HK40032023B

HK40032023B - A novel bispecific cd3/cd20 polypeptide complex

Info

Publication number: HK40032023B
Application number: HK42020021971.5A
Authority: HK
Inventors: 陈蕴颖; 梅芹; 徐建清; 王卓智; 李竞; 吕鹏
Original assignee: 正大天晴药业集团股份有限公司
Priority date: 2019-01-28
Filing date: 2020-12-11
Publication date: 2023-03-24

Description

Novel bispecific CD3/CD20 polypeptide complex

PRIORITY INFORMATION

This application claims priority from chinese invention patent application 201910080405.3 filed on day 1, 28, 2019 and PCT application PCT/CN2019/073418 filed on day 28, 1, 2019, which are fully incorporated by reference in this disclosure.

Sequence listing

The present application also submits a sequence listing file in electronic form. The entire contents of the sequence listing are incorporated by reference in this disclosure.

Technical Field

The present invention generally relates to bispecific anti-CD 3xCD20 polypeptide complexes.

Background

Bispecific antibodies are a novel class of therapeutic antibodies. They can bind to two different targets or two different epitopes on one target, resulting in additive or synergistic effects over the effects of a single antibody. Many attempts have been made to engineer new bispecific formats (e.g., DVD-Ig, Crossmab, BiTE, etc.) (Spiess et al, Molecular Immunology, 67(2), pp.95-106 (2015)). However, these bispecific antibody formats have potential limitations in terms of stability, solubility, short half-life, and immunogenicity.

Among these bispecific antibody formats, an IgG-like bispecific antibody is a universal format: one arm binds to target a and the other arm binds to target B. Structurally it consists of half of antibody a and half of antibody B, with a size and shape similar to native IgG. For ease of downstream development, it is desirable that such bispecific molecules can be easily produced and correctly assembled from a single host cell at high expression levels, like normal IgG. Unfortunately, the pairing of homologous light-heavy chains and the assembly of two different half-antibodies cannot be controlled automatically. Various mismatches in a random fashion can lead to significant product heterogeneity.

Heterodimeric assembly of two different heavy chains is achieved by introducing mutations into the Fc region, such as "hand-intro-holes" (Ridgway et al, Protein Engineering, 9(7), pp.617-21 (1996); Merchant et al, Nature Biotechnology, 16(7), pp.677-681 (1998), Electrostatic (Gunasekalan et al, Journal of Biological Chemistry, 285(25), pp.19637-19646 (2010)), or negative state design (Von Kreudenstein et al, mAbs, 5(5), pp.646-654 (2013); Leaver-Fay et al, Structure, 24(4), pp.641-651 (2016)). However, selective pairing of individual light-heavy chains of individual antibodies remains challenging. The interface between the light-heavy chains comprises a variable domain (VH-VL) and a constant domain (CH 1-CL). Several schemes have been applied to the design of orthogonal interfaces to facilitate homologous pairing. Roche exchanged the domains of CH1 and CL and created the Cross Mab platform (Schaefer et al, Proceedings of the National Academy of Sciences of the United States of America, 108(27), pp.11187-11192 (2011)), Medmemune alternatively introduced disulfide bonds (Mazor et al, mAbs, 7(2), pp.377-389 (2015)), Amgen further electrostatically modified in the CH1-CL region (Liu et al, Journal of Biological Chemistry, 290(12), pp.7535-7562 (2015)), and Lilly (Lewis et al, Nature technology, 32 (2015)), 191-198 (2014), (9-213), (230) and Genencoch (Dillon et al, mAh et al, 9-213, pp.230) introduced constant mutations in both domains.

CD20 is an activated glycosylated phosphoprotein expressed on the surface of B lymphocytes. FDA approved antibody therapy with Rituximab (Rituximab), a chimeric anti-CD20 monoclonal antibody (hereinafter also referred to as "mAb"), in 1997 represents one of the most important advances in the treatment of lymphoproliferative disorders over the last 30 years. Rituximab significantly improves various indicators of survival statistics in patients with B cell lymphomas and Chronic Lymphoid Lymphomas (CLL), especially in combination with various chemotherapeutic/radiation regimens (Chu TW, Zhang R, Yang J, et al. A Two-Step Pretargeted Nanotherapy for CD20 cross linking May Achieve Superior Anti-Lymphoma Efficacy to Rituximab. Theransotics. 2015Apr 26; 5(8): 834-46).

During the last three decades, with significant advances in the study of the protein structure and molecular function of CD20, a new generation of therapeutic antibodies targeting CD20 was generated and approved for clinical use. Ofatumumab (Ofatumumab) is a fully human anti-CD20 therapeutic antibody that Targets a different CD20 epitope closer to the cell surface than rituximab, resulting in a slower off-rate and more stable binding than rituximab (Laurenti L, Innocenti I, auto F, et al. new definitions in the management of chronic lymphocytic leukemia: roll of atomumab. on co Targets the ther. However, the clinically new generation of anti-CD20 monoclonal antibodies is not significantly superior to rituximab in terms of efficacy and safety. For anti-CD20 mAb treatment, nearly all patients with follicular lymphoma and CLL as well as about half of those with aggressive B cell lymphoma (e.g., diffuse large B cell lymphoma) will still develop disease recurrence or recurrence (Lim SH, Beers SA, French RR, et al, anti-CD20 monoclonal antibodies: pathological and clinical surgery 0. Haematologica.201Jan; 95(1): 135-43). Therefore, there remains an urgent medical need to develop new B cell targeted therapeutic strategies with different mechanisms of action (MOAs), such as bispecific antibodies and Chimeric Antigen Receptor (CAR) -T cell therapies.

The T cell CD3 receptor is a protein complex composed of four distinct chains (CD3 γ chain, CD3 δ chain and two CD3 epsilon chains). These four chains associate with a molecule called the T Cell Receptor (TCR) and the intracellular zeta chain to generate an activation signal in T lymphocytes. The TCR, zeta chain and CD3 molecules constitute a TCR complex, in which TCR is used as a subunit for recognition and binding of antigen, and CD3 is responsible as a subunit for the transmission of antigenic stimuli to the signaling pathway and ultimately the regulation of T cell activity. CD3 protein is present in almost all T cells. CD3-TCR complexes modulate T cell function in innate and adaptive immune responses, as well as cellular and humoral immune functions, including elimination of pathogenic organisms and control of tumor growth through cytotoxic effects. Mouse monoclonal antibodies targeting human CD3, such as OKT3(Kung et al, Science, 206: 347-9(1979)), were the first generation CD3 antibodies developed for therapy. Despite its strong immunosuppressive potency, OKT3 has been hampered in its clinical use by serious side effects associated with its immunogenicity and mitogenic potential (Chatenoud, Nature Reviews, 3: 123-. OKT3 induced an anti-antibody response, promoting rapid clearance and neutralization of itself (Chatenoud et al, Eur. J. Immunol., 137: 830-8 (1982)). In addition, OKT3 induced large-scale release of cytokines in vivo (Hirsch et al, J.Immunol, 142: 737-43 (1989)). These serious side effects limit the clinical use of OKT 3.

Bispecific antibodies targeting CD3 and CD20 can bind to both T cells and B cells. Once the bispecific antibody binds to both CD3 positive T cells and CD20 positive B cells simultaneously, cytolytic synapse formation is promoted, cytotoxic T cells thereby releasing perforin and granzyme which induce apoptosis of the B cells.

In view of the above, there is still a great medical need for the invention of novel potent bispecific molecules targeting CD3 and CD20 to be better used for the treatment of diseases associated with targeting CD20, including tumors.

Disclosure of Invention

In one aspect, the invention provides a bispecific polypeptide complex comprising a first antigen-binding moiety associated with a second antigen-binding moiety, wherein:

the first antigen-binding portion comprises:

a first polypeptide comprising from N-terminus to C-terminus a first heavy chain variable domain (VH) of a first antibody operably linked to a first T Cell Receptor (TCR) constant region (C1), and

a second polypeptide comprising from N-terminus to C-terminus a first light chain variable domain (VL) of a first antibody operably linked to a second TCR constant region (C2),

wherein:

c1 and C2 are capable of forming dimers containing at least one non-natural interchain linkage between C1 and C2, and said non-natural interchain linkage is capable of stabilizing said dimer,

and

the second antigen-binding moiety comprises:

a second heavy chain variable domain of a second antibody (VH2) operably linked to the antibody heavy chain CH1 domain, and

a second light chain variable domain of a second antibody (VL2) operably linked to an antibody light chain Constant (CL) domain,

wherein:

one of said first and said second antigen-binding moiety is an anti-CD 3 binding moiety and the other is an anti-CD20 binding moiety,

an anti-CD 3 binding moiety is derived from an anti-CD 3 antibody comprising:

a) comprising a nucleic acid sequence selected from SEQ ID NO: 1. 13, 25, 37 and 49, and a heavy chain CDR1,

b) comprises a sequence selected from SEQ ID NO: 2. 14, 26, 38 and 50, and a heavy chain CDR2,

c) comprises a sequence selected from SEQ ID NO: 3. 15, 27, 39 and 51, and a heavy chain CDR3 of the amino acid sequence,

d) comprising a nucleic acid sequence selected from SEQ ID NO: 4. 16, 28, 40 and 52, the kappa light chain CDR1 of the amino acid sequence,

e) comprising a nucleic acid sequence selected from SEQ ID NO: 5. 17, 29, 41 and 53, and the kappa light chain CDR2 of the amino acid sequence of

f) Comprises a sequence selected from SEQ ID NO: 6. 18, 30, 42 and 54, and kappa light chain CDR3,

an anti-CD20 binding moiety is derived from an anti-CD20 antibody comprising:

a) comprises a sequence selected from SEQ ID NO: 7. 19, 31, 43 and 55, and a heavy chain CDR1,

b) comprises a sequence selected from SEQ ID NO: 8. 20, 32, 44 and 56, and a heavy chain CDR2 of an amino acid sequence,

c) comprising a nucleic acid sequence selected from SEQ ID NO: 9. 21, 33, 45 and 57, and a heavy chain CDR3,

d) comprises a sequence selected from SEQ ID NO: 10. 22, 34, 46 and 58, the kappa light chain CDR1 of the amino acid sequence,

e) comprises a sequence selected from SEQ ID NO: 11. 23, 35, 47 and 59, and the kappa light chain CDR2 of the amino acid sequence of

f) Comprises a sequence selected from SEQ ID NO: 12. 24, 36, 48 and 60, and a kappa light chain CDR 3. In certain embodiments, the anti-CD 3 binding portion of the bispecific polypeptide complex is derived from an anti-CD 3 antibody comprising a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 61. 63, 65, 67 and 69 and a light chain variable domain sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 62. 64, 66, 68 and 70, or a light chain variable domain sequence.

In certain embodiments, the anti-CD20 binding portion of the bispecific polypeptide complex is derived from an anti-CD20 antibody comprising a heavy chain variable region comprising SEQ ID NO: 71. 73, 75, 77 and 79 and a light chain variable domain sequence comprising the amino acid sequence of SEQ ID NO: 72. 74, 76, 78 and 80.

In certain embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: 81, 82, 83, and 84.

In certain embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88.

in certain embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: 89, 90, 91, and 92 SEQ ID NO.

In certain embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96.

in certain embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100.

in certain embodiments, the sequence of SEQ ID NO:92, wherein one or more of the amino acids in the native glycosylation site at position 193, 182, 203, 206 or 207, preferably amino acid 193, is modified. In certain embodiments of the invention, the modifications include one or more of S193X, S182X, S203X, S206X, S207X, wherein X is any amino acid other than serine (Ser) and threonine (Thr). In certain preferred embodiments of the invention, the modification is S193X, wherein X is alanine (Ala), glycine (Gly), proline (Pro), or valine (Val). In certain embodiments of the invention, the mutation eliminates an O-glycosylation site that is an O-sugar of the Core1 configuration, and is of the NeuAc-Gal-GalNAc or NeuAc-Gal- (NeuAc) GalNAc structure.

In one aspect, the invention provides a bispecific polypeptide complex having one or more of the following properties:

(a) specifically binds to both human CD3 and CD20 proteins with moderate affinity;

(b) specifically binds to human CD3 protein and/or cynomolgus monkey CD3 protein;

(c) specifically binds to human CD20 protein and/or cynomolgus monkey CD20 protein;

(d) in vitro cytofunctional experiments, compared with other bispecific antibodies targeting CD3 and CD20, the antibody can induce more efficient T cell activation and can kill CD 20-positive tumor cells more effectively and specifically, with less cytokine release;

(e) has good thermal stability and is stable in the serum of a cynomolgus monkey or a human;

(f) provides superior anti-tumor effects in vivo compared to other bispecific antibodies targeting CD3 and CD 20; and

(g) the compound shows effective B cell clearing effect and good half-life period in cynomolgus monkeys, and has no adverse reaction such as accompanying cytokine storm and the like.

In one aspect, the invention provides a conjugate comprising a bispecific polypeptide complex provided by the invention conjugated to a moiety.

In one aspect, the invention provides an isolated polynucleotide encoding a bispecific polypeptide complex provided by the invention.

In one aspect, the invention provides an isolated vector comprising a polynucleotide provided by the invention.

In one aspect, the invention provides a host cell comprising an isolated polynucleotide provided by the invention, or an isolated vector provided by the invention.

In one aspect, the present invention provides a method of expressing a bispecific polypeptide complex provided by the present invention, the method comprising culturing a host cell provided by the present invention under conditions in which the bispecific polypeptide complex is expressed.

In one aspect, the invention provides a composition comprising a bispecific polypeptide complex provided by the invention.

In one aspect, the present invention provides a pharmaceutical composition comprising a bispecific polypeptide complex provided herein, and a pharmaceutically acceptable carrier.

In one aspect, the invention provides a method of treating a disease or condition associated with CD20 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific polypeptide complex provided by the invention. In certain embodiments, when both the first antigen and the second antigen are modulated, the disease or condition may be alleviated, eliminated, treated, or prevented.

In certain embodiments, the first antigen-binding moiety is linked to a first dimerization domain and the second antigen-binding moiety is linked to a second dimerization domain, wherein the first and the second dimerization domains are associated. In certain embodiments, the association is via a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, or a hydrophobic-hydrophilic interaction, or a combination thereof.

In certain embodiments, the first and/or the second dimerization domain comprises at least a portion of an antibody hinge region, which is optionally derived from IgG1, IgG2, or IgG4.

In certain embodiments, the second dimerization domain is operably linked to the heavy chain variable domain of the second antigen binding portion.

In certain embodiments, the first and the second dimerization domains are different and are associated in a manner that hinders homodimerization and/or contributes to heterodimerization.

In certain embodiments, the first and the second dimerization domains are capable of associating into a heterodimer via handle-access holes, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility.

In another aspect, the invention provides a kit for detecting, diagnosing, prognosing or treating a disease or condition, comprising a polypeptide complex provided by the invention.

In another aspect, the invention provides the use of a bispecific polypeptide complex according to the invention in the preparation of a pharmaceutical composition for the treatment of a disease or condition associated with CD20 in a subject.

In one embodiment, the disease or condition associated with CD20 is a cancer, preferably, the cancer is selected from, but not limited to, lymphoma, lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer, or gastric cancer.

In one embodiment, the disease or condition associated with CD20 is B-cell lymphoma, optionally hodgkin's lymphoma or non-hodgkin's lymphoma, wherein the non-hodgkin's lymphoma comprises: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), or Mantle Cell Lymphoma (MCL), Acute Lymphocytic Leukemia (ALL), or Waldenstrom's Macroglobulinemia (WM).

In summary, the present invention provides the following technical solutions:

1. a bispecific polypeptide complex comprising a first antigen-binding moiety associated with a second antigen-binding moiety, wherein:

the first antigen-binding portion comprises:

wherein:

c1 and C2 are capable of forming dimers containing at least one non-natural interchain linkage between C1 and C2, and said non-natural interchain linkage is capable of stabilizing said dimers, and

the second antigen-binding moiety comprises:

wherein:

the anti-CD 3 binding portion is derived from an anti-CD 3 antibody comprising:

a) comprises a sequence selected from SEQ ID NO: 1. 13, 25, 37 and 49, and a heavy chain CDR1 of an amino acid sequence,

c) comprises a sequence selected from SEQ ID NO: 3. 15, 27, 39 and 51, and the amino acid sequence of CDR3,

d) comprises a sequence selected from SEQ ID NO: 4. 16, 28, 40 and 52, and a kappa light chain CDR1,

e) comprises a sequence selected from SEQ ID NO: 5. 17, 29, 41 and 53, and the kappa light chain CDR2 of the amino acid sequence

f) Comprises a sequence selected from SEQ ID NO: 6. 18, 30, 42 and 54, and a kappa light chain CDR3,

the anti-CD20 binding portion is derived from an anti-CD20 antibody comprising:

b) comprising a nucleic acid sequence selected from SEQ ID NO: 8. 20, 32, 44 and 56, and the amino acid sequence of CDR2,

c) comprises a sequence selected from SEQ ID NO: 9. 21, 33, 45 and 57, and a heavy chain CDR3,

d) comprises a sequence selected from SEQ ID NO: 10. 22, 34, 46 and 58, and a kappa light chain CDR1,

e) comprising a nucleic acid sequence selected from SEQ ID NO: 11. 23, 35, 47 and 59, and

f) comprises a sequence selected from SEQ ID NO: 12. 24, 36, 48 and 60, and a kappa light chain CDR 3.

2. The bispecific polypeptide complex of embodiment 1, wherein the anti-CD 3 binding moiety comprises a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 61. 63, 65, 67 and 69 and a light chain variable domain sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 62. 64, 66, 68, and 70, or a light chain variable domain sequence of the amino acid sequence of seq id no.

3. The bispecific polypeptide complex of any one of the preceding embodiments, wherein the anti-CD20 binding moiety comprises a heavy chain variable region comprising SEQ ID NO: 71. 73, 75, 77 and 79 and a light chain variable domain sequence comprising SEQ ID NO: 72. 74, 76, 78 and 80.

4. The bispecific polypeptide complex of any one of the preceding embodiments, wherein the first antigen-binding moiety is linked to a first dimerization domain and the second antigen-binding moiety is linked to a second dimerization domain, wherein the first and second dimerization domains are in association.

5. The bispecific polypeptide complex of embodiment 4, wherein the association is achieved via a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, or a hydrophobic-hydrophilic interaction, or a combination thereof.

6. The bispecific polypeptide complex of embodiment 5, wherein the first and/or the second dimerization domain comprises at least a portion of an antibody hinge region, which optionally is derived from IgG1, IgG2 or IgG4.

7. The bispecific polypeptide complex of embodiment 6, wherein the first and/or the second dimerization domain comprises an antibody CH2 domain, and/or an antibody CH3 domain.

8. The bispecific polypeptide complex of embodiment 6, wherein the first dimerization domain is operably linked to the first TCR constant region at a third engagement domain (C1).

9. The bispecific polypeptide complex of embodiment 6, wherein the second dimerization domain is operably linked to the heavy chain variable domain of the second antigen binding portion.

10. The bispecific polypeptide complex of any one of the preceding embodiments, wherein the first and the second dimerization domains are different and are associated in a manner that does not favor homodimerization and/or favors heterodimerization.

11. The bispecific polypeptide complex of embodiment 10, wherein the first and the second dimerization domains are capable of associating into a heterodimer via handle-access holes, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility.

12. The bispecific polypeptide complex of any one of the preceding embodiments, wherein the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO: 84.

13. the bispecific polypeptide complex according to any one of embodiments 1-11, wherein the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88.

14. the bispecific polypeptide complex according to any one of embodiments 1-11, wherein the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO: 92.

15. the bispecific polypeptide complex according to any one of embodiments 1-11, wherein the bispecific polypeptide complex comprises a combination of four polypeptide sequences: the amino acid sequence of SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96.

16. the bispecific polypeptide complex according to any one of embodiments 1-11, wherein the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100.

17. the bispecific polypeptide complex of embodiment 14, wherein SEQ ID NO:92 is selected from the group consisting of amino acids 193, 182, 203, 206, or 207 modified to any amino acid other than serine and threonine to eliminate glycosylation sites, preferably, amino acid 193 is modified.

18. The bispecific polypeptide complex of embodiment 17, wherein SEQ ID NO:92 is modified to alanine, glycine, proline or valine at amino acid position 193.

19. A conjugate comprising a bispecific polypeptide complex according to any one of the preceding embodiments conjugated to a moiety.

20. An isolated polynucleotide encoding a bispecific polypeptide complex according to any one of embodiments 1-18.

21. An isolated vector comprising the polynucleotide according to embodiment 20.

22. A host cell comprising the isolated polynucleotide according to embodiment 20, or the isolated vector according to embodiment 21.

23. A method of expressing a bispecific polypeptide complex according to any one of embodiments 1 to 18, comprising culturing a host cell according to embodiment 22 under conditions in which the bispecific polypeptide complex is expressed.

24. A method of producing a bispecific polypeptide complex, the method comprising:

a) introducing into a host cell one or more polynucleotides encoding a bispecific polypeptide complex according to any one of embodiments 1-18; and

b) allowing the host cell to express the bispecific polypeptide complex.

25. The method of any one of embodiments 23-24, further comprising isolating the bispecific polypeptide complex.

26. A composition comprising a bispecific polypeptide complex according to any one of embodiments 1-18.

27. A pharmaceutical composition comprising a bispecific polypeptide complex according to any one of embodiments 1-18, and a pharmaceutically acceptable carrier.

28. Use of a bispecific polypeptide complex according to any one of embodiments 1-18 in the preparation of a pharmaceutical composition for the treatment of a disease or condition associated with CD20 in a subject.

29. The use according to embodiment 28, wherein the disease or condition is cancer.

The use of embodiment 29, wherein the cancer is lymphoma, lung cancer, liver cancer, cervical cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, glioblastoma, prostate cancer, esophageal cancer, or gastric cancer.

31. The use of embodiment 28, wherein the disease or condition is B-cell lymphoma, optionally hodgkin's lymphoma or non-hodgkin's lymphoma, wherein the non-hodgkin's lymphoma comprises: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), or Mantle Cell Lymphoma (MCL), Acute Lymphocytic Leukemia (ALL), or Waldenstrom's Macroglobulinemia (WM).

32. A kit comprising a bispecific polypeptide complex according to any one of embodiments 1-18.

33. The kit of embodiment 32 for use in detecting, diagnosing, prognosing or treating a CD 20-associated disease or disorder.

The foregoing and other features and advantages of the invention will be apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

Drawings

FIG. 1 presents a schematic representation of the antibody format studied, in which "E17R-1", "F16-1" and "F17R-1" schematically represent the formats of the bispecific antibodies W3278-T2U3.E17R-1.uIgG4.SP, W3278-T3U2.F16-1.uIgG4.SP and W3278-T3U2.F17R-1.uIgG4.SP, respectively, and "F18R-1" schematically represents the formats of the bispecific antibodies W3278-U2T3.1F8R-1. uIgG4.SP and W3278-U3T2.F81R-1. uIg4. SP. In the context of the present disclosure, "U" in the nomenclature of bispecific antibodies refers to an anti-CD20 antibody or an anti-CD20 binding moiety and "T" in the nomenclature of bispecific antibodies refers to an anti-CD 3 antibody or an anti-CD 3 binding moiety. The constant regions of the "T" (CL and CH1) were replaced by the constant domains of the TCR to design a unique light-heavy chain interface orthogonal to conventional antibodies. The TCR-modified "T" and native "U" were used in conjunction with the "hand-access" mutations in the Fc domain to design bispecific antibody formats "E17R-1", "F16-1", "F17R-1" and "F18R-1".

Figure 2 shows target binding as detected by FACS: (A) the detection of binding of the bispecific antibody of the present disclosure (WBP3278 BsAb) to Jurkat cells, (B) the detection of binding of the bispecific antibody of the present disclosure (WBP3278 BsAb) to Raji cells.

Figure 3 shows simultaneous dual target binding as detected by FACS.

Fig. 4 shows T cell killing.

Fig. 5A and 5B show T cell activation as indicated by CD25 expression and CD69 expression, respectively.

FIG. 6 shows CD4⁺IL-2(A) and TNF- α (B) release from T cells.

Figure 7 shows the results of serum stability.

Figure 8 shows the dose-dependent anti-tumor activity of BsAb of the present invention in an in vivo therapeutic treatment model.

FIG. 9 shows the depletion of peripheral circulating B cells following administration of WBP3278 lead Ab (i.e., W3278-U2T3.F18R-1.uIgG4) to naive male cynomolgus monkeys.

Fig. 10A, 10B, and 10C show changes in peripheral circulating T cell levels following treatment with WBP3278 lead Ab.

Figure 11 shows the change in circulating cytokine levels following treatment with WBP3278 lead Ab.

Fig. 12 shows the serum concentration of WBP3278 lead Ab as a function of time.

Detailed Description

The following description of the invention is merely illustrative of various embodiments of the invention.

Definition of

The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "a polypeptide complex" refers to one polypeptide complex or more than one polypeptide complex.

The term "about" or "approximately" as used herein refers to a quantity, level, value, amount, frequency, percentage, dimension, size, amount, weight, or length that differs by 30, 25, 20, 15, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1% of a reference quantity, level, value, amount, frequency, percentage, dimension, size, amount, weight, or length. In particular embodiments, when the term "about" or "approximately" precedes a numerical value, it means a range of the value plus or minus 15%, 10%, 5%, or 1%.

Throughout this application, unless the context dictates otherwise, the words "comprise", "comprises" and "comprising" will be understood to mean that a stated step or element or group of steps or elements is included, but not to the exclusion of any other step or element or group of steps or elements. What is meant by "consisting of … …" includes and is limited to what follows the phrase "consisting of … …". Thus, the phrase "consisting of … …" means that the listed elements are required or necessary and that no other elements may be present. What is meant by "consisting essentially of … …" is to include any element listed after this phrase and is not limited to other elements that do not interfere with or contribute to the activity or function of the listed element as detailed in this application. Thus, the phrase "consisting essentially of … …" means that the listed elements are required or necessary, but that other elements are optional and may or may not be present depending on whether they affect the activity or effect of the listed elements.

Reference throughout this application to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "some embodiment," "another embodiment," or "further embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present application. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to a polymer of amino acid residues, or a collection of polymers of multiple amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term "amino acid" refers to both naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that have been subsequently modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid (i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Alpha-carbon refers to the first carbon atom attached to a functional group, such as a carbonyl group. The beta carbon refers to the second carbon atom attached to the alpha carbon, and this system continues to name carbon atoms in the alphabetical order of the greek letters. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The term "protein" generally refers to large polypeptides. The term "peptide" generally refers to short polypeptides. Polypeptide sequences are generally described as having the amino terminus (N-terminus) at the left-hand end of the polypeptide sequence; the right hand end of the polypeptide sequence is the carboxyl terminus (C-terminus). "polypeptide complex" as used herein refers to a complex comprising one or more polypeptides involved in performing certain functions. In certain embodiments, the polypeptide is immunologically relevant.

The term "antibody" as used herein encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds a particular antigen. A natural intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region ("HCVR") and first, second and third constant regions (CH1, CH2, CH3, respectively), while each light chain consists of a variable region ("LCVR") and a constant region (CL). Mammalian heavy chains can be classified as α, δ, ε, γ, and μ, and mammalian light chains as λ or κ. Antibodies are "Y" -shaped, the stem of the "Y" -shaped structure consisting of the second and third constant regions of two heavy chains, which are bound by disulfide bonds. Each arm of the "Y" structure comprises the variable and first constant regions of one of the heavy chains, which are associated with the variable and constant regions of one of the light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable region of each chain contains three hypervariable loops, called Complementarity Determining Regions (CDRs) (the CDRs for the light (L) chain comprise LCDR1, LCDR2, LCDR3 and the CDRs for the heavy (H) chain comprise HCDR1, HCDR2, HCDR 3). CDR boundaries of antibodies can be named or identified by Kabat, Chothia, or Al-Lazikani nomenclature (Al-Lazikani, B., Chothia, C., Lesk, A.M., J.mol.biol., 273 (4)), 927 (1997); Chothia, C. et Al, J Mol biol.Dec 5; 186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J.mol.biol., 196,901 (1987); Chothia, C. et Al, Nature.Dec 21-342; 6252: 877-83 (1989); Kabat E.A. et Al, National Institutes of Health, Bethesda, Md. (1991)). Where three CDRs are separated by flanking continuous portions called Framework Regions (FRs), which are more highly conserved than CDRs and form a scaffold-supported hypervariable loop. The HCVR and LCVR each comprise 4 FRs, and the CDRs and FRs are arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The constant regions of the heavy and light chains are not involved in antigen binding, but have multiple effector functions. Antibodies can be classified into several classes depending on the amino acid sequence of the heavy chain constant region. Depending on whether it contains alpha, delta, epsilon, gamma and mu heavy chains, antibodies can be divided into five major classes or isotypes, respectively: IgA, IgD, IgE, IgG and IgM. Several major antibody classes can also be divided into subclasses, such as IgG1(γ 1 heavy chain), IgG2(γ 2 heavy chain), IgG3(γ 3 heavy chain), IgG4(γ 4 heavy chain), IgA1(α 1 heavy chain), or IgA2(α 2 heavy chain), among others.

The term "variable domain" as used herein with respect to an antibody refers to an antibody variable region or fragment thereof comprising one or more CDRs. While the variable domain may comprise the entire variable region (e.g., HCVR or LCVR), it may also comprise less than the entire variable region while still retaining the ability to bind to or form an antigen binding site.

The term "antigen-binding portion" as used herein refers to an antibody fragment formed from an antibody portion containing one or more CDRs or any other antibody fragment that binds an antigen but does not have a complete antibody structure. Examples of antigen-binding portions include, but are not limited to, variable domains, variable regions, bifunctional antibodies (diabodies), Fab ', F (ab')₂Fv fragment, disulfide bond-stabilized Fv fragment (dsFv), (dsFv)₂Bispecific dsFvs (dsFvs-dsFvs'), disulfide stabilized bifunctional antibodies (ds diabodies), multispecific antibodies, camelized single domain antibodies (camelized single domain antibodies), nanobodies, domain antibodies, and bivalent domain antibodies. The antigen binding portion may bind to the same antigen as the maternal antibody. In certain embodiments, the antigen-binding portion can comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies. More detailed forms of antigen-binding portions are described in Spiess et al, 2015 (supra) and Brinkman et al, mAbs, 9(2), pp.182-212 (2017), the entire contents of which are incorporated herein by reference.

The "Fab" of an antibody refers to the portion of the antibody that is disulfide bonded to the variable and first constant regions of one light chain (comprising the variable and constant regions) and one heavy chain. In certain embodiments, the constant regions of both the light and heavy chains are replaced by TCR constant regions.

"Fab'" refers to a Fab fragment that contains a portion of the hinge region.

“F(ab')₂"refers to a dimer of Fab.

The "fragment diffcult (Fd)" of an antibody refers to the amino-terminal half of a heavy chain fragment that can be combined with a light chain to form a Fab.

The "Fc" of an antibody refers to the portion of an antibody consisting of the second (CH2), third (CH3) constant region of a first heavy chain disulfide bonded to the second and third constant regions of a second heavy chain. The Fc portion of an antibody is responsible for a variety of different effector functions, such as ADCC and CDC, but is not involved in antigen binding.

The "hinge region" in the case of antibodies comprises the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region comprises about 25 amino acid residues and is flexible, thereby allowing the two N-terminal antigen-binding regions to move independently.

The term "CH 2 domain" as used herein refers to a portion comprising a heavy chain molecule extending from, for example, about amino acid 244 to amino acid 360 of an IgG antibody, using conventional numbering schemes (amino acids 244 to 360, Kabat numbering system; and amino acids 231-340, EU numbering system; see Kabat, E.et al, U.S. department of Health and Human Services (1983)).

The "CH 3 domain" extends from the CH2 domain of an IgG molecule to the C-terminus and comprises approximately 108 amino acids. Certain immunoglobulin classes, such as IgM, further comprise a CH4 region.

"Fv" of an antibody refers to the smallest antibody fragment that contains the entire antigen-binding site. The Fv fragment consists of the variable region of one light chain in combination with the variable region of one heavy chain. Several Fv designs are provided, including dsfvs, in which the linkage between the two domains is enhanced by an introduced disulfide bond; and a peptide linker may be used to join the two domains together into a single polypeptide to form an scFv. Fv constructs have been produced that contain the variable domain of a heavy or light immunoglobulin chain linked to the variable and constant domains of the corresponding immunoglobulin heavy or light chain. Fv have also been multimerized to form bifunctional and trifunctional antibodies (Maynard et al, Annu Rev Biomed Eng 2339-376 (2000)).

"ScFab" refers to a fusion polypeptide having an Fd connected to a light chain via a polypeptide linker, resulting in a single chain Fab fragment (ScFab).

"TriFab" refers to a trivalent, bispecific fusion protein consisting of 3 units with Fab function. Trilab accommodates the fusion of two common fabs to an asymmetric Fab-like moiety.

"Fab-Fab" refers to a fusion protein formed by fusing the Fd chain of a first Fab arm to the N-terminus of the Fd chain of a second Fab arm.

"Fab-Fv" refers to a fusion protein formed by fusing an HCVR to the C-terminus of the Fd chain and an LCVR to the C-terminus of the light chain. "Fab-dsFv" molecules can be formed by introducing an interdomain disulfide bond between the HCVR domain and the LCVR domain.

"MAb-Fv" or "IgG-Fv" refers to fusion proteins formed by fusion of an HCVR domain to the C-terminus of one Fc chain and expression of an LCVR domain alone or fused to the C-terminus of the other Fc chain, thereby producing a bispecific, trivalent IgG-Fv (mAb-Fv) fusion protein whose Fv is stabilized by interdomain disulfide bonds.

“ScFab-Fc-scFv₂"and" ScFab-Fc-scFv "refer to a fusion protein of a single-chain Fab fused to an Fc and disulfide-stabilized Fv domain.

By "attaching IgG" is meant that the Fab arm is fused to the IgG to form a bispecific (Fab)₂-a fusion protein in the form of an Fc. It may form an "IgG-Fab" or "Fab-IgG" with Fab fused to the C-or N-terminus of the IgG molecule, whether or not a linker is present. In certain embodiments, the attached IgG may be further modified to an IgG-Fab₄Of (see Brinkman et al, 2017, supra).

"DVD-Ig" refers to a dual variable domain antibody formed by fusing additional HCVR and LCVR domains of a second specificity to IgG heavy and light chains. "CODV-Ig" refers to a related form in which two HCVRs and two LCVR domains are linked in a manner that allows cross-pairing of the variable HCVR-LCVR domains, arranged (from N to C-terminal) in the order of HCVRA-HCVRB and LCVRB-LCVRA, or in the order of HCVRB-HCVRA and LCVRA-LCVRB.

"CrossMab" refers to a technique in which an unmodified light chain is paired with a corresponding unmodified heavy chain, and a modified light chain is paired with a corresponding modified heavy chain, thereby producing an antibody with reduced mismatches in the light chain.

A "BiTE" is a bispecific T-cell engaging molecule comprising a first scFv having a first antigen specificity in the direction of LCVR-HCVR linked to a second scFv having a second antigen specificity in the direction of HCVR-LCVR.

"WuXiBody" is a bispecific antibody comprising a soluble chimeric protein having the variable domain of the antibody and the constant domain of the TCR, wherein the subunits (e.g., the a and β domains) of the TCR constant domain are linked by engineered disulfide bonds.

"percent sequence identity," when used with respect to an amino acid sequence (or nucleic acid sequence), refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to a reference sequence in that candidate sequence after alignment and, if necessary, introduction of a spacer to maximize the number of identical amino acids (or nucleic acids). Conservative substitutions of such amino acid residues may or may not be considered identical residues. Sequences can be aligned to determine percent sequence identity of amino acid (or Nucleic acid) sequences by tools disclosed in the art, such as BLASTN, BLASTp (national center for Biotechnology information website (NCBI), see also Altschul S.F. et al, J.mol.biol., 215: 403-. The skilled person can use default parameters for the tool or can adjust the parameters appropriately according to the needs of the alignment, e.g. by choosing a suitable algorithm.

As used herein, "antigen" or "Ag" refers to a compound, composition, peptide, polypeptide, protein, or substance that can stimulate the production of an antibody or T cell response in cell culture or in an animal, including compositions that are added to cell cultures (e.g., hybridomas), or injected or absorbed into an animal (e.g., compositions that include cancer-specific proteins). The antigen is reacted with a product of specific humoral or cellular immunity (e.g., an antibody, including a product induced by a heterologous antigen).

An "epitope" or "antigenic determinant" refers to a region of an antigen to which a binding agent (e.g., an antibody) binds. Epitopes can be formed from contiguous amino acids (also known as linear or sequential epitopes) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (also known as conformational or conformational epitopes). Epitopes formed from contiguous amino acids are typically linearly arranged along primary amino acid residues on proteins, and small segments of contiguous amino acids can be digested by antigen binding to Major Histocompatibility Complex (MHC) molecules or retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, more typically at least 5, about 7, or about 8-10 amino acids in a unique spatial configuration.

"specific binding" or "specific binding" in this application refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen. In certain embodiments, the polypeptide complexes and bispecific polypeptide complexes provided herein bind specifically to antigens and their binding affinities (K)_D)≤10^-6M (e.g.. ltoreq.5X 10)^-7M、≤2×10^-7M、≤10^-7M、≤5×10^-8M、≤2×10^-8M、≤10^-8M、≤5×10^-9M、≤2×10^-9M、≤10^-9M is equal to or less than 10^-10M). K for the present application_DRefers to the ratio of the dissociation rate to the association rate (k)_off/k_on) This can be determined by using surface plasmon resonance methods, for example using instruments such as Biacore.

The term "operably linked" or "operably linked" refers to the juxtaposition of two or more biological sequences of interest in a relationship permitting them to function in their intended manner, regardless of the presence of a spacer or linker. When used with respect to polypeptides, the term is intended to mean that the polypeptide sequences are linked in a manner such that the linked product possesses the desired biological function. For example, an antibody variable region can be operably linked to a constant region to form a stable product with antigen binding activity. The term may also apply to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to regulatory sequences (e.g., promoters, enhancers, silencer sequences, etc.), the term is intended to mean that the polynucleotide sequences are linked in a manner that allows for the regulated expression of the polypeptide by the polynucleotide.

The term "fusion" or "fused" when used with respect to an amino acid sequence (e.g., a peptide, polypeptide, or protein) refers to the combination of two or more amino acid sequences into a single amino acid sequence that does not occur in nature, e.g., by chemical bonding or recombinant means. The fused amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

The term "spacer" as used herein refers to an artificial amino acid sequence having 1, 2, 3, 4, or 5 amino acid residues, or 5 to 15, 20, 30, 50 or more amino acid residues in length, which are linked by peptide bonds and used to link one or more polypeptides. The spacer may or may not have a secondary structure. Spacer sequences are well known in the art, see, e.g., Holliger et al, Proc. Natl. Acad. Sci. USA 90: 6444-; poljak et al, Structure 2:1121- > 1123 (1994). Any suitable spacer known in the art may be used.

The term "antigen specificity" refers to a particular antigen or epitope thereof that is selectively recognized by an antigen binding molecule.

The term "substitution" as used herein with respect to an amino acid residue refers to the substitution of one or more amino acids, naturally occurring or introduced, with another in a peptide, polypeptide or protein. Substitutions in a polypeptide can result in the reduction, enhancement, or elimination of the function of the polypeptide.

Substitutions may also be "conservative" with respect to an amino acid sequence, where one amino acid residue is replaced with a different amino acid residue from another side chain having similar physicochemical properties, or where the substitution is of an amino acid that is not critical to the activity of the polypeptide. For example, conservative substitutions may be made between non-polar side chain amino acid residues (e.g., Met, Ala, Val, Leu and Ile, Pro, Phe, Trp), between uncharged polar side chain residues (e.g., Cys, Ser, Thr, Asn, Gly and Gln), between acidic side chain residues (e.g., Asp, Glu), between basic side chain amino acids (e.g., His, Lys and Arg), between beta-branched side chain amino acids (e.g., Thr, Val and Ile), between sulfur-containing side chain amino acids (e.g., Cys and Met), or between aromatic side chain residues (e.g., Trp, Tyr, His and Phe). In certain embodiments, substitutions, deletions, or additions may also be considered "conservative substitutions. The number of amino acids inserted or deleted may range from about 1 to 5. Conservative substitutions generally do not cause significant changes in the conformational structure of the protein and, therefore, maintain the biological activity of the protein.

The term "mutation" or "mutated" as used herein in reference to an amino acid residue refers to a substitution, insertion or addition of an amino acid residue.

The term "T cell" as used herein refers to a class of lymphocytes that play an important role in cell-mediated immunity, including helper T cells (e.g., CD 4)⁺T cells, T helper type 1T cells, T helper type 2T cells, T helper type 3T cells, T helper type 17T cells), cytotoxic T cells (e.g., CD8⁺T cells), memory T cells (e.g., central memory T cells (TCM cells), effector memory T cells (TEM cells and TEMRA cells), and resident memory T cells (TRM), which are CD8+ or CD4+), natural killer T (nkt) cells, and suppressor T cells.

A native "T cell receptor" or native "TCR" is a heterodimeric T cell surface protein that binds to an invariant CD3 chain to form a complex capable of mediating signal transduction. The TCR belongs to the immunoglobulin superfamily and is similar to half antibodies with a single heavy chain and a single light chain. Native TCRs have an extracellular portion, a transmembrane portion, and an intracellular portion. The extracellular domain of the TCR has a membrane-proximal constant region and a membrane-distal variable region.

The term "subject" or "individual" or "animal" or "patient" as used herein refers to a human or non-human animal, including mammals or primates, in need of diagnosis, prognosis, amelioration, prophylaxis and/or treatment of a disease or disorder. Mammalian subjects include humans, livestock animals, farm animals, and zoo, sports, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, and the like.

Bispecific polypeptide complexes

In one aspect, the present application provides bispecific polypeptide complexes. The term "bispecific" as used in this application means that there are two antigen binding moieties, each capable of specific binding to a different antigen or to different epitopes on the same antigen. Bispecific polypeptide complexes provided herein comprise a first antigen-binding moiety associated with a second antigen-binding moiety, and one of which specifically binds CD3 and the other of which specifically binds CD 20. That is, the first antigen-binding portion can specifically bind to CD3, and the second antigen-binding portion can specifically bind to CD 20. Alternatively, the first antigen-binding portion may specifically bind to CD20 and the second antigen-binding portion may specifically bind to CD 3. In the present invention, the term "bispecific anti-CD 3xCD20 polypeptide complex" is used interchangeably with "polypeptide complex targeting CD3 and CD 20" or "anti-CD 3 and CD20 polypeptide complex".

In certain embodiments, the present application provides a bispecific polypeptide complex comprising a first antigen-binding moiety associated with a second antigen-binding moiety, wherein:

the first antigen-binding portion comprises:

a second polypeptide comprising, from N-terminus to C-terminus, a first light chain variable domain (VL) of a first antibody operably linked to a second TCR constant region (C2), wherein:

the second antigen-binding moiety comprises:

wherein:

the anti-CD 3 binding portion is derived from an anti-CD 3 antibody comprising:

b) comprises a sequence selected from SEQ ID NO: 2. 14, 26, 38 and 50, and a heavy chain CDR2 of an amino acid sequence,

d) comprises a sequence selected from SEQ ID NO: 4. 16, 28, 40 and 52, the kappa light chain CDR1 of the amino acid sequence,

e) comprises a sequence selected from SEQ ID NO: 5. 17, 29, 41 and 53, and the kappa light chain CDR2 of the amino acid sequence of

f) Comprising a nucleic acid sequence selected from SEQ ID NO: 6. 18, 30, 42 and 54, and kappa light chain CDR3,

an anti-CD20 binding moiety is derived from an anti-CD20 antibody comprising:

d) comprising a nucleic acid sequence selected from SEQ ID NO: 10. 22, 34, 46 and 58, the kappa light chain CDR1 of the amino acid sequence,

f) Comprising a nucleic acid sequence selected from SEQ ID NO: 12. 24, 36, 48 and 60, and a kappa light chain CDR 3. In certain embodiments, bispecific polypeptide complexes provided herein comprise a first antigen-binding portion comprising a sequence derived from a TCR constant region, but the second antigen-binding portion does not comprise a sequence derived from a TCR constant region.

The bispecific polypeptide complexes provided herein are significantly less susceptible to having mismatched heavy and light chain variable domains. Without wishing to be bound by any theory, it is believed that the stable TCR constant regions in the first antigen-binding portion may be specifically linked to each other and thus contribute to highly specific pairing of the target VH1 and VL1, while preventing VH1 or VL1 from forming unwanted mismatches with other variable regions that do not provide a target antigen-binding site.

In certain embodiments, the second antigen-binding portion further comprises an antibody constant CH1 domain operably linked to VH2, and an antibody light chain constant domain operably linked to VL 2. Thus, the second antigen-binding portion comprises a Fab.

When the first, second, third and fourth variable domains (e.g. VH1, VH2, VL1 and VL2) are expressed in one cell, it is highly desirable that VH1 specifically pair with VL1 and VH2 specifically pair with VL2, so that the resulting bispecific protein product will have the correct antigen binding specificity. However, in the prior art, such as hybrid-hybridomas (or quadromas), random pairing of VH1, VH2, VL1 and VL2 occurs, resulting in the generation of up to 10 different molecules, only one of which is a functional bispecific antigen-binding molecule. This not only reduces the yield, but also complicates the purification of the desired product.

The bispecific polypeptide complexes provided herein are advantageous because the variable domains are less susceptible to mismatches than in the case where the first and second antigen-binding portions are both counterparts of the native Fab. In an illustrative example, the first antigen-binding domain comprises VH1-C1 paired with VL1-C2, and the second antigen-binding domain comprises VH2-CH1 paired with VL 2-CL. It has been unexpectedly found that C1 and C2 preferentially associate with each other and do not associate as readily with CL or CH1, thereby hindering and significantly reducing the formation of unwanted pairings such as C1-CH, C1-CL, C2-CH, and C2-CL. Due to the specific association of C1-C2, VH1 specifically pairs with VL1 and thereby forms the first antigen-binding site, and CH1 specifically pairs with CL, thereby allowing specific pairing of VH2-VL2 that provides the second antigen-binding site. Accordingly, the first and second antigen-binding portions are less susceptible to mismatch and mismatches, e.g. between VH 1-VL2, VH2-VL1, VH1-VH2, VL1-VL2, are significantly reduced compared to the case where the first and second antigen-binding portions are both corresponding portions of a native Fab (e.g. in the form of VH 1-CH 1, VL1-CL, VH2-CH1 and VL 2-CL).

In certain embodiments, bispecific polypeptide complexes provided herein have significantly fewer mismatch products (e.g., at least 1, 2, 3, 4,5, or more mismatch products reduced) and/or significantly higher yields (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, or more yield increased) when expressed from a cell compared to a reference molecule expressed under comparable conditions, wherein the reference molecule is otherwise identical to the bispecific polypeptide complex except for the substitution of C1 with native CH1 and the substitution of C2 with native CL.

Antigen binding moieties comprising engineered C alpha and C beta

A first antigen-binding portion provided herein comprises a first antibody heavy chain variable domain operably linked to a first T Cell Receptor (TCR) constant region, and a first antibody light chain variable domain operably linked to a second TCR constant region, wherein the first TCR constant region and the second TCR constant region are associated via at least one non-native interchain disulfide bond. The first antigen-binding portion comprises at least two polypeptide chains, each comprising a variable domain derived from an antibody and a constant region derived from a TCR. Thus, the first antigen binding portion comprises a heavy chain variable domain and a light chain variable domain, each of which is operably linked to a pair of TCR constant regions. In certain embodiments, the pairing of TCR constant regions in the first antigen-binding portion is an α/β TCR constant region. The TCR constant regions of the polypeptide complexes provided herein can associate with each other through at least one non-native disulfide bond to form a dimer.

It has been surprisingly found that a first antigen binding moiety provided herein having at least one non-native disulfide bond can be recombinantly expressed and assembled into a desired conformation that stabilizes TCR constant region dimers and provides good antigen binding activity of antibody variable regions. In addition, the first antigen binding moiety was found to be well tolerated by conventional antibody engineering, such as modification of glycosylation sites and removal of some native sequences. In addition, the polypeptide complexes provided herein can be incorporated into a heterozygotic form that can be readily expressed and assembled with minimal or little mismatch of the antigen binding sequence due to the presence of the TCR constant region in the first antigen binding portion. Other advantages of the first antigen-binding portions and constructs provided herein will become more apparent in the disclosure that follows.

In summary, the present application provides a first antigen-binding portion comprising a first polypeptide comprising from N-terminus to C-terminus a first heavy chain variable domain (VH) of a first antibody operably linked to a first T Cell Receptor (TCR) constant region (C1), and a second polypeptide comprising from N-terminus to C-terminus a first light chain variable domain (VL) of the first antibody operably linked to a second TCR constant region (C2), wherein: c1 and C2 are capable of forming dimers, and the non-natural interchain disulfide bond between C1 and C2 is capable of stabilizing the dimers.

TCR constancyZone(s)

The first antigen binding portion described herein comprises an alpha or beta constant region derived from a TCR.

The human TCR α chain constant region is called TRAC with NCBI accession number P01848.

There are two different variants of the constant region of the human TCR β chain, known as TRBC1 and TRBC2 (IMGT nomenclature) (see also Toyonaga B et al, PNAs, Vol.82, pp.8624-8628, Immunology (1985)).

In this application, the first and the second TCR constant regions of the first antigen-binding portion provided herein are capable of forming a dimer comprising at least one non-native interchain disulfide bond between the TCR constant regions capable of stabilizing the dimer.

The term "dimer" as used herein refers to a structure of two molecules (e.g., polypeptides or proteins) that associate via covalent or non-covalent interactions. Homodimers or homodimers are formed from two identical molecules, whereas heterodimers or heterodimers are formed from two different molecules. The dimer formed by the first and the second TCR constant regions is a heterodimer.

A "mutated" amino acid residue refers to an amino acid residue that is substituted, inserted, or added and that is different from the corresponding native residue in its corresponding native TCR constant region. For example, if an amino acid residue at a particular position in the wild-type TCR constant region is referred to as a "native" residue, then its corresponding mutant residue is any residue that is different from the native residue but located at the same position on the TCR constant region. A mutated residue may be a different residue that replaces the native residue at the same position, or a different residue that is inserted before the native residue and thus occupies its original position.

In the polypeptide complexes provided herein, the first and/or the second TCR constant regions have been engineered to comprise one or more mutated amino acid residues responsible for forming the non-native interchain disulfide bond. To introduce such mutated residues into the TCR constant region, the coding sequence of the TCR region may be manipulated, for example, to replace the codon encoding the native residue with a codon encoding the mutated residue, or to insert the codon encoding the mutated residue before the codon encoding the native residue.

In the polypeptide complexes provided herein, the first and/or the second TCR constant regions have been engineered to comprise one or more mutated cysteine residues such that, upon substitution of a cysteine residue, a non-native interchain disulfide bond can be formed between the two TCR constant regions.

The non-natural interchain disulfide bond is capable of stabilizing the first antigen binding moiety. The effect of this stabilizing aspect can be embodied in various ways. For example, the presence of mutated amino acid residues or non-natural interchain disulfide bonds may enable stable expression of the polypeptide complex, and/or expression at high levels, and/or association into a stable complex having a desired biological activity (e.g., antigen binding activity), and/or expression and assembly into a high level of a desired stable complex having a desired biological activity. The ability of the interchain disulfide to stabilize the first and second TCR constant regions can be assessed using suitable methods known in the art, such as displaying molecular weight on SDS-PAGE, or measuring thermal stability by Differential Scanning Calorimetry (DSC) or Differential Scanning Fluorescence (DSF). In an illustrative example, if the product shows a molecular weight comparable to the combined molecular weight of said first and said second polypeptide, the formation of a stable first antigen-binding portion as provided herein can be confirmed by SDS-PAGE. In certain embodiments, the first antigen-binding portion described herein is stable in that it is not less than 50%, 60%, 70%, 80%, or 90% thermostable of a native Fab. In certain embodiments, the first antigen-binding portion described herein is stable in that it is thermostable comparable to a native Fab.

Without wishing to be bound by any theory, it is believed that the non-native interchain disulfide bond formed between the first and the second TCR constant regions in the first antigen-binding portion is capable of stabilizing heterodimers of TCR constant regions, thereby enhancing the correct folding level, structural stability and/or expression level of the heterodimers and the first antigen-binding portion. Unlike native TCRs anchored on the cell membrane on the surface of T cells, heterodimers of the extracellular domains of native TCRs were found to be less stable, although similar in 3D structure to antibody Fab. Indeed, the instability of native TCRs in solution has been a significant impediment to making their crystal structures difficult to elucidate (see Wang, Protein Cell,5(9), pp.649-652 (2014)). By introducing a pair of cysteine (Cys) mutations into the TCR constant region and thereby enabling interchain non-native disulfide bond formation, the first antigen-binding moiety can be stably expressed while retaining the antigen-binding capacity of the antibody variable region.

TCR constant regions comprising mutated residues are also referred to herein as "engineered" TCR constant regions. In certain embodiments, the first TCR constant region (C1) of the polypeptide complex comprises an engineered TCR alpha chain (ca) and the second TCR constant region (C2) comprises an engineered TCR beta chain (cp). In the polypeptide complexes provided herein, C1 comprises an engineered C β, and C2 comprises an engineered C α.

In the polypeptide complexes provided herein, the engineered TCR constant regions comprise one or more mutated cysteine residues comprised within the contact interface of the first and/or the second engineered constant regions.

The term "contact interface" as used herein refers to a specific region on the polypeptide where the polypeptides interact/associate with each other. The contact interface comprises one or more amino acid residues that are capable of interacting with a corresponding amino acid residue in contact or association when the interaction occurs. The amino acid residues in the contact interface may or may not be in contiguous sequence. For example, when the interface is three-dimensional, the amino acid residues within the interface can be separated from each other at different positions on the linear sequence.

In certain embodiments, one or more disulfide bonds may be formed between the engineered ca and the engineered cp. In certain embodiments, the pairing of cysteine residues is capable of forming a non-natural interchain disulfide bond.

As used throughout this application, "XnY" with respect to the TCR constant region is intended to mean that the nth amino acid residue X on the TCR constant region is substituted with amino acid residue Y, where X and Y are each single letter abbreviations for the particular amino acid residue.

In the polypeptide complexes provided herein, the engineered C β comprises or is SEQ ID NO:121 and the engineered C α comprises or is SEQ ID NO: 122.

The sequences of SEQ ID NO 121 and SEQ ID NO 122 are provided below:

SEQ ID NO:121

LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWW VNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRN HFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEA

SEQ ID NO:122

PDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITD KCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPEDTFFPSPESS

in the polypeptide complexes provided herein, one or more native glycosylation sites present in native TCR constant regions can be modified (e.g., removed) in the first antigen-binding portion provided herein. The term "glycosylation site" as used herein with respect to a polypeptide sequence refers to an amino acid residue with a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of polypeptides (such as antibodies) is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue, for example an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, wherein X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of the native glycosylation site is conveniently accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences (for N-linked glycosylation sites) or one or more of the serine or threonine residues (for O-linked glycosylation sites) present in the sequence is substituted.

In the first antigen-binding portions provided herein, at least one native glycosylation site is not present in the engineered TCR constant region (e.g., in the first and/or the second TCR constant region). Without wishing to be bound by any theory, it is believed that the first antigen-binding portion described herein may allow for the removal of all or part of the glycosylation site without affecting protein expression and stability, in contrast to the prior teaching that the presence of an N-linked glycosylation site on TCR constant regions such as ca (i.e., N34, N68 and N79) and cp (i.e., N69) is essential for protein expression and stability (see Wu et al, Mabs, 7:2, 364-.

In a first antigen-binding portion provided herein, a constant region derived from a TCR is operably linked to a variable region derived from an antibody.

In certain embodiments, the first antibody variable domain (VH) is fused to the first TCR constant region (C1) at a first junction domain and the first antibody variable domain (VL) is fused to the second TCR constant region (C2) at a second junction domain.

The term "junction domain" as used herein refers to an interface or boundary region where two amino acid sequences are fused or combined. In certain embodiments, the first engagement domain comprises at least a portion of a C-terminal fragment of an antibody V/C engager and the second engagement domain comprises at least a portion of an N-terminal fragment of a TCR V/C engager.

The term "antibody V/C binder" as used herein refers to the interface of an antibody variable domain and a constant domain, for example between a heavy chain variable domain and a CH1 domain or between a light chain variable domain and a light chain constant domain. Similarly, the term "TCR V/C engager" refers to the interface between a TCR variable domain and a constant domain, e.g., between a TCR α variable domain and a constant domain or between a TCR β variable domain and a constant domain.

In certain embodiments, the first polypeptide comprises a sequence comprising a domain operably linked according to formula (I): VH-HCJ-C1, and the second polypeptide comprises a sequence comprising a domain operably linked as in formula (II): VL-LCJ-C2, wherein:

VH is the heavy chain variable domain of an antibody;

HCJ is a first binding domain as defined above;

c1 is a first TCR constant domain as defined above;

VL is the light chain variable domain of an antibody;

LCJ is a second engagement domain as defined above;

c2 is a second TCR constant domain as defined above.

Antibody variable regions

Bispecific polypeptide complexes provided herein comprise a first antigen-binding moiety associated with a second antigen-binding moiety, and one of which specifically binds to CD3 and the other specifically binds to CD 20. In polypeptide complexes provided herein, the first antigen-binding moiety comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1) of a first antibody, and the second antigen-binding moiety comprises a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2) of a second antibody, wherein the first antibody and the second antibody are different and are selected from the group consisting of an anti-CD 3 antibody and an anti-CD20 antibody. In certain embodiments, the first antibody is an anti-CD 3 antibody and the second antibody is an anti-CD20 antibody. In certain other embodiments, the first antibody is an anti-CD20 antibody and the second antibody is an anti-CD 3 antibody.

In a conventional natural antibody, the variable region comprises 3 CDR regions separated by flanking Framework (FR) regions, for example as set out by the following formula from N-terminus to C-terminus: FR1-CDR 1-FR 2-CDR2-FR3-CDR3-FR 4.

a) anti-CD 3 binding moieties

In the polypeptide complexes provided herein, the first antigen-binding portion or the second antigen-binding portion is an anti-CD 3 binding portion.

In certain embodiments, the anti-CD 3 binding moiety is derived from the antibody W3278-t2u3.e17r-1.u igg4.sp shown in table a below. The CDR sequences of the anti-CD 3 binding portion of the W3278-t2u3.e17r-1.u igg4.sp antibody are provided below.

TABLE A

The heavy and kappa light chain variable region sequences of the anti-CD 3-binding portion of the W3278-t2u3.e17r-1.u igg4.sp antibody are provided below.

VH-amino acid sequence (SEQ ID NO: 61):

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGLE WMGWIFPGNDNIKYSEKFKGRVTITADKSTSTAYMELSSLRSEDTAV YYCAIDSVSIYYFDYWGQGTLVTVSS

VH-nucleic acid sequence (SEQ ID NO: 101):

caggtgcaactcgtgcagtctggagctgaagtgaagaagcctgggtcttcagtcaaggtcagttgcaaggcc agtgggtattccttcactacctactacatccactgggtgcggcaggcaccaggacaggggcttgagtggatg ggctggatctttcccggcaacgataatattaagtacagcgagaagttcaaagggagggtcaccattaccgcc gacaaatccacttccacagcctacatggagttgagcagcctgagatccgaggatacagccgtgtactactgt gccattgacagcgtgtccatctactactttgactactggggccagggcacactggtcacagtgagcagc

VK-amino acid sequence (SEQ ID NO: 62):

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPG QPPKLLIYWASTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCT QSFILRTFGGGTKVEIK

VK-nucleic acid sequence (SEQ ID NO: 102):

gacatcgtcatgacccagtccccagactctttggcagtgtctctcggggaaagagctaccatcaactgcaaga gcagccagtcccttctgaacagcaggaccaggaagaattacctcgcctggtaccaacagaagcccggaca gcctcctaagctcctgatctactgggcctcaacccggaagagtggagtgcccgatcgctttagcgggagcg gctccgggacagatttcacactgacaatttcctccctgcaggccgaggacgtcgccgtgtattactgtactcag agcttcattctgcggacatttggcggcgggactaaagtggagattaag

in certain embodiments, the anti-CD 3 binding moiety is derived from the antibody W3278-tj3u 2.f16-1. uggg4. sp shown in table B below. The CDR sequences of the anti-CD 3 binding portion of the W3278-t3u2.f16-1.uigg4.sp antibody are provided below.

TABLE B

Heavy and kappa light chain variable region sequences of the anti-CD 3-binding portion of the W3278-t3u2.f16-1.u igg4.sp antibody are provided below.

VH-amino acid sequence (SEQ ID NO: 63):

QVQLVQSGAEVKKPGSSVKVSCKASGFAFTDYYIHWVRQAPGQGLE WMGWISPGNVNTKYNENFKGRVTITADKSTSTAYMELSSLRSEDTA VYYCARDGYSLYYFDYWGQGTLVTVSS

VH-nucleic acid sequence (SEQ ID NO: 103):

caggtgcagcttgtgcagtctggggcagaagtgaagaagcctgggtctagtgtcaaggtgtcatgcaaggct agcgggttcgcctttactgactactacatccactgggtgcggcaggctcccggacaagggttggagtggatg ggatggatctccccaggcaatgtcaacacaaagtacaacgagaacttcaaaggccgcgtcaccattaccgc cgacaagagcacctccacagcctacatggagctgtccagcctcagaagcgaggacactgccgtctactact gtgccagggatgggtactccctgtattactttgattactggggccagggcacactggtgacagtgagctcc

VK-amino acid sequence (SEQ ID NO: 64):

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPG QPPKLLIYWASTRQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCT QSHTLRTFGGGTKVEIK

VK-nucleic acid sequence (SEQ ID NO: 104):

gatatcgtgatgacccagagcccagactcccttgctgtctccctcggcgaaagagcaaccatcaactgcaag agctcccaaagcctgctgaactccaggaccaggaagaattacctggcctggtatcagcagaagcccggcca gcctcctaagctgctcatctactgggcctccacccggcagtctggggtgcccgatcggtttagtggatctggg agcgggacagacttcacattgacaattagctcactgcaggccgaggacgtggccgtctactactgtactcag agccacactctccgcacattcggcggagggactaaagtggagattaag

in certain embodiments, the anti-CD 3 binding moiety is derived from the antibody W3278-u2t3.f18r-1.uigg4.sp shown in table C below. The CDR sequences of the anti-CD 3 binding portion of the W3278-u2t3.f18r-1.uigg4.sp antibody are provided below.

Watch C

The heavy and kappa light chain variable region sequences of the anti-CD 3 binding portion of the W3278-U2T3.F18R-1.uIgG4.SP antibody are provided below.

VH-amino acid sequence (SEQ ID NO: 65):

VH-nucleic acid sequence (SEQ ID NO: 105):

VK-amino acid sequence (SEQ ID NO: 66):

VK-nucleic acid sequence (SEQ ID NO: 106):

in certain embodiments, the anti-CD 3 binding moiety is derived from the antibody W3278-u3t2.f18r-1.uigg4.sp shown in table D below. The CDR sequences of the anti-CD 3-binding portion of the W3278-U3T2.F18R-1.uIgG4.SP antibody are provided below.

Table D

The heavy and kappa light chain variable region sequences of the anti-CD 3-binding portion of the W3278-U3T2.F18R-1.uIgG4.SP antibody are provided below.

VH-amino acid sequence (SEQ ID NO: 67):

VH-nucleic acid sequence (SEQ ID NO: 107):

VK-amino acid sequence (SEQ ID NO: 68):

VK-nucleic acid sequence (SEQ ID NO: 108):

in certain embodiments, the anti-CD 3 binding moiety is derived from the antibody W3278-t3u2.f17r-1. uggg4. sp shown in table E below. The CDR sequences of the anti-CD 3-binding portion of the W3278-t3u2.f17r-1. uggg4. sp antibody are provided below.

TABLE E

Heavy and kappa light chain variable region sequences of the anti-CD 3-binding portion of the W3278-t3u2.f17r-1.u igg4.sp antibody are provided below.

VH-amino acid sequence (SEQ ID NO: 69):

VH-nucleic acid sequence (SEQ ID NO: 109):

VK-amino acid sequence (SEQ ID NO: 70):

VK-nucleic acid sequence (SEQ ID NO: 110):

the anti-CD 3 binding moieties provided herein also comprise suitable Framework Region (FR) sequences, so long as the anti-CD 3 binding moiety can specifically bind to CD 3.

The anti-CD 3 antibodies of the present application have specific binding affinity for CD3 expressing cells (e.g., CD 4T cells) and can activate human T cells and trigger cytokine release of TNF α and IFN γ.

The binding affinity of the anti-CD 3 binding moieties provided herein can be defined by K_DValue representation, which represents when the antigen and antigen binding componentRatio of dissociation rate to association rate (k) at which association between subunits reaches equilibrium_off/k_on). Antigen binding affinity (e.g., K) can be appropriately determined using suitable methods known in the art, including, for example, flow cytometry assays_D). In some embodiments, binding of antibody to antigen at different concentrations can be determined by flow cytometry, the determined Mean Fluorescence Intensity (MFI) can be first plotted against antibody concentration, and then the dependence of specific binding fluorescence intensity (Y) on antibody concentration (X) can be fitted to a single-site saturation equation by using Prism version 5(GraphPad Software, san diego, california): y is B_max*X/(K_D+ X), thereby calculating K_DValue of, wherein B_maxIs the most specific binding of the antibody to be tested to the antigen.

In certain embodiments, the anti-CD 3 binding moieties provided herein are capable of specifically binding to human CD3 or recombinant human CD3 expressed on the surface of a cell. CD3 is a receptor expressed on cells. Recombinant CD3 is soluble CD3 that is recombinantly expressed and does not associate with the cell membrane. Recombinant CD3 can be prepared by a variety of recombinant techniques. In one example, a CD3 DNA sequence encoding the extracellular domain of human CD3 (NP _000724.1) (Met1-Asp126) can be fused to a polyhistidine tag at the C-terminus in an expression vector and subsequently transfected and expressed in 293E cells and purified by nickel affinity chromatography.

In some embodiments, the anti-CD 3 binding moieties provided herein can be at least 5x10^-9M, not more than 4X 10^-9M, not more than 3X 10^-9M, not more than 2X10^-9M, not more than 10^-9M, not more than 5X10^-10M, not more than 4X 10^-10M, not more than 3X 10^-10M, not more than 2X10^-10M, is not more than 10^-10M, not more than 5X10^-11M or not more than 4X 10^-11M, not more than 3X 10^-11M is not more than 2X10^-11M is or not more than 10^-11Binding affinity (K) of M_D) Specifically binds to human CD3 expressed on the cell surface_DThe value is obtained by flow cytometryAnd (4) measuring.

In certain embodiments, the anti-CD 3 binding moieties provided herein cross-react with cynomolgus monkey CD3 (e.g., cynomolgus monkey CD3 expressed on the cell surface, or soluble recombinant cynomolgus monkey CD 3).

Binding of the anti-CD 3 binding moiety to recombinant CD3 or CD3 expressed on the surface of a cell can be determined by methods well known in the art, such as sandwich assays (e.g., ELISA), Western blots, flow cytometry, and other binding assays. In certain embodiments, an anti-CD 3 binding moiety as provided herein has an EC of no more than 0.01nM, no more than 0.02nM, no more than 0.03nM, no more than 0.04nM, no more than 0.05nM, no more than 0.06nM, no more than 0.07 nM, or no more than 0.08nM₅₀(i.e., 50% binding concentration) specifically binds to recombinant human CD3, the EC₅₀Values were determined by ELISA. In some embodiments, the anti-CD 3 binding moiety provided herein has an EC of no more than 0.5nM, no more than 0.6nM, no more than 0.7nM, no more than 0.8nM, no more than 0.9nM, no more than 1nM, no more than 2nM, no more than 3nM, no more than 4nM, no more than 5nM, no more than 6nM, no more than 7nM, no more than 8nM, no more than 9nM, or no more than 10nM₅₀Specifically binds to human CD3 expressed on the cell surface₅₀Values were determined by flow cytometry.

In certain embodiments, the anti-CD 3 binding moiety binds cynomolgus monkey CD3 with similar binding affinity as human CD 3.

In certain embodiments, an anti-CD 3 binding moiety provided herein has an EC of no more than 0.001nM, no more than 0.005nM, no more than 0.01nM, no more than 0.02nM, no more than 0.03nM, no more than 0.04nM, or no more than 0.05nM₅₀Specifically binds to recombinant cynomolgus monkey CD3, the EC₅₀Values were determined by ELISA.

In certain embodiments, the anti-CD 3 binding moieties provided herein have specific binding affinity for human CD3 sufficient for diagnostic and/or therapeutic applications. Many treatment regimens modulate T cell immunity by targeting TCR signaling, particularly through the use of monoclonal antibodies against human CD3 in the clinic.

b) anti-CD20 antibodies

In the polypeptide complexes provided herein, the first antigen-binding portion or the second antigen-binding portion is an anti-CD20 binding portion.

In certain embodiments, the anti-CD20 binding moiety is derived from the antibody W3278-t2u3.e17r-1.u igg4.sp shown in table a' below. The CDR sequences of the anti-CD20 binding portion of the W3278-t2u3.e17r-1.u igg4.sp antibody are provided below.

Watch A'

The heavy and kappa light chain variable region sequences of the anti-CD 20-binding portion of the W3278-t2u3.e17r-1.u igg4.sp antibody are provided below.

VH-amino acid sequence (SEQ ID NO: 71):

EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLE WVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTAL YYCAKDIQYGNYYYGMDVWGQGTTVTVSS

VH-nucleic acid sequence (SEQ ID NO: 111):

gaggtgcaattggtggagagcggaggagggctcgtgcagcctggaagatctcttaggctgagttgcgctgc atctgggttcacattcaacgactacgccatgcactgggtgaggcaggctcccggcaaagggctggaatggg tgtcaactatctcctggaactccggcagcatcggctacgccgatagcgtcaagggccggtttacaatttcccg cgataacgccaagaagtccctgtacctgcagatgaacagcctgcgggccgaggatactgccctctactactg tgccaaggacattcagtacgggaattactattacgggatggacgtctggggccaggggaccaccgtgacag tcagctcc

VK-amino acid sequence (SEQ ID NO: 72):

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLI YDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPI TFGQGTRLEIK

VK-nucleic acid sequence (SEQ ID NO: 112):

gaaatcgtgctgacccagtccccagcaaccctctccctttctcctggagagagagctaccctcagctgtaggg cctcacagtctgtctccagttacctggcttggtaccagcagaaacccgggcaggcccctaggttgctgatcta cgacgccagcaatagggccactggcatcccagcccggttttccggaagcggcagcgggacagatttcaca ctcactattagcagcctggagcccgaggacttcgccgtgtactattgccagcagcggtccaactggcccatta catttggccaagggacacgcctggagattaag

in certain embodiments, the anti-CD20 binding moiety is derived from the antibody W3278-tj3u 2.f16-1. uggg4. sp, shown in table B', below. The CDR sequences of the anti-CD 20-binding portion of the W3278-t3u2.f16-1.u igg4.sp antibody are provided below.

Watch B'

Heavy and kappa light chain variable region sequences of the anti-CD 20-binding portion of the W3278-t3u2.f16-1.u igg4.sp antibody are provided below.

VH-amino acid sequence (SEQ ID NO: 73):

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGL EWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSA VYYCARSTYYGGDWYFNVWGAGTTVTVSA

VH-nucleic acid sequence (SEQ ID NO: 113):

caggtccagctgcagcagcccggagccgaactggtcaaacccggggctagcgtgaaaatgtcttgcaaag caagtggttacacattcacttcctataacatgcactgggtgaagcagacacctgggcgaggtctggaatggat cggcgccatctacccaggcaacggagacactagctataatcagaagtttaaaggaaaggccaccctgacag ctgataagtccagctctaccgcttacatgcagctgagttcactgacaagtgaggactcagcagtgtactattgc gcccgttctacctactatggcggagattggtatttcaatgtgtggggcgccggtaccacagtcaccgtgtccg cc

VK-amino acid sequence (SEQ ID NO: 74):

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIY ATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPP TFGGGTKLEIK

VK-nucleic acid sequence (SEQ ID NO: 114):

cagattgtcctgagccagagccctgccatcctgtctgctagtcccggcgagaaggtgaccatgacatgcagg gcatccagctctgtctcctacatccactggttccagcagaagcccgggagttcacctaaaccatggatctacg ctacatccaacctggcaagcggtgtgcctgtcaggttttcaggttccggcagcggaacatcttacagtctgact atttctcgggtggaggccgaagacgccgctacctactattgccagcagtggacctccaatccccctacattcg gcggagggactaagctggagatcaaa

in certain embodiments, the anti-CD20 binding moiety is derived from the antibody W3278-u2t3.f18r-1.uigg4.sp shown in table C' below. The CDR sequences of the anti-CD20 binding portion of the W3278-u2t3.f18r-1.uigg4.sp antibody are provided below.

Watch C'

The heavy and kappa light chain variable region sequences of the anti-CD 20-binding portion of the W3278-u2t3.f18r-1.uigg4.sp antibody are provided below.

VH-amino acid sequence (SEQ ID NO: 75):

VH-nucleic acid sequence (SEQ ID NO: 115):

VK-amino acid sequence (SEQ ID NO: 76):

VK-nucleic acid sequence (SEQ ID NO: 116):

in certain embodiments, the anti-CD20 binding moiety is derived from the antibody W3278-U3T2.F18R-1.uIgG4.SP shown in Table D' below. The CDR sequences of the anti-CD20 binding portion of the W3278-u3t2.f18r-1.uigg4.sp antibody are provided below.

Watch D'

The heavy and kappa light chain variable region sequences of the anti-CD 20-binding portion of the W3278-U3T2.F18R-1.uIgG4.SP antibody are provided below.

VH-amino acid sequence (SEQ ID NO: 77):

VH-nucleic acid sequence (SEQ ID NO: 117):

VK-amino acid sequence (SEQ ID NO: 78):

VK-nucleic acid sequence (SEQ ID NO: 118):

in certain embodiments, the anti-CD20 binding moiety is derived from the antibody W3278-t3u2.f17r-1. uggg4. sp shown in table E' below. The CDR sequences of the anti-CD20 binding portion of the W3278-t3u2.f17r-1.u igg4.sp antibody are provided below.

TABLE E'

Heavy and kappa light chain variable region sequences of the anti-CD 20-binding portion of the W3278-t3u2.f17r-1.u igg4.sp antibody are provided below.

VH-amino acid sequence (SEQ ID NO: 79):

VH-nucleic acid sequence (SEQ ID NO: 119):

VK-amino acid sequence (SEQ ID NO: 80):

VK-nucleic acid sequence (SEQ ID NO: 120):

the anti-CD20 binding moieties provided herein also comprise suitable Framework Region (FR) sequences, so long as the anti-CD20 binding moieties can specifically bind CD 20.

In some casesIn embodiments, the anti-CD20 binding moieties provided herein can be no more than 5x10^-9M, not more than 1X10^-9M, not more than 9X 10^-10M, not more than 8X 10^-10M, not more than 7X 10^-10M, not more than 6X 10^-10M, not more than 5X10^-10M, not more than 4X 10^-10M, not more than 3X 10^-10M, not more than 2X10^-10M is not more than 1X10^-10Binding affinity (K) of M_D) Specifically binds to human CD20 expressed on the cell surface, the K_DValues were determined by flow cytometry.

In certain embodiments, the anti-CD20 binding moieties provided herein cross-react with cynomolgus monkey CD20 (e.g., cynomolgus monkey CD20 or soluble recombinant cynomolgus monkey CD20 expressed on the cell surface).

Binding of the anti-CD20 binding moiety to CD20 expressed on the cells can be determined by methods well known in the art, such as sandwich assays (e.g., ELISA), Western blots, flow cytometry assays, and other binding assays. In certain embodiments, an anti-CD20 binding moiety provided herein has an EC of no more than 0.01nM, no more than 0.02nM, no more than 0.03nM, no more than 0.04nM, no more than 0.05nM, no more than 0.1nM, no more than 0.2nM, no more than 0.3nM, no more than 0.4nM, no more than 0.5nM, no more than 0.6nM, no more than 0.7nM, no more than 0.8nM, no more than 0.9nM, or no more than 1nM₅₀Specifically binds to human CD20 expressed on cells, the EC₅₀Values were determined by flow cytometry.

In certain embodiments, the anti-CD20 binding moiety binds cynomolgus monkey CD20 with similar binding affinity as human CD 20. In certain embodiments, an anti-CD20 binding moiety provided herein has an EC of no more than 0.2nM, no more than 0.5nM, no more than 0.8nM, no more than 1nM, no more than 2nM, or no more than 3nM₅₀Specifically binds to cynomolgus monkey CD20 expressed on cells, the EC₅₀Values were determined by flow cytometry.

In certain embodiments, the anti-CD20 binding moieties described herein are present at no more than 1pM, no more than 2pM, no more than 3pM, no more thanMore than 4pM, not more than 5pM, not more than 6pM, not more than 7pM, not more than 8pM, not more than 9pM, not more than 10pM, not more than 11pM, not more than 12pM, not more than 13pM, not more than 14pM, not more than 15pM, not more than 16pM, not more than 17pM, not more than 18pM, not more than 19pM, not more than 20pM, not more than 21pM, not more than 22pM, not more than 23pM, not more than 24pM, not more than 25pM, not more than 30pM, not more than 35pM, not more than 40pM, not more than 45pM or not more than 50pM of EC₅₀Internalized by CD 20-expressing cells, the EC₅₀Values were determined by the Fab-Zap assay.

Bispecific polypeptide complexes

In certain embodiments, the first and/or the second antigen-binding moiety is multivalent, such as bivalent, trivalent, tetravalent. The term "valency" as used herein means the presence of a specified number of antigen binding sites in a given molecule. Thus, the terms "divalent," "tetravalent," and "hexavalent" indicate the presence of two binding sites, four binding sites, and six binding sites, respectively, in the antigen binding molecule. A bivalent molecule may be monospecific if both binding sites are for specific binding to the same antigen or the same epitope. Likewise, trivalent molecules may be bispecific, for example when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope). In certain embodiments, the first and/or the second antigen-binding moiety in a bispecific polypeptide complex described herein can be bivalent, trivalent, or tetravalent, having at least two binding sites for the same antigen or epitope. In certain embodiments, this provides for stronger binding to the antigen or epitope than a monovalent counterpart antibody. In certain embodiments, in a divalent antigen-binding moiety, the first valence of the binding site and the second valence of the binding site are structurally the same (i.e., have the same sequence) or structurally different (i.e., have different sequences but are specifically the same).

In certain embodiments, the first and/or the second antigen-binding moiety is multivalent, and comprises two or more antigen-binding sites that are operably associated with each other (with or without a spacer).

In certain embodiments, the second antigen-binding portion comprises two or more fabs of the second antibody. The two fabs may be operably associated with each other, e.g., a first Fab may be covalently attached to a second Fab via a heavy chain, with or without a spacer in between.

In certain embodiments, the first antigen-binding moiety is linked to a first dimerization domain and the second antigen-binding moiety is linked to a second dimerization domain. The term "dimerization domain" as used herein refers to a peptide domain that is capable of associating with one another to form a dimer, or in some instances, a peptide domain that enables two peptides to spontaneously dimerize.

In certain embodiments, the first dimerization domain may be associated with the second dimerization domain. The association may be via any suitable interaction or linkage or bonding (e.g., via a linker, disulfide bond, hydrogen bond, electrostatic interaction, salt bridge, or hydrophobic-hydrophilic interaction, or a combination thereof). Exemplary dimerization domains include, but are not limited to, an antibody hinge region, an antibody CH2 domain, an antibody CH3 domain, and other suitable protein monomers that are capable of dimerizing and associating with each other. The hinge region, CH2, and/or CH3 domain may be derived from any antibody isotype, such as IgG1, IgG2, and IgG4.

"disulfide bond" refers to a covalent bond having the structure R-S-S-R'. The amino acid cysteine contains a thiol group which can form a disulfide bond with a second thiol group, for example from another cysteine residue. The disulfide bond may be formed between the sulfhydryl groups of two cysteine residues present on two polypeptide chains, respectively, thereby forming an interchain bridge or an interchain bond.

Hydrogen bonds are formed by electrostatic interactions between two polar groups when a hydrogen atom is covalently bonded to a highly electronegative atom such as nitrogen, oxygen, or fluorine. Hydrogen bonds can be formed between the backbone oxygen (e.g., a chalcogen group) and the amide hydrogen (nitrogen group) of each of the two residues in the polypeptide, such as a nitrogen group in Asn with an oxygen group in His, or an oxygen group in Asn with a nitrogen group in Lys. Hydrogen bonds are stronger than van der waals interactions, but weaker than covalent or ionic bonds, and are critical for maintaining secondary and tertiary structure. For example, when the spacing of amino acid residues regularly occurs between positions i and i +4, an alpha helix is formed, while the beta sheet is a peptide segment of 3-10 amino acids in length that is formed when two peptide segments are hydrogen bonded by at least two or three backbone bonds, which form a twisted, folded sheet.

Electrostatic interactions are non-covalent interactions and are important in protein folding, stability, flexibility and function, including ionic interactions, hydrogen bonding and halogen bonding. Electrostatic interactions may form in polypeptides, for example between Lys and Asp, between Lys and Glu, between Glu and Arg, or between Glu, Trp on the first strand and Arg, Val, or Thr on the second strand.

Salt bridges are close range electrostatic interactions which arise mainly from the anionic carboxylate of Asp or Glu and the cationic ammonium from Lys or guanidine from ArgA root, which is a pair of oppositely charged residues that are spatially close together in the native protein structure. Charged and polar residues in the mainly hydrophobic interface can act as hot spots for binding. Other residues, including residues with ionizable side chains (e.g., His, Tyr, and Ser) may also participate in the formation of salt bridges.

Hydrophobic interactions may be formed between one or more Val, Tyr and Ala on the first strand and one or more Val, Leu and Trp on the second strand, or between His and Ala on the first strand and Thr and Phe on the second strand (see Brinkmann et al, 2017, supra).

In certain embodiments, the first and/or the second dimerization domain comprises at least a portion of an antibody hinge region. In certain embodiments, the first and/or the second dimerization domain may further comprise an antibody CH2 domain and/or an antibody CH3 domain. In certain embodiments, the first and/or the second dimerization domain comprises at least a portion of a hinge-Fc region, i.e., the hinge-CH 2-CH3 domain. In certain embodiments, the first dimerization domain may be operably linked to the C-terminus of the first TCR constant region. In certain embodiments, the second dimerization domain may be operably linked to the C-terminus of the antibody CH1 constant region of the second antigen binding portion.

In the polypeptide complexes provided herein, the first dimerization domain is operably linked to the C-terminus of the engineered TCR constant region and together form a chimeric constant region. That is, the chimeric constant region comprises the first dimerization domain operably linked to the engineered TCR constant region.

In certain embodiments, the chimeric constant region comprises an engineered C β attached to a first hinge-Fc region derived from IgG1, IgG2, or IgG4.

In certain embodiments, the chimeric constant region further comprises a first antibody CH2 domain and/or a first antibody CH3 domain. For example, the chimeric constant region further comprises a first antibody CH2-CH3 domain attached to the C-terminus of the third engagement domain.

These chimeric constant regions and second TCR constant regions are useful because they can be manipulated to fuse to the desired antibody variable region to provide a polypeptide complex as described herein. For example, an antibody heavy chain variable region can be fused to the chimeric constant region (including C1) to provide the first polypeptide chain of a polypeptide complex described herein; and similarly, an antibody light chain variable region can be fused to said second TCR constant region (including C2) to provide said second polypeptide chain of a polypeptide complex as described herein.

In certain embodiments, the second dimerization domain comprises a hinge region. The hinge region may be derived from an antibody, such as IgG1, IgG2, or IgG4. In certain embodiments, the second dimerization domain optionally may further comprise an antibody CH2 domain and/or an antibody CH3 domain, such as a hinge-Fc region. The hinge region may be attached to an antibody heavy chain (e.g., Fab) of the second antigen binding site.

In the bispecific polypeptide complex, the first and the second dimerization domains are capable of associating as a dimer. In certain embodiments, the first and the second dimerization domains are different and are associated in a manner that is not conducive to homodimerization and/or is conducive to heterodimerization. For example, the first and the second dimerization domains may be selected such that they are not identical and they preferentially form heterodimers with each other rather than homodimers with themselves. In certain embodiments, the first and the second dimerization domains are capable of associating into a heterodimer via formation of a handle-access hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased flexibility.

In certain embodiments, the first and the second dimerization domains comprise a CH2 and/or a CH3 domain mutated to be capable of forming a handle-access hole, respectively. The handle may be obtained by substituting a small amino acid residue for a large amino acid residue in the first CH2/CH3 polypeptide, and the pore may be obtained by substituting a large amino acid residue for a small amino acid residue. For details on the site of handle-to-hole mutation see Ridgway et al, 1996, supra, Spiess et al, 2015, supra, and Brinkmann et al, 2017, supra.

Bispecific formats

In the polypeptide complexes described herein, the first antigen-binding moiety and the second binding moiety may associate into an Ig-like structure. Ig-like structures resemble natural antibodies, with a Y-shaped configuration, having two arms for antigen binding and a stem for association and stabilization. Similarity to natural antibodies can provide a number of advantages, such as good in vivo pharmacokinetics, desirable immune response and stability, and the like. It has been found that an Ig-like structure comprising a first antigen-binding moiety as provided herein associated with a second antigen-binding moiety as provided herein has a thermostability comparable to an Ig (e.g., IgG). In certain embodiments, an Ig-like structure provided herein is at least 70%, 80%, 90%, 95%, or 100% of a native IgG.

In certain embodiments, the bispecific polypeptide complex comprises 4 polypeptide chains: i) VH 1-C1-hinge-CH 2-CH 3; ii) VL 1-C2; iii) VH2-CH 1-hinge-CH 2-CH3, and iv) VL2-CL, wherein C1 and C2 are capable of forming a dimer comprising at least one non-natural interchain bond, and two hinge regions and/or two CH3 domains are capable of forming one or more interchain bonds that may facilitate dimerization.

The bispecific polypeptide complexes provided herein have a longer in vivo half-life and are relatively easy to manufacture when compared to other forms of bispecific polypeptide complexes.

Bispecific Complex sequences

In some embodiments, the first antigen-binding portion of the bispecific complex is capable of specifically binding to CD3 and the second antigen-binding portion is capable of specifically binding to CD 20. In other embodiments, the first antigen-binding portion of the bispecific complex is capable of specifically binding to CD20 and the second antigen-binding portion is capable of specifically binding to CD 3.

In certain embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83 and SEQ ID NO:84 (W3278-T2U3.E17R-1.uIgG4.SP antibody), as shown in example 2. In certain embodiments, a bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88(W3278-T3U2.F16-1.uIgG4.SP antibody), as described in example 2. In certain embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: the amino acid sequence of SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92 as in example 2. In a certain embodiment, the bispecific polypeptide complex comprises five polypeptide chains: a) has the sequence shown in SEQ ID NO: 89; b) has the sequence shown in SEQ ID NO: 90; c) has the sequence shown in SEQ ID NO: 91; d) has the sequence shown in SEQ ID NO: 91; and e) has the sequence shown in SEQ ID NO: 92. For example, in a specific embodiment, the bispecific polypeptide complex is a W3278-u2t3.f18r-1.u igg4.sp antibody comprising two anti-CD20 binding moieties, wherein the heavy chain VH-CH 1 domain of one is operably linked to the heavy chain VH domain of the anti-CD 3 binding moiety, as shown in example 2. In certain embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: the amino acid sequence of SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 and SEQ ID NO: 96(W3278-U3T2.F18R-1.uIgG4.SP antibody), as described in example 2. In certain embodiments, the bispecific polypeptide complex comprises a combination of four polypeptide sequences: SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100(W3278-T3U2.F17R-1.uIgG4.SP antibody), as described in example 2. In such embodiments, the first antigen-binding moiety binds to CD3 and the second antigen-binding moiety binds to CD 20.

In certain embodiments, the bispecific polypeptide complex comprises 4 polypeptide chains comprising: i) VH1 operably linked to a first chimeric constant region; ii) VL1 operably linked to a second chimeric constant region; iii) VH2 operably linked to a conventional antibody heavy chain constant region, and iv) VL2 operably linked to a conventional antibody light chain constant region. In certain embodiments, the first chimeric constant region may comprise C1-hinge-CH 2-CH3, portions of which are as defined above. In certain embodiments, the second chimeric constant region may comprise C2, as defined above. In certain embodiments, the conventional antibody heavy chain constant region may comprise CH 1-hinge-CH 2-CH3, portions of which are defined above. In certain embodiments, the conventional antibody light chain constant region may comprise CL, as defined above.

In certain embodiments of the invention, the sequence of SEQ ID NO:92, preferably amino acid 193, 182, 203, 206, 207, in the native glycosylation site, preferably in position 193. In certain embodiments of the invention, the modifications include one or more of S193X, S182X, S203X, S206X, S207X, wherein X is any amino acid other than serine (Ser) and threonine (Thr). In certain preferred embodiments of the invention, the modification is S193X, wherein X is alanine (Ala), glycine (Gly), proline (Pro), or valine (Val). In certain embodiments of the invention, the mutation eliminates an O-glycosylation site, the O-glycosylation pattern being an O-sugar of the Core1 configuration and having the structural formula NeuAc-Gal-GalNAc or NeuAc-Gal- (NeuAc) GalNAc.

As described above, in the bispecific antibody of the present invention, the natural glycosylation site in the corresponding polypeptide chain is modified, and the obtained bispecific antibody is closer to the natural antibody than the unmodified bispecific antibody, significantly reduced in immunogenicity, increased in half-life, and improved in drug potency.

Preparation method

The present application provides isolated nucleic acids or polynucleotides encoding the polypeptide complexes and bispecific anti-CD 3x CD20 polypeptide complexes described herein.

The term "nucleic acid" or "polynucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single or double stranded form. Unless specifically limited, the term encompasses polynucleotides containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, homologous genes, SNPs and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (see Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-.

Nucleic acids or polynucleotides encoding the polypeptide complexes and bispecific polypeptide complexes described herein can be constructed using recombinant techniques. To this end, DNA encoding the antigen binding portion (e.g., CDR or variable region) of a parent antibody may be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). DNA encoding the TCR constant region can be similarly obtained. Illustratively, a polynucleotide sequence encoding the variable domain (VH) and a polynucleotide sequence encoding the first TCR constant region (C1) are obtained and operably linked to allow transcription and expression in a host cell to produce the first polypeptide. Similarly, the polynucleotide sequence encoding VL is operably linked to the polynucleotide sequence encoding C1 such that expression of the second polynucleotide in the host cell is permitted. If desired, the polynucleotide sequence encoding one or more spacers may also be operably linked to other coding sequences to allow expression of the desired product.

The encoding polynucleotide sequence may further be operably linked to one or more regulatory sequences, optionally in an expression vector, such that expression or production of the first and the second polypeptide is feasible and is under suitable control.

The encoding polynucleotide sequence may be inserted into a vector for further cloning (amplification of the DNA) or for expression using recombinant techniques well known in the art. In another embodiment, the polypeptide complexes and bispecific polypeptide complexes described herein can be produced by homologous recombination as is well known in the art. Various carriers can be selected. The carrier component typically includes, but is not limited to, one or more of the following: a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter (e.g. SV40, CMV, EF-1. alpha.) and a transcription termination sequence.

The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted and the protein expressed. Typically, the construct will also include appropriate regulatory sequences. For example, the polynucleotide molecule may comprise a regulatory sequence located 5' to the flanking region of the nucleic acid sequence encoding the guide RNA and/or the nucleic acid sequence encoding the site-directed modifying polypeptide, operably linked to the coding sequence in a manner enabling expression of the transcript/gene in the host cell. The vector may be used to transform, transduce, or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, the vector comprises: plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phages or M13 bacteriophages, animal viruses, and the like. Animal virus species useful as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma virus vacuolium (e.g., SV 40). The vector may contain a variety of elements that control expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site. The carrier may also contain components that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein coats.

In some embodiments, the vector system comprises a mammalian, bacterial, yeast system, or the like, and comprises plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, or the like, as well as other laboratory and commercial vectors. Suitable vectors may comprise plasmid or viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses).

Vectors comprising a polynucleotide sequence described herein may be introduced into host cells for cloning or gene expression. The term "host cell" as used herein refers to a cell into which an exogenous polynucleotide and/or vector has been introduced.

Suitable host cells for cloning or expressing the DNA in the vectors described herein are prokaryotic cells, yeast or higher eukaryotic cells as described above. Prokaryotic cells suitable for use in the present invention include eubacteria, such as gram-negative or gram-positive bacteria, for example Enterobacteriaceae, such as E.coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella, and bacilli, such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas, such as Pseudomonas aeruginosa and Streptomyces.

In addition to prokaryotic cells, eukaryotic microorganisms such as filamentous fungi or yeast are suitable host cells for cloning or expressing the vectors encoding the polypeptide complex and the bispecific polypeptide complex. Saccharomyces cerevisiae, or Saccharomyces cerevisiae, is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are more commonly used and are suitable for use in the present invention, such as Schizosaccharomyces pombe; kluyveromyces hosts, such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces williamsii (ATCC 24,178), Kluyveromyces lactis (ATCC 56,500), Kluyveromyces drosophilus (ATCC 36,906), Kluyveromyces thermotolerans, and Kluyveromyces marxianus; yarrowia lipolytica (EP 402,226); pichia pastoris (EP 183,070); candida species; trichoderma reesei (EP 244,234); performing Neurospora; western schwann yeasts, such as: schwann yeast western; and filamentous fungi, such as: neurospora, Penicillium, Tolypocladium and Aspergillus, such as: aspergillus nidulans and Aspergillus niger.

Host cells suitable for expression of glycosylated polypeptide complexes and bispecific polypeptide complexes described herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Various baculovirus strains (bacterial strains) and variants thereof, and corresponding permissive insect host cells (permissive insect host cells), have been found to be derived from hosts such as: spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of viral strains for transfection are publicly available, such as Autographa californica nuclear polyhedrosis virus and Bm-5 variants of Bombyx mori nuclear polyhedrosis virus, all of which can be used in the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

However, vertebrate cells are of most interest, and culture of vertebrate cells (tissue culture) has become a routine procedure. As examples of mammalian host cells which may be used, there are SV40 transformed monkey kidney cell CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cell subclones in suspension culture, Graham et al, J.Gen Virol. 36:59(1977)), such as Expi 293; baby hamster kidney cells (BHK, ATCC CCL 10); chain hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse testicular support cells (TM4, Mather, biol. reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human liver cancer cell line (Hep G2).

Host cells are transformed with the above-described expression or cloning vectors and can be cultured in conventional nutrient media modified as necessary for inducing promoters, selecting transformed cells, or amplifying cloning vectors.

For the production of the polypeptide complexes and bispecific polypeptide complexes described herein, host cells transformed with the expression vectors described herein can be cultured in a variety of media. Commercially available culture media such as Ham's F10(Sigma), minimal essential Medium (MEM, (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing the host cells. In addition, any of the methods described in Ham et al, meth.Enz.58:44 (1979), Barnes et al, anal. biochem.102:255(1980), U.S. Pat. No. 4,767,704; 4,657,866, respectively; 4,927,762, respectively; 4,560,655 or 5,122,469; WO 90/03430; the medium described in WO 87/00195 or U.S. patent application Re.30,985 can be used as the medium for the host cells. These media may be supplemented, as necessary, with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium chloride, magnesium chloride and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds at final concentrations usually in the micromolar range), and glucose or an equivalent energy source. The medium may also contain any other necessary additives at appropriate concentrations known in the art. The conditions of the medium, such as temperature, pH, and the like, which have been previously used to select host cells for expression, are well known to those of ordinary skill.

In one aspect, the disclosure provides a method of expressing the polypeptide complexes and bispecific polypeptide complexes described herein comprising culturing the host cell described herein under conditions in which the polypeptide complex or the bispecific polypeptide complex is expressed.

In certain embodiments, the disclosure provides a method of producing a bispecific polypeptide complex described herein comprising a) introducing into a host cell one or more polynucleotides encoding a first antigen-binding moiety comprising a first polynucleotide encoding a first polypeptide comprising from N-terminus to C-terminus a first heavy chain variable domain (VH) of a first antibody operably linked to a first T Cell Receptor (TCR) constant region (C1), a second polynucleotide encoding a second polypeptide comprising from N-terminus to C-terminus a first light chain variable domain (VL) of a first antibody operably linked to a second TCR constant region (C2), and one or more additional polynucleotides encoding a second antigen-binding moiety, wherein: c1 and C2 are capable of forming a dimer, and the non-natural inter-chain linkages are capable of stabilizing the dimer of C1 and C2, the first antigen-binding moiety and the second antigen-binding moiety have reduced mismatches relative to when both the first antigen-binding moiety and the second antigen-binding moiety are corresponding portions of a natural Fab, and the first antibody has a first antigen-specificity and the second antibody has a second antigen-specificity, b) allowing the host cell to express the bispecific polypeptide complex.

In certain embodiments, the method further comprises isolating the bispecific polypeptide complex.

When recombinant technology is used, the bispecific polypeptide complexes provided herein can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, the particulate debris of the host cells or cleaved fragments is first removed, for example by centrifugation or sonication. Carter et al, Bio/Technology 10: 163-167 (1992) describes a method for isolating antibodies secreted into the membrane space of E.coli walls. Briefly, the cell paste (cell paste) was thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethanesulfonyl fluoride (PMSF) for about 30 minutes or more. Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant of the expression system is typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration device. Protease inhibitors (e.g., PMSF) may be added in any of the foregoing steps to inhibit protein degradation, and antibiotics may be added to prevent the growth of adventitious contaminants.

The bispecific polypeptide complex of the present application prepared from the cell can be purified by methods such as hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique.

When the bispecific polypeptide complex described herein comprises an immunoglobulin Fc domain, then protein a may be used as an affinity ligand, depending on the type and isotype of Fc domain present in the polypeptide complex. Protein A can be used for purification of polypeptide complexes based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, J.Immunol. meth.62:1-13 (1983)). Protein G is applicable to all mouse isoforms and human gamma 3(Guss et al, EMBO J.5: 15671575 (1986)). Agarose is the most commonly used affinity ligand attachment matrix, but other matrices may be used. Mechanically stable matrices such as controlled pore glass or poly (styrene) benzene can achieve faster flow rates and shorter processing times than can be achieved with agarose.

When the bispecific polypeptide complexes described herein comprise a CH3 domain, purification can be performed using Bakerbond abx. tm. resin (j.t. baker, new jersey philippi burgh). Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica gel chromatography, anion or cation exchange resin based heparin sepharose chromatography (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation may also be used, depending on the antibody to be recovered.

After any preliminary purification step, the mixture containing the polypeptide complex of interest and impurities may be treated by low pH hydrophobic interaction chromatography, using an elution buffer at a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., from about 0 to 0.25M salt concentration).

In certain embodiments, bispecific polypeptide complexes described herein can be readily purified in high yield using conventional methods. One advantage of the bispecific polypeptide complex is a significant reduction in mismatches between heavy and light chain variable domain sequences. This reduces the production of unwanted by-products and makes it possible to obtain a high purity product in high yield using a relatively simple purification process.

Derivatives of the same

In certain embodiments, the bispecific polypeptide complex can be used as a basis for conjugation to a desired conjugate.

It is contemplated that the polypeptide complexes or bispecific polypeptide complexes described herein may be linked to a variety of conjugates (see, e.g., "Conjugate Vaccines", constraints to Microbiology and Immunology, j.m.cruse and r.e.lewis, Jr. (eds.), Carger Press, new york (1989)). These conjugates can be linked to the polypeptide complex or bispecific polypeptide complex by covalent binding, affinity binding, intercalation, coordination binding, complexation, association, mixing or addition, and other means.

In certain embodiments, bispecific polypeptide complexes described herein can be engineered to contain specific sites outside of the epitope-binding moiety that can be used to bind one or more conjugates. For example, such sites may comprise one or more reactive amino acid residues, such as cysteine residues or histidine residues, for facilitating covalent attachment to the conjugate.

In certain embodiments, the bispecific polypeptide complex may be attached to the conjugate directly, or indirectly, for example, through another conjugate or through a linker.

For example, bispecific polypeptide complexes having reactive residues such as cysteine may be linked to a thiol-reactive reagent, wherein the reactive groups are, for example, maleimides, iodoacetamides, pyridine disulfides or other thiol-reactive conjugation partners (Haughland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem.3: 2; Garman, 1997, Non-Radioactive laboratory: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem.1: 2; Hermanson, G.in Bioconjugate Techniques (1996) Academic Press, san Diego pp.40-55, 643).

For another example, the bispecific polypeptide complex may be conjugated to biotin, followed indirectly by a second conjugate conjugated to avidin. For another example, the polypeptide complex or the bispecific polypeptide complex can be linked to a linker that is further linked to the conjugate. Examples of linkers include bifunctional coupling agents (e.g., N-succinimide-3- (2-pyridyldithio) propionate (SPDP), succinimide-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT)), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), diazide compounds (e.g., bis (p-azidobenzoyl) hexamethylenediamine), diazide derivatives (e.g., bis (p-diazobenzoyl) -ethylenediamine), diisocyanates (e.g., toluene 2, 6-diisocyanate), and histidine-reactive fluorine compounds (e.g., 1, 5-difluoro-2, 4-dinitrobenzene). Particularly preferred coupling agents include N-succinimide-3- (2-pyridyldithio) propionate (SPDP) (Carlsson et al, biochem. J.173:723-737(1978)) and N-succinimide-4- (2-pyridylthio) pentanoate (SPP) to provide disulfide linkages.

The conjugate may be a detectable label, a pharmacokinetic modifying moiety, a purifying moiety, or a cytotoxic moiety. Examples of detectable labels may include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin or texas red), enzyme-substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, carbohydrate oxidase or beta-D-galactosidase), radioisotopes (e.g., beta-D-galactosidase), and the like¹²³I、¹²⁴I、¹²⁵I、 ¹³¹I、³⁵S、³H、¹¹¹In、¹¹²In、¹⁴C、⁶⁴Cu、⁶⁷Cu、⁸⁶Y、⁸⁸Y、⁹⁰Y、¹⁷⁷Lu、 ²¹¹At、¹⁸⁶Re、¹⁸⁸Re、¹⁵³Sm、²¹²Bi and³²p, other lanthanides, luminescent labels), chromophore moieties, digoxin, biotin/avidin, DNA molecules, or gold for detection. In certain embodiments, the conjugate can be a pharmacokinetic modifying moiety such as PEG, which helps to extend the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, ethylene glycol/propylene glycol copolymers, and the like. In certain embodiments, the conjugate can be a purification moiety such as a magnetic bead. A "cytotoxic moiety" may be any agent that is harmful to or may damage or kill a cell. Examples of cytotoxic moieties include, but are not limited to, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax dione, mitoxantrone, mithramycin, actinomycesHormone D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin and analogues thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil (thioepa chlorramucil), melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly known as actinomycin), bleomycin, mithramycin and amsacromycin) and antimitotic agents (e.g., vincristine and vinblastine).

Methods for conjugating conjugates to proteins (such as antibodies, immunoglobulins, or fragments thereof) are described in, for example, U.S. Pat. nos. 5,208,020; U.S. patent nos. 6,4411,163; WO 2005037992; WO2005081711 and WO2006/034488, the entire contents of which are incorporated herein by reference.

Pharmaceutical composition

The present application also provides a pharmaceutical composition comprising a bispecific polypeptide complex described herein and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" means that the carrier, vehicle, diluent, excipient, and/or salt in question is, generally speaking, chemically and/or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

By "pharmaceutically acceptable carrier" is meant an ingredient in a pharmaceutical formulation that is distinct from the active ingredient, is biologically acceptable and is not toxic to the subject. The pharmaceutically acceptable carrier for use in the pharmaceutical compositions disclosed herein may comprise, for example, a pharmaceutically acceptable liquid, gel or solid carrier, aqueous vehicle, non-aqueous vehicle, antimicrobial substance, isotonic substance, buffer, antioxidant, anesthetic, suspending/dispersing agent, chelating agent, diluent, adjuvant, excipient, or nontoxic auxiliary substance, other components well known in the art, or various combinations thereof.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, emulsifying agents, or stabilizing agents such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in the pharmaceutical compositions described herein will reduce oxidation of the polypeptide complex or bispecific polypeptide complex. Reduction of oxidation prevents or reduces the reduction of binding affinity, thereby improving protein stability and extending shelf life. Thus, in certain embodiments, the compositions described herein comprise a polypeptide complex or bispecific polypeptide complex as described herein, and one or more antioxidants, such as methionine.

To further illustrate, a pharmaceutically acceptable carrier can comprise, for example, an aqueous medium such as sodium chloride injection, ringer's solution injection, isotonic glucose injection, sterile water injection, or dextrose and lactated ringer's injection, a non-aqueous medium such as: plant-derived fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil, antibacterial substances at bacteriostatic or fungistatic concentrations, isotonic agents such as: sodium chloride or glucose, buffers such as: phosphate or citrate buffers, antioxidants such as: sodium bisulfate, local anesthetics such as: procaine hydrochloride, suspending and dispersing agents such as: sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifiers such as: polysorbate 80 (tween-80), chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. The antimicrobial agent as a carrier may be added to the pharmaceutical composition in a multi-dose container comprising phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may comprise, for example, water, salt, glucose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, emulsifiers, pH buffers, stabilizers, solubilizers, or substances such as sodium acetate, sorbitan laurate, triethanolamine oleate or cyclodextrins.

The pharmaceutical composition may be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.

In certain embodiments, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions may be prepared in any conventional form, for example, as liquid solvents, suspending agents, emulsifying agents, or solid forms suitable for the production of liquid solvents, suspending agents or emulsifying agents. Injectable preparations can comprise sterile and/or pyrogen-free solutions ready for injection, sterile dried solubles such as lyophilized powders that are combined with solvents immediately prior to use, sterile dried insoluble products that are combined with vehicles immediately prior to use, and sterile and/or pyrogen-free emulsions. The solvent may be aqueous or non-aqueous.

In certain embodiments, a unit dose of an injectable formulation is packaged in an ampoule, a vial, or a syringe with a needle. It is well known in the art that all formulations for injectable administration should be sterile pyrogen free.

In certain embodiments, sterile lyophilized powders can be prepared by dissolving a polypeptide complex or bispecific polypeptide complex as disclosed herein in a suitable solvent. The solvent may contain a compound that enhances the stability of the powder or reconstituted solution prepared from the powder, or improves the pharmacological properties of the powder or reconstituted solution. Excipients that may be used include, but are not limited to, water, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable materials. The solvent may contain a buffer, such as a citric acid buffer, a sodium or potassium phosphate buffer, or other buffers known to those skilled in the art, and in one embodiment, the buffer is neutral in pH. The subsequent sterile filtration of the solubilization is carried out under standard conditions well known in the art, and then lyophilized to produce the desired formulation. In one embodiment, the resulting solvent is dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of a polypeptide complex, bispecific polypeptide complex, or composition thereof described herein. The loading per vial may be slightly higher (e.g., 10% excess) than is required for each dose or for multiple doses, thereby facilitating accurate sampling and accurate administration. The lyophilized powder may be stored under suitable conditions, such as in the range of about 4 ℃ to room temperature.

And (4) re-dissolving the freeze-dried powder with water for injection to obtain a preparation for injection administration. In one embodiment, the lyophilized powder can be reconstituted by addition to sterile pyrogen-free water or other suitable liquid carrier. The precise amount is determined by the selected therapy and may be determined based on empirical values.

Method of treatment

Also provided are methods of treatment comprising administering a therapeutically effective amount of a polypeptide complex or bispecific polypeptide complex described herein to a subject in need thereof, thereby treating or preventing a condition or disorder. In certain embodiments, the subject has been identified as having a disorder or condition that is likely to respond to a polypeptide complex or bispecific polypeptide complex described herein.

"treating" or "treatment" of a condition includes preventing or alleviating the condition, reducing the rate at which a condition develops or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination thereof.

The therapeutically effective dose of the bispecific polypeptide complexes described herein will depend on a variety of factors well known in the art, such as body weight, age, past medical history, current therapy, the health condition and potential for cross-reactions in the subject, allergies, hypersensitivity and side effects, as well as the route of administration and the extent of disease development. One skilled in the art (e.g., a physician or veterinarian) can proportionately lower or raise the dosage in accordance with these and other circumstances or requirements.

In certain embodiments, a bispecific polypeptide complex described herein can be administered at a therapeutically effective dose of between about 0.01mg/kg and about 100mg/kg (e.g., about 0.01mg/kg, about 0.5mg/kg, about 1mg/kg, about 2mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 55mg/kg, about 60mg/kg, about 65mg/kg, about 70mg/kg, about 75mg/kg, about 80mg/kg, about 85mg/kg, about 90mg/kg, about 95mg/kg, or about 100 mg/kg). In certain of these embodiments, the polypeptide complex or bispecific polypeptide complex described herein is administered at a dose of about 50mg/kg or less, and in certain of these embodiments, the dose is 10mg/kg or less, 5mg/kg or less, 1mg/kg or less, 0.5mg/kg or less, or 0.1mg/kg or less. In certain embodiments, the dosage administered may vary with the course of treatment. For example, in certain embodiments, the initial administration dose may be higher than the subsequent administration dose. In certain embodiments, the dosage administered may be adjusted during the course of treatment according to the subject's response.

The dosage regimen may be adjusted to achieve an optimal response (e.g., therapeutic response). For example, a single dose may be administered, or multiple divided doses may be administered over a period of time.

The bispecific polypeptide complexes described herein can be administered by routes well known in the art, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous drip, intramuscular, or intradermal) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

In certain embodiments, the condition or disorder treated by a bispecific polypeptide complex described herein is cancer or a cancerous condition, an autoimmune disease, infectious and parasitic diseases, cardiovascular diseases, neuropathy, neuropsychiatric conditions, injury, inflammation, or coagulation disorders.

As used herein, "cancer" or "cancerous condition" refers to a medical condition mediated by neoplastic or malignant cell growth, proliferation or metastasis, and encompasses both solid and non-solid cancers, such as leukemia. As used herein, "tumor" refers to a solid mass of neoplastic and/or malignant cells.

For cancer, "treating" or "therapy" may refer to inhibiting or slowing the growth, proliferation, or metastasis of neoplastic or malignant cells, preventing or delaying the progression of neoplastic or malignant cell growth, proliferation, or metastasis, or some combination thereof. For tumors, "treating" or "therapy" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination thereof.

For example, for use of a bispecific polypeptide complex disclosed herein for treating cancer, a therapeutically effective amount is a dose or concentration of the polypeptide complex that is capable of removing all or a portion of a tumor, inhibiting or slowing tumor growth, inhibiting growth or proliferation of cells that mediate a cancerous condition, inhibiting metastasis of tumor cells, ameliorating any symptoms or markers associated with a tumor or a cancerous condition, preventing or delaying the development of a tumor or a cancerous condition, or some combination thereof.

In certain embodiments, the condition or disorder comprises tumors and cancers, such as non-small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer, leukemia, lymphoma, myeloma, mycosis, meckel cell carcinoma, and other hematologic malignancies, such as Classical Hodgkin's Lymphoma (CHL), primary mediastinal large B-cell lymphoma, T cell/histiocytic-rich B-cell lymphoma, EBV positive and negative PTLD, EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablast lymphoma, extralymph node NK/T-cell lymphoma, nasopharyngeal carcinoma, and HHV 8-associated primary effusion lymphoma, B-cell lymphoma, c-T-cell lymphoma, and HHV 8-associated primary effusion lymphoma, Hodgkin's lymphoma, Central Nervous System (CNS) tumors, such as primary CNS lymphoma, spinal cord tumor, brain stem glioma.

In certain embodiments, the conditions and disorders comprise a CD 20-associated condition, such as B cell lymphoma, optionally hodgkin's lymphoma or non-hodgkin's lymphoma, wherein the non-hodgkin's lymphoma comprises: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (chronic lymphocytic leukemia, CLL), or Mantle Cell Lymphoma (MCL), Acute Lymphocytic Leukemia (ALL), or Waldenstrom's Macroglobulinemia (WM).

The bispecific polypeptide complex may be administered alone or in combination with one or more other therapeutic agents or agents.

In certain embodiments, when used to treat cancer or a tumor or proliferative disease, the bispecific polypeptide complexes described herein may be administered in combination with chemotherapy, radiation therapy, surgery for the treatment of cancer (e.g., tumor resection), one or more antiemetics, or other therapies for complications arising from chemotherapy, or any other therapeutic agent for the treatment of cancer or any medical condition associated therewith. "co-administration" as used herein encompasses simultaneous administration as part of the same pharmaceutical composition, simultaneous administration as different pharmaceutical compositions, or administration at different times as different pharmaceutical compositions. The term "in combination" as used in this application also includes that a composition administered before or after another therapeutic agent is also considered to be administered "in combination" with that therapeutic agent, even if the composition and the second agent are administered via different routes. Where possible, the other therapeutic agent administered in combination with the polypeptide complex or bispecific polypeptide complex described herein is administered with Reference to the protocol listed in the product specification for the other therapeutic agent, or with Reference to the surgeon's protocol Reference (Physicians' Desk Reference, 70 th edition (2016)), or with Reference to other protocols known in the art.

In certain embodiments, the therapeutic agent may induce or promote an immune response against the cancer. For example, tumor vaccines can be used to induce immune responses to certain tumors or cancers. Cytokine therapy may also be used to increase presentation of tumor antigens to the immune system. Examples of cytokine therapies include, but are not limited to, interferons (e.g., interferon alpha, beta, and gamma), colony stimulating factors (e.g., macrophage CSF, granulocyte macrophage CSF, and granulocyte CSF), interleukins (e.g., IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12), tumor necrosis factors (e.g., TNF-alpha and TNF-beta). Agents that inactivate immunosuppressive targets, such as TGF-beta inhibitors, IL-10 inhibitors, and Fas ligand inhibitors, can also be used. Another group of agents comprises agents that activate immune responsiveness to tumor or cancer cells, such as agents that enhance T cell activation (e.g., agonists of T cell co-stimulatory molecules, such as CTLA-4, ICOS, and OX-40), and agents that enhance dendritic cell function and antigen presentation.

Reagent kit

The present disclosure further provides kits comprising bispecific polypeptide complexes described herein. In some embodiments, the kit can be used to detect its presence or level, or to capture or enrich one or more targets of interest in a biological sample. The biological sample may comprise a cell or tissue.

In some embodiments, the kit comprises a bispecific polypeptide complex described herein conjugated to a detectable label. In certain other embodiments, the kit comprises an unlabeled bispecific polypeptide complex described herein, and further comprises a labeled second antibody capable of binding to the unlabeled bispecific polypeptide complex described herein. The kit may further comprise instructions for use and packaging separating each component in the kit.

In certain embodiments, the bispecific polypeptide complexes described herein are bound to a substrate or an apparatus. Useful substrates or instruments may be, for example, magnetic beads, microwell plates, or test strips, which may be used for binding assays (e.g., ELISA), immunograph assays, capture, or enrichment of target molecules in biological samples.

The following examples are provided to better illustrate the invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials and methods described below, in whole or in part, are within the scope of the invention. These specific compositions, materials and methods are not intended to limit the invention but merely to illustrate specific embodiments that fall within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials and methods without the exercise of inventive faculty and without departing from the scope of the invention. It will be appreciated that various modifications to the method of the invention may still be included within the scope of the invention. The inventors intend such variations to be included within the scope of the present invention.

Examples

Example 1

Material preparation and reference antibody

1. Material preparation

Information on commercially available materials used in the examples is provided in table 1.

TABLE 1 commercially available materials

2. Generation of reference antibodies

Two reference antibodies, W327-BMK1 and W327-BMK4, were used as reference antibodies in the examples.

The anti-human CD20 reference antibody BMK1 (rituximab) was generated based on the sequence of clone C2B8 from US patent application US 20140004037a 1. The anti-CD 3x CD20 reference bispecific antibody BMK4 (REGN1979) gene was synthesized according to the sequence in US patent application US 20150266966 a 1. BMK antibodies were expressed by Expi293 cells and then purified using protein a chromatography.

Example 2

Preparation of bispecific antibodies of the present application

1. Design and engineering of antibodies and TCR chimeric proteins

TCR sequence

TCRs are heterodimeric proteins consisting of two chains. About 95% of human T cells have a TCR consisting of alpha and beta chains. Considering that more crystal structures are available for beta chain TRBC1, the TRBC1 sequence was chosen as the backbone for designing the polypeptide complex ("WuXiBody") disclosed herein. A typical amino acid sequence of TRBC1 can be found in Protein Data Bank (PDB) structure 4L 4T.

Interchain disulfide bond of TCR

The TCR crystal structure was used to guide our WuXiBody design. Unlike native TCRs anchored on the surface membrane of T cells, soluble TCR molecules are less stable, although their 3D structure closely approximates that of antibody Fab. Indeed, the instability of TCRs in solution has been a major impediment to making their crystal structures difficult to elucidate (see Wang, Protein Cell,5(9), pp.649-652 (2014)). We have adopted a scheme to introduce a pair of Cys mutations in the TCR constant region and found that it can significantly improve chain assembly and enhance expression.

The junction region linking the antibody variable domain and the TCR constant domain, their relative fusion orientation, and the junction region linking the Fc are carefully fine-tuned. Since the TCR structure closely approximates that of antibody Fab, we superimposed an antibody Fv homology model on the TCR variable region (PDB 4L 4T). The superimposed structure indicates that the antibody Fv is structurally compatible with the TCR constant domain. All relevant engineering parameters are based on this structural alignment and the corresponding sequence design.

2. Preparation of bispecific antibodies of the present application

W3278 BsAbs (S.Atwell, J.B. Ridgway, J.A.wells, P.Carter, Stable magnetodiimides from remodelling the domain interface of a homomodimer using a phase display library.J. mol.biol.270, 26-35 (1997)) was produced as human IgG4 in a hand-in-hole formatArchangel, A.Huang, et al.D. G.Yansura, J.M.Scheer, Bispecific antibodies with natural architecture produced by co-culture of bacterial expressing two derivatives of alpha-antibodies, Nat.Biotechnol.31, 753-758 (2013)). The sequence of human IgG4 Fc region was designed with the S228P mutation. anti-CD 3 monoclonal antibodies were generated by immunizing mice with human CD3 epsilon and CD3 delta ECD proteins via hybridoma technology by an internal protocol. The anti-CD20 arm variable region sequences are based on the sequences of ofatumumab (clone 2F2 of PCT publication No. WO 2010083365a 1) or rituximab (clone C2B8 of U.S. patent application US 20140004037a 1). The sequences of the W3278 BsAb candidates are listed in Table 2, their DNA sequences were synthesized in Genewiz (Shanghai) and cloned into the modified pcDNA3.3 expression vector. The anti-CD20 arm and anti-CD 3 arm expression vectors were co-transfected into Expi293(Invitrogen-A14527) using the Expifactamine 293 transfection kit (Invitrogen-A14524). Cells were incubated in a medium containing 8% CO₂In a 37 ℃ incubator with a humid atmosphere at 135rpm on an orbital shaker platform in Expi293 expression medium (Invitrogen-A1435101). The culture supernatant was collected and protein purification was performed using a protein a column (GE Healthcare, 17543802). Protein concentration was measured by UV-Vis Spectrophotometer (NanoDrop 2000, Thermo Scientific). Protein purity was assessed by SDS-PAGE and analytical HPLC-SEC. FIG. 1 depicts a schematic of the W3278-BsAb candidate.

TABLE 2 sequences of heavy and/or light chains in each BsAb

Note: TCR sequences are shown in italics.

Further, in the bispecific antibody W3278-u2t3.f18r-1.u igg4.sp, the present inventors performed a heavy chain reaction on SEQ ID NO:92, wherein one or more amino acids of the natural glycosylation sites at positions 193, 182, 203, 206, 207, preferably amino acid 193, are modified. In certain embodiments, the modifications include one or more of S193X, S182X, S203X, S206X, S207X, wherein X is any amino acid other than serine (Ser) and threonine (Thr). In certain preferred embodiments, the modification is S193X, wherein X is alanine (Ala), glycine (Gly), proline (Pro), or valine (Val). The mutation eliminates O-glycosylation sites, and the O-glycosylation type is an O-sugar with Core1 configuration and has a structural formula of NeuAc-Gal-GalNAc or NeuAc-Gal- (NeuAc) GalNAc.

Example 3

In vitro characterization

1. Cell lines and primary cell isolation

The following cell lines cultured in complete medium (RPMI 1640 supplemented with 10% FBS, 100U/ml penicillin, 100. mu.g/ml streptomycin) were used: jurkat (CD3+/CD 20-cells); raji, Ramos and NAMALWA (CD20+/CD 3-cells), SU-DHL-1(CD 20-/CD 3-cells).

Human Peripheral Blood Mononuclear Cells (PBMC) were freshly isolated from heparinized venous blood from healthy normal donors by Ficoll-Paque PLUS (GE Healthcare-17-1440-03) density centrifugation. Isolation of Primary human CD8 from fresh human PBMC by EasySep kit (Stemcell-19053)⁺T cells, and CD4 was purified by EasySep (Stemcell-19052) column⁺T cells.

Binding of W3278 BsAb to target cells

Binding of W3278 BsAb to target cells was determined by flow cytometry. Briefly, 1 × 10⁵Target cells per well (CD3+/CD 20-cells or CD20+/CD 3-cells) were incubated with serial dilutions of W3278 BsAb or human IgG4 isotype control antibody for 60 minutes at 4 ℃. After warm-up, cells were washed twice with cold 1% BSA/1XPBS, and then Alexa Fluor647 conjugated goat anti-human IgG Fc (Jackson-109-. After two washes, the geometric Mean Fluorescence (MFI) of stained cells was measured using a FACS Canto II hemocytometer (BD Biosciences). Wells containing no antibody or only fluorescent secondary antibody were used to establish background fluorescence. Cell-bound EC was determined using GraphPad Prism 5 Software (GraphPad Software, La Jolla, Calif.)₅₀Values, where each value was calculated using four parameter non-linear regression analysis. FACS binding of W3278 BsAb to target cells is shown in fig. 2, and binding EC50 is shown in table 3 below.

Table 3 FACS binding of W3278 BsAbs and parental antibodies to cell surface targets EC 50.

To detect simultaneous binding of W3278 BsAb to CD3 and CD20 expressing cells, 1X10 cells were used⁶Raji cells and 1X10 cells/ml⁶Each/ml Jurkat cell was labeled with 50nM Calcein-AM (Invitrogen-C3099) and 20nM FarRed (Invitrogen-C34572), respectively. After washing with cold 1% BSA/1XPBS, the labeled Raji and Jurkat cells were resuspended and mixed at a 1:1 ratio to a final concentration of 1X10⁶One per ml. With 1x10⁵The mixed cells/well were plated and then serial dilutions of W3278 BsAb were added. Percentage of Calcein-AM and FarRed double positive cells was analyzed by FACS after incubation at 4 ℃ for 60 minutes.

The results in figure 3 indicate that W3278 lead BsAb exhibited dose-dependent simultaneous dual-target binding that was more effective than BMK 4.

3. In vitro cytotoxicity assay

Determination of BsAb by CD8 via FACS-based cytotoxicity assay⁺T lymphocyte regulates tumor cell lysis. Briefly, freshly isolated human CD8 was isolated⁺T cells were cultured in complete medium containing 50IU/ml recombinant human IL-2 and 10ng/ml OKT-3 for 3 to 5 days. On the following day, target cells, Raji, Ramos, NAMALWA and SU-DHL-1 (1X 10)⁶Individual cells/ml) were labeled with 20nM Far-Red (Invitrogen-C34572) in DPBS at 37 ℃ for 30 min and then washed twice with assay buffer (RPMI 1640 medium without phenol Red + 10% FBS). Far-Red labeled target cells (2X 10)⁴One/well) coating at 110. mu.l/well contained effect CD8⁺T cells (effector/target ratio 5:1) and BsAbs or hIgG4 isotype controls were diluted in complete medium and incubated overnight at 37 ℃. Finally, Propidium Iodide (PI) (Invitrogen-P3566) was added and incubated at room temperature for 15 minutes, then analyzed by flow cytometry. The percent cytotoxicity was calculated using the following equation: cytotoxicity ═ 100 × Far Red⁺PI⁺/(Far Red⁺PI⁺+Far Red⁺PI^-) 100%. Nonlinear regression using Prism four parametersAnalysis of EC for in vitro cytotoxicity₅₀The value is obtained.

The results in FIG. 4 show that W3278 lead Ab "W3278-U2T3. F18R-1.uIgG 4" does not kill CD20 negative SU-DHL-1 cells. W3278 lead Ab "W3278-u 2t3.f18r-1.uIgG 4" induced cell killing of CD20 positive cells more efficiently than BMK4, and the killing efficiency EC50 increased in proportion to the cell surface CD20 expression level. BsAb-mediated cytotoxicity EC50 and% maximal cytotoxicity (Max Cyto) are shown in table 4.

Table 4 cytotoxicity EC50 and% maximal cytotoxicity of W3278 lead BsAb and BMK4 BsAb on different B cell lines.

4. Cell activation and cytokine release assays

BsAb-mediated T cell activation was assessed by flow cytometry measuring expression of effector cells CD69 or CD 25. Freshly isolated purified CD4⁺T cells and CD8⁺T cells were examined as effector cells, respectively. Briefly, 5x10⁴A CD4⁺Or CD8⁺T cells were plated in 110. mu.l/well complete medium containing serial dilutions of BsAbs or hIgG4 isotype control antibody at 1X10⁴In the presence of Raji or SU-DHL-1 cells/well at 37 ℃ for 24 hours. After incubation, cells were washed twice with 1% BSA/1XDPBS and then stained with anti-human Ab group (FITC-labeled anti-human CD4(BD Pharmingen-550628); PerCP-Cy5.5-labeled anti-human CD8(BD Pharmingen-565310); PE-labeled anti-human CD69(BD Pharmingen-555531) and APC-labeled anti-human CD25(BD Pharmingen-555434)) at 4 ℃ for 30 minutes. T cell activation assessed by FACS analysis via CD69 or CD25 expression. EC50 for T cell activation was determined by using a four parameter non-linear regression analysis.

In the absence of target cells, W3278 lead Ab did not induce T cell activation. W3278 lead Ab induced CD4+ and CD8+ T cell activation, shown by CD25 expression (fig. 5A) and CD69 expression (fig. 5B), and was more potent than BMK4, only in the presence of target cells. Activation EC50 is shown in tables 5A and 5B.

Table 5a. expression of EC50 by T cell CD25 mediated by W3278 BsAb and BMK4 BsAb in the presence of Raji cells.

Table 5b. expression of EC50 by T cell CD69 mediated by W3278 BsAb and BMK4 BsAb in the presence of Raji cells.

For cytokine release (TNF-. alpha.and IL-2) assays, 5X10⁴A freshly isolated CD4⁺T cells were plated in 110. mu.l/well complete medium containing serial dilutions of BsAbs or hIgG4 isotype control antibody at 1X10⁴In the presence of Raji or SU-DHL-1 cells/well at 37 ℃ for 24 hours. After 24 hours incubation, plates were centrifuged and supernatants were collected and stored at-80 ℃ for cytokine concentration measurements by ELISA.

For TNF-. alpha.detection by ELISA, 96-well ELISA plates (Nunc MaxiSorp, ThermoFisher) were plated with 50. mu.l of carbonate-bicarbonate buffer (20mM Na₂CO₃,180mM NaHCO₃Ph9.2) purified anti-human TNF (BD Pharmingen-51-26371E) was coated overnight at 4 ℃. The following day, plates were washed with wash buffer (1X PBST buffer, 0.05% tween-20) and then blocked with 200 μ l assay diluent (PBS + 10% FBS). After blocking, 50. mu.l of test sample and recombinant human TNF standard (BD Pharmingen-51-26376E) were added and the plates were incubated for 2 hours at room temperature. TNF- α binding to the plate was detected by detecting antibody biotinylated anti-human TNF (BD Pharmingen-51-26372E). streptavidin-HRP reagent (BD Pharmingen-51-9002813)) and Tetramethylbenzidine (TMB) substrate (Sigma-860336-5G) were used for the color reaction. Wash with wash buffer between steps. The color reaction was stopped with 2M HCl after about 30 minutes. Using a multifunctional plate reader (M5e) at 450 nm.

Similarly, the IL-2 concentration in the culture supernatant was measured by ELISA. Anti-human IL-2 antibody mAb (R & D-MAB602) was used as the capture antibody and biotinylated anti-human IL-2 antibody (R & D-BAF 202) was used as the detection antibody.

As shown in FIG. 6, W3278 lead Ab did not induce cytokine release from T cells in the presence of CD20 negative SU-DHL-1 cells. W3278 lead Ab was able to induce lower levels of cytokine release compared to BMK4 only in the presence of target Raji cells. And the EC50 window (shown as the EC50 ratio between cytokine release and cell killing) elicited by W3278 Ab was greater than BMK4 (table 6).

TABLE 6 BsAb mediated CD4⁺The cytokine release EC50 and maximum levels of T cells and the ratio of cytokine release to EC50 for Raji cell killing.

5. Serum stability assay

The antibodies were mixed with freshly collected human serum and the serum proportion in the mixed sample was ensured to be > 95%. Aliquots of the mixed sample were dispensed and incubated at 37 ℃ for 0-14 days. At each time point shown in fig. 7, the samples were snap frozen in liquid nitrogen and stored at-80 ℃ prior to analysis. Each sample was analyzed by FACS for binding to Raji or Jurkat cells.

As shown in fig. 7, human serum-treated W3278 Ab bound to both Jurkat (fig. 7A) and Raji (fig. 7B) cells similarly to the Ab that had just been thawed (day 0). These results indicate that W3278 Ab is stable in human serum for at least 14 days (fig. 7).

DSF determination and thermal stability testing

DSF assays were performed using real-time fluorescent quantitative PCR (QuantStaudio 7Flex, Thermo Fisher Scientific). Briefly, 19 μ L of antibody solution was mixed with 1 μ L of 62.5X SYPRO Orange solution (Invitrogen) and added to a 96-well plate(Biosystems). The plate was heated from 26 ℃ to 95 ℃ at a rate of 2 ℃/min and the resulting fluorescence data collected. The negative derivatives of the fluorescence change with respect to different temperatures were calculated and the maximum was defined as the melting temperature T_h. If the protein has multiple unfolding transitions, the first two T's are reported_hIs called T_m1And T_m2。T_m1Always interpreted as formal melting temperature T_mTo facilitate comparison between different proteins. Data acquisition and T_hThe calculation was performed automatically by the operating Software (QuantStaudio Real-Time PCR Software v 1.3). T of W3278 Ab in different buffers_m1And T_m2The values are shown in table 7. T of W3278_mAbout 61 ℃ indicates good thermal stability of W3278 Ab.

TABLE 7 thermal stability (T) by DSF determination_mThe value is represented by T in the table_m1Shown in

Example 4

In vivo antitumor efficacy

Antibody anti-tumor efficacy in vivo was tested in Raji tumor model of PBMC humanized NOG mice. Female NOG mice (Experimental animal technology, Inc., Wei Tony, Beijing) 6-8 weeks old were used in the study. Mixing Raji tumor cells (CCL-86^TM) 5% CO at 37 ℃₂And a single layer culture in 1640 medium containing 10% fetal calf serum, 100U/ml penicillin and 100. mu.g/ml streptomycin. Tumor cells were routinely passaged twice a week. Cells grown in the exponential growth phase were collected and counted for tumor inoculation. Human PBMC were isolated from heparin whole blood of healthy blood donated individuals using Ficoll-Paque Plus according to the manufacturer's instructions.

For the treatment model, each mouse was co-injected subcutaneously in the right upper flank with pre-mixed Raji tumor cells(2.0×10⁶Seed) and PBMC (3.0X 10)⁶One). When the mean tumor volume reached about 60mm³At time, animals were randomized and received a first injection of antibody. For efficacy studies, mice received twice weekly intravenous injections of indicated amounts of antibody for a total of 3 weeks. All procedures involved in animal handling, care and treatment in the study were conducted in accordance with the Wuxi ApTec institutional animal Care and application Committee (IACUC) following the guidelines of the southern approval of the laboratory animal Care assessment and evaluation Association (AAALAC). For all tumor studies, mice were weighed and tumor growth was measured twice weekly using calipers. Tumor volume was 1/2 (length x width)²) And (6) estimating.

As shown in fig. 8, W3278 lead Ab treatment demonstrated dose-dependent antitumor activity, which was more effective than BMK4 (fig. 8A). Mice were normal in weight during the experiment (fig. 8B).

Example 5

Single dose study with WBP3278 bispecific antibody in cynomolgus monkeys first for immunization

To determine whether treatment with a WBP3278 bispecific antibody can deplete circulating B cells in primate animals and determine whether any unexpected toxicity results, the inventors conducted an exploratory, non-GLP pharmacological study in cynomolgus monkeys (Macaca Fascicularis). 4 Male cynomolgus monkeys (age 3-4 years, weight about 4kg) first used for immunization were provided by Zhaoqing Chuang Biotech, Inc., Guangdong. All procedures related to animal handling, care and treatment in the study were performed according to the pharma legacy laboratory Institution Animal Care and Use Committee (IACUC) following the guidelines approved by the institutional animal care assessment and approval committee (AAALAC).

Four animals were divided into two groups (2 animals per group) and administered once with WBP3278 lead antibody (i.e., W3278-u2t3.f18r-1.uIgG4, hereinafter abbreviated WBP3278 lead Ab) at 1mg/kg (group 1) and 10mg/kg (group 2), respectively, by slow intravenous injection over 60 seconds. Levels of peripheral circulating B and T cells were monitored by FACS for 4 weeks for the following lymphocytes: b lymphocytes (CD45+/CD20 +); t lymphocytes (CD45+/CD3 +);CD4+ T lymphocytes (CD4+/CD45+/CD3+) and CD8+ T lymphocytes (CD8+/CD45+/CD3 +). By using BD^TMCell Counting Bead Array (CBA) non-human primate Th1/Th2 cytokine kit (BD Bioscience, Cat:557800) analyzed the levels of circulating inflammatory cytokines. Treatment with WBP3278 lead Ab resulted in immediate and complete depletion of circulating B cells for at least 4 weeks (fig. 9). Levels of circulating T cells were also initially reduced following WBP3278 lead Ab treatment, returning to baseline levels or slightly higher levels after 72 hours and lasting for at least 4 weeks (fig. 10A). After 72 hours, CD8+ T cell levels were elevated (fig. 10B), CD4+ T cell levels were returned to normal levels (fig. 10C), and all continued for at least 4 weeks. After treatment with WBP3278 lead Ab, a rapid increase in circulating cytokine levels was observed, and all cytokines returned to normal levels after 24 hours (fig. 11).

The concentration of WBP3278 lead Ab in serum was determined by ELISA. Briefly, ELISA plates were coated with anti-human IgG (southern biotech, #2049-01) and then serial dilutions of serum samples were added. The binding signal was detected with goat anti-human IgG biotin (southern Biotech, #2049-08) and streptavidin-HRP (Life, # SNN 1004). Using a multi-well plate reader (M5e) at (450-540) nm. Non-compartmental pharmacokinetic analyses were performed on WBP3278 lead Ab concentrations in cynomolgus monkeys using Phoenix WinNonlin software (version 8.1, Pharsight, Mountain View, CA). PK parameters were obtained using linear/logarithmic trapezoidal rule. Individual BLQ were excluded when calculating the mean concentration. Normal dose levels and nominal sampling times were used in calculating all pharmacokinetic parameters. A summary of PK parameters is listed in table 8 and shown in figure 12. It can be seen that the system is exposed to C when the dose is increased from 1mg/kg to 10mg/kg₀Increase from 19.9. mu.g/mL to 282. mu.g/mL (about 14-fold), AUC_0-lastIncreased from 259. mu.g.h/mL to 5788. mu.g.h/mL (about 22-fold). Serum half-life (T) of WBP3278 bispecific antibody_1/2) At 1mg/kg and 10mg/kg, 43.3 hours and 89.8 hours, respectively.

TABLE 8 PK parameter summary of WBP3278 lead Ab

PK parameters	1mg/kg (average)	10mg/kg (average)
			C₀(μg/mL)	19.9	282
T_1/2(h)	43.3	89.8
			AUC_0-last(μg.h/mL)	259	5788

In addition, cage-side observations during the experiment showed that no unexpected toxicity was observed for both the high dose and low dose levels of WBP3278 lead Ab (data not shown).

The result of the experiment shows that the WBP3278 lead Ab can effectively eliminate B cells in vivo, has no adverse reactions such as cytokine storm and the like, and has enough half-life in cynomolgus monkeys. These experimental results support the advancement of WBP3278 lead Ab as a preclinical drug development.

It will be further understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or central attributes thereof. In the foregoing specification, the invention has been disclosed in terms of exemplary embodiments only, and it is to be understood that other variations are encompassed within the scope of the invention. Accordingly, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims for their scope and content.

SEQUENCE LISTING

<110> Ningda Ningqing pharmaceutical industry group GmbH

<120> novel bispecific CD3/CD20 polypeptide complex

<130> IDC186026

<150> 201910080405.3

<151> 2019-01-28

<150> PCT/CN2019/073418

<151> 2019-01-28

<160> 122

<170> PatentIn version 3.5

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Thr Tyr

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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Trp Ile Phe Pro Gly Asn Asp Asn Ile Lys Tyr Ser Glu Lys Phe

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Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Ile Asp Ser Val Ser Ile Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ser

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Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

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Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

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Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

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Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Lys Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

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Ser Phe Ile Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ala Phe Thr Asp Tyr

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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Trp Ile Ser Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Ser Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ser

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Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

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Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

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Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

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Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gln Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

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Ser His Thr Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ala Phe Thr Asp Tyr

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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Trp Ile Ser Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Ser Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ser

115

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<223> VK

<400> 66

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gln Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

85 90 95

Ser His Thr Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 67

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> VH

<400> 67

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Phe Pro Gly Asn Asp Asn Ile Lys Tyr Ser Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile Asp Ser Val Ser Ile Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 68

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> VK

<400> 68

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Lys Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

85 90 95

Ser Phe Ile Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 69

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> VH

<400> 69

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ala Phe Thr Asp Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Ser Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 70

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> VK

<400> 70

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gln Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

85 90 95

Ser His Thr Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 71

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> VH

<400> 71

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 72

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VK

<400> 72

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 73

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> VH

<400> 73

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala

115 120

<210> 74

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> VK

<400> 74

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 75

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> VH

<400> 75

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala

115 120

<210> 76

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> VK

<400> 76

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 77

<211> 122

<212> PRT

<213> Artificial sequence

<220>

<223> VH

<400> 77

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 78

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> VK

<400> 78

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 79

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> VH

<400> 79

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala

115 120

<210> 80

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> VK

<400> 80

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 81

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 1

<400> 81

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Thr Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Phe Pro Gly Asn Asp Asn Ile Lys Tyr Ser Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile Asp Ser Val Ser Ile Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Cys Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

<210> 82

<211> 474

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 2

<400> 82

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Leu Glu Asp Leu Lys Asn Val Phe

115 120 125

Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His

130 135 140

Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp

145 150 155 160

His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly

165 170 175

Val Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Gln Asp

180 185 190

Ser Arg Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp

195 200 205

Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu

210 215 220

Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln

225 230 235 240

Ile Val Ser Ala Glu Ala Trp Gly Arg Tyr Gly Pro Pro Cys Pro Pro

245 250 255

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

260 265 270

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

275 280 285

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

290 295 300

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

305 310 315 320

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

325 330 335

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

340 345 350

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

355 360 365

Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu

370 375 380

Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe

385 390 395 400

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

405 410 415

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

420 425 430

Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

435 440 445

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

450 455 460

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

465 470

<210> 83

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<223> light chain 1

<400> 83

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Lys Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

85 90 95

Ser Phe Ile Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 84

<211> 202

<212> PRT

<213> Artificial sequence

<220>

<223> light chain 2

<400> 84

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Pro Asp Ile Gln Asn

100 105 110

Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys

115 120 125

Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Gln Val Ser Gln

130 135 140

Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met

145 150 155 160

Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Gln Lys

165 170 175

Ser Asp Phe Ala Cys Ala Asn Ala Phe Gln Asn Ser Ile Ile Pro Glu

180 185 190

Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200

<210> 85

<211> 471

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 1

<400> 85

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ala Phe Thr Asp Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Ser Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

115 120 125

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

130 135 140

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

145 150 155 160

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr

165 170 175

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Gln Asp Ser Arg Tyr

180 185 190

Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

195 200 205

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

210 215 220

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

225 230 235 240

Ala Glu Ala Trp Gly Arg Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

245 250 255

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

260 265 270

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

275 280 285

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

290 295 300

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

305 310 315 320

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

325 330 335

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

340 345 350

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

355 360 365

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr

370 375 380

Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser

385 390 395 400

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

405 410 415

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

420 425 430

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

435 440 445

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

450 455 460

Ser Leu Ser Leu Ser Leu Gly

465 470

<210> 86

<211> 676

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 2

<400> 86

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser

210 215 220

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu

225 230 235 240

Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

245 250 255

Thr Phe Thr Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Gly Arg

260 265 270

Gly Leu Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser

275 280 285

Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

290 295 300

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

305 310 315 320

Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr

325 330 335

Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser

340 345 350

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr

355 360 365

Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

370 375 380

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

385 390 395 400

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

405 410 415

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr

420 425 430

Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val

435 440 445

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

450 455 460

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

465 470 475 480

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

485 490 495

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

500 505 510

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

515 520 525

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

530 535 540

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

545 550 555 560

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

565 570 575

Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

580 585 590

Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

595 600 605

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

610 615 620

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu

625 630 635 640

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

645 650 655

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

660 665 670

Leu Ser Leu Gly

675

<210> 87

<211> 207

<212> PRT

<213> Artificial sequence

<220>

<223> light chain 1

<400> 87

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gln Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

85 90 95

Ser His Thr Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

115 120 125

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

130 135 140

Thr Gln Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

145 150 155 160

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

165 170 175

Ala Trp Ser Gln Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Gln Asn

180 185 190

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200 205

<210> 88

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> light chain 2

<400> 88

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 89

<211> 700

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 1

<400> 89

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser

210 215 220

Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val

225 230 235 240

Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe

245 250 255

Ala Phe Thr Asp Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln

260 265 270

Gly Leu Glu Trp Met Gly Trp Ile Ser Pro Gly Asn Val Asn Thr Lys

275 280 285

Tyr Asn Glu Asn Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser

290 295 300

Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr

305 310 315 320

Ala Val Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Leu Tyr Tyr Phe Asp

325 330 335

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Leu Glu Asp Leu Lys Asn

340 345 350

Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile

355 360 365

Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr

370 375 380

Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His

385 390 395 400

Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu

405 410 415

Gln Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr

420 425 430

Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr

435 440 445

Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val

450 455 460

Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Tyr Gly Pro Pro Cys

465 470 475 480

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

485 490 495

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

500 505 510

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

515 520 525

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

530 535 540

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

545 550 555 560

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

565 570 575

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

580 585 590

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys

595 600 605

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

610 615 620

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

625 630 635 640

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

645 650 655

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

660 665 670

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

675 680 685

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

690 695 700

<210> 90

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 2

<400> 90

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

210 215 220

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys

355 360 365

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

<210> 91

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> light chain 1

<400> 91

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 92

<211> 207

<212> PRT

<213> Artificial sequence

<220>

<223> light chain 2

<400> 92

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gln Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

85 90 95

Ser His Thr Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

115 120 125

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

130 135 140

Thr Gln Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

145 150 155 160

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

165 170 175

Ala Trp Ser Gln Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Gln Asn

180 185 190

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200 205

<210> 93

<211> 701

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 1

<400> 93

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly

210 215 220

Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu

225 230 235 240

Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly

245 250 255

Tyr Ser Phe Thr Thr Tyr Tyr Ile His Trp Val Arg Gln Ala Pro Gly

260 265 270

Gln Gly Leu Glu Trp Met Gly Trp Ile Phe Pro Gly Asn Asp Asn Ile

275 280 285

Lys Tyr Ser Glu Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys

290 295 300

Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

305 310 315 320

Thr Ala Val Tyr Tyr Cys Ala Ile Asp Ser Val Ser Ile Tyr Tyr Phe

325 330 335

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Leu Glu Asp Leu Lys

340 345 350

Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu

355 360 365

Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe

370 375 380

Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val

385 390 395 400

His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala

405 410 415

Leu Gln Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser Ala

420 425 430

Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe

435 440 445

Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro

450 455 460

Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Tyr Gly Pro Pro

465 470 475 480

Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

485 490 495

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

500 505 510

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

515 520 525

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

530 535 540

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

545 550 555 560

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

565 570 575

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

580 585 590

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

595 600 605

Cys Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

610 615 620

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

625 630 635 640

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

645 650 655

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

660 665 670

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

675 680 685

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

690 695 700

<210> 94

<211> 448

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 2

<400> 94

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

115 120 125

Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr

130 135 140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155 160

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

165 170 175

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185 190

Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp

195 200 205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr

210 215 220

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr

340 345 350

Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

<210> 95

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> light chain 1

<400> 95

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 96

<211> 207

<212> PRT

<213> Artificial sequence

<220>

<223> light chain 2

<400> 96

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Lys Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

85 90 95

Ser Phe Ile Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

115 120 125

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

130 135 140

Thr Gln Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

145 150 155 160

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

165 170 175

Ala Trp Ser Gln Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Gln Asn

180 185 190

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200 205

<210> 97

<211> 703

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 1

<400> 97

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Ala Phe Thr Asp Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Gly Tyr Ser Leu Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu

115 120 125

Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys

130 135 140

Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu

145 150 155 160

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr

165 170 175

Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Gln Asp Ser Arg Tyr

180 185 190

Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro

195 200 205

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

210 215 220

Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser

225 230 235 240

Ala Glu Ala Trp Gly Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

260 265 270

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

275 280 285

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

290 295 300

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

305 310 315 320

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

325 330 335

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

340 345 350

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

355 360 365

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

370 375 380

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

385 390 395 400

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

405 410 415

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

420 425 430

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

435 440 445

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

450 455 460

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

465 470 475 480

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

485 490 495

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

500 505 510

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

515 520 525

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

530 535 540

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

545 550 555 560

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

565 570 575

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

580 585 590

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

595 600 605

Pro Pro Cys Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys

610 615 620

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

625 630 635 640

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

645 650 655

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

660 665 670

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

675 680 685

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

690 695 700

<210> 98

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain 2

<400> 98

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

210 215 220

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys

355 360 365

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Val Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

<210> 99

<211> 207

<212> PRT

<213> Artificial sequence

<220>

<223> light chain 1

<400> 99

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gln Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Thr Gln

85 90 95

Ser His Thr Leu Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

115 120 125

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

130 135 140

Thr Gln Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

145 150 155 160

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

165 170 175

Ala Trp Ser Gln Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Gln Asn

180 185 190

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200 205

<210> 100

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> light chain 2

<400> 100

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 101

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> VH

<400> 101

caggtgcaac tcgtgcagtc tggagctgaa gtgaagaagc ctgggtcttc agtcaaggtc 60

agttgcaagg ccagtgggta ttccttcact acctactaca tccactgggt gcggcaggca 120

ccaggacagg ggcttgagtg gatgggctgg atctttcccg gcaacgataa tattaagtac 180

agcgagaagt tcaaagggag ggtcaccatt accgccgaca aatccacttc cacagcctac 240

atggagttga gcagcctgag atccgaggat acagccgtgt actactgtgc cattgacagc 300

gtgtccatct actactttga ctactggggc cagggcacac tggtcacagt gagcagc 357

<210> 102

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> VK

<400> 102

gacatcgtca tgacccagtc cccagactct ttggcagtgt ctctcgggga aagagctacc 60

atcaactgca agagcagcca gtcccttctg aacagcagga ccaggaagaa ttacctcgcc 120

tggtaccaac agaagcccgg acagcctcct aagctcctga tctactgggc ctcaacccgg 180

aagagtggag tgcccgatcg ctttagcggg agcggctccg ggacagattt cacactgaca 240

atttcctccc tgcaggccga ggacgtcgcc gtgtattact gtactcagag cttcattctg 300

cggacatttg gcggcgggac taaagtggag attaag 336

<210> 103

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> VH

<400> 103

caggtgcagc ttgtgcagtc tggggcagaa gtgaagaagc ctgggtctag tgtcaaggtg 60

tcatgcaagg ctagcgggtt cgcctttact gactactaca tccactgggt gcggcaggct 120

cccggacaag ggttggagtg gatgggatgg atctccccag gcaatgtcaa cacaaagtac 180

aacgagaact tcaaaggccg cgtcaccatt accgccgaca agagcacctc cacagcctac 240

atggagctgt ccagcctcag aagcgaggac actgccgtct actactgtgc cagggatggg 300

tactccctgt attactttga ttactggggc cagggcacac tggtgacagt gagctcc 357

<210> 104

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> VK

<400> 104

gatatcgtga tgacccagag cccagactcc cttgctgtct ccctcggcga aagagcaacc 60

atcaactgca agagctccca aagcctgctg aactccagga ccaggaagaa ttacctggcc 120

tggtatcagc agaagcccgg ccagcctcct aagctgctca tctactgggc ctccacccgg 180

cagtctgggg tgcccgatcg gtttagtgga tctgggagcg ggacagactt cacattgaca 240

attagctcac tgcaggccga ggacgtggcc gtctactact gtactcagag ccacactctc 300

cgcacattcg gcggagggac taaagtggag attaag 336

<210> 105

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> VH

<400> 105

caggtgcagc ttgtgcagtc tggggcagaa gtgaagaagc ctgggtctag tgtcaaggtg 60

tcatgcaagg ctagcgggtt cgcctttact gactactaca tccactgggt gcggcaggct 120

cccggacaag ggttggagtg gatgggatgg atctccccag gcaatgtcaa cacaaagtac 180

aacgagaact tcaaaggccg cgtcaccatt accgccgaca agagcacctc cacagcctac 240

atggagctgt ccagcctcag aagcgaggac actgccgtct actactgtgc cagggatggg 300

tactccctgt attactttga ttactggggc cagggcacac tggtgacagt gagctcc 357

<210> 106

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> VK

<400> 106

gatatcgtga tgacccagag cccagactcc cttgctgtct ccctcggcga aagagcaacc 60

atcaactgca agagctccca aagcctgctg aactccagga ccaggaagaa ttacctggcc 120

tggtatcagc agaagcccgg ccagcctcct aagctgctca tctactgggc ctccacccgg 180

cagtctgggg tgcccgatcg gtttagtgga tctgggagcg ggacagactt cacattgaca 240

attagctcac tgcaggccga ggacgtggcc gtctactact gtactcagag ccacactctc 300

cgcacattcg gcggagggac taaagtggag attaag 336

<210> 107

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> VH

<400> 107

caggtgcaac tcgtgcagtc tggagctgaa gtgaagaagc ctgggtcttc agtcaaggtc 60

agttgcaagg ccagtgggta ttccttcact acctactaca tccactgggt gcggcaggca 120

ccaggacagg ggcttgagtg gatgggctgg atctttcccg gcaacgataa tattaagtac 180

agcgagaagt tcaaagggag ggtcaccatt accgccgaca aatccacttc cacagcctac 240

atggagttga gcagcctgag atccgaggat acagccgtgt actactgtgc cattgacagc 300

gtgtccatct actactttga ctactggggc cagggcacac tggtcacagt gagcagc 357

<210> 108

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> VK

<400> 108

gacatcgtca tgacccagtc cccagactct ttggcagtgt ctctcgggga aagagctacc 60

atcaactgca agagcagcca gtcccttctg aacagcagga ccaggaagaa ttacctcgcc 120

tggtaccaac agaagcccgg acagcctcct aagctcctga tctactgggc ctcaacccgg 180

aagagtggag tgcccgatcg ctttagcggg agcggctccg ggacagattt cacactgaca 240

atttcctccc tgcaggccga ggacgtcgcc gtgtattact gtactcagag cttcattctg 300

cggacatttg gcggcgggac taaagtggag attaag 336

<210> 109

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> VH

<400> 109

caggtgcagc ttgtgcagtc tggggcagaa gtgaagaagc ctgggtctag tgtcaaggtg 60

tcatgcaagg ctagcgggtt cgcctttact gactactaca tccactgggt gcggcaggct 120

cccggacaag ggttggagtg gatgggatgg atctccccag gcaatgtcaa cacaaagtac 180

aacgagaact tcaaaggccg cgtcaccatt accgccgaca agagcacctc cacagcctac 240

atggagctgt ccagcctcag aagcgaggac actgccgtct actactgtgc cagggatggg 300

tactccctgt attactttga ttactggggc cagggcacac tggtgacagt gagctcc 357

<210> 110

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> VK

<400> 110

gatatcgtga tgacccagag cccagactcc cttgctgtct ccctcggcga aagagcaacc 60

atcaactgca agagctccca aagcctgctg aactccagga ccaggaagaa ttacctggcc 120

tggtatcagc agaagcccgg ccagcctcct aagctgctca tctactgggc ctccacccgg 180

cagtctgggg tgcccgatcg gtttagtgga tctgggagcg ggacagactt cacattgaca 240

attagctcac tgcaggccga ggacgtggcc gtctactact gtactcagag ccacactctc 300

cgcacattcg gcggagggac taaagtggag attaag 336

<210> 111

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> VH

<400> 111

gaggtgcaat tggtggagag cggaggaggg ctcgtgcagc ctggaagatc tcttaggctg 60

agttgcgctg catctgggtt cacattcaac gactacgcca tgcactgggt gaggcaggct 120

cccggcaaag ggctggaatg ggtgtcaact atctcctgga actccggcag catcggctac 180

gccgatagcg tcaagggccg gtttacaatt tcccgcgata acgccaagaa gtccctgtac 240

ctgcagatga acagcctgcg ggccgaggat actgccctct actactgtgc caaggacatt 300

cagtacggga attactatta cgggatggac gtctggggcc aggggaccac cgtgacagtc 360

agctcc 366

<210> 112

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> VK

<400> 112

gaaatcgtgc tgacccagtc cccagcaacc ctctcccttt ctcctggaga gagagctacc 60

ctcagctgta gggcctcaca gtctgtctcc agttacctgg cttggtacca gcagaaaccc 120

gggcaggccc ctaggttgct gatctacgac gccagcaata gggccactgg catcccagcc 180

cggttttccg gaagcggcag cgggacagat ttcacactca ctattagcag cctggagccc 240

gaggacttcg ccgtgtacta ttgccagcag cggtccaact ggcccattac atttggccaa 300

gggacacgcc tggagattaa g 321

<210> 113

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> VH

<400> 113

caggtccagc tgcagcagcc cggagccgaa ctggtcaaac ccggggctag cgtgaaaatg 60

tcttgcaaag caagtggtta cacattcact tcctataaca tgcactgggt gaagcagaca 120

cctgggcgag gtctggaatg gatcggcgcc atctacccag gcaacggaga cactagctat 180

aatcagaagt ttaaaggaaa ggccaccctg acagctgata agtccagctc taccgcttac 240

atgcagctga gttcactgac aagtgaggac tcagcagtgt actattgcgc ccgttctacc 300

tactatggcg gagattggta tttcaatgtg tggggcgccg gtaccacagt caccgtgtcc 360

gcc 363

<210> 114

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> VK

<400> 114

cagattgtcc tgagccagag ccctgccatc ctgtctgcta gtcccggcga gaaggtgacc 60

atgacatgca gggcatccag ctctgtctcc tacatccact ggttccagca gaagcccggg 120

agttcaccta aaccatggat ctacgctaca tccaacctgg caagcggtgt gcctgtcagg 180

ttttcaggtt ccggcagcgg aacatcttac agtctgacta tttctcgggt ggaggccgaa 240

gacgccgcta cctactattg ccagcagtgg acctccaatc cccctacatt cggcggaggg 300

actaagctgg agatcaaa 318

<210> 115

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> VH

<400> 115

caggtccagc tgcagcagcc cggagccgaa ctggtcaaac ccggggctag cgtgaaaatg 60

tcttgcaaag caagtggtta cacattcact tcctataaca tgcactgggt gaagcagaca 120

cctgggcgag gtctggaatg gatcggcgcc atctacccag gcaacggaga cactagctat 180

aatcagaagt ttaaaggaaa ggccaccctg acagctgata agtccagctc taccgcttac 240

atgcagctga gttcactgac aagtgaggac tcagcagtgt actattgcgc ccgttctacc 300

tactatggcg gagattggta tttcaatgtg tggggcgccg gtaccacagt caccgtgtcc 360

gcc 363

<210> 116

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> VK

<400> 116

cagattgtcc tgagccagag ccctgccatc ctgtctgcta gtcccggcga gaaggtgacc 60

atgacatgca gggcatccag ctctgtctcc tacatccact ggttccagca gaagcccggg 120

agttcaccta aaccatggat ctacgctaca tccaacctgg caagcggtgt gcctgtcagg 180

ttttcaggtt ccggcagcgg aacatcttac agtctgacta tttctcgggt ggaggccgaa 240

gacgccgcta cctactattg ccagcagtgg acctccaatc cccctacatt cggcggaggg 300

actaagctgg agatcaaa 318

<210> 117

<211> 366

<212> DNA

<213> Artificial sequence

<220>

<223> VH

<400> 117

gaggtgcaat tggtggagag cggaggaggg ctcgtgcagc ctggaagatc tcttaggctg 60

agttgcgctg catctgggtt cacattcaac gactacgcca tgcactgggt gaggcaggct 120

cccggcaaag ggctggaatg ggtgtcaact atctcctgga actccggcag catcggctac 180

gccgatagcg tcaagggccg gtttacaatt tcccgcgata acgccaagaa gtccctgtac 240

ctgcagatga acagcctgcg ggccgaggat actgccctct actactgtgc caaggacatt 300

cagtacggga attactatta cgggatggac gtctggggcc aggggaccac cgtgacagtc 360

agctcc 366

<210> 118

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> VK

<400> 118

gaaatcgtgc tgacccagtc cccagcaacc ctctcccttt ctcctggaga gagagctacc 60

ctcagctgta gggcctcaca gtctgtctcc agttacctgg cttggtacca gcagaaaccc 120

gggcaggccc ctaggttgct gatctacgac gccagcaata gggccactgg catcccagcc 180

cggttttccg gaagcggcag cgggacagat ttcacactca ctattagcag cctggagccc 240

gaggacttcg ccgtgtacta ttgccagcag cggtccaact ggcccattac atttggccaa 300

gggacacgcc tggagattaa g 321

<210> 119

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> VH

<400> 119

caggtccagc tgcagcagcc cggagccgaa ctggtcaaac ccggggctag cgtgaaaatg 60

tcttgcaaag caagtggtta cacattcact tcctataaca tgcactgggt gaagcagaca 120

cctgggcgag gtctggaatg gatcggcgcc atctacccag gcaacggaga cactagctat 180

aatcagaagt ttaaaggaaa ggccaccctg acagctgata agtccagctc taccgcttac 240

atgcagctga gttcactgac aagtgaggac tcagcagtgt actattgcgc ccgttctacc 300

tactatggcg gagattggta tttcaatgtg tggggcgccg gtaccacagt caccgtgtcc 360

gcc 363

<210> 120

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> VK

<400> 120

cagattgtcc tgagccagag ccctgccatc ctgtctgcta gtcccggcga gaaggtgacc 60

atgacatgca gggcatccag ctctgtctcc tacatccact ggttccagca gaagcccggg 120

agttcaccta aaccatggat ctacgctaca tccaacctgg caagcggtgt gcctgtcagg 180

ttttcaggtt ccggcagcgg aacatcttac agtctgacta tttctcgggt ggaggccgaa 240

gacgccgcta cctactattg ccagcagtgg acctccaatc cccctacatt cggcggaggg 300

actaagctgg agatcaaa 318

<210> 121

<211> 126

<212> PRT

<213> Artificial sequence

<220>

<223> modified TCRBeta

<400> 121

Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu

1 5 10 15

Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys

20 25 30

Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val

35 40 45

Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu

50 55 60

Lys Glu Gln Pro Ala Leu Gln Asp Ser Arg Tyr Ala Leu Ser Ser Arg

65 70 75 80

Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg

85 90 95

Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln

100 105 110

Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala

115 120 125

<210> 122

<211> 95

<212> PRT

<213> Artificial sequence

<220>

<223> modified TCRAlpha

<400> 122

Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

1 5 10 15

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

20 25 30

Thr Gln Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

35 40 45

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

50 55 60

Ala Trp Ser Gln Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Gln Asn

65 70 75 80

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

85 90 95

Claims

the first antigen-binding portion comprises:

wherein:

c1 and C2 are capable of forming a dimer comprising at least one unnatural inter-chain bond between C1 and C2, and which unnatural inter-chain bond is capable of stabilizing the dimer, wherein C1 comprises an engineered TCR β chain (C β), said engineered C β being shown in SEQ ID NO:121, and C2 comprises an engineered TCR α chain (C α), said engineered C α being shown in SEQ ID NO:122, and

the second antigen-binding moiety comprises:

wherein:

an anti-CD 3 binding moiety is derived from an anti-CD 3 antibody comprising:

a) SEQ ID NO:1, the heavy chain CDR1 shown in figure 1,

b) SEQ ID NO: 2, and a heavy chain CDR2 shown in figure 2,

c) SEQ ID NO: 3, a heavy chain CDR3 shown in figure 3,

d) SEQ ID NO: 4, the kappa light chain CDR1 shown in figure 4,

e) SEQ ID NO: 5 the kappa light chain CDR2, and

f) the amino acid sequence of SEQ ID NO:6, the kappa light chain CDR3 shown in FIG. 6,

an anti-CD20 binding moiety is derived from an anti-CD20 antibody comprising:

a) the amino acid sequence of SEQ ID NO:7, heavy chain CDR1 shown in figure 7,

b) SEQ ID NO: the heavy chain CDR2 shown in figure 8,

c) SEQ ID NO:9, and a heavy chain CDR3 shown in figure 9,

d) SEQ ID NO:10, the kappa light chain CDR1 shown in,

e) SEQ ID NO:11 the kappa light chain CDR2, and

f) SEQ ID NO:12, the kappa light chain CDR 3;

or

An anti-CD 3 binding moiety is derived from an anti-CD 3 antibody comprising:

a) the amino acid sequence of SEQ ID NO: 25, and a heavy chain CDR1 shown in SEQ ID NO,

b) SEQ ID NO: 26, and a heavy chain CDR2 shown in FIG. 26,

c) SEQ ID NO: 27, a heavy chain CDR3 shown in figure 27,

d) SEQ ID NO: 28, the kappa light chain CDR1 shown in FIG. 28,

e) SEQ ID NO: 29, and the kappa light chain CDR2 shown in

f) SEQ ID NO: 30, the kappa light chain CDR3 shown in figure 30,

an anti-CD20 binding moiety is derived from an anti-CD20 antibody comprising:

a) SEQ ID NO: 31, and a heavy chain CDR1 shown in figure 31,

b) SEQ ID NO: 32, and a heavy chain CDR2 shown in figure 32,

c) SEQ ID NO: 33, and a heavy chain CDR3 shown in figure 33,

d) SEQ ID NO: 34, the kappa light chain CDR1 shown in FIG. 34,

e) SEQ ID NO: 35 the kappa light chain CDR2, and

f) the amino acid sequence of SEQ ID NO: 36, kappa light chain CDR3 shown.

2. The bispecific polypeptide complex of claim 1, wherein the anti-CD 3 binding moiety comprises SEQ ID NO:61 and the heavy chain variable domain sequence set forth in SEQ ID NO: 62; or

The anti-CD 3 binding portion comprises SEQ ID NO:65 and the heavy chain variable domain sequence set forth in SEQ ID NO:66, or a light chain variable domain sequence set forth in seq id no.

3. The bispecific polypeptide complex of claim 1, wherein the anti-CD20 binding moiety comprises SEQ ID NO:71 and the heavy chain variable domain sequence of SEQ ID NO:72, a light chain variable domain sequence; or

The anti-CD20 binding portion comprises SEQ ID NO:75 and the heavy chain variable domain sequence set forth in SEQ ID NO: 76.

4. The bispecific polypeptide complex of any one of claims 1-3, wherein the first antigen-binding moiety is linked to a first dimerization domain and the second antigen-binding moiety is linked to a second dimerization domain, wherein the first and second dimerization domains are in association.

5. The bispecific polypeptide complex of claim 4, wherein the association is via a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, or a hydrophobic-hydrophilic interaction, or a combination thereof.

6. The bispecific polypeptide complex of claim 5, wherein the first dimerization domain and/or the second dimerization domain comprise at least a portion of an antibody hinge region.

7. The bispecific polypeptide complex of claim 6, wherein the antibody hinge region is derived from IgG1, IgG2, or IgG4.

8. The bispecific polypeptide complex of claim 6, wherein the first dimerization domain and/or the second dimerization domain comprises an antibody CH2 domain and/or an antibody CH3 domain.

9. The bispecific polypeptide complex of claim 6, wherein the first dimerization domain is operably linked to the first TCR constant region at a third engagement domain (C1).

10. The bispecific polypeptide complex of claim 6, wherein the second dimerization domain is operably linked to the C-terminus of the antibody CH1 constant region of the second antigen-binding moiety.

11. The bispecific polypeptide complex of claim 4, wherein the first dimerization domain and the second dimerization domain are different and are associated in a manner that does not favor homodimerization and/or favors heterodimerization.

12. The bispecific polypeptide complex of claim 11, wherein the first dimerization domain and the second dimerization domain are capable of associating into a heterodimer via handle-access holes, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility.

13. The bispecific polypeptide complex of claim 1, wherein the bispecific polypeptide complex consists of SEQ ID NO: 81. SEQ ID NO: 82. SEQ ID NO:83 and SEQ ID NO:84, and four polypeptides as shown in the figure.

14. The bispecific polypeptide complex of claim 1, wherein the bispecific polypeptide complex consists of SEQ ID NO: 85. the amino acid sequence of SEQ ID NO: 86. SEQ ID NO: 87. SEQ ID NO: 88 and SEQ ID NO: 88, and the polypeptide is shown in the specification.

15. The bispecific polypeptide complex of claim 1, wherein the bispecific polypeptide complex consists of SEQ ID NO: 89. SEQ ID NO: 90. SEQ ID NO: 91. SEQ ID NO:91 and SEQ ID NO:92, or a pharmaceutically acceptable salt thereof.

16. The bispecific polypeptide complex of claim 1, wherein the bispecific polypeptide complex consists of SEQ ID NO: 93. SEQ ID NO: 94. SEQ ID NO: 95. SEQ ID NO: 95 and SEQ ID NO: 96 is shown in the specification.

17. The bispecific polypeptide complex of claim 1, wherein the bispecific polypeptide complex consists of SEQ ID NO: 97. SEQ ID NO: 98. SEQ ID NO: 99. SEQ ID NO: 100 and SEQ ID NO: 100, and consists of five polypeptides shown in the specification.

18. A conjugate comprising the bispecific polypeptide complex of any one of claims 1-17.

19. An isolated polynucleotide encoding the bispecific polypeptide complex of any one of claims 1-17.

20. An isolated vector comprising the polynucleotide of claim 19.

21. A host cell comprising the isolated polynucleotide of claim 19 or the isolated vector of claim 20.

22. A method of expressing a bispecific polypeptide complex of any one of claims 1-17, comprising culturing the host cell of claim 21 under conditions in which the bispecific polypeptide complex is expressed.

23. A method of producing a bispecific polypeptide complex, the method comprising:

a) introducing into a host cell one or more polynucleotides encoding a bispecific polypeptide complex of any one of claims 1-17; and

b) allowing the host cell to express the bispecific polypeptide complex.

24. The method of any one of claims 22-23, further comprising isolating the bispecific polypeptide complex.

25. A composition comprising the bispecific polypeptide complex of any one of claims 1-17.

26. A pharmaceutical composition comprising the bispecific polypeptide complex of any one of claims 1-17, and a pharmaceutically acceptable carrier.

27. Use of a bispecific polypeptide complex of any one of claims 1-17 in the manufacture of a pharmaceutical composition for treating a CD 20-associated disease or condition in a subject, wherein the CD 20-associated disease or condition is B cell lymphoma.

28. The use of claim 27, wherein the B cell lymphoma is hodgkin's lymphoma or non-hodgkin's lymphoma.

29. The use of claim 28, wherein the non-hodgkin's lymphoma comprises: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma, Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma (MCL), Acute Lymphocytic Leukemia (ALL), or Waldenstrom's Macroglobulinemia (WM).

30. A kit comprising a bispecific polypeptide complex of any one of claims 1-17.

31. The kit of claim 30 for use in detecting, diagnosing, prognosing or treating a disease or condition associated with CD20, wherein the disease or condition associated with CD20 is B cell lymphoma.