US010906982B2
( 12 ) United States Patent ( 10 ) Patent No .: US 10,906,982 B2 Faustman ( 45 ) Date of Patent : Feb. 2 , 2021
( 54 ) ANTAGONISTIC ANTI - TUMOR NECROSIS ( 58 ) Field of Classification Search FACTOR RECEPTOR 2 ANTIBODIES None See application file for complete search history . ( 71 ) Applicant: The General Hospital Corporation , Boston , MA (US ) ( 56 ) References Cited ( 72 ) Inventor: Denise L. Faustman , Boston , MA ( US ) U.S. PATENT DOCUMENTS ( 73 ) Assignee : The General Hospital Corporation , 4,309,418 A 1/1982 Green Boston , MA (US ) 4,457,916 A 7/1984 Hayashi et al . ( * ) Notice : Subject to any disclaimer , the term of this ( Continued ) patent is extended or adjusted under 35 U.S.C. 154 ( b ) by 276 days . FOREIGN PATENT DOCUMENTS EP 0612529 A2 8/1994 ( 21 ) Appl . No .: 15 /573,747 EP 2295588 A1 3/2011 ( Continued ) ( 22 ) PCT Filed : May 13 , 2016 ( 86 ) PCT No .: PCT / US2016 / 032547 OTHER PUBLICATIONS $ 371 ( c ) ( 1 ) , MacCallum et al . , Antibody - antigen interactions : Contact analysis ( 2 ) Date : Nov. 13 , 2017 and binding siite topography, J. Mol . Biol 262 : 732-745 , 1996. * (Continued ) ( 87 ) PCT Pub . No .: WO2016 / 187068 PCT Pub . Date : Nov. 24 , 2016 Primary Examiner Claire Kaufman ( 65 ) Prior Publication Data ( 74 ) Attorney, Agent, or Firm Clark & Elbing LLP US 2018/0194850 A1 Jul . 12 , 2018 ( 57 ) ABSTRACT Related U.S. Application Data Antagonistic TNFR superfamily polypeptides , such as anti ( 60 ) Provisional application No. 62 / 276,073 , filed on Jan. bodies and antigen - binding fragments thereof, and the use of 7 , 2016 , provisional application No. 62 / 162,449 , filed these polypeptides to inhibit the proliferation of regulatory on May 15 , 2015 . T cells ( T -regs ). For example, antibodies of the invention include antagonistic TNFR2 antibodies and antigen -binding ( 51 ) Int. Ci . fragments thereof, and can be used to suppress the T -reg CO7K 16/28 ( 2006.01 ) mediated deactivation of tumor reactive T - lymphocytes, as A61K 39/395 ( 2006.01 ) well as to treat a wide variety of cancers and infectious (Continued ) diseases . ( 52 ) U.S. Ci . CPC CO7K 16/2878 ( 2013.01 ) ; A61K 39/395 ( 2013.01 ) ; A61K 38/00 ( 2013.01 ) ; 16 Claims , 44 Drawing Sheets ( Continued ) Specification includes a Sequence Listing .
TNFR2 agonist TNFTNFR2 agonist TNFR2 antagonist 1 TNF TNFR2 antagonist TNFR2 antagonist 2 fum TNF INFR2 antagonist 2
20 80 Treg change relative to IL - 2 US 10,906,982 B2 Page 2
( 51 ) Int . Ci . 2004/0031066 A9 2/2004 Faustman et al . A61K 38/00 2004/0229785 Al 11/2004 Faustman ( 2006.01 ) 2005/0043514 A1 2/2005 Fukui et al . A61K 39/00 ( 2006.01 ) 2005/0158288 A1 7/2005 Faustman ( 52 ) U.S. Cl . 2005/0158302 Al 7/2005 Faustman et al . 2005/0244386 Al 11/2005 Habener et al . CPC .. A61K 2039/572 ( 2013.01 ) ; A61K 2039/585 2006/0062769 Al 3/2006 Habener et al . ( 2013.01 ) ; CO7K 2317/34 ( 2013.01 ) ; CO7K 2007/0116688 A1 5/2007 Faustman 2317/54 ( 2013.01 ) ; CO7K 2317/565 ( 2013.01 ) ; 2008/0102054 A1 5/2008 Faustman CO7K 2317/567 ( 2013.01 ) ; CO7K 2317/73 2008/0175830 Al 7/2008 Steinman et al . ( 2013.01 ) ; CO7K 2317/732 ( 2013.01 ) ; CO7K 2009/0028877 Al 1/2009 Iida et al . 2010/0068177 Al 3/2010 Faustman 2317/76 ( 2013.01 ) ; CO7K 2317/92 ( 2013.01 ) 2010/0298232 Al 11/2010 Liu References Cited 2011/0236374 A1 9/2011 Shitara et al . ( 56 ) 2012/0066777 A1 3/2012 Kawamura et al . 2012/0115739 A1 5/2012 Schmittling et al . U.S. PATENT DOCUMENTS 2012/0196919 A1 8/2012 Brown et al. 2013/0064831 A1 3/2013 Humphrey 4,495,282 A 1/1985 Ohnishi et al . 2014/0096274 Al 4/2014 Quax et al . 4,677,063 A 6/1987 Mark et al . 2014/0121123 Al 5/2014 Wang et al. 4,677,064 A 6/1987 Mark et al . 2015/0366909 A1 12/2015 Faustman 4,681,760 A 7/1987 Fathman 2019/0202925 Al * C07K 16/2878 4,791,101 A 12/1988 Adolf 7/2019 Thompson 4,816,567 A 3/1989 Cabilly et al . 4,879,226 A 11/1989 Wallace et al . FOREIGN PATENT DOCUMENTS 4,963,354 A 10/1990 Shepard et al . 4,985,241 A 1/1991 Zimmerman et al . WO WO - 92 / 04033 A1 3/1992 5,002,876 A 3/1991 Sreekrishna et al . WO WO - 93 / 02690 A1 2/1993 5,059,530 A 10/1991 Oshima et al. WO WO - 94 / 09137 Al 4/1994 5,139,481 A 8/1992 Faustman et al. WO WO - 95 / 24914 Al 9/1995 5,215,743 A 6/1993 Singh et al . WO WO - 95 / 25533 Al 9/1995 5,283,058 A 2/1994 Faustman WO WO - 97 / 08328 A1 3/1997 5,288,852 A 2/1994 Yamada et al . WO WO - 97 / 13844 Al 4/1997 5,370,870 A 12/1994 Wong WO WO - 97 / 21802 Al 6/1997 5,487,984 A 1/1996 Allet et al . WO WO - 99 / 53953 A2 10/1999 5,538,854 A 7/1996 Faustman WO WO - 99 / 59632 A1 11/1999 5,560,908 A 10/1996 Satoh et al . WO WO - 00 / 53209 A1 9/2000 5,593,698 A 1/1997 Weiner et al . WO WO - 01 / 44472 A1 6/2001 5,783,216 A 7/1998 Faustman WO WO - 01 / 91793 A1 12/2001 5,821,337 A 10/1998 Carter et al . WO WO - 02 / 26819 A2 4/2002 5,843,425 A 12/1998 Sachs et al . WO WO - 2004 / 003164 A2 1/2004 5,843,452 A 12/1998 Wiedmann et al . WO WO - 2005 / 042727 A2 5/2005 5,874,306 A 2/1999 Beattie et al . WO WO - 2006 / 109044 A2 10/2006 5,919,452 A 7/1999 Le et al . WO WO - 2008 / 157394 A2 12/2008 5,939,532 A 8/1999 Nakamura et al . WO WO - 2010 / 124259 Al 10/2010 6,046,031 A 4/2000 Ni et al . WO WO - 2012 / 122464 A1 9/2012 6,056,952 A 5/2000 Rosenberg WO WO - 2014 / 124134 Al 8/2014 6,159,461 A 12/2000 Besmer et al . WO WO - 2015 / 145360 A1 10/2015 6,165,737 A 12/2000 Wang et al . WO WO - 2016 / 187068 A1 11/2016 6,177,076 B1 1/2001 Lattime et al. WO WO - 2017 / 040312 A1 3/2017 6,284,879 B1 9/2001 Faustman WO WO - 2017 / 083525 A1 5/2017 6,414,218 B1 7/2002 Faustman et al. 6,420,139 B1 7/2002 Classen 6,491,908 B1 12/2002 Rosenberg OTHER PUBLICATIONS 6,599,710 B1 7/2003 Faustman 6,617,171 B2 9/2003 Faustman et al . MacCallum et al. , Antibody - antigen interactions : contact analysis 6,660,487 B2 12/2003 Faustman and binding site topography, J. Mol . Biol . 262 : 732-745 , 1996. * 6,709,833 B2 3/2004 Fukui et al . Lamminmaki et al . , Chrystal structure of a recombinant anti 6,773,705 B1 8/2004 Faustman et al . estradiol Fab fragment in complex with the 17beta - estradiol, J. Blol . 6,844,011 B1 1/2005 Faustman Chem . 276 : 36687-94 , 2001. * 6,866,843 B2 3/2005 Habener et al . Chen et al.Enhancement and destruction of antibody function by 6,923,959 B2 8/2005 Habener et al . somatic mutation : unequal occurrence is controlled by V gene 6,984,380 B1 1/2006 Faustman combinatorial associations EMBO J. , 14 ( 12 ) : 2784-2794 , 1995. * 7,015,037 B1 3/2006 Furcht et al . Parsons, J.A. , Peptide Hormones ( University Press :Baltimre ), pp . 7,438,902 B2 10/2008 Habener et al . 1-7 , 1976. * 7,485,293 B1 2/2009 Faustman Ji et al . , “ Cutting Edge : The Natural Ligand for Glucocorticoid 7,510,877 B2 3/2009 Yilmaz et al . Induced TNF Receptor -Related Protein Abrogates Regulatory T 7,537,756 B2 5/2009 Habener et al . Cell Suppression ," J Immunol. 172 ( 10 ) : 5823-7 ( 2004 ) . 7,582,313 B2 9/2009 Faustman Chen et al . , “ The phenotypic and functional consequences of 7,628,988 B2 12/2009 Faustman tumour necrosis factor receptor type 2 expression on CD4 ( + ) 8,173,129 B2 5/2012 Faustman 8,314,213 B2 11/2012 Bernett et al . FoxP3 ( + ) regulatory T cells ,” Immunology. 133 ( 4 ) : 426-33 ( 2011 ) . 9,676,862 B2 * 6/2017 Ellmark A61K 9/0019 International Preliminary Report on Patentability for International 9,821,010 B2 11/2017 Faustman Application No. PCT / US2017 / 032513 , dated Nov. 22 , 2018 ( 8 2002/0106689 Al 8/2002 Faustman et al. pages ). 2002/0123472 A1 9/2002 Faustman Santee et al. , “ Human tumor necrosis factor receptor p75 / 80 ( CD 120b ) 2002/0187548 A1 12/2002 Keller et al . gene structure and promoter characterization , ” J Biol Chem . 2003/0005469 Al 1/2003 Faustman et al . 271 ( 35 ) : 21151-9 ( 1996 ) . 2003/0031657 Al 2/2003 Habener et al . Extended European Search Report for European Application No. 2004/0028658 Al 2/2004 Faustman 18199457.5 , dated May 2 , 2019 ( 7 pages ). US 10,906,982 B2 Page 3
( 56 ) References Cited Bernabeu et al . , “ Beta2 -microglobulin from serum associates with MHC class I antigens on the surface of cultured cells, ” Nature. OTHER PUBLICATIONS 308 : 642-645 ( 1984 ) ( Abstract only ) ( 2 pages ) . Bill et al . , “ Use of soluble MHC class II /peptide multimers to detect International Search Report and Written Opinion for International antigen - specific T cells in human disease, ” Arthritis Res . 4 : 261-265 Application No. PCT / US18 / 59779 , dated Apr. 18 , 2019 ( 19 pages ). ( 2002 ) . Al- Awqati et al. , “ Stem cells in the kidney , ” Kidney Int . 61 (2 ) :387 Bjornson et al . , “ Turning brain into blood : A hematopoietic fate 95 ( 2002 ) . Aldrich et al . , “ Positive selection of self- and alloreactive CD8 + T adopted by adult neural stem cells in vivo ,” Science. 283 (5401 ): 534 cells in Tap - 1 mutant mice , ” Proc Natl Acad Sci USA . 91 ( 14 ) : 6525-8 537 ( 1999 ) . ( 1994 ) . Bleumink et al . , “ Etanercept - induced subacute cutaneous lupus Alison et al . , " Hepatocytes from non -hepatic adult stem cells, ” erythematosus ” Rheumatology. 40 : 1317-1319 ( 2001 ) . Nature . 406 ( 6793 ) : 257 ( 2000 ) . Boches et al . , “ Role for the adenosine triphosphate -dependent Allen et al . , “ Effect of bacillus Calmette -Guerin vaccination on proteolytic pathway in reticulocyte maturation ,” Science . 215: 978 new - onset Type 1 Diabetes , " Diabetes Care . 22 ( 10 ) : 1703-1707 980 ( 1982 ) . ( 1999 ) . Brayer et al. , " Alleles from chromosomes 1 and 3 of NOD mice Altomonte et al . , " Serum levels of interleukin - 1b , tumour necrosis combine to influence Sjögren's syndrome - like autoimmune factor- a and interleukin - 2 in rheumatoid arthritis. Correlation with exocrinopathy, ” J. Rheumatol . 27 ( 8 ) : 1896-1904 ( 2000 ) . disease activity ," Clin Rheumatol. 11 ( 2 ) : 202-205 ( 1992 ) . Brazelton et al . , “ From marrow to brain : expression of neuronal Anderson et al . , “ Can stem cells cross lineage boundaries ? ” Nat phenotypes in adult mice ,” Science . 290 : 1775-1779 ( 2000 ) . Med . 7 ( 4 ) : 393-5 ( 2001 ) . Brod et al . , “ Ingested interferon alpha suppresses Type I diabetes in Anderson et al . , “ Studies on the cytophilic properties of human non - obese diabetic mice, " Diabetologia. 41 : 1227-1232 ( 1998 ) . beta2 microglobulin ,” J Immunol. 114 ( 3 ) : 997-1000 ( 1975 ) . Brod et al . , “ New clinical trial in newly diagnosed type 1 diabetes ," Anderson et al . , “ The NOD mouse : a model of immune dysregula < http://www.diabetesstation.org/articles/brod.htm > , retrieved Jun . tion ," Annu Rev Immunol. 23 : 447-485 ( 2005 ) . 19 , 2001 ( 2 pages ). Aranda et al . , “ Analysis of intestinal lymphocytes in mouse colitis Brodbeck et al . , “ Genetic determination of nephrogenesis: the mediated by transfer of CD4 + , CD45RBhigh T Cells to SCID Pax / Eya /Six gene network , ” Pediatr Nephrol. 19 ( 3 ) : 249-255 ( 2004 ) recipients ," J Immunol. 158 ( 7 ) : 3464-3473 ( 1997 ) . ( Abstract Only ). Aristarkhov et al . , “ E2 - C , a cyclin -selective ubiquitin carrier protein Brás et al . , “ Diabetes -prone NOD mice are resistant to mycobacterium required for the destruction of mitotic cyclins ,” Proc Natl Acad Sci avium and the infection prevents autoimmune disease , ” Immunol USA . 93 ( 9 ) : 4294-9 ( 1996 ) . ogy . 89 : 20-25 ( 1996 ) . Ashton - Rickardt et al. , “ Evidence for a differential avidity model of Bunting et al . , “ Enforced P - glycoprotein pump function in murine T Cell selection in the thymus , ” Cell . 76 ( 4 ) : 651-63 ( 1994 ) . bone marrow cells results in expansion of side population stem cells Ashton - Rickardt et al . , “ Peptide contributes to the specificity of in vitro and repopulating cells in vivo , ” Blood . 96 ( 3 ) : 902-909 positive selection of CD8 + T Cells in the thymus, ” Cell . 73 ( 5 ) : 1041-9 ( 2000 ) . ( 1993 ) . Burnham et al . , " Oral BCG vaccine in Crohn's disease ,” Gut. Atkinson et al . , “ The NOD mouse model of Type 1 Diabetes : As 20 : 229-233 ( 1979 ) . good as it gets ? ” Nat Med . 5 ( 6 ) : 601-604 ( 1999 ) . Caetano et al . , “ Effect of methotrexate ( MTX ) on Nad ( P ) + Baeuerle et al . , “ NF -kappaB : Ten years after, ” Cell . 87 ( 1 ) : 13-20 dehydrogenases of HeLa cells : malic enzyme, 2 - oxoglutarate and ( 1996 ) . isocitrate dehydrogenases , " Cell Biochem Funct . 15 ( 4 ) : 259-264 Baeza et al . , “ Pancreatic regenerating gene overexpression in the ( 1997 ) . nonobese diabetic mouse during active diabetogenesis ,” Diabetes. Cairns et al . , " New onset systemic lupus erythematosus in a patient 45 ( 1 ) : 67-70 ( 1996 ) ( 5 pages ) . receiving etanercept for rheumatoid arthritis, ” Ann Rheum Dis . Baeza et al . , “ Reg protein : a potential beta -cell - specific growth 61 ( 11 ) : 1031-2 ( 2002 ) . factor? " Diabetes Metab . 22 ( 4 ) : 229-234 ( 1996 ) . Cavallo et al . , “ BCG vaccine with and without nicotinamide in Baeza et al . , “ Specific reg II gene overexpression in the non - obese recent onset IDDM : a multicenter randomized trial, " Second Con diabetic mouse pancreas during active diabetogenesis, ” FEBS Let gress of the Immunology of Diabetes Society. 18 , A063 ( 1996 ) . ters . 416 ( 3 ) : 364-8 ( 1997 ) . Cebrián et al . , “ MHC - I expression renders catecholaminergic neu Baik et al . , “ BCG vaccine prevents insulitis in low dose streptozotocin rons susceptible to T - cell -mediated degeneration ,” Nat Commun . induced diabetic mice ," Diabetes Res Clin Pract. 46 ( 2 ) : 91-97 ( 1999 ) . 5 : 3633 ( 2014 ) ( Abstract only ) ( 1 page ). Baldwin , “ The NF -kappa B and IkappaB proteins : new discoveries and Charles et al . , “ Assessment of antibodies to double - stranded DNA insights, ” Ann Rev Immunol. 14 :649-683 ( 1996 ) . induced in rheumatoid arthritis patients following treatment with Ban et al . , " Selective death of autoreactive T Cells in human infliximab , a monoclonal antibody to tumor necrosis factor alpha : diabetes by TNF or TNF receptor 2 agonism , ” Proc Natl Acad Sci findings in open - label and randomized placebo -controlled trials. ” USA . 105 ( 36 ) : 13644-13649 ( 2008 ) . Arthritis Rheum . 43 ( 11 ) : 2383-90 ( 2000 ) . Barres, “ A new role for glia : generation of neurons ! ” Cell . 97 ( 6 ) : Chatenoud et al . , “ CD3 antibody - induced dominant self tolerance in 667-70 ( 1999 ) . overtly diabetic NOD mice , ” J Immunol. 158 ( 6 ) : 2947-2954 ( 1997 ) . Baxter et al . , “ Mycobacteria precipitate an SLE - like syndrome in Choi et al ., “ Prevention of Encephalomyocarditis virus -induced diabetes -prone NOD mice, " Immunology . 83 ( 2 ) : 227-231 ( 1994 ) . diabetes by live recombinant Mycobacterium bovis Bacillus Calmette Beers et al . Disorders of Carbohydrate Metabolism : Diabetes Mel Guérin in susceptible mice , " Diabetes. 49 : 1459-1467 ( 2000 ) . litus. The Merck Manual of Diagnosis and Therapy, 17th Ed . Merck Chopra et al . “ Exogenous TNFR2 activation protects from acute Research Laboratories, 165-171 , 1999 . GvHD via host Treg cell expansion ” , J Exp Med 213 ( 9 ) : 1881-1900 Beg et al . , “ An essential role for NF - kappaB in preventing TNF ( 2016 ) ( 21 pages) . alpha -induced cell death , ” Science . 274 ( 5288 ) : 782-784 ( 1996 ) . Christen et al . , “ A dual role for TNF - alpha in type 1 diabetes : Bendelac et al ., " Syngeneic transfer of autoimmune diabetes from islet - specific expression abrogates the ongoing autoimmune process diabetic NOD mice to healthy neonates . Requirement for both when induced late but not early during pathogenesis , ” J Immunol. L3T4 + and Lyt - 2 + T Cells, ” J Exp Med . 166 ( 4 ) : 823-832 ( 1987 ) . 166 ( 12 ) : 7023-32 ( 2001 ) . Benkler et al . , “ Parkinson's disease, autoimmunity, and olfaction , ” Cole et al . , “ Two ParaHox genes, SpLox and SpCdx, interact to Int J Neurosci . 119 ( 12 ) : 2133-43 ( 2009 ) ( Abstract only ) ( 1 page ). partition the posterior endoderm in the formation of a functional Bercovici et al . , “ Systemic administration of agonist peptide blocks gut , ” Development. 136 ( 4 ) : 541-549 ( 2009 ) . the progression of spontaneous CD8 -mediated autoimmune diabe Colucci et al . , " Programmed cell death in the pathogenesis of tes in transgenic mice without bystander damage , ” J Immunol. murine IDDM : resistance to apoptosis induced in lymphocytes by 165 ( 1 ) : 202-10 ( 2000 ) . cyclophosphamide, ” J Autoimmunity 9 : 271-276 ( 1996 ) . US 10,906,982 B2 Page 4
( 56 ) References Cited EPO Communication pursuant to Article 94 ( 3 ) and Rule 71 ( 1 ) EPC for European Application No. 00914899.0 , dated Mar. 6 , 2015 ( 3 OTHER PUBLICATIONS pages ). EPO Communication pursuant to Article 94 ( 3 ) EPC for European Communication pursuant to Article 94 ( 3 ) EPC for European Patent Application No. 00914899.0 , dated May 25 , 2012 ( 9 pages ) . Application No. 14189654.8 , dated Oct. 19 , 2016 ( 5 pages ) . EPO Communication Pursuant to Article 94 ( 3 ) EPC issued in Communication pursuant to Rules 70 ( 2 ) and 70a ( 2 ) EPC for Euro European Patent Application No. 03762242.0 , dated Dec. 1 , 2011 ( 4 pean Application No. 14748807.6 , dated Aug. 2 , 2016 ( 1 page ). pages ). Corbett et al . , “ Nitric oxide mediates cytokine - induced inhibition of EPO Communication Pursuant to Article 94 ( 3 ) EPC issued in insulin secretion by human islets of Langerhans, ” Proc Natl Acad European Patent Application No. 03762242.0 , dated Oct. 30 , 2009 Sci USA . 90 ( 5 ) : 1731-1735 ( 1993 ) . ( 2 pages) . Coux et al . , “ Enzymes catalyzing ubiquitination and proteolytic EPO Communication Pursuant to Article 94 ( 3 ) EPC issued in processing of the p105 precursor of nuclear factor kappaB1, ” J Biol European Patent Application No. 04817543.4 , dated Jan. 22 , 2010 Chem . 273 ( 15 ) : 8820-8828 ( 1998 ) . ( 5 pages ) . > > EPO Communication Pursuant to Article 94 ( 3 ) EPC issued in Couzin , “ Diabetes studies conflict on power of spleen cells ,” European Patent Application No. 11008889.5 , dated Mar. 19 , 2014 Science . 311 : 1694 ( 2006 ) . ( 4 pages ). Creasey et al . , “ Biological effects of recombinant human tumor EPO Communication Pursuant to Article 94 ( 3 ) EPC issued in necrosis factor and its novel muteins on tumor and normal cell European Patent Application No. 11008889.5 , dated Mar. 4 , 2013 ( 4 lines, ” Cancer Res . 47 ( 1 ) : 145-9 ( 1987 ) . pages ). D'Andrea , “ Add Alzheimer's disease to the list of autoimmune EPO Communication Pursuant to Article 94 ( 3 ) EPC issued in diseases , ” Med Hypotheses. 64 ( 3 ) : 458-63 ( 2005 ) ( Abstract only ) ( 2 European Patent Application No. 11008889.5 , dated Oct. 27 , 2014 pages) . ( 5 pages ) . Darzynkiewicz et al . , “ Use of flow and laser scanning cytometry to EPO Communication pursuant to Rule 69 EPC for European study mechanisms regulating cell cycle and controlling cell death ,” Application No. 12005556.1 , dated Oct. 7 , 2014 ( 2 pages ) . Clinics in Laboratory Medicine . 21 ( 4 ) : 857-873 ( 2001 ) . EPO Communication under Rule 71 ( 3 ) EPC for European Appli Dear et al . , “ The Hoxll gene is essential for cell survival during cation No. 00914899.0 , dated Jun . 23 , 2015 ( 6 pages ) . spleen development , ” Development. 121 : 2909-2915 ( 1995 ) . EPO Invitation pursuant to Article 94 ( 3 ) and Rule 71 ( 1 ) EPC for Declaration of Dr. Denise Faustman from U.S. Appl. No. 10 / 358,644 , European Application No. 00914899.0 , dated Jun . 2 , 2014 ( 4 dated on May 2009 ( 4 pages ). pages ). EPO Summons to attend oral proceedings pursuant to Rule 115 ( 1 ) Declaration of Dr. Denise Faustman from U.S. Appl. No. 10 / 775,487 , EPC for European Application No. 00914899.0 , dated Nov. 12 , dated on Jun . 14 , 2007 ( 13 pages ) . 2014 ( 6 pages) . Declaration of Dr. Denise Faustman from U.S. Appl. No. 10 / 851,983 , EPO Summons to attend oral proceedings pursuant to Rule 115 ( 1 ) dated on Jul. 3 , 2007 ( 7 pages ). EPC for European Application No. 12005556.1 , dated Dec. 18 , Diaw et al . , “ Structural and affinity studies of IgM polyreactive 2015 ( 4 pages ). natural autoantibodies , " J Immunol . 158 ( 2 ) : 968-76 (1997 ) . Examination Report issued in Australian Patent Application No. Dieguez - Acuna et al . , " Characterization of mouse spleen cells by 2003247840 , dated Jan. 31 , 2008 ( 4 pages ) . subtractive proteomics ,” Mol Cell Proteomics . 4 ( 10 ) : 1459-1470 Examiner's Report for Canadian Application No. 2,543,745 , dated ( 2005 ) . Jul. 15 , 2011 ( 4 pages ). Dieguez - Acuña et al ., " Proteomics identifies multipotent and low Extended European Search Report for European Application No. oncogenic risk stem cells of the spleen ,” Int J Biochem Cell Biol . 12005556.1 , dated Sep. 2 , 2014 ( 8 pages ). 42 ( 10 ) : 1651-1660 ( 2009 ) ( 10 pages ) . Extended European Search Report for European Application No. Dilts et al . , “ Autoimmune diabetes: The involvement of benign and 14748807.6 , dated Jul. 15 , 2016 ( 10 pages ) . malignant autoimmunity , ” J Autoimmun . 12 : 229-232 ( 1999 ) . Extended European Search Report for European Patent Application Dinarello , “ Interleukin - 1 , Interleukin - 1 receptors and Interleukin - 1 No. 11008889.5 , dated Apr. 12 , 2012 ( 10 pages ) . receptor antagonist , ” Intern Rev Immunol . 16 : 457-499 ( 1998 ) . Extended European Search Report issued in European Application Driscoll et al ., “ The proteasome ( multicatalytic protease ) is a No. 14189654.8 , dated Feb. 16 , 2015 ( 7 pages ) . component of the 1500 -kDa proteolytic complex which degrades Eytan et al ., " ATP -dependent incorporation of 20S protease into the ubiquitin - conjugated proteins , " J Biol Chem . 265 ( 9 ) : 4789-4792 26S complex that degrades proteins conjugated to ubiquitin ,” Proc ( 1990 ) . Natl Acad Sci USA . 86 : 7751-7755 ( 1989 ) . Durand et al . , “ Mesenchymal lineage potentials of aorta - gonad Fan et al . , “ Generation of p50 subunit of NF - kappaB by processing mesonephros stromal clones ,” Heamatologica . 91 ( 9 ) : 1172-1179 ( 2006 ) . of p105 through an ATP - dependent pathway, ” Nature . 354 : 395-398 Eglitis et al . , “ Hematopoietic cells differentiate into both microglia ( 1991 ) . and macroglia in the brains of adult mice, ” Proc Natl Acad Sci USA . Faustman et al . , “ Abnormal T -lymphocyte subsets in Type I Dia 94 : 4080-4085 ( 1997 ) . betes , ” Diabetes. 38 : 1462-1468 ( 1989 ) . Elliott et al . , “ Effect of Bacille Calmette -Guérin vaccination on Faustman et al . , " Linkage of faulty major histocompatibility com C - Peptide secretion in children newly diagnosed with IDDM , ” plex class I to autoimmune diabetes, " Science . 254 : 1756-1761 Diabetes Care . 21 ( 10 ) : 1691-1693 ( 1998 ) . ( 1991 ) . Enayati et al . , “ Association of anti - tumor necrosis factor therapy Faustman et al . , “ Murine pancreatic beta - Cells express H - 2K and with the development of multiple sclerosis, ” J Clin Gastroenterol. H - 2D but not la antigens, ” J Exp Med . 151 : 1563-1568 ( 1980 ) . 39 ( 4 ) : 303-6 ( 2005 ) ( Abstract only ) . Faustman et al . , “ Prevention of xenograft rejection by masking Engleman et al . , " Treatment of NZB / NZW F1 hybrid mice with donor HLA class I antigens, ” Science . 252 : 1700-1702 ( 1991 ) . Mycobacterium bovis strain BCG or type II interferon preparations Faustman et al . , “ Stem cells in the spleen : Therapeutic potential for accelerates autoimmune disease, ” Arthritis Rheum . 24 ( 11) : 1396 Sjogren's syndrome, type I diabetes, and other disorders , ” available 1402 ( 1981) . in PMC Jul. 21 , 2014 , published in final edited form as : Int J EPO Communication Enclosing Supplementary European Search Biochem Cell Biol . 42 ( 10 ) : 1576-9 ( 2010 ) ( 8 pages ). Report for EP Application No. 03762242.0 , dated Jun . 8 , 2009 ( 8 Faustman et al ., “ T - lymphocyte changes linked to autoantibodies. pages ). Association of insulin autoantibodies with CD4 + CD45R + lympho EPO Communication for International Application No. PCT /US2014 / cyte subpopulation in prediabetic subjects ," Diabetes. 40 : 590-597 015101 , dated Oct. 15 , 2015 ( 2 pages ) . ( 1991 ) . EPO Communication pursuant to 94 ( 3 ) for European Application Faustman et al . , “ TNF receptor 2 pathway : drug target for autoim No. 12005556.1 , dated Jul. 2 , 2015 ( 7 pages ). mune diseases , ” Nat Rev Drug Discov . 9 ( 6 ) : 482-93 ( 2010 ) . US 10,906,982 B2 Page 5
( 56 ) References Cited Grewal et al . , “ Local expression of transgene encoded TNFalpha in islets prevents autoimmune diabetes in nonobese diabetic ( NOD ) OTHER PUBLICATIONS mice by preventing the development of auto - reactive islet -specific T Cells, ” J Exp Med . 184 : 1963-1974 ( 1996 ) . Feldman et al ., “ Anti - TNFalpha therapy is useful in rheumatoid Grilli et al . , “ Neuroprotection by aspirin and sodium salicylate arthritis and Crohn's disease : Analysis of the mechanism of action through blockade of NF -kappaB activation ,” Science . 274 : 1383 predicts utility in other diseases , ” Transplant Proc . 30 ( 8 ) : 4126-4127 1385 ( 1996 ). ( 1998 ) . Gronostajski et al . , “ The ATP dependence of the degradation of Feldmann et al . , “ Role of cytokines in rheumatoid arthritis, ” Annu short- and long - lived proteins in growing fibroblasts ,” J Biol Chem . Rev Immunol . 14 : 397-440 ( 1996 ) ( abstract only ) . 260 ( 6 ) : 3344-3349 ( 1985 ) . Ferrando et al . , " Adult T - Cell ALL patients whose lymphoblasts Gueckel et al . , “ Mutations in the yeast proteasome beta - Type express the HOX11 oncogene have an excellent prognosis when subunit Pre3 uncover position -dependent effects on proteasomal peptidase activity and in vivo function , ” J Biol Chem . 273 (31 ) : 19443 treated with chemotherapy and are not candidates for allogeneic 19452 ( 1998 ) . bone marrow transplantation in first remission , ” Blood . 11 :Abstract Gupta , “ Molecular steps of tumor necrosis factor receptor -mediated 578 ( 2002 ) . apoptosis , " Curr Mol Med . 1 ( 3 ) : 317-324 ( 2001 ) . Fischer et al . , “ A TNF receptor 2 selective agonist rescues human Haas et al . , “ Pathways of ubiquitin conjugation , ” FASEB J. 11: 1257 neurons from oxidative stress - induced cell death ,” PloS One . 1268 ( 1997 ) . 6 ( 11 ) : e27621 ( 2011 ) ( 11 pages ) . Hao et al . , “ Effect of mycophenolate mofetil on islet allografting to Fischer et al ., “ An improved flow cytometric assay for the deter chemically induced or spontaneously diabetic animals, ” Transplant mination of cytotoxic T lymphocyte activity, ” J Immunol Methods. Proc . 24 ( 6 ) : 2843-2844 ( 1992 ) . 259 : 159-169 ( 2002 ) . Harada et al . , “ Prevention of overt diabetes and insulitis in NOD Foulis , “ C.L. Oakley lecture ( 1987 ) . The pathogenesis of beta cell mice by a single BCG vaccination , " Diabetes Res Clin Pract . destruction in Type I ( insulin -dependent ) diabetes mellitus, ” J 8 : 85-89 ( 1990 ) . Pathol . 152 ( 3 ) : 141-148 ( 1987 ) . Hartwell et al ., “ Aberrant cytokine regulation in macrophages from Fu et al . , “ Antigen processing and autoimmunity: Evaluation of young autoimmune -prone mice: Evidence that the intrinsic defect in mRNA abundance and function of HLA - Linked genes, ” Ann NY MRL macrophage IL - 1 expression is transcriptionally controlled ,"22 Acad Sci . 842 : 138-155 ( 1998 ) . Mol Immunol. 32 ( 10 ) : 743-751 ( 1995 ) . Fu et al . , “ Defective major histocompatibility complex class I Hayashi et al. , “ Essential role of human leukocyte antigen -encoded expression on lymphoid cells in autoimmunity ,” J Clin Invest . proteasome subunits in NF - kappaB activation and prevention of 91 : 2301-2307 ( 1993 ) . tumor necrosis factor -alpha - induced apoptosis ,” J Biol Chem . Fukada et al. , “ Two signals are necessary for cell proliferation 275 ( 7 ) : 5238-5247 ( 2000 ) . induced by a cytokine receptor gp130 : Involvement of STAT3 in Hayashi et al . , “ NOD mice are defective in proteasome production anti- apoptosis , ” Immunity . 5 : 449-460 ( 1996 ) . and activation of NF -kappaB ,” Mol Cell Biol . 19 ( 12 ) : 8646-8659 Gage et al . , “ Multipotent progenitor cells in the adult dentate ( 1999 ) . gyrus, " J Neurobiol . 36 : 249-266 ( 1998 ) . Hershko et al . , “ The ubiquitin system for protein degradation , ” Gage , “ Mammalian neural stem cells, ” Science . 287 : 1433-1438 Annu Rev Biochem . 61 : 761-807 ( 1992 ) . ( 2000 ) . Hester et al . , “ Studies on the cytophilic properties of human Galaria et al . , “ Leukocytoclastic vasculitis due to etanercept , ” J beta2 -microglobulin . II . The role of histocompatibility antigens , ” Rheumatol. 27 ( 8 ) : 2041-4 ( 2000 ) ( Abstract only ) . Scand J Immunol. 9 ( 2 ) : 125-134 ( 1979 ) . Ganoth et al . , “ A multicomponent system that degrades proteins Hoffmann et al . “ Large - scale in vitro expansion of polyclonal conjugated to ubiquitin . Resolution of factors and evidence for human CD4 ( + ) CD25high regulatory T cells, ” Blood . 104 ( 3 ): 895 ATP - dependent complex formation ,” J Biol Chem . 263( 25 ): 12412 903 ( 2004 ) . 12419 ( 1988 ) . Horsfall et al ., “ Characterization and specificity of B - cell responses Gaur et al . , “ Induction of islet allotolerance in nonhuman primates , " in lupus induced by Mycobacterium bovis in NOD / Lt mice ," Ann NY Acad Sci . 958 : 199-203 ( 2002 ) . Immunology 95 : 8-17 ( 1998 ) . Gazda et al . , “ Diabetes results from a late change in the autoimmune Horwitz et al . , “ Recombinant bacillus Calmette -Guérin ( BCG ) response of NOD mice ,” J Autoimmun . 10 : 261-270 ( 1997 ) . vaccines expressing the Mycobacterium tuberculosis 30 - kDa major Gazda et al . , “ Regulation of autoimmune diabetes : characteristics of secretory protein induce greater protective immunity against tuber non - islet - antigen specific therapies , ” Immunol Cell Biol . 74 : 401 culosis than conventional BCG vaccines in a highly susceptible 407 ( 1996 ) . animal model, " Proc Natl Acad Sci USA . 97 ( 35 ) : 13853-13858 Genestier et al . , “ Immunosuppressive properties of methotrexate: ( 2000 ) . Apoptosis and clonal deletion of activated peripheral T Cells , ” J Hostikka et al . , “ The mouse Hoxcll gene: genomic structure and Clin Invest. 102 ( 2 ) : 322-328 ( 1998 ). expression pattern , ” Mech Dev . 70 ( 1-2 ) : 133-145 ( 1998 ) ( Abstract Gerich et al ., “ Advances in diabetes for the millenium : Understand Only ). ing insulin resistance, " MedGenMed . 6 ( 3 Suppl ): 11 ( 2004 ) ( 9 Hsu et al . , “ TRADD - TRAF2 and TRADD - FADD interactions define pages ). two distinct TNF receptor 1 signal transduction pathways , ” Cell . Ghosh et al. , “ Activation in vitro of NF - kappaB by phosphorylation 84 : 299-308 ( 1996 ) . of its inhibitor IkappaB , ” Nature . 344 : 678-682 ( 1990 ) . Humphreys -Beher et al . , “ New concepts for the development of Glas et al . , “ The CD8 + T Cell repertoire in beta2 -microglobulin autoimmune exocrinopathy derived from studies with the NOD deficient mice is biased towards reactivity against self -major his mouse model, ” Arch Oral Biol . 44 (Suppl 1 ) : S21 - S25 ( 1999 ) ( Abstract tocompatibility class I, " J Exp Med . 179 ( 2 ) : 661-672 ( 1994 ) . Only ) ( 2 pages ) . Goldberg , “ Functions of the proteasome : The lysis at the end of the Hyafil et al . , “ Dissociation and exchange of the beta2 -micoglobulin tunnel, ” Science . 268 : 522-523 ( 1995 ) . subunit of HLA - A and HLA - B antigens , ” Proc Natl Acad Sci USA . Goldberg , “ The mechanism and functions of ATP -dependent proteases 76 ( 11 ) : 5834-5838 ( 1979 ) . in bacterial and animal cells ,” Eur J Biochem . 203 : 9-23 ( 1992 ) . Hymowitz et al . , “ Toward small - molecule agonists of TNF recep Gottlieb et al . , “ Cell acidification in apoptosis: Granulocyte colony tors , ” Nat Chem Biol . 1 ( 7 ) : 353-354 ( 2005) . stimulating factor delays programmed cell death in neutrophils by International Preliminary Report on Patentability for International up -regulating the vacuolar H +-ATPase ,” Proc Natl Acad Sci USA . Application No. PCT /US2014 / 015101, dated Aug. 11 , 2015 ( 9 92 : 5965-5968 ( 1995 ) . pages) . Graves et al . , “ Lack of association between early childhood immu International Preliminary Report on Patentability for International nizations and beta - Cell autoimmunity , " Diabetes Care 22: 1694 Application No. PCT/ US2016 /032547 , dated Nov. 21 , 2017 ( 16 1697 ( 1999 ) . pages) . US 10,906,982 B2 Page 6
( 56 ) References Cited Kodama et al . , “ The therapeutic potential of tumor necrosis factor for autoimmune disease : A mechanistically based hypothesis, ” Cell OTHER PUBLICATIONS Mol Life Sci . 62 : 1850-1862 ( 2005 ) . Kopp et al . , “ Inhibition of NF -kappaB by sodium salicylate and International Search Report and Written Opinion for International aspirin ,” Science . 265 : 956-959 ( 1994 ) . Application No. PCT /US16 / 32547 , dated Aug. 31 , 2016 ( 24 pages ). Kouskoff et al . , “ Organ - specific disease provoked by systemic International Search Report and Written Opinion for International autoimmunity , ” Cell . 87 ( 5 ) : 811-822 ( 1996 ) ( Abstract Only ). Application No. PCT / US17 / 32513 , dated Oct. 25 , 2017 ( 17 pages ). Koyama et al . , “ Hoxll genes establish synovial joint organization International Search Report and Written Opinion for International and phylogenetic characteristics in developing mouse zeugopod Patent Application No. PCT / US14 / 15101 , dated Jun . 24 , 2014 ( 14 skeletal elements ,” Development . 137 ( 22 ) : 3795-800 ( 2010 ) (Abstract pages ) Only ). International Search Report for International Application No. PCT / Krause et al . , “ Multi - organ , multi- lineage engraftment by a single US00 / 06239 dated Jul . 31 , 2000 ( 2 pages ) . bone marrow - derived stem cell ,” Cell . 105 : 369-377 ( 2001 ) . International Search Report for International Application No. PCT / Kretschmer et al ., " Strong antigenic selection shaping the immu US2004 /037998 , dated Feb. 28 , 2008 ( 2 pages ). noglobulin heavy chain repertoire of B - la lymphocytes in lambda International Search Report for International Patent Application No. 2 ( 315 ) transgenic mice, ” Eur J Immunol. 32 ( 8 ) : 2317-27 ( 2002 ) . PCT /US03 / 20578 , dated Apr. 27 , 2004 ( 1 page ). Kuehnle et al . , “ The therapeutic potential of stem cells from adults, " International Search Report for International Patent Application No. BMJ . 325 : 372-376 ( 2002 ) . PCT /US03 / 36531 , dated Jul. 14 , 2004 ( 1 page ) . Kwon et al . , “ Evidence for involvement of the proteasome complex Jackson et al ., “ Hematopoietic potential of stem cells isolated from ( 26S ) and NFkappaB in IL - 1 beta - induced nitric oxide and prosta murine skeletal muscle, ” Proc Natl Acad Sci USA . 96 (25 ) : 14482 glandin production by rat islets and RINm5F Cells ” Diabetes . 14486 ( 1999 ) . 47 : 583-591 ( 1998 ). Jacob et al. , “ Monoclonal anti -tumor necrosis factor antibody Kwon et al . , “ Interleukin - lbeta - induced nitric oxide synthase expres renders non - obese diabetic mice hypersensitive to irradiation and sion by rat pancreatic beta - cells : Evidence for the involvement of enhances insulitis development, ” Int Immunol. 4 ( 5 ) : 611-614 ( 1992 ) . nuclear factor kappaB in the signaling mechanism ,” Endocrinology . Jacob et al . , “ Prevention of diabetes in nonobese diabetic mice by 136 ( 11 ) : 4790-4795 ( 1995 ) . tumor necrosis factor ( TNF ) : Similarities between TNF -alpha and Laakko et al . , “ Versatility of merocyanine 540 for the flow cytometric interleukin 1 ,” Proc Natl Acad Sci USA . 87 : 968-972 ( 1990 ) . detection of apoptosis in human and murine cells, ” J Immunol Jacob et al . , “ Tumour necrosis factor - alpha in murine autoimmune Methods . 261 : 129-139 ( 2002 ) . * lupus ' nephritis ," Nature . 331 : 356-358 ( 1988 ) . Lahav - Baratz et al . , “ Reversible phosphorylation controls the activ Jakubowski et al . , " Phase I trial of intramuscularly administered ity of cyclosome -associated cyclin -ubiquitin ligase , ” Proc Natl tumor necrosis factor in patients with advanced cancer, " J Clin Acad Sci USA . 92 : 9303-9307 ( 1995 ). Oncol . 7 ( 3 ) : 298-303 ( 1989 ) . Lakey et al . , “ BCG immunotherapy prevents recurrence of diabetes Jarrett et al . , “ Anti - tumor necrosis factor - alpha therapy - induced in islet grafts transplanted into spontaneously diabetic NOD mice , " vasculitis : case series ,” J Rheumatol. 30 ( 10 ) : 2287-91 ( 2003 ) ( Abstract Transplantation . 57 ( 8 ) : 1213-1217 ( 1994 ) . only ) . Lammert et al ., “ Induction of pancreatic differentiation by signals Jiang et al . , “ Pluripotency of mesenchymal stem cells derived from from blood vessels ,” Science . 294 : 564-567 ( 2001 ) . adult marrow , " Nature . 418 : 41-49 ( 2002 ) . Lanza et al . , “ Transplantation of encapsulated canine islets into Johansson et al . , “ Identification of a neural stem cell in the adult spontaneously diabetic BB /Wor rats without immunosuppression ,” mammalian central nervous system ,” Cell . 96 : 25-34 ( 1999 ) . Endocrinology. 131 ( 2 ) : 637-642 ( 1992 ) . Juang et al . , “ Beneficial influence of glycemic control upon the Lapchak et al . , “ Tumor necrosis factor production is deficient in growth and function of transplanted islets, ” Diabetes 43 : 1334-1339 diabetes -prone BB rats and can be corrected by complete Freund's ( 1994 ) . adjuvant: A possible immunoregulatory role of tumor necrosis Kaijzel et al . , “ Functional analysis of a human tumor necrosis factor factor in the prevention of diabetes, " Clin Immunol Immunopathol. alpha ( TNF -alpha ) promoter polymorphism related to joint damage 65 ( 2 ) : 129-134 ( 1992 ) . in rheumatoid arthritis, ” Mol Med . 4 : 724-733 ( 1998 ) . Lawrence et al . , “ Differential hepatocyte toxicity of recombinant Kanzler et al ., “ Hox11 acts cell autonomously in spleen develop Apo2L / TRAIL versions, ” Nat Med . 7 ( 4 ) : 383-385 ( 2001 ) . ment and its absence results in altered cell fate of mesenchymal Lewis et al . , “ Integrins regulate the apoptotic response to DNA spleen precursors , ” Devel Biol. 234 : 231-243 ( 2001 ). damage through modulation of p53 ,” Proc Natl Acad Sci USA . Kaufman et al . , “ Patterns of hemopoietic reconstitution in nonobese 99 ( 6 ) : 3627-3632 ( 2002 ) . diabetic mice : dichotomy of allogeneic resistance versus competi Li et al . , “ Abnormal class I assembly and peptide presentation in the tive advantage of disease -resistant marrow , " J Immunol. 158 ( 5 ) : 2435 nonobese diabetic mouse , " Proc Natl Acad Sci USA . 91 : 11128 2442 ( 1997 ) . 11132 ( 1994 ) . Kawasaki et al . , “ Prevention of type 1 diabetes: from the view point Li et al ., “ Reduced expression of peptide - loaded HLA class I of beta cell damage, " Diabetes Res Clin Pract . 66 : S27 - S32 ( 2004 ) . molecules on multiple sclerosis lymphocytes, ” Ann Neurol . 38 : 147 Kieran et al . , “ The DNA binding subunit of NF - kappaB is identical 154 ( 1995 ) . to factor KBF1 and homologous to the rel oncogene product, ” Cell . Li et al ., “ Use of Donor beta2 - Microglobulin - Deficient Transgenic 62 : 1007-1018 ( 1990 ) . Mouse Liver Cells for Isografts, Allografts, and Xenografts, ” Trans Klingensmith et al . , “ Vaccination with BCG at diagnosis does not plantation . 55 ( 4 ) : 940-6 ( 1993 ) . alter the course of IDDM ,” Diabetes 57th Annual Meeting and Lipsky et al . , “ Infliximab and methotrexate in the treatment of Scientific Sessions , Jun . 21-24 , Boston MA . 40 (Suppl1 ) : 193A , rheumatoid arthritis ," N Eng J Med . 343 : 1594-1602 ( 2000 ) . 0744 ( 1997 ) ( 3 pages ) ( Abstract Only ). Ljunggren et al ., “ MHC class I expression and CD8 + T cell Klinkhoff , “ Biological agents for rheumatoid arthritis : targeting development in TAP1 /beta2 -microglobulin double mutant mice , " both physical function and structural damage, ” Drugs. 64 ( 12 ): 1267 Int Immunol. 7 ( 6 ) : 975-984 ( 1995 ). 83 ( 2004 ) ( Abstract only ) . Loetscher et al . , “ Human tumor necrosis factor alpha ( TNF alpha ) Koarada et al . , “ B Cells lacking RP105 , A novel B cell antigen, in mutants with exclusive specificity for the 55 -kDa or 75 - kDa TNF systemic lupus erythematosus, ” Arthritis & Rheumatism . 42( 12 ): 2593 receptors ,” J Biol Chem . 268 ( 35 ) : 26350-26357 ( 1993 ) . 2600 ( 1999 ) . Macchi et al . , “ Impaired apoptosis in mitogen - stimulated lympho Kodama et al . , “ Islet regeneration during the reversal of autoim cytes ofpatients with multiple sclerosis, ” NeuroReport. 10 ( 25 ) :399 mune diabetes in NOD mice , ” Science . 302 : 1223-1227 ( 2003 ) . 402 ( 1999 ) . Kodama et al . , “ Regenerative medicine : A radical reappraisal of the Mak et al . , “ Signaling for survival and apoptosis in the immune spleen ,” Trends Mol Med . 11 ( 6 ) : 271-276 ( 2005 ) . system , ” Arthritis Res . 4 (Suppl 3 ) : S243-8252 ( 2002 ) . US 10,906,982 B2 Page 7
( 56 ) References Cited Paolillo et al . , “ The effect of Bacille Calmette -Guérin on the evolution of new enhancing lesions to hypointense T1 lesions in OTHER PUBLICATIONS relapsing remitting MS, ” J Neurol . 250 : 247-248 ( 2003 ) . Pestano et al . , “ Inactivation of misselected CD8 T cells by CD8 Markiewicz et al . , “ Long - term T cell memory requires the surface gene methylation and cell death , ” Science . 284 ( 5417 ) : 1187-91 expression of self - peptide /major histocompatibility complex mol ( 1999 ) . ecules , ” Proc Natl Sci USA . 95 : 3065-3070 ( 1998 ) . Petersen et al . , “ Bone marrow as a potential source of hepatic oval Markmann et al . , “ Indefinite survival of MHC class I - deficient cells , " Science . 284 ( 5417 ) : 1168-70 ( 1999 ) . murine pancreatic islet allografts , ” Transplantation. 54 (6 ): 1085 Pillay et al . , “ Antibodies in oncology, " N Biotechnol. 28 ( 5 ) : 518-529 1089 ( 1992 ) . ( 2011 ) . Marriott , “ TNF - alpha antagonists: Monoclonal antibodies, soluble Pontesilli et al ., “ Circulating lymphocyte populations and autoantibod receptors, thalidomide and other novel approaches , ” Expert Opin ies in non - obese diabetic ( Nod ) mice: a longitudinal study, " Clin Exp Immunol. 70 ( 1 ) : 84-93 ( 1987 ). Invest Drugs . 6 ( 8 ) : 1105-1108 ( 1997 ) . Pozzilli , “ BCG vaccine in insulin -dependent diabetes mellitus , ” Matsumoto et al . , “ Liver organogenesis promoted by endothelial Lancet . 349 ( 9064 ) : 1520-1 ( 1997 ) . cells prior to vascular function ,” Science . 294 : 559-563 ( 2001 ) . Prieto et al ., “ Apoptotic rate : A new indicator for the quantification Mayer -Proschel et al . , “ Isolation of lineage - restricted neuronal ofthe incidence of apoptosis in cell cultures ,” Cytometry . 48 ( 4 ): 185 precursors from multipotent neuroepithelial stem cells, ” Neuron . 93 ( 2002 ) . 19 : 773-785 ( 1997 ) . Product Data Sheet for TNF RII / TNFRSF1 B Inhibition of TNP McGuire et al . , “ An enzyme related to the high molecular weight alpha - induced Cyototoxicity and Neutralization by Human TNF multicatalytic proteinase , macropain , participates in a ubiquitin RII / TNFRSF1B Antibody. Retrieved Aug. 1 , 2017. R & D Systems mediated , ATP - stimulated proteolytic pathway in soluble extracts of Inc. (3 pages) . BHK 21 / C13 fibroblasts , " Biochim Biophys Acta . 967 : 195-203 Qin et al ., " BCG vaccination prevents insulin -dependent diabetes ( 1988 ) . mellitus ( IDDM ) in NOD mice after disease acceleration with McInerney et al . , “ Prevention of insulitis and diabetes onset by cyclophosphamide, ” J Autoimmun . 10 : 271-278 ( 1997 ) . treatment with complete Freund's adjuvant in NOD mice, ” Diabe Qin et al . , “ Complete Freund's adjuvant - induced T cells prevent the tes . 40 : 715-725 ( 1991 ). development and adoptive transfer of diabetes in nonobese diabetic McKay, “ Mammalian deconstruction for stem cell reconstruction , " mice ," J Immunol . 150 ( 5 ) : 2072-80 ( 1993 ) . Nat Med . 6 ( 7 ) : 747-748 ( 2000 ) . Quintana et al . , “ Experimental autoimmune myasthenia gravis in Mercurio et al . , “ p105 and p98 precursor proteins play an active role naïve non - obese diabetic ( NOD /LTJ ) mice : Susceptibility associ in NF -Kappa B -mediated signal transduction ,” Genes Dev . 7 :705 ated with natural IgG antibodies to the acetylcholine receptor , ” Int Immunol. 15 ( 1 ) : 11-16 ( 2003 ) . 718 ( 1993 ) . Raab et al . , “ In vitro evaluation of methotrexate and azathioprine for Mestas et al . , “ Of mice and not men : Differences between mouse antipsoriatic activity, ” Arch Derm Res . 253 : 77-84 ( 1975 ) . and human immunology, " J Immunol. 172 : 2731-2738 ( 2004 ) . Rabinovitch et al . , “ TNF - alpha down - regulates type 1 cytokines and Mezey et al . , “ Turning blood into brain : Cells bearing neuronal prolongs survival of syngeneic islet grafts in nonobese diabetic antigens generated in vivo from bone marrow , " Science . 290 : mice ,” J Immunol. 159 ( 12 ) : 6298-6303 ( 1997 ) . 1779-1782 ( 2000 ). Rabinovitch et al. , “ Tumor necrosis factor mediates the protective Miller et al . , “ Both the Lyt - 2 + and L3T4 + T cell subsets are required effect of Freund's adjuvant against autoimmune diabetes in BB for the transfer of diabetes in nonobese diabetic mice ,” J Immunol. rats ,” J Autoimmun . 8 ( 3 ) : 357-366 ( 1995 ) . 140 ( 3 ) : 52-58 ( 1988 ) . Rajagopalan et al ., “ Pathogenic anti -Dna autoantibody - inducing T Mittelman et al . , “ A phase I pharmacokinetic study of recombinant helper cell lines from patients with active lupus nephritis: Isolation human tumor necrosis factor administered by a 5 -day continuous of CD4-8- T helper cell lines that express the gamma delta T -cell infusion , ” Invest New Drugs . 10 ( 3 ) : 183-190 ( 1992 ) . antigen receptor, " Proc Natl Acad Sci USA . 87 : 7020-7024 ( 1990 ) . Miyazaki et al ., “ Predominance of T lymphocytes in pancreatic Raju et al ., " Characterization and developmental expression of islets and spleen of pre -diabetic non - obese diabetic ( NOD ) mice : A Tlx - 1 , the murine homolog of HOX11 , " Mech Dev . 44 ( 1 ) : 51-64 longitudinal study , ” Clin Exp Immunol. 60 : 622-630 ( 1985 ) . ( 1993 ) . Moreland et al . , “ Etanercept therapy in rheumatoid arthritis: a Ramiya et al . , " Reversal of insulin -dependent diabetes using islets randomized , controlled trial, ” Ann Intern Med . 130 ( 6 ) : 478-486 generated in vitro from pancreatic stem cells , ” Nat Med . 6 ( 3 ) : 278 ( 1999 ) . 282 ( 2000 ) . Morrison , " Stem cell potential : Can anything make anything ? ” Curr Rath et al . , “ TNF - induced signaling in apoptosis , ” J Clin Immunol. Biol . 11 :R7 -RO ( 2001 ) . 19 ( 6 ) : 350-364 ( 1999 ) . Murthi et al . , “ Novel homeobox genes are differentially expressed Rechsteiner, “ Ubiquitin -mediated pathways for intracellular proteolysis, ” in placental microvascular endothelial cells compared with Annu Rev Cell Biol . 3 : 1-30 ( 1987 ). macrovascular cells ,” Placenta . 29 ( 7 ) : 624-630 ( 2008 ) ( Abstract Rietze et al . , “ Purification of a pluripotent neural stem cell from the Only ) . adult mouse brain ,” Nature . 412 ( 6848 ) : 736-739 ( 2001 ) . Nomikos et al . , “ Combined treatment with nicotinamide and desfer Ristori et al. , “ Use of Bacille Calmette -Guérin ( BCG ) in multiple rioxamine prevents islet allograft destruction in NOD mice, " Dia sclerosis, ” Neurology . 53 : 1588-1589 ( 1999 ) . betes. 35 : 1302-1304 ( 1986 ) . Roberts et al . , “ Developmental expression of Hoxll and specifica Offield et al . , " PDX - 1 is required for pancreatic outgrowth and tion of splenic cell fate , ” Am J Pathol. 146 ( 5 ) : 1089-1101 ( 1995 ) . differentiation of the rostral duodenum , ” Development. 122 :983 Roberts et al . , “ Hox11 controls the genesis of the spleen ,” Nature . 995 ( 1996 ) . 368 : 747-749 ( 1994 ) . Ono et al . , “ IDDM in BB rats . Enhanced MHC Class I heavy - chain Robertson et al . , “ Preservation of insulin mRNA levels and insulin gene expression in pancreatic islets, " Diabetes. 37 : 1411-1418 (1988 ) . secretion in HIT cells by avoidance of chronic exposure to high Orlowski , “ The multicatalytic proteinase complex , a major glucose concentrations , " J Clin Invest . 90 : 320-325 ( 1992 ) . extralysosomal proteolytic system , ” Biochemistry . 29( 45 ): 10289 Robinson et al . , “ A novel NOD - derived murine model of primary 10297 ( 1990 ). Sjögren's Syndrome, ” Arth Rheum . 41 ( 1 ) : 150-156 ( 1998 ) . Osorio et al . , “ Beta - 2 microglobulin gene disruption prolongs Robinson et al . , " Elevated levels of cysteine protease activity in murine islet allograft survival in NOD mice ," Transplant Proc . saliva and salivary glands of the nonobese diabetic ( NOD ) mouse 26 ( 2 ) : 752 ( 1994 ) . model for Sjögren Syndrome,” Proc Natl Acad Sci USA . 94 :5767 Palombella et al ., “ The ubiquitin - proteasome pathway is required 5771 ( 1997 ) . for processing the NF- kappaB1 precursor protein and the activation Rolfe et al . , “ The ubiquitin - mediated proteolytic pathway as a of NF - kappaB , ” Cell . 78 :773-785 ( 1994 ) . therapeutic area ,” J Mol Med . 75 : 5-17 ( 1997 ) . US 10,906,982 B2 Page 8
( 56 ) References Cited Song et al ., “ Tumor necrosis factor - related apoptosis -inducing ligand ( TRAIL ) is an inhibitor of autoimmune inflammation and cell cycle OTHER PUBLICATIONS progression ,” J Exp Med . 191 ( 7 ) : 1095-1103 ( 2000 ) . Speiser et al . , “ Loss of ATP - dependent proteolysis with maturation Rosenthal, “ Prometheus's vulture and the stem - cell promise , ” N of reticulocytes and erythrocytes, ” J Biol Chem . 257 (23 ): 14122 Engl J Med . 349 ( 3 ) : 267-74 ( 2003 ) . 14127 ( 1982 ) . Ryu et al . , “ Reversal of established autoimmune diabetes by resto Sreenan et al . , “ Increased beta -Cell proliferation and reduced mass ration of endogenous beta cell function ," J Clin Invest . 108 ( 1 ) : 63-72 before diabetes onset in the nonobese diabetic mouse, " Diabetes. ( 2001 ) . 48 : 989-996 ( 1999 ) . Sadelain et al ., “ Prevention of type 1 diabetes in NOD mice by Stephens et al . , “ Protection of NIT - 1 pancreatic beta - cells from adjuvant immunotherapy , ” Diabetes . 39 : 583-589 ( 1990 ). immune attack by inhibition of NF -kappaB ,” J Autoimmun . 10 : 293 Sandborn et al . , “ Antitumor necrosis factor therapy for inflamma 298 ( 1997 ) . tory bowel disease : a review of agents, pharmacology , clinical Storms et al . , “ Hoechst dye efflux reveals a novel CD7 + CD34 results , and safety ,” Inflamm Bowel Dis . 5 ( 2 ) : 119-33 ( 1999 ) (Abstract lymphoid progenitor in human umbilical cord blood , ” Blood . only ). 96 ( 6 ) : 2125-2133 ( 2000 ) . Sandborn , “ Strategies targeting tumor necrosis factor in Crohn's Sun et al . , “ MHC class I multimers , ” Arthritis Res . 3 ( 5 ) : 265-269 disease ,” Acta Gastroenterol Belg . 64 ( 2 ) : 170-2 ( 2001 ) ( Abstract ( 2001 ) . only ). Supplementary Partial European Search Report for European Appli Sarin et al . , “ Cytotoxic effect of TNF and lymphotoxin on T cation No. 04817543 , dated Oct. 6 , 2009 ( 4 pages ) . lymphoblasts, ” J Immunol . 155 : 3716-3718 ( 1995 ) . Swale et al . , “ Etanercept- induced systemic lupus erythematosus, " Satoh et al . , “ Inhibition of type 1 diabetes in BB rats with recom Clin Exp Dermatol. 28 : 604-607 ( 2003 ) . binant human tumor necrosis factor - alpha , ” J Immunol. 145 ( 5 ) : 1395 Swirski et al . , “ Identification of splenic reservoir monocytes and 1399 ( 1990 ) . their deployment to inflammatory sites ," available in PMC Jan. 7 , Satoh et al . , “ Recombinant human tumor necrosis factor alpha 2010 , published in final edited form as : Science . 325 ( 5940 ) : 612-616 suppresses autoimmune diabetes in nonobese diabetic mice, ” J Clin ( 2009 ) ( 12 pages ). Invest . 84 : 1345-1348 ( 1989 ) . Schaible , “ Long term safety of infliximab ,” Can J Gastroenterol. Szodoray et al ., “ Programmed cell death in rheumatoid arthritis 14 ( Suppl C ) : 29C - 32C ( 2000 ) ( Abstract only ). peripheral blood T - cell subpopulations determined by laser scan Schatz et al . , “ Defective inducer T - cell function before the onset of ning cytometry , ” Lab Invest. 83 ( 12 ) : 1839-1848 ( 2003 ) . insulin -dependent diabetes mellitus , ” J Autoimmun. 4 : 125-136 ( 1991 ) . Tamura et al . , “ In vivo differentiation of stem cells in the aorta Schmidt et al ., “ Interspecies exchange of beta2 - microglobulin and gonad -mesonephros region of mouse embryo and adult bone mar associated MHC and differentiation antigens , " Immunogenetics . row , ” Exp Hematol . 30 ( 8 ) : 957-966 ( 2002 ) ( Abstract Only ) . 13 ( 6 ) : 483-91 ( 1981 ) . Tartaglia et al . , “ The two different receptors for tumor necrosis Schuppan , “ Current concepts of Celiac Disease pathogenesis, " factor mediate distinct cellular responses, ” Proc Natl Acad Sci USA . Gastroenterology . 119 ( 1 ) : 234-242 ( 2000 ) . 88 : 9292-9296 ( 1991 ) . Sears et al . , “ NF - kappaB p105 processing via the ubiquitin Tavernier et al . , “ Analysis of the structure -function relationship of proteasome pathway, ” J Biol Chem . 273 ( 3 ) : 1409-1419 ( 1998 ) . tumour necrosis factor. Human /mouse chimeric TNF proteins: gen Sedger et al. , “ Poxvirus tumor necrosis factor receptor ( TNFR )-like eral properties and epitope analysis ,” J Mol Biol . 211 ( 2 ) : 493-501 T2 proteins contain a conserved preligand assembly domain that ( 1990 ) . inhibits cellular TNFR1 - induced cell death ,” J Virol. 80 ( 18 ) : 9300-9 Technical Data Sheet for Purified Rat Anti - Human CD120b , BD ( 2006 ) . PharmingenTM ( 2011 ) ( 2 pages ) . Serrano et al . , “ Non - HLA associations with autoimmune diseases, " Terada et al . , “ Bone marrow cells adopt the phenotype of other cells Autoimmun Rev. 5 : 209-214 ( 2006 ) . by spontaneous cell fusion , ” Nature . 416 : 542-545 ( 2002 ) . Serreze et al . , “ Th1 to Th2 cytokine shifts in nonobese diabetic Thomas et al ., “ Demyelination during anti - tumor necrosis factor mice : Sometimes an outcome , rather than the cause , of diabetes alpha therapy with infliximab for Crohn's disease , ” Inflamm Bowel resistance elicited by immunostimulation , ” J Immunol. 166 : 1352 Dis . 10 ( 1 ) : 28-31 ( 2004 ) ( Abstract only ). 1359 ( 2001 ) . Toma et al . , “ Isolation of multipotent adult stem cells from the Serup et al . , “ Islet and stem cell transplantation for treating diabe dermis of mammalian skin ," Nat Cell Bio . 3 : 778-784 ( 2001 ) . tes, ” BMJ . 322 : 29-32 (2001 ) . Totpal et al . , “ TNF and its receptor antibody agonist differ in Serup , “ Panning for pancreatic stem cells ,” Nat Genet . 25 : 134-135 mediation of cellular responses, ” J Immunol. 153 : 2248-2257 ( 1994 ) . ( 2000 ) . Townsley et al. , “ Dominant -negative cyclin - selective ubiquitin car Shakoor et al . , “ Drug - induced systemic lupus erythematosus asso rier protein E2 - C /UbcH10 blocks cells in metaphase , " Proc Natl ciated with etanercept therapy, ” Lancet. 359 ( 9306 ) : 579-80 ( 2002 ) Acad Sci USA . 94 : 2362-2367 ( 1997 ) . ( Absract only ) . Trad et al . , “ Clonal Progression during the T Cell -Dependent B Cell Shehadeh et al. , “ Effect of adjuvant therapy on development of Antibody Response Depends on the Immunoglobulin DH Gene diabetes in mouse and man , ” Lancet . 343 : 706-707 ( 1994 ) . Segment Repertoire, ” Front Immunol. 5 ( 385 ) : 1-11 ( 2014 ) . Shehadeh et al ., “ Repeated BCG vaccination is more effective than Tran et al . , “ Reversal of Sjögren's - like syndrome in non - obese a single dose in preventing diabetes in non - obese diabetic ( NOD ) diabetic mice , ” Ann Rheum Dis . 66 : 812-814 ( 2007 ) . mice , ” Isr J Med Sci . 33 ( 11 ) : 711-715 ( 1997 ) . Trowsdale et al . , “ Sequences encoded in the class II region of the Shihabuddin et al . , " Adult spinal cord stem cells generate neurons MHC related to the ‘ ABC'superfamily of transporters , ” Nature . after transplantation in the adult dentate gyrus , ” J Neurosci. 20 (23 ) :8727 348 : 741-744 ( 1990 ) . 8735 ( 2000 ) . Ulaeto et al . , “ A T -cell dormant state in the autoimmune process of Shohami et al . , “ Dual role of tumor necrosis factor alpha in brain nonobese diabetic mice treated with complete Freund's adjuvant ," injury ,” Cytokine Growth Factor Rev. 10 : 119-130 ( 1999 ) . Proc Natl Acad Sci USA . 89 : 3927-3931 ( 1992 ) . Silva et al . , “ Prevention of autoimmune diabetes through Van der Kooy et al . , “ Why stem cells ?” Science . 287 : 1439-1441 immunostimulation with Q fever complement - fixing antigen , ” Ann ( 2000 ) . NY Acad Sci . 1005 : 423-430 ( 2003 ) . Van Nocker et al . , “ The multiubiquitin - chain -binding protein Mcb1 Singh et al . , " Can progression of IDDM be prevented in newly is a component of the 26S proteasome in Saccharomyces cerevisiae diagnosed patients by BCG immunotherapy ?" Diabetes Metab Rev. and plays a nonessential , substrate - specific role in protein turnover , " 13 ( 4 ) : 320-321 ( 1997 ) . Mol Cell Biol. 16 ( 11 ) :6020-6028 ( 1996 ) . Slack , “ Stem cells in epithelial tissues ,” Science . 287 : 1431-1433 Van Noort et al . , " Cell biology of autoimmune diseases , ” Int Rev ( 2000 ) . Cytol. 178 : 127-206 ( 1998 ) . US 10,906,982 B2 Page 9
( 56 ) References Cited Winston , “ Embryonic stem cell research : the case for ... > > Nat Med . 7 ( 4 ) : 396-397 ( 2001 ) . OTHER PUBLICATIONS Wong et al . , “ Identification of an MHC class I - restricted autoantigen in Type I Diabetes by screening an organ - specific cDNA library, " Van Zee et al . , “ A human tumor necrosis factor ( TNF ) alpha mutant Nat Med . 5 ( 9 ) : 1026-1031 ( 1999 ) . that binds exclusively to the p55 TNF receptor produces toxicity in Written Opinion for International Application No. PCT /US2004 / the baboon , ” J Exp Med . 179 ( 4 ) : 1185-1191 ( 1994 ) . 037998 , dated Feb. 28 , 2008 ( 3 pages ) . Vermeire et al . , “ Autoimmunity associated with anti -tumor necrosis Xu et al ., " MHC / peptide tetramer - based studies of T cell function , " factor alpha treatment in Crohn's disease: a prospective cohort J Immunol Methods . 268 : 21-28 ( 2002 ) . study, ” Gastroenterology. 125 ( 1 ) : 32-9 ( 2003 ) ( Abstract only ) . Yagi et al. , “ Possible mechanism of the preventive effect of BCG Vidal- Puig et al . , “ Tolerance to peripheral tissue Is transient and against diabetes mellitus in NOD Mouse . I. Generation of suppres maintained by tissue - specific class I expression , ” Transplant Proc . sor macrophages in spleen cells of BCG -vaccinated mice ,” Cell 26 ( 6 ) : 3314-6 ( 1994 ) . Immunol. 138 : 130-141 ( 1991 ) . Vogel, “ Stem cell research . Studies cast doubt on plasticity of adult Yagi et al. , “ Possible mechanism of the preventive effect of BCG cells, ” Science . 295 : 1989 & 1991 ( 2002 ) . against diabetes mellitus in NOD Mouse . II . Suppression of patho Von Herrath et al ., “ In vivo treatment with a MHC class I - restricted genesis by macrophage transfer from BCG -vaccinated mice ,” Cell blocking peptide can prevent virus - induced autoimmune diabetes , ” Immunol. 138 : 142-149 ( 1991 ) . J Immunol. 161 : 5087-5096 ( 1998 ). Yan et al ., “ Reduced expression of Tapl and Lmp2 antigen Wagner, “ Making and using antibodies, ” < http://www-users.med . processing genes in the nonobese diabetic ( NOD ) mouse due to a cornell.edu/~jawagne/Antibody_Approaches.html> , accessed Aug. mutation in their shared bidirectional promoter, ” J Immunol. 159 :3068 8 , 2016 ( 7 pages ). 3080 ( 1997 ) . Wang et al . , “ Prevention of recurrence of IDDM in islet Yang et al ., “ Effect of tumor necrosis factor alpha on insulin transplanted diabetic NOD mice by adjuvant immunotherapy , ” dependent diabetes mellitus in NOD Mice . I. The early development Diabetes. 41 : 114-117 ( 1992 ) . of autoimmunity and the diabetogenic process , " J Exp Med . 180 : 995 Watt et al . , “ Out of eden : stem cells and their niches , " Science . 1004 ( 1994 ) . 287 : 1427-1430 ( 2000 ) . Ying et al . , " Changing potency by spontaneous fusion ,” Nature. Watt et al . , “ Specific alternative HOX11 transcripts are expressed in 416 : 545-548 ( 2002 ) . paediatric neural tumours and T - cell acute lymphoblastic leukaemia , " Yu et al. , " Complex Interplay between Epitope Specificity and Gene . 323 : 89-99 ( 2003 ) ( Abstract only ). Isotype Dictates the Biological Activity of Antihuman CD40 Anti Waxman et al . , “ Demonstration of two distinct high molecular bodies , ” Cancer Cell . 33 : 1-12 ( 2018 ) ( 17 pages ) . weight proteases in rabbit reticulocytes, one of which degrades Zulewski et al . , “ Multipotential nestin - positive stem cells isolated ubiquitin conjugates , ” J Biol Chem . 262 ( 6 ) : 2451-2457 ( 1987 ) . from adult pancreatic islets differentiate ex vivo into pancreatic Weissman , “ Translating stem and progenitor cell biology to the endocrine , exocrine , and hepatic phenotypes , " Diabetes . 50 : 521 clinic : barriers and opportunities, ” Science . 287 : 1442-1446 ( 2000 ) . 533 ( 2001 ) . Welborn et al . , “ A human tumor necrosis factor p75 receptor agonist Zöller et al . , “ Apoptosis resistance in peripheral blood lymphocytes stimulates in vitro T cell proliferation but does not produce inflam of alopecia areata patients, ” Retrieved from Science Direct , pub mation or shock in the baboon , ” J Exp Med . 184 ( 1 ) : 165-171 ( 1996 ) . lished in : J Autoimmun . 23 ( 3 ) : 241-256 ( 2004 ) ( 30 pages ). Wellik et al. , “ Hox11 paralogous genes are essential for metanephric Okubo et al ., “ Homogeneous Expansion of Human T - regulatory kidney induction , ” Genes Dev. 16 : 1423-1432 ( 2002 ) . Cells via Tumor Necrosis Factor Receptor 2 , ” Sci Rep . 3 : 3153 ( 11 Wellik , “ The role of Hox11 paralogous genes in prostate develop pages ) ment , ” Grant Detail. ( 2009 ) ( 1 page ) ( Abstract only ) . Faustman et al. , “ TNF Receptor 2 and Disease : Autoimmunity and Weringer et al . , “ Identification of T cell subsets and Class I and Regenerative Medicine, ” Front Immunol. 4 : 478 ( 2013 ) ( 8 pages ). Class II antigen expression in islet grafts and pancreatic islets of Ban et al . , “ Strategic Internal Covalent Cross -Linking of TNF diabetic BioBreeding /Worcester rats , ” Am J Pathol. 132 ( 2 ) : 292-303 Produces a Stable TNF Trimer With Improved TNFR2 Signaling ," ( 1988 ) . Mol Cell Ther . 3 : 7 ( 2015 ) ( 6 pages ) . Wicker et al . , “ Transfer of autoimmune diabetes mellitus with Torrey et al . , “ Targeting TNFR2 With Antagonistic Antibodies splenocytes from nonobese diabetic ( NOD ) mice, ” Diabetes . 35 :855 Inhibits Proliferation of Ovarian Cancer Cells and Tumor 860 ( 1986 ) . Associated Tregs , ” Sci Signal. 10 (462 ) : eaaf8608 ( 2017 ) ( 13 pages ). Willis et al . , “ Type 1 Diabetes in insulin -treated adult -onset diabetic Extended European Search Report for European Application No. subjects ,” Diabetes Res Clin Pract . 42 : 49-53 ( 1998 ) . 17796980.5 , dated Mar. 5 , 2020 ( 12 pages ) . Wilson et al ., “ Bone -marrow haematopoietic - stem -cell niches, ” Nat Rev Immunol . 6 ( 2 ) : 93-106 ( 2006 ) . * cited by examiner U.S. Patent Feb. 2 , 2021 Sheet 1 of 44 US 10,906,982 B2
FIG . 1A . DNA and Amino Acid Sequences of TNFRAB1 Heavy Chain
TNFRAB1 Heavy Chain DNA Sequence GACCAGGCATCCCAGGGTCACCATGGAGTTAGTTTGGGCAGCAGATCCAGGGGCCAGTGGATAGA CAGATGGGGGTGTCGTTTTGGCTGAGGAGACGGTGACCGCGGTCCOTGCGCCCCAGACATCGAAG TACCAGTAGOTGGAGTAACCATCAACCCTCTGTCGTGCACAGTA4 TACATGGCCGTGTCCTCAGACC CACACTGTCTGGATAGTAGGTGT4ACTACCACCACTACTAATGGTTGCG4CCCACTCCAGCCTCTTC TCCGGAGTCTGGCGAACCCAAGACATGACATAACTACTGAAAGTGAATCCAGAGGCTGCACAGGAG AGTITCAGGGACCCTCCAGGCTTCACTAAGCCTCCCCCTGACTCCTGCAGCTGCACCTCCGGAAGC CTGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCG - AGCATGCATCTAGAGGCCCAT (SEQ ID NO : 1 )
Amino acid sequence EVOLQESGGGLVKPGGSLKLSCAASGETFSSYVMSWVROTREKRLEWVATISSGGSYTYYPOSVKGRF TISRDNAKNTLYLOMSSLRSEDTAMYYCARQRVOGYSSYWYFDWWGAGTATVSS ( SEQ ID NO : 2 ) U.S. Patent Feb. 2 , 2021 Sheet 2 of 44 US 10,906,982 B2
FIG . 1B . DNA and Amino Acid Sequences of TNFRAB1 Light Chain
TNFRA81 Light Chan
CTCCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCAGGGGGACATTGTGCTGACCCAGTGT CCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAT ATTACATGTACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCCAAC CTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGCTCTGGGACCTCTTACTCTCTCACAATCA GCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGGAATTACCCGTACACGTT CGGAGGGGGGACCAAGTTGGAAATAAAACGGGCTGATGCTCCACCAACTGTATCCATCTTCCCACC ATCCAGTGAGCAGTTACCTGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTA CCAAGCTTGATGCATAGCTTGAGTATTCTAACGCGTCACCT 7/16 ( SEQ ID NO : 3 )
DIVLTOSPAIMSASPGEKVTITCSASSSVYYMYWFOOKPGTSPKLWAYSTSNLASGVPVRFSGSGSGTSY SLTISRMEAEDAATYYCQQRRNYPYTFGGGTKLEIKRA ( SEQ ID NO : 4 ) U.S. Patent Feb. 2 , 2021 Sheet 3 of 44 US 10,906,982 B2
FIG . 2A . Epitopes within TNFR2 bound by TNFRAB1
MAPVAVWAAL AVGLELWAAA HALPAOVAFT PYAPEPGSTC RLREYYDOTA OMOCSKCSPG QHAKVFCTKT SDTVCDSCED STYTOLKNWV PECLSCGSRC SSDOVETOAC TREQNRICTO RPGWYCALSK QEGCRLCAPL PROPEA RPGTETSDVV DIESIS NTTSSTDICR PHQICNVVAI PGNASMDAVO TSTSPTRSMA PGAVHLPQPV STRSQHTQPT PEPSTAPSTS FLLPMGPSPP AEGSTGDFAL PVGLIVGVTA LGLLIIGVVN CVIMTOVKKK PICLOREAKV PHLPADKARG TOGPEQOHLL ITAPSSSSSS LESSASALDR RAPTRNQPQA PGVEASGAGE ARASTGSSDS SPGGHGTQVN VICIVNVCSS SDHSSOCSSO ASSTMGDIDS SPSESPKDEO VPFSKEECAF RSQLETPETL LGSTEEKFIP
LGVPDAGMKP S U.S. Patent Feb. 2 , 2021 Sheet 4 of 44 US 10,906,982 B2
FIG . 2B . Epitopes within TNFR2 bound by TNFRAB2
MAPVAVWAAL AVGLELWAAA HALPAQVAFT PYAPEPGSTC RLREYYDQTA OMCCSKCSPG QHAKVFCTKT SDTVCDSCED STYTOINNWV PEOLOGSRC SSDOVETOAC TREQNRICIO PEGWYCALSK QEGCRLCEFL CBPGFGVA RPGTETSDVV CKPCAPGTFS NITSSTDICR PHQICNVVAI PGNASMDAVC TSTSPTRSMA PGAVHLPQPV STRSQHIQPT PEPSTAPSTS FLLPMGPSPP AEGSIGDFAL PVGLIVGVTA IGLIIIGVVN CVIMTOVKKK PLCLOREAKV PHLPADKARG TOGPEQQHIL ITAPSSSSSS LESSASALDR RAPTRNOPQA PGVERSGAGE ARASTGSSDS SPGGHGTQVN VICIVNVCSS SDHSSOCSsQ ASSTMGDTDS SPSESPKDEQ VPFSKEECAF RSOLETPETL LGSTEEKPLP U.S. Patent Feb. 2 , 2021 Sheet 5 of 44 US 10,906,982 B2
FIG . 3A . Epitope mapping of TNFRAB1
Raw Structural SEQ ID NO . Peptide Sequence Group luminescence data 134 SRCSSDQVETQGGSGGTREQNRICT2R CYS.S 177 135 RCSSDOVETQAGGSGGTREQNRICT2R CYS.S 136 CSSDQVETQAZGGSGGTREQNRICT2R CYS.S 179 137 VPE2LS2GSRCGGSGGREQNRICTZRP CYS.S 318 138 PE2LS2GSRCSGGSGGREQNRICT2RP CYS.S 291 139 E2LSZGSRCSSGGSGGREONRICTZRP CYS.S 323 140 2LS2GSRCSSDGGSGGREQNRICT2RP CYS.S 262 141 LS2GSRCSSDOGGSGGREONRICT2RP CYS.S 261 142 S2GSRCSSDQVGGSGGREQNRICT2RP CYS.S 247 143 2GSRCSSDQVEGGSGGREQNRICT2RP CYS.S 228 144 GSRCSSDQVETGGSGGREQNRICT2RP CYS.S 166 145 SRCSSDQVETQGGSGGREQNRICTZRP CYS.S 157 146 RCSSDQVETOAGGSGGREQNRICT2RP CYS.S 130 147 CSSDQVETQAZGGSGGREQNRICTZRP CYS.S 250 148 VPEZLS2GSRCGGSGGEQNRICT2RPG CYS.S 225 149 PE2LS2GSRCSGGSGGEQNRICT2RPG CYS.S 229 U.S. Patent Feb. 2 , 2021 Sheet 6 of 44 US 10,906,982 B2
FIG . 3A ( Continued )
150 E2L52GSRCSSGGSGGEQNRICTZRPG 151 2LSZGSACSSOGGSGGEQNRICTZRPG CYS.S
154 2GSRCSSDQVEGGSGGEQNRICTZRPG 271 155 GSRCSSDQVETGGSGGEQNRICTZRPG 156 SACSSDQVETOGGSGGEQNRICT2RPG
248
16 % 21.92GSRCSSDGGSGGQNRICT RPGW CYS.S
CYSS U.S. Patent Feb. 2 , 2021 Sheet 7 of 44 US 10,906,982 B2
FIG . 3A ( Continued )
170 VPEZLSZGSRCGGSGGNRICT2RPGWY CYS.S 853 171 PE2LSZGSRCSGGSGGNRICT2RPGWY CYS.S 928 172 E2LS2GSRCSSGGSGGNRICTZRPGWY CYS.S 389 173 | 2LS2GSRCSSDGGSGGNRICTZRPGWY CYS.S 878 174 LS2GSRCSSDQGGSGGNRICT2RPGWY CYS.S 822 175 S2GSRCSSDQVGGSGGNRICT2RPGWY CYS.S 666 176 2GSRCSSDQVEGGSGGNRICT2RPGWY CYS.S 588 177 GSRCSSDQVETGGSGGNRICT2RPGWY CYS.S 587 178 SRCSSDQVETQGGSGGNRICT2RPGWY CYS.S 482 179 RCSSDQVETQAGGSGGNRICT2RPGWY CYS.S 180 CSSDQVETQAZGGSGGNRICT2RPGWY CYS.S 487 181 VPE2LS2GSRCGGSGGRICT2RPGWY2 CYS.S 478 182 PE2LS2GSRCSGGSGGRICT2RPGWY2 CYS.S 585 183 E2LS2GSRCSSGGSGGRICT2RPGWY2 CYS.S 478 184 2LS2GSRCSSDGGSGGRICT2RPGWY2 CYS.S 436 185 LS2GSRCSSDOGGSGGRICTZRPGWY2 CYS.S 546 186 S2GSRCSSDQVGGSGGRICT2RPGWY2 CYS.S 465 187 | 2GSRCSSDQVEGGSGGRICT2RPGWYZ CYS.S 614 188 GSRCSSDQVETGGSGGRICT2RPGWY2 CYS.S 594 189 SRCSSDQVETOGGSGGRICT2RPGWY2 CYS.S 568 190 RCSSDQVETRAGGSGGRICT2RPGWY2 CYS.S 543 U.S. Patent Feb. 2 , 2021 Sheet 8 of 44 US 10,906,982 B2
FIG . 3A ( Continued )
191 CSSDQVETQA2GGSGGRICTZRPGWYZ 597
193 PEZSZGSRCSGGSGGICTZRPGWY2A 194 EZS2GSRCSSGGSGGICTARPGWY2A 195 2GSRCSSDQVEGGSGGICTZRPGWYZA 196 GSRCSSDQVETGGSGGICTZRPGWY2A 388 198 RCSSDQVETOAGGSGGICTZRPGWYZA CYSS 454 200 VPEZLS2GSRCGGSGGCT2RPGWYZAL 697 202 | E2LS2GSRCSSGGSGGCT2RPGWY2AL 203 2192GSRCSSDGGSGGCT2RPGWYPAL CYS.S 204 LS2GSRCSSDOGGSGGCTZÁPGWYPAL CYSS 205 SZGSRCSSDQVGGSGGCTURPGWYPAL 206 2GSRCSSDQVEGGSGGCTZRPGWYQAL 207 GSRCSSDQVEYGOSGGCTZRPGWYQAL
209 RCSSDQVETQAGGSGGCTZRPGWYZAL U.S. Patent Feb. 2 , 2021 Sheet 9 of 44 US 10,906,982 B2
FIG . 3A ( Continued )
211 2TREQNRI2TCGGSGGRPGWYCALSKO CYS.S 212 TREQNRI2TCRGGSGGRPGWYCALSKO CYS.S 188 213 REQNRI2TCRPGGSGGRPGWYCALSKO CYS.S 228 214 EQNRIZTCRPGGGSGGRPGWYCALSKQ CYS.S 263 215 ONRIZTCRPGWGGSGGRPGWYCALSKO CYS.S 240 216 NRI2TCRPGWYGGSGGRPGWYCALSKO CYS.S 426 217 WY2ALSKQEGCGGSGG2APLRKCRPGF CYS.S 327 218 Y?ALSKQEGCRGGSGGZAPLRKCRPGF CYS.S 308 219 2ALSKQEGCRLGGSGGZAPLRKCRPGF CYS.S 439 220 ALSKQEGCRLZGGSGG2APLRKCRPGF CYS.S 560 221 LSKQEGCRLZAGGSGG2APLRKCRPGF CYS.S 529 222 SKQEGCRL2APGGSGGZAPLRKCRPGF CYS.S 507 223 KOEGCRLZAPLGGSGGZAPLRKCRPGF CYS.S 528 224 QEGCRLZAPLRGGSGGZAPLRKCRPGF CYS.S 570 225 EGCRLZAPLRKGGSGGZAPLRKCRPGF CYS.S 604 226 WYZALSKQEGCGGSGGAPLRKCRPGFG CYS.S 534 227 Y2ALSKQEGCRGGSGGAPLRKCRPGFG CYS.S 553 228 2ALSKOEGCRLGGSGGAPLRKCRPGFG CYS.S 418 229 ALSKQEGCRLZGGSGGAPLRKCRPGFG CYS.S 321 230 LSKOEGCRLZAGGSGGAPLRKCRPGFG CYS.S 253 231 SKQEGCRL2APGGSGGAPLRKCRPGFG CYS.S 220 232 KQEGCRLZAPLGGSGGAPLRKCAPGFG CYS.S 242 U.S. Patent Feb. 2 , 2021 Sheet 10 of 44 US 10,906,982 B2
FIG . 3A ( Continued )
ALSKQEGCRLZGGSGGPLRKCRPGFGV
245 YŽALSKQEGCRGGSGGRKCRPGFGVAR
SKQEGCRLAGGSGGRKCRPGFGVAR
252 EGCRLZAPLRKGGSGGRKCRPGFGVAR CYS.S 358 U.S. Patent Feb. 2 , 2021 Sheet 11 of 44 US 10,906,982 B2
Fig . 3B . Epitope mapping of TNFRAB2
Structural Raw SEQ ID NO . Peptide Sequence Group luminescence data 34 PE2LS2GSACSGGSGGNRICTZRPGWY CYS.S 928 35 2LS2GSRCSSDGGSGGNRICT2RPGWY CYS.S 878 36 VPE2LSZGSRCGGSGGNRICT2RPGWY CYS.S 853 37 LSZGSRCSSDQGGSGGNRICT2RPGWY CYS.S 822 38 PE2LSZGSRCSGGSGGCT2RPGWY2AL CYS.S 697 39 S2GSRCSSDQVGGSGGNRICT2RPGWY CYS.S 666 40 2GSRCSSDQVEGGSGGRICT2RPGWYZ CYS.S 614 41 EZ LS2GSRCSSGGSGGCT2RPGWYPAL CYS.S 605 42 EGCRL2APLRKGGSGGZAPLRKCRPGF CYSS 604 432LS2GSRCSSDGGSGGCT2RPGWYQAL CYS.S 603 44 CSSDQVETQAZGGSGGRICT2RPGWY2 CYS.S 597 45 GSRCSSDQVETGGSGGRICTZRPGWY2 CYS.S 594 46 2GSRCSSDQVEGGSGGNRICTZRPGWY CYS.S 588 47 GSRCSSDQVETGGSGGNRICT2RPGW CYS.S 587 48 PE2LSZGSRCSGGSGGRICT2RPGWY2 CYS.S 585 49 VPE2LS2GSRCGGSGGQNRICT2RPGW CYS.S 577 50 QEGCRLZAPLRGGSGGZAPLRKCRPGF CYS , S 570 51 SRCSSDQVETOGGSGGRICT2RPGWY2 CYS.S 568 U.S. Patent Feb. 2 , 2021 Sheet 12 of 44 US 10,906,982 B2
FIG . 3B ( Continued ) 52. ALSKOEGCRLÆGGSC Why
54 YQALSKQEGCRGGSGGAPLRKCRPGFG 553 55 LSŽGSRCSSDQGGSGGRICTZRPGWYZ 56 P8ZLSZOSRCSGOSGGONRICTZAPGW 57 RCSSDOVETOAGGSGGRICT2RPGWY2
59 VPELS GSRCGGSGGICTZRPGWYŽA 60 $ 2GSRCSSDQVGGSGGCTZRPGWYPAL CYS.S $ 38
529 U.S. Patent Feb. 2 , 2021 Sheet 13 of 44 US 10,906,982 B2
FIG . 3B ( Continued )
69 CSSDQVETQAZGGSGGNRICT2RPGWY CYS.S 70 VPE2LS2GSRCGGSGGCT2RPGWYPAL CYS , S 483 71 E2LS2GSRCSSGGSGGQNRICT2RPGW CYS.S 482 72 SRCSSDQVETOGGSGGNRICT2RPGWY CYS , S 482 73 2GSRCSSDOVEGGSGGCTZRPGWY2AL CYS.S 480 74 VPE2LS2GSRCGGSGGRICT2RPGWY2 CYS.S 478 75 E2LSZGSRCSSGGSGGRICTZRPGWY2 CYS.S 478 76 LSKOEGCRL2AGGSGGRKCRPGFGVAR CYS.S 77 GSRCSSDQVETGGSGGQNRICT2RPGW CYS.S 466 78 S2GSRCSSDOVGGSGGRICT2RPGWYZ CYS.S 465 79 GSRCSSDQVETGGSGGCT2RPGWYPAL CYS.S 464 80 Y2ALSKQEGCRGGSGGRKCRPGFGVAR CYS.S 81 KOEGCRLZAPLGGSGGPLRKCRPGFGV CYS , S 457 82 ALSKQEGCRL2GGSGGRKCRPGFGVAR 455 83 E2LSZGSRCSSGGSGGICT2RPGWYZA CYS.S 454 84 CSSDQVETQAZGGSGGICT2RPGWY2A CYS.S 454 85 | EGCRL2APLRKGGSGGPLRKCRPGFGV CYSS 450 86 ALSKQEGCRL2GGSGGPLRKCRPGFGV CYS.S 446 87 2ALSKOEGCRLGGSGGRKCRPGFGVAR CYS.S 88 2ALSKQEGCRLGGSGG2APLRKCRPGF CYS.S U.S. Patent Feb. 2 , 2021 Sheet 14 of 44 US 10,906,982 B2
FIG . 3B ( Continued )
90 ISKQEGCRLZAGGSGGPIRKCRPGFGV
YS.S 24 2ALSKQEGCRLGGSGGAPLRKCRPGFG 95 2ALSKQEGCRLGGSGGPLRKCRPGFGV CYS.S
99 EZLSZGSRCSSGGSGGNRICTZRPGWY LYSS U.S. Patent Feb. 2 , 2021 Sheet 15 of 44 US 10,906,982 B2
FIG . 3B ( Continued )
110 ALSKOEGCRL2GGSGGAPLRKCRPGFG CYS.S 321 111 | EGCRLZAPLRKGGSGGAPLRKCRPGFG CYS , S 312 112 Y2ALSKQEGCRGGSGGZAPLRKCRPGF CYS.S 308 113 LSZGSRCSSDQGGSGGQNRICTZRPGW CYS.S 291 114 LSKQEGCALZAGGSGGAPLRKCRPGFG CYS.S 253 115 RCSSDQVETQAGGSGGNRICTZRPGWY CYS.S 247 116 KQEGCRLZAPLGGSGGAPLRKCRPGFG CYS , S 242 117 SKQEGCRLZAPGGSGGAPLRKCRPGFG CYS , S 220 U.S. Patent Feb. 2 , 2021 Sheet 16 of 44 US 10,906,982 B2
FIG . 4. Visualization of epitopes bound by TNFR2 antibodies
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FIG . 5 T - reg Suppression Assay
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TNFR2 antagonist 1 F ( ab ) 2 2
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FIG . 12A Cytokines Relativeexpression
FIG . 12B
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FIG . 13A Binding affinity of antagonists to TNFR2 Tamantagonist k ( 1/8 ) 1 4.98E + 06 2.21E - 04 4.44E - 11 2 3.609 ( 9E + 05 2.24E - 04 6.21 ( 2 ) E - 10 km ka - association rate constant Ko Ky - dissociation rate constant Kp - equilibrium dissociation constant FIG . 13B Linear peptide mapping of TNFR2 antagonists Bosbon Within 38 Acco SER : D :NO : 3690?? INFR2 HINFR2 (23-207 ) HINFR2 ( 1-203 LPAQUAFIPYAPEPGSTCAL 9Q3933 HYNFR2 ( 9-28 ) PYAPEPGSTCRLREYYOQTA NINFR2 ( 17-36 ) ICRLREYYXTAQUCCSKS S03933 HINFR2 { 26-45 ) QTAQMCCSKCSPGQHAKVFC 0.1 BINFR2 353( ) KCSPGQHAKVFCTXTSONVO HINFR2 ( 4251 ) KVFCTKISOIVCOSCEDSTY HTNFR2 ( 50-59 ) DIVCOSCEDSTYTOLWAWP NTNFR2 ( 58-77 ) OSTYNQLWNWYPECLSCSSR 0 NINFR2 ( 6635 ) NWVPECLSCGSRCSSDQVET NINFR2 ( 74-93 ) CGSRCSSCOVEQACTREQN 584370 INFA2 ( 82-101 ) QVETQACTREQNRCTCRPG 504371 NINFR2 ( 93-109 ) REQNFICTCRPGWYCALSKQ HINFR2 (98-117 ) CRPGWYCALSKQEGCRLCAP S04073 INFR2 105-825 ) LSKQEGCRLCAPLRKCRPGF 1.2 * $ 03935 } TNFR2 ( 1080-727 ) KQEGCRLCAPLRKCAPGFGV 0 S04074 INFR24134-133 ) LCAPLRKCRPGFGVARPGTE 1.2 0 S04075 HINFR2 ( 122-141 ) RPGFGVAREGTETSOVVCKP S04076 KINFR2 ( 139-149 ) PGTETSOVVCKPCAPG TFSN HINFR2 ( 138-157 ) VCKPCAPGIFSNTTSSJDXC S04078 HINFR2 ( 146-165 ) TFSNITSSIXCRPHOXCNV S04079 HINFR2 ( 154-174 ) DICRPHCCNVVAPGNAS SCHOBO BINFR2 ( 162-181) ICHWAIPGNASMDAVCISI S04081 WINFR2 ( 170-189 ) GNASMDAVCTSTSPTRSMAP * Ratio ( 2x ) FIG . 13C Linear peptide mapping of TNFR2 antagonists with TNF competition
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FIG . 18A
Prior Art
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FIG . 18B Prior Art
FIG . 18C
Prior Art US 10,906,982 B2 1 2 ANTAGONISTIC ANTI - TUMOR NECROSIS activity of tumor -reactive T lymphocytes. The development FACTOR RECEPTOR 2 ANTIBODIES of chemical modulators of T - reg cell activity has been the subject of many pharmacological investigations, as access to FIELD OF THE INVENTION an agent capable of inhibiting T -reg -mediated T -cell sup 5 pression could vastly improve the scope and efficacy of The invention relates to polypeptides, such as antibodies adoptive cancer immunotherapy, as well as improve the and antigen - binding fragments thereof, capable of antago ability of the immune system to eradicate pathogenic organ nizing tumor necrosis factor receptor superfamily members, isms that give rise to infectious diseases. such as tumor necrosis factor receptor 2. The polypeptides of Tumor necrosis factor receptor ( TNFR ) subtypes 1 and 2 the invention can be used to modulate the activity of 10 have been identified on the T -reg cell surface as signal transduction molecules that dictate cell fate . The activation regulatory T - cells , such as in the field of immunotherapy for of TNFR1 , for instance, potentiates the caspase signaling the treatment of cell proliferation disorders and infectious cascade and terminates in T - reg apoptosis , while activation diseases . of TNFR2 induces signaling through the mitogen - activated BACKGROUND OF THE INVENTION 15 protein kinase ( MAPK ) signaling pathway, which orches trates signaling through TRAF2 / 3 and the NFKB -mediated The use of naturally -occurring and genetically engineered transcription of genes that promote escape from apoptosis T -lymphocytes is a prominent paradigm for ameliorating and cell proliferation . Due to its role in directing cell survival and growth , TNFR2 represents an attractive target therapeuticvarious human platforms pathologies for the. For treatment instance ,of while cancer traditional include 20 for preventing immune detection of tumor -reactive T lym surgical removal of tumor mass , radiation therapy, and phocytes. As such , there is currently a need for therapies that administration of chemotherapeutics ( Shewach , Chem . Rev., can prevent T - reg cell survival and proliferation for use in 109 : 2859-2861 , 2009 ) , the last decade has witnessed a treatments targeting cell proliferation disorders , such as resurgence in the application of adoptive immunotherapy to cancer, and a wide array of infectious diseases. cancer treatment regimens. With the advent of chimeric 25 antigen receptor ( CAR - T ) therapy, new methods have SUMMARY OF THE INVENTION emerged for the infusion of autologous and allogeneic The invention provides antagonistic tumor necrosis factor tumor - reactive T -cells to patients ( June, J. Clin . Invest. , receptor 2 ( TNFR2 ) antibodies and antigen -binding frag 117 : 1466-1476 , 2007 ) . CAR - T therapies harness the ments thereof, such as those that specifically bind epitopes resources of the adaptive immune response in order to 30 within human TNFR2 that contain one or more residues of promote cancer cell cytotoxicity and eradicate tumor mate- the KCRPG sequence ( SEQ ID NO : 19 ) or equivalent rial . A common motif in adoptive immunotherapy is the use epitopes in TNFR2 of non - human primates ( e.g. , bison or of T - cells that exhibit the ability to selectively potentiate cattle , as described herein ) and that do not specifically bind cytotoxicity in cells that display distinct tumor antigens. epitopes within human TNFR2 containing the KCSPG Examples of this technique include the administration of 35 sequence ( SEQ ID NO : 12 ) or equivalent epitopes in TNFR2 tumor - infiltrating lymphocytes ( Dudley et al . , J. Immu- of non - human primates . The invention additionally features antibodies and antigen -binding fragments thereof that nother ., 26 : 332-342 , 2003 ) , as well as autologous or allo inhibit the activity of other TNFR superfamily members , geneic T - cells that have been genetically re - engineered so as such as antibodies and antigen - binding fragments thereof to exhibit reactivity with a tumor - specific antigen ( Yee et al . , that bind these proteins in an anti -parallel dimer configura PNAS ., 99 : 16168-16173 , 2002 ) . 40 tion . The antibodies and antigen - binding fragments thereof Despite the promise of T -lymphocyte - based cancer immu described herein can be used for the treatment of a variety notherapy, the development of this therapeutic platform has of pathologies, including cancers and infectious diseases . been hindered by the natural propensity of the immune Disclosed herein are polypeptides, such as single - chain system to suppress immune attacks mounted on self cells . polypeptides, antibodies, or antigen -binding fragments Cancer cells , like all nucleated human cells , express class I 45 thereof, capable of specifically binding human TNFR2 that major histocompatability complex ( MHC ) proteins that dis contain a complementarity determining region -heavy chain tinguish these cells from foreign cells . In order to prevent 1 ( CDR - H1 ) and a CDR - H2 derived from a neutral TNFR2 cell fratricide , regulatory T - cells ( T - reg cells ) have evolved antibody and a CDR -H3 having the amino acid sequence that suppress the activity of T - cells that exhibit reactivity JZ JZ ŽAIZ JZ ( J) , Z ZZ , JZ JZºZºZPZ ( I) , ZZZ ( 1 ) ,, against “ self " ' MHC antigens. T - reg cells represent a hetero- 50 JRJDGJSJY ( J) FDJ ( SEQ ID NO : 278 ) , JRJDGSY ( J) ,FD geneous class of T - cells that can be distinguished based on ( J )3 ( SEQ ID NO : 279 ) , QZ?VZ2Z4YZSZWYZSZ - 75 their unique surface protein presentation. The most well- ( SEQ ID NO : 265 ) , or AZ DZ Z + ZZ SPZ Z ZWG ( SEQ understood populations of T - reg cells include CD4 + , ID NO : 266 ) , wherein CD25 + , FoxP3 + T - reg cells and CD17 + T - reg cells . The each J is independently a naturally occurring amino acid ; precise mechanisms by which these cells mediate suppres 55 each Z ' is independently a naturally occurring amino acid sion of autoreactive T - cells is the subject of ongoing inves- containing a cationic side -chain at physiological pH , such as tigations, though it has been shown that certain classes of lysine , arginine, and histidine ; T - reg cells inhibit production of the proliferation - inducing each Z ? is independently a naturally occurring amino acid cytokine IL - 2 in target T -cells and may additionally seques containing an anionic side - chain at physiological pH , such ter IL - 2 from autoreactive cells by virtue of the affinity of 60 as aspartic acid and glutamic acid ; CD25 ( a subdomain of the IL - 2 receptor ) for IL - 2 ( Josefo each Z is independently a naturally occurring amino acid wicz et al . , Ann . Rev. Immun ., 30 : 531-564 , 2012 ) . containing a polar, uncharged side - chain at physiological Although T - reg cells play an important role in maintaining pH , such as serine, threonine, asparagine, and glutamine ; peripheral tolerance, the same biochemical features that each Z4 is independently a glycine or alanine ; and underlie the ability of these cells to modulate autoreactive 65 each Z is independently a naturally occurring amino acid T - cell activity also serve to undermine adoptive immuno- containing a hydrophobic side- chain , such as alanine, valine, therapy and the natural immune response by suppressing the leucine , isoleucine, proline, and methionine , tryptophan , US 10,906,982 B2 3 4 phenylalanine, and tyrosine. As used herein in the context of thereof may contain a non - native constant region ( e.g. , a a polypeptide formula, numeric characters in subscript nota- human constant region ) , lack all or a portion of an Fc tion designate the quantity of the preceding amino acid domain , lack all or a portion of a native Fc domain , or lack present in the formula, and numeric characters in superscript an Fc domain altogether. notation designate the type of the preceding amino acid 5 The invention also features polypeptides ( e.g., single present in the formula . chain polypeptides , antibodies, and antigen - binding frag Disclosed herein are polypeptides , such as single - chain polypeptides , antibodies, or antigen - binding fragments ments thereof) capable of specifically binding human thereof, capable of specifically binding human TNFR2. The TNFR2 . The polypeptide ( e.g. , antibody or antigen -binding polypeptides ( e.g. , antibodies and antigen - binding frag- 10 fragment thereof) may contain the following CDRs : ments thereof) may contain one or more , or all , of the ( a ) a CDR - H1 having the amino acid sequence following CDRs : Z4FZZ SSZ or Z4YZZ TDZX; ( a ) a CDR - H1 having the amino acid sequence ZAJZZ ( b ) a CDR - H2 having the amino acid sequence ( J) .Z or Z4JZZ ( I) , 21 ; SSGZ ZY ( SEQ ID NO : 263 ) or VDPEYZAZT ( SEQ ID ( b ) a CDR - H2 having the amino acid sequence ( J )3247J 15 NO : 264 ) ; or ( J) .Z4ZJ ; ( c ) a CDR - H3 having the amino acid sequence ( c ) a CDR - H3 having the amino acid sequence QZ VZZ YZSZWYZ ZZ ( SEQ ID NO : 265 ) or JZ'JZZJZ JZ ( 1) ZZZS or JZ JZ 7 ( 1) , Z 77 AZ'DZ2Z4ZZ SPZ ZZWG ( SEQ ID NO : 266 ) ; ( d ) a CDR - Li having the amino acid sequence ( J )( 2d ; ) a CDR - L1 having the amino acid sequence ( J) or 20 SASSSVYYMZ ( SEQ ID NO : 267 ) or QNINKZ ( SEQ ID ( J ) , Z ; NO : 268 ) ; ( e ) a CDR - L2 having the amino acid sequence ( J) .Z or ( e ) a CDR - L2 having the amino acid sequence STSN ( J ) , Z ?; and LAZ ( SEQ ID NO : 269 ) , TYZ , or YTZ ; and (f ) a CDR - L3 having the amino acid sequence ( J ) 3Zº ( f) a CDR - L3 having the amino acid sequence ( ) .Z or (I ) Z (J ).Z ; 25 QORRNZ PYZ ( SEQ ID NO : 270 ) or CLQZ VNLXZ wherein each J is independently a naturally occurring ( SEQ ID NO : 271 ) ; amino acid ; wherein each Z is independently an amino acid contain each Z ' is independently a naturally occurring amino acid ing a cationic side - chain at physiological pH ; containing a cationic side - chain at physiological pH , such as each Z ? is independently an amino acid containing an lysine , arginine, and histidine; 30 anionic side - chain at physiological pH ; each Z is independently a naturally occurring amino acid each Z is independently an amino acid containing a polar, containing an anionic side - chain at physiological pH , such uncharged side - chain at physiological pH ; as aspartic acid and glutamic acid ; each Z4 is independently a glycine or alanine; each Zº is independently a naturally occurring amino acid each Z is independently an amino acid containing a containing a polar, uncharged side - chain at physiological 35 hydrophobic side - chain ; pH , such as serine , threonine , asparagine , and glutamine ; each X is independently leucine or isoleucine . The anti each Z4 is independently a glycine or alanine ; and body or antigen - binding fragment thereof may contain a each Z is independently a naturally occurring amino acid non - native constant region ( e.g. , a human constant region ), containing a hydrophobic side - chain , such as alanine , valine, lack all or a portion of an Fc domain , lack all or a portion leucine , isoleucine, proline , and methionine , tryptophan , 40 of a native Fc domain , or lack an Fc domain altogether. phenylalanine, and tyrosine. In another aspect , the invention features a polypeptide , The antibody or antigen - binding fragment thereof may such as a single - chain polypeptide , antibody, or antigen contain a non - native constant region ( e.g. , a human constant binding fragment thereof, capable of specifically binding region ) , lack all or a portion of an Fc domain , lack all or a human TNFR2 , wherein the polypeptide ( e.g. , single - chain portion of a native Fc domain , or lack an Fc domain 45 polypeptide, antibody, or antigen - binding fragment thereof) altogether. contains the following CDRs : Additionally disclosed herein are polypeptides ( e.g. , ( a ) a CDR - H1 having the amino acid sequence GFTFSSY single - chain polypeptides, antibodies, and antigen -binding ( SEQ ID NO : 23 ) , GYTFTDYX ( SEQ ID NO : 257 ) , or an fragments thereof) capable of specifically binding human amino acid sequence having up to two amino acid substi TNFR2 , wherein the polypeptide ( e.g. , antibody or antigen- 50 tutions ( e.g. , one or two amino acid substitutions, such as binding fragment thereof) contains one or more , or all , of the conservative amino acid substitutions ) relative to these following CDRs : sequences; ( a ) a CDR - H1 having the amino acid sequence GJTF (J ) Y ( b ) a CDR - H2 having the amino acid sequence SSGGSY ( SEQ ID NO : 276 ) or GJTF ( I) YJ ( SEQ ID NO : 277 ) ; ( SEQ ID NO : 24 ) , VDPEYGST ( SEQ ID NO : 258 ) , or an ( b ) a CDR - H2 having the amino acid sequence ( J ) 3GSJ or 55 amino acid sequence having up to two amino acid substi ( J ) , GSJ ; tutions ( e.g. , one or two amino acid substitutions, such as ( c ) a CDR - H3 having the amino acid sequence conservative amino acid substitutions) relative to these JRJDGJSJY (1 ) FDJ ( SEQ ID NO : 278 ) or JRJDGSY ( J ) , FD sequences ; ( J ) 3 ( SEQ ID NO : 279 ) ; ( c ) a CDR - H3 having the amino acid sequence ( d ) a CDR - L1 having the amino acid sequence (J ) , Y or 60 QRVDGYSSYWYFDV ( SEQ ID NO : 25 ) , ARDDGSYS ( J ) Y ; PFDYWG ( SEQ ID NO : 259 ) , or an amino acid sequence ( e ) a CDR - L2 having the amino acid sequence ( J ) .S or having up to two amino acid substitutions ( e.g. , one or two ( J ) , S ; and amino acid substitutions, such as conservative amino acid ( f) a CDR - L3 having the amino acid sequence ( I ) Y ( J ) 2T substitutions) relative to these sequences; or ( J ) 3Y ( J ) 4T ; 65 ( d ) a CDR - L1 having the amino acid sequence wherein each J is independently a naturally occurring SASSSVYYMY ( SEQ ID NO : 26 ) , QNINKY ( SEQ ID NO : amino acid . The antibody or antigen - binding fragment 260 ) , or an amino acid sequence having up to two amino US 10,906,982 B2 5 6 acid substitutions ( e.g. , one or two amino acid substitutions , In some embodiments , the CDR - H1 has the amino acid such as conservative amino acid substitutions ) relative to sequence GYTFTDYL ( SEQ ID NO : 274 ) . In some embodi these sequences; ments , the CDR - H1 has the amino acid sequence ( e ) a CDR - L2 having the amino acid sequence STSNLAS GYTFTDYI ( SEQ ID NO : 275 ) . ( SEQ ID NO : 27 ) , TYS , YTS , or an amino acid sequence 5 In some embodiments, the polypeptide ( e.g., single - chain having up to two amino acid substitutions ( e.g. , one or two polypeptide, antibody, or antigen -binding fragment thereof) amino acid substitutions, such as conservative amino acid contains: substitutions) relative to SEQ ID NO : 27 ; and ( a ) a CDR - Li having the amino acid sequence ( f) a CDR - L3 having the amino acid sequence QQRRNY- SASSSVYYMY ( SEQ ID NO : 26 ) ; PYT ( SEQ ID NO : 28 ) , CLQYVNLXT ( SEQ ID NO : 261 ) , 10 ( b ) a CDR - L2 having the amino acid sequence STSNLAS or an amino acid sequence having up to two amino acid ( SEQ ID NO : 27 ) ; and . substitutions ( e.g. , one or two amino acid substitutions, such ( c ) a CDR - L3 having the amino acid sequence QQRRNY as conservative amino acid substitutions ) relative to these PYT ( SEQ ID NO : 28 ) . sequences ; The polypeptide single - chain polypeptide, antibody, or wherein each X is independently leucine or isoleucine . 15 antigen - binding fragment thereof) may contain : The antibody or antigen - binding fragment thereof may con- ( a ) a CDR - H1 having the amino acid sequence GFTFSSY tain a non - native constant region ( e.g. , a human constant ( SEQ ID NO : 23 ) ; region ) , lack all or a portion of an Fc domain , lack all or a ( b ) a CDR - H2 having the amino acid sequence SSGGSY portion of a native Fc domain , or lack an Fc domain ( SEQ ID NO : 24 ) ; and altogether. In some embodiments, the amino acid substitu- 20 ( c ) a CDR - H3 having the amino acid sequence tions are conservative substitutions. In some embodiments , QRVDGYSSYWYFDV ( SEQ ID NO : 25 ) . the amino acid substitutions are non - conservative substitu- In some embodiments , the polypeptide single - chain poly tions . peptide, antibody , or antigen -binding fragment thereof) con In some embodiments, the polypeptide ( e.g. , single - chain tains : polypeptide , antibody, or antigen -binding fragment thereof) 25 ( a ) a CDR - L1 having the amino acid sequence QNINKY contains one or more of the following CDRs : ( SEQ ID NO : 260 ) ; ( a ) a CDR - L1 having the amino acid sequence ( b ) a CDR - L2 having the amino acid sequence TYS or SASSSVYYMY ( SEQ ID NO : 26 ) ; YTS ; and ( b ) a CDR - L2 having the amino acid sequence STSNLAS ( c ) a CDR - L3 having the amino acid sequence ( SEQ ID NO : 27 ) ; and 30 CLQYVNLXT ( SEQ ID NO : 261 ) . ( c ) a CDR - L3 having the amino acid sequence QQRRNY- In some embodiments , the polypeptide ( e.g. , single - chain PYT ( SEQ ID NO : 28 ) . polypeptide, antibody, or antigen -binding fragment thereof) In some embodiments, the polypeptide single - chain poly- contains : peptide, antibody, or antigen -binding fragment thereof) con- (a ) a CDR - H1 having the amino acid sequence tains one or more of the following CDRs : 35 GYTFTDYX ( SEQ ID NO : 257 ) ; ( a ) a CDR - L1 having the amino acid sequence QNINKY ( b ) a CDR - H2 having the amino acid sequence ( SEQ ID NO : 260 ) ; VDPEYGST ( SEQ ID NO : 258 ) ; and . ( b ) a CDR - L2 having the amino acid sequence TYS or ( c ) a CDR - H3 having the amino acid sequence ARDDG YTS ; and SYSPFDYWG ( SEQ ID NO : 259 ) . ( c ) a CDR - L3 having the amino acid sequence 40 In some embodiments , the polypeptide ( e.g. , single - chain CLQYVNLXT ( SEQ ID NO : 261 ) . polypeptide , antibody, or antigen -binding fragment thereof) In some embodiments, the CDR - L2 of the antibody or includes a framework region that contains the amino acid antigen -binding fragment thereof has the amino acid sequence LLIR ( SEQ ID NO : 262 ) bound to the N - terminus sequence TYS . In some embodiments, the CDR - L2 of the of the CDR - L2 region . In some embodiments, the polypep antibody or antigen - binding fragment thereof has the amino 45 tide ( e.g. , antibody or antigen - binding fragment thereof) acid sequence YTS . In some embodiments, the CDR - L3 has includes a framework region that contains the amino acid the amino acid sequence CLQYVNLLT ( SEQ ID NO : 272 ) . sequence TLE bound to the C - terminus of the CDR - L2 In some embodiments, the CDR - L3 has the amino acid region . sequence CLQYVNLIT ( SEQ ID NO : 273 ) . The invention also features a polypeptide ( e.g. , single In some embodiments, the polypeptide ( e.g. , single - chain 50 chain polypeptide , antibody, or antigen - binding fragment polypeptide , antibody, or antigen -binding fragment thereof) thereof) that specifically binds TNFR2 and that contains a contains one or more of the following CDRs : light chain amino acid sequence having at least 85 % ( a ) a CDR - H1 having the amino acid sequence GFTFSSY sequence identity to the amino acid sequence of SEQ ID ( SEQ ID NO : 23 ) ; NO : 4 , and a non - native constant region . In some embodi ( b ) a CDR - H2 having the amino acid sequence SSGGSY 55 ments, the polypeptide ( e.g. , antibody or antigen - binding ( SEQ ID NO : 24 ) ; and fragment thereof) contains a light chain amino acid sequence ( c ) a CDR - H3 having the amino acid sequence having at least 90 % ( e.g. , 95 % , 97 % , 99 % , or 100 % ) QRVDGYSSYWYFDV ( SEQ ID NO : 25 ). sequence identity to the amino acid sequence of SEQ ID In some embodiments , the polypeptide single - chain poly- NO : 4. The polypeptide ( e.g. , antibody or antigen - binding peptide, antibody, or antigen - binding fragment thereof) con- 60 fragment thereof) may additionally contain a heavy chain tains one or more of the following CDRs : sequence having at least 85 % sequence identity to SEQ ID ( a ) a CDR -H1 having the amino acid sequence NO : 2. In some embodiments, the polypeptide ( e.g. , anti GYTFTDYX ( SEQ ID NO : 257 ) ; body or antigen - binding fragment thereof) may contain a ( b ) a CDR - H2 having the amino acid sequence heavy chain having at least 90 % sequence identity to the VDPEYGST ( SEQ ID NO : 258 ) ; and 65 amino acid sequence of SEQ ID NO : 2. For example, the ( c ) a CDR - H3 having the amino acid sequence ARDDG- polypeptide ( e.g. , antibody or antigen -binding fragment SYSPFDYWG ( SEQ ID NO : 259 ) . thereof) may contain a light chain having at least 85 % US 10,906,982 B2 7 8 sequence identity to the amino acid sequence of SEQ ID an epitope within amino acids 120-139 of SEQ ID NO : 7 NO : 4 and a heavy chain having at least 85 % sequence ( CRPGWYCALSKQEGCRLCAP ). Additionally or alterna identity to the amino acid sequence of SEQ ID NO : 2 . tively , the antibody or antigen - binding fragment thereof may Optionally , the polypeptide ( e.g. , antibody or antigen -bind- bind an epitope within amino acids 128-147 of SEQ ID NO : ing fragment thereof) may contain a light chain having at 5 7 ( LSKQEGCRLCAPLRKCRPGF ) . In some embodiments, least 90 % sequence identity to the amino acid sequence of the antibody or antigen - binding fragment thereof binds an SEQ ID NO : 4 and a heavy chain having at least 90 % epitope within amino acids 136-155 of SEQ ID NO : 7 sequence identity to the amino acid sequence of SEQ ID (LCAPLRKCRPGFGVARPGTE ). NO : 2 . The antibodies and antigen - binding fragments of the In another aspect , the invention provides constructs con- 10 invention can inhibit TNFR2 signaling . In some embodi taining a first polypeptide domain and a second polypeptide ments, the antibody or antigen - binding fragment thereof domain , each of which contains a single - chain polypeptide reduces or inhibits the expression of one or more genes of the invention . The first and second polypeptide domains selected from the group consisting of CHUK , NFKBIE , may be the same . In some embodiments , the first and second NFKBIA , MAP3K11, TRAF2 , TRAF3 , relB , and cIAP2 / polypeptide domains may be different. The first and second 15 BIRC3 . In some embodiments , the antibody or antigen polypeptide domains may be bound by a linker, such as a binding fragment thereof inhibits NFkB activation . For linker containing an amide bond or a disulfide bridge . The instance , antagonistic TNFR2 antibodies or antigen - binding constructs may lack a murine Fc domain . fragments thereof of the invention may reduce or inhibit the Embodiments of the invention also feature an antibody or expression or post - translational modification ( e.g. , phospho antigen - binding fragment thereof that specifically binds to a 20 rylation ) of one or more of CHUK , NFKBIE , NFKBIA , peptide comprising the amino acid sequence of any one of MAP3K11 , TRAF2 , TRAF3 , relB , or CIAP2 / BIRC3 , e.g. , by SEQ ID NOs : 11 , 19 , 20 , and 34-117 with a Ky of less than 1 % , 2 % , 3 % , 4 % , 5 % , 6 % , 7 % , 8 % , 9 % , 10 % , 15 % , 20 % , about 100 nM and does not bind a peptide comprising amino 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , acids 56-60 ( KCSPG ) of SEQ ID NO : 7 , and contains a 75 % , 80 % , 85 % , 90 % , 95 % , or 100 % relative to the expres non - native constant region ( e.g. , contains a human constant 25 sion or post -translational modification ( e.g. , phosphory region ) , lacks all or a portion of an Fc domain , lacks all or lation ) of one or more of these molecules isolated from a a portion of a native Fc domain , or lacks an Fc domain sample not treated with an antagonistic TNFR2 antibody or altogether. The invention also features an antibody or anti- antigen -binding fragment thereof of the invention . Exem gen - binding fragment thereof that is capable of specifically plary assays that can be used to determine expression level binding human TNFR2 , wherein the antibody or antigen- 30 and phosphorylation state are known in the art and include binding fragment thereof specifically binds a peptide com- Western blot assays to determine protein content and quan prising one or more of amino acids 142-146 of SEQ ID NO : titative reverse transcription polymerase chain reaction (RT 7 ( KCRPG ) and does not bind a peptide comprising amino PCR ) experiments to determine mRNA content . In preferred acids 56-60 of SEQ ID NO : 7 ( KCSPG ) , and contains a embodiments , anti - TNFR2 polypeptides ( e.g. , single - chain non - native constant region ( e.g. , contains a human constant 35 polypeptides, antibodies, or antigen - binding fragments region ) , lacks all or a portion of an Fc domain , lacks all or thereof) are dominant TNFR2 antagonists and are thus a portion of a native Fc domain , or lacks an Fc domain capable of inhibiting TNFR2 activation even in the presence altogether. The antibody or antigen -binding fragment of a TNFR2 agonist ( such as TNFa ) or a growth -promoting thereof may specifically bind the peptide containing one or agent, such as IL -2 . more of amino acids 142-146 of SEQ ID NO : 7 ( KCRPG ) 40 Antagonistic TNFR2 polypeptide ( e.g., antibodies or anti with a K , of less than about 10 nM . Optionally, the antibody gen -binding fragments thereof) of the invention may bind or antigen -binding fragment thereof may specifically bind an TNFR2 with a Ky ofno greater than about 10 nM , no greater epitope within amino acids 142-149 of SEQ ID NO : 7 than 1 nM , or, in particular cases , with a Ky of about 621 (KCRPGFGV ). Alternatively or additionally, the antibody PM . For instance , antagonistic TNFR2 polypeptides ( e.g. , or antigen - binding fragment thereof may specifically bind an 45 antibodies or antigen - binding fragments thereof) of the epitope within amino acids 137-144 of SEQ ID NO : 7 invention may bind TNFR2 or an epitope thereof as ( CAPLRKCR ) . In some cases , the antibody or antigen- described herein with a Ky of from about 1 PM to about 900 binding fragment thereof may specifically bind an epitope PM ( e.g . , about 1 PM , 2 PM , 3 PM , 4 PM , 5 PM , 6 PM , 7 PM , containing at least five discontinuous or continuous residues 8 PM , 9 PM , 10 PM , 20 PM , 30 PM , 40 PM , 50 PM , 60 pM , within amino acids 150-190 of SEQ ID NO : 7 5070 PM , 80 PM , 90 PM , 100 PM , 110 PM , 120 PM , 130 PM , (RPGTETSDVVCKPCAPGTFSNTTSSTDI 140 PM , 150 PM , 160 PM , 170 PM , 180 PM , 190 PM , or 200 CRPHQICNVVAI) . The antibody or antigen -binding frag- PM , 300 PM , 310 PM , 320 PM , 330 PM , 340 PM , 350 PM , ment thereof may optionally bind an epitope within amino 360 PM , 370 PM , 380 PM , 390 PM , 400 PM , 410 PM , 420 acids 161-169 of SEQ ID NO : 7 ( CKPCAPGTF ) . In other PM , 430 PM , 440 PM , 450 PM , 460 PM , 470 PM , 480 PM , cases , the antibody or antigen - binding fragment thereof may 55 490 PM , 500 PM , 510 PM , 520 PM , 530 PM , 540 PM , 550 bind an epitope containing at least five discontinuous or pM , 560 PM , 570 PM , 580 PM , 590 PM , 600 PM , 610 PM , continuous residues within amino acids 75-128 of SEQ ID 620 PM , 630 PM , 640 PM , 650 PM , 660 PM , 670 PM , 680 NO : 7 ( CDSCEDSTYTQLWNWVPE- PM , 690 PM , 700 PM , 710 PM , 720 PM , 730 PM , 740 PM , CLSCGSRCSSDQVETQACTREQNRICTCRPGWY- 750 PM , 760 PM , 770 PM , 780 PM , 790 PM , 800 PM , 810 CAL ) . Particularly, the antibody or antigen -binding frag- 60 PM , 820 PM , 830 PM , 840 PM , 850 PM , 860 PM , 870 PM , ment thereof may bind epitope within one or more of amino 880 PM , 890 PM , or 900 PM ) . In some embodiments, the acids 80-86 ( DSTYTQL ) , 91-98 ( PECLSCGS ) , and 116-123 polypeptide ( e.g. , antibody or antigen - binding fragment ( RICTCRPG ) of SEQ ID NO : 7 . thereof) binds TNFR2 with a K , of about 44 PM , e.g. , 44.4 In some embodiments, the antibody or antigen - binding pM . In certain instances , the TNFR2 antibodies or antigen fragment thereof binds an epitope within amino acids 112- 65 binding fragments thereof of the invention can bind TNFR2 131 of SEQ ID NO : 7 (REQNRICTCRPGWYCALSKQ ). to form an antibody - antigen complex with a kon of at least The antibody or antigen -binding fragment thereof may bind about 104 M - ls - 1 , and in some embodiments , with a kon of US 10,906,982 B2 9 10 about 3.6x10 % M - s - 7. For instance, antagonistic TNFR2 thereof, of the invention may also bind TNFR2 on the antibodies or antigen - binding fragments thereof of the surface of a myeloid - derived suppressor cell ( MDSC ; e.g. , a invention may bind TNFR2 to form an antibody - antigen cell that expresses all or a subset of proteins and small complex with a kon of from about 1x104 M - s - 1 to about molecules selected from the group consisting of B7-1 1x107 M - ' s - 1 ( e.g., about 1x104 M - 1 5-1 , 5x104 M-? s ?, 5 ( CD80 ) , B7 - H1 (PD -L1 ) , CCR2, CDid , CDidi , CD2 , 1x105 M - ' s - 1 , 5x105 M - s - , 1x10 M - 5-4 , 5x10 M - 1 CD31 ( PECAM - 1 ) , CD43 , CD44 , complement component s - 1 , or 1x10 ' M - s - 1 ). In some embodiments, the antibody or antigen - binding fragment thereof can bind TNFR2 to C5a R1 , F4 / 80 ( EMR1 ) , Fcy RII ( CD16 ) , Fcy RII ( CD32 ) , form an antibody - antigen complex with a kon of about 5x10 % Fcy RIIA ( CD32a ) , Fcy RIIB ( CD32b ) , Fcy RIIB / C ( CD32b / M - ' s - 1 , e.g. , 4.98x106 M - ' s - 1 . Antibodies or antigen - bind- 10 c ) , Fey RIIC ( CD32c ), Fcy RIIIA ( CD16A ) , Fcy RIIIB ing fragments thereof of the invention may bind TNFR2 to ( CD16b ) , galectin - 3 , GP130 , Gr - 1 ( Ly -6G ), ICAM - 1 form an antibody - antigen complex , wherein the complex ( CD54 ) , IL - 1RI , IL - 4Ra , IL - 6Ra , integrin a4 (CD49d ), dissociates with a koff of no greater than about 10-35-1 . In integrin aL ( CD11a ) , integrin aM ( CD11b ) , M - CSFR , some embodiments, the complex dissociates with a kofpof no MGL1 ( CD301a ) , MGL1 / 2 ( CD301a /b ), MGL2 ( CD301 b ) , greater than about 10-45-1, e.g. , with a koffof about 3.0x10-5 15 nitric oxide, PSGL - 1 ( CD162 ) , L - selectin ( CD62L ) , siglec - 3 s - 1 . For instance , antagonistic TNFR2 antibodies or antigen ( CD33 ) , transferrin receptor ( TfR ), VEGFR1 ( Flt - 1 ) , and binding fragments thereof of the invention may bind TNFR2 VEGFR2 ( KDR or Fik - 1 ) . Particularly, MDSCs do not to form an antibody - antigen complex , wherein the complex express proteins selected from the group consisting of B7-2 dissociates with a koff of from about 1x10-5 s - l to about ( CD86 ) , B7 - H4 , CD11c , CD14 , CD21 , CD23 ( FceRII ) , 1x10-35-1 ( e.g. , about 1x10-5 55 , 5x10-5 s - 1 , 1x10-4 5- ?, 20 CD34 , CD35 , CD40 ( TNFRSF5 ) , CD117 ( c - kit ) , HLA - DR , 5x10-4 5-1 , or 1x10-3 s- ) . In some embodiments, the and Sca - 1 ( Ly6 ). Binding of TNFR2 on the MDSC may antibodies or antigen - binding fragment thereof binds inhibit or reduce proliferation of the MDSC or may promote TNFR2 to form an antibody -antigen complex, wherein the the apoptosis of the MDSC . complex dissociates with a koff of no greater than about Antagonistic TNFR2 polypeptides , such as single - chain 2.3x10-4 s , e.g. , about 2x10-4 S . In some embodiments, 25 polypeptides, antibodies, or antigen -binding fragments the complex dissociates with a kon of about 2.21x10-4 S. thereof, of the invention may demonstrate the ability to Antagonistic TNFR2 polypeptides ( e.g. , antibodies and attenuate T - reg and / or cancer cell proliferation in the pres antigen - binding fragments thereof) of the invention may be ence of a TNFR2 agonist , such as TNFa or an agonistic capable of reducing or inhibiting the proliferation of a TNFR2 antibody, or growth - promoting molecules , such as population of T - reg cells , and may do so optionally in the 30 IL - 2 . These antibodies or antigen - binding fragments thereof presence of a TNFR2 agonist , such as TNFa . In some may bind TNFR2 and stabilize the dimeric, anti -parallel embodiments , the antibody or antigen - binding fragment dimer conformation of this receptor and prevent phospho thereof is capable of reducing or inhibiting the proliferation rylation and other post -translational modifications that occur of a population of cancer cells that express TNFR2, and may during NFkB signaling. do so optionally in the presence of a TNFR2 agonist , such 35 Antagonistic TNFR2 polypeptides , such as single -chain as TNFa . For instance, the cancer cells may be Hodgkin's polypeptides, antibodies, or antigen - binding fragments lymphoma cells , cutaneous non - Hodgkin's lymphoma cells , thereof, of the invention may reduce the total quantity of T cell lymphoma cells , ovarian cancer cells , colon cancer T -reg or cancer cells in a patient ( such as a human patient ) cells , multiple myeloma cells , or renal cell carcinoma cells . or within a sample ( e.g. , a sample isolated from a patient, In some embodiments , the antibody or antigen - binding 40 such as a human patient undergoing treatment for cancer or fragment thereof is capable of reducing or inhibiting the an infectious disease as described herein relative to a sample proliferation of a population of myeloid - derived suppressor isolated from a patient not undergoing such treatment or cells , and may optionally do so in the presence of a TNFR2 relative to a sample isolated from the patient prior to agonist , such as TNFa . In some embodiments, the antibody receiving this treatment ). or antigen -binding fragment thereof is capable of selectively 45 In some embodiments, the antagonistic TNFR2 polypep reducing or inhibiting the proliferation of a population of tide ( e.g. , single - chain polypeptide, antibody , or antigen T - reg cells expressing CD25H , such as a population of T - reg binding fragment thereof) reduces expression of TNFR2 , cells expressing CD25Hi and CD45RALOW . For instance, the e.g. , by a T -reg cell or a cancer cell ( such as a Hodgkin's or antibody or antigen - binding fragment thereof may be cutaneous non - Hodgkin's lymphoma cell, T cell lymphoma capable of reducing the proliferation of a population of T - reg 50 cell , ovarian cancer cell , colon cancer cell , multiple cells expressing CD25Hi and CD45RALow ( i.e. , a population myeloma cell , or renal cell carcinoma cell ) . of activated T - reg cells , or aT- reg cells ) by, e.g. , 1 % , 2 % , Antagonistic TNFR2 polypeptides, such as single - chain 3 % , 4 % , 5 % , 6 % , 7 % , 8 % , 9 % , 10 % , 15 % , 20 % , 25 % , 30 % , polypeptides, antibodies, and antigen - binding fragments 35 % , 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , thereof of the invention may inhibit the proliferation or 85 % , 90 % , 95 % , 100 % , or more relative to a population of 55 reduce the total quantity of a population of T -reg cells in a T - reg cells that does not express the CD25Hi and patient ( e.g. , a human patient) or in a sample ( e.g. , a sample CD45RALow proteins, such as a population of T -reg cells that isolated from a human patient undergoing treatment for expresses CD25Med and CD45RAH proteins (i.e. , a popula- cancer or an infectious disease as described herein ). tion of resting T - reg cells , or rT - reg cells ) . The invention also features a method of identifying a Antagonistic TNFR2 polypeptides ( e.g., antibodies or 60 TNFR2 antagonist polypeptide ( e.g., single -chain polypep antigen - binding fragments thereof) of the invention may tide , antibody, or antigen - binding fragment thereof) by: additionally bind TNFR2 on the surface of a cancer cell , ( a ) exposing a heterogeneous mixture of antibodies or such as a tumor cell . Binding of TNFR2 on the cancer cell fragments thereof to at least one peptide having the amino may inhibit or reduce proliferation of the cancer cell or may acid sequence of any one of SEQ ID NOs : 11 , 19 , 20 , and promote the apoptosis of the cancer cell . 65 34-117 , or a peptide containing between about 10 and about Antagonistic INFR2 polypeptides , such as single - chain 30 continuous or discontinuous amino acids between posi polypeptides , antibodies , or antigen -binding fragments tions 80 and 130 of SEQ ID NO : 7 ; and US 10,906,982 B2 11 12 ( b ) retaining antibodies or fragments thereof that specifi- In some embodiments , an antibody or antigen - binding cally bind said peptide and removing antibodies or frag- fragment thereof of the invention may be conjugated to a ments thereof that do not specifically bind said peptide, therapeutic agent, such as a cytotoxic agent. thereby producing an enriched antibody mixture comprising The invention features a polynucleotide encoding an at least one TNFR2 antagonist antibody or antigen - binding 5 antibody or antigen -binding fragment thereof of the inven fragment thereof. tion , as well as a vector containing such a polynucleotide. In some embodiments , one may determine the amino acid The vector may be an expression vector, such as a eukaryotic sequence of one or more of the antibodies or antigen - binding expression vector, or a viral vector, such as an adenovirus fragments thereof in the enriched antibody mixture. Option- ( Ad , such as serotype 5 , 26 , 35 , or 48 adenovirus ), retrovirus ally, the peptide may be bound to a surface, and in some 10 ( such as a y - retrovirus or a lentivirus ), poxvirus, adeno embodiments, the antibody or antigen -binding fragment associated virus, baculovirus , herpes simplex virus, or a thereof can be expressed on the surface of a phage , bacterial vaccinia virus ( such as a modified vaccinia Ankara (MVA ). cell , or yeast cell . In alternative cases , the antibody or The invention also features host cells , such as prokaryotic antigen -binding fragment thereof is expressed as one or and eukaryotic ( e.g. , mammalian ) cells containing a vector more polypeptide chains non - covalently bound to ribosomes 15 of the invention . or covalently bound to mRNA or cDNA . In particular cases , The invention also features a method of producing a the peptide can be conjugated to a detectable label, such as polypeptide ( e.g. , single - chain polypeptide, construct , anti a fluorescent molecule , epitope tag , or radiolabel. In some body, or antigen - binding fragment) of the invention by cases , the fluorescent molecule may be green fluorescent expressing a polynucleotide encoding the single - chain poly protein , cyan fluorescent protein , yellow fluorescent protein , 20 peptide, construct , antibody, or antigen - binding fragment red fluorescent protein , phycoerythrin , allophycocyanin , thereof in a host cell and recovering the single -chain poly hoescht, 4 ', 6 - diamidino - 2 -phenylindole ( DAPI ) , propidium peptide , antibody, or antigen - binding fragment thereof from iodide , fluorescein , coumarin , rhodamine , tetramethylrhoad- host cell medium . mine, or cyanine . In certain embodiments , the epitope tag The invention also features a method of inhibiting an may be maltose - binding protein , glutathione - S - transferase , a 25 immune response mediated by a regulatory T cell , as well as poly - histidine tag , a FLAG - tag , a myc - tag , human influenza a method of treating a cell proliferation disorder in a human , hemagglutinin ( HA ) tag , biotin , or streptavidin . In addition , by administering a single - chain polypeptide, construct, anti in some embodiments steps ( a ) and ( b ) above may be body, or antigen - binding fragment thereof, polynucleotide , sequentially repeated one or more times . vector, or host cell of the invention to the human in need of The invention also features a method of producing a 30 treatment. Additionally, the invention features invention also TNFR2 antagonist antibody or antigen - binding fragment features a composition containing a single -chain polypep thereof by immunizing a non - human mammal with a peptide tide, construct , antibody, or antigen - binding fragment comprising the sequence of any one of SEQ ID NOs : 1 , 19 , thereof, polynucleotide, vector, or host cell of the invention 20 , and 34-117 , or a peptide containing between about 10 for inhibiting an immune response mediated by a regulatory and about 30 continuous or discontinuous amino acids 35 T cell , as well as for treating a cell proliferation disorder in between positions 80 and 130 of SEQ ID NO : 7 , and a human . collecting serum containing the TNFR2 antagonist antibody In some embodiments, the cell proliferation disorder is a or antigen - binding fragment thereof. Exemplary non -human cancer, such as leukemia, lymphoma, liver cancer, bone mammals that can be immunized include a rabbit, mouse , cancer, lung cancer, brain cancer , bladder cancer, gastroin rat , goat , guinea pig , hamster, horse, and sheep . In some 40 testinal cancer, breast cancer, cardiac cancer, cervical cancer , embodiments, the peptide used for immunization may con- uterine cancer , head and neck cancer, gallbladder cancer , tain the amino acid sequence KCRPG ( SEQ ID NO : 19 ) . In laryngeal cancer , lip and oral cavity cancer , ocular cancer , some cases , the peptide used for immunization may contain melanoma, pancreatic cancer, prostate cancer , colorectal the amino acid sequence CAPLRKCR ( SEQ ID NO : 11 ) . cancer, testicular cancer, or throat cancer . In particular cases , Optionally , the peptide may contain the amino acid sequence 45 the cell proliferation disorder may be a cancer selected from KCRPGFGV ( SEQ ID NO : 20 ) . the group consisting of acute lymphoblastic leukemia The invention features an antibody or antigen - binding ( ALL ), acute myeloid leukemia ( AML ), chronic lympho fragment thereof produced by any of the above - described cytic leukemia ( CLL ) , chronic myelogenous leukemia methods . In some embodiments , the antibody or antigen- ( CML ) , adrenocortical carcinoma, AIDS -related lymphoma, binding fragment thereof may be a monoclonal antibody or 50 primary CNS lymphoma, anal cancer , appendix cancer, antigen - binding fragment thereof, a polyclonal antibody or astrocytoma, atypical teratoid / rhabdoid tumor, basal cell antigen - binding fragment thereof, a humanized antibody or carcinoma, bile duct cancer , extrahepatic cancer, ewing antigen -binding fragment thereof, a primatized antibody or sarcoma family, osteosarcoma and malignant fibrous histio antigen - binding fragment thereof, a bispecific antibody or cytoma, central nervous system embryonal tumors, central antigen - binding fragment thereof, a multi- specific antibody 55 nervous system germ cell tumors, craniopharyngioma, or antigen -binding fragment thereof, a dual - variable immu- ependymoma, bronchial tumors, burkitt lymphoma, carci noglobulin domain , a monovalent antibody or antigen -bind- noid tumor, primary lymphoma, chordoma, chronic myelo ing fragment thereof, a chimeric antibody or antigen -binding proliferative neoplasms , colon cancer , extrahepatic bile duct fragment thereof, a single - chain Fv molecule ( scFv ) , a cancer, ductal carcinoma in situ ( DCIS ) , endometrial cancer, diabody, a triabody, a nanobody, an antibody - like protein 60 ependymoma, esophageal cancer , esthesioneuroblastoma, scaffold , a domain antibody, a Fv fragment, a Fab fragment, extracranial germ cell tumor, extragonadal germ cell tumor, a F ( ab ' ) 2 molecule , and a tandem scFv ( taFv ) . In some fallopian tube cancer , fibrous histiocytoma of bone , gastro embodiments , the antibody or antigen - binding fragment intestinal carcinoid tumor, gastrointestinal stromal tumors thereof is a F ( ab ' ) 2 molecule . In some embodiments, the ( GIST ) , testicular germ cell tumor, gestational trophoblastic antibody or antigen - binding fragment thereof has an isotype 65 disease , glioma , childhood brain stem glioma , hairy cell selected from the group consisting of IgG , IgA , IgM , IgD , leukemia , hepatocellular cancer , langerhans cell histiocyto and IgE . sis , hodgkin lymphoma, hypopharyngeal cancer, islet cell US 10,906,982 B2 13 14 tumors , pancreatic neuroendocrine tumors , wilms tumor and virus, Jugra virus, Saboya virus, Sepik virus, Uganda S other childhood kidney tumors, langerhans cell histiocyto- virus, Wesselsbron virus, yellow fever virus, Entebbe bat sis , small cell lung cancer , cutaneous T -cell lymphoma, virus, Yokose virus, Apoi virus, Cowbone Ridge virus , intraocular melanoma , merkel cell carcinoma, mesothe- Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus , lioma , metastatic squamous neck cancer , midline tract car cinoma , multiple endocrine neoplasia syndromes, multiple 5 Bukalasa bat virus , Carey Island virus , Dakar bat virus, myeloma / plasma cell neoplasm , myelodysplastic syn Montana myotis leukoencephalitis virus, Phnom Penh bat dromes, nasal cavity and paranasal sinus cancer, nasopha virus, Rio Bravo virus, Tamana bat virus, cell fusing agent ryngeal cancer, neuroblastoma, non - hodgkin lymphoma virus, Ippy virus, Lassa virus, lymphocytic choriomeningitis (NHL ), non - small cell lung cancer (NSCLC ) , epithelial 10 virusFlexal ( LCMVvirus, Guanarito ), Mobala virus virus, Mopeia, Junin virusvirus, , Amapari Latino virus, , ovarian cancer, germ cell ovarian cancer , low malignant Machupo virus, Oliveros virus, Paraná virus, Pichinde virus, potential ovarian cancer , pancreatic neuroendocrine tumors, Pirital virus, Sabia virus , Tacaribe virus, Tamiami virus, papillomatosis, paraganglioma, paranasal sinus and nasal Whitewater Arroyo virus, Chapare virus, Lujo virus, Han cancercavity , cancer pheochromocytoma , parathyroid cancer, pituitary , penile tumor cancer , pleuropulmo- , pharyngeal 15 taan virus, Sin Nombre virus, Dugbe virus, Bunyamwera nary blastoma , primary peritoneal cancer, rectal cancer, virus , Rift Valley fever virus, La Crosse virus, California retinoblastoma, rhabdomyosarcoma, salivary gland cancer, encephalitis virus, Crimean - Congo hemorrhagic fever kaposi sarcoma , rhabdomyosarcoma, Sézary syndrome, ( CCHF ) virus, Ebola virus, Marburg virus, Venezuelan small intestine cancer, soft tissue sarcoma , throat cancer, equine encephalitis virus ( VEE ), Eastern equine encephalitis thymoma and thymic carcinoma, thyroid cancer , transitional 20 virus ( EEE ) , Western equine encephalitis virus (WEE ) , cell cancer of the renal pelvis and ureter, urethral cancer, Sindbis virus, rubella virus, Semliki Forest virus, Ross River endometrial uterine cancer , uterine sarcoma, vaginal cancer , virus, Barmah Forest virus, O’nyong’nyong virus, and the vulvar cancer , and Waldenström macroglobulinemia . chikungunya virus, smallpox virus, monkeypox virus , vac The invention also features methods of treating Hodgkin's cinia virus , herpes simplex virus, human herpes virus, cyto or cutaneous non -Hodgkin's lymphoma, T cell lymphoma, 25 megalovirus ( CMV ), Epstein - Barr virus ( EBV ) , Varicella ovarian cancer, colon cancer, multiple myeloma, or renal Zoster virus, Kaposi's sarcoma associated - herpesvirus cell carcinoma by administration of an antagonistic TNFR2 ( KSHV ) , influenza virus, severe acute respiratory syndrome polypeptide ( e.g. , single -chain polypeptide , construct, anti- ( SARS ) virus, rabies virus, vesicular stomatitis virus ( VSV ) , body, or antigen -binding fragment thereof ), a polynucle- human respiratory syncytial virus ( RSV ) , Newcastle disease otide , vector, or host cell of the invention of the invention to 30 virus , hendravirus, nipahvirus, measles virus, rinderpest a patient ( e.g. , a mammalian patient, such as a human virus, canine distemper virus, Sendai virus, human parain patient ). For instance , the invention provides a method of fluenza virus ( e.g. , 1 , 2 , 3 , and 4 ) , rhinovirus , mumps virus , treating ovarian cancer by administration of an antagonistic poliovirus, human enterovirus ( A , B , C , and D ) , hepatitis A TNFR2 antibody or antigen -binding fragment thereof of the virus , coxsackievirus, hepatitis B virus, human papilloma invention to a patient ( e.g. , a mammalian patient, such as a 35 virus, adeno -associated virus , astrovirus, JC virus, BK virus, human patient ). The invention additionally features a com- SV40 virus, Norwalk virus , rotavirus, human immunodefi position containing a single - chain polypeptide, construct, ciency virus ( HIV ) , human T -lymphotropic virus Types I and antibody, or antigen - binding fragment thereof, polynucle- II , and transmissible spongiform encephalopathy, such as otide, vector , or host cell of the invention for treating chronic wasting disease . Hodgkin's or cutaneous non -Hodgkin's lymphoma, T cell 40 In some embodiments, bacterial infections that can be lymphoma, ovarian cancer , colon cancer , multiple myeloma, treated according to the methods of the invention include or renal cell carcinoma in a patient ( e.g. , a human patient ). those caused by a bacterium belonging to a genus selected The invention also features a method of treating an from the group consisting of Salmonella, Streptococcus, infectious disease in a patient ( e.g. , a human patient) by Bacillus, Listeria , Corynebacterium , Nocardia , Neisseria , administering a single - chain polypeptide, construct, anti- 45 Actinobacter, Moraxella , Enterobacteriacece ( e.g. , E. coli , body, or antigen - binding fragment thereof, polynucleotide, such as 0157 : H7 ) , Pseudomonas, Escherichia , Klebsiella , vector , or host cell of the invention to the human in need of Serratia , Enterobacter , Proteus, Salmonella , Shigella , treatment, as well as a composition containing a single - chain Yersinia , Haemophilus, Bordatella, Legionella , Pasteurella , polypeptide, construct , antibody, or antigen -binding frag- Francisella, Brucella , Bartonella , Clostridium , Vibrio, ment thereof, polynucleotide , vector, or host cell of the 50 Campylobacter, and Staphylococcus. In addition , parasitic invention for treating an infectious disease in a patient ( e.g. , infections that can be treated according to the methods of the a human patient ). In some embodiments, the infectious invention include those caused by Entamoeba hystolytica , disease may be caused by a virus, a bacterium , a fungus, or Giardia lamblia , Cryptosporidium muris, Trypanosomatida a parasite. For instance , viral infections that can be treated gambiense , Trypanosomatida rhodesiense, Trypanosoma according to the methods of the invention include hepatitis 55 tida crusi, Leishmania mexicana , Leishmania braziliensis , C virus, Yellow fever virus , Kadam virus, Kyasanur Forest Leishmania tropica , Leishmania donovani, Toxoplasma disease virus, Langat virus, Omsk hemorrhagic fever virus , gondii , Plasmodium vivax, Plasmodium ovale, Plasmodium Powassan virus, Royal Farm virus, Karshi virus, tick -borne malariae , Plasmodium falciparum , Trichomonas vaginalis, encephalitis virus, Neudoerfi virus, Sofjin virus, Louping ill and Histomonas meleagridis. Exemplary helminthic para virus, Negishi virus, Meaban virus , Saumarez Reef virus , 60 sites include Richuris trichiura, Ascaris lumbricoides, Tyuleniy virus , Aroa virus, dengue virus, Kedougou virus, Enterobius vermicularis, Ancylostoma duodenale , Necator Cacipacore virus, Koutango virus , Japanese encephalitis americanus , Strongyloides stercoralis, Wuchereria ban virus, Murray Valley encephalitis virus, St. Louis encepha- crofti, and Dracunculus medinensis, Schistosoma mansoni , litis virus, Usutu virus, West Nile virus, Yaounde virus, Schistosoma haematobium , Schistosoma japonicum , Fas Kokobera virus, Bagaza virus, Ilheus virus, Israel turkey 65 ciola hepatica, Fasciola gigantica , Heterophyes, Paragoni meningoencephalo -myelitis virus, Ntaya virus , Tembusu mus westermani , Taenia solium , Taenia saginata , Hyme virus , Zika virus, Banzi virus , Bouboui virus, Edge Hill nolepis nana , or Echinococcus granulosus. US 10,906,982 B2 15 16 The invention also features kits, such as a kit that contains mediated by signaling through a TNFRS member, such as a single - chain polypeptide, construct , antibody, or antigen- TNFR2 , may be a cancer. For instance, the cancer may be binding fragment of the invention ( e.g. , an antagonist Hodgkin's lymphoma, cutaneous non - Hodgkin's lym TNFR2 antibody ), a polynucleotide of the invention , a phoma, T cell lymphoma, ovarian cancer , colon cancer , vector of the invention , or a host cell of the invention . In 5 multiple myeloma, or renal cell carcinoma. some cases , kits of the invention may contain instructions for transfecting a vector of the invention into a host cell of Definitions the invention . Optionally, kits may contain instructions for ( and optionally, a reagent that can be used for) expressing a single - chain polypeptide, construct , antibody , or antigen- 10 As used herein , the term “ about ” refers to a value that is binding fragment of the invention in a host cell of the no more than 10 % above or below the value being described . invention . A kit of the invention may also contain instruc For example, the term “ about 5 nM ” indicates a range of tions for administering an antibody or antigen - binding frag from 4.5 nM to 5.5 nM . ment of the invention , a polynucleotide of the invention , a As used herein , the term “ antibody ” ( Ab ) refers to an vector of the invention , or a host cell of the invention to a 15 immunoglobulin molecule that specifically binds to , or is human patient. Optionally a kit may contain instructions for immunologically reactive with , a particular antigen, and making or using an antibody or antigen - binding fragment of includes polyclonal , monoclonal , genetically engineered and the invention, a polynucleotide of the invention , a vector of otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies , het theThe invention invention, or additionallya host cell of features the invention a polypeptide . ( e.g. , a 20 eroconjugate antibodies ( e.g. , bi- tri- and quad - specific anti single - chain polypeptide , antibody, or antigen - binding frag- bodies , diabodies, triabodies, and tetrabodies ), and antigen ment thereof) that specifically binds a tumor necrosis factor binding fragments of antibodies , including e.g. , Fab ' , receptor superfamily ( TNFRS ) member in an anti - parallel F ( ab ' ) 2 , Fab , Fv , rigG , and scFv fragments. Moreover, unless dimer conformation . The polypeptide ( e.g. , single - chain otherwise indicated , the term “ monoclonal antibody ” (mAb ) polypeptide , antibody, or antigen - binding fragment thereof) 25 is meant to include both intact molecules , as well as , may optionally contain a non - native constant region . For antibody fragments ( such as , for example, Fab and F (ab ') 2 instance , the single - chain polypeptide, antibody, or antigen- fragments ) that are capable of specifically binding to a target binding fragment thereof may inhibit trimerization of the protein . Fab and F ( ab ' ) , fragments lack the Fc fragment of TNFRS member, e.g. , in the presence of a TNFRS member an intact antibody, clear more rapidly from the circulation of agonist , such as a cognate TNFRS member ligand . The 30 the animal, and may have less non - specific tissue binding single - chain polypeptide , antibody, or antigen - binding frag- than an intact antibody ( see Wahl et al . , J. Nucl . Med . ment thereof may inhibit NFkB signaling in a cell , such as 24 : 316 , 1983 ; incorporated herein by reference ). a eukaryotic cell ( e.g. , a mammalian cell , such as a human The ter " antigen - binding fragment, ” as used herein , or bovine cell ) . In some embodiments, the single - chain refers to one or more fragments of an antibody that retain the polypeptide, antibody, or antigen -binding fragment thereof 35 ability to specifically bind to a target antigen . The antigen reduces or inhibits the expression of one or more genes binding function of an antibody can be performed by frag selected from the group consisting of CHUK , NFKBIE , ments of a full - length antibody . The antibody fragments can NFKBIA , MAP3K11 , TRAF2 , TRAF3 , relB , and cIAP2 / be a Fab , F ( ab ' ) 2 , scFv , SMIP , diabody, a triabody, an BIRC3 . The TNFRS member may, e.g. , be selected from the affibody, a nanobody, an aptamer, or a domain antibody . group consisting of TNFR1 , TNFR2 , Fas , DCR3 , DR3 , 40 Examples of binding fragments encompassed of the term TRAIL - R1 (DR4 ), TRAIL -R2 ( DR5 ) , TRAIL -R3 , TRAIL- “ antigen - binding fragment ” of an antibody include , but are R4 , DR6 , EDAR , CD271 , OPG , RANK , LTBR , TWEAK - R , not limited to : ( i ) a Fab fragment, a monovalent fragment HVEM , CD27 , CD30 , CD40 , CD137 , OX40 , GITR , consisting of the V?, VH , C?, and Chl domains; ( ii ) a F ( ab ' ) 2 BCMA , TACI, BAFFR , EDAR2 , TROY, and RELT. For fragment, a bivalent fragment comprising two Fab frag instance, polypeptides ( e.g. , single - chain polypeptides, anti- 45 ments linked by a disulfide bridge at the hinge region ; ( iii ) bodies , or antigen -binding fragments thereof) of the inven- a Fd fragment consisting of the Vh and Chl domains ; ( iv ) tion may bind TNFR2 in an anti - parallel dimer conforma- a Fv fragment consisting of the V , and Vu domains of a tion , e.g. , thereby preventing TNFa - mediated trimerization single arm of an antibody, ( v ) a dAb including Vh and V and activation of this receptor. domains ; ( vi) a dAb fragment (Ward et al . , Nature 341: 544 The invention additionally features a method of treating a 50 546 , 1989 ) , which consists of a Vh domain ; ( vii) a dAb disease mediated by signaling through a TNFRS member in which consists of a Vh or a V , domain ; ( viii) an isolated a patient ( e.g. , a human patient) by administering to the complementarity determining region ( CDR ) ; and ( ix ) a human the single -chain polypeptide, antibody , or antigen- combination of two or more isolated CDRs which may binding fragment thereof that specifically binds a TNFRS optionally be joined by a synthetic linker . Furthermore , member in an anti- parallel dimer conformation as described 55 although the two domains of the Fv fragment, V and VH : above . Additionally , the method features a composition are coded for by separate genes, they can be joined , using containing a single - chain polypeptide , antibody, or antigen- recombinant methods, by a linker that enables them to be binding fragment thereof that specifically binds a TNFRS made as a single protein chain in which the V? and VH member in an anti - parallel dimer conformation for treating regions pair to form monovalent molecules ( knownwn as a disease mediated by signaling through a TNFRS member 60 single - chain Fv ( scFv ) ; see , e.g. , Bird et al . , Science 242 : in a patient ( e.g. , a human patient ). In some embodiments, 423-426 , 1988 , and Huston et al . , Proc . Natl . Acad . Sci . USA the TNFRS member is TNFR1 , TNFR2 , Fas , DCR3 , DR3 , 85 : 5879-5883 , 1988 ) . These antibody fragments can be TRAIL -R1 ( DR4 ) , TRAIL - R2 ( DR5 ) , TRAIL -R3 , TRAIL- obtained using conventional techniques known to those of R4 , DR6 , EDAR , CD271 , OPG , RANK , LTBR , TWEAK - R , skill in the art, and the fragments can be screened for utility HVEM , CD27 , CD30 , CD40 , CD137 , OX40 , GITR , 65 in the same manner as intact antibodies . Antigen -binding BCMA , TACI, BAFFR , EDAR2 , TROY, or RELT. In some fragments can be produced by recombinant DNA tech embodiments, the TNFRS member is TNFR2 . The disease niques, enzymatic or chemical cleavage of intact immuno US 10,906,982 B2 17 18 globulins , or , in some embodiments, by chemical peptide noglobulin of one source organism , such as rat or mouse , synthesis procedures known in the art . and constant regions derived from an immunoglobulin of a As used herein , the terms “ anti - tumor necrosis factor different organism ( e.g. , a human ). Methods for producing receptor 2 antibody , " " TNFR2 antibody, " " anti - TNFR2 anti chimeric antibodies are known in the art. See , e.g. , Morri body portion ," and / or “ anti - TNFR2 antibody fragment" and 5 son , 1985 , Science 229 ( 4719 ) : 1202-7 ; Oi et al , 1986 , the like include any protein or peptide - containing molecule Bio Techniques 4 : 214-221 ; Gillies et al , 1985 , J. Immunol. that includes at least a portion of an immunoglobulin mol Methods 125 : 191-202 ; U.S. Pat . Nos . 5,807,715 ; 4,816,567 ; ecule , such as but not limited to at least one complementarity and 4,816,397 ; incorporated herein by reference . determining region ( CDR ) of a heavy or light chain or a As used herein , the term " complementarity determining ligand binding portion thereof, a heavy chain or light chain 10 region ” ( CDR ) refers to a hypervariable region found both variable region , a heavy chain or light chain constant region , in the light chain and the heavy chain variable domains . The a framework region , or any portion thereof, that is capable more highly conserved portions of variable domains are of specifically binding to TNFR2 . TNFR2 antibodies also called the framework regions ( FRs ) . As is appreciated in the fibronectininclude antibody type III - like domain protein ( 1° Fn3scaffolds ), which, such contains as the BC ,tenth DE , 15 art , the amino acid positions that delineate a hypervariable and FG structural loops similar in structure and solvent region of an antibody can vary, depending on the context and accessibility to antibody CDRs . The tertiary structure of the the various definitions known in the art . Some positions 1 ° Fn3 domain resembles that of the variable region of the within a variable domain may be viewed as hybrid hyper IgG heavy chain , and one of skill in the art can graft, e.g. , variable positions in that these positions can be deemed to be the CDRs of a TNFR2 monoclonal antibody onto the 20 within a hypervariable region under one set of criteria while fibronectin scaffold by replacing residues of the BC , DE , and being deemed to be outside a hypervariable region under a FG loops of 1 ° Fn3 with residues from the CDR - H1 , CDR- different set of criteria . One or more of these positions can H2 , or CDR - H3 regions of a TNFR2 monoclonal antibody. also be found in extended hypervariable regions . The inven As used herein , the terms antagonist TNFR2 antibody " tion includes antibodies comprising modifications in these and “ antagonistic TNFR2 antibody ” refer to TNFR2 anti- 25 hybrid hypervariable positions . The variable domains of bodies that are capable of inhibiting or reducing activation native heavy and light chains each comprise four framework of TNFR2 and / or attenuating one or more signal transduc- regions that primarily adopt a B - sheet configuration , con tion pathways mediated by TNFR2 . For example, antago- nected by three CDRs , which form loops that connect, and nistic TNFR2 antibodies can inhibit or reduce the growth in some cases form part of, the B - sheet structure . The CDRs and proliferation of a population of regulatory T - cells. 30 in each chain are held together in close proximity by the FR Antagonistic TNFR2 antibodies may inhibit or reduce regions in the order FR1- CDR1 - FR2- CDR2 - FR3 - CDR3 TNFR2 activation by blocking TNFR2 from binding TNFa . FR4 and , with the CDRs from the other antibody chains, In this way , antagonistic TNFR2 antibodies may block the contribute to the formation of the target binding site of trimerization of TNFR2 that would otherwise be induced by antibodies ( see Kabat et al , Sequences of Proteins of Immu interacting with TNFa , thus resulting in suppression of 35 nological Interest ( National Institute of Health , Bethesda , TNFR2 activity . Md . 1987 ; incorporated herein by reference ). As used herein , As used herein , the term “ bispecific antibodies ” refers to numbering of immunoglobulin amino acid residues is done monoclonal , often human or humanized antibodies that have according to the immunoglobulin amino acid residue num binding specificities for at least two different antigens. In the bering system of Kabat et al , unless otherwise indicated . invention , one of the binding specificities can be directed 40 As used herein , the terms " conservative mutation , " " con towards TNFR2, the other can be for any other antigen , e.g. , servative substitution ,” or “ conservative amino acid substi for a cell - surface protein , receptor, receptor subunit, tissue- tution ” refer to a substitution of one or more amino acids for specific antigen , virally derived protein , virally encoded one or more different amino acids that exhibit similar envelope protein , bacterially derived protein , or bacterial physicochemical properties, such as polarity , electrostatic surface protein , etc. 45 charge, and steric volume. These properties are summarized As used herein , the term “ chimeric ” antibody refers to an for each of the twenty naturally - occurring amino acids in antibody having variable sequences derived from an immu- table 1 below . TABLE 1 Representative physicochemical properties of naturally -occurring amino acids Electrostatic Side character at 3 Letter 1 Letter chain physiological pH Steric Amino Acid Code Code Polarity ( 7.4 ) Volumet Alanine Ala A nonpolar neutral small Arginine Arg R polar cationic large Asparagine Asn N polar neutral intermediate Aspartic acid Asp D polar anionic intermediate Cysteine Cys ? nonpolar neutral intermediate Glutamic acid Glu E polar anionic intermediate Glutamine Gln polar neutral intermediate Glycine Gly G nonpolar neutral small Histidine His polar Both neutral and large cationic forms in equilibrium at pH 7.4 Isoleucine Ile I nonpolar neutral large Leucine Leu L nonpolar neutral large US 10,906,982 B2 19 20 TABLE 1 -continued Representative physicochemical properties of naturally - occurring amino acids Electrostatic Side character at 3 Letter 1 Letter chain physiological pH Steric Amino Acid Code Code Polarity ( 7.4 ) Volumet Lysine Lys K polar cationic large Methionine Met M nonpolar neutral large Phenylalanine Phe F nonpolar neutral large Proline Pro P non neutral intermediate polar Serine Ser S polar neutral small Threonine Thr T polar neutral intermediate Tryptophan Trp W nonpolar neutral bulky Tyrosine Tyr Y polar neutral large Valine Val V nonpolar neutral intermediate * based on volume in A3 : 50-100 is small, 100-150 is intermediate, 150-200 is large , and > 200 is bulky From this table it is appreciated that the conservative As used herein , a “ dominant antagonist ” of TNFR2 is an amino acid families include ( i ) G , A , V , L and I ; ( ii ) D and 20 antagonist ( e.g. , an antagonistic polypeptide, such as a E ; ( iii ) C , S and T ; ( iv ) H , K and R ; ( v ) N and Q ; and ( vi ) single - chain polypeptide, antibody, or antigen -binding frag F , Y and W. A conservative mutation or substitution is ment thereof) that is capable of inhibiting TNFR2 activation therefore one that substitutes one amino acid for a member even in the presence of a TNFR2 agonist , such as TNFa , or of the same amino acid family ( e.g. , a substitution of Ser for 25 IL - 2 . For example, a TNFR2 antagonist is a dominant Thr or Lys for Arg ). antagonist if the IC50 of the antagonist increases by less than As used herein , the term “ conjugate ” refers to a com- 200 % ( e.g. , less than 200 % , 100 % , 50 % , 45 % , 40 % , 35 % , pound formed by the chemical bonding of a reactive func- 30 % , 25 % , 20 % , 15 % , 10 % , 5 % , 1 % , or less ) in the presence tional group of one molecule with an appropriately reactive of a TNFR2 agonist ( e.g. , TNFa ) or IL - 2 relative to the IC50 functional group of another molecule . 30 of the antagonist as measured in the same assay in the As used herein , the term " derivatized antibodies ” refers to absence of a TNFR2 agonist, such as TNFa , or IL - 2 . antibodies that are modified by a chemical reaction so as to Inhibition of TNFR2 activation can be assessed , for cleave residues or add chemical moieties not native to an instance, by measuring the inhibition of proliferation of a population of TNFR2 + cells , such as T - reg cells , cancer cells glycosylationisolated antibody , acetylation . Derivatized , pegylationantibodies ,can phosphorylation be obtained by, 35 that express TNFR2, or myeloid -derived suppressor cells , as well as by measuring the inhibition of NFkB signaling ( e.g. , amidation , derivatization by addition of known chemical by monitoring the reduction in expression of one or more protecting /blocking groups, proteolytic cleavage, linkage to genes selected from the group consisting of CHUK , NFK a cellular ligand or other protein . Any of a variety of BIE , NFKBIA , MAP3K11, TRAF2 , TRAF3 , relB , and chemical modifications can be carried out by known tech 40 CIAP2 / BIRC3 in a conventional gene expression assay) . niques , including, without limitation , specific chemical Cell proliferation assays and gene expression assays that can cleavage , acetylation , formylation , metabolic synthesis of be used to monitor TNFR2 activation are described herein , tunicamycin , etc. using established procedures. Addition for instance , in Examples 9 and 12 , respectively. ally, the derivative can contain one or more non -natural As used herein , a “ dual variable domain immunoglobu amino acids , e.g. , using amber suppression technology ( see, 45 lin ” ( “ DVD - Ig ” ) refers to an antibody that combines the e.g. , U.S. Pat . No. 6,964,859 ; incorporated herein by refer- target -binding variable domains of two monoclonal antibod ence ) . ies via linkers to create a tetravalent, dual - targeting single As used herein , the term “ diabodies ” refers to bivalent agent. (Gu et al . , Meth . Enzymol ., 502 : 25-41 , 2012 ; incor antibodies comprising two polypeptide chains, in which porated by reference herein ). Suitable linkers for use in the each polypeptide chain includes Vh and V , domains joined 50 light chains of the DVDs of the invention include those by a linker that is too short ( e.g. , a linker composed of five identified on Table 2.1 on page 30 of Gu et al .: the short K amino acids ) to allow for intramolecular association of VH chain linkers ADAAP ( SEQ ID NO : 118 ) ( murine ) and and VL domains on the same peptide chain . This configu- TVAAP ( SEQ ID NO : 119 ) (human ); the long K chain ration forces each domain to pair with a complementary linkers ADAAPTVSIFP ( SEQ ID NO : 120 ) (murine ) and domain on another polypeptide chain so as to form a 55 TVAAPSVFIFPP ( SEQ ID NO : 121 ) (human ); the short A homodimeric structure . Accordingly, the term “ triabodies ” chain linker QPKAAP ( SEQ ID NO : 122 ) (human ); the long refers to trivalent antibodies comprising three peptide A chain linker QPKAAPSVTLFPP ( SEQ ID NO : 123 ) chains, each of which contains one VH domain and one VL (human ); the GS - short linker GGSGG ( SEQ ID NO : 124 ) , domain joined by a linker that is exceedingly short ( e.g. , a the GS - medium linker GGSGGGGSG ( SEQ ID NO : 125 ) , linker composed of 1-2 amino acids ) to permit intramolecu- 60 and the GS - long linker GGSGGGGSGGGGS ( SEQ ID NO : lar association of VH and VL domains within the same 126 ) ( all GS linkers are murine and human ). Suitable linkers peptide chain . In order to fold into their native structure , for use in the heavy chains of the DVDs include those peptides configured in this way typically trimerize so as to identified on Table 2.1 on page 30 of Gu & Ghayur, 2012 , position the VH and VL domains of neighboring peptide Methods in Enzymology 502 : 25-41 , incorporated by refer chains spatially proximal to one another to permit proper 65 ence herein : the short linkers AKTTAP ( SEQ ID NO : 127 ) folding ( see Holliger et al . , Proc . Natl . Acad . Sci . USA (murine ) and ASTKGP ( SEQ ID NO : 128 ) (human ); the 90 : 6444-48 , 1993 ; incorporated herein by reference ). long linkers AKTTAPSVYPLAP ( SEQ ID NO : 129 ) (mu US 10,906,982 B2 21 22 rine ) and ASTKGPSVFPLAP ( SEQ ID NO : 130 ) ( human ); 03089 , Traunecker et al . , EMBO J. 10 : 3655 ( 1991 ) , Suresh the GS - short linker GGGGSG ( SEQ ID NO : 131 ) , the et al . , Methods in Enzymology 121 : 210 ( 1986 ) ; incorpo GS - medium linker GGGGSGGGGS ( SEQ ID NO : 132 ) , rated herein by reference . Heterospecific antibodies can and the GS - long linker GGGGSGGGGSGGGG ( SEQ ID include Fc mutations that enforce correct chain association NO : 133 ) ( all GS linkers are murine and human ). 5 in multi - specific antibodies, as described by Klein et al , As used herein , the term “ endogenous ” describes a mol- mAbs 4 ( 6 ) : 653-663 , 2012 ; incorporated herein by reference . ecule ( e.g. , a polypeptide , nucleic acid , or cofactor ) that is As used herein , the term “ human antibody ” refers to an found naturally in a particular organism ( e.g. , a human ) or in antibody in which substantially every part of the protein a particular location within an organism ( e.g. , an organ , a ( e.g. , CDR , framework , Cz, Ch domains ( e.g. , Chl, Ch2, tissue , or a cell , such as a human cell ) . 10 CH3 ), hinge, ( V2, VH )) is substantially non - immunogenic in As used herein , the term " exogenous ” describes a mol- humans, with only minor sequence changes or variations . A ecule ( e.g. , a polypeptide , nucleic acid , or cofactor) that is human antibody can be produced in a human cell ( e.g. , by not found naturally in a particular organism ( e.g. , a human ) recombinant expression ), or by a non -human animal or a or in a particular location within an organism ( e.g. , an organ , prokaryotic or eukaryotic cell that is capable of expressing a tissue , or a cell , such as a human cell ). Exogenous 15 functionally rearranged human immunoglobulin ( e.g. , heavy materials include those that are provided from an external chain and / or light chain ) genes . Further, when a human source to an organism or to cultured matter extracted there antibody is a single - chain antibody, it can include a linker from . peptide that is not found in native human antibodies. For As used herein , the term “ framework region ” or “ FW example, an Fv can comprise a linker peptide , such as two region ” includes amino acid residues that are adjacent to the 20 to about eight glycine or other amino acid residues , which CDRs . FW region residues may be present in , for example , connects the variable region of the heavy chain and the human antibodies, rodent -derived antibodies ( e.g. , murine variable region of the light chain . Such linker peptides are antibodies ), humanized antibodies , primatized antibodies, considered to be of human origin . Human antibodies can be chimeric antibodies, antibody fragments ( e.g. , Fab frag- made by a variety of methods known in the art including ments ), single - chain antibody fragments ( e.g. , scFv frag- 25 phage display methods using antibody libraries derived from ments ), antibody domains , and bispecific antibodies , among human immunoglobulin sequences . See U.S. Pat . Nos . others . 4,444,887 and 4,716,111 ; and PCT publications WO 1998 / As used herein , the term “ fusion protein ” refers to a 46645 ; WO 1998/50433 ; WO 1998/24893 ; WO 1998 / protein that is joined via a covalent bond to another mol- 16654 ; WO 1996/34096 ; WO 1996/33735 ; and WO 1991 / ecule . A fusion protein can be chemically synthesized by, 30 10741 ; incorporated herein by reference. Human antibodies e.g. , an amide -bond forming reaction between the N - termi- can also be produced using transgenic mice that are inca nus of one protein to the C - terminus of another protein . pable of expressing functional endogenous immunoglobu Alternatively, a fusion protein containing one protein cova- lins , but which can express human immunoglobulin genes . lently bound to another protein can be expressed recombi- See , e.g. , PCT publications WO 98/24893 ; WO 92/01047 ; nantly in a cell ( e.g. , a eukaryotic cell or prokaryotic cell ) by 35 WO 96/34096 ; WO 96/33735 ; U.S. Pat . Nos . 5,413,923 ; expression of a polynucleotide encoding the fusion protein , 5,625,126 ; 5,633,425 ; 5,569,825 ; 5,661,016 ; 5,545,806 ; for example, from a vector or the genome of the cell . A 5,814,318 ; 5,885,793 ; 5,916,771 ; and 5,939,598 ; incorpo fusion protein may contain one protein that is covalently rated by reference herein . bound to a linker, which in turn is covalently bound to As used herein , the term “ humanized ” antibodies refers to another molecule . Examples of linkers that can be used for 40 forms of non - human ( e.g. , murine) antibodies that are chi the formation of a fusion protein include peptide - containing meric immunoglobulins, immunoglobulin chains or frag linkers , such as those that contain naturally occurring or ments thereof ( such as Fv , Fab , Fab ' , F ( ab ' ) 2 or other non -naturally occurring amino acids . In some embodiments, target -binding subdomains of antibodies) which contain it may be desirable to include D - amino acids in the linker, minimal sequences derived from non - human immunoglobu as these residues are not present in naturally -occurring 45 lin . In general, the humanized antibody will comprise sub proteins and are thus more resistant to degradation by stantially all of at least one , and typically two, variable endogenous proteases. Linkers can be prepared using a domains , in which all or substantially all of the CDR regions variety of strategies that are well known in the art, and correspond to those of a non - human immunoglobulin. All or depending on the reactive components of the linker, can be substantially all of the FR regions may also be those of a cleaved by enzymatic hydrolysis, photolysis , hydrolysis 50 human immunoglobulin sequence . The humanized antibody under acidic conditions , hydrolysis under basic conditions , can also comprise at least a portion of an immunoglobulin oxidation , disulfide reduction , nucleophilic cleavage , or constant region ( Fc ) , typically that of a human immuno organometallic cleavage ( Leriche et al . , Bioorg . Med . globulin consensus sequence . Methods of antibody human Chem ., 20 : 571-582 , 2012 ) . ization are known in the art. See , e.g. , Riechmann et al . , As used herein , the term " heterospecific antibodies ” refers 55 Nature 332 : 323-7 , 1988 ; U.S. Pat . Nos . 5,530,101 ; 5,585 , to monoclonal, preferably human or humanized , antibodies 089 ; 5,693,761 ; 5,693,762 ; and 6,180,370 to Queen et al ; that have binding specificities for at least two different EP239400 ; PCT publication WO 91/09967 ; U.S. Pat . No. antigens . Traditionally, the recombinant production of het- 5,225,539 ; EP592106 ; and EP519596 ; incorporated herein erospecific antibodies is based on the co - expression of two by reference . immunoglobulin heavy chain - light chain pairs , where the 60 As used herein , the term “ hydrophobic side - chain ” refers two heavy chains have different specificities ( Milstein et al . , to an amino acid side - chain that exhibits low solubility in Nature 305 : 537 , 1983 ) . Similar procedures are disclosed , water relative due to , e.g. , the steric or electronic properties e.g. , in WO 93/08829 , U.S. Pat . Nos . 6,210,668 ; 6,193,967 ; of the chemical moieties present within the side - chain . 6,132,992 ; 6,106,833 ; 6,060,285 ; 6,037,453 ; 6,010,902 ; Examples of amino acids containing hydrophobic side 5,989,530 ; 5,959,084 ; 5,959,083 ; 5,932,448 ; 5,833,985 ; 65 chains include those containing unsaturated aliphatic hydro 5,821,333 ; 5,807,706 ; 5,643,759 , 5,601,819 ; 5,582,996 , carbons, such as alanine , valine, leucine, isoleucine , proline , 5,496,549 , 4,676,980 , WO 91/00360 , WO 92/00373 , EP and methionine, as well as amino acids containing aromatic US 10,906,982 B2 23 24 ring systems that are electrostatically neutral at physiologi- TNFR2 + cancer cells , and / or MDSCs ) and / or by measuring cal pH , such as tryptophan , phenylalanine, and tyrosine . the expression of one or more NFkB target genes, such as As used herein , the term “ monoclonal antibody ” refers to CHUK , NFKBIE , NFKBIA , MAP3K11 , TRAF2, TRAF3 , an antibody that is derived from a single clone , including relB , and /or CIAP2 / BIRC3 . Exemplary assays for measuring any eukaryotic , prokaryotic , or phage clone , and not the 5 cell proliferation and gene expression are described , e.g. , in method by which it is produced . Examples 9 and 12 , respectively. As used herein , the term “ multi - specific antibodies ” refers As used herein , the term “ non - native constant region ” to antibodies that exhibit affinity for more than one target refers to an antibody constant region that is derived from a antigen . Multi - specific antibodies can have structures simi- source that is different from the antibody variable region or lar to full immunoglobulin molecules and include Fc 10 that is a human - generated synthetic polypeptide having an regions , for example IgG Fc regions . Such structures can amino sequence that is different from the native antibody include, but not limited to , IgG - Fv, IgG - scFv ) 2, DVD - Ig , constant region sequence . For instance , an antibody con ( scFv ) 2- ( scFv ) 2 - Fc and ( scFv ) 2 - Fc-( scFv ) 2. In case of IgG- taining a non - native constant region may have a variable ( scFv ) ,, the scFv can be attached to either the N -terminal or region derived from a non -human source ( e.g. , a mouse , rat, the C - terminal end of either the heavy chain or the light 15 or rabbit ) and a constant region derived from a human source chain . Exemplary multi - specific molecules that include Fc ( e.g. , a human antibody constant region ) . regions and into which anti - TNFR2 antibodies or antigen- As used herein , the term “ percent ( % ) sequence identity " binding fragments thereof can be incorporated have been refers to the percentage of amino acid ( or nucleic acid ) reviewed by Kontermann , 2012 , mAbs 4 ( 2 ) : 182-197 , Yazaki residues of a candidate sequence that are identical to the et al , 2013 , Protein Engineering, Design & Selection 26 ( 3 ) : 20 amino acid ( or nucleic acid ) residues of a reference sequence 187-193 , and Grote et al , 2012 , in Proetzel & Ebersbach after aligning the sequences and introducing gaps, if neces ( eds . ) , Antibody Methods and Protocols, Methods in sary, to achieve the maximum percent sequence identity Molecular Biology vol . 901 , chapter 16 : 247-263 ; incorpo- ( e.g. , gaps can be introduced in one or both of the candidate rated herein by reference . In some embodiments, antibody and reference sequences for optimal alignment and non fragments can be components of multi - specific molecules 25 homologous sequences can be disregarded for comparison without Fc regions , based on fragments of IgG or DVD or purposes ). Alignment for purposes of determining percent scFv . Exemplary multi - specific molecules that lack Fc sequence identity can be achieved in various ways that are regions and into which antibodies or antibody fragments can within the skill in the art, for instance , using publicly be incorporated include scFv dimers ( diabodies ) , trimers available computer software , such as BLAST , ALIGN , or ( triabodies ) and tetramers ( tetrabodies ), Fab dimers ( conju- 30 Megalign ( DNASTAR ) software . Those skilled in the art can gates by adhesive polypeptide or protein domains ) and Fab determine appropriate parameters for measuring alignment, trimers ( chemically conjugated ), are described by Hudson including any algorithms needed to achieve maximal align and Souriau , 2003 , Nature Medicine 9 : 129-134 ; incorpo- ment over the full length of the sequences being compared . rated herein by reference . For example, a reference sequence aligned for comparison As used herein , the term “ myeloid - derived suppressor 35 with a candidate sequence may show that the candidate cell” or “ MDSC ” refers to a cell of the immune system that sequence exhibits from 50 % to 100 % sequence identity modulates the activity of a variety of effector cells and across the full length of the candidate sequence or a selected antigen - presenting cells , such as T - cells, NK cells , dendritic portion of contiguous amino acid ( or nucleic acid ) residues cells , and macrophages , among others . Myeloid derived of the candidate sequence . The length of the candidate suppressor cells are distinguished by their gene expression 40 sequence aligned for comparison purposes may be , for profile, and express all or a subset of proteins and small example, at least 30 % , ( e.g. , 30 % , 40 , 50 % , 60 % , 70 % , 80 % , molecules selected from the group consisting of B7-1 90 % , or 100 % ) of the length of the reference sequence . ( CD80 ) , B7 - H1 ( PD - L1 ) , CCR2 , CD1d , CDidi , CD2 , When a position in the candidate sequence is occupied by CD31 (PECAM - 1 ) , CD43, CD44 , complement component the same amino acid residue as the corresponding position in C5a R1 , F4 / 80 ( EMR1 ) , Fcy RII ( CD16 ) , Fcy RII ( CD32 ) , 45 the reference sequence , then the molecules are identical at Fcy RIIA ( CD32a ) , Fcy RIIB ( CD32b ) , Fcy RIIB / C ( CD32b / that position. c ) , Fcy RIIC ( CD32c ) , Fcy RIIIA ( CD16A ) , Fcy RIIIB As used herein , the term “ primatized antibody ” refers to ( CD16b ) , galectin - 3 , GP130 , Gr - 1 ( Ly -6G ), ICAM - 1 an antibody comprising framework regions from primate ( CD54 ) , IL - 1 RI , IL - 4Ra , IL - 6Ra , integrin a4 (CD49d ), derived antibodies and other regions , such as CDRs and integrin aL ( CD11a ) , integrin aM ( CD11b ) , M - CSFR , 50 constant regions , from antibodies of a non - primate source . MGL1 ( CD301a ) , MGL1 / 2 ( CD301a / b ), MGL2 ( CD301b ) , Methods for producing primatized antibodies are known in nitric oxide, PSGL - 1 ( CD162 ) , L - selectin ( CD62L ) , siglec - 3 the art. See e.g. , U.S. Pat . Nos . 5,658,570 ; 5,681,722 ; and ( CD33 ) , transferrin receptor ( TfR ), VEGFR1 ( Flt - 1 ) , and 5,693,780 ; incorporated herein by reference . VEGFR2 ( KDR or Flk - 1 ) . Particularly , MDSCs do not As used herein , the term “ operatively linked ” in the express proteins selected from the group consisting of B7-2 55 context of a polynucleotide fragment is intended to mean ( CD86 ) , B7 - H4 , CD11c , CD14 , CD21 , CD23 ( Fc?RII ) , that the two polynucleotide fragments are joined such that CD34 , CD35 , CD40 ( TNFRSF5 ) , CD117 ( c - kit ) , HLA -DR , the amino acid sequences encoded by the two polynucle and Sca - 1 ( Ly6 ). otide fragments remain in - frame. As used herein , the term “ neutral TNFR2 polypeptide ” As used herein , the term “ pharmacokinetic profile ” refers refers to a polypeptide ( such as a single - chain polypeptide , 60 to the absorption , distribution , metabolism , and clearance of an antibody, or an antibody fragment ) that binds TNFR2 and a drug over time following administration of the drug to a does not exert an antagonistic or an agonistic effect on patient. TNFR2 activation . For instance , a TNFR2 polypeptide is a As used herein , a " recessive antagonist ” of TNFR2 is an neutral TNFR2 polypeptide if the polypeptide binds TNFR2 antagonist ( e.g. , an antagonistic polypeptide, such as a and neither potentiates nor suppresses TNFR2 activation , for 65 single - chain polypeptide, antibody, or antigen - binding frag instance, as assessed by measuring the proliferation of a ment thereof) that inhibits TNFR2 activation to a signifi population of TNFR2 - expressing cells ( e.g. , T - reg cells , cantly lesser extent in the presence of a TNFR2 agonist , such US 10,906,982 B2 25 26 as TNFa , or IL - 2 relative to the extent of inhibition of the ments are described , for example, in WO 2011/084714 ; same antagonist as measured in the absence of a TNFR2 incorporated herein by reference . agonist , such as TNFa , or IL - 2 . For example, a TNFR2 As used herein , the phrase " specifically binds ” refers to a antagonist is a recessive antagonist if the IC50 of the antago- binding reaction which is determinative of the presence of nist increases by, e.g. , 10 - fold , 20 - fold , 30 - fold , 40 - fold , 5 an antigen in a heterogeneous population of proteins and 50 - fold , 60 - fold , 70 - fold , 80 - fold , 90 - fold , 100 - fold , or more other biological molecules that is recognized , e.g. , by an in the presence of a TNFR2 agonist ( e.g. , TNFa ) or IL - 2 antibody or antigen - binding fragment thereof, with particu relative to the IC50 of the antagonist as measured in the same larity. An antibody or antigen - binding fragment thereof that assay the absence of a TNFR2 agonist , such as TNFa , or specifically binds to an antigen will bind to the antigen with IL - 2 . Inhibition of TNFR2 activation can be assessed , for 10 a Ky of less than 100 nM . For example, an antibody or instance , by measuring the inhibition of proliferation of a antigen -binding fragment thereof that specifically binds to population of TNFR2 + cells , such as T - reg cells , cancer cells an antigen will bind to the antigen with a Ky of up to 100 that express TNFR2 , or myeloid - derived suppressor cells , as nM ( e.g. , between 1 pM and 100 nM ) . An antibody or well as by measuring the inhibition of NFkB signaling ( e.g. , antigen - binding fragment thereof that does not exhibit spe by monitoring the reduction in expression of one or more 15 cific binding to a particular antigen or epitope thereof will genes selected from the group consisting of CHUK , NFK- exhibit a Ky of greater than 100 nM ( e.g. , greater than 500 BIE , NFKBIA , MAP3K11 , TRAF2 , TRAF3, relB , and nm , 1 uM , 100 uM , 500 M , or 1 mM ) for that particular CIAP2 /BIRC3 in a conventional gene expression assay ) . antigen or epitope thereof. A variety of immunoassay for Cell proliferation assays and gene expression assays that can mats may be used to select antibodies specifically immu be used to monitor TNFR2 activation are described herein , 20 noreactive with a particular protein or carbohydrate. For for instance , in Examples 9 and 12 , respectively. example, solid - phase ELISA immunoassays are routinely As used herein, the term “ regulatory sequence ” includes used to select antibodies specifically immunoreactive with a promoters , enhancers and other expression control elements protein or carbohydrate . See , Harlow & Lane , Antibodies , A ( e.g. , polyadenylation signals ) that control the transcription Laboratory Manual, Cold Spring Harbor Press, New York or translation of the antibody chain genes . Such regulatory 25 ( 1988 ) and Harlow & Lane , Using Antibodies , A Laboratory sequences are described , for example, in Goeddel, Gene Manual, Cold Spring Harbor Press, New York ( 1999 ) , for a Expression Technology : Methods in Enzymology 185 ( Aca- description of immunoassay formats and conditions that can demic Press , San Diego , Calif ., 1990 ) ; incorporated herein be used to determine specific immunoreactivity . by reference . As used herein , the terms “ subject ” and “ patient ” refer to As used herein , the term “ scFv ” refers to a single - chain Fv 30 an organism that receives treatment for a particular disease antibody in which the variable domains of the heavy chain or condition as described herein ( such as cancer or an and the light chain from an antibody have been joined to infectious disease ). Examples of subjects and patients form one chain . scFv fragments contain a single polypeptide include mammals, such as humans, primates , pigs , goats , chain that includes the variable region of an antibody light rabbits, hamsters, cats , dogs , guinea pigs , members of the chain ( VL ) ( e.g. , CDR - L1 , CDR - L2 , and / or CDR - L3 ) and 35 bovidae family ( such as cattle , bison , buffalo , elk , and yaks , the variable region of an antibody heavy chain ( VH ) ( e.g. , among others ), cows , sheep, horses , and bison , among CDR - H1 , CDR - H2 , and /or CDR - H3 ) separated by a linker. others, receiving treatment for diseases or conditions, for The linker that joins the VL and VH regions of a scFv example , cell proliferation disorders , such as cancer or fragment can be a peptide linker composed of proteinogenic infectious diseases . amino acids. Alternative linkers can be used to so as to 40 As used herein , the term “ transfection ” refers to any of a increase the resistance of the scFv fragment to proteolytic wide variety of techniques commonly used for the introduc degradation ( e.g. , linkers containing D - amino acids ), in tion of exogenous DNA into a prokaryotic or eukaryotic host order to enhance the solubility of the scFv fragment ( e.g. , cell, e.g. , electroporation, lipofection, calcium - phosphate hydrophilic linkers such as polyethylene glycol -containing precipitation , DEAE — dextran transfection and the like . linkers or polypeptides containing repeating glycine and 45 As used herein , the terms “ treat ” or “ treatment ” refer to serine residues ), to improve the biophysical stability of the therapeutic treatment, in which the object is to prevent or molecule ( e.g. , a linker containing cysteine residues that slow down ( lessen ) an undesired physiological change or form intramolecular or intermolecular disulfide bonds ) , or to disorder, such as the progression of a cell proliferation attenuate the immunogenicity of the scFv fragment ( e.g. , disorder , such as cancer , or an infectious disease . Beneficial linkers containing glycosylation sites ) . scFv molecules are 50 or desired clinical results include , but are not limited to , known in the art and are described , e.g. , in U.S. Pat . No. alleviation of symptoms, diminishment of extent of disease, 5,892,019 , Flo et al . , ( Gene 77:51 , 1989 ) ; Bird et al . , stabilized ( i.e. , not worsening ) state of disease , delay or ( Science 242 : 423 , 1988 ) ; Pantoliano et al . , ( Biochemistry slowing of disease progression , amelioration or palliation of 30 : 10117 , 1991 ) ; Milenic et al . , ( Cancer Research 51 : 6363 , the disease state , and remission (whether partial or total ) , 1991 ) ; and Takkinen et al . , ( Protein Engineering 4 : 837 , 55 whether detectable or undetectable . Those in need of treat 1991 ) . The VL and VH domains of a scFv molecule can be ment include those already with the condition or disorder, as derived from one or more antibody molecules . It will also be well as those prone to have the condition or disorder or those understood by one of ordinary skill in the art that the in which the condition or disorder is to be prevented . variable regions of the scFv molecules of the invention can As used herein , the terms “ tumor necrosis factor receptor be modified such that they vary in amino acid sequence from 60 superfamily ,” “ TNFR superfamily, ” or “ TNFRS ” refer to a the antibody molecule from which they were derived . For group of type I transmembrane proteins with a carboxy example, in one embodiment, nucleotide or amino acid terminal intracellular domain and an amino - terminal extra substitutions leading to conservative substitutions or cellular domain characterized by a common cysteine rich changes at amino acid residues can be made ( e.g. , in CDR domain ( CRD ) . The TNFR superfamily includes receptors and / or framework residues ). Alternatively or in addition , 65 that mediate cellular signaling as a consequence of binding mutations are made to CDR amino acid residues to optimize to one or more ligands in the TNF superfamily. The TNFR antigen binding using art recognized techniques. scFv frag- superfamily can be divided into two subgroups: receptors US 10,906,982 B2 27 28 containing the intracellular death domain and those lacking light chain , including the light chain of an Fv , scFv, dsFv or this domain . The death domain is an 80 amino acid motif Fab . Antibodies ( Abs) and immunoglobulins ( Igs ) are gly that propagates apoptotic signal transduction cascades fol coproteins having the same structural characteristics . While lowing receptor activation . Exemplary TNFR super family antibodies exhibit binding specificity to a specific target, members that contain the intracellular death domain include 5 immunoglobulins include both antibodies and other anti TNFR1 , while TNFR2 represents a TNFR super family body -like molecules which lack target specificity . Native protein that does not contain this domain . Members of the antibodies and immunoglobulins are usually heterotetra TNFR superfamily include TNFR1 , TNFR2, RANK , CD30 , meric glycoproteins of about 150,000 Daltons , composed of CD40 , Lymphotoxin beta receptor ( LT - 13R ), OX40 , Fas two identical light ( L ) chains and two identical heavy ( H ) receptor, Decoy receptor 3 ( DCR3 ) , CD27 , 4-1BB , Death 10 chains. Each heavy chain of a native antibody has at the receptor 4 ( DR4 ) , Death receptor 5 ( DR5 ) , Decoy receptor amino terminus a variable domain ( VH ) followed by a 1 ( DCR1 ) , Decoy receptor 2 (DCR2 ), Osteoprotegrin , number of constant domains . Each light chain of a native TWEAK receptor, TACI, BAFF receptor, Herpesvirus entry antibody has a variable domain at the amino terminus ( VL ) mediator, Nerve growth factor receptor, B - cell maturation and a constant domain at the carboxy terminus . antigen , Glucocorticoid - induced TNFR - related , TROY, 15 Death receptor 6 ( DR6 ) , Death receptor 3 ( DR3 ) , and BRIEF DESCRIPTION OF THE FIGURES Ectodysplasin A2 receptor. As used herein the term “ variable region CDR ” includes FIGS . 1A and 1B show the DNA and amino acid amino acids in a CDR or complementarity determining sequences of the heavy and light chains of the antagonistic region as identified using sequence or structure based meth- 20 TNFR2 antibody TNFRAB1. FIG . 1A shows the DNA ods . As used herein , the term “ CDR ” or “ complementarity sequence that encodes the heavy chain of TNFRAB1 ( top ) determining region ” refers to the noncontiguous antigen- and amino acid sequence of the heavy chain ( bottom ) . The binding sites found within the variable regions of both heavy amino acid seque es of the three complementarity -deter and light chain polypeptides. These particular regions have mining regions ( CDRs ) are shown in bold . FIG . 1B shows been described by Kabat et al . , J. Biol . Chem . 252 : 6609- 25 the DNA sequence that encodes the light chain of TNFRAB1 6616 , 1977 and Kabat , et al . , Sequences of Proteins of ( top ) and amino acid sequence of the light chain (bottom ). Immunological Interest, Fifth Edition , U.S. Department of The amino acid sequences of the three CDRs are shown in Health and Human Services, NIH Publication No. 91-3242 , bold . 1991 ; by Chothia et al . , ( J. Mol . Biol . 196 : 901-917 , 1987 ) , FIGS . 2A and 2B show the amino acid sequence of human and by MacCallum et al . , ( J. Mol . Biol . 262 : 732-745 , 1996 ) 30 TNFR2 ( SEQ ID NO : 7 ) . Notably , human TNFR2 is num where the definitions include overlapping or subsets of bered herein starting with an N - terminal methionine at amino acid residues when compared against each other. In position 1 and concluding with a C - terminal serine at certain embodi its , the term " CDR ” is a CDR as defined position 461 ( SEQ ID NO : 7 ) . All references to amino acid by Kabat based on sequence comparisons . positions within TNFR2 are made in the context of the As used herein , the term “ vector ” includes a nucleic acid 35 TNFR2 numbering scheme shown in FIGS . 2A and 2B . vector, e.g. , a DNA vector, such as a plasmid , a RNA vector, ( FIG . 2A ) Shaded residues KCRPGFGV ( SEQ ID NO : 20 ) virus or other suitable replicon ( e.g. , viral vector ) . A variety define an epitope that is specifically bound by the antago of vectors have been developed for the delivery of poly- nistic TNFR2 antibody TNFRAB1. Significantly, the ability nucleotides encoding exogenous proteins into a prokaryotic of TNFRAB1 to selectively bind residues within this region or eukaryotic cell . Examples of such expression vectors are 40 without binding underlined residues KCSPG ( SEQ ID NO : disclosed in , e.g. , WO 1994/11026 ; incorporated herein by 12 ) promotes antagonism of TNFR2 signaling . The poor ( or reference . Expression vectors of the invention contain a lack of) affinity of the antibodies of the invention for polynucleotide sequence as well as , e.g. , additional sequence residues within the region of, or near, the underlined resi elements used for the expression of proteins and / or the dues is consistent with the antagonistic activity of these integration of these polynucleotide sequences into the 45 antibodies, as binding to epitopes containing the underlined genome of a mammalian cell . Certain vectors that can be residues has been correlated with attenuation of the inhibi used for the expression of antibodies and antibody fragments tory activity among TNFR2 antibodies. TNFRAB1 addition of the invention include plasmids that contain regulatory ally binds an epitope that includes shaded residues CKP sequences, such as promoter and enhancer regions, which CAPGTF ( SEQ ID NO : 21 ) . Though these residues are not direct gene transcription . Other useful vectors for expression 50 consecutive in primary sequence with the KCRPG motif, of antibodies and antibody fragments contain polynucleotide they are likely spatially proximal in the three dimensional sequences that enhance the rate of translation of these genes tertiary structure of TNFR2 and may be appropriately posi or improve the stability or nuclear export of the mRNA that tioned for interaction with an antagonistic TNFR2 antibody results from gene transcription . These sequence elements of the invention ( see FIG . 4 ) . ( FIG . 2B ) Shaded residues include , e.g. , 5 ' and 3 ' untranslated regions, an internal 55 CAPLRKRCR ( SEQ ID NO : 11 ) define an epitope that is ribosomal entry site ( IRES ) , and polyadenylation signal site specifically bound by the antagonistic TNFR2 antibody in order to direct efficient transcription of the gene carried on TNFRAB2 . TNFRAB2 may additionally bind one or more the expression vector . The expression vectors of the inven- regions that include the residues DSTYTQL ( SEQ ID NO : tion may also contain a polynucleotide encoding a marker 8 ) , PECLSCGS ( SEQ ID NO : 9 ) , and RICTCRPG ( SEQ ID for selection of cells that contain such a vector . Examples of 60 NO : 10 ) , which may be part of a discontinuous epitope . a suitable marker include genes that encode resistance to Though these residues are not consecutive in primary antibiotics, such as ampicillin , chloramphenicol, kanamycin , sequence , they are likely spatially proximal in the three or nourseothricin . dimensional tertiary structure of TNFR2 and may be appro As used herein , the term “ VH ” refers to the variable priately positioned for interaction with an antagonistic region of an immunoglobulin heavy chain of an antibody, 65 TNFR2 antibody of the invention . including the heavy chain of an Fv , scFv, or Fab . References FIGS . 3A and 3B are tables showing the raw data obtained to “ VL ” refer to the variable region of an immunoglobulin from enzyme- linked immunosorbant assay ( ELISA ) experi US 10,906,982 B2 29 30 ments that were conducted to determine the affinity of showing the effect of TNFa and dominant antagonistic TNFRAB1 and TNFRAB2 for various continuous and dis- TNFR2 antibodies TNFRAB1 and TNFRAB2 on T - reg cell continuous epitopes within TFNR2 ( see Example 1 ) . Raw proliferation . Values on the x - axis represent the percent luminescence values are shown in the fourth column of the change in the quantity of T - reg cells relative to a sample tables ( right ). The peptide sequences shown represent those 5 treated with IL - 2 . FIGS . 6D and 6E are graphs showing the that contain a portion of the conformational epitope within results of duplicate experiments conducted in order to deter TNFR2 that interacts with TNFRAB1 ( FIG . 3A ) or mine the effect of TNFRAB2 on T -reg cell proliferation . TNFRAB2 ( FIG . 3B ) . Amino acid residues with the single- FIGS . 6F and 6G are graphs showing the results of duplicate digit code “ 2 ” designate cysteine residues that were chemi- experiments conducted in order to determine the effect of cally protected at the thiol position with an acetamidomethyl 10 TNFRAB1 on T - reg cell proliferation . ( ACM ) moiety during peptide synthesis. These residues are FIG . 7A is a graph showing the effect of TNFa on the not reactive with bromomethyl- containing electrophiles and proliferation of CD4 + T - reg cells . Values on the x - axis indi were therefore not cross - linked during the cyclization and cate the percent change in the quantity of T -reg cells upon bicyclization phases of peptide synthesis. The third column treatment with TNFa relative to treatment with IL - 2 . FIG . in the tables indicates the general structure of the peptide 15 7B is a graph showing the ability of TNFRAB1 to domi scaffold . “ CYS.S” indicates a 27 -residue peptide in which nantly inhibit T - reg cell proliferation in the presence and positions 1-11 and 17-27 of the peptide represent 11 - residue absence of TNFa . FIG . 7C is a graph showing the ability of peptides derived from TNFR2 that contain cysteine residues TNFRAB2 to inhibit T -reg cell proliferation in the presence that form disulfide bridges in the native protein based on and absence of TNFa . FIGS . 7D and 7E are graphs showing information available for UniProt entry P20333 . The 20 the effect of TNFR2 dominant antagonists on proliferation sequence Gly -Gly -Ser -Gly -Gly ( SEQ ID NO : 124 ) was of T - reg cells in the presence and absence of TNFa . incorporated into positions 12-16 of peptides of this group . FIG . 8A is a graph showing the ability of TNFRAB1 and Native Cys residues that do not form disulfide bridges were TNFRAB2 to inhibit the proliferation of T - reg cells gener protected with acetamidomethyl ( ACM ) protecting groups ally . FIG . 8B is a graph showing the effect of TNFRAB1 and and are designated with the single - digit code “ 2 ” . 25 TNFRAB2 on T - reg cell proliferation relative to the effect FIG . 4 is a schematic illustrating the conformational induced by treatment with IL - 2 . FIG . 8C is a graph showing epitopes within TNFR2 that may interact with antagonist the effect of TNFRAB1 and TNFRAB2 on total T -reg TNFR2 antibodies, such as TNFRAB1 and TNFRAB2, as quantity . FIG . 8D is a graph showing the effect of well as residues that do not interact with antagonist TNFR2 TNFRAB1 and TNFRAB2 on total T - reg quantity relative to antibodies . The KCSPG motif ( SEQ ID NO : 12 ) is shown in 30 the effect induced by treatment with IL - 2 . FIG . 8E is a graph the expansion at the top left of the figure ; the KCRPG ( SEQ demonstrating the effect of TNFRAB1 and TNFRAB2 on ID NO : 19 ) motif is shown in the expansion at the right of activated T - reg cells ( aT -reg cells ) that express CD25 in a the figure. Exterior surface of the protein designates the van high - affinity state ( CD25Hi) and CD45RA in a low - affinity der Waals surface of TNFR2 . FIG . 4 is a rendering of a state (CD45RALOW ). FIG . 8F is a graph showing the effect of monomer of TNFR2 isolated from the X -ray crystal struc- 35 TNFRAB1 and TNFRAB2 on a population of T - reg cells ture of TNFR2 ( PDB ID : 3ALQ , Mukai, et al . , Sci . Signal . , that express CD45ROHi and CD25Hi. FIG . 8G is a graph 3 : ra83 , 2010 ) . showing the effect of TNFRAB2 on CD25Hi- expressing FIG . 5 is a graph showing that TNFRAB1 suppresses the CD4 + T cells . FIG . 8H is a graph demonstrating the effect growth of cultured T -reg cells in vitro . Values represent the of TNFRAB2 on CD25Hi– expressing T -reg cells . These fraction of viable T -reg cells relative to untreated control 40 data demonstrate that, while the proliferation of both acti cells that remained in culture after exposure of the cells to vated ( aT -reg ) and resting (rT - reg ) cells is inhibited upon a particular condition . Bars shown on the far left represent treatment with TNFRAB1 or TNFRAB2 , antagonistic T - reg cells treated with either IL - 2 at 200 U ml/ ( control ), TNFR2 antibodies preferentially inhibit the proliferation of TNFa ( 20 ng /ml ), TNFR2 agonist ( 2.5 pg /ml ) , antagonistic a T -reg cells TNFR2 antibody TNFRAB1 ( 2.5 pg /ml ) , or antagonistic 45 FIG . 9A is a series of 2 - dimensional flow cytometry plots TNFR2 antibody TNFRAB2 ( 2.5 pg /ml ) . Bars shown sec- demonstrating the effect of agents that direct the growth of ond from the left demonstrate the dose -dependent variation T -reg cells ( e.g. , TNFa , IL - 2 , and TNFR2 agonists ) as well in T -reg viability upon treatment of cells with TNFRAB1. as a TNFR2 antagonist, TNFRAB2, on the proliferation of Bars shown second from the right show the ability of T - reg cells of various phenotypes. Treatment with a TNFR2 TNFRAB1 to inhibit the growth - promoting activity of 50 antagonist antibody preferentially inhibits the proliferation TNFa . T - reg cells were treated with a constant concentration of CD25Hi- expressing T -reg cells . The proportion of CD4 + , of TNFa ( 20 ng /ml ) and varying concentrations of CD25Hi + cells expressing TNFR2 after up to 48 hours of TNFRAB1 ( from 0.0008 to 25 pg / ml) . TNFRAB1 was incubation with either IL - 2 ( 200 U /ml ) alone , with TNFa capable of suppressing TNFa - induced proliferation in a ( 20 ng /ml ) , or with TNFR2 antagonist antibodies ( 12.5 concentration - dependent manner, indicating that TNFRAB1 55 pg /ml ) is shown . FIG . 9B is a graph showing the effect of antagonizes TNFR2 . Bars shown on the far right demon- TNFRAB1 on the secretion of TNFR2 , shown in units of strate the effect of TNFa on the growth of T - reg cells . pg /ml . FIG . 9C is a series of 1 - dimensional flow cytometry Incubation of T - reg cells with 20 ng /ml TNFa resulted in plots showing the effect of TNFRAB2 on CD8 + T -cell count approximately 130 % proliferation relative to untreated cells . as measured by carboxyfluorescein ( CFSE ) labeling. FIG . 6A is a graph showing the ability of TNFa to induce 60 FIG . 10A is a graph showing the ability of full - length aT - reg cell proliferation in a dose - dependent manner. FIG . TNFRAB1 ( IgG ) as well as F ( ab ' ) 2 fragment of TNFRAB1 6B is a graph showing the ability of IL - 2 to induce aT -reg to inhibit the proliferation of T - reg cells . FIG . 10B is a graph cell proliferation in a dose -dependent manner . Incubation of showing the ability of full - length TNFRAB2 ( IgG ) , as well freshly isolated human CD4 + cells for up to 48 hours with as a F ( ab ') 2 fragment of TNFRAB2, to inhibit the prolifera TNFa and IL - 2 ( 200 U /ml ) induces T - reg cell expansion in 65 tion of T - reg cells . These data demonstrate that specific a dose - dependent manner and the presence of IL - 2 is impor- binding of the Fab regions of these antagonistic TNFR2 tant for promoting T - reg expansion . FIG . 6C is a graph antibodies to TNFR2 is likely responsible for modulating US 10,906,982 B2 31 32 T -reg cell growth , rather than non - specific binding of the Fc ug /ml ) . Phosphorylation of RelA /NFKB p65 is induced by regions of these antibodies. Incubation of freshly isolated TNFa and inhibited by the TNFR2 antagonist mAbs in a CD4 + cells for up to 48 hrs with IL - 2 ( 200 U /ml ) plus either dose -dependent manner. the full antibody or F ( ab ') 2 fragment of TNFRAB1 or FIG . 13A is a table showing the kinetic and thermody TNFRAB2 produces similar dose - dependent inhibition of 5 namic parameters of the binding of TNFRAB1 and T -reg cells in the presence or absence of TNFa ( 20 ng /ml ) . TNFRAB2 to TNFR2 . Association rate constants are shown in units of Mºls - 1 , dissociation rate constants are shown in FIG . 10C is a graph showing the results of a dose - response units of s ', and equilibrium constants are shown in units of assay in which T - reg cells were treated with TNFRAB1 in M. FIG . 13B is a table showing the affinity of TNFRAB1 the presence of anti -IgG antibodies. FIG . 10D is a graph 10 and TNFRAB2 for various linear peptide sequences within showing the results of a dose - response assay in which T -reg human TNFR2 . Relative affinity is indicated as a series of cells were treated with TNFRAB2 in the presence of anti “ +” symbols, such that higher quantities of this symbol IgG antibodies . The dose -dependent suppression of T -reg represent elevated affinity values . Raw data derived from cell growth induced by TNFR2 antagonist antibodies was ELISA binding experiments are provided in the “ Reading " unaffected by the presence of anti- IgG molecules , indicating 15 column . FIG . 13C is a table showing the effect of TNFa on that non -specific cross - linking mediated by TNFRAB1 or the affinity of TNFRAB1 and TNFRAB2 for various linear TNFRAB2 is not responsible for the inhibitory effect of peptide sequences within human TNFR2 . Relative affinity is these antibodies on T - reg cell proliferation . Co - incubation of indicated as a series of “ + ” symbols , such that higher crosslinking antibody ( 2.5 ug /ml ) with TNFR2 antagonist quantities of this symbol represent elevated affinity values . antibodies ( 0.02-25 ug /ml ) does not affect the ability of the 20 Raw data derived from ELISA binding experiments are antibodies to inhibit T -reg proliferation in a dose -dependent provided in the “ Reading " column . manner . Data are presented as a single representative FIGS . 14A and 14B are graphs showing the results of example. duplicate experiments conducted in order to determine the FIG . 11A is an image of a polyacrylamide gel showing the effect of a recessive - TNFR2 antagonist antibody that mildly results of SDS - PAGE analysis conducted following the 25 inhibits TNFR2 activity ( recessive - antagonist TNFR2 anti expression of TNFRAB1 . FIG . 11B is an image of a poly- body A ) on the proliferation of T - reg cells . FIGS . 14C and acrylamide gel showing the results of SDS - PAGE analysis 14D are graphs showing the results of duplicate experiments conducted following the expression of TNFRAB2 . Analysis conducted in order to determine the effect of a second of reduced and non - reduced TNFR2 antagonist antibodies recessive - TNFR2 antagonist that mildly inhibits TNFR2 ( 2.5 ug ) is shown . FIG . 11C is an image of a polyacrylamide 30 activity (recessive - antagonist TNFR2 antibody B ) on the gel showing the results of a SDS - PAGE analysis conducted proliferation of T - reg cells . These recessive -antagonist anti following the expression of F ( ab ') 2 fragments of TNFRAB1. bodies were raised against the exterior region of TNFR2 in FIG . 11D is an image of a polyacrylamide gel showing the order to prevent TNFR2 trimerization that leads NFKB results of a SDS - PAGE analysis conducted following the activation . expression of Fab ') , fragments of TNFRAB2 . Analysis of 35 FIG . 15A is a structural model showing a three -dimen TNFR2 antagonist antibodies before and after digestion in sional structure of the anti -parallel dimer and parallel dimer F ( ab ' ) 2 fragment preparation is shown . of human TNFR2 . FIG . 15B is a structural model showing FIG . 12A is a graph showing the effect of TNFR2 antago- the three - dimensional structure of the TNFa - TNFR2 com nist antibodies on the expression of TNFa and lymphotoxin . plex . Shaded residues represent amino acids within human FIG . 12B is a graph showing the effect of TNFR2 antagonist 40 TNFR2 that are bound by TNFRAB1 . Proteins are portrayed antibodies on the expression of FoxP3 and CD25 . FIG . 12C in ribbon form beneath a Van der Waals surface . The data is a graph showing the effect of TNFR2 antagonist antibod- described herein clearly shows that for the full antibody or ies on the expression of genes that promote NFKB activa- the F ( ab ') 2 fragment thereof to bind to TNFR2 in an anti tion : conserved helix - loop -helix ubiquitous kinase ( CHUK ) , parallel conformation , the TNFR2 receptor would bind the nuclear factor of kappa light polypeptide gene enhancer in 45 antibody or fragment thereof at the indicated amino acid B - cells inhibitor epsilon ( NFKBIE ) , nuclear factor of kappa motifs . The binding sites of the parallel dimer are too close light polypeptide gene enhancer in B - cells inhibitor alpha to one another and would thus preclude antibody binding . ( NFKBIA ) , mitogen - activated protein kinase 11 Moreover, the trimeric TNFa - TNFR2 complex masks the (MAP3K11 ), TNFa receptor - associated factor 2 ( TRAF2 ), epitopes bound by antagonistic TNFR2 antibodies, as these TNFa receptor - associated factor 3 ( TRAF3 ), transcription 50 residues are located within the interior of the trimeric factor relB , and baculoviral IAP repeat containing 3 protein structure . FIG . 15C is an image showing the known and ( CIAP2 / BIRC3 ) . Real - time PCR analysis was used to detect published trimeric structural model of TNFR2 as a trimer RNA isolated from fresh CD4 + cells after incubation with with contained trimer TNF . Also shown in this structure are IL - 2 ( 50 U /ml ) in combination with either TNFa ( 20 ng /ml ) shaded residues that represent amino acids within human or the TNFR2 antagonist ( 2.5 ug /ml ) for 3 hours . FIG . 12D 55 TNFR2 that are bound by the recessive -antagonist TNFR2 is a graph showing the ability of TNFRAB1 to suppress antibodies A and B. NFkB activation as measured using a cell - based ELISA FIG . 16A is a graph showing the effect of TNFRAB1 on assay . FIG . 12E is a graph showing the ability of TNFRAB2 the proliferation of T - reg cells isolated from a patient to suppress NFkB activation as measured using a cell - based presenting with ovarian cancer ( grey shade ) and of T - reg ELISA assay . Phosphorylated RelA /NFkB p65 was used as 60 cells isolated from a subject not presenting with ovarian a marker of NFKB activity . Treatment of T - reg cells with cancer ( black shade ) . FIG . 16B is a graph showing the effect antagonistic TNFR2 antibodies resulted in attenuated NFKB of TNFRAB2 on the proliferation of T -reg cells isolated activation relative to treatment with TNFa . Using a cell- from a patient presenting with ovarian cancer ( grey shade ) based ELISA , the phosphorylation RelA /NFKB p65 was and of T - reg cells isolated from a subject not presenting with measured after 10 minute incubation of fresh CD4 + cells 65 ovarian cancer ( black shade ) . with IL - 2 ( 200 U /ml ) and various concentrations of TNFa FIG . 17A is a graph showing the effect of TNFRAB1 on ( 0.2-20 ng /ml ) or TNFR2 antagonist antibodies ( 0.02-25 the proliferation of T -reg cells isolated from a subject not US 10,906,982 B2 33 34 presenting with ovarian cancer . FIG . 17B is a graph showing TNFR1 , in an anti -parallel dimer conformation . By binding the effect of TNFRAB2 on the proliferation of T - reg cells a TNFRS member in an anti - parallel dimer structure, anti isolated from a subject not presenting with ovarian cancer. bodies or antigen - binding fragments thereof form a complex FIG . 17C is a graph showing the effect of TNFRAB1 on the with the TNFRS member in which receptor residues that proliferation of T - reg cells isolated from a subject presenting 5 bind a cognate ligand , such as TNFa in the case of TNFR2 , with ovarian cancer. FIG . 17D is a graph showing the effect are sequestered within the interior of the complex . Thus, of TNFRAB2 on the proliferation of T -reg cells isolated antibodies or antigen - binding fragments thereof of the from a subject presenting with ovarian cancer . FIG . 17E is invention may prevent ligand -mediated trimerization , and a graph showing the effect of TNFRAB1 on the proliferation hence activation , of a TNFRS member by forming a com of T - reg cells isolated from a subject not presenting with 10 plex with the TNFRS member that sterically precludes the ovarian cancer ( duplicate data set relative to FIG . 17A ). FIG . endogenous ligand from accessing its cognate binding sites 17F is a graph showing the effect of TNFRAB2 on the within the receptor. Exemplary TNFRS members that are proliferation of T - reg cells isolated from a subject not known to adopt an anti -parallel dimer conformation include presenting with ovarian cancer ( duplicate data set relative to TNFR1 , TNFR2 , Fas , DCR3 , DR3 , TRAIL -R1 ( DR4 ) . FIG . 17B ) . FIG . 176 is a graph showing the effect of 15 TRAIL -R2 ( DR5 ) , TRAIL -R3 , TRAIL -R4 , DR6 , EDAR , TNFRAB1 on the proliferation of T - reg cells isolated from CD271 , OPG , RANK , LTBR , TWEAK - R , HVEM , CD27 , a subject presenting with ovarian cancer ( duplicate data set CD30 , CD40 , CD137 , OX40 , GITR , BCMA , TACI, relative to FIG . 17C ) . FIG . 17H is a graph showing the effect BAFFR , EDAR2 , TROY, and RELT, among others . Poly of TNFRAB2 on the proliferation of T -reg cells isolated peptides ( e.g. , single - chain polypeptides, antibodies, and from a subject presenting with ovarian cancer ( duplicate 20 antigen - binding fragments ) of the invention may therefore data set relative to FIG . 17D ) . be used to bind a TNFRS member, such as TNFR2 , in an FIG . 18A is a structural model of a TNFRS member, the anti -parallel dimer conformation in order to inhibit the human death receptor 3 ( DR3 ) , shown in an anti- parallel activity of the TNFRS member in a target cell , such as a dimer conformation . The four cysteine - rich domains of DR3 T - reg cell , a TNFR2 + cancer cell , or a myeloid - derived are indicated by Roman numerals I - IV . The structural model 25 suppressor cell . For instance , antagonistic polypeptides, is reproduced from Tengchuan , Original Archival Copy of such as single - chain polypeptides , antibodies, or antigen Thesis , Structural Characterization of TNF Receptors and binding fragments thereof of the invention may bind DR3 in Ligands, Chicago , Ill . , 2008 , the disclosure of which is an anti - parallel dimer conformation ( e.g. , as shown in FIG . incorporated herein by reference in its entirety. FIG . 18B is 18A ) and / or TNFR1 in an anti -parallel dimer conformation a structural model showing TNFR1 in an anti - parallel dimer 30 ( e.g., as shown in FIG . 18B ). Antagonistic polypeptides conformation . In this model, the N - terminus of one TNFR1 ( e.g. , single -chain polypeptides , antibodies, and antigen monomer is located near the center of the other TNFR1 binding fragments ) of the invention may bind TNFRS monomer . FIG . 18C is a structural model showing TNFR1 members such as DR3 and / or TNFR1 in an nti- parallel in an alternative anti - parallel dimer conformation . In this conformation as shown in FIGS . 18A and 18B , thus render model , the N - termini of the TNFR1 monomers and the 35 ing cognate ligand binding sites sterically inaccessible and C - termini of the TNFR1 monomers are located proximal to attenuating TNFRS member -mediated signaling . For one another. FIGS . 18B and 18C are reproduced from instance , antagonistic DR3 antibodies or antigen - binding Naismith et al . Structure 4 : 1251-1262 ( 1996 ) , the disclosure fragments thereof of the invention may bind an epitope of which is incorporated herein by reference in its entirety . within residues 138-150 of the DR3 amino acid sequence 40 (GENBANKTM Accession No. AAQ88676.1 ). Antagonistic DETAILED DESCRIPTION TNFR1 antibodies or antigen - binding fragments thereof of the invention may bind an epitope within residues 185-197 Antagonistic TNFR2 polypeptides ( e.g. , single - chain of the TNFR1 amino acid sequence (GENBANKTM Acces polypeptides, antibodies, and antigen - binding fragments ) of sion No. NP_001056 ) . the invention inhibit the activation of TNFR2 on TNFR2- 45 Polypeptides ( e.g., single -chain polypeptides, antibodies , expressing cells by binding this receptor ( e.g. , on the exte- and antigen -binding fragments ) of the invention may be rior surface of a T - reg cell , a cancer cell that expresses dominant antagonists ( e.g. , dominant TNFR2 antagonistic TNFR2 , or a myeloid -derived suppressor cell ( MDSC ) , and polypeptides, such as single - chain polypeptides, antibodies, thus prevent the protein from recruiting its cognate ligand , or antigen -binding fragments thereof ). Dominant antagonis TNFa . TNFa potentiates TNFR2 signaling by nucleating a 50 tic TNFR2 polypeptides are those that are capable of binding trimer of TNFR2 proteins . It is this trimerization event that TNFR2 ( e.g. , in an anti- parallel dimer conformation ) and brings individual TNFR2 proteins into close proximity and inhibiting TNFR2 - mediated signal transduction even in the initiates signaling via the MAPK /NFKB / TRAF2 / 3 pathway, presence of an agonist , such as TNFa , or IL - 2 . Forexample , which ultimately leads to cell growth and escape from in the presence of a TNFR2 agonist , a dominant antagonistic apoptosis . TNFR2 - binding polypeptides ( e.g. , single - chain 55 TNFR2 polypeptide may inhibit the proliferation of a popu polypeptides, antibodies , and antibody fragments ) can lation of cells , such as T - reg cells , cancer cells that express antagonize this interaction by binding the receptor and TNFR2 , or myeloid -derived suppressor cells by , e.g. , 1 % , preventing TNFa from triggering this structural change . For 2 % , 3 % , 4 % , 5 % , 6 % , 7 % , 8 % , 9 % , 10 % , 15 % , 20 % , 25 % , instance , one mechanism by which this may occur is through 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , the formation of an anti - parallel TNFR2 dimer , which is an 60 80 % , 85 % , 90 % , 95 % , 100 % , or more , relative to a popu inactive structural form of the receptor. lation of such cells that is not treated with a dominant Polypeptides ( e.g. , single - chain polypeptides, antibodies, antagonistic TNFR2 polypeptide . In contrast, recessive and antigen - binding fragments ) of the invention may be antagonistic TNFR2 polypeptides are capable of binding used to inhibit the activity of other members of the tumor TNFR2 and inhibiting TNFR2 mediated- signaling, but the necrosis factor receptor superfamily ( TNFRS ) . For instance , 65 ability of these polypeptides to do so is attenuated in the antibodies and antigen - binding fragments thereof of the presence of a TNFR2 agonist . For instance , the IC50 of a invention may bind a TNFRS member, such as DR3 or recessive antagonistic TNFR2 polypeptide as measured in US 10,906,982 B2 35 36 the presence of a TNFR2 agonist , such as TNFa , or IL - 2 , in entirety ). Similarly, the TRAIL receptor is known to form an a T - reg cell death assay , a TNFR2 + cancer cell death assay , anti- parallel dimer ( Shirley et al . Rec . Pat . Anticanc . Drug or a myeloid - derived suppressor cell death assay may be Disc . 6 : 311 ( 2011 ) , the disclosure of which is incorporated augmented , e.g. , by 1 % , 2 % , 3 % , 4 % , 5 % , 6 % , 7 % , 8 % , 9 % , herein by reference in its entirety ). TRAF6 has additionally 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , 5 been shown to adopt this structural conformation (Marien 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 100 % , or feld et al . Mol . Cell . Biol . 26 : 9209 ( 2006 ) ; Yin et al . more , relative to that of a dominant antagonistic TNFR2 Biochem . 48 : 10558 ( 2009 ) ; and Yin et al . Nat . Struct. Mol . polypeptide . Examples of cell death assays that can be used Biol . 16 : 658 ( 2009 ) , the disclosures of each of which are to measure the antagonistic effects of TNFR2 antibodies are incorporated herein by reference in their entirety ). NGFR described herein , e.g. , in Example 9 , below . Dominant 10 has been shown to adopt an anti -parallel dimer conformation antagonistic TNFR2 polypeptides, such as single - chain ( Bibel et al . Genes Dev. 14 : 2919 ( 2000 ) , the disclosure of polypeptides , antibodies, or antigen -binding fragments which is incorporated herein by reference in its entirety ). thereof of the invention may therefore be used to suppress Additionally, CD40 is known to exhibit this structural motif the proliferation of a TNFR2 - expressing cell ( e.g. , a T - reg ( Smulski et al . J. Biol . Chem . 288 : 10914 ( 2013 ) , the dis cell , a TNFR2 + cancer cell , such as an ovarian cancer cell , 15 closure of which is incorporated herein by reference in its or a myeloid -derived suppressor cell ) even in the presence of entirety ). Additionally, CD137 has been shown to exist in an a growth - inducing signal, such as TNFa or IL - 2 . anti -parallel dimer state ( Vinay et al . CD137 Pathway: Polypeptides ( e.g. , single - chain polypeptides, antibodies , Immunology and Diseases , New York , N.Y., 2006 , the and antigen - binding fragments ) of the invention can be used disclosure of which is incorporated herein by reference in its to attenuate the activity ( e.g. , proliferation ) of T - reg cells 20 entirety ). FAS , CD40 , OX40 , and CD27 have additionally that typically accompanies T - cell -mediated cytotoxicity been shown to adopt an anti - parallel dimer conformation against self cells , such as the attack of a tumor cell by a ( Tartaglia et al . J. Biol . Chem . 267 : 4304 ( 1992 ) , the disclo T - lymphocyte. Antagonistic TNFR2 polypeptides can be sure of which is incorporated herein by reference in its administered to a mammalian subject, such as a human ( e.g. , entirety ). BAFF - R has also been found to exhibit this by any of a number of routes of administration described 25 structural motif ( Kim et al . , Nat . Struct . Biol . 10 : 342 ( 2003 ) , herein ) in order to prolong the duration of an adaptive the disclosure of which is incorporated herein by reference immune response , such as a response against a cancer cell or in its entirety ). a pathogenic organism . In this way , antagonistic TNFR2 Polypeptides ( e.g. , single - chain polypeptides , antibodies , polypeptides of the invention may synergize with existing and antigen - binding fragments ) that bind amino acid techniques to enhance T - lymphocyte - based therapy for can- 30 sequences within a TNFRS member protein that share cer and for infectious diseases . For instance , TNFR2 antago- sequence or structural homology with one or more of the nists of the invention may be administered to suppress T - reg KCRPGFGV ( SEQ ID NO : 20 ) , CKPCAPGTF ( SEQ ID cell activity, thereby enhancing the cytotoxic effect of tumor NO : 21 ) , CAPLRKCR ( SEQ ID NO : 11 ) , DSTYTQL ( SEQ reactive T - cells . TNFR2 antagonists may also synergize with ID NO : 8 ) , PECLSCGS ( SEQ ID NO : 9 ) , or RICTCRPG existing strategies to promote tumor - reactive T - cell survival, 35 ( SEQ ID NO : 10 ) motifs within human TNFR2 may bind such as lymphodepletion and growth factor therapy, and in and stabilize the TNFRS member protein in an anti -parallel turn prolong the duration of anti -tumor reactivity in vivo . dimer conformation . For instance , epitopes within a TNFRS Antagonistic TNFR2 polypeptides ( e.g. , single - chain poly- member protein that have at least 85 % sequence identity peptides , antibodies, and antigen - binding fragments ) can ( e.g. , 85 % , 90 % , 95 % , 97 % , 99 % , or 100 % sequence also be used to treat a broad array of infectious diseases in 40 identity ) to one of the aforementioned amino acid sequences a mammalian subject ( e.g. , a human ), as inhibition of T - reg within human TNFR2 can be identified using conventional growth and proliferation promotes the activity of CD8 + T- protein sequence alignment techniques known in the art. lymphocytes capable of mounting an attack on pathogenic Antagonistic TNFRS member polypeptides that bind one or organisms. Additionally, antagonistic TNFR2 polypeptides , more of these homologous epitopes can then be developed such as single - chain polypeptides, antibodies, and antigen- 45 using a variety of library screening and in vitro display binding fragments thereof, of the invention can be used to techniques as described herein ( for instance, see Example treat a wide variety of infectious diseases , such as Myco- 15 , below ) . Antagonistic TNFRS member polypeptides bacterium tuberculosis, in an agricultural farm animal ( e.g. , ( e.g. , single - chain polypeptides , antibodies, and antigen a bovine mammal , pig , cow , horse, sheep, goat , cat , dog , binding fragments ) developed in this manner may bind the rabbit, hamster, guinea pig , or other non -human mammal ). 50 TNFRS member protein in an anti - parallel dimer conforma Antagonistic TNFRS Member Polypeptides tion , thereby rendering cognate ligand binding sites steri TNFRS member proteins exhibit conformational proper- cally inaccessible and thus precluding receptor trimerization ties typified by TNFR2. For instance, these receptors are and activation . In particular, single - chain polypeptides, anti known to adopt an anti- parallel dimer conformation , and are bodies , or antigen - binding fragments thereof that specifi activated upon trimerization induced by binding to a cognate 55 cally bind a TNFRS member and stabilize an anti- parallel ligand . The amino acid sequences and three - dimensional dimer confirmation have binding specificity to only a single structures of TNFRS member proteins are known in the art . TNFRS member and do not cross - react with other TNFRS For instance , structural studies of unliganded TNFa recep- members . tors have revealed that these proteins adopt an anti - parallel Antagonistic TNFR2 Polypeptides dimer conformation , as described , e.g. , in Naismith et al . J. 60 The anti - TFNR2 polypeptides ( e.g. , single - chain poly Biol . Chem . 290 : 13303 ( 1995 ) , the disclosure of which is peptides , antibodies , and antigen - binding fragments) of the incorporated herein by reference in its entirety . Additionally, invention are capable of interacting with and inhibiting the it has been shown that the human death receptor 3 ( DR3 ) activity of TNFR2 . Thus, the anti - TNFR2 antibodies of the adopts an anti- parallel dimer conformation ( Tengchuan , invention can selectively antagonize the TNFa - TNFR2 Original Archival Copy of Thesis, Structural Characteriza- 65 interaction rather than promote TNFR2 signaling . This is tion of TNF Receptors and Ligands, Chicago , Ill . , 2008 , the particularly important for therapeutic applications, e.g. , can disclosure of which is incorporated herein by reference in its cer immunotherapy, as TNFR2 activation upon association US 10,906,982 B2 37 38 with TNFa leads to propagation of the MAPK and TRAF2 / 3 cells , TNFR2 + cancer cells , and / or MDSCs within a sample signal cascade and activation of NFKB - mediated transcrip- ( e.g. , within a patient, such as a human patient ). Antagonistic tion of genes involved in T - reg cell growth and escape from TNFR2 single - chain polypeptides, antibodies, or antigen apoptosis ( Faustman , et al . , Nat. Rev. Drug Disc . , 9 : 482- binding fragments thereof of the invention may be capable 493 , 2010 ) . The TNFR2 polypeptides of the invention may 5 of reducing the total quantity of T - reg cells , cancer cells bind TNFR2 with high affinity and may sterically sequester ( such as cutaneous T cell lymphoma cells , ovarian cancer the receptor from TNFa rather than allow TNFa binding to cells , colon cancer cells , renal cell carcinoma cells or TNFR2 initiate TNFR2 signaling , e.g. , by binding TNFR2 in multiple myeloma cells , among others ), and /or MDSCs in a an anti - parallel dimer conformation in which TNFa binding sample treated with an antagonist TNFR2 polypeptide ( such sites are sterically inaccessible . The antibodies of the inven- 10 as a sample isolated from a human patient undergoing tion can therefore be used to suppress T - reg cell growth and treatment for cancer or an infectious disease as described proliferation and can be administered to a mammalian herein ) by , e.g. , 1 % , 2 % , 3 % , 4 % , 5 % , 6 % , 7 % , 8 % , 9 % , subject, such as a human patient with a cell proliferation 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , disorder or an infectious disease , in order to enhance the 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 100 % , or effectiveness of an immune response ( e.g. , an immune 15 more , relative to a sample not treated with an antagonist response against cancerous cells or pathogenic organisms) in TNFR2 antibody or antigen - binding fragment thereof. the patient. The ability of antagonistic TNFR2 polypeptides ( e.g. , Antagonistic TNFR2 single -chain polypeptides , antibod- single - chain polypeptides , antibodies , and antigen - binding ies , or antigen -binding fragments thereof of the invention fragments ) of the invention to attenuate T - reg and / or cancer may additionally bind and inactivate TNFR2 on the surface 20 cell growth may be due to the ability of these polypeptides of a cancer cell , such as a tumor cell . For instance, antago- to diminish the quantity of soluble TNFR2 within a sample nistic TNFR2 single - chain polypeptides, antibodies, and ( e.g. , a sample isolated from a human patient undergoing antigen - binding fragments thereof described herein may treatment for cancer or an infectious disease as described bind TNFR2 on the surface a Hodgkin's or cutaneous herein ). Soluble TNFR2 can be secreted by , e.g. , T - reg cells , non - Hodgkin's lymphoma cell , T cell lymphoma cell , ovar- 25 and can interfere with the ability of TNFR2 antagonists to ian cancer cell , colon cancer cell , multiple myeloma cell , or localize to TNFR2 at the surface of a T - reg cell , TNFR2 + renal cell carcinoma cell , among others. The ability of cancer cell , or MDSC by binding and sequestering such antagonistic TNFR2 single - chain polypeptides , antibodies, antagonists in the extracellular environment. By reducing and antigen - binding fragments thereof of the invention to TNFR2 secretion , antagonistic TNFR2 single - chain poly bind TNFR2 directly on a cancer cell provides another 30 peptides, antibodies , or antigen - binding fragments thereof of pathway by which these molecules may attenuate cancer cell the invention may render T - reg cells , TNFR2 + cancer cells , survival and proliferation. For instance , an antagonistic and /or MDSCs increasingly susceptible to therapeutic mol TNFR2 single - chain polypeptide, antibody , or antigen -bind- ecules , such as an antagonistic TNFR2 antibody or antigen ing fragment thereof of the invention , such as an antibody or binding fragment thereof, and / or additional anti - cancer antigen - binding fragment thereof that contains one or more 35 agents described herein or known in the art that may be used of the heavy chain and /or light chain CDRs of TNFRAB1 or in conjunction with the compositions and methods of the TNFRAB2 ( or a CDR that has at least 85 % sequence invention . identity ( e.g. , 85 % , 90 % , 95 % , 97 % , 99 % , or 100 % Antagonistic TNFR2 polypeptides ( e.g. , single - chain sequence identity ) to a heavy chain and / or light chain CDR polypeptides , antibodies, and antigen - binding fragments ) of of TNFRAB1 or TNFRAB2, may bind TNFR2 directly on 40 the invention may be capable of inhibiting the proliferation the surface of a cancer cell ( e.g. , a cutaneous T cell lym- or reducing the total quantity of a population of T - reg cells phoma cell , ovarian cancer cell , colon cancer cell , or mul- in a sample ( e.g. , a sample isolated from a human patient tiple myeloma cell , such as an ovarian cancer cell ) in order undergoing treatment for cancer or an infectious disease as to diminish the ability of the cell to proliferate . described herein ) and may act selectively on T -reg cells in an Antagonistic TNFR2 polypeptides ( e.g. , single - chain 45 actively - dividing state . Antagonistic TNFR2 single - chain polypeptides , antibodies , and antigen - binding fragments ) of polypeptides, antibodies, or antigen -binding fragments the invention may demonstrate the ability to attenuate T - reg thereof of the invention may selectively target active T - reg and / or cancer cell proliferation even in the presence of a cells that express CD25Hi and CD45RALOW, e.g. , over rest TNFR2 agonist , such as TNFa or an agonistic TNFR2 ing T - reg cells that express CD25Med and CD45RAHI. For antibody, or growth -promoting molecules, such as IL - 2 . 50 instance , antagonistic TNFR2 polypeptides ( e.g., single Without being limited by mechanism , antagonistic TNFR2 chain polypeptides, antibodies, and antigen - binding frag single -chain polypeptides, antibodies, or antigen -binding ments ) of the invention may be capable of reducing the fragments thereof of the invention may exhibit this property proliferation of a population of T - reg cells expressing due to the ability of these antibodies or antigen -binding CD25Hi and CD45RALOW by, e.g. , 1 % , 2 % , 3 % , 4 % , 5 % , fragments thereof to bind TNFR2 and stabilize the dimeric , 55 6 % , 7 % , 8 % , 9 % , 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , anti- parallel dimer conformation of this receptor. This struc- 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , tural configuration is not capable of potentiating NFkB 95 % , 100 % , or more relative to a population of T - reg cells signaling . By maintaining TNFR2 in an inactive structural that does not express the CD25Hi and CD45RALLow ' proteins, state, antagonistic TNFR2 single - chain polypeptides, anti- such as a population of T - reg cells that expresses CD25Med bodies, or antigen - binding fragments thereof of the inven- 60 and CD45RAH proteins . tion may prevent TNFR2 agonists from restoring cell Antagonistic TNFR2 antibodies of the invention may growth . inhibit, e.g. , T -reg , cancer cell , and / or MDSC growth with a Another property that may be exhibited by antagonistic similar potency as that exhibited by antigen - binding frag TNFR2 polypeptides ( e.g. , single - chain polypeptides, anti- ments of such antibodies . For instance, removal of the Fc bodies , and antigen - binding fragments) is the ability to not 65 region of an antagonistic TNFR2 antibody of the invention only reduce T - reg cell , TNFR2 + cancer cell , and / or MDSC may not alter the ability of the molecule to attenuate the proliferation , but also to reduce the total quantity of T - reg proliferation or reduce the total quantity of T -reg cells and / or US 10,906,982 B2 39 40 cancer cells in a sample ( e.g. , a sample isolated from a Polypeptides ( e.g. , single - chain polypeptides , antibodies, human patient undergoing treatment for cancer or an infec- and antigen - binding fragments ) of the invention can also be tious disease as described herein ). Antagonistic TNFR2 characterized by a variety of in vitro binding assays . antibodies and antigen - binding fragments thereof of the Examples of experiments that can be used to determine the invention may function by a pathway distinct from antibody- 5 K ) or IC50 of a TNFR2 single - chain polypeptide , antibody , dependent cellular cytotoxicity ( ADCC ) , in which an Fc or fragment thereof include , e.g. , surface plasmon reso region is required to recruit effector proteins in order to nance , isothermal titration calorimetry, fluorescence anisot induce cell death . Additionally, antagonistic TNFR2 anti- ropy , and ELISA - based assays , among others . ELISA rep bodies or antigen - binding fragments thereof may not be resents a particularly useful method for analyzing antibody susceptible to a loss of inhibitory capacity in the presence of 10 activity, as such assays typically require minimal concen cross - linking agents. Antagonistic TNFR2 antibodies or trations of antibodies. A common signal that is analyzed in antigen - binding fragments thereof of the invention may a typical ELISA assay is luminescence , which is typically therefore exhibit therapeutic activity in a variety of isotypes, the result of the activity of a peroxidase conjugated to a such as IgG , IgA , IgM , IgD , or IgE , or in a variety of forms, secondary antibody that specifically binds a primary anti such as a monoclonal antibody or antigen -binding fragment 15 body ( e.g. , a TNFR2 antibody of the invention ). Polypep thereof, a polyclonal antibody or antigen - binding fragment tides of the invention are capable of binding TNFR2 and thereof, a humanized antibody or antigen -binding fragment epitopes derived thereof, such as epitopes containing one or thereof, a primatized antibody or antigen - binding fragment more of residues 142-146 of SEQ ID NO : 7 within human thereof, a bispecific antibody or antigen - binding fragment TNFR2 ( KCRPG , as shown in FIGS . 2A and 2B ) , as well as thereof, a multi - specific antibody or antigen - binding frag- 20 isolated peptides derived from TNFR2 that structurally ment thereof, a dual - variable immunoglobulin domain , a pre - organize various residues in a manner that may simulate monovalent antibody or antigen - binding fragment thereof, a the conformation of these amino acids in the native protein . chimeric antibody or antigen - binding fragment thereof, a For instance , polypeptides of the invention may bind pep single -chain Fv molecule ( scFv ) , a diabody, a triabody, a tides containing the amino acid sequence of any one of SEQ nanobody, an antibody - like protein scaffold , a domain anti- 25 ID NOs : 11 , 19 , 20 , and 34-117 , or a peptide containing body, a Fv fragment, a Fab fragment, a F ( ab ' ) 2 molecule , and between about 10 and about 30 continuous or discontinuous a tandem scFv ( taFv ). amino acids between positions 80 and 130 of SEQ ID NO : Specific Binding Properties of Antagonistic TNFR2 Poly- 7. In a direct ELISA experiment, this binding can be peptides quantified, e.g. , by analyzing the luminescence that occurs The specific binding of a single - chain polypeptide , anti- 30 upon incubation of an HRP substrate ( e.g. , 2,2 '- azino -di - 3 body or antibody fragment of the invention to human ethylbenzthiazoline sulfonate ) with an antigen - antibody TNFR2 can be determined by any of a variety of established complex bound to a HRP - conjugated secondary antibody . methods . The affinity can be represented quantitatively by For instance , polypeptides of the invention may induce a various measurements , including the concentration of anti- luminescence response of about 400 absorbance units or body needed to achieve half -maximal inhibition of the 35 more when incubated with surface - immobilized antigen and TNFC - TNFR2 interaction in vitro ( IC50 ) and the equilib- a HRP - conjugated secondary antibody in the presence of an rium constant (KD ) of the polypeptide - TNFR2 complex HRP substrate ( see , e.g. , Example 3 ) . In some embodiments, dissociation . The equilibrium constant, Kp, that describes the luminescence observed can be from about 400 to about the interaction of TNFR2 with a polypeptide of the invention 900 absorbance units ( e.g. , 400-900 absorbance units , 500 is the chemical equilibrium constant for the dissociation 40 800 absorbance units, or 600-700 absorbance units ). In reaction of a TNFR2 - antibody complex into solvent- sepa- particular cases , the luminescence observed can be from rated TNFR2 and antibody molecules that do not interact about 600 to about 900 absorbance units ( e.g. , 600-900 with one another. absorbance units or 700-800 absorbance units ) . Polypeptides ( e.g. , single - chain polypeptides , antibodies, Kinetic Properties of Antagonistic TNFR2 Polypeptides and antigen -binding fragments ) of the invention include 45 In addition to the thermodynamic parameters of a those that specifically bind to TNFR2 with a Ky value of less TNFR2 - polypeptide interaction, it is also possible to quan than 100 nM ( e.g. , 95 nM , 90 nM , 85 nM , 80 nM , 75 nM , titatively characterize the kinetic association and dissocia 70 nM , 65 nM , 60 nM , 55 nM , 50 nM , 45 nM , 40 nM , 35 tion of a single - chain polypeptide, antibody, or antibody nM , 30 nM , 25 nM , 20 nM , 15 nM , 10 nM , 5 nM , 4 nM , 3 fragment of the invention with TNFR2 . This can be done , nM , 2 nM , or 1 nM ) . In some embodiments, antibodies of 50 e.g. , by monitoring the rate of antibody - antigen complex the invention are those that specifically bind to TNFR2 with formation according to established procedures. For example, a K , value of less than 1 nM ( e.g. , ( e.g. , 990 PM , 980 PM , one can use surface plasmon resonance ( SPR ) to determine 970 PM , 960 PM , 950 PM , 940 PM , 930 PM , 920 PM , 910 the rate constants for the formation (kon ) and dissociation PM , 900 PM , 890 PM , 880 PM , 870 PM , 860 PM , 850 PM , (kof ) of an antibody - TNFR2 complex. These data also 840 PM , 830 PM , 820 PM , 810 PM , 800 PM , 790 PM , 780 55 enable calculation of the equilibrium constant of ( KD) of PM , 770 PM , 760 PM , 750 PM , 740 PM , 730 PM , 720 PM , antibody - TNFR2 complex dissociation , since the equilib 710 PM , 700 PM , 690 PM , 680 PM , 670 pM , 660 PM , 650 rium constant of this unimolecular dissociation can be PM , 640 PM , 630 PM , 620 PM , 610 PM , 600 PM , 590 PM , expressed as the ratio of the koff to kon values . SPR is a 580 PM , 570 PM , 560 PM , 550 PM , 540 PM , 530 PM , 520 technique that is particularly advantageous for determining PM , 510 PM , 500 PM , 490 PM , 480 PM , 470 PM , 460 PM , 60 kinetic and thermodynamic parameters of receptor -antibody 450 PM , 440 PM , 430 PM , 420 PM , 410 PM , 400 PM , 390 interactions since the experiment does not require that one PM , 380 PM , 370 PM , 360 PM , 350 PM , 340 PM , 330 PM , component be modified by attachment of a chemical label . 320 PM , 310 PM , 300 PM , 290 PM , 280 PM , 270 PM , 260 Rather, the receptor is typically immobilized on a solid PM , 250 PM , 240 PM , 230 PM , 220 PM , 210 PM , 200 pM , metallic surface which is treated in pulses with solutions of 190 PM , 180 PM , 170 PM , 160 PM , 150 PM , 140 PM , 130 65 increasing concentrations of antibody. Antibody - receptor PM , 120 PM , 110 PM , 100 PM , 90 PM , 80 PM , 70 PM , 60 binding induces distortion in the angle of reflection of PM , 50 PM , 40 PM , 30 PM , 20 PM , 10 PM , 5 PM , or 1 PM ) . incident light at the metallic surface, and this change in US 10,906,982 B2 41 42 refractive index over time as antibody is introduced to the than epitopes that promote signal transduction . Various system can be fit to established regression models in order discrete peptide fragments found within the TNFR2 primary to calculate the association and dissociation rate constants of structure bind antagonistic antibodies of the invention by an antibody -receptor interaction . virtue of the spatial orientation of these residues in the native Polypeptides ( e.g., single -chain polypeptides, antibodies, 5 conformation of the receptor. Significantly, these residues and antigen -binding fragments ) of the invention exhibit high have been difficult to identify, as many isolated linear kon and low koff values upon interaction with TNFR2 , con- TNFR2 -derived peptides do not appear to interact with sistent with high - affinity receptor binding . For example , antagonistic TNFR2 polypeptides ( e.g. , single - chain poly polypeptides of the invention may exhibit kon values in the peptides, antibodies, and antibody fragments) due to the presence of TNFR2 of greater than 104 M -l s -l ( e.g. , 10 different conformations these peptides exhibit when struc 1.0x104 M - ' s - 1 , 1.5x104 M - ' s - 1 , 2.0x104 M - ' s - 1 , 2.5x104 turally pre -organized within the full - length protein and when M - 15-1 , 3.0x104 M - 1 , -1,3.5x104 M - 1 , -1, 4.0x104 M - 1 , -1, isolated in solution. Epitope mapping analysis using con 4.5x104 M -15-1 , 5.0x104 M -15-1,5.5x10 ^ M - ' s - 1 , 6.0x104 strained cyclic and bicyclic peptides derived from various M - 3-1,6.5x104 M -1-1 , 7.0x104 M - ' s - 1 , 7.5x104 M - ' s - 1 , regions of TNFR2 indicates that antagonistic TNFR2 anti 8.0x104 M - ' s - 1 , 8.5x104 M -' s - 1 , 9.0x104 M - 15-1 , 9.5x104 15 bodies of the invention bind epitopes from distinct regions M - 1 5-1 , 1.0x10 M - 1 , -1 , 1.5x105 M - s - 1 , 2.0x10 % M - 1 , -1 , of the TNFR2 amino acid sequence in a conformation 2.5x105 M - 1 , -1 , 3.0x105 M - 1 , -1 , 3.5x105 M - 1 -1 , 4.0x105 dependent manner. Particularly important epitopes that bind M - ' s - 1 , 4.5x10 % M - ' s - 1 , 5.0x10 % M - ' s - 1 , 5.5x10 % M - ' s - 1 , antagonistic TNFR2 polypeptides of the invention and pro 6.0x10 - M - ' s - 1,6.5x105 M - ' s - 1 , 7.0x10 M - ' s - 1 , 7.5x105 mote receptor antagonism are those that contain one or more M's - 1, 8.0x10 M - ' s - 1, 8.5x10 M's- 1 , 9.0x10?M - ' s - 1 , 20 residues of the KCRPG motif ( SEQ ID NO : 19 ) , located at 9.5x105 M - s - 1 , or 1.0x106 M - ' s- ). Polypeptides of the positions 142-146 of SEQ ID NO : 7 within human TNFR2 . invention exhibit low koff values when bound to TNFR2, One or more of these residues reside within larger epitopes since antibodies are capable of interacting with distinct ( e.g. , residues 142-149 of SEQ ID NO : 7 , shown in FIG . 2A , TNFR2 epitopes with a high affinity. Residues within these and residues 137-144 of SEQ ID NO : 7 , shown in FIG . 2B ) epitopes form strong intermolecular contacts with TFNR2 , 25 that may interact with antagonistic TNFR2 antibodies of the which serves to slow the dissociation of the polypeptide- invention . The knowledge of those residues that selectively TNFR2 complex . This high receptor affinity is manifested in bind antagonistic TNFR2 polypeptides can be used to iden low kvalues. For instance , antibodies of the invention may tify and design a wide array of antagonistic TNFR2 anti exhibit koff values of less than 10-35-1 when complexed to bodies and antigen - binding fragments thereof using library TNFR2 ( e.g. , 1.0x10-3 s - 4 , 9.5x10-4 S - 1 , 9.0x10-4 5-1 , 30 screening techniques, e.g. , those described herein or known 8.5x10-45-1 , 8.0x10-45-1,7.5x10-45-1 , 7.0x10-45-1 , 6.5x in the art. For instance , structurally rigidified peptides con 10-45-4 , 6.0x10-45-4 , 5.5x10-45-1 , 5.0x10-45-4 , 4.5x10- taining one or more the residues within the KCRPG s - 1 , 4.0x10-4 5-1 , 3.5x10-4 S - 1 , 3.0x10-45-1 , 2.5x10-45-1 , sequence ( e.g. , peptides having the sequence of SEQ ID 2.0x10-4 S - 1 , 1.5x10-4 5-1 , 1.0x10-4 S - 1 , 9.5x10-5 5-1 , NOs : 42 , 50 , 52-54 , and 61-63 ) can be used to screen and 9.0x10-5 5-1 , 8.5x10-5s - 1, 8.0x10-5 5-1 , 7.5x10-5 5- !, 7.0x 35 select for polypeptides ( e.g. , single - chain polypeptides , anti 10-5 5-4 , 6.5x10-5 5-4 , 6.0x10-5s - 1 , 5.5x10-5 5-1 , 5.0x10-5 bodies , and antibody - like scaffolds) that bind these epitopes 5-1 , 4.5x10-5 5-1 , 4.0x10-5 : -1, 3.5x10-5 5-1, 3.0x10-5 5-1 2 with high affinity and selectivity . 2.5x10-5s- !, 2.0x10-5 5-1 , 1.5x10-5s - 4 , or 1.0x10-5 5-1) . Several distinct residues within TNFR2 bind antagonistic Epitopes within TNFR2 Bound by Antagonistic TNFR2 TNFR2 polypeptides, such as single - chain polypeptides, Polypeptides 40 antibodies , and antibody fragments, of the invention and The high affinities of polypeptides ( e.g. , single - chain establish strong intermolecular contacts with these antibod polypeptides , antibodies, and antigen - binding fragments ) of ies . Notably , functional antagonistic TNFR2 polypeptides of the invention for TNFR2 coupled with the rapid onset of the invention selectively bind an epitope containing one or polypeptide - TNFR2 complex formation and the slow disso more residues of amino acids 142-146 of SEQ ID NO : 7 ciation of these complexes render these polypeptides well- 45 within human TNFR2 ( KCRPG , SEQ ID NO : 19 ) . The suited for therapeutic applications as suppressors of T - reg spatial orientation of this epitope is shown in FIG . 4 . cell growth and proliferation . The high kon values , for Antagonistic TNFR2 polypeptides ( e.g. , single - chain poly instance, indicate that polypeptides ( e.g. , single - chain poly- peptides, antibodies, and antigen - binding fragments ) of the peptides, antibodies , and antigen - binding fragments ) of the invention are capable of selectively binding an epitope of invention are capable of localizing to the surface of a 50 TNFR2 that contains one or more of these residues and TNFR2 - expressing cell ( e.g. , a T - reg cell ) and rapidly asso- distinctly do not exhibit specific binding to an epitope ciating with TNFR2 , thereby preventing receptor activation containing residues 56-60 of SEQ ID NO : 7 within human that may otherwise be induced by TNFa ( e.g. , by inhibiting TNFR2 ( KCSPG , SEQ ID NO : 12 ) . For instance, polypep the trimerization of TNFR2 by TNFa ) . Moreover, the slow tides ( e.g. , single - chain polypeptides, antibodies and anti dissociation of the polypeptide - TNFR2 complex can be 55 body fragments ) of the invention do not exhibit specific indicative of a long half - life of the complex in vivo , which binding to epitopes that include one or more , or all , of results in stable , sustained down -regulation of the growth of residues 48-67 of SEQ ID NO : 7 within human TNFR2 the TNFR2 -expressing cell ( e.g. , sustained down - regulation ( QTAQMCCSKCSPGQHAKVFC , SEQ ID NO : 18 ) , as of T - reg growth ). These ideal thermodynamic and kinetic well as epitopes that exhibit at least 85 % sequence identity parameters of TNFR2 binding are consistent with the strong 60 ( e.g. , 85 % , 90 % , 95 % , 97 % , 99 % , or 100 % sequence intermolecular contacts that are established upon association identity ) to this sequence and epitopes that contain conser of polypeptides of the invention with TNFR2 . vative amino acid substitutions relative to this sequence ( so Among the difficulties in developing anti - TNFR2 poly- long as the amino acid sequence KCSPG is present in the peptides ( e.g. , single -chain polypeptides , antibodies, and epitope ) . Polypeptides that exhibit the ability to bind an antigen - binding fragments ) that are capable of antagonizing 65 epitope containing one or more residues of amino acids TNFR2 has been the elucidation of epitopes within TNFR2 142-146 of SEQ ID NO : 7 within human TNFR2 and an that participate in antagonistic complex formation rather epitope containing residues 56-60 of SEQ ID NO : 7 within US 10,906,982 B2 43 44 human TNFR2 have been shown to lack inhibitory (antago- epitope containing residues 142-149 of SEQ ID NO : 7 nistic ) activity . As such , the ability of a TNFR2 antibody to within human TNFR2 ( KCRPGFGV, SEQ ID NO : 20 ) , or an discriminate among these epitopes and specifically interact epitope that exhibits at least 85 % sequence identity ( e.g. , with an epitope including one or more of residues 142-146 85 % , 90 % , 95 % , 97 % , 99 % , or 100 % sequence identity ) to of SEQ ID NO : 7 within human TNFR2 and to not engage 5 this sequence ( so long as one or more residues of the in specific binding with an epitope composed of residues KCRPG sequence is present in the epitope ) . Additionally or 56-60 of SEQ ID NO : 7 within human TNFR2 characterizes alternatively, antagonistic TNFR2 antibodies of the inven polypeptides ( e.g. , single - chain polypeptides, antibodies, tion may specifically bind an epitope including residues and antigen - binding fragments ) of the invention that antago- 137-144 of SEQ ID NO : 7 within human TNFR2 ( CA nize TNFR2 signaling. 10 PLRKCR , SEQ ID NO : 11 ) , or an epitope that exhibits at One exemplary procedure that can be used to predict the least 85 % sequence identity ( e.g. , 85 % , 90 % , 95 % , 97 % , inhibitory activity of a TNFR2 single - chain polypeptide, 99 % , or 100 % sequence identity ) to this sequence ( so long antibody, or antibody fragment of the invention is to com- as one or more residues of the KCRPG sequence is present pare the affinity of the polypeptide for a peptide containing in the epitope ). the KCRPG motif ( e.g. , a linear peptide having the sequence 15 Antagonistic TNFR2 single - chain polypeptides, antibod KQEGCRLCAPLRKCRPGFGV, SEQ ID NO : 17 ) to the ies , and antibody fragments of the invention may optionally affinity of the same antibody or antibody fragment for a bind a downstream epitope composed of at least five con peptide containing the KCSPG sequence ( e.g. , a linear tinuous or discontinuous residues from positions 150-190 of peptide having the sequence SEQ ID NO : 7 within human TNFR2 QTAQMCCSKCSPGQHAKVFC , SEQ ID NO : 18 ) . For 20 (ARPGTETSDVVCKPCAPGTFSNTTSSTDI instance, antagonistic TNFR2 antibody TNFRAB1 specifi- CRPHQICNVVAI, SEQ ID NO : 22 ) , as well as epitopes that cally binds the peptide fragment defined by residues 130- exhibit at least 85 % sequence identity ( e.g. , 85 % , 90 % , 95 % , 149 of SEQ ID NO : 7 within human TNFR2 (KQEGCRL- 97 % , 99 % , or 100 % sequence identity ) to this sequence and CAPLRKCRPGFGV, SEQ ID NO : 17 ) with a 40 - fold epitopes that contain conservative amino acid substitutions greater affinity than the peptide fragment defined by residues 25 relative to this sequence . For example , in some embodi 48-67 of SEQ ID NO : 7 within human TNFR2 ments , antagonistic TNFR2 polypeptides of the invention ( QTAQMCCSKCSPGQHAKVFC , SEQ ID NO : 18 ) . may specifically bind an epitope that includes residues from Antagonistic TNFR2 polypeptides ( e.g. , single - chain poly- positions 161-169 of SEQ ID NO : 7 within human TNFR2 peptides, antibodies, and antigen -binding fragments ) of the ( CKPCAPGTF , SEQ ID NO : 21 ) , as well as epitopes that invention bind an epitope containing one or more residues of 30 exhibit at least 85 % sequence identity ( e.g. , 85 % , 90 % , 95 % , the KCRPG sequence ( SEQ ID NO : 19 ) with an affinity that 97 % , 99 % , or 100 % sequence identity ) to this sequence and is at least 10 - fold greater than the affinity of the same epitopes that contain conservative amino acid substitutions single - chain polypeptide, antibody , or antigen - binding frag- relative to this sequence . ment for a peptide that contains the KCSPG sequence of In addition to interacting with the one or more residues of human TNFR2 ( SEQ ID NO : 12 ) . For example , antagonistic 35 the KCRPG motif ( SEQ ID NO : 19 ) , antagonistic TNFR2 TNFR2 polypeptides ( e.g. , single - chain polypeptides, anti- polypeptides of the invention may also specifically bind an bodies , and antigen - binding fragments thereof) of the inven- epitope within human TNFR2 that includes at least five tion bind an epitope containing one or more residues of the continuous or discontinuous residues from positions 75-128 KCRPG sequence ( SEQ ID NO : 19 ) of human TNFR2 with of SEQ ID NO : 7 within human TNFR2 (CDSCED an affinity that is 10 - fold , 20 - fold , 30 - fold , 40 - fold , 50 - fold , 40 STYTQLWNWVPECLSCGSRCSSDQVETQACTREQN 60 - fold , 70 - fold , 80 - fold , 90 - fold , 100 - fold , 200 - fold , 300- RICTCRPGWYCAL , SEQ ID NO : 13 ) , as well as epitopes fold , 400 - fold , 500 - fold , 600 - fold , 700 - fold , 800 - fold , 900- that exhibit at least 85 % sequence identity ( e.g. , 85 % , 90 % , fold , 1000 - fold , or more than 1000 - fold greater than the 95 % , 97 % , 99 % , or 100 % sequence identity ) to this affinity of the same single - chain polypeptide , antibody, or sequence and epitopes that contain conservative amino acid antigen - binding fragment for a peptide that contains the 45 substitutions relative to this sequence . Anti - TNFR2 poly KCSPG sequence ( SEQ ID NO : 12 ) of human TNFR2 . peptides ( e.g. , single - chain polypeptides, antibodies, and Polypeptides ( e.g. , single - chain polypeptides , antibodies, or antigen - binding fragments ) of the invention may also spe antigen -binding fragments thereof) that bind epitopes con- cifically bind an epitope within human TNFR2 that includes taining one or more residues of the KCRPG sequence at least five continuous or discontinuous residues from ( amino acids 142-146 of SEQ ID NO : 7 within human 50 positions 75-91 of SEQ ID NO : 7 within human TNFR2 TNFR2 ) and epitopes containing the KCSPG motif ( amino (CDSCEDSTYTQLWNWVP , SEQ ID NO : 14 ) , as well as acids 56-60 of SEQ ID NO : 7 within human TNFR2 ) with epitopes that exhibit at least 85 % sequence identity ( e.g. , similar affinity ( e.g. , less than a 10 - fold difference in affinity ) 85 % , 90 % , 95 % , 97 % , 99 % , or 100 % sequence identity ) to are not considered antagonistic TNFR2 polypeptides of the this sequence and epitopes that contain conservative amino invention . 55 acid substitutions relative to this sequence . In some embodi In addition to one or more residues of amino acids ments , anti - TNFR2 polypeptides ( e.g. , single - chain poly 142-146 of SEQ ID NO : 7 , polypeptides of the invention peptides, antibodies , and antigen - binding fragments ) of the may also bind one or more residues of a larger epitope that invention may specifically bind an epitope that includes includes at least five continuous or discontinuous residues residues at positions 80-86 of SEQ ID NO : 7 within human from positions 130-149 of SEQ ID NO : 7 within human 60 TNFR2 (DSTYTQL , SEQ ID NO : 8 ), as well as epitopes TNFR2 (KQEGCRLCAPLRKCRPGFGV , SEQ ID NO : that exhibit at least 85 % sequence identity ( e.g. , 85 % , 90 % , 17 ) , or an epitope that exhibits at least 85 % sequence 95 % , 97 % , 99 % , or 100 % sequence identity ) to this identity ( e.g. , 85 % , 90 % , 95 % , 97 % , 99 % , or 100 % sequence and epitopes that contain conservative amino acid sequence identity ) to this sequence and epitopes that contain substitutions relative to this sequence . Antagonistic TNFR2 conservative amino acid substitutions relative to this 65 polypeptides ( e.g. , single - chain polypeptides, antibodies, sequence. For example, antagonistic TNFR2 antibodies or and antigen - binding fragments ) of the invention also may antibody fragments of the invention may specifically bind an specifically bind an epitope that includes at least five con US 10,906,982 B2 45 46 tinuous or discontinuous residues from positions 86-103 of identity ) to this sequence and epitopes that contain conser SEQ ID NO : 7 within human TNFR2 (LWNWVPE- vative amino acid substitutions relative to this sequence . CLSCGSRCSSD , SEQ ID NO : 15 ) , as well as epitopes that Antagonistic TNFR2 polypeptides of the invention may bind exhibit at least 85 % sequence identity ( e.g. , 85 % , 90 % , 95 % , an epitope within amino acids 128-147 of SEQ ID NO : 7 97 % , 99 % , or 100 % sequence identity ) to this sequence and 5 ( LSKQEGCRLCAPLRKCRPGF ) , as well as epitopes that epitopes that contain conservative amino acid substitutions exhibit at least 85 % sequence identity ( e.g. , 85 % , 90 % , 95 % , relative to this sequence . In some embodiments, polypep tides ( e.g. , single - chain polypeptides, antibodies, and anti 97 % , 99 % , or 100 % sequence identity ) to this sequence and gen - binding fragments ) of the invention may specifically epitopes that contain conservative amino acid substitutions bind an epitope that includes residues from positions 91-98 10 relative to this sequence . Antagonistic TNFR2 polypeptides of SEQ ID NO : 7 within human TNFR2 ( PECLSCGS , SEQ of the invention may optionally bind an epitope within ID NO : 9 ) , as well as an epitope that exhibits at least 85 % amino acids 136-155 of SEQ ID NO : 7 ( LCA sequence identity ( e.g. , 85 % , 90 % , 95 % , 97 % , 99 % , or PLRKCRPGFGVARPGTE ), as well as epitopes that exhibit 100 % sequence identity ) to this sequence and epitopes that at least 85 % sequence identity ( e.g. , 85 % , 90 % , 95 % , 97 % , contain conservative amino acid substitutions relative to this 15 99 % , or 100 % sequence identity ) to this sequence and sequence . The polypeptides ( e.g. , single - chain polypeptides, epitopes that contain conservative amino acid substitutions antibodies , and antigen - binding fragments) of the invention relative to this sequence . may also specifically bind an epitope that include at least Antagonistic TNFR2 Polypeptides that Bind TNFR2 from five continuous or discontinuous residues from positions Non -Human Animals 111-128 of SEQ ID NO : 7 within human TNFR2 ( TREQN- 20 In addition to binding epitopes within human TFNR2 that RICTCRPGWYCAL , SEQ ID NO : 16 ) , as well as epitopes contain the KCRPG motif, antagonistic TNFR2 polypep that exhibit at least 85 % sequence identity ( e.g. , 85 % , 90 % , tides ( e.g. , single -chain polypeptides, antibodies, and anti 95 % , 97 % , 99 % , or 100 % sequence identity ) to this gen -binding fragments ) of the invention also include those sequence and epitopes that contain conservative amino acid that specifically bind epitopes containing the equivalent substitutions relative to this sequence. In some embodi- 25 motif within TNFR2 derived from non - human animals , such ments , polypeptides ( e.g. , single -chain polypeptides , anti as non -human mammals, e.g. , in a cow , bison , mouse , or rat, bodies , and antigen - binding fragments ) of the invention may among others . The location of sequences equivalent to the specifically bind an epitope that includes residues from human KCRPG motif in TNFR2 derived from exemplary positions 116-123 of SEQ ID NO : 7 within human TNFR2 non - human mammals is shown in Table 2 , below : ( RICTCRPG , SEQ ID NO : 10 ) , as well as epitopes that 30 exhibit at least 85 % sequence identity ( e.g. , 85 % , 90 % , 95 % , TABLE 2 97 % , 99 % , or 100 % sequence identity ) to this sequence and Location of sequences equivalent to KCRPG epitopes that contain conservative amino acid substitutions in TNFR2 from non - human mammals relative to this sequence . For example, antagonistic TNFR2 Amino acid SEQ ID Genbank single - chain polypeptides , antibodies, and antibody frag- 35 positions of NO . of Accession No. ments of the invention may specifically bind an epitope Sequence equivalent full - length of full -length containing residues 116-123 ofSEQ ID NO : 7 within human Source of equivalent sequence TNFR2 TNFR2 TNFR2 ( RICTCRPG , SEQ ID NO : 10 ) and residues 137 TNFR2 to KCRPG within TNFR2 sequence sequence 144 of SEQ ID NO : 7 within human TNFR2 ( CAPLRKCR , Human KCRPG 142-146 7 P20333.3 SEQ ID NO : 11 ). 40 Cattle KCGPG 142-146 280 AA105223 Antagonistic TNFR2 polypeptides of the invention may Bison KCGPG 142-146 281 XP 010848145 bind an epitope within amino acids 112-131 of SEQ ID NO : Mouse KCGPG 144-148 282 AAA39752.1 7 ( REQNRICTCRPGWYCALSKQ ) , as well as epitopes Rat KCGPG 144-148 283 Q8OWY6 that exhibit at least 85 % sequence identity ( e.g. , 85 % , 90 % , 95 % , 97 % , 99 % , or 100 % sequence identity ) to this 45 Epitopes within TNFR2 derived from the non -human sequence and epitopes that contain conservative amino acid mammals discussed above that may be bound by antago substitutions relative to this sequence . Additionally or alter- nistic TNFR2 polypeptides ( e.g. , single - chain polypeptides , natively, antagonistic TNFR2 single -chain polypeptides, antibodies, and antigen - binding fragments ) of the invention antibodies , or antigen -binding fragments thereof of the are summarized in the sequence alignment below . This invention may bind an epitope within amino acids 120-139 50 sequence alignment shows partial sequences of TNFR2 of SEQ ID NO : 7 (CRPGWYCALSKQEGCRLCAP ), as derived from human , cattle , bison , mouse , and rat, as well as well as epitopes that exhibit at least 85 % sequence identity epitopes ( highlighted in grey ) equivalent to the human ( e.g. , 85 % , 90 % , 95 % , 97 % , 99 % , or 100 % sequence KCRPG motif .
Alignment of partial TNFR2 sequences derived from human and select non - human mammals Human : 1 MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRL -- REYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSC 78 Cattle : 1 MAPTAFWAALAVGLQFWAAGRAVPAQAVFTPYIPEPGSSCRQ -- QEYYNQKIQMCCSKCPPGYRVQSLCNMTLDTICASC 78 Bison : 1 MAPTAFWAALAVGLQFWAAGRAVPAQAVFTPYIPEPGSSCRQ -- QEYYNHKIQMCCSKCPPGYRVQSLCNTTLDTICASC 78 Mouse : 1 MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQIS - QEYYDRKAQMCCAKCPPGQYVKHFCNKTSDTVCADC 79 Rat : 1 MAPAALWVALVVELQLWATGHTVPAKVVLTPYKPEPGNQCQIS - QEYYDKKAQMCCAKCPPGQYAKHFCNKTSDTVCADC 79
Human : 79 EDSTYTOLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEG - CRLCAPLRKCRPGFGVARPGTETS 157 Cattle : 79 ESSTYTQLWNLVTACFSCNSRCSSDQVETQACTTKQNRICTCKPGWYCTLGROEG - CRLCVALRKCGPGFGVAKPGTATT 157 Bison : 79 ESSTYTOLWNLVTACFSCNSRCSSDQVETQACTTKQNRICTCKPGWYCTLGRQEG - CRLCVALRKCGPGFGVAKPGTATT 157 Mouse : 80 EASMYTOVWNQFRTCLSCSSSCTTDQVEIRACTKOONRVCACEAGRYCALKTHSGSCRQCMRLSKCGPGFGVASSRAPNG 159 Rat : 80 AAGMFTQVWNHLHTCLSCSSSCSDDOVETHNCTKKONRVCACNADSYCALKLHSGNCRQCMKLSKCGPGFGVARSRTSNG 159 US 10,906,982 B2 47 48 - continued Human : 158 DVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCT- -STSPTRSMAPGAVHLPQPVSTRSQHTQPTP 231 Cattle : 158 NVICAPCGPGTFSDTTSYTDTCKPHRNCSSVAIPGTAS TDAVCT- --SVLPTRKVARG- --PATTRSQHMEPTL 225 Bison : 158 NVICAPCGPGTFSDTTSYTDTCKPHRNCSSVAIPGTAS TDAVCT------SVLPTRKVARG ------PATTRSQHMEPTL 225 Mouse : 160 NVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNAS TDAVCAPESPTLSAIPR ------TLYVSQPEPTRSQPLDQEP 233 Rat : 160 NVICSACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDAVCASESPTPSAVPR ------TIYVSQPEPTRSQPMDQEP 233 Human : 232 EPSTAPSTSFLLPMGPSPPA ---- EGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLOREAKVPHLPADK 307 Cattle : 226 GPSTAPSTFFLLPKVPSPPSSPVEQPNTGNISLPIELIVGVTALGLLLIVVVNCVIMTQKKKKPFCLQGDAKVPHLPANK 305 Bison : 226 GPSTAPSTFFLLPKVPSPPSSPVEQPNAGNISLPIELIVGVTALGLLLIVVVNCVIMTOKKKKPFCLQGDAKVPHLPANK 305 Mouse : 234 GPSQTPS --- ILTSLGSTPI -- IEQSTKGGISLPIGLIVGVTSLGLLMLGLVNCIILVORKKKPSCLQRDAKVPHVPDEK 308 Rat : 234 GPSQTPH --- IPVSLGSTPI -- IEPSITGGISLPIGLIVGLTTLGLLMLGLANCFILVQRKKKPSCLQRETMVPHLPDDK 308 Human : 308 ARGTOGPEQQHLLITAPSSSSSSLESSASALDRRAPTRNQPQAPGVE - ASGAGEARASTGSSDSSPGGHGTQVNVTCIVN 386 Cattle : 306 AQGAPGPEQQHLLTTAPSSSSSSLESSTSSTDKRAPTRSQLQS PGVEKASTSGEAQTGCSSSEASSGGHGTQVNVTCIVN 385 Bison : 306 AQGAPGPEQQHLLTTAPSSSSSSLESSTSSTDKRAPTRSQLQSPGVE - ANTSGEAQTGCSSSEASSGGHGTQVNVTCIVN 384 Mouse : 309 SQDAVGLEQQHLLTTAPSSSSSSLESSASAGDRRAPPGGHPQARVMAEAQGFQEARASSRISDSSHGSHGTHVNVTCIVN 388 Rat : 309 SQDAIGLEQQHLLTTAPSSSSSSLESSASAGDRRAPPGGHPQARVTAEAQGSQEACAGSRSSDSSHGSHGTHVNVTCIVN 388 The Antagonistic TNFR2 Antibody TNFRAB 1 In addition to binding an epitope contained within the A representative antagonistic TNFR2 polypeptide ( e.g. , sequence KCRPGFGV ( SEQ ID NO : 20 ) , TNFRAB1 also single - chain polypeptide , antibody, or antigen - binding frag- binds to a downstream epitope contained within a sequence ment thereof) of the invention can be based on TNFRAB1, 20 defined by positions 161-169 of SEQ ID NO : 7 within also referred to herein as TNFR2 antagonist 1 , which is a human TNFR2 ( CKPCAPGTF , SEQ ID NO : 21 ) . Anti murine antibody that antagonizes the TNFRA - TNFR2 inter- TNFR2 single - chain polypeptides , antibodies , and antibody action and is capable of suppressing TNFa - mediated T - reg fragments of the invention may also bind this epitope or a cell proliferation ( see , e.g. , FIG . 5 ) . The variable regions of larger region within TNFR2 containing this epitope ( e.g. , a TNFRAB1 ( e.g. , the heavy and light chain CDRs ) , and 25 sequence that includes at least five continuous or discon variants thereof that exhibit substantially similar specific tinuous residues from positions 150-190 of SEQ ID NO : 7 binding properties to TNFRAB1 , can be used to make an within human TNFR2 ( ARPGTETSDVVCKP antagonistic TNFR2 antibody or antigen - binding fragment CAPGTFSNTTSSTDICRPHQICNVVAI, SEQ ID NO : 22 ) . thereof of the invention, e.g. , by replacing the mouse con- 30 TNFRAB1 contains two heavy chains , as well as two light stant region of TNFRAB1 with a non - native constant region chains, as shown in FIGS . 1A and 1B . The heavy chains of ( e.g. , a constant region from a human antibody ) using TNFRAB1 contain the following amino acid sequence methods known in the art or described herein . ( CDRs are indicated in bold ) : Polypeptides ( e.g. , single - chain polypeptides , antibodies, and antigen - binding fragments) of the invention may exhibit 35 ( SEQ ID NO : 2 ) binding properties that are the same as or similar to those of EVQLQESGGGLVKPGGSLKLSCAASGFTFSSYVMSWVRQTPEKRLEWVAT TNFRAB1. These properties are as follows: In the presence ISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARQR of TNFR2, TNFRAB1 exhibits a high kon value of 4.98x106 VDGYSSYWYFDVWGAGTAVTVSS M - ls - 1 , as well as a low koff of 2.21x10-4 5-1 and a Ky of about 44.4 pM in complex with TNFR2 . The high affinity of 40 The sequence of the TNFRAB1 light chain is as TNFRAB1 for TNFR2 coupled with the rapid formation and follows ( CDRs are indicated in bold ) : the slow dissociation of the TNFRAB1 - TNFR2 complex is DIVLTOSPAIMSASPGEKVTITCSASSSVYYMYWFQQKPGTSPKLWIYST( SEQ ID NO : 4 ) consistent with the strong intermolecular contacts that SNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQRRNYPYTFGGG underlie this protein - protein interaction . TNFRAB1 binds to TKLEIKRA distinct epitopes within the primary structure of TNFR2 that 45 are spatially aligned in the native conformation of the The Antagonistic TNFR2 Antibody TNFRAB2 receptor. The KCRPGFGV motif ( SEQ ID NO : 20 ) , and A representative antagonistic TNFR2 polypeptide ( e.g. , specifically, the KCRPG sequence ( SEQ ID NO : 19 ) , has single - chain polypeptide, antibody, or antigen -binding frag been identified as a particularly important component of the ment thereof) of the invention can be based on TNFRAB2 , functional epitope that establishes strong intermolecular 50 also referred to herein as TNFR2 antagonist 2 , an antibody contacts with TNFRAB1 as determined by epitope mapping that selectively binds and inhibits TNFR2 by virtue of analysis (FIGS . 2 and 3 ) . The interaction of these residues specifically binding various epitopes within this receptor . with anti - TNFR2 polypeptides of the invention selectively For instance, antagonistic TNFR2 single - chain polypep promotes antagonistic activity . Significantly, a TNFR2 tides, antibodies, or antigen - binding fragments thereof of the epitope including amino acid residues 56-60 of SEQ ID NO : 55 invention may exhibit binding properties that are the same as 7 within human TNFR2 ( KCSPG , SEQ ID NO : 12 ) is or similar to those of TNFRAB2 . These properties are as distinctly not a part of the conformational epitope that is follows: In the presence of TNFR2 , TNFRAB2 exhibits a specifically bound by TNFRAB1 or antagonistic TNFR2 high kon value of 3.6099x109 M - ' s- , as well as a low koff polypeptides of the invention , as specific binding to both of of 2.24x10-4 5-1 and a K , of about 621 PM in complex with these epitopes has been shown to lead to a loss of, or 60 TNFR2 . An epitope containing residues 137-144 of SEQ ID significant reduction in , antagonistic activity . As such , NO : 7 within human TNFR2 ( CAPLRKCR , SEQ ID NO : TNFR2 polypeptides ( e.g. , single - chain polypeptides, anti- 11 ) has been identified as a particularly important compo bodies , and antigen - binding fragments) that specifically bind nent of the functional epitope that establishes strong inter both of these epitopes ( KCSPG and an epitope containing at molecular contacts with TNFRAB2 as determined by least the KCR sequence , and more specifically, the KCRPG 65 epitope mapping analysis ( see , e.g. , Example 1 and FIGS . sequence of human TNFR2) are not considered antagonistic 2B and 3B ) . Included in the invention are TNFR2 antibodies TNFR2 polypeptides of the invention . and antibody fragments that specifically bind this epitope . US 10,906,982 B2 49 50 In addition to binding an epitope containing residues and TNFRAB2 . An analysis of the residues common to the CAPLRKCR ( SEQ ID NO : 11 ) , TNFRAB2 also binds to CDRs of both antibodies provides insight into the molecular epitopes that include one or more residues within positions features of antibodies that bind TNFR2 and exhibit an 80-86 of SEQ ID NO : 7 within human TNFR2 ( DSTYTQL , antagonistic effect. Epitope mapping analysis has shown that SEQ ID NO : 8 ) , positions 91-98 of SEQ ID NO : 7 within 5 both TNFRAB1 and TNFRAB2 bind to TNFR2 in an human TNFR2 ( PECLSCGS , SEQ ID NO : 9 ) , as well as anti -parallel dimer configuration . These antibodies bind an positions 116-123 of SEQ ID NO : 7 within human TNFR2 epitope within TNFR2 that contains residues 142-146 of ( RICTCRPG , SEQ ID NO : 10 ) . TNFR2 single - chain poly- SEQ ID NO : 7 and do not bind epitopes containing residues peptides , antibodies , and antibody fragments of the inven- 56-60 of SEQ ID NO : 7. The structural similarities between tion may also bind one or more of these epitopes . Polypep- 10 corresponding CDR - H and CDR - L regions provide a basis tides of the invention can be designed and identified using for predicting residue substitutions that may preserve or the knowledge of the epitopes specifically bound by enhance TNFR2 affinity and antagonism . Inspection of the TNFRAB2 . For instance , one can use any of a variety of in heavy chain and light chain CDR sequences of these anti vitro peptide display techniques or combinatorial antibody library screens as described herein or known in the art in 15 bodies demonstrates that several residues and physico order to screen for polypeptides ( e.g. , single - chain polypep chemical characteristics are conserved within corresponding tides , antibodies, and antigen - binding fragments thereof) CDR sequences, while other positions within these CDRs capable of binding these epitopes with high affinity and can be varied significantly without loss of affinity and selectivity antagonistic function . For instance , the CDR - H1 sequences In addition , TNFR2 polypeptides of the invention include 20 of TNFRAB1 and TNFRAB2 are shown below : those that contain one or more of the heavy and light chain CDRs or heavy and light chain variable regions of TNFRAB2 and a constant region that is non - native to that of GFTFSSY ( TNFRAB1 CDR - H1 , SEQ ID NO : 23 ) TNFRAB2 ( e.g. , a constant region from a human antibody) . GYTFTDY ( L / I ) ( TNFRAB2 CDR - H1 , SEQ ID NO : 257 ) The heavy chain and light chain CDRs of TNFRAB2 are 25 shown below : G - TF -- Y ( Consensus sequence ) Alignment of the sequences reveals a shared GXTFXXY TNFRAB2 CDR - H1 : motif, wherein “ X ” designates any amino acid . These CDR ( SEQ ID NO : 257 ) 30 H1 sequences feature a conserved glycine residue at the first GYTFTDY ( L / I ) position and conserved threonine, phenylalanine, and tyro TNFRAB2 CDR - H2 : sine residues at the third , fourth , and seventh positions , ( SEQ ID NO : 258 ) VDPEYGST respectively. Inspection of these sequences demonstrates 35 that the CDR - H1 region is tolerant of substitutions at the TNFRAB2 CDR - H3 : remaining positions . Side - chains of varying polarity are ( SEQ ID NO : 259 ) tolerated at the second position , for example, as both phe ARDDGSYSPFDYWG nylalanine, containing an unsubstituted and hydrophobic TNFRAB2 CDR - L1 : phenyl moiety, and tyrosine , containing a protic hydroxyl ( SEQ ID NO : 260 ) 40 substituent, are found in this position in the CDR - H1 region ONINKY of TNFRAB1 and TNFRAB2, respectively . Additionally, TNFRAB2 CDR - L2 : while the fifth and sixth positions are occupied by polar TYS serine residues in the CDR -H1 of TNFRAB1, these posi or tions feature a threonine , containing an additional hydro YTS 45 phobic methyl substituent, and aspartic acid , which is TNFRAB2 CDR - L3 : anionic at physiological pH , in TNFRAB2 . This diversity ( SEQ ID NO : 261 ) demonstrates that these positions can be substituted with CLQYVNL ( L / I ) T amino acids of diverse electrostatic properties without loss of TNFR2 affinity and antagonism . As shown above, the CDR - H1 sequence of TNFRAB2 50 may contain either a leucine or isoleucine residue at the Sequence analysis of the CDR - H2 regions of TNFRAB1 eighth position of this region . Similarly, the TNFRAB2 and TNFRAB2 similarly reveals a set of conserved amino CDR - L2 may include a TYS or YTS tripeptide, and the acids at various positions throughout these regions: TNFRAB2 CDR - L3 may contain either a leucine or isoleu cine residue at the eighth position of this region . Notably, the 55 SSG -- GSY CDR - L2 of TNFRAB2 is flanked by the N - terminal frame ( TNFRAB1 CDR - H2 , SEQ ID NO : 24 ) work residues LLIR ( SEQ ID NO : 262 ) and the C -terminal VDPEYGST ( TNFRAB2 CDR - H2 , SEQ ID NO : 258 ) framework residues TLE . Accordingly , antagonistic TNFR2 antibodies or antigen -binding fragments thereof of the GS ( Consensus sequence ) invention include those that contain one or more of the 60 above CDRs of TNFRAB2, as well as N - terminal LLIR Analysis of this sequence alignment demonstrates that the ( SEQ ID NO : 262 ) and C - terminal TLE residues that flank CDR - H2 sequences exhibit a conserved GS motif at the the CDR - L2 sequence of the antagonistic TNFR2 antibody C - terminal end of the CDR - H2 region, with side - chains of or antigen -binding fragment thereof . variable molecular size , polarity, and electrostatic charge Molecular Determinants of TNFR2 Affinity and Antagonism 65 tolerated at the remaining positions . Additionally , alignment Notably, there are distinct sequence similarities between of the corresponding CDR - H3 sequences of TNFRAB1 and the CDRs of the antagonistic TNFR2 antibodies TNFRAB1 TNFRAB2 is shown below : US 10,906,982 B2 51 52 cationic side - chains ( Arg ), conformationally restricted side QRVDGYSSYWYFDV ( TNFRAB1 CDR - H3 , SEQ ID NO : 25 ) chains ( Pro ) , and side - chains of varying polarity ( e.g. , Gin , Asn , Leu , and Val) . Collectively, the shared structural fea ARDDG - S - YSPFDYWG ( TNFRAB2 CDR - H3 , SEQ ID NO : 259 ) tures of the above CDR - H and CDR - L sequences provide -R - DG - S - Y -- FD --- ( Consensus sequence ) 5 insight into those residues that are important for selectively binding one or more residues of the KCRPG epitope of A similar analysis of the CDR - H3 sequences of TNFR2 ( positions 142-146 of SEQ ID NO : 7 , shown in SEQ TNFRAB1 and TNFRAB2 reveals conserved arginine, ID NO : 19 ) in an anti- parallel dimer configuration and aspartic acid , glycine, serine, tyrosine, and phenylalanine 10 demonstrateretaining affinity that andcertain antagonistic amino acids activity can. be varied while residues throughout this CDR . Notably , residues of varying Antagonistic TNFR2 polypeptides of the invention ( e.g. , steric and electrostatic properties are tolerated in the remain single - chain polypeptides , antibodies , or antigen -binding ing positions . For instance, the first position of the CDR - H3 fragments thereof) may therefore have heavy chain and light sequence tolerates amino acid residues of contrasting size chain CDRs that contain the above consensus sequences . and hydrogen bond - forming tendencies , as the first position 15 For instance , TNFR2 antagonists of the invention may have of CDR - H3 in TNFRAB1 features a polar glutamine residue a CDR - H1 having the amino acid sequence 24JZZ ( I) , Z containing a carboxamide side - chain with hydrogen bond or Z4JZºZ ( 1 ), ZJ ; a CDR - H2 having the amino acid donor and acceptor moieties, while an alanine residue bear- sequence ( J ) 3Z4ZJ or ( J ) ; z47 ) ; a CDR - H3 having the ing an unfunctionalized methyl side - chain is found at the amino acid sequence JZ JZ2Z4JZ JZ ( J) ,ZZ zor corresponding position in TNFRAB2. Additionally, the third 20 JZ + JZZ4ZZ ( ) ? ZZ ( ) 2; a CDR - Li having the amino position in the above CDR - H3 sequences features a hydro- acid sequence (J ) .Z or ( I) ; Z ; a CDR - L2 having the amino phobic valine in TNFRAB1 and an anionic aspartic acid acid sequence (J ) .Z or ( J )2Z "; and a CDR - L3 having the moiety in the corresponding position of TNFRAB2. amino acid sequence (J ) 3Z ( 1) ,Zor (J ) , Z (1 ) Z ; wherein A similar analysis reveals molecular features common to each J is independently a naturally occurring amino acid ; the CDR - L sequences of TNFRAB1 and TNFRAB2 . For 25 each Z ' is independently a naturally occurring amino acid instance, the CDR - L1 sequences of TNFRAB1 and containing a cationic side - chain at physiological pH ; each Z2 TNFRAB2 are shown below : is independently a naturally occurring amino acid containing an anionic side - chain at physiological pH ; each Z is inde pendently a naturally occurring amino acid containing a SASSSVYYMY ( TNFRAB1 CDR - L1 , SEQ ID NO : 26 ) 30 polar, uncharged side -chain at physiological pH ; each Z is independently a glycine or alanine ; and each Z is indepen Q - N -- INK - Y ( TNFRAB2 CDR - L1 , SEQ ID NO : 260 ) dently a naturally occurring amino acid containing a hydro Y ( Consensus residue ) phobic side -chain . In some embodiments , antagonistic TNFR2 polypeptides 35 of the invention ( e.g. , single - chain polypeptides, antibodies, Inspection of these sequences reveals that a hydroxyl or antigen - binding fragments thereof) may have a CDR - H1 containing tyrosine residue is featured at the final position of having the amino acid sequence GJTF ( J ) 2Y ( SEQ ID NO : CDR - L1 , while residues of varying physicochemical prop 276 ) or GJTF ( J ) 2YJ ( SEQ ID NO : 277 ) ; a CDR - H2 having erties are tolerated at the remaining positions . Similarly, the amino acid sequence ( J ) 3GSJ or ( I ) ; GSJ ; a CDR - H3 TNFRAB2analysis of revealsthe CDR a conserved - L2 regions amino of acidTNFRAB1 at the finaland 40 having the amino acid sequence JRJDGJSJY ( J) FDJ ( SEQ ID NO : 278 ) or JRJDGSY ( J) FD ( I ) , ( SEQ ID NO : 279 ) ; a position in both regions: CDR - L1 having the amino acid sequence ( I ) , Y or ( J ) Y ; a CDR - L2 having the amino acid sequence ( J ) .S or ( I ) 2S ; and STSNLAS a CDR - L3 having the amino acid sequence ( J ) ; Y ( J ) 2T or ( TNFRAB1 CDR - L2 , SEQ ID NO : 27 ) 45 ( J ) 3 Y ( J ) 4T ; wherein each J is independently a naturally TY ---- S or ( TNFRAB2 CDR - L2 ) occurring amino acid . YT ---- S Antagonistic TNFR2 polypeptides of the invention ( e.g. , S single - chain polypeptides, antibodies , or antigen - binding ( Consensus residue ) fragments thereof) may have a CDR - H1 having the amino 50 acid sequence Z4FZZSSZ or Z4YZZTDZX ; a CDR Analysis of the above sequence alignment demonstrates H2 having the amino acid sequence SSGZAZY ( SEQ ID that serine residues are featured at the third position of these NO : 263 ) or VDPEYZAZT ( SEQ ID NO : 264 ) ; a CDR - H3 CDR - L2 sequences , while substitutions are widely tolerated having the amino acid sequence at the remaining residues. Similarly, the CDR - L3 sequences OZ VZ2Z4YZ SZ WYZZZ ( SEQ ID NO : 265 ) or of TNRAB1 and TNFRAB2 are as follows : 55 AZ'DZ Z4ZZ SPZZPZ WG ( SEQ ID NO : 266 ) ; a CDR L1 having the amino acid sequence SASSSVYYMZ ( SEQ -T ID NO : 267 ) or QNINKZ ( SEQ ID NO : 268 ) ; a CDR - L2 Q - QRRNYPY ( TNFRAB1 CDR - L3 , SEQ ID NO : 28 ) having the amino acid sequence STSNLAZ ( SEQ ID NO : CLQ --- YVNL ( L / I ) T ( TNFRAB2 CDR - L3 , SEQ ID NO : 261 ) 269 ) , TYZ , or YTZ ?; and a CDR - L3 having the amino acid 60 sequence QQRRNZ PYZ ( SEQ ID NO : 270 ) or Y -T ( Consensus sequence ) CLQZ - VNLXZ ( SEQ ID NO : 271 ) ; wherein each Z ' is independently an amino acid containing a cationic side Analysis of the CDR - L3 sequences of TNFRAB1 and chain at physiological pH ; each Z is independently an TNFRAB2 reveals a preference for tyrosine and threonine amino acid containing an anionic side -chain at physiological residues at distinct positions within these regions , while 65 pH ; each Zº is independently an amino acid containing a amino acids of a wide range of physicochemical character- polar, uncharged side - chain at physiological pH ; each Z * is istics are tolerated at other positions , including residues with independently a glycine or alanine ; each Z is independently US 10,906,982 B2 53 54 an amino acid containing a hydrophobic side - chain ; and of an antagonistic TNFR2 antibody, such as TNFRAB1 or each X is independently leucine or isoleucine. TNFRAB2 , with the heavy chain variable region and light In some embodiments , antagonistic TNFR2 polypeptides chain variable region of a consensus human antibody. Con of the invention ( e.g. , single - chain polypeptides, antibodies, sensus human antibody heavy chain and light chain or antigen - binding fragments thereof) may have a CDR - H1 5 sequences are known in the art ( see e.g. , the “ VBASE ” having the amino acid sequence GFTFSSY ( SEQ ID NO : human germline sequence database ; see also Kabat, et al . , 23 ) , GYTFTDYX ( SEQ ID NO : 257 ) , or an amino acid Sequences of Proteins of Immunological Interest, Fifth sequence having up to two amino acid substitutions relative Edition , U.S. Department of Health and Human Services , to these sequences ; a CDR - H2 having the amino acid NIH Publication No. 91-3242 , 1991 ; Tomlinson et al . , J. sequence SSGGSY ( SEQ ID NO : 24 ) , VDPEYGST ( SEQ 10 Mol . Biol . 227 : 776-98 , 1992 ; and Cox et al , Eur. J. Immu ID NO : 258 ) , or an amino acid sequence having up to two nol . 24 : 827-836 , 1994 ; incorporated herein by reference ). In amino acid substitutions relative to these sequences ; a this way, the variable domain framework residues and CDRs CDR - H3 having the amino acid sequence can be identified by sequence alignment ( see Kabat , supra ) . QRVDGYSSYWYFDV ( SEQ ID NO : 25 ) , ARDDGSYS- One can substitute one or more CDRs of the heavy chain PFDYWG ( SEQ ID NO : 259 ) , or an amino acid sequence 15 and / or light chain variable domains of consensus human having up to two amino acid substitutions relative to these antibody with one or more corresponding CDRs of an sequences ; a CDR - L1 having the amino acid sequence antagonistic TNFR2 antibody, such as TNFRAB1 or SASSSVYYMY ( SEQ ID NO : 26 ) , QNINKY ( SEQ ID NO : TNFRAB2 , in order to produce a humanized TNFR2 anti 260 ) , or an amino acid sequence having up to two amino body. Exemplary variable domains of a consensus human acid substitutions relative to these sequences ; a CDR - L2 20 antibody include the heavy chain variable domain : having the amino acid sequence STSNLAS ( SEQ ID NO : EVOLVESGGGLVOPGGSLRLSCAASGFTFSDYAM 27 ) , TYS , YTS , or an amino acid sequence having up to two SWVRQAPGKGLEWVAVISENGSDTYYADSVKGR amino acid substitutions relative to SEQ ID NO : 27 ; and a FTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRG CDR - L3 having the amino acid sequence QQRRNYPYT GAVSYFDVWGQGTLVTVSS ( SEQ ID NO : 32 ) and the ( SEQ ID NO : 28 ) , CLQYVNLXT ( SEQ ID NO : 261 ) , or an 25 light chain variable domain : DIQMTQSPSSL amino acid sequence having up to two amino acid substi- SASVGDRVTITCRASQDVSSY tutions relative to these sequences . LAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGT Humanized , Primatized , and Chimeric Antibodies Derived DFTLTISSLQPEDFATYYCQQYNSLPY from TNFRAB1 and / or TNFRAB2 TFGQGTKVEIKRT ( SEQ ID NO : 33 ) identified in U.S. Antibodies of the invention include human , humanized , 30 Pat . No. 6,054,297 ; incorporated herein by reference ( CDRs primatized , and chimeric antibodies that contain one or more are shown in bold were determined according to the method of the CDRs of TNFRAB1, or a CDR that exhibits at least of Chothia , et al . , J. Mol . Biol , 196 : 901-917 , 1987 ) . These 85 % sequence identity ( e.g. , 90 % , 95 % , 97 % , 99 % , or 100 % amino acid substitutions can be de, for example , by sequence identity ) to any of these CDRs or sequences that recombinant expression of polynucleotides encoding the contain conservative mutations relative to these CDRs . 35 heavy and light chains of a humanized antibody in a host cell Antibodies of the invention also include human , humanized , using methods known in the art or described herein . primatized , and chimeric antibodies that contain one or more Similarly , this strategy can also be used to produce of the CDRs of TNFRAB2 , or a CDR that exhibits at least primatized anti - TNFR2 antibodies, as one can substitute the 85 % sequence identity ( e.g. , 90 % , 95 % , 97 % , 99 % , or 100 % CDRs of the heavy and / or light chain variable domains of a sequence identity ) to any of these CDRs or sequences that 40 primate antibody consensus sequence with one or more contain conservative mutations relative to these CDRs . For corresponding CDRs of TNFRAB1 and / or TNFRAB2. Con instance, antibodies of the invention also include human , sensus primate antibody sequences known in the art ( see humanized , primatized , and chimeric antibodies that contain e.g. , U.S. Pat . Nos . 5,658,570 ; 5,681,722 ; and 5,693,780 ; one or more CDRs that are identical to the above CDRs incorporated herein by reference ) . except for conservative amino acid substitutions. In some 45 In some embodiments, it may be desirable to import embodiments, a humanized , primatized , or chimeric anti- particular framework residues in addition to CDR sequences body may contain one or more of the CDRs of TNFRABI , from a TNFR2 antibody, such as TNFRAB1 or TNFRAB2 , or a CDR that exhibits at least 85 % sequence identity ( e.g. , into the heavy and / or light chain variable domains of a 90 % , 95 % , 97 % , 99 % , or 100 % sequence identity ) to any of human antibody. For instance , U.S. Pat . No. 6,054,297 the CDRs of TNFRAB1 or sequences that contain conser- 50 identifies several instances when it may be advantageous to vative mutations relative to these CDRs , and one or more retain certain framework residues from a particular antibody CDRs of TNFRAB2, or a CDR that exhibits at least 85 % heavy chain or light chain variable region in the resulting sequence identity ( e.g. , 90 % , 95 % , 97 % , 99 % , or 100 % humanized antibody. In some embodiments, framework sequence identity ) to any of the CDRs of TNFRAB2 or residues may engage in non - covalent interactions with the sequences that contain conservative mutations relative to 55 antigen and thus contribute to the affinity of the antibody for these CDRs . For example , antagonistic TNFR2 antibodies of the target antigen. In other cases , individual framework the invention can be generated by incorporating any of the residues may modulate the conformation of a CDR , and thus above CDRs into the framework regions ( e.g. , FW1 , FW2 , indirectly influence the interaction of the antibody with the FW3 , and FW4 ) of a human antibody. Exemplary frame- antigen . Alternatively, certain framework residues may form work regions that can be used for the development of a 60 the interface between VH and VL domains , and may there humanized anti - TNFR2 antibody containing one or more of fore contribute to the global antibody structure . In other the above CDRs include , without limitation , those described cases , framework residues may constitute functional glyco in U.S. Pat. Nos . 7,732,578 , 8,093,068 , and WO 2003 / sylation sites ( e.g. , Asn - X - Ser / Thr) which may dictate anti 105782 ; incorporated herein by reference . body structure and antigen affinity upon attachment to One strategy that can be used to design humanized 65 carbohydrate moieties. In cases such as those described antibodies of the invention is to align the sequences of the above , it may be beneficial to retain certain framework heavy chain variable region and light chain variable region residues of a TNFR2 antibody ( e.g. , TNFRAB1 or US 10,906,982 B2 55 56 TNFRAB2 ) in the antagonistic antibodies and antigen- known in the art, such as a scFv fragment. Single chain binding fragments thereof of the invention , such as human- polypeptides may alternatively contain one or more CDRs ized antibodies , as various framework residues may promote described herein covalently bound to one another using high epitope affinity and improved biochemical activity of conventional bond - forming techniques known in the art, for the antibody or antigen -binding fragment thereof. 5 instance , by an amide bond, a thioether bond , a carbon Antibodies of the invention also include antibody frag carbon bond , or by a linker, such as a peptide linker or a ments, Fab domains, F (ab ') molecules , F ( ab ' ) , molecules , multi- valent electrophile (e.g. , a bis ( bromomethyl ) arene single - chain variable fragments ( scFvs ) , tandem scFv frag derivative , such as a bis ( bromomethyl )benzene or bis (bro ments , diabodies , triabodies , dual variable domain immu momethyl )pyridine ) described herein or known in the art . noglobulins , multi- specific antibodies, bispecific antibodies , 10 For instance , antagonistic TNFR2 single - chain polypep and heterospecific antibodies that contain one or more of the tides of the invention may have one or more heavy chain and CDRs of TNFRAB1 or TNFRAB2 ( e.g. , a CDR containing light chain CDRs that contain the above - described consen the amino acid sequence of any one of SEQ ID NOs : 23-28 ) sus sequences that promote selective binding to TNFR2 or90 %a ,CDR 95 % ,that 97% exhibits , 99 % , orat 100least % 85 sequence % sequence identity identity ) to any ( e.g. of , 15 epitopes , such as the KCRPG motif ( SEQ ID NO: 19 ) , and these CDRs . Antibodies and antigen - binding fragments induce TNFR2 antagonism . For instance , antagonistic thereof of the invention include those that also contain TNFR2 single - chain polypeptides of the invention may have CDRs having between one and three amino acid substitu a CDR - H1 having the amino acid sequence Z4JZZ (1 ) , Z tions ( e.g. , conservative or nonconservative substitutions) or Z4JZZ ( J ) 2ZJ; a CDR - H2 having the amino acid relative to the CDR sequences of TNFRAB1 or TNFRAB2. 20 sequence ( J) 3Z4ZJ or ( I) ; Z4ZJ; a CDR - H3 having the These molecules can be expressed recombinantly, e.g. , by amino acid sequence JZ'JZ ZAJZJZ ( I ), Z Zºzor incorporating polynucleotides encoding these proteins into JZ JZPZ4ZZ (J )2ZzZ ( I ) 2; a CDR - L1 having the amino expression vectors for transfection in a eukaryotic or pro- acid sequence ( J) , Z or ( J ) 3Z ; a CDR - L2 having the amino karyotic cell using techniques described herein or known in acid sequence ( J) .Z or ( J) , Z ?, and / or a CDR -L3 having the the art, or synthesized chemically, e.g. , by solid -phase 25 amino acid sequence ( J) 3Z (J ).Z or (J ) 3Z ( J ) .Zº ; wherein peptide synthesis methods described herein or known in the each J is independently a naturally occurring amino acid ; art . each Z is independently a naturally occurring amino acid Antibodies of the invention additionally include antibody- containing a cationic side - chain at physiological pH ; each Z ? like scaffolds that contain one or more of the CDRs of is independently a naturally occurring amino acid containing TNFRAB1 or TNFRAB2 , or a CDR that exhibits at least 30 an anionic side - chain at physiological pH ; each Zº is inde 85 % sequence identity ( e.g. , 90 % , 95 % , 97 % , 99 % , or 100 % pendently a naturally occurring amino acid containing a sequence identity ) to any of these CDRs or sequences that polar, uncharged side - chain at physiological pH ; each 2 * is contain between one and three amino acid substitutions independently a glycine or anine ; and each Z® is indepen ( e.g. , conservative or nonconservative substitutions) relative dently a naturally occurring amino acid containing a hydro to the CDR sequences of TNFRAB1 or TFNRAB2. 35 phobic side - chain . Examples of antibody -like scaffolds include proteins that In some embodiments, antagonistic TNFR2 single - chain contain a tenth fibronectin type III domain ( ° Fn3 ) , which polypeptides of the invention may have a CDR - H1 having contains BC , DE , and FG structural loops analogous to the amino acid sequence GJTF ( J ) 2Y ( SEQ ID NO : 276 ) or canonical antibodies. It has been shown that the tertiary GJTF ( I) YJ ( SEQ ID NO : 277 ) ; a CDR - H2 having the structure of the 1 ° Fn3 domain resembles that of the variable 40 amino acid sequence ( I ) 2GSJ or ( I ) , GSJ ; a CDR - H3 having region of the IgG heavy chain , and one of skill in the art can the amino acid sequence JRJDGJSJY ( J ) 2FDJ ( SEQ ID NO : graft, e.g. , one or more CDRs of TNFRAB1 and / or 278 ) or JRJDGSY ( 1 ) FD ( I ) , ( SEQ ID NO : 279 ) ; a CDR - L1 TNFRAB2, or sequences having least 85 % sequence having the amino acid sequence ( I ) , Y or ( I) sY; a CDR - L2 identity ( e.g. , 90 % , 95 % , 97 % , 99 % , or 100 % sequence having the amino acid sequence (1 ) S or ( J ) 2S ; and /or a identity ) to any of these CDRs or sequences containing 45 CDR - L3 having the amino acid sequence ( J ) , Y ( J ) 2T or conserved amino acid substitutions relative to these CDRs ( J ) 3Y ( J ) 4T ; wherein each J is independently a naturally onto the fibronectin scaffold by replacing residues of the BC , occurring amino acid . DE , and FG loops of 1° Fn3 with residues of TNFRABI Antagonistic TNFR2 single - chain polypeptides of the and / or TNFRAB2 CDRs. This can be achieved by recom- invention may have a CDR - H1 having the amino acid binant expression of a modified 1 ° Fn3 domain in a prokary- 50 sequence ZFZZ SSZ or Z4YZZSTDZX; a CDR - H2 otic or eukaryotic cell ( e.g. , using the vectors and techniques having the amino acid sequence SSGZZY ( SEQ ID NO : described herein ). Examples of using the lºFn3 domain as an 263 ) or VDPEYZAZT ( SEQ ID NO : 264 ) ; a CDR - H3 antibody - like scaffold for the grafting of CDRs from anti- having the amino acid sequence bodies onto the BC , DE , and FG structural loops are reported OZ VZ2Z4YZ SZ WYZZZ ( SEQ ID NO : 265 ) or in WO 2000/034784 , WO 2009/142773 , WO 2012/088006 , 55 AZ DZ²Z4ZZ SPZZZWG ( SEQ ID NO : 266 ) ; a CDR and U.S. Pat . No. 8,278,419 ; incorporated herein by refer- L1 having the amino acid sequence SASSSVYYMZ ( SEQ ence . ID NO : 267 ) or QNINKZ ( SEQ ID NO : 268 ) ; a CDR - L2 Antagonistic TNFR2 Single - Chain Polypeptides having the amino acid sequence STSNLAZ ( SEQ ID NO : TNFR2 antagonists of the invention may be in the form of 269 ) , TYZ , or YTZ ?; and / or a CDR - L3 having the amino a single -chain polypeptide, such as a single - chain polypep- 60 acid sequence QQRRNZ PYZ ( SEQ ID NO : 270 ) or tide that contains one, two , or three heavy chain CDRs of a CLQZSVNLXZ ( SEQ ID NO : 271 ) ; wherein each 2 is monoclonal TNFR2 antagonist antibody described herein independently an amino acid containing a cationic side ( e.g. , TNFRAB1 or TNFRAB2 ), and / or one , two , or three chain at physiological pH ; each Zº is independently an light chain CDRs of a monoclonal TNFR2 antagonist anti- amino acid containing an anionic side -chain at physiological body described herein ( e.g. , TNFRAB1 or TNFRAB2) . 65 pH ; each Z is independently an amino acid containing a Single -chain polypeptides may be in the form of an antibody polar, uncharged side - chain at physiological pH ; each Z * is fragment, e.g. , an antibody fragment described herein or independently a glycine or alanine ; each Z is independently US 10,906,982 B2 57 58 an amino acid containing a hydrophobic side - chain ; and introduce the vectors into host cells , such as those described each X is independently leucine or isoleucine . in Molecular Cloning; A Laboratory Manual, Second Edi In some embodiments, antagonistic TNFR2 single - chain tion ( Sambrook , Fritsch and Maniatis ( eds ) , Cold Spring polypeptides of the invention may have a CDR - H1 having Harbor, N.Y., 1989 ) , Current Protocols in Molecular Biol the amino acid sequence GFTFSSY ( SEQ ID NO : 23 ) , 5 ogy (Ausubel et al . , eds . , Greene Publishing Associates, GYTFTDYX ( SEQ ID NO : 257 ) , or an amino acid sequence 1989 ) , and in U.S. Pat . No. 4,816,397 ; incorporated herein having up to two amino acid substitutions relative to these by reference. sequences ; a CDR - H2 having the amino acid sequence Vectors for Expression of Antagonistic TNFR2 Polypeptides SSGGSY ( SEQ ID NO : 24 ) , VDPEYGST ( SEQ ID NO : Viral genomes provide a rich source of vectors that can be 258 ) , or an amino acid sequence having up to two amino 10 used for the efficient delivery of exogenous genes into the acid substitutions relative to these sequences; a CDR - H3 genome of a cell ( e.g. , a eukaryotic or prokaryotic cell ) . having the amino acid sequence QRVDGYSSYWYFDV Viral genomes are particularly useful vectors for gene deliv ( SEQ ID NO : 25 ) , ARDDGSYSPFDYWG ( SEQ ID NO : ery because the polynucleotides contained within such 259 ) , or an amino acid sequence having up to two amino genomes are typically incorporated into the genome of a acid substitutions relative to these sequences; a CDR -L1 15 target cell by generalized or specialized transduction. These having the amino acid sequence SASSSVYYMY ( SEQ ID processes occur as part of the natural viral replication cycle , NO : 26 ) , QNINKY ( SEQ ID NO : 260 ) , or an amino acid and do not require added proteins or reagents in order to sequence having up to two amino acid substitutions relative induce gene integration . Examples of viral vectors include a to these sequences ; a CDR - L2 having the amino acid retrovirus, adenovirus ( e.g. , Ad5 , Ad26 , Ad34 , Ad35 , and sequence STSNLAS ( SEQ ID NO : 27 ) , TYS , YTS , or an 20 Ad48 ) , parvovirus ( e.g. , adeno - associated viruses ), corona amino acid sequence having up to two amino acid substi- virus, negative strand RNA viruses such as orthomyxovirus tutions relative to SEQ ID NO : 27 ; and /or a CDR - L3 having ( e.g. , influenza virus ), rhabdovirus ( e.g. , rabies and vesicular the amino acid sequence QQRRNYPYT ( SEQ ID NO : 28 ) , stomatitis virus ), paramyxovirus ( e.g. measles and Sendai ), CLQYVNLXT ( SEQ ID NO : 261 ) , or an amino acid positive strand RNA viruses , such as picornavirus and sequence having up to two amino acid substitutions relative 25 alphavirus, and double stranded DNA viruses including to these sequences . adenovirus, herpesvirus ( e.g. , Herpes Simplex virus types 1 Single - chain polypeptides can be produced by a variety of and 2 , Epstein -Barr virus, cytomegalovirus ), and poxvirus recombinant and synthetic techniques, such as by recombi- ( e.g. , vaccinia , modified vaccinia Ankara (MVA ), fowlpox nant gene expression or solid -phase peptide synthesis pro- and canarypox ). Other viruses useful for delivering poly cedures described herein or known in the art. For instance , 30 nucleotides encoding antibody light and heavy chains or one of skill in the art can design polynucleotides encoding, antibody fragments of the invention include Norwalk virus , e.g. , two or more of the above CDRs operably linked to one togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, another in frame so as to produce a continuous , single - chain and hepatitis virus, for example. Examples of retroviruses peptide containing these CDRs . Optionally, the CDRs may include: avian leukosis - sarcoma, mammalian C -type , B - type be separated by a spacer, such as by a framework region 35 viruses, D - type viruses, HTLV -BLV group , lentivirus, spu ( e.g. , a framework sequence described herein or a frame- mavirus ( Coffin , J. M. , Retroviridae : The viruses and their work region of a germline consensus sequence of a human replication , In Fundamental Virology , Third Edition , B. N. antibody ) or a flexible linker, such as a poly - glycine or Fields , et al . , Eds . , Lippincott- Raven Publishers, Philadel glycine / serine linker described herein or known in the art. phia, 1996 ) . Other examples include murine leukemia When produced by chemical synthesis methods, native 40 viruses, murine sarcoma viruses, mouse mammary tumor chemical ligation can optionally be used as a strategy for the virus, bovine leukemia virus, feline leukemia virus , feline synthesis of long peptides ( e.g. , greater than 50 amino sarcoma virus, avian leukemia virus, human T - cell leukemia acids ) . Native chemical ligation protocols are known in the virus, baboon endogenous virus, Gibbon ape leukemia virus, art and have been described , e.g. , by Dawson et al . ( Science , Mason Pfizer monkey virus, simian immunodeficiency 266 : 776-779 , 1994 ) ; incorporated herein by reference. A 45 virus, simian sarcoma virus, Rous sarcoma virus and lenti detailed description of techniques for the production of viruses . Other examples of vectors are described , for single - chain polypeptides, full - length antibodies , and anti- example , in McVey et al . , ( U.S. Pat . No. 5,801,030 ) ; incor body fragments is provided in the sections that follow . porated herein by reference . Nucleic Acids and Expression Systems Genome Editing Techniques Antagonistic INFR2 polypeptides ( e.g. , single - chain 50 In addition to viral vectors , a variety of additional meth polypeptides , antibodies , or antigen - binding fragments ods have been developed for the incorporation of genes, e.g. , thereof) of the invention can be prepared by any of a variety those encoding antibody light and heavy chains, single of established techniques. For instance , an antagonistic chain polypeptides, single -chain variable fragments ( scFvs ) , TNFR2 polypeptides ( e.g., single -chain polypeptides , anti- tandem scFvs , Fab domains, F ( ab ' ) , domains , diabodies , and bodies , or antigen - binding fragments thereof) of the inven- 55 triabodies, among others, into the genomes of target cells for tion can be prepared by recombinant expression of one or polypeptide expression. One such method that can be used more immunoglobulin light and heavy chain genes in a host for incorporating polynucleotides encoding anti - TNFR2 cell . For instance , to express an antibody recombinantly, a polypeptides ( e.g. , single - chain polypeptides, antibodies, or host cell can be transfected with one or more recombinant antigen - binding fragments thereof) into prokaryotic or expression vectors carrying DNA fragments encoding the 60 eukaryotic cells includes transposons. Transposons are poly immunoglobulin light and heavy chains of the antibody such nucleotides that encode transposase enzymes and contain a that the light and heavy chains are expressed in the host cell polynucleotide sequence or gene of interest flanked by and , optionally , secreted into the medium in which the host excision sites at the 5 ' and 3 ' positions . Once a transposon cells are cultured , from which medium the antibodies can be has been delivered into a cell , expression of the transposase recovered . Standard recombinant DNA methodologies are 65 gene commences and results in active enzymes that cleave used to obtain antibody heavy and light chain genes , incor- the gene of interest from the transposon . This activity is porate these genes into recombinant expression vectors and mediated by the site -specific recognition of transposon exci US 10,906,982 B2 59 60 sion sites by the transposase . In some embodiments, these invention into the genome of a prokaryotic or eukaryotic cell excision sites may be terminal repeats or inverted terminal include the use of ARCUSTM meganucleases that can be repeats . Once excised from the transposon , the gene of rationally designed so as to site -specifically cleave genomic interest can be integrated into the genome of a prokaryotic DNA . The use of these enzymes for the incorporation of or eukaryotic cell by transposase - catalyzed cleavage of 5 polynucleotides encoding antagonistic TNFR2 polypeptides similar excision sites that exist within nuclear genome of the ( e.g. , single - chain polypeptides , antibodies, or antibody cell . This allows the gene encoding , e.g. , an anti - TNFR2 fragments ) of the invention into the genome of a prokaryotic antibody or fragment or domain thereof to be inserted into or eukaryotic cell is particularly advantageous in view of the the cleaved nuclear DNA at the excision sites , and subse- structure -activity relationships that have been established quent ligation of the phosphodiester bonds that join the gene 10 for such enzymes. Single - chain meganucleases can thus be of interest to the DNA of the prokaryotic or eukaryotic cell modified at certain amino acid positions in order to create genome completes the incorporation process . In some nucleases that selectively cleave DNA at desired locations . embodiments, the transposon may be a retrotransposon, such These single -chain nucleases have been described exten that the gene encoding the antibody is first transcribed to an sively, e.g. , in U.S. Pat . Nos . 8,021,867 and 8,445,251 ; RNA product and then reverse - transcribed to DNA before 15 incorporated herein by reference. incorporation in the prokaryotic or eukaryotic cell genome. Polynucleotide Sequence Elements Exemplary transposon systems include the piggybac trans- To express antagonistic TNFR2 polypeptides ( e.g. , single poson ( described in detail in WO 2010/085699 ) and the chain polypeptides, antibodies, or antibody fragments sleeping beauty transposon ( described in detail in thereof) of the invention , polynucleotides encoding partial US20050112764 ) ; incorporated herein by reference . 20 or full- length light and heavy chains , or CDRs thereof, e.g. , Another useful method for the integration of nucleic acid obtained as described above , can be inserted into expression molecules encoding anti - TNFR2 polypeptides ( e.g. , single- vectors such that the genes are operatively linked to tran chain polypeptides , antibodies, or antigen - binding frag- scriptional and translational control sequences . The expres ments thereof) into the genome of a prokaryotic or eukary- sion vector and expression control sequences are chosen to otic cell is the clustered regularly interspaced short 25 be compatible with the expression host cell used . Polynucle palindromic repeats ( CRISPR ) / Cas system , which is a sys- otides encoding , e.g. , the light chain gene and the heavy tem that originally evolved as an adaptive defense mecha- chain of a TNFR2 antibody can be inserted into separate nism in bacteria and archaea against infection by viruses. vectors, or , optionally, both polynucleotides can be incor The CRISPR /Cas system consists of palindromic repeat porated into the same expression vector using established sequences within plasmid DNA and an associated Cas9 30 techniques described herein or known in the art. nuclease . This ensemble of DNA and protein directs site In addition to polynucleotides encoding the heavy and specific DNA cleavage of a target sequence by first incor- light chains of an antibody ( or a polynucleotide encoding a porating foreign DNA into CRISPR loci . Polynucleotides single - chain polypeptide or an antibody fragment, such as a containing these foreign sequences and the repeat -spacer scFv molecule ) , the recombinant expression vectors of the elements of the CRISPR locus are in turn transcribed in a 35 invention may carry regulatory sequences that control the host cell to create a guide RNA , which can subsequently expression of the antibody chain genes in a host cell . The anneal to a target sequence and localize the Cas9 nuclease to design of the expression vector , including the selection of this site . In this manner , highly site -specific cas9 - mediated regulatory sequences , may depend on such factors as the DNA cleavage can be engendered in a foreign polynucle- choice of the host cell to be transformed or the level of otide because the interaction that brings cas9 within close 40 expression of protein desired . For instance , suitable regula proximity of the target DNA molecule is governed by tory sequences for mammalian host cell expression include RNA : DNA hybridization . As a result, one can theoretically viral elements that direct high levels of protein expression in design a CRISPR /Cas system to cleave any target DNA mammalian cells , such as promoters and / or enhancers molecule of interest. This technique has been exploited in derived from cytomegalovirus ( CMV ) ( such as the CMV order to edit eukaryotic genomes ( Hwang et al . , Nat . Bio- 45 promoter / enhancer ), Simian Virus 40 ( SV40 ) ( such as the tech . , 31 : 227-229 , 2013 ) and can be used as an efficient SV40 promoter / enhancer ), adenovirus, ( e.g. , the adenovirus means of site - specifically editing eukaryotic or prokaryotic major late promoter ( AdMLP )) and polyoma . For further genomes in order to cleave DNA prior to the incorporation description of viral regulatory elements , and sequences of a polynucleotide encoding an anti - TNFR2 polypeptide of thereof, see e.g. , U.S. Pat . Nos . 5,168,062 , 4,510,245 , and the invention . The use of CRISPR / Cas to modulate gene 50 4,968,615 . expression has been described in U.S. Pat . No. 8,697,359 , In addition to the antibody chain or CDR genes and which is incorporated herein by reference . regulatory sequences , the recombinant expression vectors of Alternative methods for site - specifically cleaving the invention can carry additional sequences , such as genomic DNA prior to the incorporation of a polynucleotide sequences that regulate replication of the vector in host cells encoding a TNFR2 antibody or antibody fragment of the 55 ( e.g. , origins of replication ) and selectable marker genes . A invention include the use of zinc finger nucleases and selectable marker gene facilitates selection of host cells into transcription activator - like effector nucleases ( TALENs ) . which the vector has been introduced ( see e.g. , U.S. Pat. Unlike the CRISPR / Cas system , these enzymes do not Nos . 4,399,216 , 4,634,665 and 5,179,017 ) . For example, contain a guiding polynucleotide to localize to a specific typically the selectable marker gene confers resistance to target sequence . Target specificity is instead controlled by 60 cytotoxic drugs, such as G418 , puromycin, blasticidin , DNA binding domains within these enzymes . Zinc finger hygromycin or methotrexate , to a host cell into which the nucleases and TALENs for use in genome editing applica- vector has been introduced . Suitable selectable marker genes tions are described in Urnov et al . (Nat . Rev. Genet ., include the dihydrofolate reductase ( DHFR ) gene ( for use in 11 : 636-646 , 2010 ) ; and in Joung et al . , (Nat . Rev. Mol . Cell . DHFR host cells with methotrexate selection / amplification ) Bio . 14 : 49-55 , 2013 ) ; incorporated herein by reference . 65 and the neo gene ( for G418 selection ) . In order to express the Additional genome editing techniques that can be used to light and heavy chains of a TNFR2 antibody or a TNFR2 incorporate polynucleotides encoding antibodies of the antibody fragment, the expression vector ( s ) containing poly US 10,906,982 B2 61 62 nucleotides encoding the heavy and light chains can be sized and used as a template for mutagenesis to generate a transfected into a host cell by standard techniques. variant as described herein using routine mutagenesis tech Polynucleotides Encoding Modified Antagonistic TNFR2 niques. Alternatively, a DNA fragment encoding the variant Polypeptides can be directly synthesized ( e.g. , by established solid -phase In some embodiments, antagonistic TNFR2 polypeptides 5 nucleic acid chemical synthesis procedures ). ( e.g. , single - chain polypeptides, antibodies, or antibody Once DNA fragments encoding TNFRAB1, TNFRAB2, fragments of the invention ) may be similar to TNFRAB1 or TNFRAB1- related , or TFNRAB2 - related CDRs or VH and TNFRAB2 but feature differences in the sequence of one or VL segments are obtained , these DNA fragments can be more CDRs . In other cases , the polypeptides of the invention further manipulated by standard recombinant DNA tech may be similar to TNFRAB1 or TNFRAB2 but feature 10 niques, e.g. , to convert the variable region genes to full differences in one or more framework regions. For instance , length antibody chain genes , to Fab fragment genes or to a one or more framework regions of TNFRAB1 or TNFRAB2 scFv gene. In these manipulations, a VL - or VH -encoding may be substituted with the framework region of a human DNA fragment is operatively linked to another DNA frag antibody. Exemplary framework regions include , for ment encoding another protein , such as an antibody constant example, human framework regions described in U.S. Pat . 15 region or a flexible linker. No. 7,829,086 , and primate framework regions as described The isolated DNA encoding the Vh region of an anti in EP 1945668 ; incorporated herein by reference . Alterna- TNFR2 antibody of the invention can be converted to a tively , polypeptides ( e.g. , single - chain polypeptides, anti- full - length heavy chain gene ( as well as a Fab heavy chain bodies, or antigen - binding fragments thereof) of the inven- gene ) , e.g. , by operatively linking the Vy - encoding DNA to tion may be similar to TNFRAB1 or TNFRAB2 but exhibit 20 another DNA molecule encoding heavy chain constant differences in the sequence of one or more CDRs and region domains ( CH1 , CH2 , CH3 , and , optionally , CH4 ) . differences in one or more framework regions . To generate The sequences of human heavy chain constant region genes nucleic acids encoding such TNFR2 antagonist polypep- are known in the art ( see e.g. , Kabat et al . , Sequences of tides , DNA fragments encoding , e.g. , one or more CDRs , or Proteins of Immunological Interest, Fifth Edition , U.S. at least one , or both , of the light chain variable regions and 25 Department of Health and Human Services, NIH Publication the heavy chain variable regions can be produced by chemi- No. 91-3242 , 1991 ) and DNA fragments encompassing cal synthesis ( e.g. , by solid - phase polynucleotide synthesis these regions can be obtained by standard PCR amplifica techniques ), in vitro gene amplification ( e.g. , by polymerase tion . The heavy chain constant region can be an IgG1 , IgG2 , chain reaction techniques ), or by replication of the poly- IgG3 , IgG4 , IgA , IgE , IgM or IgD constant region , and in nucleotide in a host organism . For instance, nucleic acids 30 certain embodiments is an IgG1 constant region . For a Fab encoding anti - TNFR2 polypeptides of the invention may be fragment heavy chain gene , the VH - encoding DNA can be obtained by amplification and modification of germline operatively linked to another DNA molecule encoding only DNA or cDNA encoding light and heavy chain variable the heavy chain CH1 domain . sequences so as to incorporate the CDRs of TNFRAB1 or The isolated DNA encoding the VL region of an anti TNFRAB2 into the framework residues of a consensus 35 TNFR2 polypeptide ( e.g. , single - chain polypeptide, anti antibody. In some embodiments, a humanized antagonistic body , or antigen - binding fragment thereof) of the invention TNFR2 antibody may include one or more CDRs of TFN- can be converted to a full -length light chain gene ( as well as RAB1 , or a variant thereof that has at least 85 % sequence a Fab light chain gene ) by operatively linking the VL identity ( e.g. , 90 % , 95 % , 97 % , 99 % , or 100 % sequence encoding DNA to another DNA molecule encoding the light identity ) to any of these CDRs or sequences that contain 40 chain constant region , CL . The sequences of human light between one and three amino acid substitutions ( e.g. , con- chain constant region genes are known in the art ( see e.g. , servative or nonconservative substitutions) relative to the Kabat et al . , Sequences of Proteins of Immunological Inter CDR sequences of TNFRAB1, and one or more CDRs of est , Fifth Edition ( U.S. Department of Health and Human TNFRAB2, or a variant thereof that has at least 85 % Services , NIH Publication No. 91-3242 , 1991 ) ) and DNA sequence identity ( e.g. , 90 % , 95 % , 97 % , 99 % , or 100 % 45 fragments encompassing these regions can be obtained , e.g. , sequence identity ) to any of these CDRs or sequences that by amplification in a prokaryotic or eukaryotic cell of a contain between one and three amino acid substitutions polynucleotide encoding these regions, by PCR amplifica ( e.g. , conservative or nonconservative substitutions) relative tion , or by chemical polynucleotide synthesis . The light to the CDR sequences of TFNRAB2. This can be achieved , chain constant region can be a kappa ( K ) or lambda ( a ) for example , by performing site - directed mutagenesis of 50 constant region , but in certain embodiments is a kappa germline DNA or cDNA and amplifying the resulting poly- constant region . To create a scFv gene , the VH and VL nucleotides using the polymerase chain reaction ( PCR ) encoding DNA fragments are operatively linked to another according to established procedures. Germline DNA fragment encoding a flexible linker, e.g. , a polynucleotide sequences for human heavy and light chain variable region encoding a flexible , hydrophilic amino acid sequence , such genes are known in the art ( see , e.g. , the “ VBASE ” human 55 as the amino acid sequence (Gly Ser )z , such that the Vh and germline sequence database ; see also Kabat et al . , Sequences V sequences can be expressed as a contiguous single - chain of Proteins of Immunological Interest, Fifth Edition , U.S. protein , with the V , and Vð regions joined by the linker ( see Department of Health and Human Services, NIH Publication e.g. , Bird et al . , Science 242 : 423-426 , 1988 ; Huston et al . , No. 91-3242 , 1991 ; Tomlinson et al . , J. Mol . Biol . 227 : 776- Proc . Natl. Acad . Sci . USA 85 : 5879-5883 , 1988 ; McCafferty 798 , 1992 ; and Cox et al . , Eur. J. Immunol. 24 : 827-836 , 60 et al . , Nature 348 : 552-554 , 1990 ) . 1994 ; incorporated herein by reference ). Chimeric nucleic Recombinant DNA technology can also be used to acid constructs encoding human heavy and light chain remove some or all of the DNA encoding either or both of variable regions containing one or more of the CDRs of the light and heavy chains that is not necessary for binding TNFRAB1 and TNFRAB2 can be produced , e.g. , using to TNFR2 . The molecules expressed from such truncated established cloning techniques known in the art. Addition- 65 DNA molecules are also encompassed by the polypeptides ally , a polynucleotide encoding the heavy or light chain of the invention . In addition , bifunctional polypeptides ( e.g. , variable region of TNFRAB1 or TFNRAB2 can be synthe- bifunctional antibodies) can be produced in which one heavy US 10,906,982 B2 63 64 and one light chain are derived from TNFRAB1 and /or Polypeptides ( e.g., single -chain polypeptides, antibodies , TNFRAB2 and the other heavy and light chain are specific and antigen -binding fragments thereof) can be recovered for an antigen other than TNFR2 . Such antibodies can be from the culture medium using standard protein purification generated, e.g. , by crosslinking a heavy chain and light chain methods. Host cells can also be used to produce portions of of TNFRAB1 and / or TNFRAB2 to a heavy chain and light 5 intact antibodies , such as Fab fragments or scFv molecules . chain of a second antibody by standard chemical crosslink The invention also includes methods in which the above ing methods ( e.g. , by disulfide bond formation ). Bifunc procedure is varied according to established protocols known in the art. For example , it can be desirable to transfect tional antibodies can also be made by expressing a nucleic a host cell with DNA encoding either the light chain or the acid molecule engineered to encode a bifunctional antibody 10 heavy chain ( but not both ) of an anti - TNFR2 antibody of this in a prokaryotic or eukaryotic cell . invention in order to produce an antigen -binding fragment of Dual specific antibodies, i.e. , antibodies that bind TNFR2 the antibody. and a different antigen using the same binding site , can be Once an anti - TNFR2 polypeptide ( e.g. , single -chain poly produced by mutating amino acid residues in the light chain peptide , antibody , or antigen - binding fragment) thereof of and / or heavy chain CDRs . In some embodiments, dual 15 the invention has been produced by recombinant expression , specific antibodies that bind two antigens, such as TNFR2 it can be purified by any method known in the art , such as and a second cell - surface receptor, can be produced by a method useful for purification of an immunoglobulin mutating amino acid residues in the periphery of the antigen molecule , for example , by chromatography ( e.g. , ion binding site (Bostrom et al . , Science 323 : 1610-1614 , 2009 ) . exchange, affinity, particularly by affinity for TNFR2 after Dual functional antibodies can be made by expressing a 20 Protein A or Protein G selection , and sizing column chro polynucleotide engineered to encode a dual specific anti- matography ), centrifugation , differential solubility, or by body. any other standard technique for the purification of proteins. Modified antagonistic TNFR2 antibodies and antibody Further , the anti - TNFR2 polypeptides ( e.g. , single -chain fragments of the invention can also be produced by chemical polypeptides, antibodies, and antigen - binding fragments synthesis ( e.g. , by the methods described in Solid Phase 25 thereof) of the invention can be fused to heterologous Peptide Synthesis, 2nd ed . , 1984 The Pierce Chemical Co. , polypeptide sequences described herein or otherwise known Rockford , 111 ; incorporated herein by reference ). Variant in the art to facilitate purification or to produce therapeutic antibodies can also be generated using a cell - free synthetic conjugates ( see " Antibody conjugates, " below ). platform ( see , e.g. , Chu et al . , Biochemia No. 2 , 2001 Once isolated , an anti - TNFR2 antibody or antigen -bind ( Roche Molecular Biologicals ) ; incorporated herein by ref- 30 ing fragments thereof can , if desired , be further purified , erence ). e.g. , by high performance liquid chromatography ( see , e.g. , Host Cells for Expression of Antagonistic TNFR2 Polypep- Fisher, Laboratory Techniques in Biochemistry and Molecu tides lar Biology ( Work and Burd S., 1980 ); incor It is possible to express the polypeptides ( e.g. , single- porated herein by reference ), or by gel filtration chromatog chain polypeptides , antibodies, and antigen - binding frag- 35 raphy, such as on a SuperdexTM 75 column ( Pharmacia ments thereof) of the invention in either prokaryotic or Biotech AB , Uppsala, Sweden ). eukaryotic host cells . In some embodiments , expression of Platforms for Generating and Affinity -Maturing Antagonis polypeptides ( e.g. , single - chain polypeptides , antibodies, or tic Anti - TNFR2 Polypeptides antigen -binding fragments thereof ) is performed in eukary- Mapping Epitopes of TNFR2 that Promote Receptor otic cells , e.g. , mammalian host cells , for optimal secretion 40 Antagonism of a properly folded and immunologically active antibody . Antagonistic TNFR2 polypeptides ( e.g. , single -chain Exemplary mammalian host cells for expressing the recom- polypeptides, antibodies and antigen - binding fragments binant polypeptides ( e.g. , single - chain polypeptides, anti- thereof) of the invention can be produced by screening bodies , or antigen - binding fragments thereof) of the inven- libraries of polypeptides ( e.g. , single - chain polypeptides, tion include Chinese Hamster Ovary ( CHO cells ) ( including 45 antibodies , and antigen - binding fragments thereof) for func DHFR CHO cells , described in Urlaub and Chasin ( 1980 , tional molecules that are capable of binding epitopes within Proc . Natl . Acad . Sci . USA 77 : 4216-4220 ) , used with a TNFR2 that selectively promote receptor antagonism rather DHFR selectable marker, e.g. , as described in Kaufman and than receptor activation . Linear peptides isolated from the Sharp ( 1982 , Mol . Biol . 159 : 601-621 ) , NSO myeloma cells , TNFR2 protein may not adopt the same three dimensional COS cells , 293 cells , and SP2 / 0 cells . Additional cell types 50 conformations as those peptide sequences located within the that may be useful for the expression of single - chain poly- protein . TNFR2 provides a structurally rigidified framework peptides, antibodies, and fragments thereof include bacterial that biases the conformations of individual peptide frag cells , such as BL - 21 ( DE3 ) E. coli cells , which can be ments and reinforces these spatial orientations by establish transformed with vectors containing foreign DNA according ing intramolecular contacts ( e.g. , hydrogen bonds, dipole to established protocols. Additional eukaryotic cells that 55 dipole interactions, salt bridges) and by differentially may be useful for expression of polypeptides include yeast positioning various regions for exposure to solvent depend cells , such as auxotrophic strains of S. cerevisiae , which can ing on the relative hydrophilicity and lipophilicity of these be transformed and selectively grown in incomplete media areas (Mukai et al . , Sci . Signal ., 3 : ra83 - ra83, 2010 ) . The according to established procedures known in the art . When conformational constraint of a peptide fragment within recombinant expression vectors encoding antibody genes 60 TNFR2 can be achieved by incorporating the amino acid ( e.g. , genes encoding one or more CDRs , an antibody heavy residues of a TNFR2 epitope ( e.g. , an epitope that promotes chain , or an antibody light chain ) are introduced into mam- receptor antagonism ) into a structurally pre - organized pep malian host cells , the antibodies are produced by culturing tide scaffold , such as a cyclic , bicyclic , tricyclic, or tetrayclic the host cells for a period of time sufficient to allow for peptide . Cyclic and polycyclic peptides such as these con expression of the antibody in the host cells or secretion of 65 fine a peptide fragment to a distinct three - dimensional the antibody into the culture medium in which the host cells conformation . This can be achieved by synthesizing peptide are grown . epitopes isolated from TNFR2 by established chemical US 10,906,982 B2 65 66 synthetic methods ( e.g. , by solid - phase peptide synthesis as preferentially bind TNFRAB1 and /or TNFRAB2 and may described herein ) and incorporating cysteine residues into structurally pre- organize these residues such that they the sequence at the N- and C - terminal positions or at various resemble the conformations of the corresponding peptide in internal positions within the peptide chain . It may be advan- the native protein . Cyclic and polycyclic peptides thus tageous to incorporate cysteine residues that are chemically 5 obtained ( e.g. , peptides having the sequence of any one of protected at the thiol moiety with a protecting group that can SEQ ID NOs : 11 , 19 , 20 , and 34-117 , or a peptide containing be removed under conditions different from those used to between about 10 and about 30 continuous or discontinuous remove other protecting groups within the peptide being amino acids between positions 80 and 130 of SEQ ID NO : synthesized and different from those used to assemble the 7 ) can be used to screen libraries of antibodies and antigen peptide chain . Exemplary orthogonal protecting groups for 10 binding fragments thereof in order to identify anti - TNFR2 the cysteine thiol include the 4 -methyltrityl group and polypeptides of the invention . Moreover, since these con 4 -methoxtrityl group , each of which can be removed using strained peptides act as surrogates for epitopes within dilute trifluoracetic acid ( examples are described , e.g. , in TNFR2 that promote receptor antagonism , polypeptides Isidro - Llobet et al . , Chem Rev. , 109 : 2455-2504 , 2009 ) . ( e.g., single - chain polypeptides, antibodies, and antigen After introducing a cysteine residue into a synthetic 15 binding fragments thereof) generated using this screening peptide fragment derived from an epitope within TNFR2, technique may bind the corresponding epitopes in TNFR2 the peptide can be cyclized by treating the peptide with a and are expected to be antagonistic of receptor activity . multivalent electrophile, such as a bis (bromomethyl ) or Screening of Libraries for Antagonistic TNFR2 Polypep tris (bromomethyl )arene derivative . Alternative multivalent tides thiol - reactive electrophiles can be used , e.g. , 1,5 - difluoro- 20 Methods for high throughput screening of polypeptide 2,4 - dinitrobenzene, acyclic dibromoalkanes, and others ( see , ( e.g. , single - chain polypeptide, antibody , or antibody frag e.g. , Jo et al . , J. Am . Chem . Soc . , 134 : 17704-17713 , 2012 ; ment) libraries for molecules capable of binding epitopes incorporated herein by reference ). In some embodiments, it within TNFR2 ( e.g. , epitopes presented by peptides having may be advantageous to prevent the participation of a the sequence of any one of SEQ ID NOs : 11 , 19 , 20 , and cysteine residue in the synthetic peptide fragment in a 25 34-117 , or a peptide containing between about 10 and about cyclization reaction . For instance , it may be desirable to 30 continuous or discontinuous amino acids between posi synthesize a polycyclic peptide containing multiple cysteine tions 80 and 130 of SEQ ID NO : 7 ) include , without residues such that only select cysteine thiols participate in limitation , display techniques including phage display , bac the intramolecular crosslinking process. To prevent terial display, yeast display, mammalian display, ribosome unwanted participation of these additional Cys thiol groups 30 display, mRNA display, and cDNA display . The use of phage in the coupling reaction , a simple approach is for instance to display to isolate ligands that bind biologically relevant use Fmoc -Cys ( Acm ) (Fmoc - acetamidomethyl- L - cysteine) molecules has been reviewed , e.g. , in Felici et al . (Biotech for introduction of a protected Cys residue during the course nol . Annual Rev. 1 : 149-18 1995 ) , Katz ( Annual Rev. of peptide synthesis . Alternatively, Fmoc - Cys ( StBu ) -OH Biophys. Biomol . Struct . 26 : 27-45 , 1997 ) , and Hoogenboom can be used , and / or the corresponding t- butyloxycarbonyl 35 et al. ( Immunotechnology 4 : 1-20 , 1998 ) . Several random ( Boc ) -protected amino acids . The Acm or StBu group is not ized combinatorial peptide libraries have been constructed to removed during the course of a normal TFA deprotection- select for polypeptides that bind different targets, e.g. , cell cleavage reaction but requires oxidative ( e.g. , iodine , 12 ) surface receptors or DNA ( reviewed by Kay ( Perspect . Drug treatment in case of Acm group , or reductive treatment ( e.g. , Discovery Des . 2 , 251-268 , 1995 ) , Kay et al . , ( Mol . Divers . 3 -mercaptoethanol ( excess ) or 1,4 - dithiothreiotol ( excess )) 40 1 : 139-140 , 1996 ) ) . Proteins and multimeric proteins have in case of the StBu group to give the reduced sulfhydryl been successfully phage -displayed as functional molecules form of the peptide, which can either be used directly or ( see EP 0349578A , EP 4527839A , EP 0589877A ; Chiswell subsequently oxidized to the corresponding cystinyl peptide. and McCafferty ( Trends Biotechnol. 10 , 80-84 1992 ) ) . In In one embodiment, a peptide is used which contains at least addition , functional antibody fragments ( e.g. Fab , single one Cys derivative , such as Cys ( Acm ) or Cys ( StBu ) , to 45 chain Fv [ scFv ] ) have been expressed (McCafferty et al . allow selective deprotection of a Cys - thiol group . Selective ( Nature 348 : 552-554 , 1990 ) , Barbas et al . ( Proc. Natl. Acad deprotection of a Cys - thiol group renders the Cys - thiol Sci . USA 88 : 7978-7982 , 1991 ) , Clackson et al . ( Nature group available for reacting at a desired moment, such as 352 : 624-628 , 1991 ) ) . These references are hereby incorpo following completion of peptide chain assembly and prior to rated by reference in their entirety . the deprotection of other residues within the peptide ( see , 50 ( i ) Phage Display Techniques e.g. , WO 2008/013454 ; incorporated herein by reference ). As an example, phage display techniques can be used in As an example , libraries of cyclic and polycyclic peptides order to screen libraries of polypeptides , such as single containing individual fragments isolated from TNFR2 and chain polypeptides, antibodies, and antigen -binding frag combinations of fragments from distinct regions of TNFR2 ments thereof, for functional molecules capable of binding can be synthesized by techniques such as those described 55 cyclic or polycyclic peptides containing epitopes within above in order to incorporate cysteine residues at various TNFR2 that promote receptor antagonism ( e.g. , peptides positions within the peptide scaffold and using different having the sequence of any one of SEQ ID NOs : 11 , 19 , 20 , electrophilic crosslinking reagents ( see , e.g. , Example 1 and and 34-117 , and particularly those that contain the KCRPG FIG . 3 , SEQ ID NOs : 34-117 ) . These peptides can be motif, as in SEQ ID NOs : 42 , 50 , 52-54 , and 61-63 ) . For immobilized on a solid surface and screened for molecules 60 instance, libraries of polynucleotides encoding single - chain that bind antagonistic TNFR2 polypeptides ( e.g. , single- antibody fragments, such as scFv fragments, that contain chain polypeptides , antibodies , or antigen - binding frag- randomized hypervariable regions can be obtained using ments thereof) such as TNFRAB1 or TNFRAB2, using an established procedures ( e.g., solid -phase polynucleotide ELISA - based screening platform using established proce- synthesis or error - prone PCR techniques, see McCullum et dures. Using this assay , for instance , peptides that specifi- 65 al . (Meth . Mol . Biol . , 634 : 103-109 , 2010 ) ; incorporated cally bind TNFRAB1 and / or TNFRAB2 with high affinity herein by reference ). These randomized polynucleotides can therefore contain residues within epitopes of TNFR2 that subsequently be incorporated into a viral genome such that US 10,906,982 B2 67 68 the randomized antibody chains encoded by these genes are scFv fragments, tandem scFv fragments, and other antigen expressed on the surface of filamentous phage , e.g. , by a binding fragments of the invention that can be used as covalent bond between the antibody chain and a coat protein antagonists of TNFR2 . Exemplary phage display protocols ( e.g. , pill coat protein on the surface of M13 phage ). This for the identification of antibody chains and antigen -binding provides a physical connection between the genotype and 5 fragments thereof that bind a particular antigen with high phenotype of the antibody chain . In this way , libraries of affinity are well - established and are described , e.g. , in U.S. phage that display diverse antibody chains containing ran- Pat. No. 7,846,892 , WO 1997/002342 , U.S. Pat. No. 8,846 , dom mutations in hypervariable regions can be screened for 867 , and WO 2007/132917 ; incorporated herein by refer the ability of the exterior antibody chains to bind TNFR2 ence . Similar phage display techniques can be used to epitopes ( e.g. , peptides having the sequence of any one of 10 generate antibody - like scaffolds ( e.g. , 1 ° Fn3 domains ) of the SEQ ID NOs : 11 , 19 , 20 , and 34-117 , or a peptide containing invention that bind epitopes within TNFR2 that promote between about 10 and about 30 continuous or discontinuous receptor antagonism ( e.g. , epitopes presented by peptides amino acids between positions 80 and 130 of SEQ ID NO : with the sequence of any one of SEQ ID NOs : 11 , 19 , 20 , 7 ) that are immobilized to a surface using established and 34-117 , and particularly those that contain the KCRPG procedures. For instance , cyclic peptides such as those 15 motif, as in SEQ ID NOs : 42 , 50 , 52-54 , and 61-63 ) . represented by SEQ ID NOs : 42 , 50 , 52-54 , and 61-63 , Exemplary phage display protocols for the identification of which contain the KCRPG motif, can be physically bound to antibody - like scaffold proteins are described , e.g. , in WO the surface of a microtiter plate by forming a covalent bond 2009/086116 ; incorporated herein by reference ). between the peptide and an epitope tag ( e.g. , biotin ) and ( ii ) Cell - Based Display Techniques incubating the peptide in wells of a microtiter plate that have 20 Other in vitro display techniques that exploit the linkage been previously coated with a complementary tag ( e.g. , between genotype and phenotype of a solvent - exposed poly avidin ) that binds the tag attached to the peptide with high peptide ( e.g. , single -chain polypeptide, antibody, or antigen affinity. Suitable epitope tags include , without limitation , binding fragment thereof) include yeast and bacterial dis maltose - binding protein , glutathione - S - transferase, a poly- play . Yeast display techniques are established in the art and histidine tag , a FLAG -tag , a myc - tag, human influenza 25 are often advantageous in that high quantities of antibodies hemagglutinin ( HA ) tag , biotin , streptavidin . Peptides con- ( often up to 30,000 ) can be presented on the surface of an taining the epitopes presented by these molecules are individual yeast cell ( see , e.g. , Boder et al . ( Nat Biotechno . capable of being immobilized on surfaces containing such 15 : 553 , 1997 ) ; incorporated herein by reference ). The larger complementary molecules as maltose , glutathione, a nickel- size of yeast cells over filamentous phage enables an addi containing complex , an anti - FLAG antibody, an anti- myc 30 tional screening strategy, as one can use flow cytometry to antibody , an anti - HA antibody, streptavidin , or biotin , both analyze and sort libraries of yeast . For instance , estab respectively . In this way , phage can be incubated with a lished procedures can be used to generate libraries of surface containing an immobilized TNFR2 - derived peptide bacterial cells or yeast cells that express polypeptides, such for a time suitable to allow binding of the antibody to the as single - chain polypeptides, antibodies, or antibody frag constrained peptide and in the presence of an appropriate 35 ments, containing randomized hypervariable regions ( see , buffer system ( e.g. , one that contains physiological salt e.g. , see U.S. Pat . No. 7,749,501 and US 2013/0085072 ; the concentration , ionic strength , and is maintained at physi- teachings of each which are incorporated herein by refer ological pH by a buffering agent ). The surface can then be ence ). For instance , large libraries of yeast cells that express washed ( e.g. , with phosphate buffer containing 0.1 % Tween- polynucleotides encoding naïve scFv fragments can be made 20 ) so as to remove phage that do not present antibody 40 using established procedures ( de Bruin et al . , Nat Biotechnol chains that interact with the TNFR2 -derived peptides with 17 : 397 , 1999 ; incorporated herein by reference ). Yeast cells an affinity greater than a particular threshold value. expressing these polynucleotides can then be incubated with The affinity of the polypeptides that remain after this two different fluorescent molecules during the panning initial panning ( i.e. , screening ) step can be modulated by steps : one dye that binds conserved residues within the adjusting the conditions of the washing step ( e.g. , by includ- 45 antibody and thus reflects the amount of antibody displayed , ing mildly acidic or basic components, or by including other and another dye that fluoresces at a different wavelength and TNFR2 -derived peptides at a low concentration in order to binds the antigen and thus indicates the amount of antigen compete with immobilized peptides for antigen -binding bound . In these cases , it is useful to use a cyclic or polycyclic sites ) . In this way , the population of phage that remains peptide containing the sequence of any one of SEQ ID NOs : bound to the surfaces of the microtiter plate following the 50 11 , 19 , 20 , and 34-117 ( and particularly those that contain washing step is enriched for phage that bind TNFR2 -derived the KCRPG motif, as in SEQ ID NOs : 42 , 50 , 52-54 , and peptide epitopes that promote receptor antagonism . The 61-63 ) that has been conjugated to an epitope tag ( e.g. , remaining phage can then be amplified by eluting the phage biotin ), optionally at a residue that is not expected to from the surface containing these peptides ( e.g. , by altering interfere with antibody - antigen binding . This enables a the ambient pH , ionic strength , or temperature) so as to 55 fluorescent dye labeled with a complementary tag ( e.g. , diminish protein - protein interaction strength . The isolated avidin ) to localize to the antibody -antigen complex . This phage can then be amplified , e.g. , by infecting bacterial results in great flexibility and immediate feedback on the cells , and the resulting phage can optionally be subjected to progress of a selection . In contrast to phage display , by panning by additional iterations of screening so as to further normalizing to antibody display levels , antibodies with enrich the population of phage for those harboring higher- 60 higher affinities, rather than greater expression levels can affinity anti - TNFR2 polypeptides. Following these panning easily be selected . In fact, it is possible to distinguish and stages , phage that display high - affinity antibodies or anti- sort antibodies whose affinities differ by only two - fold gen -binding fragments thereof can subsequently be isolated ( VanAntwerp and Wittrup ( Biotechnol Prog 16:31 , 2000 ) ) . and the genomes of these phage can be sequenced in order ( iii ) Nucleotide Display Techniques to identify the polynucleotide and polypeptide sequences of 65 Display techniques that utilize in vitro translation of the encoded antibodies. Phage display techniques such as randomized polynucleotide libraries also provide a powerful this can be used to generate , e.g. , antibody chains , such as approach to generating anti - TNFR2 antibodies of the inven US 10,906,982 B2 69 70 tion . For instance , randomized DNA libraries encoding binding fragments thereof that encode random mutations single - chain polypeptides , antibodies , or antigen - binding only at particular sites within hypervariable regions. These fragments thereof that contain mutations within designated polynucleotides can then be expressed in , e.g. , filamentous hypervariable regions can be obtained , e.g. , using estab- phage , bacterial cells , yeast cells , mammalian cells , or in lished PCR - based mutagenesis techniques as described 5 vitro using , e.g. , ribosome display, mRNA display, or cDNA herein . The polynucleotides of these libraries may contain display techniques in order to screen for polypeptides , such transcription regulating sequences, such as promoters and as single -chain polypeptides, antibodies, or antigen -binding transcription terminating sequences, and may additionally fragments thereof that specifically bind TNFR2 epitopes encode sequences that increase the rate of translation of the ( e.g., peptides containing the sequence of any one of SEQ ID resulting mRNA construct ( e.g. , IRES sequences, 5 ' and 3 ' 10 NOs : 11 , 19 , 20 , and 34-117 , and particularly those that UTRs , a poly -adenylation tract, etc ) . These polynucleotide contain the KCRPG motif, as in SEQ ID NOs : 42 , 50 , 52-54 , libraries can be incubated in an appropriately buffered and 61-63 ) with improved binding affinity. Yeast display is solution containing RNA polymerase and RNA nucleoside particularly well - suited for affinity maturation , and has been triphosphates (NTPs ) in order to enable transcription of the used previously to improve the affinity of a single - chain DNA sequences to competent mRNA molecules , which can 15 antibody to a Ky of 48 fM (Boder et al . ( Proc Natl Acad Sci subsequently be translated by large and small ribosomal USA 97 : 10701 , 2000 ) ) . subunits , aminoacyl tRNA molecules , and translation initia- Additional in vitro techniques that can be used for the tion and elongation factors present in solution ( e.g. , using generation and affinity maturation of antagonistic TNFR2 the PURExpress® In Vitro Protein Synthesis Kit , New antibodies of the invention include the screening of combi England Biolabs® ) . Designed mRNA modifications can 20 natorial libraries of polypeptides, such as single - chain poly enable the antibody product to remain covalently bound to peptides, antibodies, or antigen -binding fragments thereof the mRNA template by a chemical bond to puromycin ( e.g. , for functional molecules capable of specifically binding see Keefe ( Curr. Protoc . Mol . Biol . , Chapter 24 , Unit 24.5 , TNFR2 - derived peptides ( e.g. , a peptide having the amino 2001 ) ; incorporated herein by reference ). This genotype- acid sequence of any one of SEQ ID NOs : 11 , 19 , 20 , and phenotype linkage can thus be used to select for antibodies 25 34-117 , such as SEQ ID NOs : 42 , 50 , 52-54 , and 61-63 ) . that bind a TNFR2 - derived peptide ( e.g. , a peptide that has Combinatorial polypeptide libraries , such as antibody or the sequence of any one of SEQ ID NOs : 11 , 19 , 20 , and antibody fragment libraries , can be obtained , e.g. , by expres 34-117 , and particularly those that contain the KCRPG sion of polynucleotides encoding randomized hypervariable motif, as in SEQ ID NOs : 42 , 50 , 52-54 , and 61-63 ) by regions of an antibody or antigen -binding fragment thereof incubating mRNA : antibody fusion constructs with a peptide 30 in a eukaryotic or prokaryotic cell . This can be achieved , immobilized to a surface and panning in a fashion similar to e.g. , using gene expression techniques described herein or phage display techniques ( see , e.g. , WO 2006/072773 ; known in the art. Heterogeneous mixtures of antibodies can incorporated herein by reference ). be purified, e.g. , by Protein A or Protein G selection , sizing Optionally, polypeptides ( e.g. , single - chain polypeptides , column chromatography ), centrifugation, differential solu antibodies, or antigen - binding fragments thereof) of the 35 bility , and / or by any other standard technique for the puri invention can be generated using a similar technique , except fication of proteins. Libraries of combinatorial libraries thus the polypeptide product may be bound non - covalently to the obtained can be screened , e.g. , by incubating a heteroge ribosome -mRNA complex rather than covalently via a puro- neous mixture of these antibodies with a peptide derived mycin linker. This platform , known as ribosome display, has from TNFR2 that has been immobilized to a surface ( e.g. , a been described , e.g. , in U.S. Pat . No. 7,074,557 ; incorpo- 40 peptide having the amino acid sequence of any one of SEQ rated herein by reference . Alternatively, antibodies can be ID NOs : 11 , 19 , 20 , and 34-117 immobilized to the surface generated using cDNA display, a technique analogous to of a solid - phase resin or a well of a microtiter plate ) for a mRNA display with the exception that cDNA , rather than period of time sufficient to allow antibody -antigen binding. mRNA , is covalently bound to an antibody product via a Non -binding antibodies or fragments thereof can be puromycin linker. cDNA display techniques offer the advan- 45 removed by washing the surface with an appropriate buffer tage of being able to perform panning steps under increas- ( e.g. , a solution buffered at physiological pH ( approximately ingly stringent conditions, e.g. , under conditions in which 7.4 ) and containing physiological salt concentrations and the salt concentration , ionic strength , pH , and / or temperature ionic strength , and optionally containing a detergent, such as of the environment is adjusted in order to screen for anti- TWEEN - 20 ) . Antibodies that remain bound can subse bodies with particularly high affinity for TNFR2 -derived 50 quently be detected , e.g. , using an ELISA - based detection peptides. This is due to the higher natural stability of protocol ( see , e.g. , U.S. Pat . No. 4,661,445 ; incorporated double - stranded cDNA over single - stranded mRNA . cDNA herein by reference ). display screening techniques are described , e.g. , in Ueno et Additional techniques for screening combinatorial librar al . (Methods Mol . Biol . , 805 : 113-135 , 2012 ) ; incorporated ies of polypeptides for those that specifically bind TNFR2 herein by reference . 55 derived peptides ( e.g. , a peptide containing the amino acid In addition to generating anti - TNFR2 polypeptides of the sequence of any one of SEQ ID NOs : 11 , 19 , 20 , and 34-117 , invention , in vitro display techniques ( e.g. , those described such as SEQ ID NOs : 42 , 50 , 52-54 , and 61-63 ) include the herein and those known in the art) also provide methods for screening of one - bead - one - compound libraries of single improving the affinity of an anti - TNFR2 polypeptide of the chain polypeptides or antibody fragments. Single - chain invention . For instance , rather than screening libraries of 60 polypeptides and antibody fragments can be chemically single - chain polypeptides, antibodies, and fragments thereof synthesized on a solid bead ( e.g. , using established split containing completely randomized hypervariable regions, and -pool solid - phase peptide synthesis protocols) composed one can screen narrower libraries of single - chain polypep- of a hydrophilic , water - swellable material such that each tides , antibodies, and antigen - binding fragments thereof that bead displays a single antibody fragment. Heterogeneous feature targeted mutations at specific sites within hypervari- 65 bead mixtures can then be incubated with a TNFR2 - derived able regions . This can be accomplished , e.g. , by assembling peptide that is optionally labeled with a detectable moiety libraries of polynucleotides encoding antibodies or antigen- ( e.g. , a fluorescent dye ) or that is conjugated to an epitope US 10,906,982 B2 71 72 tag ( e.g. , biotin , avidin , FLAG tag , HA tag ) that can later be immunizing primates are known in the art ( see , e.g. , WO detected by treatment with a complementary tag ( e.g. , avi- 1986/6004782 ; incorporated herein by reference ). Immuni din , biotin , anti - FLAG antibody, anti -HA antibody , respec- zation represents a robust method of producing monoclonal tively ) . Beads containing antibody fragments that specifi- antibodies by exploiting the antigen specificity of B lym cally bind a TNFR2 - derived peptide ( e.g. , a peptide 5 phocytes. For example , monoclonal antibodies can be pre containing the amino acid sequence of any one of SEQ ID pared by the Kohler -Millstein procedure ( described , e.g. , in NOs : 11 , 19 , 20 , and 34-117 , such as SEQ ID NOs : 42 , 50 , EP 0110716 ; incorporated herein by reference ), wherein 52-54 , and 61-63 ) can be identified by analyzing the fluo- spleen cells from a non - human animal ( e.g. , a primate ) rescent properties of the beads following incubation with a immunized with a peptide that presents a TNFR2 - derived fluorescently - labeled antigen or complementary tag ( e.g. , by 10 antigen that promotes receptor antagonism ( e.g. , a peptide confocal fluorescent microscopy or by fluorescence- acti- containing the amino acid sequence of any one of SEQ ID vated bead sorting; see , e.g. , Muller et al . ( J. Biol . Chem . , NOs : 11 , 19 , 20 , and 34-117 , such as SEQ ID NOs : 42 , 50 , 16500-16505 , 1996 ) ; incorporated herein by reference ). 52-54 , and 61-63 ) . A clonally -expanded B lymphocyte pro Beads containing antibody fragments that specifically bind duced by immunization can be isolated from the serum of TNFR2 - derived peptides can thus be separated from those 15 the animal and subsequently fused with a myeloma cell in that do not contain high - affinity antibody fragments. The order to form a hybridoma . Hybridomas are particularly sequence of an antibody fragment that specifically binds a useful agents for antibody production , as these immortalized TNFR2 - derived peptide can be determined by techniques cells can provide a lasting supply of an antigen -specific known in the art, including, e.g. , Edman degradation , tan- antibody. Antibodies from such hybridomas can subse dem mass spectrometry, matrix - assisted laser -desorption 20 quently be isolated using techniques known in the art , e.g. , time -of - flight mass spectrometry (MALDI - TOF MS ) , by purifying the antibodies from the cell culture medium by nuclear magnetic resonance ( NMR ) , and 2D gel electropho- affinity chromatography, using reagents such as Protein A or resis , among others ( see , e.g. , WO 2004/062553 ; incorpo- Protein G. rated herein by reference ). Antagonistic TNFR2 Polypeptide Conjugates Negative Screens of Polypeptides 25 Prior to administration of antagonistic TNFR2 polypep In addition to the above - described methods for screening tides of the invention to a mammalian subject ( e.g. , a for a single - chain polypeptide , antibody, or antibody frag- human ), it may be desirable to conjugate the polypeptide ment that specifically binds to an epitope derived from ( e.g., single - chain polypeptide, antibody , or antigen -binding human TNFR2 containing the KCR or KCRPG motif, one fragment thereof) to a second molecule , e g . , to modulate the can additionally perform negative screens in order to elimi- 30 activity of the polypeptide in vivo . Antagonistic TNFR2 nate single - chain polypeptides, antibodies, or antibody frag- single - chain polypeptides, antibodies , and fragments thereof ments that may also bind an epitope that contains the can be conjugated to other molecules at either the N -termi KCSPG sequence . For instance , mixtures of single - chain nus or C -terminus of a light or heavy chain of the polypep polypeptides , antibodies , or antibody fragments isolated as a tide using any one of a variety of established conjugation result of any of the above - described screening techniques 35 strategies that are well -known in the art . Examples of pairs can be screened for single - chain polypeptides, antibodies, or of reactive functional groups that can be used to covalently antibody fragments that also specifically bind to a peptide tether an antagonistic TNFR2 single - chain polypeptide, anti derived from human TNFR2 that contains the KCSPG motif, body , or fragment thereof to another molecule include , such as a peptide containing residues 48-67 of SEQ ID NO : without limitation , thiol pairs , carboxylic acids and amino 7 ( QTAQMCCSKCSPGQHAKVFC , SEQ ID NO : 18 ) . This 40 groups, ketones and amino groups, aldehydes and amino can be accomplished using any of the above - described groups, thiols and alpha ,beta - unsaturated moieties ( such as methods or variations thereof, e.g. , such that the single - chain maleimides or dehydroalanine ), thiols and alpha - halo polypeptides , antibodies, or antibody fragments being amides , carboxylic acids and hydrazides, aldehydes and screened are those that were previously identified as being hydrazides, and ketones and hydrazides. capable of specifically binding a peptide containing one or 45 Antagonistic TNFR2 single - chain polypeptides, antibod more residues of the KCRPG sequence ( e.g. , at least the ies , and fragments thereof can be covalently appended KCR sequence ). Exemplary techniques useful for a negative directly to another molecule by chemical conjugation as screen include those described above or known in the art, described . Alternatively, fusion proteins containing antago such as phage display, yeast display, bacterial display, ribo- nistic TNFR2 single - chain polypeptides, antibodies, and some display, mRNA display , cDNA display, or surface- 50 fragments thereof can be expressed recombinantly from a based combinatorial library screens ( e.g. , in an ELISA cell ( e.g. , a eukaryotic cell or prokaryotic cell ) . This can be format ). This screening technique represents a useful strat- accomplished , for example, by incorporating a polynucle egy for identifying an antagonistic TNFR2 single - chain otide encoding the fusion protein into the nuclear genome of polypeptides, antibodies , and antibody fragments, as poly- a cell ( e.g. , using techniques described herein or known in peptides capable of binding TNFR2 epitopes containing the 55 the art ). Optionally , single - chain polypeptides, antibodies, KCSPG sequence and one or more residues of the KCRPG and fragments thereof of the invention can be joined to a sequence have been shown to lack , or to have significantly second molecule by forming a covalent bond between the reduced , antagonistic activity . antibody and a linker. This linker can then be subsequently Immunization of a Non - Human Mammal conjugated to another molecule , or the linker can be conju Another strategy that can be used to produce antagonistic 60 gated to another molecule prior to ligation to the anti TNFR2 antibodies or antibody fragments of the invention TNFR2 single -chain polypeptide, antibody, or fragment includes immunizing a non - human mammal. Examples of thereof. Examples of linkers that can be used for the non -human mammals that can be immunized in order to formation of a conjugate include polypeptide linkers, such produce antagonistic TNFR2 antibodies and fragments as those that contain naturally occurring or non - naturally thereof of the invention include rabbits , mice , rats , goats , 65 occurring amino acids . In some embodiments, it may be guinea pigs , hamsters, horses , and sheep , as well as non- desirable to include D - amino acids in the linker, as these human primates . For instance , established procedures for residues are not present in naturally - occurring proteins and US 10,906,982 B2 73 74 are thus more resistant to degradation by endogenous pro- ride; plicamycin ; plomestane ; porfimer sodium ; teases . Fusion proteins containing polypeptide linkers can be porfiromycin ; prednimustine; procarbazine hydrochloride ; made using chemical synthesis techniques, such as those puromycin ; puromycin hydrochloride; pyrazofurin ; described herein , or through recombinant expression of a rhizoxin ; rhizoxin d ; riboprine ; rogletimide; safingol; safin polynucleotide encoding the fusion protein in a cell ( e.g. , a 5 gol hydrochloride; semustine ; simtrazene; sparfosate prokaryotic or eukaryotic cell ) . Linkers can be prepared sodium ; sparsomycin ; spirogermanium hydrochloride ; using a variety of strategies that are well known in the art, spiromustine; spiroplatin ; streptonigrin ; streptozocin ; stron and depending on the reactive components of the linker, can tium chloride sr 89 ; sulofenur; talisomycin ; taxane ; taxoid ; be cleaved by enzymatic hydrolysis, photolysis , hydrolysis tecogalan sodium ; tegafur; teloxantrone hydrochloride; under acidic conditions , hydrolysis under basic conditions , 10 temoporfin ; teniposide ; teroxirone; testolactone ; thia oxidation, disulfide reduction , nucleophilic cleavage , or miprine ; thioguanine; thiotepa; thymitaq; tiazofurin ; tira organometallic cleavage ( Leriche et al . , Bioorg . Med . pazamine; tomudex ; top53; topotecan hydrochloride ; Chem ., 20 : 571-582 , 2012 ) . toremifene citrate ; trestolone acetate ; triciribine phosphate ; Drug -Polypeptide Conjugates trimetrexate ; trimetrexate glucuronate; triptorelin ; tubulo An antagonistic TNFR2 polypeptide ( e.g. , single - chain 15 zole hydrochloride; uracil mustard ; uredepa; vapreotide; polypeptide, antibody , or antigen - binding fragment thereof) verteporfin ; vinblastine ; vinblastine sulfate; vincristine ; of the invention can additionally be conjugated to , admixed vincristine sulfate ; vindesine ; vindesine sulfate ; vinepidine with , or administered separately from a therapeutic agent, sulfate ; vinglycinate sulfate; vinleurosine sulfate ; vinorel such as a cytotoxic molecule . Conjugates of the invention bine tartrate ; vinrosidine sulfate ; vinzolidine sulfate ; voro may be applicable to the treatment or prevention of a disease 20 zole; zeniplatin ; zinostatin ; zorubicin hydrochloride; 2 - chlo associated with aberrant cell proliferation , such as a cancer rodeoxyadenosine ; 2 deoxyformycin ; described herein . Exemplary cytotoxic agents that can be 9 - aminocamptothecin ; raltitrexed ; N -propargyl - 5,8 -dide conjugated to , admixed with , or administered separately azafolic acid ; 2chloro - 2' - arabino -fluoro - 2' - deoxyadenosine ; from an antagonistic TNFR2 polypeptide include, without 2 - chloro - 2 ' -deoxyadenosine ; anisomycin ; trichostatin A ; limitation , antineoplastic agents such as : acivicin ; aclarubi- 25 hPRL - G129R ; CEP - 751 ; linomide ; sulfur mustard ; nitrogen cin ; acodazole hydrochloride ; acronine; adozelesin ; adri- mustard (mechlorethamine ); cyclophosphamide; mel amycin ; aldesleukin ; altretamine ; ambomycin ; a . metan- phalan ; chlorambucil; ifosfamide; busulfan ; N -methyl - N trone acetate ; aminoglutethimide ; amsacrine; anastrozole ; nitrosourea (MNU ); N , N ' - Bis ( 2 - chloroethyl )-N - nitrosourea anthramycin ; asparaginase ; asperlin ; azacitidine ; azetepa ; ( BCNU ); N- ( 2 -chloroethyl ) -N ' cyclohexyl- N -nitrosourea azotomycin ; batimastat; benzodepa ; bicalutamide ; bisant- 30 ( CCNU ) ; N- ( 2 -chloroethyl ) -N '- trans - 4 -methylcyclohexyl rene hydrochloride ; bisnafide dimesylate; bizelesin ; bleo- N - nitrosourea (MeCCNU ); N-( 2 - chloroethyl) -N '- (diethyl ) mycin sulfate ; brequinar sodium ; bropirimine; busulfan ; ethylphosphonate - N - nitrosourea ( fotemustine ); streptozoto cactinomycin ; calusterone; camptothecin ; caracemide ; car- diacarbazine ( DTIC ) ; mitozolomide ; temozolomide ; betimer; carboplatin ; carmustine; carubicin hydrochloride; thiotepa ; mitomycin C ; AZQ ; adozelesin ; cisplatin ; carbo carzelesin ; cedefingol; chlorambucil ; cirolemycin ; cisplatin ; 35 platin ; ormaplatin ; oxaliplatin ; C1-973 ; DWA 2114R ; cladribine; combretestatin a - 4 ; crisnatol mesylate ; cyclo- JM216 ; JM335 ; Bis ( platinum ); tomudex ; azacitidine ; cyt phosphamide; cytarabine; dacarbazine ; daca (n- [ 2-( dim- arabine ; gemcitabine ; 6 -mercaptopurine ; 6 -thioguanine ; ethyl - amino ) ethyl ]acridine - 4 - carboxamide ); dactinomycin ; hypoxanthine; teniposide 9 - amino camptothecin ; topotecan ; daunorubicin hydrochloride; daunomycin ; decitabine ; CPT - 11 ; Doxorubicin ; Daunomycin , Epirubicin ; darubicin ; dexormaplatin ; dezaguanine ; dezaguanine mesylate ; diazi- 40 mitoxantrone; losoxantrone ; Dactinomycin (Actinomycin quone ; docetaxel; dolasatins ; doxorubicin ; doxorubicin D ) ; amsacrine ; pyrazoloacridine; all - trans retinol; 14 -hy hydrochloride ; droloxifene ; droloxifene citrate ; dromo- droxy - retro - retinol; all - trans retinoic acid ; N-( 4 -hydroxy stanolone propionate; duazomycin ; edatrexate ; eflornithine phenyl) retinamide ; 13 - cis retinoic acid ; 3 -methyl TTNEB ; hydrochloride ; ellipticine ; elsamitrucin ; enloplatin ; enpro- 9 - cis retinoic acid ; fludarabine ( 2 - F - ara -AMP ); or 2 - chloro mate ; epipropidine; epirubicin hydrochloride; erbulozole; 45 deoxyadenosine ( 2 - Cda ) . esorubicin hydrochloride; estramustine; estramustine phos- Other therapeutic compounds that can be conjugated to , phate sodium ; etanidazole ; ethiodized oil i 131 ; etoposide; admixed with , or administered separately from an antago etoposide phosphate ; etoprine; fadrozole hydrochloride; nistic TNFR2 single - chain polypeptide , antibody, or anti fazarabine ; fenretinide; floxuridine; fludarabine phosphate; gen - binding fragment thereof of the invention in order to fluorouracil; 5 - fdump; flurocitabine ; fosquidone; fostriecin 50 treat, prevent, or study the progression of a disease associ sodium ; gemcitabine ; gemcitabine hydrochloride; gold au ated with aberrant cell proliferation include , but are not 198 ; homocamptothecin ; hydroxyurea ; idarubicin hydro- limited to , cytotoxic agents such as 20 -pi - 1,25 dihydroxyvi chloride; ifosfamide; ilmofosine; interferon alfa - 2a ; inter- tamin D3 ; 5 - ethynyluracil; abiraterone ; acylfulvene; adecy feron alfa - 2b ; interferon alfa - nl; interferon alfa - n3; inter- penol; adozelesin ; aldesleukin ; ALL - TK antagonists ; altret feron beta - i a ; interferon gamma -i b ; iproplatin ; irinotecan 55 amine ; ambamustine; amidox ; amifostine; aminolevulinic hydrochloride ; lanreotide acetate ; letrozole ; leuprolide acid ; amrubicin ; amsacrine ; anagrelide; anastrozole ; acetate ; liarozole hydrochloride ; lometrexol sodium ; lomus- andrographolide ; angiogenesis inhibitors ; antagonist D ; tine ; losoxantrone hydrochloride; masoprocol ; maytansine ; antagonist G ; antarelix ; anti -dorsalizing morphogenetic pro mechlorethamine hydrochloride; megestrol acetate ; tein - 1 ; antiandrogen , prostatic carcinoma; antiestrogen ; anti melengestrol acetate ; melphalan ; menogaril; mercaptopu- 60 neoplaston ; antisense oligonucleotides; aphidicolin glyci rine ; methotrexate ; methotrexate sodium ; metoprine; nate ; apoptosis gene modulators; apoptosis regulators ; meturedepa; mitindomide ; mitocarcin ; mitocromin ; mitogil- apurinic acid ; ara -CDP -DL -PTBA ; argininedeaminase ; asu lin ; mitomalcin ; mitomycin ; mitosper ; mitotane; mitoxan- lacrine; atamestane; atrimustine; axinastatin 1 ; axinastatin 2 ; trone hydrochloride; mycophenolic acid ; nocodazole; axinastatin 3 ; azasetron ; azatoxin ; azatyrosine; baccatin III nogalamycin ; ormaplatin ; oxisuran ; paclitaxel ; pegaspar- 65 derivatives ; balanol; batimastat; BCR / ABL antagonists; gase ; peliomycin ; pentamustine; peploycinsulfate ; perfosf- benzochlorins; benzoylstaurosporine; beta lactam deriva amide ; pipobroman ; piposulfan ; piroxantrone hydrochlo- tives ; beta - alethine ; betaclamycin B ; betulinic acid ; bFGF US 10,906,982 B2 75 76 inhibitor ; bicalutamide ; bisantrene; bisaziridinylspermine; oral cytokine inducer ; ormaplatin ; osaterone ; oxaliplatin ; bisnafide ; bistratene A ; bizelesin ; breflate ; bleomycin A2 ; oxaunomycin ; paclitaxel analogues; paclitaxel derivatives ; bleomycin B2 ; bropirimine ; budotitane ; buthionine sulfoxi- palauamine; palmitoylrhizoxin ; pamidronic acid ; panaxyt mine ; calcipotriol ; calphostin C ; camptothecin derivatives riol; panomifene; parabactin ; pazelliptine; pegaspargase ; ( e.g. , 10 - hydroxy - camptothecin ); canarypox IL - 2 ; capecit- 5 peldesine; pentosan polysulfate sodium ; pentostatin ; pentro abine ; carboxamide - amino - triazole ; carboxyamidotriazole; zole ; perflubron ; perfosfamide ; perillyl alcohol ; phenazino CaRest M3 ; CARN 700 ; cartilage derived inhibitor; carze- mycin ; phenylacetate; phosphatase inhibitors; picibanil; lesin ; casein kinase inhibitors ( ICOS ) ; castanospermine ; pilocarpine hydrochloride; pirarubicin ; piritrexim ; placetin cecropin B ; cetrorelix ; chlorins ; chloroquinoxaline sulfona- A ; placetin B ; plasminogen activator inhibitor; platinum mide; cicaprost; cis -porphyrin ; cladribine ; clomifene ana- 10 complex ; platinum compounds; platinum - triamine complex ; logues ; clotrimazole; collismycin A ; collismycin B ; com- podophyllotoxin ; porfimer sodium ; porfiromycin ; propyl bretastatin A4 ; combretastatin analogue ; conagenin ; bis -acridone ; prostaglandin J2 ; proteasome inhibitors ; pro crambescidin 816 ; crisnatol; cryptophycin 8 ; cryptophycin tein A - based immune modulator ; protein kinase C inhibitor ; A derivatives; curacin A ; cyclopentanthraquinones ; cyclo- protein kinase C inhibitors, microalgal; protein tyrosine platam ; cypemycin ; cytarabine ocfosfate; cytolytic factor; 15 phosphatase inhibitors; purine nucleoside phosphorylase cytostatin ; dacliximab ; decitabine; dehydrodidemnin B ; inhibitors; purpurins; pyrazoloacridine ; pyridoxylated 2'deoxycoformycin (DCF ) ; deslorelin ; dexifosfamide ; hemoglobin polyoxyethylene conjugate ; raf antagonists; dexrazoxane; dexverapamil, diaziquone ; didemnin B ; didox; raltitrexed ; ramosetron ; ras farnesyl protein transferase diethylnorspermine ; dihydro - 5 -azacytidine ; dihydrotaxol, inhibitors; ras inhibitors; ras -GAP inhibitor; retelliptine 9- ; dioxamycin ; diphenyl spiromustine ; discodermolide ; 20 demethylated ; rhenium Re 186 etidronate; rhizoxin ; docosanol ; dolasetron ; doxifluridine ; droloxifene; dronabi- ribozymes ; RII retinamide; rogletimide; rohitukine; nol ; duocarmycin SA ; ebselen ; ecomustine; edelfosine ; romurtide ; roquinimex ; rubiginone B1 ; ruboxyl; safingol; edrecolomab ; eflornithine ; elemene ; emitefur; epirubicin ; saintopin ; SarCNU ; sarcophytol A ; sargramostim ; Sdi 1 epothilones ( A , R = H ; B , R =Me ) ; epithilones; epristeride; mimetics; semustine; senescence derived inhibitor 1 ; sense estramustine analogue ; estrogen agonists ; estrogen antago- 25 oligonucleotides; signal transduction inhibitors ; signal trans nists ; etanidazole ; etoposide ; etoposide 4 ' - phosphate (etopo- duction modulators ; single -chain antigen binding protein ; fos ); exemestane; fadrozole ; fazarabine ; fenretinide; fil- sizofiran ; sobuzoxane ; sodium borocaptate ; sodium pheny grastim ; finasteride; flavopiridol; flezelastine; fluasterone; lacetate ; solverol; somatomedin binding protein , sonermin ; fludarabine; fluorodaunorunicin hydrochloride; forfenimex ; sparfosic acid ; spicamycin D ; spiromustine; splenopentin ; formestane; fostriecin ; fotemustine; gadolinium texaphyrin ; 30 spongistatin 1 ; squalamine; stem cell inhibitor; stem - cell gallium nitrate ; galocitabine; ganirelix ; gelatinase inhibitors; division inhibitors; stipiamide; stromelysin inhibitors ; gemcitabine; glutathione inhibitors ; hepsulfam ; heregulin; sulfinosine ; superactive vasoactive intestinal peptide antago hexamethylene bisacetamide; homoharringtonine ( HHT ) ; nist ; suradista ; in ; swainsonine ; synthetic glycosami hypericin ; ibandronic acid ; idarubicin ; idoxifene ; idraman- noglycans; tallimustine; tamoxifen methiodide; tauromus tone ; ilmofosine; ilomastat; imidazoacridones ; imiquimod ; 35 tine; tazarotene; tecogalan sodium ; tegafur; tellurapyrylium ; immunostimulant peptides ; insulin - like growth factor - 1 telomerase inhibitors ; temoporfin ; temozolomide; tenipo receptor inhibitor, interferon agonists ; interferons; inter- side ; tetrachlorodecaoxide; tetrazomine ; thaliblastine; tha leukins ; iobenguane; iododoxorubicin ; ipomeanol ; irinote- lidomide ; thiocoraline; thrombopoietin ; thrombopoietin can ; iroplact ; irsogladine ; isobengazole ; isohomohalicon- mimetic ; thymalfasin ; thymopoietin receptor agonist ; thy drin B ; itasetron; jasplakinolide; kahalalide F ; lamellarin - N 40 motrinan ; thyroid stimulating hormone; tin ethyl etiopurpu triacetate ; lanreotide ; leinamycin ; lenograstim ; lentinan sul- rin ; tirapazamine ; titanocene dichloride ; topotecan ; topsen fate; leptolstatin ; letrozole; leukemia inhibiting factor; leu- tin ; toremifene; totipotent stem cell factor ; translation kocyte alpha interferon ; leuprolide + estrogen + progesterone ; inhibitors; tretinoin ; triacetyluridine ; triciribine; trimetrex leuprorelin ; levamisole; liarozole ; linear polyamine ana- ate ; triptorelin ; tropisetron; turosteride ; tyrosine kinase logue; lipophilic disaccharide peptide; lipophilic platinum 45 inhibitors; tyrphostins; UBC inhibitors; ubenimex ; urogeni compounds; lissoclinamide 7 ; lobaplatin ; lombricine; lom- tal sinus -derived growth inhibitory factor; urokinase recep etrexol; lonidamine; losoxantrone; lovastatin ; loxoribine ; tor antagonists ; vapreotide; variolin B ; vector system , eryth lurtotecan ; lutetium texaphyrin ; lysofylline; lytic peptides; rocyte gene therapy ; velaresol; veramine ; verdins; maytansine ; mannostatin A ; marimastat; masoprocol ; mas- verteporfin ; vinorelbine ; vinxaltine; vitaxin ; vorozole ; zan pin ; matrilysin inhibitors ; matrix metalloproteinase inhibi- 50 oterone; zeniplatin ; zilascorb ; and zinostatin stimalamer . tors ; menogaril; rnerbarone; meterelin ; methioninase ; meto- Labeled Anti - TNFR2 Polypeptides clopramide ; MIF inhibitor ; ifepristone; miltefosine; In some embodiments, antagonistic TNFR2 single - chain mirimostim ; mismatched double stranded RNA ; mithracin ; polypeptides, antibodies , or antigen - binding fragments mitoguazone; mitolactol ; mitomycin analogues; mitonafide; thereof may be conjugated to another molecule ( e.g. , an mitotoxin fibroblast growth factor - saporin ; mitoxantrone; 55 epitope tag ) for the purpose of purification or detection . mofarotene; molgramostim ; monoclonal antibody, human Examples of such molecules that are useful in protein chorionic gonadotrophin ; monophosphoryl lipid A +myo- purification include those that present structural epitopes bacterium cell wall sk ; mopidamol; multiple drug resistance capable of being recognized by a second molecule . This is gene inhibitor ; multiple tumor suppressor 1 - based therapy ; a common strategy that is employed in protein purification mustard anticancer agent; mycaperoxide B ; mycobacterial 60 by affinity chromatography, in which a molecule is immo cell wall extract; myriaporone; N - acetyldinaline; N - substi- bilized on a solid support and exposed to a heterogeneous tuted benzamides; nafarelin ; nagrestip ; naloxone + pentazo- mixture containing a target protein conjugated to a molecule cine; napavin ; naphterpin ; nartograstim ; nedaplatin ; nemo- capable of binding the immobilized compound . Examples of rubicin ; neridronic acid ; neutral endopeptidase ; nilutamide ; epitope tag molecules that can be conjugated to antagonistic nisamycin ; nitric oxide modulators ; nitroxide antioxidant; 65 TNFR2 single - chain polypeptides, antibodies, or fragments nitrullyn ; 06 -benzylguanine ; octreotide ; okicenone ; oligo- thereof for the purposes of molecular recognition include , nucleotides ; onapristone ; ondansetron ; ondansetron ; oracin ; without limitation , maltose - binding protein , glutathione - S US 10,906,982 B2 77 78 transferase, a poly - histidine tag , a FLAG -tag , a myc - tag , Bioluminescent proteins can also be incorporated into a human influenza hemagglutinin ( HA ) tag , biotin , streptavi- fusion protein for the purposes of detection and visualization din . Conjugates containing the epitopes presented by these of an antagonistic anti - TNFR2 polypeptide. Bioluminescent molecules are capable of being recognized by such comple- proteins, such as Luciferase and aequorin , emit light as part mentary molecules as maltose , glutathione, a nickel - con- 5 of a chemical reaction with a substrate ( e.g. , luciferin and taining complex, an anti - FLAG antibody, an anti- myc anti coelenterazine ). Exemplary bioluminescent proteins suitable body , an anti -HA antibody, streptavidin , or biotin , for use as a diagnostic sequence and methods for their use respectively . For example, one can purify an antagonistic are described in , e.g. , U.S. Pat . Nos . 5,292,658 , 5,670,356 , TNFR2 single - chain polypeptide, antibody, or fragment 6,171,809 , and 7,183,092 , each of which is herein incorpo thereof of the invention that has been conjugated to an 10 rated by reference . Antagonistic TNFR2 single - chain poly epitope tag from a complex mixture of other proteins and peptides, antibodies, or fragments thereof labeled with bio biomolecules ( e.g. , DNA , RNA , carbohydrates, phospholip luminescent proteins are a useful tool for the detection of ids , etc ) by treating the mixture with a solid -phase resin antibodies of the invention following an in vitro assay . For containing an complementary molecule that can selectively is thatinstance has ,been the conjugatedpresence of to an a antagonisticbioluminescent TNFR2 protein antibody can be recognize and bind the epitope tag of the antagonistic detected among a complex mixture of additional proteins by anti- TNFR2 antibody or fragment thereof . Examples of separating the components of the mixture using gel electro solid - phase resins include agarose beads , which are com phoresis methods known in the art ( e.g. , native gel analysis ) patible with purifications in aqueous solution . and subsequently transferring the separated proteins to a An antagonistic TNFR2 polypeptide of the invention can 20 membrane in order to perform a Western blot . Detection of also be covalently appended to a fluorescent molecule , e.g. , the antagonistic TNFR2 antibody among the mixture of to detect the antibody or antigen - binding fragment thereof other proteins can be achieved by treating the membrane by fluorimetry and / or by direct visualization using fluores- with an appropriate Luciferase substrate and subsequently cence microscopy. Exemplary fluorescent molecules that visualizing the mixture of proteins on film using established can be conjugated to polypeptides of the invention include 25 protocols. green fluorescent protein , cyan fluorescent protein , yellow The polypeptides ( e.g. , single - chain polypeptides, anti fluorescent protein , red fluorescent protein , phycoerythrin , bodies , and fragments thereof) of the invention can also be allophycocyanin , hoescht, 4 ' , 6 - diamidino - 2 - phenylindole conjugated to a molecule comprising a radioactive nucleus, ( DAPI ) , propidium iodide, fluorescein , coumarin , rhod- such that an antibody or fragment thereof of the invention amine , tetramethylrhoadmine, and cyanine. Additional 30 can be detected by analyzing the radioactive emission pat examples of fluorescent molecules suitable for conjugation tern of the nucleus. Alternatively, an antagonistic TNFR2 to polypeptides of the invention are well - known in the art antibody or fragment thereof can be modified directly by and have been described in detail in , e.g. , U.S. Pat . Nos . incorporating a radioactive nucleus within the antibody 7,417,131 and 7,413,874 , each of which is incorporated by during the preparation of the protein . Radioactive isotopes reference herein . 35 of methionine ( 35S ) , nitrogen ( 15N ) , or carbon ( 13C ) can be Antagonistic TNFR2 polypeptides containing a fluores- incorporated into antibodies or fragments thereof of the cent molecule are particularly useful for monitoring the invention by, e.g. , culturing bacteria in media that has been cell - surface localization properties of polypeptides, such as supplemented with nutrients containing these isotopes . single - chain polypeptides, antibodies, and fragments thereof Optionally , tyrosine derivatives containing a radioactive of the invention . For instance, one can expose cultured 40 halogen can be incorporated into an antagonistic TNFR2 mammalian cells ( e.g. , T -reg cells ) to antagonistic TNFR2 antibody or fragment thereof by, e.g. , culturing bacterial single - chain polypeptides, antibodies, or fragments thereof cells in media supplemented with radiolabeled tyrosine. It of the invention that have been covalently conjugated to a has been shown that tyrosine functionalized with a radioac fluorescent molecule and subsequently analyze these cells tive halogen at the C2 position of the phenol system are using conventional fluorescent microscopy techniques 45 rapidly incorporated into elongating polypeptide chains known in the art . Confocal fluorescent microscopy is a using the endogenous translation enzymes in vivo (U.S. Pat. particularly powerful method for determining cell - surface No. 4,925,651 ; incorporated herein by reference ). The halo localization of antagonistic anti - TNFR2 single - chain poly- gens include fluorine, chlorine, bromine , iodine , and asta peptides, antibodies, or fragments thereof, as individual tine . Additionally , antagonistic TNFR2 antibodies or frag planes of a cell can be analyzed in order to distinguish 50 ments thereof can be modified following isolation and antibodies or fragments thereof that have been internalized purification from cell culture by functionalizing antibodies into a cell's interior, e.g. , by receptor -mediated endocytosis, or fragments thereof of the invention with a radioactive from those that are bound to the external face of the cell isotope . The halogens represent a class of isotopes that can membrane . Additionally, cells can be treated with antago- be readily incorporated into a purified protein by aromatic nistic TNFR2 antibodies conjugated to a fluorescent mol- 55 substitution at tyrosine or tryptophan , e.g. , via reaction of ecule that emits visible light of a particular wavelength ( e.g. , one or more of these residues with an electrophilic halogen fluorescein , which fluoresces at about 535 nm ) and an species. Examples of radioactive halogen isotopes include additional fluorescent molecule that is known to localize to 18F, 75Br. 77Br, 1221 , 1231 , 1241 , 1251 , 1201 , 1311 , or 211 At. a particular site on the T -reg cell surface and that fluoresces Another alternative strategy for the incorporation of a at a different wavelength ( e.g. , a molecule that localizes to 60 radioactive isotope is the covalent attachment of a chelating CD25 and that fluoresces at about 599 nm ). The resulting group to the antagonistic anti - TNFR2 polypeptide ( e.g. , emission patterns can be visualized by confocal fluorescence single - chain polypeptide , antibody, or fragment thereof) . microscopy and the images from these two wavelengths can Chelating groups can be covalently appended to an antago be merged in order to reveal information regarding the nistic TNFR2 polypeptide by attachment to a reactive func location of the antagonistic TNFR2 single - chain polypep- 65 tional group , such as a thiol, amino group , alcohol, or tide , antibody, or antigen -binding fragment thereof on the carboxylic acid . The chelating groups can then be modified T - reg cell surface with respect to other receptors . to contain any of a variety of metallic radioisotopes, includ US 10,906,982 B2 79 80 ing, without limitation , such radioactive nuclides as 1251, molecule and prevent aberrant crosslinking . Conversely, 67Ga , 111 In , 99Tc, 169Yb , 186Re, 1231 , 1241, 1251 , 1311 , 99m Tc, cystine bond ( s ) may be added to the antibody or fragment 11111 In , 64 Cu, 67 Cu , 186Re, 188Re , 177Lu , 90Y, 77As , 72 As, 86Y, thereof to improve its stability ( particularly where the anti 89Zr , 211 At , 212 Bi, 213Bi , or 225Ac . body is an antibody fragment, such as an Fv fragment) . This In some embodiments , it may be desirable to covalently 5 can be accomplished , e.g. , by altering a polynucleotide conjugate the polypeptides ( e.g. , single - chain polypeptides, encoding the antibody heavy and light chains or a poly antibodies , or fragments thereof) of the invention with a nucleotide encoding an antibody fragment so as to encode chelating group capable of binding a metal ion from heavy one or more additional pairs of cysteine residues that can elements or rare earth ions , such as Gd3 + , Fe3 + , Mn3 +, or Cr2 + . Conjugates containing chelating groups that are coor- 10 reinforceform disulfide antibody bonds tertiary under structure oxidative ( seeconditions , e.g. , U.S. in Patorder. No. to dinated to such paramagnetic metals are useful as in MRI 7,422,899 ; incorporated herein by reference ). imaging applications. Paramagnetic metals include , but are Another useful modification that may be made to anti not limited to , chromium ( III ) , manganese ( II ) , iron ( II ) , iron TNFR2 polypeptides ( e.g. , single - chain polypeptides, anti neodymium( III ) , cobalt (( II III ) , )nickel , samarium ( II ) , copper (III ), gadolinium ( II ) , praseodymium ( III) , terbium ( III ) , 15 bodies, or fragments thereof ) of the invention includes ( III ) , dysprosium ( III ) , holmium ( III ) , erbium ( III ) , and altering the glycosylation profile of these antibodies and ytterbium ( III ) . In this way, antagonistic TNFR2 antibodies fragments thereof. This can be achieved , e.g. , by substitut can be detected by MRI spectroscopy. For instance , one can ing , inserting, or deleting amino acids in an antagonistic administer antagonistic TNFR2 antibodies or fragments TNFR2 antibody so as to insert or remove a glycosylation thereof conjugated to chelating groups bound to paramag- 20 site . Glycosylation of antibodies typically occurs in netic ions to a mammalian subject ( e.g. , a human patient) in N - linked or O - linked fashion . N - linked glycosylation is a order to monitor the distribution of the antibody following process whereby the attachment of a carbohydrate moiety to administration . This can be achieved by administration of an antibody occurs at the side - chain of an asparagine resi the antibody to a patient by any of the administration routes due . Consensus amino acid sequences for N - linked glyco described herein , such as intravenously, and subsequently 25 sylation include the tripeptide sequences asparagine- X - ser analyzing the location of the administered antibody by ine (NXS ) and asparagine - X - threonine (NXT ), where X is recording an MRI of the patient according to established any amino acid except proline . The insertion of either of protocols. these tripeptide sequences in a polypeptide ( e.g. , an anti Antagonistic TNFR2 polypeptides ( e.g. , single - chain TNFR2 antibody) creates potential glycosylation site . polypeptides , antibodies, or fragments thereof) can addition- 30 O - linked glycosylation refers to the attachment of one of the ally be conjugated to other molecules for the purpose of sugars N -acetylgalactosamine , galactose , or xylose to a improving the solubility and stability of the protein in hydroxyamino acid , most commonly serine or threonine, aqueous solution . Examples of such molecules include PEG , although 5 - hydroxyproline or 5 -hydroxylysine are also com PSA , bovine serum albumin ( BSA ) , and human serum petent substrates for glycoside formation . Addition of gly albumin ( HSA ) , among others . For instance , one can con- 35 cosylation sites to an anti- TNFR2 antibody can thus be jugate an antagonistic TNFR2 antibody or fragment thereof accomplished by altering the amino acid sequence of the to carbohydrate moieties in order to evade detection of the antibody ( e.g. , using recombinant expression techniques as antibody or fragment thereof by the immune system of the described herein ) such that it contains one or more of the patient receiving treatment. This process of hyperglycosy- above - described tripeptide sequences to promote N - linked lation reduces the immunogenicity of therapeutic proteins by 40 glycosylation , or one or more serine or threonine residues to sterically inhibiting the interaction of the protein with B - cell the sequence of the original antibody engender O - linked receptors in circulation . Alternatively, antagonistic TNFR2 glycosylation ( see , e.g. , U.S. Pat. No. 7,422,899 ; incorpo antibodies or fragments thereof can be conjugated to mol- rated herein by reference ). ecules that prevent clearance from human serum and In alternative cases , it may be desirable to modify the improve the pharmacokinetic profile of antibodies of the 45 antibody or fragment thereof of the invention with respect to invention . Exemplary molecules that can be conjugated to or effector function , e.g. , so as to enhance antigen - dependent inserted within anti - TNFR2 antibodies or fragments thereof cell- mediated cytotoxicity ( ADCC ) and / or complement of the invention so as to attenuate clearance and improve the dependent cytotoxicity ( CDC ) of the antibody. This may be pharmacokinetic profile of these antibodies and fragments achieved by introducing one or more amino acid substitu include salvage receptor binding epitopes . These epitopes 50 tions in an Fc region of the antibody . For instance , cysteine are found within the Fc region of an IgG immunoglobulin residues may be introduced in the Fc region of an anti and have been shown to bind Fc receptors and prolong TNFR2 antibody or fragment thereof ( e.g. , by recombinant antibody half - life in human serum . The insertion of salvage expression techniques as described herein ), so as to facilitate receptor binding epitopes into anti - TNFR2 antibodies or additional inter - chain disulfide bond formation in this fragments thereof can be achieved, e.g. , as described in U.S. 55 region . The homodimeric antibody thus generated may have Pat . No. 5,739,277 ; incorporated herein by reference. increased conformational constraint, which may foster Modified Antagonistic TFNR2 Polypeptides improved internalization capability and / or increased In addition to conjugation to other therapeutic agents and complement- mediated cell killing and antibody -dependent labels for identification or visualization , anti - TNFR2 poly- cellular cytotoxicity ( ADCC ) . Homodimeric antibodies with peptides ( e.g. , single - chain polypeptides , antibodies, or frag- 60 enhanced anti- tumor activity may also be prepared using ments thereof) of the invention can also be modified so as to heterobifunctional cross - linkers as described, for example , improve their pharmacokinetic profile, biophysical stability, in Wolff et al . ( Canc. Res . , 53 : 2560-2565 , 1993 ) ; incorpo or inhibitory capacity . For instance , any cysteine residue not rated herein by reference . Alternatively , an antibody can be involved in maintaining the proper conformation of the engineered which has dual Fc regions and may thereby have anti- TNFR2 antibody or fragment thereof may be substi- 65 enhanced complement lysis and ADCC capabilities ( see tuted with an isosteric or isolectronic amino acid ( e.g. , Stevenson et al . (Anti - Canc. Drug Des . , 3 : 219-230 , 1989 ) ; serine ) in order to improve the oxidative stability of the incorporated herein by reference ). US 10,906,982 B2 81 82 The serum half -life of anti - TNFR2 polypeptides ( e.g. , about 20 % , 30 % , 40 % , or 50 % , or more ) relative to an single -chain polypeptides, antibodies, or fragments thereof) untreated population of T - reg cells . of the invention can be improved in some embodiments by Antagonistic TNFR2 polypeptides of the invention can be incorporating one more amino acid modifications, such as administered to a mammalian subject ( e.g. , a human ) suf by altering the CH1 or CL region of the Fab domain to 5 fering from cancer in order to improve the condition of the introduce a salvage receptor motif, e.g. , that found in the two patient by promoting the immune response against cancer loops of a CH2 domain of an Fc region of an IgG . Such cells and tumorogenic material. Antibodies of the invention alterations are described , for instance , in U.S. Pat . Nos . can be administered to a subject, e.g. , via any of the routes 5,869,046 and 6,121,022 ; incorporated herein by reference . of administration described herein . Polypeptides of the Additional framework modifications can also be made to 10 inventioncally acceptable can also carriers be formulated , and may withbe optionally excipients conjugated, biologi reduce immunogenicity of the antibody or fragment thereof to , admixed with , or co - administered separately ( e.g. , or to reduce or remove T cell epitopes that reside therein , as sequentially ) with additional therapeutic agents , such as described for instance in US2003 /0153043 ; incorporated anti- cancer agents. Cancers that can be treated by adminis herein by reference . 15 tration of polypeptides ( e.g. , single - chain polypeptides , anti Methods of Treatment bodies , or fragments thereof ) of the invention include such Methods of Treating Cell Proliferation Disorders cancers as leukemia, lymphoma, liver cancer , bone cancer , Antagonistic TNFR2 polypeptides ( e.g. , single - chain lung cancer , brain cancer, bladder cancer, gastrointestinal polypeptides , antibodies, or fragments thereof) of the inven cancer, breast cancer, cardiac cancer, cervical cancer, uterine tion are useful therapeutics for the treatment of a wide array 20 cancer , head and neck cancer, gallbladder cancer , laryngeal of cancers and cell proliferation disorders . Antagonistic cancer, lip and oral cavity cancer , ocular cancer , melanoma, TNFR2 polypeptides ( e.g. , single - chain polypeptides, anti- pancreatic cancer, prostate cancer , colorectal cancer , testicu bodies, or fragments thereof) can be administered to a lar cancer , and throat cancer . Particular cancers that can be mammalian subject, such as a human , suffering from a cell treated by administration of antibodies or antigen - binding proliferation disorder, such as cancer , e.g. , to enhance the 25 fragments thereof of the invention include , without limita effectiveness of the adaptive immune response against the tion , acute lymphoblastic leukemia ( ALL ) , acute myeloid target cancer cells . In particular, antagonistic TNFR2 poly- leukemia ( AML ), chronic lymphocytic leukemia ( CLL ) , peptides ( e.g. , single - chain polypeptides, antibodies , or frag- chronic myelogenous leukemia ( CML ) , adrenocortical car ments thereof) of the invention can be administered to a cinoma , AIDS - related lymphoma, primary CNS lymphoma, mammalian subject, such as a human , to reduce or inhibit 30 anal cancer, appendix cancer, astrocytoma, atypical teratoid / T - reg cell growth and activation , which allows tumor- infil- rhabdoid tumor, basal cell carcinoma, bile duct cancer, trating T -lymphocytes to localize to cells presenting tumor- extrahepatic cancer, ewing sarcoma family, osteosarcoma associated antigens and promote cytotoxicity . In addition , and malignant fibrous histiocytoma , central nervous system polypeptides of the invention may synergize with existing embryonal tumors, central nervous system germ cell tumors, adoptive T -cell therapy platforms, as one of the limitations 35 craniopharyngioma, ependymoma, bronchial tumors, burkitt on the effectiveness of this strategy has been the difficulty of lymphoma, carcinoid tumor, primary lymphoma, chordoma, prolonging cytotoxicity of tumor - reactive T -cells following chronic myeloproliferative neoplasms , colon cancer , extra infusion into a mammalian subject ( e.g. , a human ). Poly- hepatic bile duct cancer , ductal carcinoma in situ ( DCIS ) , peptides of the invention may also promote the activity of endometrial cancer , ependymoma, esophageal cancer , esthe allogeneic T - lymphocytes, which may express foreign MHC 40 sioneuroblastoma, extracranial germ cell tumor, extrago proteins and may be increasingly susceptible to inactivation nadal germ cell tumor, fallopian tube cancer , fibrous histio by the host immune system . For example, antibodies and cytoma of bone, gastrointestinal carcinoid tumor, antigen - binding fragments thereof of the invention can miti- gastrointestinal stromal tumors ( GIST ) , testicular germ cell gate the T - reg -mediated depletion of tumor - reactive T - cells tumor, gestational trophoblastic disease , glioma, childhood by suppressing the growth and proliferation of T - reg cells 45 brain stem glioma , hairy cell leukemia , hepatocellular can that typically accompanies T - cell infusion . For instance , cer, langerhans cell histiocytosis, hodgkin lymphoma, hypo polypeptides ( e.g. , single - chain polypeptides , antibodies, or pharyngeal cancer, islet cell tumors , pancreatic neuroendo fragments thereof) of the invention may be capable of crine tumors , wilms tumor and other childhood kidney reducing the growth of a population of T - reg cells by about tumors , langerhans cell histiocytosis , small cell lung cancer , 50 % to about 200 % relative to untreated cells ( e.g. , 50 % , 50 cutaneous T - cell lymphoma, intraocular melanoma, merkel 75 % , 100 % , 125 % , 150 % , 175 % , or 200 % ) . The reduction cell carcinoma, mesothelioma, metastatic squamous neck in cellular growth occurs even in the presence of TNFa . In cancer , midline tract carcinoma, multiple endocrine neopla some embodiments, polypeptides ( e.g. , single - chain poly- sia syndromes, multiple myeloma/ plasma cell neoplasm , peptides, antibodies, or fragments thereof) of the invention myelodysplastic syndromes, nasal cavity and paranasal may be capable of restricting the growth of a population of 55 sinus cancer, nasopharyngeal cancer , neuroblastoma, non T - reg cells in the presence of TNFa to between 90 % and hodgkin lymphoma ( NHL ) , non - small cell lung cancer 150 % relative to untreated cells ( e.g. , 90 % , 100 % , 110 % , (NSCLC ), epithelial ovarian cancer, germ cell ovarian can 120 % , 130 % , 140 % , or 150 % , as described , e.g. , in Example cer , low malignant potential ovarian cancer , pancreatic neu 4 ) . Antagonistic TNFR2 polypeptides ( e.g. , single - chain roendocrine tumors, papillomatosis , paraganglioma, parana polypeptides , antibodies, or fragments thereof) of the inven- 60 sal sinus and nasal cavity cancer, parathyroid cancer, penile tion are also capable of restricting the proliferation of a cancer , pharyngeal cancer , pheochromocytoma, pituitary population of T -reg cells to less than 70 % ( e.g. , 60 % , 50 % , tumor, pleuropulmonary blastoma , primary peritoneal can 40 % , 30 % , 20 % , 10 % , 5 % , or 1 % ) of that of an untreated cer , rectal cancer , retinoblastoma, rhabdomyosarcoma, sali population of T - reg cells . Antagonistic TNFR2 polypeptides vary gland cancer, kaposi sarcoma , rhabdomyosarcoma, ( e.g. , single - chain polypeptides, antibodies, or fragments 65 sézary syndrome, small intestine cancer , soft tissue sarcoma , thereof) of the invention are also capable of decreasing the throat cancer, thymoma and thymic carcinoma, thyroid survival of a population of T - reg cells by about 10 % ( e.g. , by cancer, transitional cell cancer of the renal pelvis and ureter, US 10,906,982 B2 83 84 urethral cancer, endometrial uterine cancer, uterine sarcoma , it may also be administered as a pharmaceutical formulation vaginal cancer , vulvar cancer, and Waldenström macro- in combination with excipients, carriers, and optionally, globulinemia . additional therapeutic agents. For example , antagonistic TNFR2 polypeptides ( e.g. , Polypeptides ( e.g. , single - chain polypeptides , antibodies , single - chain polypeptides , antibodies, or fragments thereof ) 5 or fragments thereof) of the invention can be monitored for of the invention, such as variants of TNFRAB1 and their ability to attenuate the progression of a cell prolifera TNFRAB2 having a non - native constant region , e.g. , tion disease , such as cancer, by any of a variety of methods humanized TNFRAB1 and TNFRAB2 antibodies , and frag- known in the art . For instance , a physician may monitor the ments thereof ( e.g. , Fab fragments ), can be administered to response of a mammalian subject ( e.g. , a human ) to treat a patient ( e.g. , a mammalian patient, such as a human 10 ment with an antibody, antibody fragment, or single - chain patient) in order to treat Hodgkin's or cutaneous non- polypeptide of the invention by analyzing the volume of one Hodgkin's lymphoma, T cell lymphoma, ovarian cancer, or more tumors in the patient. For example, polypeptides colon cancer , multiple myeloma, or renal cell carcinoma. ( e.g. , single - chain polypeptides , antibodies , or fragments An anti - TNFR2 polypeptide ( e.g. , single -chain polypep- thereof) of the invention may be capable of reducing tumor tide , antibody, or antigen - binding fragment thereof) of the 15 volume by between 1 % and 100 % ( e.g. , 1 % , 5 % , 10 % , 20 % , invention can also be co - administered with a therapeutic 30 % , 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , 95 % , 99 % , or antibody that exhibits reactivity towards a cancer cell . In this 100 % ) . Alternatively, a physician may monitor the respon way , antagonistic TNFR2 polypeptides of the invention may siveness of a subject ( e.g. , a human ) t to treatment with synergize not only with the adaptive immune response , e.g. , antagonistic TNFR2 single - chain polypeptides , antibodies , by prolonging T - lymphocyte tumor reactivity, but also with 20 or antigen -binding fragments thereof of the invention by other inhibitors of tumor cell growth . Examples of addi- analyzing the T - reg cell population in the lymph of a tional therapeutic antibodies that can be used to treat cancer particular subject. For instance , a physician may withdraw a and other cell proliferation disorders include those that sample of blood from a mammalian subject ( e.g. , a human ) exhibit reactivity with a tumor antigen or a cell - surface and determine the quantity or density of a population of protein that is overexpressed on the surface of a cancer cell . 25 T -reg cells ( e.g. , CD4 + CD25 + FOXP3 + T - reg cells or CD17 + Exemplary antibodies that can be admixed , co - administered , T -reg cells ) using established procedures , such as fluores or sequentially administered with antagonistic TNFR2 poly- cence activated cell sorting. peptides of the invention include , without limitation , Trastu- Methods of Treating Infectious Diseases zamb ( HERCEPTIN® ) , Bevacizumab (AVASTIN® ), Antagonistic TNFR2 polypeptides ( e.g. , single -chain Cetuximab ( ERBITUX® ), Panitumumab ( VECTIBIX® ) , 30 polypeptides, antibodies, or fragments thereof) of the inven Ipilimumab ( YERVOY® ), Rituximab (RITUXAN® and tion can also be used for treating infectious diseases , such as MABTHERA® ), Alemtuzumab (CAMPATH® ), Ofatu- those caused by any one or more of a virus , a bacterium , a mumab ( ARZERRA® ), Gemtuzumab ozogamicin (MY- fungus, or a parasite. For instance , antagonistic TNFR2 LOTARG® ), Brentuximab vedotin ( ADCETRIS® ), 90Y- polypeptides ( e.g. , single - chain polypeptides, antibodies, or Ibritumomab Tiuxetan ( ZEVALIN® ), and 1311- 35 fragments thereof) can be administered to a mammalian Tositumomab ( BEXXAR® ), which are described in detail in subject ( e.g. , a human ) suffering from an infectious disease Scott et al . ( Cancer Immun ., 12 : 14-21 , 2012 ) ; incorporated in order to treat the disease , as well as to alleviate one or herein by reference . more symptoms of the disease . A physician having ordinary skill in the art can readily For example, antagonistic TNFR2 polypeptides ( e.g. , determine an effective amount of an antagonistic TNFR2 40 single - chain polypeptides, antibodies, or fragments thereof) polypeptide for administration to a mammalian subject ( e.g. , of the invention can be used for treating, or alleviating one a human ) in need thereof. For example , a physician could or more symptoms of, viral infections in a mammalian start prescribing doses of a polypeptide of the invention at subject, such as a human , that are caused by , e.g. , a member levels lower than that required in order to achieve the desired of the Flaviviridae family ( e.g. , a member of the Flavivirus, therapeutic effect and gradually increase the dosage until the 45 Pestivirus, and Hepacivirus genera ), which includes the desired effect is achieved . Alternatively, a physician may hepatitis C virus, Yellow fever virus ; Tick -borne viruses, begin a treatment regimen by administering an antagonistic such as the Gadgets Gully virus, Kadam virus , Kyasanur TFNR2 antibody or antibody fragment at a high dose and Forest disease virus, Langat virus, Omsk hemorrhagic fever subsequently administer progressively lower doses until a virus , Powassan virus, Royal Farm virus, Karshi virus, therapeutic effect is achieved ( e.g. , a reduction in the volume 50 tick - borne encephalitis virus, Neudoerfl virus, Sofjin virus, of one or more tumors , a decrease in the population of T - reg Louping ill virus and the Negishi virus ; seabird tick -borne cells , or remission of a cell proliferation disorder ). In viruses , such as the Meaban virus, Saumarez Reef virus, and general, a suitable daily dose of an antibody or antigen- the Tyuleniy virus ; mosquito -borne viruses, such as the Aroa binding fragment thereof of the invention will be an amount virus, dengue virus, Kedougou virus, Cacipacore virus , of the antibody which is the lowest dose effective to produce 55 Koutango virus, Japanese encephalitis virus, Murray Valley a therapeutic effect. A single - chain polypeptide, antibody, or encephalitis virus, St. Louis encephalitis virus, Usutu virus , antigen - binding fragment thereof of the invention may be West Nile virus, Yaounde virus, Kokobera virus, Bagaza administered by injection , e.g. , by intravenous, intramuscu- virus, Ilheus virus, Israel turkey meningoencephalo -myelitis lar, intraperitoneal, or subcutaneous injection , optionally virus, Ntaya virus, Tembusu virus, Zika virus, Banzi virus , proximal to the site of the target tissue ( e.g. , a tumor ) . A 60 Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus , daily dose of a therapeutic composition of an antibody or Sepik virus , Uganda S virus, Wesselsbron virus , yellow antigen - binding fragment thereof of the invention may be fever virus; and viruses with no known arthropod vector, administered as a single dose or as two , three , four, five , six such as the Entebbe bat virus, Yokose virus, Apoi virus, or more doses administered separately at appropriate inter- Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja vals throughout the day, week , month , or year, optionally , in 65 virus , San Perlita virus, Bukalasa bat virus, Carey Island unit dosage forms. While it is possible for an antibody or virus, Dakar bat virus, Montana myotis leukoencephalitis fragment thereof of the invention to be administered alone, virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat US 10,906,982 B2 85 86 virus, and the Cell fusing agent virus; a member of the dues 56-60 of SEQ ID NO : 7 ) and has a non -native constant Arenaviridae family , which includes the Ippy virus, Lassa region, such as a TNFR2 antibody that contains one or more virus ( e.g. , the Josiah , LP, or GA391 strain ), lymphocytic CDRs or a variant thereof of TNFRAB1 and / or TNFRAB2 ) choriomeningitis virus ( LCMV ), Mobala virus, Mopeia to a human in order to treat an HIV infection ( such as a virus, Amapari virus, Flexal virus, Guanarito virus, Junin 5 human suffering from AIDS ) . virus, Latino virus, Machupo virus, Oliveros virus, Parané Antagonistic TNFR2 polypeptides ( e.g. , single - chain virus, Pichinde virus, Pirital virus, Sabia virus, Tacaribe polypeptides, antibodies, or fragments thereof) of the inven virus , Tamiami virus , Whitewater Arroyo virus, Chapare virus, and Lujo virus; a member of the Bunyaviridae family tion can also be used for treating, or alleviating one or more ( e.g. , a member of the Hantavirus, Nairovirus, Orthobunya- 10 symptoms of, bacterial infections in a mammalian subject virus, and Phlebovirus genera ), which includes the Hantaan ( e.g. , a human ). Examples of bacterial infections that may be virus , Sin Nombre virus, Dugbe virus , Bunyamwera virus, treated by administration of an antagonistic TNFR2 anti Rift Valley fever virus , La Crosse virus, California encepha body or antibody fragment of the invention include , without litis virus, and Crimean - Congo hemorrhagic fever ( CCHF ) limitation , those caused by bacteria within the genera Strep virus; a member of the Filoviridae family, which includes 15 tococcus, Bacillus, Listeria, Corynebacterium , Nocardia , the Ebola virus ( e.g. , the Zaire , Sudan , Ivory Coast , Reston , Neisseria , Actinobacter, Moraxella , Enterobacteriacece and Uganda strains ) and the Marburg virus ( e.g. , the Angola , ( e.g. , E. coli , such as 0157 : H7 ) , Pseudomonas ( such as Ci67 , Musoke, Popp , Ravn and Lake Victoria strains ); a Pseudomonas aeruginosa ), Escherichia , Klebsiella, Serra member of the Togaviridae family ( e.g. , a member of the tia , Enterobacter, Proteus, Salmonella, Shigella, Yersinia , Alphavirus genus ), which includes the Venezuelan equine 20 Haemophilus, Bordetella ( such as Bordetella pertussis ), encephalitis virus ( VEE ) , Eastern equine encephalitis virus Legionella, Pasteurella, Francisella , Brucella , Bartonella , ( EEE ) , Western equine encephalitis virus ( WEE ) , Sindbis Clostridium , Vibrio , Campylobacter, Staphylococcus, Myco virus, rubella virus, Semliki Forest virus, Ross River virus, bacterium ( such as Mycobacterium tuberculosis and Myco Barmah Forest virus, O'nyong’nyong virus, and the chikun- bacterium avium paratuberculosis, and Helicobacter ( such gunya virus ; a member of the Poxviridae family ( e.g. , a 25 as Helicobacter pylori and Helicobacter hepaticus ). Particu member of the Orthopoxvirus genus ), which includes the larly, methods of the invention include administering an smallpox virus, monkeypox virus, and vaccinia virus; a antagonistic TNFR2 antibody ( e.g. , a TNFR2 antibody that member of the Herpesviridae family, which includes the specifically binds an epitope containing one or more resi herpes simplex virus ( HSV ; types 1 , 2 , and 6 ) , human herpes dues of the KCRPG sequence of TNFR2 ( residues 142-146 virusBarr virus( e.g. , ( types EBV 7) , andVaricella 8 ) , cytomegalovirus - Zoster virus, and ( CMV Kaposi's ), Epstein- sar 30 of SEQ ID NO : 7 ) and that does not exhibit specific binding coma associated -herpesvirus ( KSHV ) ; a member of the to an epitope containing the KCSPG sequence of TNFR2 Orthomyxoviridae family, which includes the influenza ( residues 56-60 of SEQ ID NO : 7 ) and has a non - native virus ( A , B , and C ) , such as the H5N1 avian influenza virus constant region, such as a TNFR2 antibody that contains one or H1N1 swine flu ; a member of the Coronaviridae family, 35 or more CDRs or a variant thereof of TNFRAB1 and / or which includes the severe acute respiratory syndrome TNFRAB2) to a human or a non - human mammal in order to ( SARS ) virus; a member of the Rhabdoviridae family, which treat a Mycobacterium tuberculosis infection . Particular includes the rabies virus and vesicular stomatitis virus methods of the invention include administering an antago ( VSV ) ; a member of the Paramyxoviridae family, which nistic TNFR2 antibody ( e.g. , a TNFR2 antibody that spe includes the human respiratory syncytial virus ( RSV ) , 40 cifically binds an epitope containing one or more residues of Newcastle disease virus, hendravirus , nipahvirus, measles the KCRPG sequence of TNFR2 ( residues 142-146 of SEQ virus, rinderpest virus, canine distemper virus, Sendai virus, ID NO : 7 ) and that does not exhibit specific binding to an human parainfluenza virus ( e.g. , 1 , 2 , 3 , and 4 ) , rhinovirus, epitope containing the KCSPG sequence of TNFR2 ( resi and mumps virus; a member of the Picornaviridae family, dues 56-60 of SEQ ID NO : 7 ) and has a non - native constant which includes the poliovirus, human enterovirus ( A , B , C , 45 region , such as a TNFR2 antibody that contains one or more and D ) , hepatitis A virus, and the coxsackievirus; a member CDRs or a variant thereof of TNFRAB1 and / or TNFRAB2 ) of the Hepadnaviridae family, which includes the hepatitis B to bovine mammals or bison in order to treat a Mycobacte virus; a member of the Papillamoviridae family, which rium tuberculosis infection . Additionally, methods of the includes the human papilloma virus; a member of the invention include administering an antagonistic TNFR2 Parvoviridae family, which includes the adeno - associated 50 antibody ( e.g. , a TNFR2 antibody that specifically binds an virus ; a member of the Astroviridae family, which includes epitope containing one or more residues of the KCRPG the astrovirus ; a member of the Polyomaviridae family, sequence of TNFR2 ( residues 142-146 of SEQ ID NO : 7 ) which includes the JC virus, BK virus, and SV40 virus; a and that does not exhibit specific binding to an epitope member of the Calciviridae family, which includes the containing the KCSPG sequence of TNFR2 ( residues 56-60 Norwalk virus; a member of the Reoviridae family, which 55 of SEQ ID NO : 7 ) and has a non - native constant region, such includes the rotavirus; and a member of the Retroviridae as a TNFR2 antibody that contains one or more CDRs or a family , which includes the human immunodeficiency virus variant thereof of TNFRAB1 and / or TNFRAB2) to a human (HIV ; e.g. , types 1 and 2 ) , and human T - lymphotropic virus or a non - human mammal in order to treat a Mycobacterium Types I and II (HTLV - 1 and HTLV - 2 , respectively ) ; Friend avium paratuberculosis infection . Particular methods of the Leukemia Virus ; and transmissible spongiform encepha- 60 invention include administering an antagonistic TNFR2 lopathy, such as chronic wasting disease . Particularly , meth- antibody ( e.g. , a TNFR2 antibody that specifically binds an ods of the invention include administering an antagonistic epitope containing one or more residues of the KCRPG TNFR2 antibody ( e.g. , a TNFR2 antibody that specifically sequence of TNFR2 ( residues 142-146 of SEQ ID NO : 7 ) binds an epitope containing one or more residues of the and that does not exhibit specific binding to an epitope KCRPG sequence of TNFR2 ( residues 142-146 of SEQ ID 65 containing the KCSPG sequence of TNFR2 ( residues 56-60 NO : 7 ) and that does not exhibit specific binding to an of SEQ ID NO : 7 ) and has a non -native constant region , such epitope containing the KCSPG sequence of TNFR2 ( resi- as a TNFR2 antibody that contains one or more CDRs or a US 10,906,982 B2 87 88 variant thereof of TNFRAB1 and / or TNFRAB2) to bovine characterized by a certain degree of purity after isolating the mammals or bison in order to treat a Mycobacterium avium antibody from cell culture media or after chemical synthesis, paratuberculosis infection . e.g. , of a single - chain antibody fragment ( e.g. , scFv ) by Antagonistic TNFR2 polypeptides ( e.g. , single - chain established solid - phase peptide synthesis methods or native polypeptides , antibodies, or fragments thereof) of the inven- 5 chemical ligation as described herein . An antagonistic tion can also be administered to a mammalian subject ( e.g. , TNFR2 antibody of the invention may be at least 10 % pure a human ) for treating, or alleviating one or more symptoms prior to incorporating the antibody into a pharmaceutical of, parasitic infections caused by a protozoan parasite ( e.g. , composition ( e.g. , 10 % , 20 % , 30 % , 40 % , 50 % , 60 % , 70 % , an intestinal protozoa, a tissue protozoa , or a blood proto- 80 % , 90 % , 95 % , 97 % , 98 % , 99 % , 99.5 % , 99.9 % , 99.99 % , zoa ) or a helminthic parasite ( e.g. , a nematode , a helminth , 10 or 100 % pure ). an adenophorea, a secementea, a trematode, a fluke (blood Pharmaceutical compositions of anti - TNFR2 polypep flukes, liver flukes, intestinal flukes, and lung flukes ), or a tides ( e.g. , single - chain polypeptides, antibodies, or frag cestode ) . Exemplary protozoan parasites that can be treated ments thereof) of the invention can be prepared for storage according to the methods of the invention include, without as lyophilized formulations or aqueous solutions by mixing limitation, Entamoeba hystolytica , Giardia lamblia , Cryp- 15 the antibody having the desired degree of purity with tosporidium muris , Trypanosomatida gambiense, Trypano- optional pharmaceutically acceptable carriers , excipients or somatida rhodesiense, Trypanosomatida crusi, Leishmania stabilizers typically employed in the art, e.g. , buffering mexicana , Leishmania braziliensis, Leishmania tropica , agents, stabilizing agents, preservatives, isotonifiers, non Leishmania donovani, Leishmania major, Toxoplasma gon- ionic detergents, antioxidants, and other miscellaneous addi dii , Plasmodium vivax , Plasmodium ovale, Plasmodium 20 tives . See , e.g. , Remington's Pharmaceutical Sciences, 16th malariae, Plasmodium falciparum , Plasmodium yoelli, edition ( Osol , ed . 1980 ; incorporated herein by reference ). Trichomonas vaginalis, and Histomonas meleagridis. Exem- Such additives must be nontoxic to the recipients at the plary helminthic parasites include richuris trichiura , dosages and concentrations employed . Ascaris lumbricoides, Enterobius vermicularis, Ancylo- Buffering Agents stoma duodenale, Necator americanus, Strongyloides ster- 25 Buffering agents help to maintain the pH in the range coralis, Wuchereria bancrofti, and Dracunculus medinensis, which approximates physiological conditions. They can be Schistosoma mansoni , Schistosoma haematobium , Schisto- present at concentration ranging from about 2 mM to about soma japonicum , Fasciola hepatica , Fasciola gigantica , 50 mM . Suitable buffering agents for use with antagonistic Heterophyes, Paragonimus westermani, Taenia solium , Tae- TNFR2 polypeptides ( e.g. , single - chain polypeptides , anti nia saginata, Hymenolepis nana , and Echinococcus granu- 30 bodies , or fragments thereof) of the invention include both losus. Additional parasitic infections that can be treated organic and inorganic acids and salts thereof such as citrate according to the methods of the invention include Onchocer- buffers { e.g. , monosodium citrate - disodium citrate mixture, cas volvulus. citric acid - trisodium citrate mixtu citric acid -monoso Antagonistic TNFR2 polypeptides ( e.g. , single - chain dium citrate mixture , etc. ), succinate buffers { e.g. , succinic polypeptides, antibodies, or fragments thereof) can also be 35 acid -monosodium succinate mixture, succinic acid - sodium administered to a mammalian subject ( e.g. , a human ) in hydroxide mixture , succinic acid - disodium succinate mix order to treat, or to alleviate one or more symptoms of, ture , etc. ) , tartrate buffers ( e.g. , tartaric acid - sodium tartrate fungal infections . Examples of fungal infections that may be mixture , tartaric acid - potassium tartrate mixture, tartaric treated according to the methods of the invention include , acid - sodium hydroxide mixture , etc. ) , fumarate buffers without limitation, those caused by, e.g. , Aspergillus, Can- 40 { e.g. , fumaric acid - monosodium fumarate mixture, fumaric dida, Malassezia , Trichosporon, Fusarium , Acremonium , acid - disodium fumarate mixture, monosodium fumarate Rhizopus, Mucor, Pneumocystis, and Absidia . Exemplary disodium fumarate mixture , etc. ), gluconate buffers { e.g. , fungal infections that can be treated according to the meth- gluconic acid - sodium glyconate mixture, gluconic acid ods of the invention also include Pneumocystis carinii , sodium hydroxide mixture , gluconic acid -potassium glyu Paracoccidioides brasiliensis and Histoplasma capsulatum . 45 conate mixture, etc.) , oxalate buffer { e.g. , oxalic acid Pharmaceutical Compositions sodium oxalate mixture , oxalic acid - sodium hydroxide Therapeutic compositions containing an antagonistic mixture, oxalic acid - potassium oxalate mixture , etc. ), lactate TNFR2 polypeptide, such as a single -chain polypeptide, buffers { e.g. , lactic acid - sodium lactate mixture , lactic acid antibody, or antigen -binding fragment thereof of the inven- sodium hydroxide mixture , lactic acid -potassium lactate tion can be prepared using methods known in the art. For 50 mixture, etc.) and acetate buffers { e.g. , acetic acid - sodium example , such compositions can be prepared using, e.g. , acetate mixture, acetic acid - sodium hydroxide mixture , physiologically acceptable carriers, excipients or stabilizers etc. ) . Additionally, phosphate buffers, histidine buffers and ( Remington's Pharmaceutical Sciences 16th edition , Osol , trimethylamine salts such as Tris can be used . A. Ed . ( 1980 ) ; incorporated herein by reference ), and in a Preservatives desired form , e.g. , in the form of lyophilized formulations or 55 Preservatives can be added to a composition of the aqueous solutions . The compositions can also be prepared so invention to retard microbial growth , and can be added in as to contain the active agent ( e.g. , an antagonistic anti- amounts ranging from 0.2 % -1 % ( w / v ). Suitable preserva TNFR2 antibody or fragment thereof) at a desired concen- tives for use with antagonistic TNFR2 polypeptides ( e.g. , tration . For example, a pharmaceutical composition of the single - chain polypeptides , antibodies, or fragments thereof) invention may contain at least 10 % ( e.g. , 10 % , 20 % , 30 % , 60 of the invention include phenol, benzyl alcohol , meta - cresol, 40 % , 50 % , 60 % , 70 % , 80 % , 90 % , 95 % , 97 % , 98 % , 99 % , methyl paraben , propyl paraben, octadecyldimethylbenzyl 99.5 % , 99.9 % , or 100 % ) active agent by weight ( w / w ). ammonium chloride , benzalconium halides { e.g. , chloride , Additionally, an active agent ( e.g. , an antagonistic TNFR2 bromide, and iodide ) , hexamethonium chloride, and alkyl antibody or fragment thereof of the invention ) that can be parabens such as methyl or propyl paraben , catechol, resor incorporated into a pharmaceutical formulation can itself 65 cinol , cyclohexanol, and 3 - pentanol. Isotonicifiers some have a desired level of purity . For example , an antibody or times known as “ stabilizers " can be added to ensure isoto antigen -binding fragment thereof of the invention may be nicity of liquid compositions of the invention and include US 10,906,982 B2 89 90 polhydric sugar alcohols , for example trihydric or higher CAR - T therapy ). For instance , an antagonistic TNFR2 sugar alcohols, such as glycerin , arabitol, xylitol, sorbitol single - chain polypeptide, antibody, antibody fragment, or and mannitol. Stabilizers refer to a broad category of excipi- therapeutic conjugate thereof ( e.g. , a drug -antibody conju ents which can range in function from a bulking agent to an gate described herein ), may be admixed with one or more additive which solubilizes the therapeutic agent or helps to 5 additional active agents that can be used to treat cancer or prevent denaturation or adherence to the container wall . another cell proliferation disorder ( e.g. , neoplasm ). Alterna Typical stabilizers can be polyhydric sugar alcohols ( enu- tively , pharmaceutical compositions of the invention may be merated above ); amino acids such as arginine, lysine , gly- formulated for co - administration or sequential administra cine, glutamine , asparagine , histidine , alanine , ornithine, tion with one or more additional active agents that can be L - leucine , 2 -phenylalanine , glutamic acid , threonine, etc. , 10 used to treat cancer or other cell proliferation disorders . organic sugars or sugar alcohols , such as lactose , trehalose , Examples of additional active agents that can be used to treat stachyose , mannitol, sorbitol, xylitol, ribitol, myoinisitol , cancer and other cell proliferation disorders and that can be galactitol, glycerol and the like, including cyclitols such as conjugated to , admixed with , or administered separately inositol ; polyethylene glycol; amino acid polymers; sulfur from an antagonistic TNFR2 single - chain polypeptide , anti containing reducing agents, such as urea , glutathione, thio- 15 body, or antibody fragment of the invention include cyto ctic acid , sodium thioglycolate , thioglycerol, a -monothio- toxic agents ( e.g. , those described herein ), as well as anti glycerol and sodium thio sulfate; low molecular weight bodies that exhibit reactivity with a tumor antigen or a polypeptides ( e.g. , peptides of 10 residues or fewer ); pro- cell - surface protein that is overexpressed on the surface of a teins such as human serum albumin , bovine serum albumin , cancer cell . Exemplary antibodies that can be conjugated to , gelatin or immunoglobulins; hydrophilic polymers , such as 20 admixed with , or administered separately from antagonistic polyvinylpyrrolidone monosaccharides, such as xylose , TNFR2 antibodies of the invention include, without limita mannose , fructose, glucose ; disaccharides such as lactose , tion , Trastuzamb ( HERCEPTIN? ) , Bevacizumab (AVAS maltose , sucrose and trisaccharides such as raffinose ; and TIN® ) , Cetuximab ( ERBITUX® ), Panitumumab polysaccharides such as dextran . Stabilizers can be present ( VECTIBIX® ), Ipilimumab ( YERVOY® ), Rituximab ( RIT in the range from 0.1 to 10,000 weights per part of weight 25 UXAN® and MABTHERA® ), Alemtuzumab (CAM active protein . PATH® ), Ofatumumab ( ARZERRA® ), Gemtuzumab ozo Detergents gamicin (MYLOTARG® ), Brentuximab vedotin Non - ionic surfactants or detergents ( also known as “ wet- ( ADCETRIS® ), 90Y - Ibritumomab Tiuxetan (ZEVALIN® ), ting agents ” ) can be added to help solubilize the therapeutic and 1311 - Tositumomab ( BEXXAR® ), which are described agent as well as to protect the therapeutic protein against 30 in detail in Scott et al . ( Cancer Immun ., 12 : 14-21 , 2012 ) ; agitation - induced aggregation , which also permits the for- incorporated herein by reference . mulation to be exposed to shear surface stressed without Additional agents that can be conjugated to , admixed causing denaturation of the protein . Suitable non - ionic sur- with , or administered separately from antagonistic TNFR2 factants include polysorbates ( 20 , 80 , etc. ) , polyoxamers polypeptides ( e.g. , single - chain polypeptides, antibodies, or ( 184 , 188 etc. ) , Pluronic polyols , polyoxyethylene sorbitan 35 fragments thereof) of the invention include T - lymphocytes monoethers ( TWEEN® - 20 , TWEEN® - 80 , etc. ). Non - ionic that exhibit reactivity with a specific antigen associated with surfactants can be present in a range of about 0.05 mg /mL a particular pathology. For instance , antagonistic TNFR2 to about 1.0 mg /mL , for example about 0.07 mg/ mL to about polypeptides ( e.g. , single - chain polypeptides, antibodies, or 0.2 mg /mL . fragments thereof) of the invention can be formulated for Additional miscellaneous excipients include bulking agents 40 administration with a T - cell that expresses a chimeric anti ( e.g. , starch ), chelating agents ( e.g. , EDTA ), antioxidants gen receptor ( CAR - T ) in order to treat a cell proliferation ( e.g. , ascorbic acid , methionine, vitamin E ) , and cosolvents . disorder, such as a cancer described herein . Antagonistic Other Pharmaceutical Carriers TNFR2 polypeptides ( e.g. , single - chain polypeptides , anti Alternative pharmaceutically acceptable carriers that can bodies , or fragments thereof) can synergize with CAR - T be incorporated into a composition of the invention may 45 therapy by preventing T - reg cells from deactivating T - lym include dextrose , sucrose , sorbitol, mannitol, starch , rubber phocytes that have been genetically modified so as to arable, potassium phosphate, arginate, gelatin , potassium express tumor - reactive antigen receptors . In this way , silicate , microcrystalline cellulose , polyvinylpyrrolidone, CAR - T cells can be administered to a patient prior to , cellulose , water , syrups , methyl cellulose , methylhydroxy concurrently with , or after administration of an antagonistic benzoate , propylhydroxy benzoate, talc , magnesium stear- 50 TNFR2 single - chain polypeptide , antibody, or antigen -bind ate , and mineral oils , but not limited to . A composition ing fragment thereof in order to treat a mammalian subject containing an antagonistic TNFR2 polypeptide of the inven- ( e.g. , a human ) suffering from a cell proliferation disorder, tion may further include a lubricant, a humectant, a sweet- such as cancer . ener , a flavoring agent, an emulsifier, a suspending agent, CAR - T therapy is a particularly robust platform for tar and a preservative. Details of suitable pharmaceutically 55 geting cancer cells in view of the ability to genetically acceptable carriers and formulations can be found in Rem- engineer T - lymphocytes to express an antigen receptor spe ington's Pharmaceutical Sciences ( 19th ed . , 1995 ) , which is cific to a tumor - associated antigen . For instance , identifica incorporated herein by reference . tion of antigens overexpressed on the surfaces of tumors and Compositions for Combination Therapy other cancer cells can inform the design and discovery of Pharmaceutical compositions of the invention may 60 chimeric T -cell receptors , which are often composed of optionally include more than one active agent. For instance , cytoplasmic and transmembrane domains derived from a compositions of the invention may contain an antagonistic naturally occurring T - cell receptor operatively linked to an TNFR2 polypeptide, such as a single -chain polypeptide, extracellular scFv fragment that specifically binds to a antibody, or fragment thereof conjugated to , admixed with , particular antigenic peptide. T - cells can be genetically modi or administered separately from another pharmaceutically 65 fied in order to express an antigen receptor that specifically active molecule , e.g. , a cytotoxic agent, an antibiotic , or a binds to a particular tumor antigen by any of a variety of T -lymphocyte ( e.g. , a gene - edited T - lymphocyte for use in genome editing techniques described herein or known in the US 10,906,982 B2 91 92 art. Exemplary techniques for modifying a T - cell genome so Blood - Brain Barrier Penetration as to incorporate a gene encoding a chimeric antigen recep- In certain embodiments , antagonistic TNFR2 polypep tor include the CRISPER / Cas, zinc finger nuclease , TALEN , tides ( e.g. , single - chain polypeptides, antibodies, or frag ARCUSTM platforms described herein . Methods for the ments thereof) of the invention can be formulated to ensure genetic engineering of CAR - T lymphocytes have been 5 proper distribution in vivo . described , e.g. , in WO 2014/127261 , WO 2014/039523 , WO For example, the blood - brain barrier ( BBB ) excludes 2014/099671 , and WO 20120790000 ; the disclosures of many highly hydrophilic compounds. To ensure that the each of which are incorporated by reference herein . therapeutic compositions of the invention cross the BBB ( if desired ) , they can be formulated , for example, in lipososomes . theCAR invention - T cells include useful those in the that compositions have been genetically and methods modi- of 10 Methods of manufacturing liposomes have been described, fied such that the cell does not express the endogenous T - cell e.g. , U.S. Pat . Nos . 4,522,811 ; 5,374,548 ; and 5,399,331 . receptor. For instance , a CAR - T cell may be modified by The liposomes may comprise one or more moieties that are genome - editing techniques, such as those described herein , selectively transported into specific cells or organs , thereby so as to suppress expression of the endogenous T - cell 15 enhancingClin . Pharmacol targeted. 29 drug :685 ,delivery 1989 )) . (Exemplary see , e.g. , V. targetingV. Ranade moi ( J. receptor in order to prevent graft- versus -host reactions in a eties include , e.g. , folate or biotin ( see , e.g. , U.S. Pat. No. patient receiving a CAR - T infusion . Additionally or alter- 5,416,016 ) ; mannosides (Umezawa et al . ( Biochem . Bio natively, CAR - T cells can be genetically modified so as to phys. Res . Commun . 153 : 1038 , 1988 ) ) ; antibodies ( P. G. reduce the expression of one or more endogenous MHC Bloeman et al . ( FEBS Lett . 357 : 140 , 1995 ) ; M. Owais et al . proteins. This is a particularly useful technique for the 20 ( Antimicrob . Agents Chemother. 39 : 180 , 1995 ) ) ; surfactant infusion of allogeneic T - lymphocytes, as recognition of protein A receptor ( Briscoe et al . ( Am . J. Physiol. 1233 : 134 , foreign MHC proteins represents one mechanism that pro- 1995 ) ) ; the disclosures of each of which are incorporated motes allograft rejection. One of skill in the art can also herein by reference . modify a T - lymphocyte so as to suppress the expression of Routes of Administration and Dosing immune suppressor proteins, such as programmed cell death 25 Antagonistic TNFR2 polypeptides ( e.g. , single - chain protein 1 ( PD - 1 ) and cytotoxic T - lymphocyte -associated polypeptides, antibodies , or fragments thereof) of the inven protein 4 ( CTLA - 4 ) . These proteins are cell surface recep- tion can be administered to a mammalian subject ( e.g. , a tors that, when activated , attenuate T -cell activation . Infu- human ) by a variety of routes such as orally , transdermally , sion of CAR - T cells that have been genetically modified so subcutaneously, intranasally , intravenously, intramuscularly, as to diminish the expression of one or more immunosu- 30 intraocularly , intratumorally, parenterally, topically, intrath pressor proteins represents one strategy that can be used to ecally and intracerebroventricularly . The most suitable route prolong the T -lymphocyte -mediated cytotoxicity in vivo . for administration in any given case will depend on the In addition to deleting specific genes , one can also modify particular antibody or antigen -binding fragment adminis CAR - T cells in order to express a T -cell receptor with a tered , the patient, pharmaceutical formulation methods, desired antigen specificity. For instance , one can genetically 35 administration methods ( e.g. , administration time and modify a T - lymphocyte in order to express a T - cell receptor administration route ), the patient's age , body weight, sex , that specifically binds to a tumor - associated antigen in order severity of the diseases being treated , the patient's diet, and to target infused T - cells to cancerous cells . An exemplary the patient's excretion rate . T - cell receptor that may be expressed by a CAR - T cell is one The effective dose of an anti - TNFR2 single -chain poly that binds PD - L1 , a cell surface protein that is often over- 40 peptide, antibody, or antigen - binding fragment thereof of the expressed on various tumor cells . As PD - L1 activates PD - 1 invention can range from about 0.0001 to about 100 mg/ kg on the surface of T - lymphocytes, targeting this tumor anti- of body weight per single ( e.g. , bolus ) administration , mul gen with CAR - T therapy can synergize with antagonistic tiple administrations or continuous administration, or to TNFR2 antibodies or antibody fragments of the invention in achieve a serum concentration of 0.0001-5000 ug /mL serum order to increase the duration of an immune response 45 concentration per single ( e.g. , bolus ) administration , mul mediated by a T - lymphocyte in vivo . CAR - T cells can also tiple administrations or continuous administration , or any be modified so as to express a T - cell receptor that specifi- effective range or value therein depending on the condition cally binds an antigen associated with one or more infectious being treated , the route of administration and the age , disease , such as an antigen derived from a viral protein , a weight, and condition of the subject. In certain embodi bacterial cell , a fungus, or other parasitic organism . 50 ments , e.g. , for the treatment of cancer , each dose can range Other pharmaceutical compositions of the invention from about 0.0001 mg to about 500 mg /kg of body weight . include those that contain an antagonistic TNFR2 antibody For instance , a pharmaceutical composition of the invention or antibody fragment, interferon alpha , and / or one or more may be administered in a daily dose in the range of 0.001 antibiotics that can be administered to a patient ( e.g. , a 100 mg /kg (body weight ). The dose may be administered human patient) suffering from an infectious disease . For 55 one or more times ( e.g. , 2-10 times ) per day, week , month , instance , an antagonistic TNFR2 antibody or antibody frag- or year to a mammalian subject ( e.g. , a human ) in need ment can be conjugated to , admixed with , or administered thereof. separately from an antibiotic useful for treating one or more Therapeutic compositions can be administered with medi infectious diseases, such as amikacin , gentamicin , kanamy- cal devices known in the art . For example , in an embodi cin , neomycin, netilmicin , tobramycin, paromomycin , strep- 60 ment , a therapeutic composition of the invention can be tomycin , spectinomycin , geldanamycin , herbimycin , rifaxi- administered with a needleless hypodermic injection device , min , loracarbef, ertapenem , doripenem , imipenem , such as the devices disclosed in U.S. Pat . Nos . 5,399,163 ; meropenem , cefadroxil, cefazolin , cefazlexin , cefaclor, 5,383,851 ; 5,312,335 ; 5,064,413 ; 4,941,880 ; 4,790,824 ; or cefoxitin , cefprozil, cefuroxime, cefdinir , cefditoren , cefop- 4,596,556 . Examples of well - known implants and modules erazone , clindamycin, lincomycin , daptomycin , erythromy- 65 useful in the invention include : U.S. Pat. No. 4,487,603 , cin , linezolid , torezolid , amoxicillin , ampicillin , bacitracin , which discloses an implantable micro - infusion pump for ciprofloxacin , doxycycline , and tetracycline , among others . dispensing medication at a controlled rate ; U.S. Pat . No. US 10,906,982 B2 93 94 4,486,194 , which discloses a therapeutic device for admin- quantity of T - reg cells in a blood sample withdrawn from a istering medicaments through the skin ; U.S. Pat . No. 4,447 , subject ( e.g. , a human ) that is undergoing treatment with an 233 , which discloses a medication infusion pump for deliv- antibody of the invention . Such a kit may contain , e.g. , ering medication at a precise infusion rate; U.S. Pat . No. antibodies that selectively bind cell - surface antigens pre 4,447,224 , which discloses a variable flow implantable 5 sented by T -reg cells , such as CD4 and CD25 . Optionally , infusion apparatus for continuous drug delivery; U.S. Pat . these antibodies may be labeled with a fluorescent dye, such No. 4,439,196 , which discloses an osmotic drug delivery as fluorescein or tetramethylrhodamine, in order to facilitate system having multi - chamber compartments; and U.S. Pat . analysis of a population of T -reg cells by fluorescence No. 4,475,196 , which discloses an osmotic drug delivery activated cell sorting ( FACS ) methods known in the art . Kits system . These patents are incorporated herein by reference . 10 of the invention may optionally contain one or more Many other such implants, delivery systems , and modules reagents that can be used to quantify a population of are known to those skilled in the art . tumor -reactive T - lymphocytes in order to determine the Kits Containing Antagonistic Anti - TNFR2 Polypeptides effectiveness of an antagonistic TNFR2 polypeptide of the This invention also includes kits that contain antagonistic invention in restoring tumor - infiltrating lymphocyte prolif anti - TNFR2 polypeptides ( e.g. , single -chain polypeptides, 15 eration . For instance , kits of the invention may contain an antibodies, or fragments thereof ). The kits provided herein antibody that selectively binds cell - surface markers on the may contain any of the antagonistic TNFR2 polypeptides surface of a cytotoxic T - cell, such as CD8 or CD3 . Option described above, as well as any of the polynucleotides ally , these antibodies may be labeled with fluorescent mol encoding these polypeptides, vectors containing these poly- ecules so as to enable quantitation by FACS analysis . peptides, or cells engineered to express and secrete poly- 20 A kit of the invention may also contain one or more peptides of the invention ( e.g. , prokaryotic or eukaryotic reagents useful for determining the affinity and selectivity of cells ) . A kit of this invention may include reagents that can an antagonistic TNFR2 single - chain polypeptide, antibody, be used to produce the compositions of the invention ( e.g. , or antigen - binding fragment thereof of the invention for one antagonistic anti- TNFR2 polypeptides , such as single - chain or more peptides derived from TNFR2 ( e.g. , a peptide polypeptides , antibodies, or fragments thereof, conjugates 25 containing the sequence of any one of SEQ ID NOs : 11 , 19 , containing antagonistic anti - TNFR2 polypeptides , poly- 20 , and 34-117 , or a peptide containing between about 10 nucleotides encoding antagonistic anti - TNFR2 polypep- and about 30 continuous or discontinuous amino acids tides , vectors containing these polypeptides ). Optionally, between positions 80 and 130 of SEQ ID NO : 7 ) . For kits of the invention may include reagents that can induce instance , a kit may contain an antagonistic TNFR2 antibody the expression of antagonistic TNFR2 antibodies within 30 and one or more reagents that can be used in an ELISAassay a cells ( e.g. , mammalian cells ) , such as doxycycline or tetra- to determine the Ky of an antibody of the invention for one cycline . In other cases , a kit of the invention may contain a or more peptides that present a TNFR2 epitope in a confor compound capable of binding and detecting a fusion protein mation similar to that of the epitope in the native protein . A that contains an antagonistic TNFR2 polypeptide and an kit may contain , e.g. , a microtiter plate containing wells that epitope tag . For instance , in such cases a kit of the invention 35 have been previously conjugated to avidin , and may contain may contain maltose , glutathione, a nickel - containing com- a library of TNFR2 - derived peptides , each of which conju plex , an anti - FLAG antibody, an anti -myc antibody, an gated to a biotin moiety . Such a kit may optionally contain anti -HA antibody, biotin , or streptavidin . a secondary antibody that specifically binds to the Fc region Kits of the invention may also include reagents that are of an antagonistic TNFR2 antibody of the invention , and the capable of detecting an antagonistic TNFR2 single - chain 40 secondary antibody may be conjugated to an enzyme ( e.g. , polypeptide , antibody, or fragment thereof directly . horseradish peroxidase ) that catalyzes a chemical reaction Examples of such reagents include secondary antibodies that that results in the emission of luminescent light. selectively recognize and bind particular structural features Kits of the invention may also contain antagonistic within the Fc region of an anti - TNFR2 antibody of the TNFR2 polypeptides ( e.g. , single -chain polypeptides, anti invention . Kits of the invention may contain secondary 45 bodies , or fragments thereof) of the invention and reagents antibodies that recognize the Fc region of an antagonistic that can be conjugated to such a polypeptide, including those TNFR2 antibody and that are conjugated to a fluorescent previously described ( e.g. , a cytotoxic agent, a fluorescent molecule . These antibody - fluorophore conjugates provide a molecule , a bioluminescent molecule , a molecule containing tool for analyzing the localization of antagonistic anti- a radioactive isotope , a molecule containing a chelating TNFR2 antibodies, e.g. , in a particular tissue or cultured 50 group bound to a paramagnetic ion , etc ). These kits may mammalian cell using established immunofluorescence additionally contain instructions for how the conjugation of techniques. In some embodiments , kits of the invention may an antagonistic TNFR2 polypeptide of the invention to a include additional fluorescent compounds that exhibit second molecule , such as those described above , can be known sub - cellular localization patterns. These reagents can achieved . be used in combination with another antibody - fluorophore 55 A kit of the invention may also contain a vector containing conjugate , e.g. , one that specifically recognizes a different a polynucleotide that encodes an antagonistic anti - TNFR2 receptor on the cell surface in order to analyze the localiza- single - chain polypeptide, antibody, or fragment thereof, tion of an anti - TNFR2 antibody relative to other cell - surface such as any of the vectors described herein . Alternatively, a proteins. kit may include mammalian cells ( e.g. , CHO cells ) that have Kits of the invention may also contain a reagent that can 60 been genetically altered to express and secrete antagonistic be used for the analysis of a patient's response to treatment TNFR2 polypeptides ( e.g. , single - chain polypeptides , anti by administration of antagonistic TNFR2 polypeptides ( e.g. , bodies , or fragments thereof) from the nuclear genome of the single - chain polypeptides, antibodies, or fragments thereof) cell . Such a kit may also contain instructions describing how of the invention . For instance , kits of the invention may expression of the antagonistic TNFR2 single - chain polypep include an antagonistic TNFR2 polypeptide , such as a 65 tide , antibody , or fragment thereof from a polynucleotide single - chain polypeptide, antibody , or antibody fragment, can be induced , and may additionally include reagents ( such and one or more reagents that can be used to determine the as , e.g. , doxycycline or tetracycline) that can be used to