CD3 BINDING MOLECULES

Abstract

The invention relates to heavy chain variable regions, binding domains and antibodies specific for human CD3, and CD3 binding proteins. The invention further relates to the use of a CD3 binding protein, preferably an antibody, of the invention in the treatment of cancer or autoimmune disease.

Claims

1. An antigen-binding protein that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region wherein the heavy chain variable region comprises: a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00073 (SEQ ID NO: 42) CDR1: SFGIS (SEQ ID NO: 43) CDR2: GFIPVLGTANYAQKFQG  (SEQ ID NO: 44) CDR3: RGNWNPFDP; or comprising the amino acid sequence: TABLE-US-00074 (SEQ ID NO: 6) CDR1: SX.sub.1TFTIS; (SEQ ID NO: 7) CDR2: GIIPX.sub.2FGTITYAQKFQG; (SEQ ID NO: 8) CDR3: RGNWNPFDP; in particular wherein X.sub.1=K or R; X.sub.2=L or I; or a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00075 (SEQ ID NO: 84) CDR1: SKTLTIS; (SEQ ID NO: 85) CDR2: GIIPIFGSITYAQKFQD; (SEQ ID NO: 86) CDR3: RGNWNPFDP; or comprising the amino acid sequence: TABLE-US-00076 (SEQ ID NO: 100) CDR1: GSGIS; (SEQ ID NO: 101) CDR2: GFIPFFGSANYAQKFRD; (SEQ ID NO: 8) CDR3: RGNWNPX.sub.13DP; wherein X.sub.13=or L or F; or the amino acid sequence: TABLE-US-00077 (SEQ ID NO: 45) EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGG FIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRG NWNPFDPWGQGTLVTVSS; or (SEP ID NO: 54) QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWL GGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTR RGNWNPFDPWGQGTLVTVSS; or (SEQ ID NO: 63) EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWL GSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTR RGNWNPFDPWGQGTLVTVSS; or (SEP ID NO: 71) QVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGG FIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRG NWNPLDPWGQGTLVTVSS; or (SEP ID NO: 87) QVQLVQSGAEVKKPGSSVKVSCKASGVTFKSKTLTISWVRQAPGQGLEWL GGIIPIFGSITYAQKFQDRVSITADKSTNTAYLELNSLRSEDTAIYYCAR RGNWNPFDPWGQGTLVTVSS; or (SEP ID NO: 103) EVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGG FIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRG NWNPFDPWGQGTLVTVSS; with 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or a combination thereof at one or more positions other than the CDRs; or a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00078 (SEQ ID NO: 9) CDR1: RX.sub.3WIG; (SEQ ID NO: 200) CDR2: IIYPGDSDTRYSPSFQG; (SEQ ID NO: 10) CDR3: X.sub.4IRYFX.sub.5WSEDYHYYX.sub.6DV; wherein X.sub.3=F or Y; X.sub.4=H or N; X.sub.5=D or V; X.sub.6=L or M; or the amino acid sequence TABLE-US-00079 (SEQ ID NO: 202) EVQLVQSGAEVKKPGESLKISCKGSGYSFTRFWIGWVRQMPGKGLEWMGI IYPGDSDTRYSPSFQGQVTISADKSTSTAYLQWSSLKASDTGMYYCVRHI RYFDWSEDYHYYLDVWGKGTTVTVSS; or (SEQ ID NO: 211) EVQLVESGAEVKKPGESLKISCKGSGYSFTRYWIGWVRQMPGKGLEWMGI IYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRNI RYFVWSEDYHYYMDVWGKGTTVTVSS; with 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or a combination thereof at one or more positions other than the CDRs; or a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00080 (SEQ ID NO: 117) CDR1: SYALS; (SEQ ID NO: 118) CDR2: GISGSGRTTWYADSVKG; (SEQ ID NO: 119) CDR3: DGGYSYGPYWYFDL; or a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00081 (SEQ ID NO: 125) CDR1: SYALS; (SEQ ID NO: 126) CDR2: AISGSGRTTWYADSVKG; (SEQ ID NO: 127) CDR3: DGGYTYGPYWYFDL; or the amino acid sequence TABLE-US-00082 (SEQ ID NO: 120) QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSG ISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDG GYSYGPYWYFDLWGRGTLVTVSS; or (SEQ ID NO: 128) QVQLVESGGGLVQPGGSLRLSCATSGFTFISYALSWVRQAPGKGLEWVSA ISGSGRTTWYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDG GYTYGPYWYFDLWGRGTLVTVSS; with 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or a combination thereof at one or more positions other than the CDRs, or a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00083 (SEQ ID NO: 142) CDR1: DYTMH; (SEQ ID NO: 143) CDR2: DISWSSGSIGYADSVKG; (SEQ ID NO: 144) CDR3: DHRGYGDYEGGGFDY; the amino acid sequence TABLE-US-00084 (SEQ ID NO: 145) EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSD ISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDH RGYGDYEGGGFDYWGQGTLVTVSS; or (SEQ ID NO: 153) EVQLVESGGGLVQPGRSLRLSCATSGFTFDDYTMHWVRQAPGKGLEWVSD ISWSSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYFCAKDH RGYGDYEGGGFDHWGQGTLVTVSS; or (SEP ID NO: 185) EVQLVESGGGLVQPGRSLRLSCVTSGFTFDDYTMHWVRQAPGKGLEWVSD ISWSSGTTGYADSVKGRFTISRDNAKDSLYLQMNSLRTEDTALYYCAKDH MGYGDYEGGGFDYWGQGTLVTVSS; or (SEQ ID NO: 169) EVQLVESGGVVVQPGGSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSD ISWSGGSIYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAKDH RGYGDYEGGGFDYWGRGTLVTVSS; with 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or a combination thereof at one or more positions other than the CDRs, or a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00085 (SEQ ID NO: 182) CDR1: DYTMH; (SEQ ID NO: 11) CDR2: DISWSX.sub.7GX.sub.8X.sub.9X.sub.10YADSVKG; (SEQ ID NO: 12) CDR3: DHX.sub.11GYGDYEGGGFDX.sub.12; wherein X.sub.7=S or G; X.sub.8=S or T; X.sub.9=I or T; X.sub.10=G or Y; X.sub.11=R or M; X.sub.12=H or Y, preferably X.sub.7, X.sub.8, X.sub.9 and X.sub.10 are S, S, I and G, and X.sub.11 and X.sub.2 are R and H, or preferably X.sub.7, X.sub.8, X.sub.9 and X.sub.10 are G, S, I and Y, and X.sub.11 and X.sub.2 are R and Y, or preferably X.sub.7, X.sub.8, X.sub.9 and X.sub.10 are S, T, T and G, and X.sub.11 and X.sub.2 are M and Y.

2-13. (canceled)

14. The antigen-binding protein of claim 1, wherein said light chain variable region comprises a common light chain variable region.

15. The antigen-binding protein of claim 1, wherein said common light chain variable region comprises an IgVκ1-39 light chain variable region.

16. The antigen-binding protein of claim 1, wherein said light chain variable region is a germline IgVκ1-39*01 variable region.

17. The antigen-binding protein of claim 1, wherein the light chain variable region comprises the kappa light chain IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ5*01.

18. The antigen-binding protein of claim 1, wherein the light chain variable region comprises the germline kappa light chain IgVκ1-39*01/IGJκ1*01 or IgVκ1-39*01/IGJκ5*01.

19. The antigen-binding protein of claim 1, wherein said light chain variable region comprises the amino acid sequence TABLE-US-00086 (SEQ ID NO: 16) DIQMT[[ ]]QSPSS[[ ]]LSASV[[ ]]GDRVT[[ ]]ITCRA[[ ]] SQSIS[[ ]]SYLNW[[ ]]YQQKP[[ ]]GKAPK[[ ]]LLIYA[[ ]] ASSLQ[[ ]]SGVPS[[ ]]RFSGS[[ ]]GSGTD[[ ]]FTLTI[[ ]] SSLQP[[ ]]EDFAT[[ ]]YYCQQ[[ ]]SYSTP[[ ]]PTFGQ[[ ]] GTKVE[[ ]]IK or (SEQ ID NO: 19) DIQMT[[ ]]QSPSS[[ ]]LSASV[[ ]]GDRVT[[ ]]ITCRA[[ ]] SQSIS[[ ]]SYLNW[[ ]]YQQKP[[ ]]GKAPK[[ ]]LLIYA[[ ]] ASSLQ[[ ]]SGVPS[[ ]]RFSGS[[ ]]GSGTD[[ ]]FTLTI[[ ]] SSLQP[[ ]]EDFAT[[ ]]YYCQQ[[ ]]SYSTP[[ ]]PITFG[[ ]] QGTRL[[ ]]EIK with 0-5 amino acid variations, insertions, deletions, substitutions, additions or a combination thereof.

20. The antigen-binding protein of claim 1, which is an antibody.

21-22. (canceled)

23. The antigen-binding protein of claims claim 20, which wherein the antibody is a human or humanized antibody.

24-33. (canceled)

34. A bispecific antibody comprising an antigen-binding protein of claim 1.

35. The bispecific antibody of claim 34, further comprising a H/L chain combination that binds a tumor-antigen.

36. The bispecific antibody of claim 35, wherein said H/L chain combination that binds a tumor-antigen binds human BCMA, CD19, CD20, CD30, CD33, CD38, CD44, CD123, CD138, CEA, CLEC12A, CS-1, EGFR, EGFRvIII, EPCAM, DLL3, LGR5, MSLN, FOLR1, FOLR3, HER2, HM1.24, MCSP, PD-L1, PSMA protein or a variant thereof.

37. The bispecific antibody of claim 34, which is a human or humanized antibody.

38. The bispecific antibody of claim 34, comprising two different immunoglobulin heavy chains with compatible heterodimerization domains.

39. The bispecific antibody of claim 38, wherein said compatible heterodimerization domains are compatible immunoglobulin heavy chain CH3 heterodimerization domains.

40. The bispecific antibody of claim 34, wherein said bispecific antibody is an IgG antibody with a mutant CH2 and/or lower hinge domain such that interaction of the bispecific IgG antibody to a Fc-gamma receptor is reduced.

41. The bispecific antibody of claim 40, wherein the mutant CH2 and/or lower hinge domain comprise an amino substitution at position 235 and/or 236 (according to EU numbering).

42. The bispecific antibody of claim 34, comprising a common light chain.

43. A method for treating a subject in need thereof, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antigen-binding protein that binds human CD3 comprising an antibody variable domain comprising a heavy chain variable region and a light chain variable region wherein the heavy chain variable region comprises: a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00087 (SEQ ID NO: 42) CDR1: SFGIS (SEQ ID NO: 43) CDR2: GFIPVLGTANYAQKFQG (SEQ ID NO: 44) CDR3: RGNWNPFDP; or comprising the amino acid sequence: TABLE-US-00088 (SEQ ID NO: 6) CDR1: SX.sub.1TFTIS; (SEQ ID NO: 7) CDR2: GIIPX.sub.2FGTITYAQKFQG; (SEQ ID NO: 8) CDR3: RGNWNPFDP;  wherein X.sub.1=K or R; X.sub.2=L or I; or a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00089 (SEQ ID NO: 84) CDR1: SKTLTIS; (SEQ ID NO: 85) CDR2: GIIPIFGSITYAQKFQD; (SEQ ID NO: 86) CDR3: RGNWNPFDP or comprising the amino acid sequence: TABLE-US-00090 (SEQ ID NO: 100) CDR1: GSGIS; (SEQ ID NO: 101) CDR2: GFIPFFGSANYAQKFRD; (SEQ ID NO: 8) CDR3: RGNWNPX.sub.13DP; wherein X.sub.13=or L or F; or the amino acid sequence: TABLE-US-00091 (SEQ ID NO: 45) EVQLVQSGAEVKKPGSSVKVSCKASGGTFRSFGISWVRQAPGQGLEWMGG FIPVLGTANYAQKFQGRVTIIADKSTNTAYMELSSLRSEDTAVYYCARRG NWNPFDPWGQGTLVTVSS; or (SEQ ID NO: 54) QVQLVQSGAEVKKPGSSVKVSCKASGDAFKSKTFTISWVRQAPGQGLEWL GGIIPLFGTITYAQKFQGRVTITADKSTNTAFMELSSLRSEDTAMYYCTR RGNWNPFDPWGQGTLVTVSS; or (SEQ ID NO: 63) EVQLVQSGSELKKPGSSVKVSCKASGVTFNSRTFTISWVRQAPGQGLEWL GSIIPIFGTITYAQKFQGRVTITADKSTSTAFMELTSLRSEDTAIYYCTR RGNWNPFDPWGQGTLVTVSS; or (SEQ ID NO: 71) QVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGG FIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRG NWNPLDPWGQGTLVTVSS; or (SEQ ID NO: 87) QVQLVQSGAEVKKPGSSVKVSCKASGVTFKSKTLTISWVRQAPGQGLEWL GGIIPIFGSITYAQKFQDRVSITADKSTNTAYLELNSLRSEDTAIYYCAR RGNWNPFDPWGQGTLVTVSS; or (SEQ ID NO: 103) EVQLVQSGAEVKKPGSSVKVSCKASGGTFRGSGISWVRQAPGQGLEWVGG FIPFFGSANYAQKFRDRVTITADKSATTAYMELSSLRSEDTAIYYCAKRG NWNPFDPWGQGTLVTVSS; with 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or a combination thereof at one or more positions other than the CDRs; or a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00092 (SEQ ID NO: 9) CDR1: RX.sub.3WIG; (SEQ ID NO: 200) CDR2: IIYPGDSDTRYSPSFQG; (SEQ ID NO: 10) CDR3: X.sub.4IRYFX.sub.5WSEDYHYYX.sub.6DV; wherein X.sub.3=F or Y; X.sub.4=H or N; X.sub.5=D or V; X.sub.6=L or M; or the amino acid sequence TABLE-US-00093 (SEQ ID NO: 202) EVQLVQSGAEVKKPGESLKISCKGSGYSFTRFWIGWVRQMPGKGLEWMGI IYPGDSDTRYSPSFQGQVTISADKSTSTAYLQWSSLKASDTGMYYCVRHI RYFDWSEDYHYYLDVWGKGTTVTVSS; or (SEQ ID NO: 211) EVQLVESGAEVKKPGESLKISCKGSGYSFTRYWIGWVRQMPGKGLEWMGI IYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCVRNI RYFVWSEDYHYYMDVWGKGTTVTVSS; with 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or a combination thereof at one or more positions other than the CDRs; or a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00094 (SEQ ID NO: 117) CDR1: SYALS; (SEQ ID NO: 118) CDR2: GISGSGRTTWYADSVKG; (SEQ ID NO: 119) CDR3: DGGYSYGPYWYFDL; or a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00095 (SEQ ID NO: 125) CDR1: SYALS; (SEQ ID NO: 126) CDR2 AISGSGRTTWYADSVKG; (SEQ ID NO: 127) CDR3: DGGYTYGPYWYFDL; or the amino acid sequence TABLE-US-00096 (SEQ ID NO: 120) QVQLVQSGGGLVQPGGSLRLSCATSGFKFSSYALSWVRQAPGKGLEWVSG ISGSGRTTWYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDG GYSYGPYWYFDLWGRGTLVTVSS; or (SEQ ID NO: 128) QVQLVESGGGLVQPGGSLRLSCATSGFTFISYALSWVRQAPGKGLEWVSA ISGSGRTTWYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDG GYTYGPYWYFDLWGRGTLVTVSS; with 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or a combination thereof at one or more positions other than the CDRs; or a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00097 (SEQ ID NO: 142) CDR1: DYTMH; (SEQ ID NO: 143) CDR2: DISWSSGSIGYADSVKG; (SEQ ID NO: 144) CDR3: DHRGYGDYEGGGFDY; or the amino acid sequence TABLE-US-00098 (SEQ ID NO: 145) EVQLVESGGGLVQPGRSLRLSCATSGFNFDDYTMHWVRQAPGKGLEWVSD ISWSSGSIGYADSVKGRFTISRDNAKNSLWLQMNSLRTEDTALYFCAKDH RGYGDYEGGGFDYWGQGTLVTVSS; or (SEQ ID NO: 153) EVQLVESGGGLVQPGRSLRLSCATSGFTFDDYTMHWVRQAPGKGLEWVSD ISWSSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYFCAKDH RGYGDYEGGGFDHWGQGTLVTVSS; or (SEQ ID NO: 185) EVQLVESGGGLVQPGRSLRLSCVTSGFTFDDYTMHWVRQAPGKGLEWVSD ISWSSGTTGYADSVKGRFTISRDNAKDSLYLQMNSLRTEDTALYYCAKDH MGYGDYEGGGFDYWGQGTLVTVSS; or (SEQ ID NO: 169) EVQLVESGGVVVQPGGSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSD ISWSGGSIYYADSVKGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAKDH RGYGDYEGGGFDYWGRGTLVTVSS; with 0-10, preferably 0-5 amino acid variations, insertions, deletions, substitutions, additions or a combination thereof at one or more positions other than the CDRs; or a CDR1, CDR2 and CDR3 comprising the amino acid sequence: TABLE-US-00099 (SEQ ID NO: 182) CDR1: DYTMH; (SEQ ID NO: 11) CDR2: DISWSX.sub.7GX.sub.8X.sub.9X.sub.10YADSVKG; (SEQ ID NO: 12) CDR3: DHX.sub.11GYGDYEGGGFDX.sub.12; wherein X.sub.7=S or G; X.sub.8=S or T; X.sub.9=I or T; X.sub.10=G or Y; X.sub.11=R or M; X.sub.12=H or Y, preferably X.sub.7, X.sub.8, X.sub.9 and X.sub.10 are S, S, I and G, and X.sub.11 and X.sub.12 are R and H, or preferably X.sub.7, X.sub.8, X.sub.9 and X.sub.10 are G, S, I and Y, and X.sub.11 and X.sub.12 are R and Y, or preferably X.sub.7, X.sub.8, X.sub.9 and X.sub.10 are S, T, T and G, and X.sub.11 and X.sub.12 are M and Y.

44. The method of claim 43, wherein the subject has cancer or is treated for cancer.

45. The method of claim 43, wherein the subject has an over-active immune system.

46. The method of claim 45, wherein the subject has an auto-immune disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0336] FIG. 1

[0337] Evaluation of functional activity: T cell cytotoxicity assay with BxPC3 target cells upon treatment with EGFRxCD3 bispecific antibodies. Each bispecific antibody comprises a CD3 binding domain comprised of a heavy chain variable region designated by MF number, and an EGFR binding domain comprising a heavy chain variable region MF8233. These variable regions are paired with a common light chain to form an EGFRxCD3 bispecific antibody. Affinity (HPB-ALL binding) versus BxPC3 lysis. Certain antibodies of the invention exhibit a relative low level of binding to HPB-ALL cells indicating that the CD3 binding domain of the antibody binds human CD3 with a comparatively low affinity. It is clear that the relative lower affinity does not necessarily prohibit tumor antigen mediated T cell cytotoxicity of BxPC3 cells (BxPC3 lysis, vertical axis). The bispecific antibodies MF8233×MF8508 and MF8233×MF8057 can efficiently lyse BxPC3 cells whereas the bispecific antibodies MF8233×MF8397 and MF8233×MF9249 do not do so efficiently while having similar binding. Further, a comparison of the bispecific antibody MF8233×MF6955 binds HPB-ALL (i.e. human CD3) with a higher affinity but does not lyse BxPC3 cells more efficiently than the bispecific antibodies MF8233×MF8508 and MF8233×MF8057 that bind CD3 to a lesser extent. MF6955 is a heavy chain variable region combined with a common light chain and used as comparator sequences, and corresponds to H1 H7232B (1129) VH, in US2014/0088295 A1. The comparator bispecific antibody having MF6955 and the same EGFR binding domain separately has a higher affinity for human CD3 than MF9267, and exhibits more efficient killing than MF9267. In contrast, other antibodies incorporating a CD3 binding domain of the invention, such as MF8058, have approximately the same binding activity as MF6955, yet demonstrate more efficient killing of BxPC3 cells, such as MF8233×MF8058. Other bispecific antibodies comprising a binding domain of the invention capable of binding CD3 demonstrate relative high binding and more efficient killing, such as MF8233×MF8078, which are useful for particular applications described herein. Whereas other bispecific antibodies comprising a binding domain of the invention capable of binding CD3 demonstrate relative low affinity and low killing, such as MF8233×MF9249 and MF8233×MF8397, which are useful for alternative applications described herein.

[0338] FIG. 2

[0339] Antibody titration curves indicating their capacity to induce T cell mediated % killing of BxPC3 target cells compared to no antibody control. Curves for the antibodies MF8233×MF8078, MF8233×MF8397; and MF8233×MF8508 are shown.

[0340] FIG. 3

[0341] Summary of the titration curve data of various bispecific antibodies in T cell cytotoxicity with BxPC3 target cells. The CD3 Fab column indicates the MF number of the CD3 binding arm. The EGFR arm has the indicated MF8233 number. The column indicates the supercluster numbers to set out variants based on the same VH gene segment. The column indicating CD3 binding reflect the results of the HBP-ALL binding experiment.

The results of two independent cytotoxicity assays to determine capacity to induce T cell mediated lysis of BxPC3 target cells are shown.

[0342] FIG. 4

[0343] T cell activation in T cell cytotoxicity assay with BxPC3 target cells on CD8+T cells with the expression. Antibody titration curves of various supercluster numbers, which set out variant CD3 binding domains. The other arm of the bispecific antibody has the heavy chain binding domain of MF8233. For comparison the bispecific antibodies MF8233×MF6955 and MF8233×MF6964 were tested as well, where MF6955 and MF6964 are heavy chain variable regions combined with a common light chain and used as comparator sequences, and correspond to H1 H7232B (1129) VH and HH7241 B (1145) respectively, in US2014/0088295 A1

[0344] FIG. 5

[0345] Summary of the titration curve data of various antibodies in T cell activation T cell cytotoxicity assay with BxPC3 target cells. The MF nr. column indicates the MF number of the CD3 binding domain. The EGFR binding domain has the indicated MF8233 number. The supercluster information of the various CD3 binding domain sequences is indicated in column “supercluster”; The column indicating CD3 affinity reflects the results of the HBP-ALL binding experiment.

The results of CD4+and CD8+cells are shown for the markers CD69 and CD25. The indicated bispecific antibodies are examples from a larger pool of bispecific antibodies.

[0346] FIG. 6

[0347] Evaluation of functional activity: T cell cytotoxicity assay with BxPC3 target cells. Affinity (HPB-ALL binding) versus CD8+T cell activation measured by CD69 expression. Certain antibodies of the invention exhibit a relative low level of binding to HPB-ALL cells indicating that the CD3 binding domain of the antibody binds human CD3 with a relative low affinity. Such affinity does not necessarily prohibit tumor antigen mediated T-cell activation as exemplified by the results of the CD8 positive CD69 activation analysis. The bispecific antibodies MF8233×MF8508 and MF8233×MF8057 can efficiently activate T cells whereas the bispecific antibodies MF8233×MF8397 and MF8233×MF9249 do not do so as efficiently. Certain CD3 binding domains that do not bind efficiently to these cells also do not activate T cells (see lower left corner). Whereas other CD3 binding domains MF8508 and MF8057, which bind HPB-ALL cells less than comparator CD3 binding domain MF6955, for example, activate T cells to a similar degree. Other bispecific antibodies comprising a binding domain of the invention capable of binding CD3 demonstrate relative high binding and high levels of activation, such as MF8078, which has use in particular applications described herein. Whereas other bispecific antibodies comprising a binding domain of the invention capable of binding CD3 demonstrates relative low affinity and low activation, such as MF9249 and MF8397, which are useful for alternative applications described herein.

[0348] FIG. 7

[0349] Evaluation of functional activity: T cell cytotoxicity assay with HCT116 target cells. Affinity (HPB-ALL binding) versus HCT-116 lysis.

Certain bispecific antibodies of the invention exhibit a low level of binding to HPB-ALL cells indicating that the CD3 binding domain of the antibody binds human CD3 with a comparatively low affinity. It is clear that the low affinity does not necessarily prohibit tumor antigen mediated cell lysis of HCT-116 cells (vertical axis). The bispecific antibodies MF8233×MF8508 and MF8233×MF8057 can efficiently lyse HCT-116 cells whereas the bispecific antibodies MF8233×x MF8397 and MF8233×MF9249 do not do so efficiently. For comparison the bispecific antibodies MF8233×MF6955 and MF8233×MF6964 bind HPB-ALL (i.e. human CD3) with a higher affinity than, for example, MF8233×MF8508, MF8233×MF8057 and MF8233×MF9267, but do not lyse HCT-116 cells more efficiently than MF8233×MF8508 or MF8233×MF9267, or significantly more than MF8233×MF8057 relative to the difference in binding, in a test as presented here. Another bispecific antibodies comprising a binding domain of the invention capable of binding CD3 demonstrates relative high binding and high levels of killing, such as MF8078, which has use in particular applications described herein. Whereas other bispecific antibodies comprising a binding domain of the invention capable of binding CD3 demonstrate relative low affinity and low killing, such as MF9249 and MF8397, which are useful for alternative applications described herein.

[0350] FIG. 8

[0351] Antibody titration curves in T cell cytotoxicity assay with HCT-116 target cells indicating the % killing of HCT-116 cells compared to no antibody control. Curves for various bispecific antibodies are shown.

[0352] FIG. 9

[0353] Summary of the titration curve data of various antibodies in T cell cytotoxicity assay with HCT-116 target cells. The CD3 Fab column indicates the MF number of the CD3 binding domain. The EGFR binding domain has the indicated MF8233 number. The column indicates the supercluster numbers to set out variants based on the same VH gene segment. The column indicating CD3 binding reflect the results of the HBP-ALL binding experiment. Percentage lysis of HCT-116 cells and EC50 values for lysis (ng/mL) are indicated in the next columns. The indicated bispecific antibodies are examples from a larger pool of bispecific antibodies.

[0354] FIG. 10

[0355] FIGS. 10a and 10b set out a schematic diagram of the MV1624 expression vector and the MV1625 expression vector.

[0356] FIG. 11

[0357] Common light chain used in mono- and bispecific IgG.

FIG. 11A: Common light chain amino acid sequence. FIG. 11B: Common light chain variable domain DNA sequence and translation (IGKV1-39/jk1). FIG. 11C: Common light chain constant region DNA sequence and translation. FIG. 11D: IGKV1-39/jk5 common light chain variable domain translation. FIG. 11E: V-region IGKV1-39A; FIG. 11F: CDR1, CDR2 and CDR3 of the common light chain.

[0358] FIG. 12

[0359] IgG heavy chains for the generation of bispecific molecules. FIG. 12A: CH1 region. FIG. 12B: hinge region. FIG. 12C: CH2 region. FIG. 12D: CH2 containing L235G and G236R silencing substitutions. FIG. 12E: CH3 domain containing substitutions L351 K and T366K (KK). FIG. 12F; CH3 domain containing substitutions L351 D and L368E (DE).

[0360] FIG. 13

[0361] Sequences of various DNA encoding and amino acid sequences of the heavy chain variable regions and parts thereof described in the specification.

[0362] FIG. 14

[0363] Characterization of additional clones from supercluster 1 in comparison with clones MF8057 and MF8058. A: Binding of selected MF clones with HPB-ALL human cells expressing a human CD3-TCR complex in a FACS assay. B: T-cell cytotoxicity assay with HCT-116 cells indicating the % killing of HCT-116 cells. C-E: Quantification of activation markers CD25 and CD69 in FACS indicating T-cell activation. F-G: Cytokine production in the supernatants from the cytotoxicity assay.

[0364] FIG. 15

[0365] Characterization of clones from supercluster 4. A: Binding of the selected MF clones to HPB-ALL human cells. B: T cell cytotoxicity assay with BxPC3 cells indicating % killing of BXP3 cells. C-E: Cytokine production in the supernatants from the cytotoxicity assay.

[0366] FIG. 16

[0367] Evaluation of CD3 functional activity. A: Affinity (HPB-ALL) on X-axis versus HCT-116 Lysis on Y-axis for additional clones from supercluster 1 (MF8048, MF8101, MF8056), supercluster 3 (MF8562) and supercluster 4 (MF8998). B: Antibodies belonging to supercluster 1 and supercluster 4 which show similar activity in cytotoxicity assay and different binding affinity. C: Antibodies belonging to supercluster 1 and supercluster 3 which exhibit similar binding affinity and differential lysis activity.

[0368] FIG. 17

[0369] Activity of CD3 Fabs MF8998 and MF8058 in bispecific CD3xEGFR format.

[0370] FIG. 18

[0371] FACS binding data of a large panel of IgGs specific for CD3. For antibodies MF5196, MF6955 and MF6964, binding was determined by BlAcoreTM on CD36s-Fc antigen, whereas FACS binding data to HPB-ALL cells is shown for the remainder of the clones.

[0372] FIG. 19

[0373] Nucleotide sequence of human CLEC12A.

[0374] FIG. 20

[0375] Amino acid sequences of the human CD3γ-, δ, ε- and ζ-chain.

[0376] The following Examples illustrate the invention:

EXAMPLES

[0377] Cell lines

[0378] BxPC3 human pancreatic cancer cell line.

[0379] HCT-116 human colon carcinoma cell line.

[0380] Immunization of Memo® mice with CD3

[0381] For generation of human antibodies binding to CD3, mice transgenic for the human common light chain and for a human heavy chain (HC) minilocus (comprising a selection of human V gene segments, all human Ds and all human Js) (see W02009/157771 incorporated herein by reference) were immunized with TCR/CD3 containing lipoparticles (Intergral Molecular). These mice are referred to as ‘MeMo®’ mice. For specific heavy chain variable regions, or trivalent multimers having the sequences disclosed herein, they can be produced by any means known to persons of ordinary skill in the art.

[0382] MeMo® mice were immunized with Hek293T-derived human 5D5M TCR/CD3 containing lipoparticles, followed by human T-cells for the generation of an anti-TCR/CDR3 immune response and anti-TCR/CD3 antibody panel generation.

[0383] Lipoparticles concentrate conformationally intact membrane proteins directly from the cell surface, permitting these complex proteins to be manipulated as soluble, high-concentration proteins for antibody immunization and screening

[0384] The lipoparticles used in the present study for immunisation contain the 5D5M TCRαβ combination. Vectors comprising the 5D5M TCRαβ combination were synthesized, cloned and used to generate lipoparticles containing this TCR/CD3 combination by transient transfection into HEK293T cells (Intergral Molecular).

TABLE-US-00067 5D5M TCRα MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLF WYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDS ASYLCAVMDSNYQLIWGAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCL FTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACA NAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILL LKVAGFNLLMTLRLWSS 5D5M TCRβ MRIRLLCCVAFSLLWAGPVIAGITQAPTSQILAAGRRMTLRCTQDMRHNA MYWYRQDLGLGLRLIHYSNTAGTTGKGEVPDGYSVSRANTDDFPLTLASA VPSQTSVYFCASSEAGGNTGELFFGEGSRLTVLEDLNKVFPPEVAVFEPS EAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQP ALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALV LMAMVKRKDF

[0385] MeMo® mice were used for immunizations using TCR/CD3 lipoparticles and primary human T cells.

[0386] The immunization schedule contains points on day 35, 56, 77 and 98, where the antigen-specific Ig serum titer was determined by ELISA using QTG-derived 3SDX TCR/CD3 positive and -negative lipoparticles using anti mouse IgG detection and by ELISA using CD3c5E-Fc fusion protein as a positive control. The reactivity was observed in sera drawn at day 35 will determine which mice developed a relevant anti-TCR/CD3 response.

[0387] For all immunized mice, lymphoid material for antibody discovery was collected and stored when:

[0388] Titers are 1/300 for human TCR/CD3 (in ELISA using lipoparticles), or:

[0389] Titers are <1/300 and >1/100 for human TCR/CD3 and did not increase during the last booster immunization.

[0390] Priming Immunisation using Lipoparticles

[0391] To prime the humoral immune response in the MeMo® mice for TCR/CD3, lipoparticles containing the human 5D5M TCRa6 combination was used for immunization. Lipoparticles were used together with Gerbu adjuvant for the first and second injection.

[0392] Booster Immunizations using polyclonal T-cells

[0393] Mice were immunised by sub-cutaneous injection of cell suspension. The first booster immunisations (day 28) comprised a mix of cells in PBS with adjuvant and all subsequent injections are only composed of cells in PBS. Mice that have developed at day 35 serum IgG titers of 1/300 against human TCR/CD3 (determined by ELISA using lipoparticles) received additional injections with cells on days 42, 43 and 44. Mice that failed to meet these criteria receive booster immunisations (day 42 and 49) with cells. All subsequent immunisations are given as sub-cutaneous injections of cells in PBS. After the final immunisation, mice are sacrificed, bled for serum and the spleen and left inguinal lymph nodes are collected.

[0394] Screening Sera from Immunised Mice in ELISA

[0395] Interim serum IgG titers were screened by ELISA using TCR/CD3-containing lipoparticles and ‘null’ lipoparticles. Serum IgG titers were determined using anti-mouse IgG staining, as this staining was shown to be the most sensitive.

[0396] Generation of ‘Immune’ Phage Antibody Repertoires by RT-PCR Cloning of VH Genes

[0397] From successfully immunized mice, the inguinal lymph nodes were used for the construction of ‘immune’ phage antibody repertoires. RNA was extracted from the lymphoid tissue using Trizol LS and 1 μg of total RNA was used in a RT reaction using an IgG-CH1 specific primer. The resulting cDNA was then used to amplify the polyclonal pool of VH-encoding cDNA using in-house developed VH-specific primers essentially as described in Marks et al. (J Mol Biol. 1991 Dec. 5;222(3):581-97). The resulting PCR product was then cloned in a phagemid vector for the display of Fab fragments on phage, as described in de Haard et al. (J Biol Chem. 1999 Jun. 25;274(26):18218-30) with the exception that the light chain was the same for every antibody and was encoded by the vector. After ligation, the phagemids were used to transform E.coli TG1 bacteria and transformed bacteria were plated onto LB-agar plates containing ampicillin and glucose. All phage libraries contained >10e6 transformants and had an insert frequency of >80%. Bacteria were harvested after overnight growth and used to prepare phage according to established protocols (de Haard et al., J Biol Chem. 1999 Jun. 25 ;274(26):18218-30).

[0398] Selection of Phage Carrying Fab Fragments Specifically Binding to Human CD3.

[0399] Phage libraries were rescued according to standardized procedures (J Mol Biol. 1991 Dec. 5;222(3):581-97; J Biol Chem. 1999 Jun. 25;274(26):18218-30) and phage were selected with one or more rounds of selection of the immune phage antibody repertoires. In the first round, recombinant CD3 protein was coated onto the wells of a maxisorp.sup.TM ELISA plate or to a NUNC immuno-tube, whereas in the second round, either recombinant CD3 protein or cells over-expressing the human CD3 protein were used. The maxisorpTM ELISA plates or immuno-tubes were blocked with 4% ELK. Phage antibody libraries were also blocked with 4% ELK and excess of human IgG to deplete for Fc region binders prior to the addition of the phage library to the coated antigen.

[0400] Incubation with the phage library with the coated protein was performed for 2 hrs at room temperature under shaking conditions. Plates or tubes were then washed with 0.05% Tween-20 in PBS followed by 5 to 10 times washing with PBS. Bound phage were eluted using 50 mM glycine (pH 2.2) and added to E. coli TG-1 and incubated at 37° C. for phage infection.

[0401] Subsequently infected bacteria were plated on agar plates containing Ampicillin, and glucose and incubated at 37° C. overnight. After the first round of selection, colonies were scraped off the plates and combined and thereafter rescued and amplified to prepare an enriched first round phage pool for the synthetic repertoires. For the ‘immune’ repertoires, single clones were screened for target binding after the first round of phage selection.

[0402] Antibody Cloning and Production

[0403] Bispecific antibodies as used herein typically differ from each other only in the particular amino acid sequence of the heavy chain variable region of one or both variable domains. The antibodies were produced by cloning the heavy chain variable regions into expression vectors for the expression of heavy and light chains. Methods for the production of bispecific antibodies are known in the art.

[0404] Briefly, DNA encoding the heavy chain variable region for the CD3 targeted variable domain was cloned into MV1624 vector (see FIG. 10a), encoding the KK residues (L351 K, T366K) in the CH3 region for the generation of IgG heavy chain heterodimers (WO2013/157954 and WO2013/157953). The Fc constant regions contains mutation in the CH2 to silence the Fc effector function. The DNA encoding the heavy chain variable region replaces the stuffer region in the construct. The variable region is preceded by an encoded HC signal peptide (not shown). The DNA encoding the heavy chain variable region for the EGFR targeted variable domain was cloned into vector MV1625 (FIG. 10b) expressing the second heavy chain of the base antibody portion of the bispecific antibodies, bearing the L351 D-L368E mutations in the CH3 region (WO2013/157954 and WO2013/157953). The DNA encoding the heavy chain variable region replaces the stuffer region in the construct. The variable region is preceded by an encoded HC signal peptide (not shown). Both constructs also contain an expression cassette for expression of the IGKV1-39/jk1 light chain. Expression of the two heavy chains together with the mentioned light chain leads to the production of the bispecific antibody.

[0405] 293-F cells were used for expression of the designed antibodies in a 24 wells plate format. Two days before transfection, 293-F cell stock was split in 293-F culture medium in a 1:1 ratio and incubated overnight at 37° C. and 8% CO.sub.2 at an orbital shaking speed of 155 rpm. Cells were diluted on the day before transfection to a density of 5×10.sup.5cells/mL. 4ml of the suspension cells were seeded into a 24 deep wells plate, covered with a breathable seal and incubated overnight at 37° C. and 8% CO2 at an orbital shaking speed of 285 rpm. On transfection day, 4.8 ml 293-F culture medium were mixed with 240 μg of polyethylenimine (PEI) linear (MW 25,000). For each IgG to be produced, 200 uL of the 293F culture medium-PEI mix was added to 8 μl of DNA (for IgG heterodimers 4 μl of DNA encoding each heavy chain). The mixture was incubated for 20 minutes at room temperature before gently adding to the cells. On the day after transfection Penicillin-Streptomycin (Pen Strep) diluted in 500 μL 293F medium was added to each well. The plates were incubated at 37° C. and 8% CO.sub.2 at an orbital shaking speed of 285 rpm until harvest seven days after transfection. Plates were centrifuged 5 min at 500 g, supernatants containing IgGs were filtered using 10-12 μm melt blown polypropylene filter plates and stored at −20° C. prior to purification.

[0406] Purification of Antibodies from Culture Supernatant

[0407] Medium containing antibodies is harvested and centrifuged to remove the cell debris. Subsequently Protein A Sepharose beads are added to the medium. Medium and Protein A Sepharose beads are incubated with the antibodies to allow binding.

[0408] After incubation the beads are isolated from the medium and washed, by a vacuum filter. The antibodies are eluted from the beads by incubation with elution buffer. Optionally, the buffer of the purified IgG is exchanged/desalted.

[0409] Buffer Exchange

[0410] In order to desalt the purified antibodies the antibody fraction is centrifuged using a filter plate or filter column. The plate or column is centrifuged to reduce the volume of the antibody fraction. Subsequently, PBS or the required buffer is added to the fraction to replace the buffer with a low salt buffer. Optionally this centrifugation step followed by adding buffer is repeated in order to further desalt the storage buffer of the antibodies.

[0411] Antibody Tumor Antigen Specific T Cell Activation and Lysis of BxPC3 Cells or of HTC-116 Cells.

[0412] The capacity of the particular CD3 x tumor antigen bispecific IgG combinations to induce tumor antigen-specific T cell activation and lysis of tumor antigen positive target cells in a cytotoxicity assay was tested. The effector cells were healthy donor-derived resting T cells and the target cells were BxPC3 cells or HTC-116 cells.

[0413] Using Ficoll and EasySep human T cell isolation kit according to standard techniques resting T cells were isolated from whole blood from healthy donors, checked for >95% T cell purity by anti-CD3 antibody using flow cytometric analysis and subsequently cryopreserved. For a cytotoxicity assay the cryopreserved T cells were thawed and used if their viability was >90% upon thawing, determined by standard Trypan Blue staining. Cytotoxicity assay in short, thawed resting T cells and BxPC3 or HCT116 target cells were co-cultured in an E:T ratio of 5:1 for 48 hours. Antibodies were tested in a dilution range. a CD3 monospecific antibody and an EGFR monospecific antibody, as well as an irrelevant IgG1 isotype control mAb are included in the assay as controls (e.g., an antibody which binds CD3 and another antigen such as tetanus toxin (TT)). T cell activation was quantified using flow cytometry; CD8 T cells were gated based on CD8 expression and subsequently analyzed for their activation status by measuring CD69 expression on T cells. Target cell lysis was determined by measuring the fraction of alive cells by measuring ATP levels assessed by CellTiterGlo (Promega). ATP levels, measured by luminescence on an Envision Microplate reader results in Relative light unit (RLU) values, which were analyzed using Graph Pad Prism.

[0414] Target cell lysis for each sample was calculated as follows:

% Killing=(100−(RLU sample/RLU no IgG)×100).

[0415] In this assay, the bispecific antibodies have two binding domains. One of the binding domains is targeted towards EGFR and the other to CD3. Both binding domains have the same (common) light chain variable region (VL) and a different heavy chain variable region (VH). The EGFR targeted binding domain has a VH with the amino acid sequence of MF8233. The CD3 targeted binding domain has a VH with an amino acid sequence of one of the MFs indicated for CD3. The bispecific antibody contain mutation in the CH2 to silence the Fc effector function.

[0416] The antibody MF8233×MF8397 induced upregulation of CD69 (FIG. 4-6) and CD25 (FIG. 4-6) on CD4 and CD8 T cells, determined after 48 hours of co-culture at an E±T ratio of 5:1. T cell-mediated lysis was measured after 48 hours.

[0417] CD3 Bispecific Antibody Characterization

[0418] A candidate EGFR/CD3 IgG bispecific antibody can be tested for binding using any suitable assay. For example, binding to membrane-expressed CD3 on HPB-ALL cells (DSMZ, ACC 483) can be assessed by flow cytometry (according to the FACS procedure as previously described in WO2014/051433). In one embodiment, the binding of a candidate EGFR/CD3 bispecific antibody to CD3 on HPB ALL cells is demonstrated by flow cytometry, performed according to standard procedures known in the art. Binding to cell expressed CD3 can be confirmed using CHO cell transfected with CD3δ/ε or CD3γ/ε. The binding of the candidate bispecific IgG1 to EGFR can be determined using BxPC3 and HCT-116 as well as CHO cells transfected with an EGFR expression construct; a CD3 monospecific antibody and an EGFR monospecific antibody, as well as an irrelevant IgG1 isotype control mAb are included in the assay as controls (e.g., an antibody which binds CD3 and another antigen such as tetanus toxin (TT)).

[0419] Generation of further clones from superclusters 1, 3 and 4 From the immune phage library screening (as described in section ‘Selection of phage carrying Fab fragments specifically binding to human CD3’), additional clones were characterized carrying Fab fragments specifically binding to human CD3. From supercluster 1, additional clones were identified, including MF8048, MF8101 and MF8056. Additional clones were identified from supercluster 3 and supercluster 4, including MF8562 of supercluster 3 and MF8998 of supercluster 4.

[0420] From supercluster 4, further new clones were identified using next-generation sequencing (NGS) analysis. NGS was performed on the VH gene pools present from MeMo® mice that were used to generate the anti-CD3 panel. To this aim, the sequence data sets obtained from different mice were compared with an MF sequence belonging to supercluster 4 MF. This led to identification of sequence variants clones MF10401 and MF10428 that belong to supercluster 4. Several different mutations were found in the HCDR1 and HCDR2 for different sequences.

[0421] VH sequences of all additional clones from supercluster 1, 3 and 4 were cloned into MV1624 (DM-KK) vector and expressed as CD3xEGFR bispecific format for further characterization, as described in section ‘Antibody cloning and Production’ above.

[0422] Characterization of Further Clones from Superclusters 1 and 4

[0423] Additional clones from supercluster 1 were characterized with respect to their functional activity in a bispecific format. The EGFR binding domain of the bispecific CD3xEGFR antibody has the amino acid sequence encoded by MF8233. As a control, these CD3 clones were also tested with another antigen (e.g. Tetanus toxin) with the amino acid sequence encoded by MF1337. Reference MFs from supercluster 1 (MF8057 and MF8058) were included to directly compare the affinity of the sequence variant to that of already characterized MF clones from supercluster 1 according to sections: ‘Antibody tumor antigen specific T cell activation and lysis of BxPC3 cells or of HTC-116 Cells’ and ‘CD3 Bispecific Antibody Characterization’ as described above. Binding affinity to HPB-All cells expressing human CD3-TCR complex using flow cytometry (FIG. 14A and FIG. 18) and T-cell activation and lysis of tumor antigen positive target cells (HCT-116) in a cytotoxicity assay (FIG. 14B-E) assays were performed. No target cell lysis was observed with bispecific antibodies having the MF1337 control arm. For the different CD3 clones tested, target cell lysis was observed in a dose dependent manner. Low target cell lysis was observed for MF8048. Expression levels of activation markers CD69 and CD25 on CD4 and CD8 T cells were measured in FACS staining for the evaluation of T-cell activation. Dose-dependent T-cell activation was observed for all clones, and no T-cell activation was observed for negative control. Finally, cytokine production of IFN-γ and TNF-α was determined in the supernatant derived from the EGFRxCD3 cytotoxicity assay with HCT-116 cells after 48 hrs using Luminex® Assays (eBiosciences™) following standard manufacturer's instructions (FIG. 14F-G).

[0424] For characterization of additional clones belonging to supercluster 4, binding affinity was determined in FACS to HPB-ALL cells (FIG. 15). PG1337, a monovalent antibody with two identical MF1337 arms specific for tetanus toxin, was used as a negative control. For cytotoxicity assay, HCT-116 cells and BxPC3 cells were used as target cells to test activity of MF8998 and BxPC3 target cells were used to test activity of MF10401 and MF10428. A CD3xTAA bispecific antibody with known high activity was included as positive control. Target cell lysis was quantified using cell viability measurements. Supernatant from the cytotoxicity assay was used to measure cytokine levels for IL-6, IFN-γ and TNF-α using Luminex® assays.

[0425] The three supercluster 4 clones tested were thus found to exhibit different binding but similar lysis activity. Although the lysis activity of these clones was similar, reduced cytokine production was observed.

TABLE-US-00068 TABLE 1 AUC values for binding of the indicated CD3 clones mentioned in FIG. 15A MF combination AUC MF8233 × MF8998 13630 MF8233 × MF10428 2700 MF8233 × MF10401 9646 MF1337 × MF1337 255

TABLE-US-00069 TABLE 2 AUC and EC50 values for target cell lysis of the indicated CD3 clones mentioned in FIG. 15A. MF combination Target AUC EC50 (ng/mL) MF8233 × MF8998 EGFR 253 6.9 MF8233 × MF10428 EGFR 216 10.7 MF8233 × MF10401 EGFR 241 4.6 CD3 × Mock: −Ctrl Mock 11 — CD3 × TAA: +Ctrl TAA 369 4.5

TABLE-US-00070 TABLE 3 AUC and EC50 values for the amount of indicated cytokines and CD3 clones mentioned in FIG. 15A. MF combination Cytokine AUC EC50 (ng/mL) MF8233 × MF8998 IL-6 858 51.2 MF8233 × MF10428 IL-6 659 8.6 MF8233 × MF10401 IL-6 657 3.1 MF8233 × MF8998 IFNγ 311 >400 MF8233 × MF10428 IFNγ 107 >4000 MF8233 × MF10401 IFNγ 114 >4000 MF8233 × MF8998 TNFα 50 — MF8233 × MF10428 TNFα 50 — MF8233 × MF10401 TNFα 50 —

[0426] As evaluated by the cytotoxicity assay, it was observed that all further identified MFs are functional. Next, a graph was plotted between lysis on Y-axis and binding affinity on X-axis (FIG. 16A) to understand the relation within and across superclusters between lysis and affinity. Overall, a diverse panel of anti-CD3 Fabs was generated composed of multiple superclusters and covering a range of affinities. Interestingly, clones were identified in supercluster 1 and 4 which exhibit similar activity but different CD3 binding (FIG. 16B). Comparison of superclusters 1 and 3 revealed clones which exhibit similar CD3 binding and differential activity (FIG. 16C).

TABLE-US-00071 TABLE 4 AUC and EC50 values for target cell lysis of the indicated CD3 clones mentioned in FIGS. 16B and 16C. EC50 MF combination AUC (ng/mL) MF8233 × MF8057 117 28.5 MF8233 × MF8058 283 4.4 MF8233 × MF8101 355 1.0 MF8233 × MF8508 267 6.4 MF8233 × MF8998 232 8.0 MF8233 × MF8397 62 >400 MF8233 × MF8562 57 >400

[0427] Characterization of CD3 Antigens

[0428] As described above, two clones from supercluster 1 and supercluster 4, i.e. MF8058 and MF8998 respectively, were found to have similar lysis activity in a bispecific format with clone MF8233 as Fab arm binding EGFR as the tumor-cell antigen (FIG. 17). For this experiment, lysis activity against HCT-116 cells was measured as described herein above.

[0429] MF9257×MF8233 was used as a positive control and MF9257×MF1337 was the negative control. As can be seen from FIG. 17 and Table 5, dose-dependent and high killing percentage were observed for multiple tested bispecific antibodies.

TABLE-US-00072 TABLE 5 AUC and EC50 values for target cell lysis for the indicated bispecific antibodies mentioned in FIG. 17. Negative controls displayed no significant measurable activity. Tumor target and Cytoxicity immune cell assay/ engaging target if EC50 target cell applicable MF combination AUC (ng/mL) HCT116 EGFR × CD3 MF8233 × MF8058 283 4.4 HCT116 EGFR × CD3 MF8233 × MF8998 232 8.0 HCT116 EGFR +control 354 0.5

Binding Affinity

[0430] As described in section ‘CD3 Bispecific antibody characterization’, the binding affinity of additional CD3 clones was analyzed in FACS on HPB-ALL cells expressing human CD3. The affinities of MF6955 and MF6964 for CD3 were measured by surface plasmon resonance (SPR) technology using a BIAcore™T100. An anti-human IgG mouse monoclonal antibody (Becton and Dickinson, cat. Nr. 555784) was coupled to the surfaces of a CM5 sensor chip using free amine chemistry (NHS/EDC). Then the CD3xTAA bispecific antibody was captured onto this sensor surface. Subsequently the recombinant purified antigen human CD3δε-Fc was run over the sensor surface in a concentration range to measure on- and off- rates. After each cycle, the sensor surface was regenerated by a pulse of HCl and CD3xTAA bispecific antibody was captured again. From the obtained sensograms, on-and off- rates were determined using the BIAevaluation software. FIG. 18 describes the binding affinity range of the CD3 panel generated.