentiviral transduction as described herein . Transgene hours at 37 C . Cells coated with immune complexes (see expression was assessed by flow cytometry as described above ) were incubated with activated U937 macrophages for herein using an anti -HA antibody (Abcam ) . The cells were 30 minutes at 37 C . The co -culture was analyzed by flow cultured to terminal differentiation as described herein . cytometry . Macrophages were identified by FSC /SSC gat Biochemical activity of the antibody scFv was assessed by ing . Presence of immune complex on macrophages was flow cytometry for binding to the target protein , in this case analyzed by detecting Dylight 650 fluorescence in the mac hepatitis B surface antigen (HBsAg ) . Recombinant HBsAg rophage population . protein (Abcam ) was labeled with Dylight- 650 fluorophore [ 0700 ] FIGS . 6A - 6B show the biochemical and biological ( Pierce ) . scFv - expressing cells were incubated with 100 nM activity of complement receptor 1 (CR1 ) exogenously over labeled protein , washed in PBS , and analyzed for Dylight expressed on cultured erythroid cells . 650 fluorescence by flow cytometry as described herein . [0701 ] FIG . 6A shows the biochemical activity of CR1, [0706 ] Biological activity of the antibody scFv was defined as the capture of immune complexes in vitro . The assessed by in vivo capture of HBsAg detected by flow black histogram shows the capture of BSA -based immune cytometry. Recombinant HBsAg protein (Abcam ) was complexes by CR1 over - expressed on cultured erythroid labeled with Dylight -650 fluorophore (Pierce ) . scFv - ex cells . The shaded histogram shows the minimal background pressing cells were fluorescently labeled with CFSE US 2018 /0271910 A1 Sep . 27 , 2018 hl

( Sigma) . Immunocompromised NSG mice ( Jackson labs ) at room temperature . Anti -biotin antibody ( Abcam ) was were injected with ~ 400 pmol of the labeled HBsAg into the fluorescently labeled with Dylight 650 (Pierce ) . Labeling tail vein . A few minutes later , the same mice were injected efficiency of the cells was assessed by flow cytometry using with 2x10 ^ 7 scFv - expressing cells . Blood was collected by the labeled anti- biotin antibody and CFSE fluorescence as submandibular puncture at regular intervals in an EDTA detection markers. 250 ug labeled antibody was injected into containing tube . Collected blood cells were washed and an NSG mouse ( Jackson Labs ) intravenously via the tail analyzed by flow cytometry as described herein . Human vein . Four hours later 1x10 ^ 8 biotinylated cells were cells were identified as those that were CFSE positive . injected intravenously via the tail vein . Blood was collected Capture of HBsAg was detected as Dylight650 fluorescence by submandibular puncture at regular intervals in an EDTA on the human cells . containing tube. Collected blood cells were washed and [0707 ] FIG . 7A -7B show the biochemical activity of anti analyzed by flow cytometry as described herein . Human body scFv , defined as the binding of its cognate antigen , cells were identified as those that were CFSE positive . hepatitis B surface antigen (HBsAg ) . In FIG . 7A , cells that Capture of anti- biotin antibody was detected as Dylight650 express (black ) or do not express ( gray shaded ) the antibody fluorescence on the human cells . Plasma from the blood scFv are incubated with 450 nM HBsAg and stained with draw was analyzed by ELISA using a biotin - coated biotinylated anti- HBsAg antibody and fluorescent strepta microplate ( Pierce ) per manufacturer ' s instructions to detect vidin . Cells that express the antibody scFv ( ~ 45 % of the the level of antibody in circulation . cells in this culture ) bind to the antigen . In FIG . 7B , cells that express the antibody scFv are incubated with various con [0714 ] 2 . Capture of Anti -HA Antibody by Transgenic centrations ofHBsAg and stained as above , showing that the Cultured Cells binding event is dose - dependent with an affinity of approxi [ 0715 ] Erythroid cells were cultured as described herein . mately 10 nM . A transgene construct encoding glycophorin A signal [0708 ] FIG . 7C -7D show the biological activity of anti sequence, an HA epitope tag, glycophorin A coding body scFv , defined as the capture of cognate antigen HBsAg sequence , viral T2A cleavable sequence and GFP was while in circulation in a mouse. In this experiment, immu assembled by Gibson assembly as described herein . The nocompromised NSG mice were injected with ~ 400 umol transgene was introduced into the erythroid cells by lenti fluorescently - labeled HBsAg via the tail vein . Five minutes viral transduction as described herein . The cells were cul later, cultured enucleated erythroid cells ( 7C ) or cultured tured to terminal differentiation as described herein . Cells enucleated erythroid cells that expressed exogenous anti were analyzed by flow cytometry as described herein , using body scFv ( 7D ) were injected via the tail vein . Prior to an anti -HA antibody (Life Technologies ) fluorescently injection , all cultured cells were labeled with CFSE fluo labeled with Dylight 650 ( Pierce ) and GFP fluorescence to rescent dye. Blood was collected 6 hours later , analyzed on detect expression of both transgenes . 250 ug labeled anti a flow cytometer, and gated on CFSE + human cells . Bare HA antibody was injected into an NSG mouse ( Jackson cultured cells did not bind to HBsAg ( 7C ), whereas antibody Labs ) intravenously via the tail vein . Four hours later 1x10 ^ 8 scFv - expressing cells do bind to HBsAg (7D ). Consistently cultured cells were injected intravenously via the tail vein . with the biochemical activity experiment, approximately Blood was collected by submandibular puncture at regular 45 % of the cells in this culture express antibody -scFv . intervals in an EDTA - containing tube . Collected blood cells 10709 These data demonstrate that the antibody scFv is were washed and analyzed by flow cytometry as described biochemically active when expressed on the surface of herein . Human cells were identified as those that were CFSE cultured erythroid cells and that the antibody scFv on the positive. Capture of anti - HA antibody was detected as erythroid cell is able to bind its target in vivo when in Dylight 650 fluorescence on the human cells . Plasma from circulation . This has profound implications for therapeutic the blood draw was analyzed by ELISA using an HA approaches in which the capture , sequestration , and clear peptide - coated microplate (Pierce ) per manufacturer' s ance of a substance in circulation is desired . instructions to detect the level of antibody in circulation . [0716 ] FIGS. 8A -8D show biochemical and biological Example 25 : Activity — Circulating Clearance activity of (8A - 8B ) the polpeptide HA expressed on the [0710 ] FIGS . 8A - 8D show both biochemical and biologi surface of cultured erythroid cells as a fusion to GPA and of cal activity for surface molecule capture agents used for (8C - 8D ) biotin chemically conjugated to the surface of circulating clearance of a target. primary erythrocytes . Biochemical activity is defined as the [0711 ] Biochemical activity of the capture agents , in this capture of a target protein in vitro . Biological activity is case HA polypeptide and biotin , was assessed by flow defined as the enhanced clearance of a target protein in vitro . cytometry for binding to the target protein , in this case [0717 ] In FIG . 8A , the HA polypeptide, expressed as a anti - HA antibody and anti -biotin antibody . Biological activ fusion to the N terminus of GPA , captures a mouse anti -HA ity of the capture agents was assessed by in vivo capture and antibody in vivo . NSG mice were injected with fluores clearance of target protein as detected by flow cytometry and cently - labeled mouse anti- HA antibody, followed by injec plasma protein quantification . tion of cultured human erythroid cells that either do not ( top ) [ 0712 ] 1 . Capture of Anti -Biotin Antibody by Chemically or do (bottom ) express HA epitope tag on their surface as a Modified Cells fusion to GPA . Blood was drawn and cells analyzed on the 10713 ] Eyrthrocytes from a normal human donor were flow cytometer . The x -axis measures CFSE fluorescence . purchased (Research Blood Components ). Cells were The y - axis measures anti -HA antibody Dylight 650 fluores labeled with CFSE (Sigma ) per manufacturer ' s instructions cence . CFSE - positive cultured human erythrocytes are for 20 minutes at 37 C . Cells were then biotinylated with observed in both samples , but only the cells expressing the NHS -biotin (Sigma ) per manufacturer 's instructions using HA epitope tag are able to capture circulating anti -HA 0 .02 volumes of 2 mm stock biotin reagent for 30 minutes antibody . US 2018 /0271910 A1 Sep . 27 , 2018

[0718 ] In FIG . 8B , mice were injected with anti -HA erythrocytes (EA ) . EA ( 100 ul) are incubated with 100 ul of antibody then then optionally with cultured human erythroid five serial twofold dilutions ( 1 :20 , 1 : 40 , 1 :80 , 1: 160 , and cells that either do not or do express HA peptide on their 1 : 320 ) of the normalhuman serum (NHS ) or similar dilution surface as a fusion to GPA . Plasma was collected at multiple of the mixture of NHS and the exogenous antigen -express time points and the level of anti -HA antibody in plasma was ing EHC at 37° C . for 1 hour. NHS incubated with GVB2 + assessed by ELISA using an HA peptide - coated plate as a buffer is used as the control. Background control is obtained substrate . Mice injected with anti -HA antibody alone (open by incubating EA with buffer alone ( serum is not added ) , and circles , solid line — this mouse died after 120 minutes of total lysis ( 100 % hemolysis ) is determined by adding dis causes unrelated to treatment) or with anti -HA antibody tilled water to EA . The reaction is stopped using 1 . 2 ml of followed by cells that do not express HA peptide on their ice -cold 0 .15M NaCl, the mixture is spun to pellet the surface (dashed line ) have significant antibody in circulation unlysed cells , and the optical density of the supernatant is out to 24 hours post injection of cells . In contrast , mice determined spectrophotometrically (412 nm ) . The percent injected with anti -HA antibody followed by cells that age of hemolysis is determined relative to the 100 % lysis express HA peptide on their surface are depleted of target control. Complement activity is quantitated by determining antibody within minutes . This data conclusively demon the serum dilution required to lyse 50 % of cells in the assay strates that the target antibody is rapidly and specifically mixture . The results are expressed as the reciprocal of this cleared from circulation by cultured erythroid cells that dilution in CH50 units /ml of serum . express exogenous antigen polypeptide on their surface . [0724 ] Briefly , the AH50 assay depends on lysis of unsen [0719 ] In FIG . 8C , the biotin molecule , conjugated to the sitized rabbit erythrocytes ( Erab ) by human serum by acti surface of erythroid cells by amine functionalization chem vation of the alternative pathway. Activation of the calcium istry, captures a mouse anti - biotin antibody. In both of these dependent classical pathway is prevented by addition of the cases capture was assessed by flow cytometry . Cells that are calcium chelator ethylene glycol tetraacetic acid ( EGTA ) to CFSE labeled and biotinylated show up as double positive the assay buffer , and magnesium , necessary for both path when stained with a fluorescent anti -biotin antibody ( lower ways , is added to the buffer. Briefly , a cell suspension of dot plot) , whereas CFSE - labeled cells that are not bioti rabbit RBC ( 2x10 ^ 8 cell /ml ) is prepared in the GVB -Mg2 + nylated only show up as single positive (upper dot plot) . EGTA buffer . A serial 1 . 5 - fold dilution ( 1 : 4 , 1 : 6 , 1 : 9 , 1 : 13 . 5 , [ 0720 ] In FIG . 8D , mice were injected with anti -biotin and 1 : 20 . 25 ) of normal human serum (NHS ) or similar antibody then then optionally with cultured human erythroid dilution of the mixture of NHS and the exogenous antigen cells that either are not or are conjugated to biotin on their expressing EHC are prepared in GVB -Mg2 + -EGTA buffer , surface. Plasma was collected at multiple time points and the and 100 ul of each serum dilution is added to 50 ul of level of anti- biotin antibody in plasma was assessed by standardized Erab . NHS incubated with GVB -Mg2 + - EGTA ELISA using a biotin -coated plate as a substrate. Mice buffer is used as the control. The mixture is then incubated injected with anti -biotin antibody alone ( open circles , solid at 60 minutes at 37° C . in a shaking water bath to keep cells line ) or with anti -biotin antibody followed by cells that are in suspension , and 1 . 2 ml of ice - cold NaCl ( 0 . 15 M ) is used not conjugated to biotin on their surface (dashed line ) have to stop the reaction . The tubes are spun at 1250 g , at 4° C ., significant antibody in circulation out to 24 hours post for 10 minutes to pellet the cells , and the optical density of injection of cells . In contrast , mice injected with anti -biotin the supernatant is determined spectrophotometrically (412 antibody followed by cells that are conjugated to biotin on nm ) . Background control has 100 ul GVB -Mg2 + - EGTA their surface are depleted of target antibody within minutes . buffer , and 50 ul Erab and does not exceed 10 % of the total This data conclusively demonstrates that target antibodies lysis . In the total lysis control tube 100 ul of distilled water are rapidly and specifically cleared from circulation by is added to 50 ul Erab suspension , and the percentage of cultured erythroid cells that contain exogenous antigen hemolysis is determined relative to 100 % lysis control. The polypeptide on their surface . results of the assay are calculated and complement activity [ 0721] Together the data conclusively demonstrate that is quantitated by determining the serum dilution required to suitable exogenous antigens on exogenous antigen - express lyse 50 % of cells in the assay mixture . The results are ing EHCs are able to bind their target molecules in vivo and expressed as the reciprocal of this dilution in AH50 units/ ml mediate rapid circulating clearance of targetmolecules when of serum . in circulation , which has profound therapeutic implications . Example 27 : Activity of Platelet - Loaded Thymidine Example 26 : Activity of Complement Regulators Phosphorylase [0722 ] The complement regulatory activity of the exog [0725 ] A transgene encoding thymidine phosphorylase enous antigen - expressing EHCs is assessed by standard with an HA tag at the C - terminus is constructed by Gibson CH50 and AH50 assays known in the art (see e .g ., Kabat et assembly as described herein . Platelets are cultured from al. 1961 Exp Immunochem pp . 133 - 239 and Platts -Mills et precursor cells as described herein . The transgene is intro al. 1974 J Immunol 113 : 348 ) . duced into the cultured platelet precursor cells by lentiviral [ 0723] Briefly , the CH50 assay utilizes sheep erythrocytes transduction as described herein . Expression of thymidine ( SRBC ) as target cells . Briefly , a suspension containing phosphorylase within the cultured platelets is assessed by 1x10 ^ 9 SRBC /ml is prepared in the GVB ( 2 + ) buffer (gela Western blotting using an anti -HA detection antibody, as tin /Veronal - buffered saline with Ca2 + and Mg2 + ) , pH 7 . 35 . described herein . Hemolysin ( rabbit anti - sheep antiserum ) is titrated to deter [ 0726 ] Thymidine phosphorylase activity is determined in mine the optimal dilution to sensitize SRBC . Diluted platelet samples by quantifying the rate of conversion of hemolysin ( 1 : 800 ) is mixed with an equal volume of SRBC thymidine to thymine . Preliminary experiments are con ( 1x109 SRBC /ml ), and the whole is incubated at 37° C . for d ucted to determine the linear metabolite formation kinetics 15 minutes . This results in 5x10 ^ 8 /ml antibody - coated with respect to time and enzyme dilution ; the method is US 2018 /0271910 A1 Sep . 27 , 2018 62 shown to be linear for up to 16 min , over a thymine flow cytometry as described herein . Antibody capture by phosphorylase range of 4 . 0 -719 nmol/ min /ml ( correspond platelets is determined by tracking the CFSE -Dylight 650 ing to a sample dilution range of 10 - 9088 ) . Lysates of double positive population . pre - dialysis samples cultured platelet and control platelet samples are prepared by diluting thawed samples 1: 710 with Example 29 : Activity In Vivo (Mouse ) 125 mM Tris - HCl, pH 7 .4 . Twenty - five ul of the platelet lysate is then added to 100 ul sodium phosphate buffer ( 100 10731 ] Mouse erythroid cells are cultured as described mM , pH 6 . 5 ) and 25 ul thymidine standard ( 10 mm ), mixed herein . Erythroid precursor cells are transduced with a and incubated at 37 C for 10 min . The reaction is terminated suitable exogenous antigen polypeptide transgene , e . g . , with 25 ul of 40 % TCA . Assay blanks are prepared by encoding complement receptor 1 (CR1 ) using a lentivirus as adding TCA to the sodium phosphate buffer / thymidine incu described herein . Cells are cultured to terminal differentia bation mixture prior to adding the platelet lysate . Samples tion as described herein . The presence of the exogenous are centrifuged at 13 , 400xg for 2 min , and the supernatant protein in the cells is assessed by flow cytometry as washed twice with water- saturated diethyl ether with 2 min described herein . The cells are labeled with a fluorescent die , on a shaker to extract the TCA . To avoid ether interfering e . g ., CFSE (Sigma Aldrich ) per manufacturer ' s instructions with HPLC separation , effective removal is achieved by to aid in their detection . The cells are injected into a exposing the matrix to the air for 5 min to allow evaporation NZBWF1/ J mouse model of lupus, or other appropriate of the ether . A sample volume of 10 ul is injected into the model of disease or activity corresponding to the suitable HPLC . exogenous antigen polypeptide , approximately 1x10 ^ 8 cells [ 0727 ] Chromatographic separation of substrate and prod injected via the tail vein . Blood is collected at multiple time uct is achieved using reversed phase chromatography with points by submandibular puncture . Immune complex levels isocratic elution using a Waters Alliance HPLC 2795 sys in the plasma are detected by Raji cell assay, see e . g . , tem . A pre -packed C18column (Spherisorb ODS 125 Theofilopoulos et al . 976 , J Clin Invest 57 ( 1 ): 169. Pharma mmx4 . 6 mm ID , 5 um particle size , Waters ) is used as the cokinetics of the cultured cells are assessed by flow cytom stationary stage . Analytes are eluted using a mobile phase of etry as described herein , by tracking the percentage of CFSE ammonium acetate ( 40 mM ) with the ion - pairing agent fluorescent cells in the drawn blood sample . Mouse overall tetrabutyl ammonium hydrogen sulphate ( 5 mM ) adjusted to health is assessed by gross necropsy, including histology of pH 2 .70 with HCl, delivered at a flow rate of 1 . 0 ml/min , kidney tissue to track reduction of immune complex depo with a run time of 8 min . UV detection is at 254 nm and 0 . 1 sition and inflammation -mediated damage . absorbance units full scale . Metabolites are identified by comparing spectra with pure standards. Example 30 : Rapid Screening [ 0732 ] Cell lines , e . g ., 293T and K562 , have shorter Example 28 : Activity of Platelet- Displayed expression and culturing cycles ( ~ 1 day ) compared to cul Goodpasture Antigen tured erythroid cells (days - weeks) . These cell lines can be [ 0728 ] A transgene encoding collagen alpha -3 (IV ) used to rapidly iterate through a gene library encoding (COL4A3 ) NC1 domain antigen fused to the N terminus of suitable exogenous antigen polypeptides to identify the CD42b (GP1B , genbank AAH27955 . 1 ) with an intervening exogenous antigen polypeptide with the highest expression HA tag is constructed by Gibson assembly as described or activity . herein . Platelets are cultured from precursor cells as [0733 ] A library of suitable exogenous antigen polypep described herein . The transgene is introduced into the cul tide transgenes , e. g ., full- length and shorter variants of tured platelet precursor cells by lentiviral transduction as complement receptor 1 (CR1 ) , are constructed by poly described herein . Expression of the exogenous antigen on merase chain reaction and Gibson assembly as described the cultured platelets is assessed by flow cytometry using an herein . The library of transgenes is transfected into anti -HA detection antibody as described herein . HEK293T cells in a parallel fashion in a microtiter plate using lipofectamine as described herein and transduced into [0729 ] Serum is collected from a patient suffering from K562 cells using lentivirus as described herein . The expres Goodpasture ' s syndrome, and the serum is tested for anti sion of the exogenous antigens is assessed by flow cytom COL4A3 antibodies by commercial ELISA (MyBioSource etry as described herein after 24 - 48 hours . The activity of COL4A3 ELISA Kit ) . The binding capacity of the antigen each of the exogenous antigens in the library is assessed by expressing platelets is assessed by flow cytometry as capture of fluorescent immune complex detected with flow described herein , using this anti- COL4A3 serum as the cytometry as described herein , and by the transfer of fluo primary detection antibody and fluorescent anti- human IgG rescent immune complexes to cultured monocytes detected as the secondary detection antibody. with flow cytometry as described herein . The exogenous [0730 ] Platelet - facilitated clearance of a circulating anti antigens from the library that are most functional — e . g . , are gen in vivo is modeled in a mouse using the antigen highest expressed , capture most immune complexes, or best expressing platelets . NSG mice are injected with 100 uL of transfer immune complexes to monocytes are then indi mouse anti- human COL4A3 antibody ( Creative BioMart) vidually transduced into parallel erythroid cell cultures as fluorescently labeled with Dylight 650 dye . CFSE - labeled described herein using lentivirus as described herein . The cultured platelets ( 10 ^ 8 per mouse ) that express the exog expression of each exogenous antigen on cultured erythroid enous antigen are then injected via the tail vein . Blood is cells is assessed by flow cytometry as described herein The drawn from a submandibular location at 10 min , 30 min , 2 activity of each exogenous antigen on cultured erythroid h , 12 h , and 24 h . Blood is centrifuged to collect the cells is assessed by capture of fluorescent immune complex platelet -rich fraction , which is then stained and analyzed by detected with flow cytometry as described herein , and by the US 2018 /0271910 A1 Sep . 27 , 2018 64 transfer of fluorescent immune complexes to cultured mono were stained by H & E staining and trichrome staining . cytes detected with flow cytometry as described herein . Microscope images were taken at 10x and 20x magnifica tion . Example 31 : Assessment of Clearance Rate of RBC [ 0740 ] For therapeutic applications, it is important that cultured erythroid cells and cultured erythroid cells that In Vivo contain exogenous proteins ( either intracellularly or on the [ 0734 ] The clearance rate of erythroid cells was assessed surface ) not induce adverse events , such as activation of the in vivo in an immunocompromised mouse model. NSG mice clotting cascade and tissue thrombus formation . were treated at day - 1 with 100 uL of clordonate liposome [0741 ] FIGS . 10A and 10B show the levels of fibrinopep ( Clodrosomes . com ) solution to selectively deplete macro tide A and B in mouse plasma for mice injected with ( 1 ) phages. Cells were labeled with the fluorescent tag CFSE human red blood cells, ( 2 ) cultured enucleated erythroid and approximately 1x10 ^ 8 cells were injected into each cells , (3 ) cultured enucleated erythroid cells expressing an mouse via the tail vein . At regular intervals blood was intracellular exogenous protein , ( 4 ) cultured enucleated collected by submandibular puncture and blood cells were erythroid cells expressing a surface exogenous protein , and collected . Cells were co -stained with anti -human GPA anti ( 5 ) recombinant protein alone. The levels of fibrinopeptide bodies and analyzed by flow cytometry . Human erythroid A and B , a marker of fibrinogen breakdown and activation cells were distinguished from mouse erythroid cells by of the clotting cascade, are substantially similar for all CFSE signal and by human GPA signal . samples. [ 0735 ] For therapeutic applications, it is important that [0742 ] FIGS. 10C and 10D show histologically stained cultured erythroid cells and cultured erythroid cells contain sections of spleen for a mouse injected with cultured enucle ing exogenous protein either intracellularly or on the surface ated erythroid cells ( 10C ) and recombinant protein ( 10D ) . circulate normally in vivo . This is shown in FIGS . 9A - 9B There is no substantial difference between the tissue , and no using a standard immunocompromised mouse model . In identifiable tissue damage in spleen , liver , lung , brain , heart, FIG . 9A , blood collected from an injected mouse is analyzed and kidney was observed between any of the samples . on the flow cytometer . Cultured human erythroid cells are 10743 ] These data conclusively demonstrate that cultured identified in the top right quadrant of the plot, double erythroid cells , with or without exogenous protein , do not positive for CFSE and human -GPA . In FIG . 9B , mice were induce any adverse events while in circulation in mice . injected with human red blood cells (solid circles ), cultured enucleated erythroid cells (dashed diamonds ), cultured Example 33 : Assessment of Exogenous Protein enucleated erythroid cells that express an intracellular exog Retention in Circulation enous protein (dotted squares ) and cultured enucleated [0744 ] The retention of exogenous proteins in and on erythroid cells that express a surface exogenous protein cultured enucleated erythroid cells was assessed by flow ( open triangles ) . The clearance rate of the human cells is cytometry and Western blotting . measured as the percentage of CFSE + cells remaining over [0745 ] 1. Retention of Exogenous Protein Assessed by time, scaled to the initial time point ( 20 minutes post Flow Cytometry injection ) . There is no significant difference in clearance rate [0746 ] Erythroid cells were cultured as described herein . between the four samples. A transgene construct encoding glycophorin A signal [ 0736 ] These data clearly demonstrates that cultured sequence , antibody scFv specific to hepatitis B surface enucleated erythroid cells have substantially similar circu antigen , HA epitope tag , and glycophorin Acoding sequence lation to normal human red blood cells . Furthermore , exog was assembled by Gibson assembly as described herein . The enous proteins expressed either in the intracellular space or transgene was introduced into the erythroid cells by lenti on the surface of the cells do not substantially affect the viral transduction as described herein . The cells were cul circulation behavior of these cells . This is an important tured to terminal differentiation as described herein . Cells result for therapeutic translation of the technology . were fluorescently labeled with CFSE and injected into an immunocompromised NSG mouse ( Jackson Labs) via the tail vein ( 1x10 ^ 8 cells per mouse ) . At regular intervals blood Example 32 : Assessment of Adverse Circulatory was collected by submandibular puncture . Collected cells Events were stained with a fluorescent anti -HA antibody ( Abcam ) , [0737 ] The incidence of adverse events caused by cultured and analyzed by flow cytometry . Human cells were identi eyrthroid cells in circulation were assessed by detection of fied as CFSE + cells , and exogenous protein retention was fibrinogen breakdown products in blood and histology in assessed by the fraction of CFSE + cells that also stained positive for the epitope tag . animals injected with cultured erythroid cells . [0747 ] 2 . Retention of Exogenous Protein Assessed by [0738 ] Detection of Fibrinogen Breakdown Products . Western Blot Mice were injected with cultured erythroid cells as described [0748 ] Erythroid cells were cultured as described herein . herein . Blood was collected from mice by submandibular A transgene construct encoding adenosine deaminase and an puncture in an EDTA - containing tube . Cells were separated HA epitope tag was assembled by Gibson assembly as by centrifugation and plasma was collected . The levels of described herein . The transgene was introduced into the fibrinogen breakdown products fibrinopeptide A and fibrino erythroid cells by lentiviral transduction as described herein . peptide B were measured in mouse plasma by ELISA The cells were cultured to terminal differentiation as (MyBiosource ) following manufacturer' s instructions. described herein . Cells were fluorescently labeled with [ 0739 ] Histology. Tissue samples from the same mice CFSE and injected into an immunocompromised NSG were collected following necropsy . Tissues were trimmed , mouse ( Jackson Labs ) via the tail vein ( 1x10 ^ 8 cells per embedded in paraffin wax , and sectioned . Tissue sections mouse ) . At regular intervals blood was collected by sub US 2018 /0271910 A1 Sep . 27 , 2018 65 mandibular puncture . Collected cells were washed , lysed , HA tag . When analyzed by Western blot, it is clear that the and analyzed by Western blot as described herein with a enzyme' s circulating half - life is significantly extended when detection antibody against the HA epitope tag . expressed within a circulating cell compared to when [ 0749 ] For therapeutic applications, it is important that injected in soluble form . cultured erythroid cells that contain exogenous proteins either intracellularly or on the surface retain these transgenes Example 35 : Assessment of Clearance Rate In when in circulation . This feat is non - trivial given that it is Vivo — Platelets widely hypothesized in the field that erythroid cells undergo [0755 ] A population of exogenous thymidine phosphory a rigorous program ofmaturation and elimination of proteins lase expressing platelets is cultured using the herein detailed unnecessary for basic function when in circulation as they procedure and is labeled with CFSE and injected into an mature (Liu J et al. ( 2010 ) Blood 115 ( 10 ): 2021- 2027 , Lod NSG mouse via the tail vein . A population of native human ish H F et al. ( 1975 ) Developmental Biology 47 ( 1 ) : 59 ) . sourced platelets is similarly labeled with CFSE and injected [0750 ] FIGS . 11A - 11B show that exogenous proteins into another mouse . Samples are taken from both mice at 10 expressed in and on cultured enucleated erythroid cells were min , 1 h , 4 h , 8 h , 24 h , and 48 h and flow cytometry is used retained in circulation . In FIG . 11A , mice were injected with cultured enucleated erythroid cells that expressed antibody to quantify platelet circulation levels . The half- life of natural scFv on their surface . The percentage of antibody scFv vs cultured platelets is compared . positive cells began and remained steadily at approximately Example 36 : Assessment of Adverse Circulatory 50 % through the duration of the multi - day circulation study. Events - Platelets In FIG . 11B , mice were injected either with cultured enucle ated erythroid cells that expressed a cytoplasmic enzyme [ 0756 ] For therapeutic applications, it is important that with an HA tag or with recombinant enzymewith an HA tag . cultured platelets and cultured platelets that contain exog When analyzed by Western blot, it is clear that the enzyme enous proteins ( either intracellularly or on the surface ) not retained within the cultured cell for the duration of the induce adverse events , such as activation of the clotting experiment. The decrease in band intensity is attributable to cascade and tissue thrombus formation . Upon injection of the clearance of cells during the experiment , not from the cultured platelets into an NSG mouse via the tail vein , removal of exogenous enzyme from said cells . fibrinogen breakdown products fibrinopeptide A and fibrino [ 0751 ] The data clearly demonstrate that exogenous pro peptide B are detected in mouse plasma by ELISA following teins expressed in and on culture enucleated erythroid cells manufacturer ' s protocol (MyBiosource ) . Tissue samples are retained in and on the cells in circulation , which has from NSG mice are collected following necropsy. Tissues tremendous and unprecedented implications for therapeutic are trimmed , embedded in paraffin wax , and sectioned . relevance . Tissue sections are stained by H & E staining and trichrome staining . Microscope images are taken at 10x and 20x Example 34 : Assessment of Half -Life Extension In magnification and assessed by a trained pathologist for any Vivo pathogenic features. [ 0752] Erythroid cells were cultured as described herein . Example 37 : Assessment of Exogenous Protein A transgene construct encoding adenosine deaminase and an Retention in Circulation — Platelets HA epitope tag was assembled by Gibson assembly as described herein . The transgene was introduced into the 10757 ]. The retention of exogenous proteins in and on erythroid cells by lentiviral transduction as described herein . cultured platelets is assessed by flow cytometry and Western The cells were cultured to terminal differentiation as blotting described herein . Cells were fluorescently labeled with 10758 ) CFSE labeled platelets that contain intracellular CFSE and injected into an immunocompromised NSG exogenous protein are injected into a mouse via the tail vein . mouse (Jackson Labs ) via the tail vein ( 1x10 ^ 8 cells per At regular intervals blood is collected by submandibular mouse ) . At regular intervals blood was collected by sub puncture . Blood is centrifuged to isolate the platelet -rich mandibular puncture . Collected cells were washed , lysed , plasma , which is then lysed , and analyzed by Western blot and analyzed by Western blot as described herein with a with staining for an epitope tag present on the exogenous detection antibody against the HA epitope tag. protein . [ 0753] A transgene encoding adenosine deaminase with an HA tag at the C -terminus was constructed by Gibson assem Example 38 : Acquisition of Donor Cells for bly as described herein . The transgene was introduced into Production HEK -293T cells by lipofectamine transfection (Life Tech [0759 ] After obtaining informed consent, healthy CD34 + nologies ) as described herein . The protein was purified from stem cell donors receive rhG - CSF (Granocyte or Neupo the cell culture supernatant after 7 days using an HA affinity gen ), 10 ug /kg /day s . c . , for 5 days for peripheral blood stem resin ( Pierce ) according to manufacturer ' s instructions. Pro cell mobilization and then undergo apheresis for 2 consecu tein concentration was assessed by absorbance of light at tive days to collect mobilized CD34 +HSC . Mononuclear 280 nm . Protein (40 ug ) was injected into an immunocom cells (MNC ) are isolated from mobilized peripheral blood by promised NSG mouse ( Jackson Labs ) via the tail vein . At Ficoll density gradient centrifugation and are split in two regular intervals blood was collected by submandibular parts . One part is used to purify CD34 + cells by using puncture . Plasma was analyzed by Western blot as described anti - CD34 - coated magnetic beads (Miltenyi Biotec , Inc. , herein with a detection antibody against the HA epitope tag . Germany ) , relative to Miltenyi protocol. The purity of the [0754 ] In FIG . 11B , mice were injected either with cul CD34 + fractions is controlled . CD34 + -enriched HSC are tured enucleated erythroid cells that expressed a cytoplasmic then used immediately in the two - step culture method or enzyme with an HA tag or with recombinant enzymewith an frozen until use in the one - step culture method . US 2018 /0271910 A1 Sep . 27 , 2018

[0760 ] Complete medium (CM ) used is RPMI 1640 (Eu - (# TSP100375 , Polymicro Technologies) with an internal robio , France ) , supplemented with 2 mM L - glutamine and diameter of 100 um . At the input end , the capillary is fed 100 IU /ml penicillin - streptomycin (Gibco , Grand Island , with a luer -lok tip stock syringe ( # 309585 BD ) connected N . Y ., USA ) and 10 % heat - inactivated FBS (Gibco ) . IMDM via a PEEK luer to a MicroTight adapter (# P -662 , Upchurch (Gibco ), supplemented with 10 % heat - inactivated FBS, is Scientific ) . The stock syringe is loaded on a Model 33 Twin used for expansion . Recombinant human stem cell factor Syringe Pump (# 553333 , Harvard Apparatus ) , kept in a ( rhSCF ), thrombopoietin ( TPO ), fetal liver tyrosine kinase 3 refrigerator at 4 C . At the output end , the capillary enters the ligand (Flt - 3L ) , GM - CSF , and TNF - alpha are purchased bioreactor: a two port FEP cell culture bag ( # 2PF -0002 , from R & D Systems (Minneapolis , Minn . , USA ). VueLife ) placed on an orbital shaker in a cell culture incubator at 37 C with 5 % CO2. The capillary is fed through Example 39: Scale -Up for Production a self- sealing rubber septa ( # B - IIS , InterLink ) with a needle , into the midpoint of the bioreactor. The opposing connector [ 0761 ] Erythroid cells are scaled up in volume progres on the bioreactor is replaced with an additional self - sealing sively , maintaining the cells at a density of between 1x10 ^ 5 rubber septa . Stock syringes and delivery capillaries are and 2x10 ^6 cells /mL in static culture. Expansion stage is blocked overnight before use with a solution of PBS with seeded at 10 ^ 5 /ml and includes 3 - 7 progressive volume 10 % fetal bovine serum to prevent protein adhesion to transfers ; 100 ml, 500 ml, 1 L , 10 L , 50 L , 100 L , 100 L . syringe and capillary walls . During the course of production the cell media includes a [0765 ] National Instruments LabVIEW 7 . 1 is used to combination of IMDM , FBS , BSA , holotransferrin , insulin , create a program to control the syringe pump 's injections. glutamine, dexamethasone , beta estradiol, IL - 3 , SCF, and The program ' s basic dosing strategy is an initial injection to erythropoietin . When the cells reach a volume appropriate concentration L1 followed by wait time tl and subsequent for seeding the production bioreactor, they are transferred to injections, each to concentration L2 and followed by wait the production bioreactor for final scale -up and differentia time t2 , repeated for n times . The user inputs the flow rate , tion . the stock concentration , the initial culture volume, the desired concentration after injections, the time between Example 40 : Culturing Cells in a Bioreactor injections, and the total number of injections. (Wave ) [0762 ] The WAVE Bioreactor 2 / 10 system is set up Example 41 : Assessment of Immunogenicity and according to the operator manual. In brief, the Cellbag is Tolerance Induction assembled on the rocking unit , which is placed on the [0766 ] 1. Tolerance Induction in Mice perfusion module . After inflating the bag with air , the weight [0767 ] In mice , tolerance can be induced by 3 sequential is set to zero . Subsequently , the bag is filled with the intravenous injections with a cell composition of the inven appropriate amount of culturing media and incubated for at tion comprising an antigenic protein , in this example oval least two hours , allowing the media to reach 37 C . The bumin (OVA ) . Naive mice are injected on days - 7 , - 3 and media and cells are transferred to the bag via a transfer flask , - 1 with either free OVA or OVA expressed within the cell a special designed DURAN glass bottle with two ports . In composition of the invention ( cell -OVA ) . Mice are then the upper part of the flask , a filter is connected to the port. immunized to OVA by two injections of the antigen mixed In the other port , by the bottom of the flask , a tube is with poly I: C adjuvant (Invivogen , San Diego , Calif. ) to assembled . The tube one the transfer flask is coupled with induce a strong immune response . the feed connection on the Cellbag . The transfer flask is [0768 ] 2 . Assessment of Antibody Titer maintained in a LAF hood , to decrease the risk of contami [07691 IgG levels in mouse serum are evaluated by stan nation . dard ELISA . Briefly , serum is obtained at various time [0763 ] Before perfusion is started , tubing and containers points from blood samples of mice that have been injected for harvest and feed are connected to the Cellbag . Tubing is with a cell composition of the invention comprising an prepared as follows; a 50 or 70 cm long Saniflex ASTP - ELP antigenic protein , e . g . Ovalbumin (OVA ) , and from mice silicone tubing (Gore /Saniflex AB ) , with an inner and outer that have been injected with free or recombinant OVA . A diameter of 3 . 2 respectively 6 . 4 mm , is equipped with male standard ELISA assay is used , with OVA as the antigen ( 1 luer lock connections in both ends. The silicone tubing is ug /ml in 50 mM carbonate buffer , pH 9 . 7 ) adsorbed onto connected to one end of a C - Flex tube , via a female luer assay plates. Serum samples are serially diluted in the range lock . At the other end of the C - Flex tube a male luer lock is of 1 : 50 - 1 : 200 for pre - treatment or no - treatment serum , and assembled and tubings are thereafter autoclaved . Luer locks 1 : 400 - 1 : 500 ,000 for post- treatment serum and tested in are held in place with zip - ties on all tubes . Prior to perfusion , duplicate. The binding of anti -OVA antibodies in serum to the silicone part is connected to the Cellbag and the C - Flex the adsorbed recombinant OVA is detected colorimetrically part to a 5 L container (Hyclone Labtainer) for both feed and with a secondary anti -mouse immunoglobulin conjugated to harvest. All connections are performed in a laminar airflow horseradish peroxidase , followed by treatment with a chro cabinet . mogenic substrate . [0764 ] Control of environmental and metabolic factors [ 0770 ] 3 . Analysis of T Cell Responses can alter the expression or activity of transcription factors [0771 ] Tolerance is induced to the antigenic protein OVA and gene regulatory proteins of erythroid cells in culture , see as described herein . Mice are euthanized 7 days after the 2nd e . g ., Csaszar et al. , 2009 Biotechnol Bioeng 103 ( 2 ) :402 ; administration of the immunization phase injection of OVA , Csaszar et al. 2012 Cell Stem Cell 10 ( 2 ) :218 . To provide and their spleens are collected . Spleen cell suspensions are control over inputs and outputs in the reactor a micro obtained by straining the organs through a 70 micron cell volume delivery system is created , a key component of strainer and after RBC lysis with a 0 . 8 % ammonium chlo which is a 60 - 80 cm long fused silica capillary ride solution (Stem - Cell Technologies, Grenoble , France ). US 2018 /0271910 A1 Sep . 27 , 2018

All samples are incubated with anti -Fc receptor antibody [0779 ] Assessing Enucleation by Microscopy (Benzidine (purified anti- CD16 / 32 , Ozyme, San Diego , Calif. ) to pre Giemsa ) . Erythroid cells were cultured as described herein . vent non - specific binding prior to incubations with antibod At various time points , cells were collected , washed with ies for analysis . The following monoclonal antibodies ( Abs ) PBS, and spun onto slides using a Cytospin ( Thermo Sci are used for spleen cell staining: PC5 - anti -CD62L (MEL14 ) entific ) . Cells were fixed cells after cytospin with - 20 C and PC7 -anti -CD8 , purchased from Biolegend . OVA pep methanol for 2 min at room temp , rinsed with water, and tide -MHC tetramers (PE -Kb - SIINFEKL tetramers (Seq . ID air - dried . A benzidine tablet (Sigma # D5905 ) was dissolved No . 30 ) ) are purchased from Beckmann Coulter . OVA with 10 mL PBS , to which 10 VaL of H , O , was added . The specific T cells are identified by flow cytometry as cells that solution was filtered with a 0 .22 um syringe filter . The cell are double positive by staining with anti -CD8 and OVA spot on the slide was covered with 300 - 500 uL of benzidine peptide- MCH tetramers. Of this cell population , the percent solution , incubated at room temperature for 1 hr, then age of OVA -specific CD8 T cells that are activated is washed with water . Giemsa stain was diluted determined by the fraction of cells that are positive by ( Sigma# GS500 ) 1 : 20 with water . The cell spot on the slide staining anti -CD62L antibody. was covered with 300 - 500 L Giemsa solution , incubated at [ 0772] 4 . In Vivo T Cell Lysis Assay room temperature for 40 minutes , washed with water , and [ 0773] Naive spleen cells are pulsed with 10 micrograms/ air - dried . Slides were then mounted and sealed before imag ml of SIINFEKL peptide (Seq . ID No . 26 ) (Genscript , ing on a microscope . Piscataway, N . J . ) at 37 C for 1 hour and then labeled with (0780 ] FIG . 12A shows the expansion rate of erythroid 0 . 4 microM CFSE (CFSE low ) . A control population of cells in culture during a seven day window of expansion and untreated splenocytes is labeled with 4 microM CFSE differentiation for cells that contain transgenes ( dashed line (CFSE high ). CFSE low and CFSE high cells are combined and dotted line) and cells that do not contain a transgene in a ratio of 1 : 1 and 1E7 cells per mouse are injected by i . v . ( solid line) . Of note, the expansion rate of cultured cells that route to mice that have previously been tolerized to OVA contain a transgene is indistinguishable from that of cells antigen as described herein or to mice that have been that do not contain a transgene. immunized with OVA antigen as described herein . Sixteen 10781 ] FIG . 12B is a collection of flow cytometry plots for hours later , spleen single -cell suspensions are prepared as cells stained with antibodies against the cell surface differ described herein and analyzed using flow cytometry to entiation markers GPA and CKIT . At this particular stage of determine the CFSE low /CFSE high cell ratio . differentiation , the culture is losing its ?KIT expression and increasing its GPA expression as the cells approach terminal Example 42: Assess Expansion and Differentiation maturation . Of note , cultured cells that contain a transgene of Cultured Erythroid Cells are indistinguishable from those that do not contain a [0774 ] It is important to assess the expansion , differentia transgene by this metric of differentiation . tion , and enucleation in vitro differentiated cells to ensure 10782 ] FIG . 12C is a collection of flow cytometry plots for that the introduction of a transgene does not negatively affect cells stained with an antibody against the surface marker the quality of the cells in culture . Expansion is assessed by GPA and a fluorescent DNA stain . Three cell populations are cell counting . Differentiation is assessed by flow cytometry , evident: ( 1 ) cells that are GPA -high and DNA -low , com Western blot , and RT- PCR . Enucleation is assessed by flow prising enucleated erythroid cells ; (2 ) cells that are GPA cytometry . high and DNA -high , comprising erythroid cells that still [0775 ] Assessing Expansion Rate by Cell Counting . contain genetic material; and ( 3 ) cells that are GPA -low and Erythroid cells are cultured as described herein . At various DNA - high , comprising pyrenocytes or the membrane - en time points , cells are collected , washed with PBS , and capsulated ejected nuclei from enucleated cells . Of note , counted using a Countess Automatic Cell Counter instru cultured cells that contain a transgene are indistinguishable ment (Life Technologies ) . The expansion rate of the cells is from those that do not contain a transgene by this metric of determined by the growth in number of cells over time . enucleation . [0776 ] Assessing Differentiation by Flow Cytometry . [0783 ] The introduction of a transgene into cell culture Erythroid cells are cultured as described herein . At various does not noticeably affect the rate of expansion , the differ time points , cells are collected , washed with PBS , and entiation , or the rate of enucleation of the cells in culture . stained with 1: 100 dilutions of fluorescent antibodies against the cell surface markers GPA (CD235a ) , CKIT (CD117 ) , and Example 43: Assess Hemoglobin Content TR (CD71 ) , purchased from Life Technologies. Labeled [0784 ] 1 . Total Hemoglobin cells were analyzed by flow cytometry as described herein . [0785 ] Erythrocyte hemoglobin content was determined [0777 ] Assessing Differentiation by Western Blot. Eryth by Drabkin ' s reagent (Sigma - Aldrich , product D5941) per roid cells are cultured as described herein . At various time manufacturer ' s instructions. Briefly , blood cells were com points , cells are collected , washed with PBS , lysed with bined with the reagent in an aqueous buffer , mixed thor RIPA buffer , and analyzed by Western Blot as described oughly , and absorbance of light at a wavelength of 540 nm herein using antibodies for differentiation markers GATAI , was measured using a standard spectrophotometer. A soluble GATA2 , Band3 , CD44 , and actin ( Abcam ) . hemoglobin standard curve was used to quantify the hemo [0778 ] Assessing Enucleation by Flow Cytometry. Eryth globin content in the cells . roid cells are cultured as described herein . At various time [0786 ] 2 . Hemoglobin Typing by RT- PCR points , cells are collected , washed with PBS , and stained [0787 ] Cells were lysed and total RNA is collected . with a fluorescent antibody against glycophorin A (Life Reverse Transcription was carried out with the SuperScript Technologies ) and the nucleic acid stain DRAQ5 (Pierce ) at First -Strand Synthesis System for RT -PCR ( Life Technolo manufacturer- recommended dilutions, and analyzed on an gies ) according to manufacturer 's protocol. Briefly, total Attune flow cytometer as described herein . RNA ( 5 ug ) was incubated with 150 ng random hexamer US 2018 /0271910 A1 Sep . 27 , 2018

primer and 10 nmol dNTP mix in 10 UL H2O for five of the presence of polyploid MKs. Differentiated CD34 + minutes at 65 C then 1 minute on ice . The reaction master cells are assessed for the presence of multinucleated mature mixture was prepared with 2 uL 10x RT buffer, 4 uL of 25 MKs by morphology . mM MgCl2 , 2 uL of 0 . 1M DTT , and 1 uL of RNAseOUT. The reaction mixture was added to the RNA / primer mixture , Example 45 : Assessment Differentiation of mixed briefly , and then placed at room temperature for 2 Cultured Platelets — qPCR min . 1 uL (50 units ) of SuperScript II RT was added to each [0792 ] The differentiation state of platelets in culture can tube, mixed , and incubated at 250C . for 10 min . The be assessed by quantitative PCR . Platelet RNA is extracted reaction was incubated at 42 C for 50 min , heat inactivated to further characterize the cultured cells . Total RNA is at 70 C for 15 min , then stored on ice . 1 UL RNase H was extracted using TRIzol reagent ( Invitrogen ). The purity of added and incubated at 37 C for 20 min . This reaction each platelet preparation is assessed by PCR analysis of product, the 1st strand cDNA , was then stored at - 20 C until platelet (GPIIIa ) and leukocyte (CD45 ) markers . The integ needed for RT- PCR reaction . rity of platelet RNA is assessed using Bioanalyzer 2100 [0788 ] Primers to amplify the different hemoglobin genes ( Agilent) prior to further analyses. and control genes were purchased from IDT- DNA . The [0793 ] Total RNA is collected from cell lysate and a primers were as follows: hHBB _ F - tcctgaggagaagtctgccgt cDNA library is generated using a commercial synthesis kit ( Seq . ID No . 9 ) ; hHBB _ R - ggagtggacagatccccaaag (Seq . ID ( Clontech ). The labeled cDNAs are quantified with the No . 10 ) ; hHBA _ F1 -tctcctgccgacaagaccaa (Seq . ID No . 11 ) ; Quant- iT PicoGreen dsDNA Kit ( Invitrogen ) and diluted to hHBA _ R1- gcagtggcttagettgaagttg ( Seq . ID No. 12 ) ; hHBA _ 3 PM for loading into a single lane and sequencing on an F2 -caacttcaagctaagccactgc (Seq . ID No . 13 ) ; hHBA R2 Illumina 16 Genome Analyzer (Solexa ). cggtgctcacagaagccag ( Seq. ID No . 14 ) ; hHBD _ F -gactgctgt [ 0794 ] Raw sequences are filtered through serial quality caatgccctgt ( Seq . ID No. 15 ) ; hHBD _ R control criteria . First, the presence of at least 6 nt of the 3 ' aaaggcacctagcaccttctt (Seq . ID No. 16 ) ; hHBG2_ F Solexa adapter is verified . The sequence reads that did not cactggagctacagacaagaaggtg (Seq . ID No . 17 ) ; hHBG2_ R comply with this criterion are discarded , whereas the others tctcccaccatagaagataccagg (Seq . ID No . 18 ) ; hHBE F are trimmed to remove the adapter sequence harbored at the aagagcctcaggatccagcac (Seq . ID No. 19 ); hHBE _ R 3 ' end . The remaining tags are further filtered regarding their tcagcagtgatggatggacac (Seq . ID No . 20 ) ; h18S - RNA - F length ( > 10 nt ) , copy number ( > 4 reads ) and readability ( < 9 cgcagctaggaataatggaatagg ( Seq . ID No . 21) ; h18S -RNA - R non - identified nucleotides , annotated N ). Reads complying catggcctcagttccgaaa (Seq . ID No . 22 ). with all those criteria are subsequently defined as usable [0789 ] An RT PCR reaction mix was prepared with 25 uL reads . SYBR Green Mix (2x ) (Applied Biosystems) , 0 . 5 uL 1st 10795 ]. All the usable reads are aligned to pre -microRNAs strand cDNA , 2 uL forward /reverse primer pair mix ( each extracted from mirBase database . Sequence tags that primer at 5 umol/ uL ) , in a total volume of 50 UL H2O . matched perfectly to more than one precursor are distributed Reactions were run in an ABI Prism SDS 7000 instrument equally among them . In order to account for Drosha and ( applied biosystems) using the following amplification Dicer imperfect cleavage, any sequence tag that perfectly cycle : 50 C 2 min , 1 cycle ; 95 C 10 min , 1 cycle ; 95 C 15 matched the pre -microRNA in themature microRNA region , S - > 60 C 30 s - > 72 C 30 s , 40 cycles ; 72 C 10 min , 1 cycle . allowing up to 4 nt shift as compared to the reference mature Dissociation curve analysis and RT -PCR results was per microRNA position , is considered as a mature microRNA . formed with the SDS 7000 instrument. The microRNA expression level is defined as the number of reads mapping each mature microRNA normalized to the Example 44 : Assessment Differentiation of total number of usable reads, considering that the overall Cultured Platelets — FACS number of small RNAs is invariant. The relative abundance [0790 ] The differentiation state of platelets in culture can of each microRNA is defined as the number of reads be assessed by flow cytometry. Megakaryocytes (MKs ) mapping each microRNA compared to the total number of represent a distinct cellular morphology that precedes ter reads mapping mature microRNAs . minal platelet differentiation . To determine the extent of Example 46 : Purification by Centrifugation maturation toward MKs, 1x10 ^ 6 cultured cells (LAMA - 84 and CD34 + cells ) are washed and then labeled with ( a ) [0796 ] Cultured cell fractions can be purified and sepa anti- CD41 - FITC (GplIb / Illa ; BD Bioscience , San Jose , rated from nuclei and contaminating alternate - density cell Calif . , USA ) or anti CD71 - FITC or ( b ) anti -CD33 - FITC , types via centrifugation . Cells are centrifuged at 200 g for 15 anti- CD41 - PE , anti -CD45 -PerCp and CD34 -APC (Beck minutes to isolate an erythrocyte and reticulocyte rich frac man Coulter , Fullerton , Calif ., USA ), and analyzed for the tion . The supernatant is pipetted off and the desirable cell percentage of CD41 cells generated . fraction is then washed in modified Tyrode ' s buffer (con [0791 ] To determine the amount of ploidy, differentiated taining 138 mM NaC1, 5 . 5 mM dextrose , 12 mM NaHCO3 , LAMA - 84 cells are fixed overnight in 75 % ethanol at 4° C . 0 . 8 mM CaC12 , 0 . 4 mM MgCl2 , 2 . 9 mM KC12 , 0 . 36 mm and labeled with propidium iodide (PI , 50 ug / ml) and Na2HPO4 and 20 mM Hepes, pH 7 . 4 ) in presence of 1 uM analyzed using the FACScalibur (Becton Dickinson ) , prostaglandin 12 , and resuspended in the same buffer. whereas day 14 differentiated CD34 + cells are analyzed quantitatively under a microscope after May -Grunwald / Example 47 : Purification by Chemical Enucleation Giemsa staining by quantitating the number of nuclei per [0797 ] Enucleation of cultured cells can be stimulated by cell and specific morphology of MKs with this stain . Only chemical additives to the culture , which can help increase cells with MK morphology are analyzed . The presence of the enucleated fraction of cells prior to purification . Eryth multinucleated cells in the cytospin preparation is indicative roid cells are cultured as described herein . 48 hours prior to US 2018 /0271910 A1 Sep . 27 , 2018 69 collection , cells are incubated with 210 mM Me2SO . Cells Example 49 : Purification by Ex Vivo Maturation are then collected by centrifugation at 350xg for 5 min at [0803 ] Erythroid cells that are not fully mature can be room temperature , resuspended at a level of 3x105 cells per driven to maturity by ex vivo incubation in a system that ml in fresh medium containing 210 mM Me280 and 5 mimics the natural in vivo maturation triggers . ug /mL of cytochalasin B ( or other actin or nucleus manipu [0804 ] 1 . Co -Culture with Stromal Cells lating molecule , ie . p38 MAPK , psoralens ) and incubated at 10805 ] In the final stage of culture , erythroid cells are 37 C . The proportion of cells without nuclei is assessed by cultured on an adherent stromal layer in fresh medium flow cytometry as described herein , using DRAQ5 as a without cytokines . The cultures are maintained at 37 C in 5 % nucleic acid stain and antibodies against glycophorin A as an CO2 in air. The adherent cell layer consists of either the erythroid surface marker of differentiation . MS - 5 stromal cell line or mesenchymal stromal cells MSCs( ) established from whole normal adult bone marrow Example 48 : Purification by Acoustophoresis ( see Prockop , DJ ( 1997 ) Science 276 :71 ) in RPMI ( Invit rogen ) supplemented with 10 % fetal calf serum . Adherent [0798 ] Several mechanical separation systems may be MSCs are expanded and purified through at least two used to obtain a uniform cell population . Free flow acous successive passages prior to use in co - culture . tophoresis represents one mechanical separation method [0806 ] 2 . Culture in Fibronectin - Coated Plates (Petersson 2007, American Chemical Society ) . While sus [0807 ] In the final stage of culture , erythroid cells are pended in saline solution ( 0 . 9 mg/ mL ) with nutrient addi cultured in plates adsorbed with human fibronectin . To tives, including CsC1 ( 0 . 22 g /mL ), is added to the saline produce these plates, fibronectin (Sigma Aldrich ) is recon solution . A sample suspension containing cultured erythroid stituted with 1 mL sterile H2O /mg of protein and allowed to cells is processed using an acoustopheresis chip (Cell - Care ) dissolve for at least 30 minutes at 37° C . A small amount of with two active outlets ( flow rate 0 . 10 mL /min per outlet ) . undissolved material may remain . This will not affect prod [0799 ] Syringe pumps (WPI SP260P , World Precision uct performance . The fibronectin solution is diluted 100x in Instruments Inc. , Sarasota , Fla . ) are used to control the flow sterile balanced salt solution and added to the culture surface rates in the chip . All outlets are individually connected to with a minimal volume. The culture surface is allowed to air high -precision glass syringes ( 1005 TLL and 1010 TLL , dry for at least 45 minutes at room temperature . Excess Hamilton Bonaduz AG , Bonaduz , Switzerland ) via the injec fibronectin is removed by aspiration . tors using Teflon tubing , allowing independent control of the outlet flow rates . The clean fluid inlet is connected to a Example 50 : Purification by Magnetophoresis syringe pump and the cell suspension inlet to a 50 -mm - long [0808 ] Strategies for separating , enriching , and /or purify piece of Teflon tubing ( 0 . 3 -mm i. d . ) with its other end ing erythroid cells by magnetophoresis are known in the art , submerged in a beaker from which the sample suspension is see e . g ., Zborowski et al. , 2003, Biophys J 84 ( 4 ) 2638 and aspirated at a rate defined by the difference between the net Jin & Chalmers 2012 , PLOS One 2012 7 (8 ): e39491 . A outlet flow and the clean fluid inlet flow . commercial magnetic separation system ( Quadro MACSTM Separator combining four MidiMACSTM separation units [ 0800 ] The ultrasound used to induce the standing wave and LD columns, Miltenyi Biotec , Auburn , Calif. ) is used between the walls of the separation channel is generated for magnetic erythrocyte enrichment from HSC - derived using a 20x20 mm piezoelectric ceramic (Pz26 , Ferroperm erythrocyte cultures . Cells are deoxygenated in a Glove Piezoceramics AS , Kvistgard , Denmark ) attached to the BagTM inflatable glove chamber ( Cole Parmer , Vernon Hills , back side of the chip . Ultrasonic gel ( Aquasonic Clear, I11 . ) , filled with nitrogen (MedipureTM nitrogen , concentra Parker Laboratories Inc ., Fairfield , N . J . ) ensures a good tion > 99 % , Praxair , Inc . , Danbury , Conn . ) . Before deoxy acoustic coupling between the two . The piezoelectric genation , all materials and equipment including the separa ceramic is actuated via a power amplifier (model 75A250 , tion system , degassed sterile buffer ( PBS + 2 mM EDTA Amplifier Research , Souderton , Pa. ) connected to a function + 0 . 5 % BSA ) , and sterile collection tubes are placed in the generator (HP 3325A , Hewlett -Packard Inc. , Palo Alto , glove bag , which is then tightly sealed . Deoxygenated Calif .) . Even though the acoustic waves enter the chip from cultures are loaded directly into a MACS® LD column the back side , a standing wave is induced between the side which was placed in the QuadroMACSTM separator kept walls of the separation channel as a result of the coupling of under anoxic conditions inside an inflatable glove chamber the mechanical vibrations along the three axes of the crystal filled with N2 gas . Cells which pass through the column structure . contained within the magnet are labeled as negative fraction [ 0801] The separation process is monitored using a stan and they are expected to be “ non -magnetic ” , including dard microscope and a wattmeter ( 43 Thruline Wattmeter , HSCs and erythroid cells before final maturation . The cells Bird Electronic Corp . , Cleveland , Ohio ) . The process can retained in the separation column are labeled as positive subsequently be controlled by tuning the signal frequency , fraction , which is “ magnetic ” and consist of maturing RBC the actuation power, and the flow rates . like cells nearly full of functional hemoglobin . They are [ 0802] The cell size distributions in the samples are ana eluted from LD column after its removal from the magnet . lyzed using a Coulter counter (Multisizer 3 , Beckman Once separation is finished , oxygenated cells are reversibly Coulter Inc . , Fullerton , Calif . ) . Each sample is mixed with recovered by exposing the collected cells to air . an electrolyte ( Isoton II , Beckman Coulter Inc . ) and ana lyzed using a 100 -um aperture . The level of hemolysis , i. e ., Example 51 : Purification by FACS the concentration of free hemoglobin from damaged red [0809 ] A population of erythroid cultured cells is sorted cells , is measured using a photometer (Plasma / low HB using a Becton -Dickinson Aria Ilu cell sorter. Prior to Photometer, HemoCue AB , Angelholm , Sweden ). sorting , cells are collected , washed with PBS, and stained US 2018 /0271910 A1 Sep . 27 , 2018 70 with a fluorescent antibody against glycophorin A (Life washing , RBC are reacted with FITC - labeled anti -human Technologies ) and the nucleic acid stain DRAQ5 (Pierce ) at IgG / IgM (DAKO , Glostrup , Denmark ) ( x30 , 100 uL ) for 30 manufacturer -recommended dilutions . A 100 m nozzle is min at 4° C . and then subjected to flow cytometric analysis . used with a drop drive frequency of 28 , 000 drops /second . [ 0815 ] The inhibitory effect of enzyme treatment on The sample threshold rate is approximately 4000 events / complement activation is also evaluated by the change of second . The temperature control option is used to maintain C3d deposition . After RBC are incubated with 50 % human sample and collection tubes at 4° C . the entire duration of sera in the presence of complement activity for 15 min at 37° sorting . Additionally , the sample agitation feature is used at C . , RBC are reacted with FITC - labeled rabbit anti- human 200 rpm to prevent the sample from sedimenting throughout C3d Ab (DAKO , Glostrup , Denmark ) (x100 , 100 uL ) for 30 the sort . The sample is sorted in aliquots of approximately min at 4° C . and then applied to flow cytometric analysis . 750 ul dispensed from the syringe . Meanwhile , during these The percentage of the control level ( in the absence of pauses the collection tubes are kept at 4° C . , protected from enzyme) is calculated based on MFI to evaluate the inhibi the light , and gently mixed prior to resuming sort . The sorted tory effect of enzyme treatment on Ab binding and C3d samples are collected into a 12x75 mm borosilicate glass deposition . collection tube containing 250 ul DMEM supplemented with 10 % FCS . Example 52 : Purification of Platelets by Centrifugation Example 51 : Purification by Enzymatic Treatment of Cells [ 0816 ] Platelets can be purified from mixed cell suspen sions by centrifugation . Some 40 ml of whole blood is [ 0810 ) Allogeneic erythrocyte sourcing may benefit from distributed in blood collection tubes with sodium citrate at A and B antigen removal to generate a universally compat 3 . 2 % used as an anticoagulant. The tubes are centrifuged at ible product. This may be facilitated by a set of enzymes 400xg for 10 min . After this stage , three layers are clearly capable of selectively cleaving the galactose groups, ren demarcated : plasma, red blood cells , and an intermediate dering the erythroid cells more immunogenically favorable . zone . The plasma is at the top with the platelets, the red [ 0811 ] Two types of recombinant proteins of endo - B blood cells are at the bottom because of their heavier galactosidase , which are originally identified from density ; and the fine , whitish intermediate zone consists of Clostridium perfringens, are produced in E . coli BL -21 larger platelets and leukocytes and is called the buffy coat. using standard cloning methods . ABase is prepared for Using a Jelco 18G needle, the upper portion of plasma with releasing A / B Ag and endo - B - galactosidase C (EndoGalC ) platelets is drawn off , and the buffy coat is placed into two for releasing Gala1 - 3Galß1 - 4GlcNAc (Gal Ag ) , which is other tubes , this time with no additives: one tube to produce known to be highly immunogenic in xenotransplantation , plasma ( P tube ) and the other to produce thrombin ( T tube ). and has a carbohydrate structure resembling the A / B Ag . Only 1 . 5 ml of plasma is used to produce thrombin , to which ABase cleaves Galß1 -4GlcNAc linkage in blood type A 0 . 5 ml of calcium gluconate at 10 % is added , with 15 min [GalNAca1 - 3 (Fuca1 - 2 ) Galß1 - 4GlcNAc ) and in blood type in a double boiler at 37° C . The two tubes are then centri B [Gala1 - 3 ( Fuca1- 2 ) Galß1- 4GlcNAc ] . fuged again , this time at 800xg , for the same length of time [0812 ] Briefly , after cloning of ABase , an expression plas ( T = 10 min ) . After this final centrifugation , the T tube mid with a C - terminal His tag is constructed in the PET- 15b contains a thrombin - rich liquid while the P tube contains the vector eabC without signal peptide . This exogenous gene is platelet sedimentation and some red blood cells ( erythro transformed into E . coli BL -21 cells . The enzyme produced cyte - platelet clump ) . The volume is reduced at this stage by in the cells as a soluble protein fraction is purified over a removing two - thirds of the total plasma volume. The portion nickel- nitrilotriacetic acid column (QIAGEN GmbH , removed is platelet poor , while the remaining portion with Hilden , Germany ) . Finally , 5 mL of purified recombinant the sedimented platelets ( that are easily dispersible by ABase is obtained at the concentration of 3 . 6 mg/ mL with stirring ) is platelet rich . the specific activity of 1500 U /mg . One unit of the enzy matic activity is defined as the amount of the enzyme Example 53: Thymidine Incorporation required to hydrolyze 1 umol of the substrate per min . [ 0813] The effect of ABase treatment on Ag presence, Ab [0817 ] Self - replication potential of a cell population can binding and complement activation is examined . Human be assessed using a thymidine incorporation assay known in A / B RBC are digested with ABase and subjected to flow the art , see e . g . , Harkonen et al. 1991 Exp Cell Res 186L288 cytometric analysis after incubation with cross - reactive and Tanaka et al. 1992 PNAS 89 :8928 . (anti - A or anti- B or anti- A and B containing ; type B , type A [0818 ] Briefly , uniformly 13C - and 15N - enriched thymi or type O respectively ) human sera . The mean fluorescence dine [ U - 13C , 15N - TAR ] is obtained from Martek Biosci intensity (MFI ) is used to quantitate the expression level of ences ( Columbia , Md . ) , and 3 H - TdR ( 80 Ci/ mmol ) is blood type A , B and Gal Ag . Digestion level is expressed as purchased from ICN Radiochemicals ( Irvine , Calif .) . Media a percentage of blood type A or B Ag expressed on RBC and buffers are obtained from Fisher Scientific ( Pittsburgh , after incubation in the absence of ABase . Pa . ) . All enzymes except phosphodiesterase are from Boe [0814 ] Fresh blood type o sera are pooled from three hinger Mannheim ( Indianapolis , Ind . ) . Phosphodiesterase II healthy human volunteers and frozen at - 80° C . to preserve is obtained from Worthington Biochemical Corporation endogenous complement activity until used . Heat- inacti (Lakewood , N . J . ) . High - performance liquid chomatography vated ( for 30 min at 56° C .) sera are used for analysis of Ab (HPLC ) solvents are from EM Science (Gibbstown , N . J. ) binding . RBC with and without enzyme (ABase ) digestion and contained < 0 . 1 ppm evaporation residue . are incubated with 50 % blood type O sera ( 100 uL ) diluted [0819 ] Erythroid cells are cultured as described herein . with phosphate - buffered saline containing 0 . 2 % bovine Following the culture , cells are collected for use in the serum albumin ( PBS /BSA ) for 30 min at 37° C . After thymidine incorporation assay . US 2018 /0271910 A1 Sep . 27 , 2018

[0820 ] Cells are labeled with [U - 13C , 15N ]- TdR at 1 .6 The enrichment of CO2 evolved from each DNA -derived ug /ml for 18 h , with the addition of unenriched thymidine to deoxynucleoside is computed by the equation ( 13 )CO2 ( per achieve a final thymidine concentration of 1 uM . After they mil ) = 1000x (IR experimental - IR std )/ IRstd . To maintain the are washed with phosphate -buffered saline , the cells are highest level of internal consistency and avoid any interex cultured in supplemented DMEM for 6 h more before 3 perimental drift , the isotope ratio for dG is subtracted across H - TdR is added at the indicated concentrations ( 0 . 1 - 10 all experiments from the isotope ratio for T . The data from uCi/ml ) for another 18 - h incubation . Unlabeled thymidine is the end of the stable - isotope labeling period (day 0 ) to the added to the samples to bring the final thymidine concen end of the washout ( day 3 ) are evaluated . tration to 0 . 13 uM , which is equivalent to the concentration of 3 H - TdR in the samples receiving 10 uCi radiolabel/ ml . Example 54 : Quantification of Nuclear Material After removal of 3 H - TR, the cells are incubated in [0826 ] The number of cells in a mixed population that supplemented DMEM for an additional 6 - 54 h before iso contain DNA is assessed by flow cytometry using the DNA lation of DNA . stain DRAQ5 ( Pierce ) . Cells are incubated with the stain per [ 0821 ] DNA is extracted using the modified Puregene manufacturer ' s instructions and analyzed on a flow cytom DNA isolation kit (Gentra Systems, Minneapolis , Minn . ). eter , e .g ., an Attune cytometer (Life Technologies ). The Based on the number of cells in the sample , a scale - up / percentage of cells above a predefined threshold of nuclear scale -down procedure is used to determine the added reagent material content is quantified . volumes . For example , when 1x10 ^ 7 cells are used , 21 ul containing 328 ug of proteinase K is added to 3 ml of cell Example 55 : Tumorigenicity Assay In Vitro lysis solution . After mixing , the sample is left overnight at room temperature . The following day , 10 ug of RNase is [ 0827 ] To assess the replication potential of cells , a soft added and the sample is mixed and incubated for 2 h at 37 agar colony formation assay can be performed . In brief , a C . Protein precipitation solution ( 1 ml) is added , and the base agar layer is made by making a 0 . 5 % Agar + 1xRPMI+ sample is incubated on ice for 5 min . After centrifugation for 10 % FCS solution , all components warmed to 40 C , and 10 min at 2000 g , the supernatant containing DNA is mixed adding 1 . 5 mL of the solution to a 35 mm petri dish . The agar with 3 ml 100 % 2 - propanol and gently inverted 50 times or is allowed to solidify for 30 min at room temp before use . until white threads of DNA became visible . The sample is [0828 ] The top agarose layer is prepared by melting 0 . 7 % then centrifuged at 2000 g for 5 min . The resultant DNA agarose in a microwave and cooling to 40 C . A 2xRPMI+ pellet is dried for 5 min before washing in 3 ml of 70 % 20 % FCS solution is heated to 40 C . Cells are counted and ethanol and recentrifugation for 5 min at 2000 g . The final prepared for plating at 5000 cells per plate at a density of pellet is air - dried and then rehydrated in deionized H20 and 200 , 000 cells per mL . 0 . 1 mL of cell suspension is added to quantitated by absorption at 260 nm . The same procedure is 10 mL tubes, followed by 3 mL of the warm 0 . 7 % Agarose applied to CD34 + stem cells as a control for replicative and 3 mL of the warm RPMI/FCS solution . The solution is ability . mixed gently by swirling and added ( 1 . 5 mL ) to each of [0822 ] Any DNA is denatured by boiling for 3 min , then three or four replicate base agar plates . chilled rapidly on ice . The enzymatic hydrolysis procedure (0829 ] Plates are incubated at 37 C in a humidified incu is carried out with a DNA concentration of 0 .5 mg/ml . The bator for 10 - 30 days. Cells are fed 1 - 2 times per week with following protocol describes volume of reagent added per cell culture media , 0 . 75 mL /plate . milliliter of DNA solution . DNA is hydrolyzed with 10 ul of 0830 ] To assess colony formation , plates are stained with nuclease P1 ( 0 . 5 U / ul) and 5 ul of DNase I ( 4 U / ul) in 10 ul 0 . 5 mL of 0 .005 % Crystal Violet for > 1 hr. Colonies are of buffer containing 200 mM MgCl2 , 100 mM ZnCl2 , and counted using a dissecting microscope . 1M Tris , pH 7 . 2 , for 2 h at 45° C ., followed by addition of 20 ul phosphodiesterase ( 4 mU /ul ) and further incubation for Example 56 : Tumorigenicity Assay In Vivo 2 h at 37° C . Finally , 5 ul of 10M ammonium acetate (pH [ 0831] Terminally - differentiated cultured erythroid cells 9 .0 ) and 10 ul of alkaline phosphatase ( 1 U /ul ) are added , are implanted in various animal models to evaluate the and the samples incubated for another 2 h at 37° C . potential for tumorigenicity . Several tissues are collected 10823 ] The digested DNA sample is filtered with a 0 . 22 from the various models and analyzed with histological , um nylon filter . This sample is analyzed with the HPLC / immunochemical, and fluorescent assays to quantify tum CRI/ IRMS system , using a 4 .6x250 mm Supelcosil LC - 18 - S origenicity . HPLC column (Supelco , Bellefonte , Pa . ) . The same solvent [0832 ] Animals receive daily intraperitoneal injections of system is used at 1 ml/min and a linear gradient of 5 % to CSA ( 10 mg/kg , Sandimmune , Novatis Pharma, Nirnberg ) 25 % B in 15 min . starting two days before grafting . For the depletion of NK [ 0824 ] After separation by HPLC , the deoxynucleosides cells , some rats receive, in addition to CSA intraperitoneal are analyzed using chemical reaction interface mass spec injections of the monoclonal antibody (mAb ), anti -NKR trometry (CRIMS ) . In this process , the deoxynucleosides P1A ( clone 10 / 78 , mouse IgG , , BD Biosciences, Heidel flow into a nebulization and desolvation system driven by a berg , Germany ) or the respective isotype control ( clone stream of helium , where they emerge as a dry particle beam . PPV -06 , mouse IgG , Exbio , Prague , Czech Republic ) . The The 13C02 / 12C02 abundances from this in - line generated anti -NKR - P1A mAb ( clone 10 /78 ) is directed against the CO2 are determined with a Finnigan /MAT Delta S isotope same epitope as the mAb (clone 3 . 2 . 3 ) . One mg of the ratio mass spectrometer ( ThermoFinnigan , San Jose , Calif. ) respective antibodies are given one day before the injection and its accompanying Isodat data system . 5 -Fluorodeoxyu of erythroid cells followed by 0 .5 mg at day 4 after cell ridine ( Sigma) is used as an internal isotope ratio standard . transplantation . [ 0825 ] Isotope ratios ( IR in equation that follows) for three 10833 ] Blood samples are taken before starting these nucleosides are obtained from each sample : T , DA , and dG . experiments , at day 0 and 4 days after erythroid cell trans US 2018 /0271910 A1 Sep . 27 , 2018 72 plantation , and at autopsy ( day 92 ) in order to determine the of factors, such as surface area, volume, internal viscosity , proportion of NK cells in the blood by flow cytometry . For and mechanical properties of the cell membrane. the analysis of subcutaneous tumor growth erythroid cells [ 0842 ] Onyp is the osmolality at which the DI reaches half are injected in 100 ul phosphate -buffered saline (PBS ) into of its maximum value . This gives an indication of the the flank of the animals . Tumor growth is monitored every position of the hypertonic part of the curve, which is related second day by palpation and size is recorded using linear to the internal viscosity of the cell as well as mechanical calipers . Animals are sacrificed before day 100 when a properties of the membrane, such as how it will bend under tumor volume of 1 cm in mice and 5 cm in rats is reached , force ( stiffness ) . when a weight loss of more than 10 % occurs , or when any [0843 ] The parameters obtained for the cultured erythroid behavioral signs of pain or suffering are observable . Autop cells are compared to the same values for primary erythroid sies of all animals are performed . cells . [0834 ] Murine tissue near the site of injection is immedi ately frozen in liquid nitrogen or placed in phosphate Example 58 : Deformability by LORCA buffered 4 % formalin for 16 h and then embedded in paraffin . Spleens and lymph nodes are removed for subse [0844 ] The deformability of purified CRBC populations is quent immunological analyses. The transplantation of eryth measured by a laser diffraction technique (LORCA , laser roid cells into the striatum of unilaterally 6 -OHDA - lesioned assisted optical rotational cell analyzer , R & R Mechano rats is performed . These animals are sacrificed 6 weeks after trics ). In brief, a highly diluted suspension of cells is sheared transplantation . in a Couette system with a gap of 0 . 3 mm between 2 [ 0835 ] Animal tissue is analyzed by flow cytometry . cylinders , one of which is able to rotate to induce shear Appropriate fluorescent and PE -conjugated antibodies stresses . A laser beam is passed through the suspension , and against established cancer cell biomarkers of CD133 , CD3, the diffraction pattern is measured at 37° C . At low shear CD , CD16 , CD19 , CD20 , CD56 , CD44 , CD24 , and CD133 stress , the cells are circular disks, whereas at high shear are added to the excised tissues samples and analyzed to stress , the cells become elliptical. The cell deformability is quantify tumorigenic potential. expressed in terms of the elongation index (EI ) , which depends on the ellipticity of the deforming cells. Aliquots Example 57 : Deformability by EKTA containing 12 . 5 uL of pelleted RBC pellets are diluted in 5 [ 0836 ] Erythroid cells cultured as described herein are mL of polyvinylpyrrolidone solution (molecular weight 360 assessed for deformability characteristics relative to natural 000 ) . The EI values at 30 Pa (referred to as Elmax ) and 3 Pa erythrocyte samples via ektacytometry . are selected as representative values of the deformability for [ 0837 ). The ektacytometer consists of a Couette - type vis easy comparison between samples at various shear stresses. cometer combined with a helium -neon laser used to produce a diffraction image of red cells suspended in a viscous fluid Example 59: Assessment of Vascular between the two cylinders . When the viscometer rotates, Occlusion - Ex Vivo Rat Vasculature normal red cells elongate in the shear field , causing the [0845 ] The potential for vascular occlusion of erythroid diffraction image to become elliptical. The ellipticity of the cells can be assessed with isolated artificially perfused rat image is measured by quantifying the light intensity along vasculature using methods known in the art , see e . g . , Kaul the major ( A ) and minor ( B ) axes of the diffraction pattern et al. 1983 , J Clin Invest 72 :22 . Briefly , in anesthetized and expressing this as a ratio ( A - B )/ ( A + B ) , the deformabil ( sodium pentabarbitol 30 mg/ kg ) rats of the Wistar strain , ity index ( DI) or elongation index (EI ) . The viscosity of the 120 - 150 g , the right ileocolic artery and vein are cannulated medium is chosen to be greater than the internal viscosity of with heparinized ( 100 L /mL ) silastic tubing at a site 3 cm the densest erythroid cells . A 31 g / liter solution of polyvi distant from the ileocolic junction . Under a steady - state nylpyrrolidone (PVP ) , mw = 360 , 000 , in a phosphate buffer perfusion with Ringer ' s containing 1 % bovine serum albu of 0 .04M composed of K2HPO4 and KH2P04 in distilled min , the ascending colon and terminal ileum ( 3 cm each ) are water yields a viscosity of 0 . 20 poise at 25° C . and 12 poise sectioned between ties . After hemostatic ties of all vascular at 37 C . connections is achieved , the tissue is isolated . The isolated [0838 ] Osmolarity is adjusted with NaCl to the desired mesoappendix is gently spread on an optically clear Lucite level and measured in a Roebling freezing - point osmometer. block on a microscope stage . The entire preparation is The final pH is varied by using small additions of 1- M covered with a plastic saran wrap except for outlets of solutions of NaOH and HCl and is measured in a Technicon cannulas and the microscope objective . BG II blood gas analyzer. Sodium azide is added as a 10846 ] The control arterial perfusion pressure ( Ppa ) and preservative to stock solutions to obtain 0 . 4 g / l . venous outflow pressures (Pv ) are kept constant at 80 and [ 0839] The ektacytometer collects three primary metrics 3 . 8 mmHg, respectively , and monitored via Statham -Gould from the erythroid cell samples and compares them to native P - 50 pressure transducers (Stathan Instruments Inc , Oxnard erythrocytes; Osmolality minimum (Omin ), deformability Calif .) . The venous outflow (Fv ) rate is monitored using a index (Dim ) , and the osmolality at which the DI reaches photoelectric dropcounter and expressed in mL /min . A lapse half of its maximum value (Ohyp ). of 10 - 12 min is allowed for tissue equilibration and stabi [0840 ] Omin is related to the surface area to volume ratio lization of Fv. Only preparations exhibiting mesoappendix of the cell and has been found to equal the 50 % hemolysis microvasculature free of host blood cells and with a steady point in the classical osmotic fragility test . Fv of 4 . 6 + / - 0 . 5 (mean + / - SD ) are used . The experiments are [ 0841 ] Dimax is the maximum value of the deformability done at 37 C . index , normally reached at 290 mosmol (the physiologic [0847 ] Erythroid cells are isolated as described herein . osmolality value ). This indicates the maximum deformabil After control measurements of Ppa and Fv, erythroid cells ity of the cell under shear stress and is related to a number (0 .2 mL , Hct 30 % ) are gently delivered via an injection port