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US 20170152480A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0152480 A1 Jensen (43) Pub. Date: Jun. 1, 2017

(54) TRUNCATED EPIDERMAL GROWTH (52) U.S. Cl. FACTOR RECEPTOR (EGFRT) FOR CPC ...... CI2N5/0636 (2013.01); C07K 14/71 TRANSDUCED T CELL SELECTION (2013.01); A61K 35/17 (2013.01); C12N 2510/00 (2013.01) (71) Applicant: City of Hope, Duarte, CA (US) (72) Inventor: Michael C. Jensen, Duarte, CA (US) (57) ABSTRACT (21) Appl. No.: 15/431,248 A non-immunogenic selection epitope may be generated by (22) Filed: Feb. 13, 2017 removing certain amino acid sequences of the protein. For example, a gene encoding a truncated human epidermal Related U.S. Application Data receptor polypeptide (EGFRt) that lacks the (60) Division of application No. 14/340,512, filed on Jul. membrane distal EGF-binding domain and the cytoplasmic 24, 2014, now Pat. No. 9,580,685, which is a division signaling tail, but retains an extracellular epitope recognized of application No. 13/463,247, filed on May 3, 2012, by an anti-EGFR antibody is provided. Cells may be geneti now Pat. No. 8,802.374, which is a continuation of cally modified to express EGFRt and then purified without application No. PCT/US2010/055329, filed on Nov. the immunoactivity that would accompany the use of full 3, 2010. length EGFR immunoactivity. Through flow cytometric analysis, EGFRt was successfully utilized as an in vivo (60) Provisional application No. 61/257.567, filed on Nov. tracking marker for genetically modified human T cell 3, 2009. engraftment in mice. Furthermore, EGFRt was demon strated to have cellular depletion potential through cetux Publication Classification imab mediated antibody dependent cellular cytotoxicity (51) Int. C. (ADCC) pathways. Thus, EGFRt may be used as a non CI2N 5/0783 (2006.01) immunogenic selection tool, tracking marker, a depletion A 6LX 35/7 (2006.01) tool or a Suicide gene for genetically modified cells having C07K I4/7 (2006.01) therapeutic potential.

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Baseline Evaluations Derivation and Cryopreservation of Autologous Tcm ala Leukapheresis Derived CD9R CAR"/CD8"/EGFRt "Ce Salvage/Priming Il Product & Rituxan' w Mobilization with G-CSF W

Apheresis and Salvage Therapy, HPC Cryopreservation of Mobilization and Autologous HPC Product Collection, Myeloablative Preparative Regimen, HPC w Rescue, and Supportive Care per COH Heme/BMT Myeloablative Program Standard Practice Conditioning Regimen w Infusion of CD34+ selected HSCs (Day 0)

------D T Cell Infusion (Day +2 or +3) < 1.5 x 10° cells/m? US 2017/0152480 A1 Jun. 1, 2017

TRUNCATED EPIDERMAL GROWTH signaling or trafficking domains and/or any extracellular FACTOR RECEPTOR (EGFRT) FOR domains unrecognized by the known antibody. The removal TRANSDUCED T CELL SELECTION of the signaling or trafficking domains and/or any extracel lular domains unrecognized by the known antibody renders PRIORITY CLAIM the endogenous cell-surface molecule inert, which is a 0001. This application is a divisional of U.S. application desired property for the molecule. The non-immunogenic Ser. No. 14/340,512, filed Jul. 24, 2014, which is a divisional selection epitope may also be used for as a selection tool or of U.S. application Ser. No. 13/463,247, filed May 3, 2012, tracking marker. issued as U.S. Pat. No. 8,802.374, which is a continuation of 0006. The modified endogenous cell-surface molecule International Application No. PCT/US2010/055329, filed may be, but is not limited to, any cell-surface related Nov. 3, 2010, which claims the benefit of U.S. Provisional receptor, ligand, glycoprotein, cell adhesion molecule, anti Application No. 61/257.567, filed Nov. 3, 2009, the subject gen, integrin or cluster of differentiation (CD) that is modi matter of which is incorporated by reference as if fully set fied as described herein. In some embodiments, the modified forth herein. endogenous cell-Surface molecule is a truncated tyrosine kinase receptor. In one aspect, the truncated tyrosine kinase TECHNICAL FIELD receptor is a member of the epidermal growth factor receptor family (e.g., ErbB1, ErbB2, ErbB3, ErbB4). 0002 The present products and methods relate to the 0007 Epidermal growth factor receptor, also known as fields of immunology and purification of genetically modi EGFR, ErbB1 and HER1, is a cell-surface receptor for fied cells, specifically to a truncated or otherwise modified members of the epidermal growth factor family of extracel receptor paired with a corresponding antibody, such as a lular ligands. Alterations in EGFR activity have been impli polypeptide derived from human epidermal growth factor cated in certain cancers. In a first aspect, a gene encoding an receptor (EGFR) paired with , for use in cancer EGFR polypeptide comprising human epidermal growth . factor receptor (EGFR) that is constructed by removal of nucleic acid sequences that encode polypeptides including BACKGROUND the membrane distal EGF-binding domain and the cytoplas 0003. Immune cell products with homogenous expres mic signaling tail (a “truncated EGFR’ or “EGFRt”), but sion of tumor targeting chimeric antigen receptors (CARS) retains the extracellular membrane proximal epitope recog are desirable for clinical evaluation of adoptive therapy nized by an anti-EGFR antibody. Preferably, the antibody is strategies to eliminate the product-to-product variability of a known, commercially available anti-EGFR monoclonal transgene expression otherwise intrinsic to transduction and antibody, Such as cetuximab, , or other genetic modification procedures without Subsequent . selection. Immunotherapy using genetically redirected 0008. Application of biotinylated-cetuximab to immuno immune cells is an attractive approach for treating minimal magnetic selection in combination with anti-biotin micro residual disease in a variety of cancer patients. However, beads successfully enriches T cells that have been lentivi immunologic rejection of cell products expressing antibiotic rally transduced with EGFRt-containing constructs from as selection proteins as part of the transduction strategy has low as 2% of the population to greater than 90% purity impeded this strategy. A novel selection marker that is not without observable toxicity to the cell preparation. Consti expressed on human lymphocytes, does not contain endog tutive expression of this inert EGFRt molecule does not enous signaling or trafficking function, and is recognized by affect T cell phenotype or effector function as directed by the a known, preferably commercially available, pharmaceutical coordinately expressed chimeric antigen receptor (CAR), grade antibody reagent that can be utilized for selection, in CD19R. Through flow cytometric analysis, EGFRt was Vivo tracking, and depletion of transduced cells would be a Successfully utilized as an in Vivo tracking marker for T cell significant improvement in the art. engraftment in mice. Furthermore, EGFRt was demon strated to have suicide gene potential through Erbitux(R) SUMMARY mediated antibody dependent cellular cytotoxicity (ADCC) 0004 Products and methods for purification, both in vivo pathways. Thus, EGFRt may be used as a non-immunogenic and ex vivo, of genetically modified cells are provided selection tool, tracking marker, and Suicide gene for trans herein. The genetically modified cells may be modified by duced T cells that have immunotherapeutic potential. The transduction, or any other process that adds, deletes, alters, EGFRt nucleic acid may also be detected by means well or disrupts an endogenous nucleotide sequence. The geneti known in the art. cally modified cells may be transduced T cells with altered 0009. In another embodiment, methods of discovering activity, including altered immunoactivity. and designing modified, truncated or altered endogenous 0005 According to the embodiments described herein, a cell-surface molecules which bind to antibodies, preferably non-immunogenic selection epitope compatible with immu commercially available antibodies, as described herein are nomagnetic selection facilitates immunotherapy in cancer provided. The methods include modeling the protein of patients without undesirable immunologic rejection of cell interest and truncating functional portions, while leaving the products (i.e. as seen when expressing antibiotic selection antibody-binding portions intact. The resulting modified proteins) may be generated by removing certain amino acid receptor or ligand can be sorted using a labeled antibody and sequences of the protein. In some embodiments, the non then enriched such that the concentration of the modified immunogenic selection epitope is a gene encoding an endog receptor or ligand is increased. enous cell-surface molecule that is modified or truncated to 0010 Yet another embodiment provides a method of retain an extracellular epitope recognized by a known anti selecting transduced T cells comprising transducing T cells body or functional fragment thereof, and to remove any with a modified, truncated or altered endogenous cell US 2017/0152480 A1 Jun. 1, 2017

Surface molecule gene sequence (e.g., truncated EGFR) and B occurred after 3 REM stimulations of transduced then applying an antibody that binds the modified ligand or CMVpp65-specific T-derived cells. Selection of receptor sequence to the transduced T cells. If the modified CD19CAREGFRt" Line C occurred after 2 REM stimula receptor sequence is EGFRt, the antibody is preferably a tions of transduced CD8+T-derived cells. Selection of biotinylated anti-EGFR . The T cells CD19CAREGFRt" Line D occurred after 1 REM stimula are then Sorted by adding anti-biotin microbeads and select tion of transduced T-derived cells. Selection of ing the T cells using immunomagnetic separation, adding CD19CAREGFRt IMPDH2dm Line E occurred after 1 fluorochrome-conjugated anti-biotin and selecting the T REM stimulation of transduced T-derived cells. cells using Fluorescence Activated Cell Sorting, or any other (0015 FIGS. 3a-b show that the EGFRt expressed on reliable method of sorting the cells. The modified ligand or selected T cells is inert. In FIG. 3a, EGFRt expressed on T receptor sequences, such as the EGFRt sequence, may be cells is not phosphorylated upon co-incubation with EGF. contained in a suitable transfer vehicle such as a lentiviral Negative control T cells, CD19CAREGFRt" Line A cells, Vector. or A431 cells were incubated for 5 minutes with or without 0011. These and other embodiments are further explained either 100 ng/mL EGF or cetuximab (referred to in the figure in the drawing and detailed description herein. as ErbtX) and then lysed in the presence of phosphatase inhibitor. Lysates run on Western blots were then probed BRIEF DESCRIPTION OF THE DRAWINGS using antibodies specific for either B-actin, the cytoplasmic 0012 FIG. 1 is a molecular model of EGFR vs. EGFRt domain of EGFR, or the phosphorylated tyrosine at position proteins based on the crystal structure files. The EGFR 1068 of EGFR. FIG. 3b shows that EGF does not bind to the structure on the left shows a full-length EGFR with the surface of EGFRt expressing T cells. A431, Line A, and structure of the four extracellular domains (Domains I-IV). negative control T cells were stained with PE-conjugated The middle structure shows the truncated EGFR (EGFRt), anti-EGFR, or either biotinylated cetuximab or biotinylated which is missing Domain I, Domain II, the Juxatmembrane EGF followed by PE-conjugated streptavidin (black histo Domain, and the Tyrosine Kinase Domain as compared to an gram) versus PE-conjugated isotype control Ab or strepta unmodified EGFR. The EGFRt on the right shows truncated vidin alone (open histogram) by flow cytometry. Percent structure bound to Eribitux(R) Fab, comprised of V-C and positive staining is indicated in each histogram. V-C. The domains are separated with dotted lines. 0016 FIGS. 4a-d illustrate that selected EGFRt" 0013 FIGS. 2a-d illustrate the selection of EGFRt. T CD19R T cells can be expanded with maintenance of cells using biotinylated cetuximab (referred to in the figure effector phenotype. FIG. 4a is a line graph showing expan as Erbitux(R). FIG. 2a is a schematic of the cetuximab sion of EGFRt-selected T cells, Lines A-E, over 12 or more biotinylation and reformulation process. FIG. 2b is a graph days after rapid expansion medium (REM) stimulation was showing titration of biotinylated cetuximab. 10° EGFR" initiated on the day of AutoMACSTM selection (day 0). cells were stained with either Oug (black), 1.45 Lug (red), (MACS is magnetic activated cell sorting.) Expansion of T 0.145 lug (orange), 14.5 ng (yellow), 1.45 ng (green), 0.145 cells in rapid expansion medium (REM) involved the incu ng (blue) or 14.5 ug (purple) of biotinylated cetuximab bation of 10° T cells with 30 ng/mL anti-CD3e (OKT3: followed by 0.5 Dug PE-conjugated streptavidin and ana Ortho Biotech, Raritan, N.J.), 5x107 y-irradiated PBMCs lyzed by flow cytometry. 14.5 ng or more of biotinylated (3500 cGy), and 107Y-irradiated LCLs (8000 cGy) in 50 mL cetuximab was deemed sufficient for future staining. FIG.2c CM; with addition of 50 U/mL rhIL-2 and 10 ng/ml rhIL-15 depicts Schematics of both the immunomagnetic (top) and (CellCienix) every 48 hours, beginning on day 1. T cells the fluorescence activated cell sorting (bottom) EGFRt were re-stimulated in this manner every 14 days. FIG. 4b selection procedures. shows histograms representing EGFRt-selected T cells (11 0014 FIG. 2d shows immunomagnetic selection of vari to 13 days after stimulation) that were phenotyped for ous T cell lines lentivirally transduced with CAR and EGFRt surface EGFR (i.e., EGFRt, with biotinylated cetuximab), containing constructs. Schematics of the CD19CAR-T2A Fc (i.e., CAR), and T cell markers CD4 or CD8, (black EGFRt (left) and CD19CAR-T2A-EGFRt-IMPDH2dm histogram) vs. isotype control Ab (open histogram) by flow (right) constructs contained in lentiviral vectors are shown cytometry. Percent positive staining is indicated in each above the corresponding pre- and post-selection flow cyto histogram. “N.D.” indicates no data. FIG. 4C are five lines metric analyses for surface EGFRt expression. Codon opti graphs, one for each of Lines A-E, of EGFRt-selected T cells mized sequence portions of the CD19-specific, CD28 co (within 11 to 15 days after REM stimulation) incubated for stimulatory CAR, followed by the self-cleavable T2A, 4 hours with 'Cr-labled NS0, U251T, CD19t-expressing EGFRt and IMPDH2dm selection markers are indicated, NS0, CMV pp65-expressing U251T, CD19-expressing along with the Elongation Factor 1 promoter sequences Daudi or SupB15, or OKT3-expressing LCL cells as targets (EF-1p), and the GCSFR alpha chain signal sequences at the indicated E:T ratios. Chromium release was measured (GCSFRSs, which directs surface expression). Flow cyto to determine cytotoxic activity. FIG. 4d is a graph showing metric analysis of lentivirally transduced T cell lines that had MPA resistance of the CD19CAREGFRt IMPDH2dm been stained with a biotinylated-cetuximab antibody and Line E. Control T cells that do not express IMPDH2dm and PE-conjugated anti-biotin antibody (black histograms) was EGFRt-selected IMPDH2dm-expressing Line E cells were performed on both the input T cells (PRE SLXN) and the cultured either with or without 1 uM MPA and total cell positive fraction obtained from AutoMACSTM (POS numbers were monitored. FRXN). Open histograms represent staining with PE-con (0017 FIG. 5 shows EGFRt expression can be used as a jugated anti-biotin antibody alone, and the percent positive tracking marker for in vivo T cell engraftment. Day 36 cells are indicated in each histogram. Selection of marrow harvested from a control mouse or from a mouse CD19CAREGFRt" Line A occurred 3 days after transduc that had received 107 CD19CAREGFRt" Line C at day 0 tion of T cell blasts. Selection of CD19CAREGFRt" Line was stained using PerCP-conjugated anti-human CD45 and US 2017/0152480 A1 Jun. 1, 2017 biotinylated cetuximab ("Bio-Erb') followed by PE-conju antibodies and antibody fragments that may be human, gated Streptavidin. Quadrants were created based on isotype mouse, humanized, chimeric, or derived from another spe control staining, and percent positive staining in each quad cies. A "monoclonal antibody' is an antibody obtained from rant is indicated in each histogram. a population of Substantially homogeneous antibodies that is 0018 FIG. 6 is a graph showing EGFRt expression being directed against a specific antigenic site. targets T cells for cetuximab (referred to in the figure as 0027) “Variant” refers to polypeptides having amino acid Erbitux(R) mediated ADCC. Cr-labeled Line A cells were sequences that differ to Some extent from a native sequence pre-incubated either with or without up to 20 g/mL of polypeptide. Ordinarily, amino acid sequence variants will cetuximab or the CD20-specific mAb Rituxan as a negative possess at least about 80% sequence identity, more prefer control prior to addition of human PBMC as effectors. ably, at least about 90% homologous by sequence. The 0019 FIG. 7 is the nucleotide (sense strand is SEQ ID amino acid sequence variants may possess Substitutions, NO: 1, antisense strand is SEQ ID NO: 2) and amino acid deletions, and/or insertions at certain positions within the (SEQ ID NO: 3) sequences of GMCSFR alpha chain signal reference amino acid sequence. sequence linked to EGFRt. The GMCSFR alpha chain signal (0028. “Percentage identity” or “percent identity” is sequence, which directs surface expression, is encoded by defined as the percentage of residues in the amino acid nucleotides 1-66. EGFRt is encoded by nucleotides sequence variant that are identical after best aligning the 67-1071. sequences and introducing gaps, if necessary, to achieve the 0020 FIG. 8 is the nucleotide (sense strand is SEQ ID maximum percent sequence identity. Methods and computer NO: 4, antisense strand is SEQ ID NO: 5) and amino acid programs for the alignment are well known in the art. Such (SEQ ID NO: 6) sequences of CD19R-CD28gg-Zeta(CO)- programs include GAP, BESTFIT. FASTA, BLAST or Align T2A-EGFRt. CD19R-CD28gg-Zeta(CO) is encoded by 2. nucleotides 1-2040: T2A is encoded by nucleotides 2041 0029) “Antibody-dependent cell-mediated cytotoxicity' 2112: GMCSFR is encoded by nucleotides 2113-2178: and "ADCC refer to a cell-mediated reaction in which EGFRt is encoded by nucleotides 2179-3186. nonspecific cytotoxic cells that express Fc receptors, such as 0021 FIG. 9 is a graph showing CD19R-CD28gg-Zeta natural killer cells, neutrophils, and macrophages, recognize (CO)-T2A-EGFRt expression. Transduction of anti-CD3/ bound antibody on a target cell and cause lysis of the target anti-CD28 bead stimulated primary T cell blasts with the cell. ADCC activity may be assessed using methods. Such as CD19R-CD28gg-Zeta(CO)-T2A-EGFRt epHIV7 lentiviral those described in U.S. Pat. No. 5,821,337. vector (MOI-3) results in surface detection of both the CAR 0030 “Effector cells” are leukocytes which express one (using a biotinylated anti-Fc Aband streptavidin-PE) and the or more constant region receptors and perform effector truncated EGFR molecule (using a biotinylated cetuximab functions. Ab and streptavidin-PE) by flow cytometry on day 4. The 0031. To “treat a disease or a disorder, such as cancer, white peak in each panel is non-transduced control T cell means to take either therapeutic measures or preventative blasts. measures to lessen or abate the disease or disorder. Such 0022 FIG. 10 is a schema showing a possible process treatment includes prevention, alleviation of symptoms, flow for clinical trials for testing products of the present diminishment or stabilization of Scope, and/or remission. disclosure. 0032. The term “therapeutically effective amount” refers to an amount of a compound or molecule effective to treat DETAILED DESCRIPTION a disease or disorder. 0023. Certain embodiments of the invention are 0033 “Cancer refers to cells undergoing uncontrolled described in detail, using specific examples, sequences, and cellular growth. Examples of cancer include colorectal can drawings. The enumerated embodiments are not intended to cer and head and neck cancer. A “chemotherapeutic agent' limit the invention to those embodiments, as the invention is is a chemical compound useful in the treatment of cancer. intended to cover all alternatives, modifications, and equiva 0034. A “cytokine' is a protein released by one cell to act lents, which may be included within the scope of the present on another cell as an intercellular mediator. invention as defined by the claims. One skilled in the art will 0035 “Non-immunogenic’ refers to a material that does recognize many methods and materials similar or equivalent not initiate, provoke or enhance an immune response where to those described herein, which could be used in the the immune response includes the adaptive and/or innate practice of the present invention. immune responses. 0024. Erbitux(R) is a registered trademark for the anti 0036. The term “gene' means the segment of DNA EGFR monoclonal antibody cetuximab and is intended to involved in producing a polypeptide chain; it includes independently include the trade name product formulation, regions preceding and following the coding region “leader the generic drug, and the active pharmaceutical ingredient(s) and trailer as well as intervening sequences (introns) of the trade name product. between individual coding segments (exons). Some genes 0025. The term “genetic modification” means any pro may be developed which lack, in whole or in part, introns. cess that adds, deletes, alters, or disrupts an endogenous Some leader sequences may enhance translation of the nucleotide sequence and includes, but is not limited to viral nucleic acid into polypeptides. mediated gene transfer, liposome mediated transfer, trans 0037. The term “isolated means that the material is formation, transfection and transduction, e.g., viral mediated removed from its original environment (e.g., the natural gene transfer such as the use of vectors based on DNA environment if it is naturally occurring). For example, a viruses such as lentivirus, adenovirus, retroviruses, adeno naturally-occurring polynucleotide or polypeptide present in associated virus and herpes virus. a living animal is not isolated, but the same polynucleotide 0026. The term “antibody' includes monoclonal antibod or polypeptide, separated from Some or all of the coexisting ies, polyclonal antibodies, dimers, multimers, multispecific materials in the natural system, is isolated. Such polynucle US 2017/0152480 A1 Jun. 1, 2017 otides could be part of a vector and/or such polynucleotides Surface related receptor, glycoprotein, cell adhesion mol or polypeptides could be part of a composition, and still be ecule, antigen, integrin or cluster of differentiation (CD) that isolated in that Such vector or composition is not part of its is modified as described herein. Modification of such cell natural environment. Surface molecules is accomplished by keeping an epitope 0038. As used herein, a “vector” may be any agent that is recognized by a known antibody or functional frag capable of delivering or maintaining nucleic acid in a host ment thereof, and removing any signaling or trafficking cell, and includes viral vectors (e.g. retroviral vectors, domains and/or any extracellular domains unrecognized by lentiviral vectors, adenoviral vectors, or adeno-associated a known antibody. Removal of the signaling or trafficking viral vectors), plasmids, naked nucleic acids, nucleic acids domains and/or any extracellular domains unrecognized by complexed with polypeptide or other molecules and nucleic a known antibody renders the endogenous cell-surface mol acids immobilized onto Solid phase particles. The appropri ecule non-immunogenic and/or inert. ate DNA sequence may be inserted into the vector by a 0043. Examples of endogenous cell-surface molecules variety of procedures. In general, the DNA sequence is that may be modified or truncated according to the embodi inserted into an appropriate restriction endonuclease site(s) ments described herein include, but are not limited to by procedures known in the art. Such procedures and others EpCAM, VEGFR, integrins (e.g., integrins Ov3, C4. are deemed to be within the scope of those skilled in the art. CIIb?33, C.437, C.5 B1, Civ3, Civ), TNF receptor superfamily Transcription of the DNA encoding the polypeptides of the (e.g., TRAIL-R1, TRAIL-R2), PDGF Receptor, interferon present invention by higher eukaryotes is increased by receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, inserting an enhancer sequence into the vector. Enhancers CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, are cis-acting elements of DNA, usually about from 10 to GD3, or clusters of differentiation (e.g., CD2, CD3, CD4. 300 bp that act on a promoter to increase its transcription. CD5, CD11, CD11a/LFA-1, CD15, CD18/ITGB2, CD19, Examples including the SV40 enhancer on the late side of CD20, CD22, CD23/IgE Receptor, CD25, CD28, CD30, the replication origin bp 100 to 270, a cytomegalovirus early CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, promoter enhancer, the polyoma enhancer on the late side of CD74, CD80, CD125, CD147/basigin, CD152/CTLA-4, the replication origin, and adenovirus enhancers. CD154/CD40L, CD195/CCR5, CD319/SLAMF7). 0039) “Receptor” means a polypeptide that is capable of 0044 Corresponding commercial antibodies that may be specific binding to a molecule. Whereas many receptors may used to recognize a modified or truncated endogenous typically operate on the Surface of a cell. Some receptors cell-surface molecule include, but are not limited to, , may bind ligands when located inside the cell (and prior to , , , afutuzumab, transport to the Surface) or may reside predominantly intra , altumomab pentetate, anatumomab mafena cellularly and bind ligand therein. toX, apolizumab, arcitumomab, aselizumab, atlizumab (to 0040 “Antibody or functional fragment thereof means cilizumab), basiliximab, , benralizumab, an immunoglobulin molecule that specifically binds to, or is besilesomab, , , bren immunologically reactive with a particular antigen or tuximab vedotin, , capromab pen epitope, and includes both polyclonal and monoclonal anti detide, , CC49, cedelizumab, celmoleukin, bodies. The term antibody includes genetically engineered , clenoliximab, clivatuZumab tetrax or otherwise modified forms of immunoglobulins, such as etan, CNTO-95, , , daclizumab, intrabodies, peptibodies, chimeric antibodies, fully human , , , , antibodies, humanized antibodies, and heteroconjugate anti efalizumab, , enlimomab pegol, epitumomab bodies (e.g., bispecific antibodies, diabodies, triabodies, and cituxetan, epratuZumab, erlizumab, , fanole tetrabodies). The term functional antibody fragment Somab, faralimomab, , galiximab, gavili includes antigen binding fragments of antibodies, including momab, , glembatumumab vedo e.g., Fab', F(ab'). Sub.2, Fab, Fv, rigG, and scFv fragments. tin, gomiliximab, ibalizumab, , The term schv refers to a single chain Fv antibody in which igovomab, , , inolimomab, inotu the variable domains of the heavy chain and of the light Zumab ozogamicin, , keliximab, , chain of a traditional two chain antibody have been joined to , , , lumiliximab, form one chain. , maslimomab, , minretu 0041. In one embodiment, a gene encoding a modified momab, , muromonab-CD3, naptumomab endogenous cell-surface molecule that may be used as a estafenatox, natalizumab, ocrelizumab, odulimomab, ofatu non-immunogenic selection epitope compatible with immu mumab, , , , nomagnetic selection is provided. Such a non-immunogenic otelixizumab, , priliximab, PRO 140, ritux selection epitope may facilitate immunotherapy in cancer imab, rovelizumab, ruplizumab, Satumomab pendetide, patients without undesirable immunologic rejection of cell Siplizumab, Sontuzumab, , taplitumomab pap products. The endogenous cell Surface molecule may be tox, teneliximab, teplizumab, TGN1412, ticilimumab modified or truncated to retain an extracellular epitope (tremelimumab), , tocilizumab (atlizumab), recognized by a known antibody or functional fragment toralizumab, to situmomab, tremelimumab, tucotuZumab, thereof, and to remove any signaling or trafficking domains vedolizumab, , visilizumab, , Volocix and/or any extracellular domains unrecognized by said imab, Votumumab, Zanolimumab, Ziralimumab, Zolimomab known antibody. A modified endogenous cell Surface mol aritoX. ecule which lacks a signaling or trafficking domain and/or 0045. In some embodiments, the modified endogenous any extracellular domains unrecognized by said known cell-surface molecule is encoded by a modified or truncated antibody is rendered inert. tyrosine kinase receptor gene. Examples of tyrosine kinase 0042. The modified endogenous cell-surface molecule receptors that may be modified or truncated according to the may be, but is not limited to, any non-immunogenic cell embodiments described herein include, but are not limited US 2017/0152480 A1 Jun. 1, 2017 to, members of the endothelial growth factor receptor family tively, the biotinylated-cetuximab may be used in conjunc (EGRF/ErbB1/HER1; ErbB2/HER2/neu, ErbB3/HER3; tion with Fluorochrome-conjugated anti-biotin for fluores ErbB4/HER4), hepatocyte growth factor receptor (HGFR/ cence activated cell sorting. c-MET) and insulin-like growth factor receptor-1 (IGF-1R). 0050. In another embodiment, a modified endogenous According to some embodiments, modified tyrosine kinase cell-surface molecule may be used as a marker for in vivo T receptors retain an extracellular epitope recognized by a cell engraftment. For example, when the modified endog known antibody or functional fragment thereof, and lack at enous cell-surface molecule is EGFRt, the EGFRt may be least a tyrosine kinase domain. A modified tyrosine kinase used to track the uptake of the T cells to which it is attached receptor which lacks at least a tyrosine kinase domain in vivo without affecting cellular function of the T cells or renders the receptor inert. the cells to which the T cells are targeted, such as bone marrow cells in a transplant situation. The use of cetuximab 0046 Commercial antibodies that may be used to recog conjugated to probes or reporter genes such as Sr39TK may nize a modified tyrosine kinase receptor include, but are not be used to improve the tracking potential of EGFRt-express limited to AMG-102, AMG-479, BIIB022OA-5 D5, CP-751, ing cells to patients via PET imaging techniques. 871, IMC-A12, R1507, cetuximab, , ertumax 0051. In a separate embodiment, a modified endogenous omab, , matuZumab, necitumumab, panitu cell-surface molecule may be used to induce cell suicide. For mumab, , , , example, EGFRt may be used as a Suicide gene via cetux , . imab mediated complement and/or antibody dependent cell 0047. In one embodiment, the modified endogenous cell mediated cytotoxicity (ADCC) pathways. The fact that surface molecule is a truncated EGFR (tGFR) that lacks cetuximab is a therapeutic FDA-approved antibody further the membrane distal EGF-binding domain and the cytoplas facilitates the suicide gene potential of EGFRt in the clinical mic signaling tail, but retains the extracellular membrane Setting. proximal epitope recognized by a known antibody or func 0052. In other embodiments, the truncated epidermal tional fragment thereof (e.g., cetuximab, matuZumab, neci growth factor receptor (EGFRt) selection epitope or other tumumab or panitumumab). In another embodiment, the modified cell-surface molecule is attached to other tEGFR is missing Domain I, Domain II, the Juxtamembrane sequences. One exemplar sequence is the GMCSFR alpha Domain and the Tyrosine Kinase Domain as compared to an chain signal sequence, which directs surface expression, unmodified EGFR (FIG. 1). attached to EGFRt. GMCSFR is encoded by nucleotides 1-66 and EGFRt is encoded by nucleotides 67-1071 of SEQ 0.048. A gene encoding a modified endogenous cell Sur ID NO: 1. See FIG. 7. Also in FIG. 7 is the antisense Strand face molecule may be used as a cell selection or enrichment (SEQ ID NO: 2) and amino acid (SEQ ID NO:3) sequences marker for a genetically modified population of immune of GMCSFR alpha chain signal sequence linked to EGFRt. cells (e.g., T cells). The gene encoding a modified endog Another such sequence is a codon-optimized cDNA enous cell Surface molecule may be coupled to a gene sequence encoding an anti-CD19 costimulatory chimeric encoding a tumor targeting chimeric antigen receptor antigen receptor (CD19R-CD28gg-Zeta(CO)), and a cleav (CAR). These genes may be inserted into a vector to able T2A linker. Cytotoxic T lymphocytes (CTLs) modified transduce the population of T cells to be genetically modi to express a CD19-specific chimeric antigen receptor (CAR) fied. After transduction, the cells that are successfully trans that signals via a cytoplasmic costimulatory (CD28) domain duced and express the CAR and modified endogenous fused to the cytoplasmic CD3- domain exhibits superior cell-surface molecule are enriched by any suitable purifica anti-tumor potency that can be attributed to CD28-mediated tion method, such as immunomagnetic purification with Survival and enhanced cytokine production. This construct anti-biotin microbeads or fluorochrome-conjugated anti-bio may be further modified to incorporate a C-terminal 2A tin for fluorescence activated cell sorting, using a commer cleavable linker followed by the coding sequence for a cial antibody that recognizes the modified endogenous cell truncated human EGFR (EGFRt) for the purpose of immu surface molecule expressed by the transduced cell. nomagnetic purification of CAR-expressing transductants 0049. In another embodiment, a gene encoding a trun using cetuximab-biotin/anti-biotin microbeads. See the cated human epidermal growth factor receptor (EGFRt) that CD19R-CD28gg-Zeta(CO)-T2A-EGFRt sequence attached lacks the membrane distal EGF-binding domain and the as FIG. 8, SEQ ID NOS: 4 (nucleotide sense strand), 5 cytoplasmic signaling tail, but retains the extracellular mem (nucleotide antisense strand), and 6 (protein). Lentivector brane proximal epitope recognized by the FDA-approved transduction of primary human T cells with this codon anti-EGFR monoclonal antibody (mAb) cetuximab or optimized cDNA directs the coordinated expression of the another anti-EGFR antibody, is constructed and described CAR and EGFRt (FIG. 9). herein. The EGFRt may be coupled with chimeric antigen 0053 To eliminate variability between transgene expres receptors specific for a tumor associated antigen. The tumor sion products otherwise intrinsic to transduction procedures associated antigen may be CD19, CD20, or CD22, or any without Subsequent selection, a non-immunogenic selection other tumor associated antigen, but is preferably CD19 epitope, EGFRt, compatible with immunomagnetic selec (CD19CAR). The tumor associated antigen is followed by a tion using the CliniMACS device (Miltenyi Biotec, Bergisch C-terminal 2A cleavable linker and the coding sequence for Gladbach, Germany) was developed. For example, EGFRt is EGFRt. The biotinylated-cetuximab may be used in con a truncated human epidermal growth factor receptor that junction with commercially available anti-biotin microbeads lacks the membrane distal EGF-binding domain and the for the purpose of immunomagnetic purification of the ectoplasmic signaling tail, but retains the extracellular mem tumor associated antigen/CAR-expressing transductants. In brane proximal epitope recognized by the commercial anti the instance where the tumor associated antigen is CD19 the EGFR mAb cetuximab. See FIG. 1. Biotinylated-cetuximab product is CD19CAR-expressing transductants. Alterna is applied to immunomagnetic selection in combination with US 2017/0152480 A1 Jun. 1, 2017

anti-biotin microbeads (Miltenyi). Human OKT3 blasts that PE-conjugated anti-IFNY, PerCP-conjugated anti-CD45 and had been lentivirally transduced with CD19R-CD28gg-Zeta PE-conjugated streptavidin were obtained from BD Biosci (CO)-T2A-EGFRt were subjected to immunomagnetic ences (San Jose, Calif.). Biotinylated anti-Fc was purchased selection using the Miltenyi AutoMACS device, and the from Jackson ImmunoResearch Laboratories, Inc. (West frequency of EGFRt--CAR+ T cells was enriched from 22% grove, Pa.). PE-conjugated anti-Biotin was purchased from (pre-selection) to 99% (post-selection) without observable Miltenyi Biotec (Auburn, Calif.). Biotinylated EGF was toxicity to the cell preparation. It is also possible that, purchased from Molecular Probes(R Invitrogen (Carlsbad, instead of or in addition to immunomagnetic sorting, the Calif.). PE-conjugated anti-EGFR was purchased from EGFRt can be purified using fluorescence-based cell sorting Abcam Inc. (Cambridge, Mass.). All antibodies and biotin techniques. EGF were used according to the manufacturer's instructions. 0054 Due to the absence of the EGF-binding domains Flow cytometric data acquisition was performed on a FAC and intracellular signaling domains, EGFRt is inactive when Scalibur (BD Biosciences), and the percentage of cells in a expressed by T cells. Importantly, the EGFRt-selected T region of analysis was calculated using FCS Express V3 (De cells maintain their desired effector phenotype—including Novo Software, Los Angeles, Calif.). anti-tumor cyotoxic activity mediated by the chimeric anti 0059 For generation of the biotinylated-cetuximab, 200 gen receptor that is coordinately expressed with the mg of cetuximab (Erbitux(R) was buffer exchanged (19 EGFRt—and remain amenable to established expansion hours) to PBS (D-PBS, pH 7.5+0.1) using a MidCee Hoop protocols. Cartridge (UFP-30-E-H42LA) with 527 mL. The material at 0055. Overall, this EGFRt has various advantages for 2 mg/mL was then modified at a 20:1 ratio using Sulfo immunotherapeutic cell products compared to other selec NHS-LC-Biotin in a reaction that was carried out for 1 hour tion markers that have been previously reported. Specifi at room temperature and then diafiltered to remove the cally, unlike truncated CD4 and CD19, it is not endog excess biotin. The 200 mg of biotinylated cetuximab was enously expressed by Subpopulations of lymphocytes. then buffer exchanged (18 hours) to PBS (D-PBS, pH Furthermore, in contrast to truncated CD34 and low affinity 7.5+0.1) using MidCee Hoop Cartridge (UFP-30-E-H42LA) nerve growth factor receptor, it does not have any activity with 533 mL. Glycerol was added to a final concentration of that might negatively affect the immune cell product (i.e., in 20% and then the material was frozen in vials. terms of signaling or trafficking). Lastly, it alone can be bound/recognized by a known, preferably commercially Cell Lines available, pharmaceutical grade antibody reagent, i.e., cetuximab. Together, these attributes make EGFRt a supe 0060. Unless otherwise indicated, all cell lines were rior selection marker for any transfection/transduction sys maintained in RPMI 1640 (Irvine Scientific, Santa Ana, tem that can be applied to the generation of cell products for Calif.) supplemented with 2 mM L-glutamine (Irvine Sci adoptive immunotherapy. Thus, EGFRt is well suited to be entific), 25 mM N-2-hydroxyethylpiperazine-N'-2-ethane used as a selection marker for lentivirally transduced T cells sulfonic acid (HEPES, Irvine Scientific), 100 U/mL peni of immunotherapeutic relevance. cillin, 0.1 mg/mL streptomycin (Irvine Scientific), and 10% 0056. Also provided are methods for identifying new heat-inactivated fetal calf serum (FCS, Hyclone, Logan, therapeutic cell products having the following criteria: a Utah), hereafter referred to as culture media (CM). modified endogenous cell-surface molecule, ligand or recep 0061. To generate T cells, human peripheral blood mono tor that is not, as modified, endogenously expressed in the nuclear cells (PBMC) were isolated by density gradient subject in which it is intended to be therapeutically utilized, centrifugation over Ficoll-Paque (Pharmacia Biotech, Pis does not have any immunoactivity or other functional activ cataway, N.J.) from heparinized peripheral blood obtained ity that would hinder the functioning of the product or the from consented healthy donors participating on a City of subject into which the product is administered, and that it Hope National Medical Center Internal Review Board can be recognized by a known antibody. approved protocol. For generation of Line A, washed PBMC 0057. Having described the invention with reference to were stimulated with 25 U/mL, IL-2 and a 1:1 (cell: bead) the embodiments and illustrative examples, those in the art ratio of Dynabeads(R Human T expander CD3/CD28 (Invit may appreciate modifications to the invention as described rogen, Carlsbad, Calif.). For generation of the other lines, and illustrated that do not depart from the spirit and scope of washed PBMC were first autoMACSTM depleted using anti the invention as disclosed in the specification. The examples CD45RA beads (Miltenyi Biotec) per the manufacturers are set forth to aid in understanding the invention but are not protocol, and in some cases also depleted with PE-conju intended to, and should not be construed to limit its scope in gated anti-CD4 (BDBiosciences) with anti-PE beads (Milte any way. The examples do not include detailed descriptions nyi Biotec). The resulting cells then underwent of conventional methods. Such methods are well known to autoMACSTM positive selection using biotinylated DREG56 those of ordinary skill in the art and are described in (anti-CD62L) and anti-biotin beads (Miltenyi Biotec) to numerous publications. produce purified CD62L"CD45RO" T. CD8" cells were further selected in some cases using AutoMACSTM (Milte Example 1: Generation of EGFRt and nyi Biotec) per the manufacturer's protocol. CMV-specific Immunomagnetic Selection of EGFRt Expressing T cells were generated by stimulating T cells with 5 U/ml Cells rhIL-2 (Chiron, Emeryville, Calif.) and autologous irradi ated viral antigen presenting cells at a 4:1 (responder: Materials & Methods stimulator) ratio once a week for three weeks, using 10% human serum instead of FCS to avoid non-specific stimu Antibodies and Flow Cytometry lation. The viral antigen presenting cells were derived from 0058 FITC-, PE- and PerCP-conjugated isotype controls, PBMC that had been genetically modified to express PerCP-conjugated anti-CD8, FITC conjugated anti-CD4. CMVpp65 antigen. US 2017/0152480 A1 Jun. 1, 2017

0062 PBMC were resuspended in nucleofection solution (Wake Forest, N.C.). U251T-pp65 were generated by lenti using the Human T cell Nucleofector kit (Amaxa Inc., viral transduction of U251T with pp65-2A-eGFP-fluc Gaithersberg, Md.), and 5x107 cells were aliquoted into epHIV7 (p.J01928) at an MOI of 1. The resulting U251T - 0.2-cm cuvettes containing 10 ug HygroR-pp65 pEK (or pp65-2A-eGFP-fluc epHIV7 were then FACS sorted for pmaxGFP from Amaxa Inc., as a transfection control) in a the GFP" population (c.J05058). The Daudi lymphoma line final volume of 100 uL/cuvette, and electroporated using the was purchased from ATCC and grown in media consisting of Amaxa Nucleofector I (Amaxa Inc.), program U-14, after RPMI 1640 (Irvine Scientific), 2 mM L-Glutamine (Irvine which cells were allowed to recover for 6 hours at 37° C. Scientific), 10% heat-inactivated FCS (Hyclone). SupB15 prior to Y-irradiation (1200 cGy). acute lymphoblastic leukemia cells and A431 epidermoid 0063. The CD19CAR-T2A-EGFRt epHIV7 (p.J02104) carcinoma cells were purchased from ATCC. and CD19CAR-T2A-EGFRt-T2A-IMPDH2dm epHIV7 (p.J02111) lentiviral constructs contain a) the chimeric anti Protein Analysis gen receptor (CAR) sequences consisting of the V and V. gene segments of the CD19-specific FmC63 mAb, an IgG1 0065 Cells (up to 107) were lysed with 804 of 1% hinge-C-C, the transmembrane and cytoplasmic signal Triton-X lysis buffer containing phosphatase inhibitor cock ing domains of the costimulatory molecule CD28, and the tail II (Sigma-Aldrich Corp., St. Louis, Mo.) (1:20 of cytoplasmic domain of the CD3 chain 10; b) the self inhibitor to buffer by volume). 50 g of protein was loaded cleaving T2A sequence 11; c) the truncated EGFR in each lane, and Western blots were probed with antibodies sequence (See FIG. 1); and d) the IMPDH2 double mutant from the Phospho-EGF receptor antibody sampler kit (Cell that confers MPA-resistance, as indicated. Lentiviral trans Signaling Technology, Inc., Danvers, Mass.) followed by duction was carried out on T cells that were stimulated with IRDyeTM 680 CW or 800CW conjugated goat anti-rabbit either 30 ng/mL anti-CD3e (OKT3: Ortho Biotech, Raritan, antibodies (LI-COR, Lincoln, Nebr.), as well as the N.J.) (i.e., for Line A) or human CD3/CD28Dynal beads at IRDyeTM 800 conjugated anti-beta-Actin antibody (LI a 1:10 ratio (i.e., for Lines B, C, D and E) and 25 UIL2/ml. COR) as per the manufacturers instructions. Blots were Cells were cultured for up to 2 hours at 37° C. on RetroNec imaged on the Odyssey Infrared Imaging System (LI-COR). tin R (50 ug/ml) coated plates prior to addition of the lentivirus at an MOI of 3 and 5 lug/ml polyybrene. After 4 Chromium-Release Assays hours, warm medium was added to triple to Volume, and the cells were then washed and plated in fresh media after 48 0066. The cytolytic activity of T cells was determined by hours. AutoMACSTM sorting of EGFRt-expressing cells was 4-hour chromium-release assay (CRA), where effector cells carried out with biotinylated cetuximab and anti-biotin were seeded into triplicate wells of V-bottom 96-well micro microbeads (Miltenyi Biotec) as per the manufacturers plates containing 5x10 "Cr-labeled targeT cells instructions. Expansion of T cells in rapid expansion (NaCrO; (5 mCi/mL); Amersham Pharmacia, Piscat medium (REM) involved the incubation of 10 T cells with away, N.J.) at various E:T ratios in 200 uL of CM and 30 ng/mL anti-CD3e (OKT3: Ortho Biotech, Raritan, N.J.), incubated for 4 hours at 5% CO., 37° C. Plates were 5x107 y-irradiated PBMCs (3500 cGy), and 107 y-irradiated centrifuged, and 100 ul of Supernatant was removed from LCLs (8000 cGy) in 50 mL CM; with addition of 50 U/mL each well to assess chromium release using a y-counter rhIL-2 and 10 ng/ml rhIL-15 (CellCienix) every 48 hours, (Packard Cobra II, Downer's Grove, Ill.). The percent spe beginning on day 1. T cells were re-stimulated in this cific lysis was calculated as follows: 100x(experimental manner every 14 days. release-spontaneous release)/(maximum release-spontane 0064 EBV-transformed lymphoblastoid cell lines ous release). Maximum release was determined by measur (LCLs) were made from PBMC as previously described ing the 'Cr content of wells containing labeled targets lysed 13. LCL-OKT3 cells were generated by resuspending LCL with 2% SDS. in nucleofection Solution using the Amaxa Nucleofector kit T, adding OKT3-2A-Hygromycin pRK (p.JO1609) plasmid 0067. Antibody dependent cell mediated cytotoxicity was at 5ug/10" cells, and electroporating cells using the Amaxa determined by chromium release as above using 5x10 Nucleofector I, program T-20. The resulting LCL OKT3 51Cr-labeled targeT cells that had been pre-incubated for 90 2A-Hygro p K (c.J03987) were grown in CM containing min with up to 10 g/mL of either cetuximab or 0.4 mg/ml hygromycin. The mouse myeloma line NSO (gift (a CD20-specific mAb), washed and then co-incubated with from Andrew Raubitschek, City of Hope National Medical 5x10 freshly isolated PBMC. Center, Duarte, Calif.) was resuspended in nucleofection solution using the Nucleofector kit T (Amaxa Inc., Gaith T Cell Engraftment and Cetuximab Mediated Suicide In ersberg, Md.), CD19t-DHFRdm-2A-IL12 pEK (p.J.01607) Vivo or GFP-IMPDH2dm-2A-IL 15 pcDNA3.1 (+) (p.J01043) plasmid was added at 5 g/5x10° cells, and cells were 0068. For T cell engraftment, six- to ten-week old NOD/ electroporated using the Amaxa Nucleofector I, program Scid IL-2RYC" mice are injected i.v. on day 0 with 107 T T-27. The resulting NS0 CD19t-DHFRdm-2A-IL 12 pEK cells (Line C). 2x107 irradiated (8000 rads) NS0 GFP. (c.J03935) and NS0 GFP:IMPDH2-IL15(IL2ss) pcDNA3. IMPDH2-IL15(IL2ss) pcDNA3.1(+) (c.J02096) cells are 1(+) (c.J02096) were grown in DMEM (Irvine Scientific, administered i.p. 3 times a week starting on day 0 to provide Santa Ana, Calif.) supplemented with 10% heat-inactivated a systemic Supply of human IL-15 in vivo. Bone marrow FCS, 25 mM HEPES, and 2 mM L-glutamine in the pres was harvested from euthanized animals and analyzed by ence of either 0.05 uM methotrexate (MTX) or 6 uM flow cytometry. Antibody dependent cell mediated cytotox mycophenolic acid (MPA). The tumorigenic strain of U251, icity assays are performed to determine the activity of termed U251T, was a kind gift of Dr. Waldemar Debinski cetuximab against EGFRt T cells. US 2017/0152480 A1 Jun. 1, 2017

Results (FIG. 4c). A direct comparison of the CD19-specific reac tivity of Line E versus its non-selected or parental coun Immunomagnetic Selection of EGFRt Expressing T Cells terpart shows that there is enhanced CD19CAR-mediated cytotoxicity upon EGFRt-selection. In addition, the CMV 0069. A truncated human EGFR (EGFRt), which con specific T-derived CD19CAR"EGFRt" Line B cells also tains only the transmembrane domain and extracellular show cytotoxic activity through their endogenous T cell domains III and IV of the full length EGFR, was generated receptor against targets expressing CMV-pp65 antigen. as a non-immunogenic selection epitope compatible with 0073. For the CD19CAREGFRt"IMPDH2dm" Line E, immunomagnetic selection. As shown in the FIG. 1 molecu the ability of the inosine monophosphate dehydrogenase 2 lar model, the EGFRt retains the ability to be bound by double mutant (IMPDH2dm) to confer resistance to the cetuximab, but not have any signaling capacity due to the IMPDH2-inhibitor mycophenolic acid (MPA; a common absence of the intracellular domains. Furthermore, it lacks immunosuppressant used to prevent rejection in organ trans the N-terminal domain required for EGF-binding. plantation) was also tested. Upon culture in 1 uMMPA, the 0070. To immunomagnetically select for EGFRt-express survival and/or proliferation of Line E cells is not inhibited ing cells, biotinylated-cetuximab was generated (FIG. 2a, b) (FIG. 4d). This is in contrast to the inhibition seen with a to be used in conjunction with commercially available control T cell line that lacks expression of the IMPDH2dn anti-biotin microbeads and an AutoMACSTM separator gene. These data provide further evidence that EGFRt (Miltenyi Biotec) (FIG.2c). Lentiviral transduction of vari mediated selection results in the corresponding selection of ous T cell lines with EGFRt-containing constructs, where the other genes present in the lentiviral construct used to the EGFRt gene was separated from other genes of interest transduce T cells. on either one or both ends with the self-cleaving T2A sequence, consistently resulted in Surface detection of the Tracking of EGFRt" T Cells In Vivo EGFRt molecule on less than 40% of the cells (FIG. 2d). Surface detection may also be accomplished with a EGFRt 0074 To test the potential for detecting in vivo engrafted Sr39TK fusion. Immunomagnetic selection allowed for T cells, bone marrow cells collected from mice that had been recovery of EGFRt' T cell populations with greater than engrafted with CD19CAREGFRt" Line C was analyzed by 90% purity. T cell populations that underwent this transduc flow cytometry using biotinylated cetuximab (FIG. 5). Con tion and selection procedure included anti-CD3/anti-CD28 trol mice that did not receive T cells revealed that there was bead stimulated T cell blasts (for Line A), central memory Some cross-reaction of the cetuximab against murine EGFR. (CD45RO"CD62L. T.) derived T cells (for Lines B, C Thus, it was determined that successful detection of and E), which in some cases were also pre-selected for CMV engrafted Line C cells required double staining for both specificity (via the endogenous TCR; for Line B) or CD8 human CD45 and EGFRt. Cells may also analyzed using expression (for Line C), as well as effector memory immunohistochemistry to determine potential for Screening (CD62L CD45RO"T) derived T cells (for line D). These biopsy material. data show that EGFRt can successfully be used as a selection marker for various sources of T cell transductants, even Cetuximab Mediated Cytotoxicity of EGFRt" T Cells when the original transduction efficiency was as low a 2%. 0075. Because cetuximab is known to lyse EGFR-ex pressing cells via antibody dependent cell mediated cyto Inactivity of EGFRt on Selected T Cells toxicity (ADCC), assays were performed to determine the (0071. To confirm that the EGFRt is inactive, Western ADCC activity of cetuximab against EGFRt" T cells (FIG. immunoblot analyses for EGFR phosphorylation were car 6). Using "Cr-labeled Line A cells as targeted and freshly ried out on the EGFRt-selected T cells after culture with isolated human PBMC as effectors, cetuximab was found to either EGF or cetuximab. As expected, cetuximab did not significantly mediate chromium-release above that seen induce EGFR phosphorylation above background even in when using the CD20-specific humanized mAb Rituxan. the EGFR" cell line A431 (FIG. 3a). Furthermore, in con trast to that seen with the A431 cells, no phosphorylation Example of Therapeutic Use of EGFRt" T. Cells was seen in lysates of Line A after co-incubation with EGF. 0076 Adult subjects with high-risk intermediate grade Indeed, using biotinylated EGF, flow cytometric analysis B-cell lymphomas who are candidates for an autologous confirmed that EGF cannot bind the EGFRt-selected T cells myeloablative stem cell transplant procedure may receive (FIG. 3b), as expected due to the truncation in its N-termi post-transplant immunotherapy with adoptively transferred nus. These EGFRt' T cells were also not recognized by autologous Tcm-derived CD19R" CD8" EGFRt" T cell another anti-EGFR antibody distinct from cetuximab. grafts. A leukapheresis product collected from each patient Maintenance of Effector Phenotype in Expanded EGFRt" undergoes selection of Tcm, transduction with clinical grade CD19CAR T Cells CD19CAR-T2A-EGFRt epHIV7, and then selection and 0072 Directly after AutoMACSTM separation, the expansion of the EGFRt" cells in a closed system. After the selected T cells were expanded 30-fold or greater within 12 resulting cell products have undergone quality control test days after REM stimulation with OKT3, irradiated PBMC ing (including sterility and tumor specific cytotoxicity tests), feeders and LCL, IL-2 and IL-15 (FIG. 4a). Flow cytometric they are cryopreserved. Meanwhile, following leukaphere analysis of the resulting expanded EGFRt' T cells further sis, study participants commence with standard Salvage confirmed that that they express the CD19CAR and T cell chemotherapy, with mobilization for auto HSC collection markers such as CD8, TCR, CD3, perforin, granzyme, etc. with cytoreductive chemotherapy and G-CSF. Since the (FIG. 4b). Furthermore, CD19CAR-directed cytotoxic EGFRt-selected, CD19-specific T cells will also target nor activity of these EGFRt-selected lines is evident in chro mal CD20" (CD19) B cells, the B cell numbers can first be mium release assays using CD19-expressing tumor targets lowered using RituximabtM to reduce the recipients inflam US 2017/0152480 A1 Jun. 1, 2017

matory response upon receiving the genetically modified 0081. 3. Fehse, B. Richters, A, Putimtseva-Scharf, K, CTL and also increase availability of infused T cells to Klump, H. Li, Z. Ostertag, W., et al. (2000). CD34 splice immediately target lymphoma cells. Furthermore, Ritux variant: an attractive marker for selection of gene-modi imabtM may blunt a humoral immune response against the fied cells. Mol Ther 1: 448-56. genetically modified T cells. If Rituximab TM is not given as I0082) 4. Gaines, P. and Wojchowski, D M (1999), pIRES part of the Salvage/Priming chemotherapy regimen, research CD4t, a dicistronic expression vector for MACS- or participants may receive a single intravenous infusion of FACS-based selection of transfected cells. Biotechniques RituximabTM (chimeric anti-CD20 antibody) at 375 mg/m 26: 683-8. within 4-weeks of the planned auto-HSCT procedure. Ritux 0083) 5. Fehse, B, Uhde. A. Fehse, N. Eckert, H G, imabtM infusion would be carried out per standard practice Clausen, J. Ruger, R. et al. (1997). Selective immunoaf including premedication with diphenhydramine and acet finity-based enrichment of CD34+ cells transduced with aminophen and hydrocortisone. On Day +2 or Day +3 after retroviral vectors containing an intracytoplasmatically HSCT, the autologous cryopreserved CD19R" CD8" truncated version of the human low-affinity nerve growth EGFRt' T cell product will be transported, thawed and factor receptor (deltaLNGFR) gene. Hum Gene Ther. 8: infused at the patient’s bedside. Research participants can be 1815-24. pre-medicated at least 30 minutes prior to T cell infusion 0084 6. Lemoine, F M, Mesel-Lemoine, M. Cherai, M. with 15 mg/kg of acetaminophen P.O. (max. 650 mg.) and Gallot, G. Vie, H. Leclercq, V, et al. (2004). Efficient diphenhydramine 0.5-1 mg/kg I.V. (max dose 50 mg). transduction and selection of human T-lymphocytes with Clinical and laboratory correlative follow-up studies can bicistronic Thy1/HSV1-TK retroviral vector produced by then be performed at the physician's discretion, and may include quantitative RT-PCR studies for the presence of a human packaging cell line. J Gene Med 6: 374-86. CD19-expressing lymphoma cells and/or the adoptively I0085 7. Li, S, Schmitz, KR, Jeffrey, P D, Wiltzius, JJ, transfered T cells; FDG-PET and/or CT scans; bone marrow Kussie, P, and Ferguson, KM (2005). Structural basis for examination for disease specific pathologic evaluation; inhibition of the epidermal growth factor receptor by lymph node biopsy; and/or long-term follow up per the cetuximab. Cancer Cell 7: 301-11. guidelines set forth by the FDA's Biologic Response Modi I0086 8. Dawson, J. P. Berger, M B, Lin, C C, Sch fiers Advisory Committee that apply to gene transfer studies. lessinger, J. Lemmon, MA, and Ferguson, KM (2005). FIG. 10 provides a possible schematic for clinical testing of Epidermal growth factor receptor dimerization and acti the present products and methods. Vation require ligand-induced conformational changes in 0077. The present invention is not to be limited in scope the dimer interface. Mol Cell Biol 25: 7734-42. by the specific embodiments disclosed in the examples I0087 9. Lange, C. Li, Z. Fang, L, Baum, C, and Fehse, which are intended as illustrations of a few aspects of the B (2007). CD34 modulates the trafficking behavior of invention and any embodiments that are functionally equiva hematopoietic cells in vivo. Stem Cells Dev 16:297-304. lent are within the scope of this invention. Indeed, various I0088 10. Kowolik, C M, Topp, M. S. Gonzalez, S, modifications of the invention in addition to those shown Pfeiffer, T. Olivares, S. Gonzalez, N, et al. (2006). CD28 and described herein will become apparent to those skilled costimulation provided through a CD19-specific chimeric in the art and are intended to fall within the scope of the antigen receptor enhances in vivo persistence and antitu appended claims. mor efficacy of adoptively transferred T cells. Cancer Res 0078 All patents, patent applications, and references 66: 10995-1004. cited throughout the specification are expressly incorporated I0089 11. Szymczak, A L. Workman, C J, Wang, Y. by reference. Vignali, K. M. Dilioglou, S. Vanin, E. F. et al. (2004). Correction of multi-gene deficiency in vivo using a single REFERENCES self-cleaving 2A peptide-based retroviral vector. Nat 0079 1. Berger, C, Flowers, M. E. Warren, E H, and Biotechnol 22:589-94. Riddell, S R (2006). Analysis of transgene-specific 0090 12. Yam, P. Jensen, M. Akkina, R. Anderson, J. immune responses that limit the in vivo persistence of Villacres, MC, Wu, J, et al. (2006). Ex vivo selection and adoptively transferred HSV-TK-modified donor T cells expansion of cells based on expression of a mutated after allogeneic hematopoietic cell transplantation. Blood inosine monophosphate dehydrogenase 2 after HIV vec 107: 2294-3O2. tor transduction: effects on lymphocytes, monocytes, and 0080 2. Tey, S K, Dotti, G. Rooney, CM, Heslop, H E, CD34+ Stem cells. Mol Ther 14: 236-44. and Brenner, M K (2007). Inducible caspase 9 suicide (0091. 13. Pelloquin, F, Lamelin, J P and Lenoir, G M gene to improve the safety of allodepleted T cells after (1986). Human B lymphocytes immortalization by haploidentical stem cell transplantation. Biol Blood Mar Epstein-Barr virus in the presence of cyclosporin A. In row Transplant 13: 913-24. Vitro Cell Dev Biol 22: 689-94.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 6

<21 Os SEQ ID NO 1 &211s LENGTH: 1071 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens

US 2017/0152480 A1 Jun. 1, 2017 11

- Continued

Ctgcggc.cgg tacacacggt gga cacggta ggtttgacgt ggatgcctac gtgaccc.ggt 96.O ccagaact tc. cacaggttg Cttacccgga ttctagggca ggtagcggtg accctaccac 1 O2O cc.ccgggagg agaacgacga ccaccaccgg gaccc.ctago C9gagaagta C 1071

<210s, SEQ ID NO 3 &211s LENGTH: 357 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 3 Met Lieu. Lieu. Lieu Val Thir Ser Lieu. Lieu. Lieu. Cys Glu Lieu Pro His Pro 1. 5 1O 15 Ala Phe Lieu. Lieu. Ile Pro Arg Llys Val Cys Asn Gly Ile Gly Ile Gly 2O 25 3O Glu Phe Lys Asp Ser Lieu. Ser Ile Asn Ala Thr Asn. Ile Llys His Phe 35 4 O 45 Lys Asn. Cys Thir Ser Ile Ser Gly Asp Lieu. His Ile Lieu Pro Val Ala SO 55 6 O Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Glin Glu 65 70 7s 8O Lieu. Asp Ile Lieu Lys Thr Val Lys Glu Ile Thr Gly Phe Lieu. Lieu. Ile 85 90 95 Glin Ala Trp Pro Glu ASn Arg Thr Asp Lieu. His Ala Phe Glu Asn Lieu 1OO 105 11 O Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Glin Phe Ser Lieu Ala 115 12 O 125 Val Val Ser Lieu. Asn. Ile Thir Ser Lieu. Gly Lieu. Arg Ser Lieu Lys Glu 13 O 135 14 O Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Lieu. Cys Tyr 145 150 155 160 Ala Asn. Thir Ile ASn Trp Llys Llys Lieu. Phe Gly. Thir Ser Gly Glin Lys 1.65 17O 17s Thir Lys Ile Ile Ser Asn Arg Gly Glu Asn. Ser Cys Lys Ala Thr Gly 18O 185 19 O Glin Val Cys His Ala Lieu. Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu 195 2OO 2O5 Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu. Cys 21 O 215 22O Val Asp Llys Cys Asn Lieu. Lieu. Glu Gly Glu Pro Arg Glu Phe Val Glu 225 23 O 235 24 O Asn Ser Glu. Cys Ile Glin Cys His Pro Glu. Cys Lieu Pro Glin Ala Met 245 250 255

Asn. Ile Thir Cys Thr Gly Arg Gly Pro Asp Asn. Cys Ile Glin Cys Ala 26 O 265 27 O His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val 27s 28O 285 Met Gly Glu Asn. Asn. Thir Lieu Val Trp Llys Tyr Ala Asp Ala Gly. His 29 O 295 3 OO Val Cys His Lieu. Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro 3. OS 310 315 32O

Gly Lieu. Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala

US 2017/0152480 A1 Jun. 1, 2017 15

- Continued ttgtag toga C9tgtc.ctgc ccctggtctg ttgacat agg to acacgggit gatgtaactg 294 O ccgggggtga cqcagttctg gacgggcc.gt cct cagtacc Ctcttttgtt gtgggaccag 3 OOO acct tcatgc gtctgcggcc ggtacacacg gtgga cacgg taggtttgac gtggatgcct 3 O 6 O acgtgacccg gtc.ca.gaact tcc.gacaggt tott acccg gattic taggg Cagg tagcgg 312 O tgaccct acc accc.ccggga ggagaacgac gaccaccacc gggaccc ct a gcc.ggagaag 318O tacact 3186

<210s, SEQ ID NO 6 &211s LENGTH: 1061 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 6 Met Lieu. Lieu. Lieu Val Thir Ser Lieu. Lieu. Lieu. Cys Glu Lieu Pro His Pro 1. 5 1O 15 Ala Phe Leu Lieu. Ile Pro Asp Ile Gln Met Thr Glin Thr Thr Ser Ser 2O 25 3O Lieu. Ser Ala Ser Lieu. Gly Asp Arg Val Thir Ile Ser Cys Arg Ala Ser 35 4 O 45 Glin Asp Ile Ser Llys Tyr Lieu. Asn Trp Tyr Glin Glin Llys Pro Asp Gly SO 55 6 O Thr Val Lys Lieu. Lieu. Ile Tyr His Thr Ser Arg Lieu. His Ser Gly Val 65 70 75 8O Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Lieu. Thr 85 90 95 Ile Ser Asn Lieu. Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Glin Glin 1OO 105 11 O Gly Asn Thr Lieu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile 115 12 O 125 Thr Gly Ser Thr Ser Gly Ser Gly Llys Pro Gly Ser Gly Glu Gly Ser 13 O 135 14 O Thir Lys Gly Glu Val Llys Lieu. Glin Glu Ser Gly Pro Gly Lieu Val Ala 145 150 155 160 Pro Ser Glin Ser Lieu. Ser Val Thr Cys Thr Val Ser Gly Val Ser Lieu. 1.65 17O 17s Pro Asp Tyr Gly Val Ser Trp Ile Arg Glin Pro Pro Arg Lys Gly Lieu. 18O 185 19 O Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thir Thr Tyr Tyr Asn Ser 195 2OO 2O5 Ala Lieu Lys Ser Arg Lieu. Thir Ile Ile Lys Asp Asn. Ser Lys Ser Glin 21 O 215 22O

Val Phe Lieu Lys Met Asn. Ser Lieu. Glin Thr Asp Asp Thr Ala Ile Tyr 225 23 O 235 24 O Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255

Trp Gly Glin Gly Thr Ser Val Thr Val Ser Ser Glu Ser Lys Tyr Gly 26 O 265 27 O

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 27s 28O 285

Val Phe Leu Phe Pro Pro Llys Pro Lys Asp Thr Lieu Met Ile Ser Arg 29 O 295 3 OO US 2017/0152480 A1 Jun. 1, 2017 16

- Continued

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Glin Glu Asp Pro 3. OS 310 315 32O Glu Val Glin Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 3.25 330 335 Lys Thr Llys Pro Arg Glu Glu Glin Phe Asin Ser Thr Tyr Arg Val Val 34 O 345 35. O Ser Val Lieu. Thr Val Lieu. His Glin Asp Trp Lieu. Asn Gly Lys Glu Tyr 355 360 365 Lys Cys Llys Val Ser Asn Lys Gly Lieu Pro Ser Ser Ile Glu Lys Thr 37 O 375 38O Ile Ser Lys Ala Lys Gly Glin Pro Arg Glu Pro Glin Val Tyr Thr Lieu. 385 390 395 4 OO Pro Pro Ser Glin Glu Glu Met Thr Lys Asn Glin Val Ser Lieu. Thir Cys 4 OS 41O 415 Lieu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 42O 425 43 O Asn Gly Glin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lieu. Asp 435 44 O 445

Ser Asp Gly Ser Phe Phe Lieu. Tyr Ser Arg Lieu. Thr Val Asp Llys Ser 450 45.5 460

Arg Trp Glin Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 465 470 47s 48O

Lieu. His Asn His Tyr Thr Glin Llys Ser Lieu. Ser Lieu. Ser Lieu. Gly Lys 485 490 495

Met Phe Trp Val Lieu Val Val Val Gly Gly Val Lieu Ala Cys Tyr Ser SOO 505 51O

Lieu. Lieu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg 515 52O 525 Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 53 O 535 54 O Gly Pro Thr Arg Llys His Tyr Glin Pro Tyr Ala Pro Pro Arg Asp Phe 5.45 550 555 560

Ala Ala Tyr Arg Ser Gly Gly Gly Arg Val Llys Phe Ser Arg Ser Ala 565 st O sts

Asp Ala Pro Ala Tyr Glin Glin Gly Glin Asn Glin Lieu. Tyr Asn. Glu Lieu 58O 585 59 O

Asn Lieu. Gly Arg Arg Glu Glu Tyr Asp Val Lieu. Asp Lys Arg Arg Gly 595 6OO 605

Arg Asp Pro Glu Met Gly G Llys Pro Arg Arg Lys Asn Pro Glin Glu 610 6 : 62O Gly Lieu. Tyr Asn. Glu Lieu Gln Lys Asp Llys Met Ala Glu Ala Tyr Ser 625 630 635 64 O Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 645 650 655

Lieu. Tyr Glin Gly Lieu. Ser Thr Ala Thir Lys Asp Thr Tyr Asp Ala Lieu. 660 665 67 O His Met Glin Ala Lieu Pro Pro Arg Lieu. Glu Gly Gly Gly Glu Gly Arg 675 68O 685

Gly Ser Lieu. Lieu. Thir Cys Gly Asp Val Glu Glu ASn Pro Gly Pro Arg US 2017/0152480 A1 Jun. 1, 2017 17

- Continued

69 O. 695 7 OO

Met Lieu. Lieu. Lieu Val Thir Ser Lieu. Lieu. Lieu. Cys Glu Lieu. Pro His Pro 7 Os 71O 71s 72O Ala Phe Lieu. Lieu. Ile Pro Arg Llys Val Cys Asn Gly Ile Gly Ile Gly 72 73 O 73

Glu Phe Lys Asp Ser Lieu. Ser Ile Asn Ala Thr Asn. Ile Lys His Phe 740 74. 7 O

Lys Asn. Cys Thir Ser Ile Ser Gly Asp Lieu. His Ile Lieu. Pro Wall Ala 7ss 760 765

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Glin Glu 770 775 78O

Lieu. Asp Ile Lieu Lys Thr Val Lys Glu Ile Thr Gly Phe Luell Lieu. Ile 78s 79 O 79. 8OO

Glin Ala Trp Pro Glu Asn Arg Thr Asp Lieu. His Ala Phe Glu Asn Luell 805 810 815

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Glin Phe Ser Lieu Ala 82O 825 83 O

Val Val Ser Lieu. Asn. Ile Thir Ser Lieu. Gly Lieu. Arg Ser Luell Lys Glu 835 84 O 845

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Luell 850 855 860 Ala Asn. Thir Ile ASn Trp Llys Llys Lieu. Phe Gly. Thir Ser Gly Gln Lys 865 870 875 88O

Thir Lys Ile Ile Ser Asn Arg Gly Glu Asn. Ser Cys Llys Ala Thr Gly 885 890 895

Glin Val Cys His Ala Lieu. Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu 9 OO 905 91 O Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys 915 92 O 925

Val Asp Llys Cys Asn Lieu. Lieu. Glu Gly Glu Pro Arg Glu Phe Wall Glu 93 O 935 94 O

Asn Ser Glu. Cys Ile Glin Cys His Pro Glu. Cys Lieu Pro Glin Ala Met 945 950 955 96.O

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Glin Cys Ala 965 97O 97.

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val 98O 985 99 O Met Gly Glu Asn. Asn. Thir Lieu Val Trp Llys Tyr Ala Asp Ala Gly His 995 1OOO 1005 Val Cys His Lieu. Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly 1010 1015 1 O2O Pro Gly Lieu. Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser 1025 1O3 O 1035 Ile Ala Thr Gly Met Val Gly Ala Lieu. Lieu Lleu Lleu Lieu Val Val 104 O 1045 1 OSO Ala Lieu. Gly Ile Gly Lieu. Phe Met 105.5 106 O

1. A population of human T-cells transfected with a Surface molecule, said cell Surface molecule comprising an genetically modified Epidermal Growth Factor Receptor EGFR Domain III and an EGFR Domain IV; but lacking all (EGFR) gene, said gene comprising a nucleotide sequence of the domains consisting of an EGFR Domain I, an EGFR encoding a truncated non-immunogenic endogenous cell Domain II, an EGFRJuxtamembrane Domain, and an EGFR US 2017/0152480 A1 Jun. 1, 2017

Tyrosine Kinase Domain; wherein the truncated non-immu 13. A population of human T-cells transfected with a nogenic endogenous cell Surface molecule (i) does not have genetically modified Epidermal Growth Factor Receptor endogenous signaling or trafficking function; (ii) binds a (EGFR) gene that is coupled to a CD19CAR and a C-ter therapeutic anti-EGFR antibody; (iii) does not bind an minal 2A cleavable linker, wherein the T-cells encode an endogenous EGFR ligand; and (iv) acts as a marker. amino acid sequence comprising SEQ ID NO:6. 2. The T-cells of claim 1, wherein the genetically modified 14. The T-cells of claim 13, wherein the genetically EGFR gene comprises nucleotides 67-1071 of SEQ ID modified EGFR gene is expressed by the T-cells and is used NO:2. as a marker to select or enrich cells. 3. The T-cells of claim 1, wherein the genetically modified 15. The T-cells of claim 13, wherein the genetically EGFR gene encodes an amino acid sequence comprising modified EGFR gene is expressed by the T-cells and is used residues 23-357 of SEQ ID NO:3. as a marker to induce cell Suicide. 4. The T-cells of claim 1, wherein the genetically modified 16. The T-cells of claim 13, wherein the genetically EGFR gene further comprises a GMCSFR alpha chain modified EGFR gene is expressed by the T-cells and is used signal sequence. as a marker to deplete cells. 5. The T-cells of claim 4, wherein the genetically modified 17. The T-cells of claim 13, wherein the genetically EGFR gene comprises SEQ ID NO:2. modified EGFR gene is inserted into a vector to transfect the 6. The T-cells of claim 4, wherein the genetically modified population of human T-cells. EGFR gene comprises SEQ ID NO:2. 18. A population of human T-cells transfected with a 7. The T-cells of claim 4, wherein the genetically modified genetically modified Epidermal Growth Factor Receptor EGFR gene encodes an amino acid sequence comprising at (EGFR) gene, said gene comprising a nucleotide sequence least 90% identical to SEQ ID NO:3. encoding a truncated non-immunogenic endogenous cell 8. The T-cells of claim 4, wherein the genetically modified Surface molecule, said cell Surface molecule comprising an EGFR gene encodes an amino acid sequence comprising EGFR Domain III and an EGFR Domain IV; but lacking all SEQ ID NO:3. of the domains consisting of an EGFR Domain I, an EGFR 9. The T-cells of claim 1, wherein the genetically modified Domain II, an EGFRJuxtamembrane Domain, and an EGFR EGFR gene is inserted into a vector to transfect the popu Tyrosine Kinase Domain; wherein the truncated non-immu lation of human T-cells. nogenic endogenous cell Surface molecule (i) does not have 10. The T-cells of claim 1, wherein the marker is used to endogenous signaling or trafficking function; (ii) binds a enrich cells, to select cells, to deplete cells expressing the therapeutic anti-EGFR antibody; (iii) does not bind an cell surface molecule, or to induce cell suicide in cells endogenous EGFR ligand; and (iv) acts as a marker to enrich expressing the cell Surface molecule cells, to select cells, to deplete cells expressing said cell 11. The T-cells of claim 1, wherein the gene is part of a construct which comprises the modified EGFR coupled via Surface molecule, or to induce cell Suicide in cells express a C-terminal 2A cleavable linker to a chimeric antigen ing said cell Surface molecule. receptor specific for a tumor associated antigen selected 19. The T-cells of claim 18, wherein the therapeutic from CD19, a codon-optimized anti-CD19 costimulatory anti-EGFR antibody is cetuximab. chimeric antigen receptor (CD19CAR), CD20 or CD22. 20. The T-cells of claim 18, wherein the genetically 12. The T-cells of claim 11, wherein the modified EGFR modified EGFR gene is inserted into a vector to transfect the is coupled to a CD19CAR and a C-terminal 2A cleavable population of human T-cells. linker. k k k k k