CD3-Targeting Antibodies, Polyspecific Antibodies, and Uses Thereof

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof that targets CD3, which has a moderate ability to activate T cells and can effectively reduce cellular factor release. The present invention also relates to a multispecific antibody that targets CD3 and other antigens, such as tumor-associated antigens and / or immune checkpoint molecules, which can significantly reduce cytokine release while ensuring effective and specific killing of target cells, and has significantly improved safety. The present invention also relates to the use of an antibody or antigen-binding fragment thereof that targets CD3, and a multispecific antibody, or a composition comprising the same, in the treatment of diseases.

Description

[Technical field]

[0001] Technical Field The present disclosure relates to a CD3-targeting antibody or its antigen-binding fragment, which has the ability to moderately activate T cells and can effectively reduce cytokine release. The present disclosure also relates to a multispecific antibody that targets CD3 and other antigens (e.g., tumor-associated antigens and / or immune checkpoint molecules). The multispecific antibody can efficiently and specifically kill target cells, while at the same time significantly reducing cytokine release, thereby having significantly improved safety. The present disclosure also relates to the use of the CD3-targeting antibody or its antigen-binding fragment, the multispecific antibody, or a composition comprising them in the treatment of disease. [Background technology]

[0002] Background technology CD3 is a protein complex and T cell coreceptor that is mainly expressed in T cells and participates in the activation of cytotoxic T cells (CD8+ naive T cells) and helper T cells (CD4+ naive T cells). CD3 consists of one CD3γ chain, one CD3δ chain and two CD3ε chains. These chains bind to the T cell receptor (TCR) and the ζ chain (zeta chain) to generate activation signals in T lymphocytes. CD3 antibodies in clinical development or on the market are mainly derived from humanized CD3 antibodies (such as OKT3 / UCHT1 / L2K / TR66). However, their affinity to CD3 is relatively high, which easily leads to excessive activation of T cells and release of large amounts of cytokines, thus causing cytokine storm syndrome, which may have severe side effects in humans and may even be life-threatening in severe cases. To address the risk of cytokine storm syndrome (CRS), their dosages are generally very low, which results in a narrow therapeutic window and is detrimental to the patient's benefit.

[0003] Thus, there is a need in the art to develop new CD3 antibodies and bispecific antibodies that reduce the probability of cytokine storm syndrome while providing the capacity for moderate T cell activation, thereby improving safety and providing benefits in the treatment of diseases, particularly cancer. Summary of the Invention [Means for solving the problem]

[0004] Contents of this Disclosure Through extensive research, the inventors of the present application have screened and obtained novel CD3 antibodies. These antibodies show that cytokine release is reduced and they can moderately activate T cells. The inventors of the present application have also screened and obtained novel MSLN single domain antibodies, which have good binding affinity to MSLN. Based on this, multispecific antibodies targeting CD3 and additional antigens (e.g., MSLN, CD19, CD20, Trop2, Her2 or claudin 18.2) have been further developed, which show enhanced tumor cell localization and induce effective activation of T cells, thereby significantly reducing cytokine release and specifically killing tumor cells, thus significantly improving safety. Therefore, the following aspects are provided:

[0005] In a first aspect, the present disclosure provides an antibody or antigen-binding fragment thereof capable of specifically binding to CD3, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an HCDR1 comprising the sequence set forth in SEQ ID NO: 107, an HCDR2 comprising the sequence set forth in SEQ ID NO: 108, and a HCDR3 comprising the sequence set forth in SEQ ID NO: 109, and a HCDR4 comprising the sequence set forth in SEQ ID NO: 200, and a HCDR5 comprising the sequence set forth in SEQ ID NO: 210, 10 WX 11 X 12 X 13(SEQ ID NO:112) (wherein X1 is A, H or P; X2 is A, E, G, H, K, Q or S; X3 is D, N or R; X4 is F or P; X5 is G, K, L, P, Q, R, S, V, W or Y; X6 is M, N, Q or R; X7 is G, N, S or T; X8 is A, Q, R or Y; X9 is G, I or V; X 10 is N or S;X 11 is F or W;X 12 is A, E, K or Q; X 13 is H, L, M, S or Y); with the proviso that the HCDR3 is not as set forth in SEQ ID NO: 90.

[0006] In a second aspect, the present disclosure provides a single domain antibody or antigen-binding fragment thereof capable of specifically binding to MSLN, the single domain antibody or antigen-binding fragment thereof comprising: (1) The following CDRs as defined by the IMGT numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 150 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 151 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 152 or a variant thereof; (2) the following CDRs as defined by the Kabat numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 153 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 154 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 155 or a variant thereof; (3) The following CDRs as defined by the AbM numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 156 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 157 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 155 or a variant thereof; (4) The following CDRs as defined by the Chothia numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 158 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 159 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 155 or a variant thereof; or (5) The following CDRs as defined by the Contact numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 160 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 161 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 162 or a variant thereof. Includes; The variant according to any one of (1) to (5) has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived; preferably, the substitutions are conservative substitutions; Preferably, the single domain antibody or antigen-binding fragment thereof comprises: (1) The following CDRs as defined by the IMGT numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 150, CDR2 comprising the sequence set forth in SEQ ID NO: 151, and CDR3 comprising the sequence set forth in SEQ ID NO: 152; (2) the following CDRs as defined by the Kabat numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 153, CDR2 comprising the sequence set forth in SEQ ID NO: 154, and CDR3 comprising the sequence set forth in SEQ ID NO: 155; (3) The following CDRs as defined by the AbM numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 156, CDR2 comprising the sequence set forth in SEQ ID NO: 157, and CDR3 comprising the sequence set forth in SEQ ID NO: 155; (4) The following CDRs as defined by the Chothia numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 158, CDR2 comprising the sequence set forth in SEQ ID NO: 159, and CDR3 comprising the sequence set forth in SEQ ID NO: 155; or (5) The following CDRs as defined by the Contact numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 160, CDR2 comprising the sequence set forth in SEQ ID NO: 161, and CDR3 comprising the sequence set forth in SEQ ID NO: 162. Including, Relates to single domain antibodies or antigen-binding fragments thereof.

[0007] In a third aspect, the present disclosure relates to a multispecific antibody comprising a CD3-targeting antigen binding domain and at least one antigen binding domain targeting another antigen, wherein the CD3-targeting antigen binding domain is selected from an antibody or antigen-binding fragment thereof according to the first aspect, and the other antigen is selected from a tumor-associated antigen (TAA) and / or an immune checkpoint molecule.

[0008] In a fourth aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: - an antibody or antigen-binding fragment thereof according to the first aspect, or a heavy chain variable region and / or a light chain variable region thereof; - a single domain antibody or antigen-binding fragment thereof according to the second aspect; or - a multispecific antibody, or a polypeptide chain thereof, according to the third aspect The present invention relates to an isolated nucleic acid molecule encoding

[0009] In a fifth aspect, the present disclosure relates to a vector comprising the nucleic acid molecule according to the fourth aspect.

[0010] In a sixth aspect, the present disclosure relates to a host cell comprising the nucleic acid molecule according to the fourth aspect or the vector according to the fifth aspect.

[0011] In a seventh aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect, a single domain antibody or antigen-binding fragment thereof according to the second aspect, a multispecific antibody according to the third aspect, an isolated nucleic acid molecule according to the fourth aspect, a vector according to the fifth aspect, or a host cell according to the sixth aspect, and a pharma- ceutically acceptable carrier and / or excipient.

[0012] In an eighth aspect, the present disclosure relates to the use of an antibody or antigen-binding fragment thereof according to the first aspect, a single domain antibody or antigen-binding fragment thereof according to the second aspect, a multispecific antibody according to the third aspect, an isolated nucleic acid molecule according to the fourth aspect, a vector according to the fifth aspect, a host cell according to the sixth aspect, or a pharmaceutical composition according to the seventh aspect for preventing and / or treating a disease, or in the manufacture of a medicament for preventing and / or treating a disease.

[0013] In a ninth aspect, the present disclosure relates to a method for preventing and / or treating a disease comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof according to the first aspect, the single domain antibody or antigen-binding fragment thereof according to the second aspect, the multispecific antibody according to the third aspect, the isolated nucleic acid molecule according to the fourth aspect, the vector according to the fifth aspect, the host cell according to the sixth aspect, or the pharmaceutical composition according to the seventh aspect. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows a schematic diagram of the structure of the CD3-MSLN bispecific antibody in Example 13. [Diagram 2] FIG. 2 shows the results of determining the binding activity of the CD3-MSLN bispecific antibody to MC38-MSLN cells in Example 14. [Figure 3A] 3A to 3D show the results of determining the dynamic affinity of the CD3-MSLN bispecific antibody to the human CD3εγ recombinant antigen in Example 14. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 4] FIG. 4 shows the results of determining the dynamic affinity of the CD3-MSLN bispecific antibody to the MSLN recombinant antigen in Example 14. [Figure 5A]5A to 5F show the results of detecting the stability of Jurkat-TIGIT-luc cells in Example 15. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above. [Figure 6A] 6A to 6F show the results of determining activation of the T cell activation signaling pathway by the CD3-MSLN bispecific antibody in Example 16. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 6F] Same as above. [Figure 7A] 7A to 7I show the results of determining TDCC mediated by the CD3-MSLN bispecific antibody in Example 17. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above. [Figure 7F] Same as above. [Figure 7G] Same as above. [Figure 7H] Same as above. [Figure 7I] Same as above. [Figure 8] FIG. 8 shows a schematic diagram of the structure of the CD3-CD19 bispecific antibody in Example 18. [Figure 9] FIG. 9 shows the results of determination of TDCC mediated by the CD3-CD19 bispecific antibody in Example 18. [Figure 10] FIG. 10 shows the results of determining TDCC mediated by the CD3-CD20 bispecific antibody in Example 19. [Figure 11]FIG. 11 shows the results of determination of TDCC mediated by CD3-trop2 bispecific antibody in Example 20. [Figure 12] FIG. 12 shows the results of determination of TDCC mediated by the CD3-Her2 bispecific antibody in Example 21. [Figure 13] FIG. 13 shows the results of determining TDCC mediated by CD3-claudin 18.2 bispecific antibody in Example 22. [Figure 14A] 14A to 14I show the detection results of IL-2 release levels mediated by the CD3-MSLN bispecific antibody in Example 23. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above. [Figure 14E] Same as above. [Figure 14F] Same as above. [Figure 14G] Same as above. [Figure 14H] Same as above. [Figure 14I] Same as above. [Figure 15A] 15A to 15I show the results of detection of INF-γ release levels mediated by the CD3-MSLN bispecific antibody in Example 23. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 15F] Same as above. [Figure 15G] Same as above. [Figure 15H] Same as above. [Figure 15I] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Specific Model for Carrying Out This Disclosure The present disclosure provides novel CD3 antibodies, and in particular provides the following aspects:

[0016] CD3 antibody In a first aspect, the present disclosure provides an antibody or antigen-binding fragment thereof capable of specifically binding to CD3, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an HCDR1 comprising the sequence set forth in SEQ ID NO: 107, an HCDR2 comprising the sequence set forth in SEQ ID NO: 108, and a HCDR3 comprising the sequence set forth in SEQ ID NO: 109, and a HCDR4 comprising the sequence set forth in SEQ ID NO: 200, and a HCDR5 comprising the sequence set forth in SEQ ID NO: 210, 10 WX 11 X 12 X 13 (SEQ ID NO:112) (wherein X1 is A, H or P; X2 is A, E, G, H, K, Q or S; X3 is D, N or R; X4 is F or P; X5 is G, K, L, P, Q, R, S, V, W or Y; X6 is M, N, Q or R; X7 is G, N, S or T; X8 is A, Q, R or Y; X9 is G, I or V; X 10 is N or S;X 11 is F or W;X 12 is A, E, K or Q; X 13 is H, L, M, S or Y); with the proviso that the HCDR3 is not as set forth in SEQ ID NO: 90.

[0017] In certain embodiments, the VL comprises an LCDR1 comprising the sequence set forth in SEQ ID NO:137, an LCDR2 comprising the sequence set forth in SEQ ID NO:138, and an LCDR3 comprising the sequence set forth in SEQ ID NO:139.

[0018] In certain embodiments, the HCDR3 is not represented by any one of SEQ ID NOs: 60, 64, 77, and 89.

[0019] In certain embodiments, HCDR3 comprises a sequence as set forth in X1X2X3FX4NX5YX6SWFAX7 (SEQ ID NO: 148), where X1 is H or P; X2 is G, E or A; X3 is N or R; X4 is G, K, S or P; X5 is T, S or N; X6 is V or G; and X7 is M, Y, S or L. In certain embodiments, HCDR3 comprises a sequence as set forth in any one of SEQ ID NOs: 56, 61, 69, 79, 82, 87.

[0020] In certain embodiments, HCDR3 is (1) the sequence shown in HX2NFGNSYVSWFAY (SEQ ID NO: 113) (wherein X2 is A, E, H, K, Q or S, preferably H); (2) the sequence shown in HGNFX5NSYVSWFAY (SEQ ID NO: 114), where X5 is K, L, P, Q, R, S, V, W or Y, preferably K; (3) the sequence shown in HGNFGNSX8VSWFAY (SEQ ID NO: 115), where X8 is A, Q or R, preferably R; or (4)HGNFGNSYVSWFX 12 Y (SEQ ID NO: 116) (wherein X 12 is E, K or Q) Includes.

[0021] In certain embodiments, the HCDR3 comprises a sequence set forth in any one of SEQ ID NOs: 75, 53, 72, 51, 57, 64.

[0022] In certain embodiments, HCDR3 is (1) A sequence shown in X1X2NFGNSYVSWFAY (SEQ ID NO: 117) (wherein X1 is A or P, preferably A; X2 is A, E, H, K, Q or S, preferably K, and preferably X1 is A and X2 is K); (2)X1GNFGNSYVX 10WFAY (SEQ ID NO: 118) (wherein X1 is A or P, preferably A; X 10 is N; preferably, X1 is A and X 10 is N; or (3)X1GNFGNSYVSWFX 12 Y (SEQ ID NO: 119) (wherein X1 is A or P, preferably A; X 12 is E, K or Q, preferably E or Q; preferably, X1 is A and X 12 is E or Q) Includes.

[0023] In certain embodiments, the HCDR3 comprises a sequence as set forth in any one of SEQ ID NOs: 50, 66, 55, 85.

[0024] In certain embodiments, HCDR3 is (1) A sequence represented by X1X2NFGNSYVSWFAY (SEQ ID NO: 117) (wherein X1 is A or P, preferably A; X2 is A, E, H, K, Q or S, preferably K; preferably, X1 is A and X2 is K); (2) HX2X3FGNSYVSWFAY (SEQ ID NO: 120) (wherein X2 is A, E, H, K, Q or S, preferably E; X3 is D or R, preferably R; preferably, X2 is E and X3 is R); (3) HX2NFX5NSYVSWFAY (SEQ ID NO: 121) (wherein X2 is A, E, H, K, Q or S, preferably A or S; X5 is K, L, P, Q, R, S, V, W or Y, preferably L, P, S, Q, V or R; preferably, X2 is A or S and X5 is L, P, S, Q, V or R; preferably, X2 and X5 are A / L, S / P, S / L, A / S, S / Q, S / V or S / R, respectively); (4) HX2NFGX6SYVSWFAY (SEQ ID NO: 122) (wherein X2 is A, E, H, K, Q or S, preferably S; X6 is M, Q or R, preferably R; preferably, X2 is S and X6 is R); (5) HX2NFGNX7YVSWFAY (SEQ ID NO: 123) (wherein X2 is A, E, H, K, Q or S, preferably S; X7 is G, N or T, preferably T or G; preferably, X2 is S and X7 is T or G); (6) HX2NFGNSX8VSWFAY (SEQ ID NO: 124) (wherein X2 is A, E, H, K, Q or S, preferably S; X8 is A, Q or R, preferably R; preferably, X2 is S and X8 is R); (7)HX2NFGNSYVSWX 11 AY (SEQ ID NO: 125) (wherein X2 is A, E, H, K, Q or S, preferably S; 11 is W; preferably, X2 is S and X 11 is W); (8)HX2NFGNSYVSWFX 12 Y (SEQ ID NO: 126) (wherein X2 is A, E, H, K, Q or S, preferably S; X 12 is E, K or Q, preferably E or Q; preferably, X2 is S or Q, and X 12 is E or Q; preferably, X and X 12 are S / E, Q / Q or S / Q, respectively; or (9)HX2NFGNSYVSWFAX 13 (SEQ ID NO: 127) (wherein X2 is A, E, H, K, Q or S, preferably A; X 13 is H, L, M or S, preferably S; preferably, X2 is A and X 13 is S) Includes.

[0025] In certain embodiments, the HCDR3 comprises a sequence set forth in any one of SEQ ID NOs: 50, 69, 49, 58, 59, 60, 70, 82, 86, 71, 68, 77, 78, 81, 62, 84, 89, 74.

[0026] In certain embodiments, HCDR3 is (1) HX2NFX5NSYVSWFAY (SEQ ID NO: 121) (wherein X2 is A, E, H, K, Q or S, preferably A or S; X5 is K, L, P, Q, R, S, V, W or Y, preferably L, P, S, Q, V or R; preferably, X2 is A or S and X5 is L, P, S, Q, V or R; preferably, X2 and X5 are A / L, S / P, S / L, A / S, S / Q, S / V or S / R, respectively); (2) HGNFX5X6SYVSWFAY (SEQ ID NO: 129) (wherein X5 is K, L, P, Q, R, S, V, W or Y, preferably P or K; X6 is M, Q or R, preferably M or Q; preferably, X5 is P or K and X6 is M or Q; preferably, X5 and X6 are P / M or K / Q, respectively); (3) HGNFX5NSYX9SWFAY (SEQ ID NO: 130), wherein X5 is K, L, P, Q, R, S, V, W or Y, preferably K or Q; X9 is G or I; preferably, X5 is K or Q and X9 is G or I; preferably, X5 and X9 are K / G or Q / I, respectively; (4)HGNFX5NSYVSWFX 12 X5 is K, L, P, Q, R, S, V, W or Y, preferably Q or L; 12 is E, K or Q, preferably Q; preferably, X5 is Q or L, and X 12 is Q); or (5)HGNFX5NSYVSWFAX 13(SEQ ID NO: 132) (wherein X5 is K, L, P, Q, R, S, V, W or Y, preferably Y or W; X 13 is H, L, M or S, preferably H or S; preferably, X5 is Y or W, and X 13 is H or S; preferably, X5 and X 13 are Y / H or W / S, respectively) Includes.

[0027] In certain embodiments, the HCDR3 comprises a sequence set forth in any one of SEQ ID NOs: 49, 58, 59, 60, 70, 82, 86, 76, 80, 61, 88, 52, 83, 54, 65.

[0028] In certain embodiments, HCDR3 is (1)X1GNFGNSYVSWFX 12 Y (SEQ ID NO: 119) (wherein X1 is A or P, preferably A; X 12 is E, K or Q, preferably Q; preferably, X1 is A and X 12 is Q or E); (2)HX2NFGNSYVSWFX 12 Y (SEQ ID NO: 126) (wherein X2 is A, E, H, K, Q or S, preferably S; X 12 is E, K or Q, preferably E or Q; preferably, X2 is S and X 12 is E or Q; preferably, X and X 12 are S / E, Q / Q or S / Q, respectively; (3)HGNFX5NSYVSWFX 12 X5 is K, L, P, Q, R, S, V, W or Y, preferably Q or L; 12 is E, K or Q, preferably Q; preferably, X5 is Q or L, and X 12 is Q); or (4)HGNFGNSX8VSWFX 12Y (SEQ ID NO: 134) (wherein X8 is A, Q or R, preferably A or Q; X 12 is E, K or Q, preferably Q; preferably, X8 is A or Q, and X 12 is Q) Includes.

[0029] In certain embodiments, the HCDR3 comprises a sequence set forth in any one of SEQ ID NOs: 55, 85, 62, 84, 89, 52, 83, 67, 73.

[0030] In certain embodiments, HCDR3 is (1)HX2NFGNSYVSWFAX 13 (SEQ ID NO: 127) (wherein X2 is A, E, H, K, Q or S, preferably A; X 13 is H, L, M or S, preferably S; preferably, X2 is A and X 13 is S); (2)HGNFX5NSYVSWFAX 13 (SEQ ID NO: 132) (wherein X5 is K, L, P, Q, R, S, V, W or Y, preferably Y or W; X 13 is H, L, M or S, preferably H or S; preferably, X5 is Y or W, and X 13 is H or S; preferably, X5 and X 13 are Y / H or W / S, respectively; or (3)HGNFGNX7YVSWFAX 13 (SEQ ID NO: 133) (wherein X7 is G, N or T, preferably T or N; X 13 is H, L, M or S, preferably M or S; preferably, X7 is T or N, and X 13 is M or S; preferably, X7 and X 13 are T / M or N / S, respectively) Includes.

[0031] In certain embodiments, the HCDR3 comprises a sequence set forth in any one of SEQ ID NOs: 74, 54, 65, 56, and 79.

[0032] In certain embodiments, HCDR3 is (1) HX2X3FGNSYVSWFAY (SEQ ID NO: 120) (wherein X2 is A, E, H, K, Q or S, preferably E; X3 is D or R, preferably R; preferably, X2 is E and X3 is R); or (2) HGX3X4GNSYVSWFAY (SEQ ID NO: 128) (wherein X3 is D or R, preferably D; X4 is P; preferably, X3 is D and X4 is P) Includes.

[0033] In certain embodiments, the HCDR3 comprises a sequence as set forth in any one of SEQ ID NOs: 69, 63.

[0034] In certain embodiments, HCDR3 is X1GNFX5NSYVSWFAX 13 (SEQ ID NO: 135) (wherein X1 is A or P, preferably P; X5 is K, L, P, Q, R, S, V, W or Y, preferably P; X 13 is H, L, M or S, preferably L; preferably, X1 is P, X5 is P, and X 13 is L).

[0035] In certain embodiments, the HCDR3 comprises the sequence set forth in SEQ ID NO:87.

[0036] In certain embodiments, the VH comprises an HCDR1 comprising the sequence set forth in SEQ ID NO: 107, an HCDR2 comprising the sequence set forth in SEQ ID NO: 108, and an HCDR3 comprising the sequence set forth in any one of SEQ ID NOs: 49 to 89; the VL comprises an LCDR1 comprising the sequence set forth in SEQ ID NO: 137, an LCDR2 comprising the sequence set forth in SEQ ID NO: 138, and an LCDR3 comprising the sequence set forth in SEQ ID NO: 139. Preferably, with the proviso that the HCDR3 is not represented by any one of SEQ ID NOs: 60, 64, 77, and 89.

[0037] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a framework region derived from a human immunoglobulin.

[0038] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a framework region contained in an amino acid sequence encoded by a gene derived from the human germline, In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region contained in an amino acid sequence encoded by a gene derived from the human heavy chain germline, and / or a light chain framework region contained in an amino acid sequence encoded by a gene derived from the human light chain germline.

[0039] In certain embodiments, the VH comprises HFR1, HFR2, HFR3, and HFR4, HFR1 comprises the sequence set forth in SEQ ID NO: 109; HFR2 comprises the sequence set forth in SEQ ID NO: 110; HFR3 comprises the sequence set forth in VKX1RFTISRDDSKSX2LYLQMNX3LKTEDTAX4YYCVR (SEQ ID NO: 136), where X1 is G or D, X2 is I or S, X3 is N or S, and X4 is M or V; HFR4 comprises the sequence set forth in SEQ ID NO:111.

[0040] In certain embodiments, HFR3 comprises a sequence set forth in any one of SEQ ID NOs: 91-106.

[0041] In certain embodiments, the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-46, or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0042] In certain embodiments, the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 8, 13, 22, 33, 37, 43, or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0043] In certain embodiments, the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3, 5, 9, 16, 20, 25, 28, or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0044] In certain embodiments, the VH comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 7, 18, 40, or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0045] In certain embodiments, the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2, 22, 1, 10, 11, 12, 23, 36, 37, 41, 24, 21, 30, 32, 35, 14, 31, 39, 45, 27, 46, or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0046] In certain embodiments, the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1, 10, 11, 12, 23, 36, 37, 41, 29, 34, 13, 44, 4, 38, 42, 6, 17, or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0047] In certain embodiments, the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7, 40, 14, 31, 39, 45, 4, 38, 42, 19, 26, or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0048] In certain embodiments, the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 27, 46, 6, 17, 8, 33, or a variant thereof, which has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0049] In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 22 or 15, or a variant thereof, where the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0050] In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:43 or a variant thereof, where the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0051] In certain embodiments, the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-11, 13-15, 17-19, 21-29, 31-35, 37-44, 46, or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0052] In certain embodiments, the VL comprises LFR1, LFR2, LFR3 and LFR4, LFR1 comprises the sequence set forth in SEQ ID NO: 140; LFR2 comprises the sequence set forth in WX1QQTPGQAX2RX3LIX4 (SEQ ID NO: 144), where X1 is V or Y, X2 is F or P, X3 is G or T, and X4 is G or Y; LFR3 comprises the sequence set forth in GVPARFSGSX4X5GX6KAALTITGAQADDESX7YFCA (SEQ ID NO: 147), where X4 is L or I, X5 is L or I, X6 is D or N, and X7 is I or D; LFR4 comprises the sequence shown in SEQ ID NO:141.

[0053] In certain embodiments, LFR2 comprises the sequence set forth in SEQ ID NO:142 or 143.

[0054] In certain embodiments, LFR3 comprises the sequence set forth in SEQ ID NO:145 or 146.

[0055] In certain embodiments, the VL comprises an LFR1 set forth in SEQ ID NO:140, an LFR2 set forth in SEQ ID NO:142, an LFR3 set forth in SEQ ID NO:145, and an LFR4 set forth in SEQ ID NO:141.

[0056] In certain embodiments, the VL comprises an LFR1 set forth in SEQ ID NO:140, an LFR2 set forth in SEQ ID NO:143, an LFR3 set forth in SEQ ID NO:146, and an LFR4 set forth in SEQ ID NO:141.

[0057] In certain embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 47 or 48, or a variant thereof, where the variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0058] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in any one of SEQ ID NOs: 1-46 or a variant thereof, and a VL comprising the sequence set forth in SEQ ID NO: 47 or a variant thereof; a variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0059] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in any one of SEQ ID NOs: 1-46 or a variant thereof, and a VL comprising the sequence set forth in SEQ ID NO: 48 or a variant thereof; a variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0060] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising a sequence set forth in any one of SEQ ID NOs: 1-11, 13-15, 17-19, 21-29, 31-35, 37-44, 46, or a variant thereof, and a VL comprising a sequence set forth in SEQ ID NO: 47, or a variant thereof; a variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0061] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising a sequence set forth in any one of SEQ ID NOs: 1-11, 13-15, 17-19, 21-29, 31-35, 37-44, 46, or a variant thereof, and a VL comprising a sequence set forth in SEQ ID NO: 48, or a variant thereof; a variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0062] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in any one of SEQ ID NOs: 8, 13, 22, 33, 37, 43 or a variant thereof, and a VL comprising the sequence set forth in SEQ ID NO: 47 or a variant thereof; a variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0063] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in any one of SEQ ID NOs: 8, 13, 22, 33, 37, 43 or a variant thereof, and a VL comprising the sequence set forth in SEQ ID NO: 48 or a variant thereof; a variant has one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0064] In certain embodiments, the VH comprises HCDR1-HCDR3 and HFR1-HFR4, which have the following features: Item 1: HCDR1 comprises the sequence set forth in SEQ ID NO: 107; Item 2: HCDR2 comprises the sequence set forth in SEQ ID NO: 108; Item 3: HCDR3 is (3-1) A sequence shown in X1X2NFGNSYVSWFAY (SEQ ID NO: 117) (wherein X1 is A or P, preferably A; X2 is A, E, H, K, Q or S, preferably K; preferably, X1 is A and X2 is K); (3-2) HX2X3FGNSYVSWFAY (SEQ ID NO: 120) (wherein X2 is A, E, H, K, Q or S, preferably E; X3 is D or R, preferably R; preferably, X2 is E and X3 is R); (3-3) HX2NFX5NSYVSWFAY (SEQ ID NO: 121) (wherein X2 is A, E, H, K, Q or S, preferably A or S; X5 is K, L, P, Q, R, S, V, W or Y, preferably L, P, S, Q, V or R; preferably, X2 is A or S and X5 is L, P, S, Q, V or R; preferably, X2 and X5 are A / L, S / P, S / L, A / S, S / Q, S / V or S / R, respectively); (3-4) the sequence shown in HX2NFGX6SYVSWFAY (SEQ ID NO: 122) (wherein X2 is A, E, H, K, Q or S, preferably S; X6 is M, Q or R, preferably R; preferably, X2 is S and X6 is R); (3-5) the sequence shown in HX2NFGNX7YVSWFAY (SEQ ID NO: 123) (wherein X2 is A, E, H, K, Q or S, preferably S; X7 is G, N or T, preferably T or G; preferably, X2 is S and X7 is T or G); (3-6) the sequence shown in HX2NFGNSX8VSWFAY (SEQ ID NO: 124) (wherein X2 is A, E, H, K, Q or S, preferably S; X8 is A, Q or R, preferably R; preferably, X2 is S and X8 is R); (3-7)HX2NFGNSYVSWX 11 AY (SEQ ID NO: 125) (wherein X2 is A, E, H, K, Q or S, preferably S; 11 is W; preferably, X2 is S and X 11 is W(X 11 for W)) the sequence shown; (3-8)HX2NFGNSYVSWFX 12Y (SEQ ID NO: 126) (wherein X2 is A, E, H, K, Q or S, preferably S; X 12 is E, K or Q, preferably E or Q; preferably, X2 is S and X 12 is E or Q; preferably, X and X 12 are S / E, Q / Q or S / Q, respectively; and (3-9)HX2NFGNSYVSWFAX 13 (SEQ ID NO: 127) (wherein X2 is A, E, H, K, Q or S, preferably A; X 13 is H, L, M or S, preferably S; preferably, X2 is A and X 13 is S) comprising a sequence selected from the group consisting of: Item 4: HFR1 comprises the sequence set forth in SEQ ID NO: 109; Item 5: HFR2 comprises the sequence set forth in SEQ ID NO: 110; Item 6: HFR3 comprises the sequence set forth in VKX1RFTISRDDSKSX2LYLQMNX3LKTEDTAX4YYCVR (SEQ ID NO: 136), where X1 is G or D, X2 is I or S, X3 is N or S, and X4 is M or V; Item 7: HFR4 comprises the sequence set forth in SEQ ID NO: 111; Preferably, the VL has the following features: item 8: the VL comprises the sequence shown in SEQ ID NO: 47; or item 9: the VL comprises the sequence shown in SEQ ID NO: 48.

[0065] In some embodiments, the VH has the following characteristics: item 1, item 2, item 3, item 4, item 5, item 6', item 7, where item 6': HFR3 comprises a sequence shown in any one of SEQ ID NOs: 91 to 106. The VL preferably has the following characteristics: item 8 or item 9.

[0066] In some embodiments, the VH has the following features: item 1, item 2, item 3', item 4, item 5, item 6, item 7, where item 3': HCDR3 comprises a sequence set forth in any one of SEQ ID NOs: 50, 69, 49, 58, 59, 60, 70, 82, 86, 71, 68, 77, 78, 81, 62, 84, 89, and 74. The VL preferably has the following features: item 8 or item 9.

[0067] In some embodiments, the VH has the following features: item 1, item 2, item 3', item 4, item 5, item 6', and item 7. The VL preferably has the following features: item 8 or item 9.

[0068] In certain embodiments, the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2, 22, 1, 10, 11, 12, 23, 36, 37, 41, 24, 21, 30, 32, 35, 14, 31, 39, 45, 27, and 46. The VL preferably comprises a sequence set forth in SEQ ID NO: 47 or 48, e.g., the sequence set forth in SEQ ID NO: 47.

[0069] In certain embodiments, the VH comprises HCDR1-HCDR3 and HFR1-HFR4, which have the following features: Item 11: HCDR1 comprises the sequence set forth in SEQ ID NO: 107; Item 12: HCDR2 comprises the sequence set forth in SEQ ID NO: 108; Item 13: HCDR3 is (13-1) HX2NFX5NSYVSWFAY (SEQ ID NO: 121) (wherein X2 is A, E, H, K, Q or S, preferably A or S; X5 is K, L, P, Q, R, S, V, W or Y, preferably L, P, S, Q, V or R; preferably, X2 is A or S and X5 is L, P, S, Q, V or R; preferably, X2 and X5 are A / L, S / P, S / L, A / S, S / Q, S / V or S / R, respectively); (13-2) HGNFX5X6SYVSWFAY (SEQ ID NO: 129) (wherein X5 is K, L, P, Q, R, S, V, W or Y, preferably P or K; X6 is M, Q or R, preferably M or Q; preferably, X5 is P or K and X6 is M or Q; preferably, X5 and X6 are P / M or K / Q, respectively); (13-3) HGNFX5NSYX9SWFAY (SEQ ID NO: 130) (wherein X5 is K, L, P, Q, R, S, V, W or Y, preferably K or Q; X9 is G or I; preferably, X5 is K or Q and X9 is G or I; preferably, X5 and X9 are K / G or Q / I, respectively); (13-4)HGNFX5NSYVSWFX 12 X5 is K, L, P, Q, R, S, V, W or Y, preferably Q or L; 12 is E, K or Q, preferably Q; preferably, X5 is Q or L, and X 12 is Q); and (13-5)HGNFX5NSYVSWFAX 13 (SEQ ID NO: 132) (wherein X5 is K, L, P, Q, R, S, V, W or Y, preferably Y or W; X 13 is H, L, M or S, preferably H or S; preferably, X5 is Y or W, and X 13 is H or S; preferably, X5 and X 13 are Y / H or W / S, respectively) comprising a sequence selected from the group consisting of: Item 14: HFR1 comprises the sequence set forth in SEQ ID NO: 109; Item 15: HFR2 comprises the sequence set forth in SEQ ID NO: 110; Item 16: HFR3 comprises the sequence set forth in VKX1RFTISRDDSKSX2LYLQMNX3LKTEDTAX4YYCVR (SEQ ID NO: 136), where X1 is G or D, X2 is I or S, X3 is N or S, and X4 is M or V; Item 17: HFR4 comprises the sequence set forth in SEQ ID NO: 111; Preferably, the VL has the following features: item 18: the VL comprises the sequence shown in SEQ ID NO: 47; or item 19: the VL comprises the sequence shown in SEQ ID NO: 48.

[0070] In some embodiments, the VH has the following features: item 11, item 12, item 13, item 14, item 15, item 16', item 17, where item 16': HFR3 comprises a sequence set forth in any one of SEQ ID NOs: 91 to 106. The VL preferably has the following features: item 18 or item 19.

[0071] In some embodiments, the VH has the following features: item 11, item 12, item 13', item 14, item 15, item 16, item 17, where item 13': HCDR3 comprises a sequence set forth in any one of SEQ ID NOs: 49, 58, 59, 60, 70, 82, 86, 76, 80, 61, 88, 52, 83, 54, and 65. The VL preferably has the following features: item 18 or item 19.

[0072] In some embodiments, the VH has the following features: item 11, item 12, item 13', item 14, item 15, item 16', item 17. The VL preferably has the following features: item 18 or item 19.

[0073] In certain embodiments, the VH comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1, 10, 11, 12, 23, 36, 37, 41, 29, 34, 13, 44, 4, 38, 42, 6, 17. The VL preferably comprises a sequence set forth in SEQ ID NO: 47 or 48, e.g., the sequence set forth in SEQ ID NO: 47.

[0074] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin.

[0075] In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin. In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region.

[0076] In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from human immunoglobulin. In certain embodiments, the light chain constant region is a kappa light chain constant region. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as set forth in SEQ ID NO: 165.

[0077] In certain embodiments, the Fc domain contained in the antibody or its antigen-binding fragment is a native Fc region, which comprises the same amino acid sequence as that of the Fc region found in nature. The native Fc region can have effector function. Exemplary "effector functions" include binding to Fc receptor; C1q binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cell phagocytosis (ADCP); down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation, etc.

[0078] In certain embodiments, the Fc domain contained in an antibody or antigen-binding fragment thereof may also be a variant Fc region, which may comprise one or more (e.g., 1-10, e.g., 1-5) amino acid mutations or chemical modifications compared to the native Fc region to alter one or more of the following properties of the antibodies of the disclosure: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function or complement function, etc. The functional alteration may be made by substituting at least one amino acid residue in the native Fc region with a different residue or by chemical modification, for example, alteration (e.g., reduction or enhancement) of effector function may be made by changing the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q).

[0079] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a mutated or chemically modified Fc region having reduced or enhanced antibody-dependent cellular cytotoxicity (ADCC), reduced or enhanced antibody-dependent cellular phagocytosis (ADCP), and / or reduced or enhanced complement-dependent cytotoxicity (CDC) compared to the wild-type Fc region. Methods for obtaining the above-mentioned altered (e.g., reduced) effector function are known in the art, for example, one or more (e.g., 1, 2, 3, or 4) mutations selected from the following may be introduced into the heavy chain constant region: L234A, L235A, G237A, K322A according to EU numbering. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 164.

[0080] In certain embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a Fab', a (Fab')2, an Fv, a disulfide-linked Fv, an scFv, and a diabody.

[0081] In certain embodiments, the antibody is an IgG antibody, such as an IgG1, IgG2, IgG3 or IgG4 antibody.

[0082] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to the epsilon and / or gamma chains of CD3 (e.g., human and / or cynomolgus CD3).

[0083] In certain embodiments, the antibody or antigen-binding fragment thereof binds to CD3 (e.g., human and / or cynomolgus CD3, e.g., epsilon and / or gamma chains) with a binding affinity of 6×10 -10 M or more, 6.5×10 -10 M or more, 7×10 -10 M or more, 7.5×10 -10 M or more, 8×10 -10 M or more, 8.5×10 -10 M or more, 9×10 -10 M or more, 9.5×10 -10 M or more, 1×10 -9 M or more, 1.5×10 -9 M or more, 2×10 -9 M or more, 2.5×10 -9 M or more, 3×10 -9 M or more, 3.5×10 -9 M or more, 4×10 -9 M or more, 4.5×10 -9 M or more, 5×10 -9 M or more, 5.5×10 -9 M or more, 6×10 -9 M or more, 6.5×10 -9 M or more, 7×10 -9 M or more, 7.5×10 -9 M or more, 8×10 -9 M or more, 8.5×10 -9 M or more, 9×10 -9 M or more, 9.5×10 -9 M or more, 1×10 -8 M or more, 1.5×10 -8 M or more, or 2 x 10 -8 K over M D In certain embodiments, the antibody or antigen-binding fragment thereof binds to CD3 (e.g., human and / or cynomolgus CD3, e.g., ε and / or γ chains) at 10 -7 M or less, e.g., 9.5×10-8 M or less, 9×10 -8 M or less, 8.5×10 -8 M or less, 8×10 -8 M or less, 7.5×10 -8 M or less, 7×10 -8 M or less, 6.5×10 -8 M or less, 6×10 -8 M or less, 5.5×10 -8 M or less, 5×10 -8 M or less, 4.5×10 -8 M or less, 4×10 -8 M or less, 3.5×10 -8 M or less, 3×10 -8 M or less, 2.5×10 -8 M or less, or 2 x 10 -8 K below M D In certain embodiments, the antibody or antigen-binding fragment thereof binds to CD3 (e.g., human and / or cynomolgus CD3, e.g., ε and / or γ chains) at 10 -8 ~10 -10 K of M D Combine with.

[0084] In certain embodiments, the antibody or antigen-binding fragment thereof has an EC of 0.05 nM or greater, 0.1 nM or greater, 0.2 nM or greater, 0.3 nM or greater, 0.4 nM or greater, 0.5 nM or greater, 0.6 nM or greater, 0.7 nM or greater, 0.8 nM or greater, 0.9 nM or greater, 1 nM or greater, 1.5 nM or greater, 2 nM or greater, 2.5 nM or greater, 3 nM or greater, 3.5 nM or greater, 4 nM or greater, 4.5 nM or greater, 5 nM or greater, 5.5 nM or greater, 6 nM or greater, 6.5 nM or greater, 7 nM or greater, 7.5 nM or greater, 8 nM or greater, 8.5 nM or greater, 9 nM or greater, as measured by flow cytometry. 50In certain embodiments, the antibody or antigen-binding fragment thereof binds to CD3 (e.g., human and / or cynomolgus monkey CD3, e.g., cells expressing CD3) with an EC of 20 nM or less, e.g., 15 nM or less, 10 nM or less, 9.5 nM or less, 9 nM or less, 8.5 nM or less, 8 nM or less, 7.5 nM or less, 7 nM or less, 6.5 nM or less, 6 nM or less, 5.5 nM or less, 5 nM or less, 4.5 nM or less, 4 nM or less, 3.5 nM or less, 3 nM or less, 2.5 nM or less, 2 nM or less, 1.5 nM or less, or 1 nM or less, as measured by flow cytometry. 50 Combine with.

[0085] MSLN single domain antibodies In a second aspect, the present disclosure relates to a single domain antibody or an antigen-binding fragment thereof that can specifically bind to MSLN (e.g., human MSLN). A single domain antibody is typically composed of four framework regions (FR) and three complementarity determining regions (CDRs), called FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, and the antigen-binding fragment thereof comprises at least a portion of a single domain antibody, which portion is sufficient to confer the ability to specifically bind to an antigen (e.g., MSLN). A single domain antibody may be truncated at the N- or C-terminus to comprise only a portion of FR1 and / or FR4, or to lack one or two of its framework regions, so long as it substantially retains the ability to specifically bind to an antigen.

[0086] In certain embodiments, the single domain antibody or antigen-binding fragment thereof of the present disclosure comprises the following CDRs as defined by the IMGT numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 150 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 151 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 152 or a variant thereof, the variant having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of one, two or three amino acids) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions. In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the IMGT numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 150, CDR2 comprising the sequence set forth in SEQ ID NO: 151, and CDR3 comprising the sequence set forth in SEQ ID NO: 152.

[0087] In certain embodiments, the single domain antibody or antigen-binding fragment thereof of the present disclosure comprises the following CDRs as defined by the Kabat numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 153 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 154 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 155 or a variant thereof, wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions. In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the Kabat numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 153, CDR2 comprising the sequence set forth in SEQ ID NO: 154, and CDR3 comprising the sequence set forth in SEQ ID NO: 155.

[0088] In certain embodiments, the single domain antibody or antigen-binding fragment thereof of the present disclosure comprises the following CDRs as defined by the AbM numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 156 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 157 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 155 or a variant thereof, wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions. In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the AbM numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 156, CDR2 comprising the sequence set forth in SEQ ID NO: 157, and CDR3 comprising the sequence set forth in SEQ ID NO: 155.

[0089] In certain embodiments, the single domain antibody or antigen-binding fragment thereof of the present disclosure comprises the following CDRs as defined by the Chothia numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 158 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 159 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 155 or a variant thereof, wherein the variant has one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of one, two or three amino acids) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions. In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the Chothia numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 158, CDR2 comprising the sequence set forth in SEQ ID NO: 159, and CDR3 comprising the sequence set forth in SEQ ID NO: 155.

[0090] In certain embodiments, the single domain antibody or antigen-binding fragment thereof of the present disclosure comprises the following CDRs as defined by the Contact numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 160 or a variant thereof, CDR2 comprising the sequence set forth in SEQ ID NO: 161 or a variant thereof, and CDR3 comprising the sequence set forth in SEQ ID NO: 162 or a variant thereof, the variant having one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of one, two or three amino acids) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions. In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises the following CDRs as defined by the Contact numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 160, CDR2 comprising the sequence set forth in SEQ ID NO: 161, and CDR3 comprising the sequence set forth in SEQ ID NO: 162.

[0091] In certain embodiments, the single domain antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 149 or a variant thereof, the variant having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it is derived, or having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions). In certain embodiments, the substitutions are conservative substitutions.

[0092] In certain embodiments, the single domain antibody or antigen-binding fragment thereof of the present disclosure may be a humanized VHH, i.e., a VHH in which one or more framework regions have been substantially replaced with human framework regions. In certain embodiments, the single domain antibody or antigen-binding fragment thereof further comprises a heavy chain framework region of a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline antibody gene), the heavy chain framework region optionally comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations of human residues to camelid residues.

[0093] In certain embodiments, the single domain antibody or antigen-binding fragment thereof binds to MSLN (e.g., human MSLN) at least 10 -8 M or less, 9.5×10 -9 M or less, 9×10 -9 M, 8.5×10 -9 M or less, 8×10 -9 M or less, 7.5×10 -9 M or less, 7×10 -9 M or less, 6.5×10 -9 M or less, 6×10 -9 M or less, 5.5×10 -9 M or less, 5×10 -9 M or less, 4.5×10 -9 M or less, 4×10 -9 M or less, 3.5×10 -9 M or less, 3×10 -9 M or less, 2.5×10 -9 M or less, 2×10 -9 M or less, 1.5×10 -9 M or less, 10 -9 M or less, 9.5×10 -10 M or less, 9×10 -10 M or less, 8.5×10 -10 M or less, 8×10 -10 M or less, 7.5×10 -10 M or less, 7×10 -10 K below M D Combine with.

[0094] In certain embodiments, the single domain antibody or antigen-binding fragment thereof has an EC of less than about 100 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, or less for MSLN (e.g., human MSLN, e.g., cells expressing MSLN) as measured by flow cytometry. 50 Combine with.

[0095] multispecific antibody The antibody or antigen-binding fragment thereof of the first aspect of the disclosure and / or the single domain antibody or antigen-binding fragment thereof of the second aspect of the disclosure may be used to form a multispecific antibody. A multispecific antibody is an antibody that is capable of specifically binding to at least two (e.g., two, three, or four) different antigens and thereby capable of binding to at least two different binding sites and / or target molecules.

[0096] Thus, in a third aspect, the present disclosure relates to a multispecific antibody.

[0097] Multispecific antibody 1 The present disclosure provides a multispecific antibody 1 comprising a CD3-targeting antigen binding domain and at least one antigen binding domain targeting an additional antigen, wherein the CD3-targeting antigen binding domain is selected from the antibody of the first aspect or an antigen-binding fragment thereof, and the additional antigen is selected from a tumor-associated antigen (TAA) and / or an immune checkpoint molecule.

[0098] In certain embodiments, the multispecific antibody is a bispecific antibody comprising a CD3-targeting antigen-binding domain of the first aspect and an antigen-binding domain targeting a tumor-associated antigen. When the bispecific antibody comprises a first antigen-binding region (e.g., an antibody of the first aspect) that binds to a T cell-specific target (e.g., CD3) and a second antigen-binding region that binds to a tumor-specific target, the bispecific antibody can promote the targeting and recruitment of T cells to tumor cells by binding to CD3 present on T cells and a specific target antigen on tumor cells, thereby inducing the killing activity of tumor-specific (MHC-independent) cytotoxic T cells.

[0099] In certain embodiments, the multispecific antibody is a trispecific antibody comprising a CD3-targeting antigen-binding domain of the first aspect, an antigen-binding domain that targets a tumor-associated antigen, and an antigen-binding domain that targets an immune checkpoint molecule.

[0100] Those skilled in the art will understand that all structural forms of bispecific antibodies known in the art can be used in the present disclosure. In certain embodiments, the bispecific antibodies of the present disclosure can be: (i) a single antibody with two arms containing different antigen binding domains, (ii) a single chain antibody formed by linking two scFvs in tandem, for example through an additional peptide linker, which is specific for two different epitopes; (iii) a dual variable domain antibody (DVD-Ig™), in which each of the light and heavy chains contains two variable domains linked in tandem by a short peptide; (iv) a chemically linked bispecific (Fab')2 fragment; (v) a TandAb, which is a fusion of two single chain diabodies, each of which is specific for two different epitopes. (vi) flexibodies, which are a combination of scFvs and diabodies, resulting in multivalent molecules; (vii) so-called "dock-and-lock" molecules, which are based on the "polymerization and docking domain" in protein kinase A, which when applied to Fabs, results in trivalent bispecific binding proteins consisting of two identical Fab fragments connected to different Fab fragments; (viii) so-called scorpion molecules, which contain, for example, two scFvs fused to both ends of a human Fab arm; and (ix) diabodies.

[0101] In certain embodiments, the CD3-targeting antigen-binding domain is a full-length antibody, an Fv fragment, a Fab fragment, a F(ab')2 fragment, or an scFv. In certain embodiments, the additional antigen-binding domains included in the multispecific antibody (e.g., an antigen-binding domain targeting a tumor-associated antigen, an antigen-binding domain targeting an immune checkpoint molecule) are independently selected from the group consisting of a full-length antibody, an Fv fragment, a Fab fragment, a F(ab')2 fragment, a scFv, and a VHH.

[0102] In certain embodiments, the antigen binding domains contained in the multispecific antibody are linked by a peptide linker.In certain embodiments, the antigen binding domain that targets tumor-associated antigens or immune checkpoint molecules is linked to the N-terminus and / or C-terminus of the heavy chain of the CD3-targeting antigen binding domain and / or the N-terminus and / or C-terminus of the light chain of the CD3-targeting antigen binding domain, optionally via a linker.In certain embodiments, the CD3-targeting antigen binding domain comprises at least one heavy chain and at least one light chain, and the antigen binding domain that targets tumor-associated antigens or immune checkpoint molecules is linked to the heavy chain, or the CD3-targeting antigen binding domain comprises two identical heavy chains and two identical light chains, and the antigen binding domain that targets tumor-associated antigens or immune checkpoint molecules is linked to two heavy chains.

[0103] In certain embodiments, the tumor associated antigen is CD19, BCMA, EGFR, HER2, HER3, HER4, PSMA, EpCAM, EphA2, CD33, CD123, CD38, CLDN18, MSLN, TROP2, Mucin 1, AFP, CD79b, GUCY2C, LRRC15, gp100, STEAP1, ROR1, 5T4, CEA, DLL3, CD20, CD7, PRAME, CDH19, CDH17, GPA33, HLA-A2, CD34, FAP, GPRC5D, GPC3, B7-H3, CLL-1, CLDN6, Flt3, NY-ESO- 1, PSCA, NECTIN-4, ENPP3, IGFR-1, TSA1, Melan-A, MUC16 (CA125), MUC17, SSTR2, c-Met, B7-H6, CSPG4, CAIX, MCSP, BIRC5, BIRC7, BRCA1, BORIS, CCR5, GD2, GD3, GloboH, GM3, hTERT, LMP2, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, Ras, SART-3, TRP-1, TRP-2, CD22, CD30, FOLR1, and any combination thereof.

[0104] In certain preferred embodiments, the tumor-associated antigen is selected from the group consisting of MSLN, CD19, CD20, TROP2, HER2, and claudin 18.2.

[0105] In certain embodiments, the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, Lag-3, TIGIT, CD73, VISTA, B7-H3, NKG2D, NKG2A, OX40, OX40L, CD40, CD47, LIGHT, ICOS, HVEM, BTLA, B7-H4, 4-1BB, 4-1BBL, and any combination thereof.

[0106] In certain embodiments, the multispecific antibody comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising the heavy chain of the antibody of the first aspect and an antigen-binding domain capable of specifically binding to a tumor-associated antigen (e.g., MSLN, CD19, CD20, Trop2, Her2 or claudin 18.2), linked to the N- or C-terminus of the heavy chain; and the second polypeptide chain comprising the light chain of the antibody of the first aspect.

[0107] In certain embodiments, the multispecific antibody is a CD3 / MSLN-targeting bispecific antibody, which comprises an antigen-binding domain capable of specifically binding to CD3 and an antigen-binding domain capable of specifically binding to MSLN, wherein the CD3-targeting antigen-binding domain is selected from an antibody or antigen-binding fragment thereof described in the first aspect; and the MSLN-targeting antigen-binding domain is selected from a single-domain antibody or antigen-binding fragment thereof described in the second aspect.

[0108] In certain embodiments, the CD3 / MSLN-targeting bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain having the structure shown below: [VH]-[CH]-[L]-[VHH], where [VH] is selected from the heavy chain variable region sequences provided in the first aspect (e.g., a sequence set forth in any one of SEQ ID NOs: 1-46), [CH] is selected from the heavy chain constant region sequences provided in the first aspect (e.g., a sequence set forth in SEQ ID NO: 164), and [L] is a peptide linker (e.g., one or more glycines and / or one or more cyclic amino acids). or a peptide linker containing multiple serine, e.g., the sequence shown in SEQ ID NO: 163), and [VHH] is selected from the single domain antibody sequences provided in the second aspect (e.g., the sequence shown in SEQ ID NO: 149); the second polypeptide chain comprises the structure shown below: [VL]-[CL], where [VL] is selected from the light chain variable region sequences provided in the first aspect (e.g., the sequence shown in SEQ ID NO: 47 or 48), and [CL] is selected from the light chain constant region sequences provided in the first aspect (e.g., the sequence shown in SEQ ID NO: 165).

[0109] In certain embodiments, bispecific antibodies targeting CD3 and MSLN of the present disclosure induce 6×10 IL- ... -10 M or more, 6.5×10 -10 M or more, 7×10 -10 M or more, 7.5×10 -10 M or more, 8×10 -10 M or more, 8.5×10 -10 M or more, 9×10 -10 M or more, 9.5×10 -10 M or more, 1×10 -9 M or more, 1.5×10 -9 M or more, 2×10 -9 M or more, 2.5×10 -9 M or more, 3×10 -9 M or more, 3.5×10 -9 M or more, 4×10 -9 M or more, 4.5×10-9 M or more, 5×10 -9 M or more, 5.5×10 -9 M or more, 6×10 -9 M or more, 6.5×10 -9 M or more, 7×10 -9 M or more, 7.5×10 -9 M or more, 8×10 -9 M or more, 8.5×10 -9 M or more, 9×10 -9 M or more, 9.5×10 -9 M or more, 1×10 -8 M or more, 1.5×10 -8 M or more, or 2 x 10 -8 K over M D In certain embodiments, bispecific antibodies targeting CD3 and MSLN of the present disclosure bind to CD3 (e.g., human and / or cynomolgus CD3, e.g., ε and / or γ chains) at 10 -7 M or less, e.g., 9.5×10 -8 M or less, 9×10 -8 M or less, 8.5×10 -8 M or less, 8×10 -8 M or less, 7.5×10 -8 M or less, 7×10 -8 M or less, 6.5×10 -8 M or less, 6×10 -8 M or less, 5.5×10 -8 M or less, 5×10 -8 M or less, 4.5×10 -8 M or less, 4×10 -8 M or less, 3.5×10 -8 M or less, 3×10 -8 M or less, 2.5×10 -8 M or less, or 2 x 10 -8 K below M D In certain embodiments, the bispecific antibody binds to CD3 (e.g., human and / or cynomolgus CD3, e.g., ε and / or γ chains) at 10 -8 ~10 -10 K of M D Combine with.

[0110] In certain embodiments, bispecific antibodies targeting CD3 and MSLN of the present disclosure have an EC of 0.05 nM or more, 0.1 nM or more, 0.2 nM or more, 0.3 nM or more, 0.4 nM or more, 0.5 nM or more, 0.6 nM or more, 0.7 nM or more, 0.8 nM or more, 0.9 nM or more, 1 nM or more, 1.5 nM or more, 2 nM or more, 2.5 nM or more, 3 nM or more, 3.5 nM or more, 4 nM or more, 4.5 nM or more, 5 nM or more, 5.5 nM or more, 6 nM or more, 6.5 nM or more, 7 nM or more, 7.5 nM or more, 8 nM or more, 8.5 nM or more, 9 nM or more on CD3 (e.g., human and / or cynomolgus monkey CD3, e.g., CD3 expressing cells) as measured by flow cytometry. 50 In certain embodiments, bispecific antibodies targeting CD3 and MSLN of the present disclosure bind to CD3 (e.g., human and / or cynomolgus monkey CD3, e.g., CD3 expressing cells) with an EC of 20 nM or less, e.g., 15 nM or less, 10 nM or less, 9.5 nM or less, 9 nM or less, 8.5 nM or less, 8 nM or less, 7.5 nM or less, 7 nM or less, 6.5 nM or less, 6 nM or less, 5.5 nM or less, 5 nM or less, 4.5 nM or less, 4 nM or less, 3.5 nM or less, 3 nM or less, 2.5 nM or less, 2 nM or less, 1.5 nM or less, or 1 nM or less, as measured by flow cytometry. 50 Combine with.

[0111] In certain embodiments, bispecific antibodies targeting CD3 and MSLN of the present disclosure induce 10-fold increase in MSLN (e.g., human MSLN) as measured by SPR. -8 M or less, 9.5×10 -9 M or less, 9×10 -9 M, 8.5×10 -9 M or less, 8×10 -9 M or less, 7.5×10 -9 M or less, 7×10 -9 M or less, 6.5×10 -9 M or less, 6×10 -9 M or less, 5.5×10 -9 M or less, 5×10 -9 M or less, 4.5×10 -9 M or less, 4×10 -9 M or less, 3.5×10-9 M or less, 3×10 -9 M or less, 2.5×10 -9 M or less, 2×10 -9 M or less, 1.5×10 -9 M or less, 10 -9 M or less, 9.5×10 -10 M or less, 9×10 -10 M or less, 8.5×10 -10 M or less, 8×10 -10 M or less, 7.5×10 -10 M or less, 7×10 -10 K below M D Combine with.

[0112] In certain embodiments, bispecific antibodies targeting CD3 and MSLN of the present disclosure have an EC50 of less than about 100 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, or less on MSLN (e.g., human MSLN, e.g., cells expressing MSLN) as measured by flow cytometry. 50 Combine with.

[0113] In certain embodiments, the multispecific antibody is a CD3 / CD19-targeting bispecific antibody, which comprises an antigen-binding domain capable of specifically binding to CD3 and an antigen-binding domain capable of specifically binding to CD19, wherein the CD3-targeting antigen-binding domain is selected from an antibody or antigen-binding fragment thereof described in the first aspect; the CD19-targeting antigen-binding domain comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 171 or a variant thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 170 or a variant thereof.

[0114] In certain embodiments, the multispecific antibody is a CD3 / CD20 targeted bispecific antibody, which comprises an antigen binding domain that targets CD3 and an antigen binding domain capable of specifically binding to CD20, wherein the CD3 targeted antigen binding domain is selected from an antibody or antigen binding fragment thereof described in the first aspect; the CD20 targeted antigen binding domain comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 173 or a variant thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 172 or a variant thereof.

[0115] In certain embodiments, the multispecific antibody is a CD3 / trop2-targeting bispecific antibody, which comprises an antigen-binding domain that targets CD3 and an antigen-binding domain capable of specifically binding to trop2, wherein the CD3-targeting antigen-binding domain is selected from an antibody or antigen-binding fragment thereof described in the first aspect; and the trop2-targeting antigen-binding domain is a VHH, which comprises the sequence set forth in SEQ ID NO: 174 or a variant thereof.

[0116] In certain embodiments, the multispecific antibody is a CD3 / Her2-targeting bispecific antibody, which comprises an antigen-binding domain that targets CD3 and an antigen-binding domain capable of specifically binding to Her2, wherein the antigen-binding domain that targets CD3 is selected from an antibody or antigen-binding fragment thereof described in the first aspect; and the antigen-binding domain that targets Her2 is a VHH, which comprises the sequence set forth in SEQ ID NO: 175 or a variant thereof.

[0117] In certain embodiments, the multispecific antibody is a CD3 / claudin18.2-targeting bispecific antibody, which comprises an antigen-binding domain that targets CD3 and an antigen-binding domain capable of specifically binding to claudin18.2, wherein the CD3-targeting antigen-binding domain is selected from an antibody or antigen-binding fragment thereof described in the first aspect; and the claudin18.2-targeting antigen-binding domain is a VHH, which comprises the sequence set forth in SEQ ID NO: 176 or a variant thereof.

[0118] Multispecific antibody 2 The present disclosure also provides a multispecific antibody 2 comprising a single domain antibody or antigen-binding fragment thereof as described in the second aspect; in certain preferred embodiments the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

[0119] Preparation of antibodies The antibody of the first embodiment of the present disclosure, the single domain antibody of the second embodiment, or the multispecific antibody of the third embodiment of the present disclosure can be prepared by various methods known in the art, for example, by genetic engineering and recombinant technology.For example, the DNA molecules encoding them can be obtained by chemical synthesis or PCR amplification.The obtained DNA molecules are inserted into an expression vector and then transfected into a host cell.The transfected host cell is then cultured under defined conditions to express the antibody, single domain antibody, or multispecific antibody of the present disclosure.

[0120] In a fourth aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: - an antibody or antigen-binding fragment thereof according to the first aspect, or a heavy chain variable region and / or a light chain variable region thereof; - a single domain antibody or antigen-binding fragment thereof according to the second aspect; or - a multispecific antibody 1 or a multispecific antibody 2, or a polypeptide chain thereof, according to the third aspect The present invention provides an isolated nucleic acid molecule encoding

[0121] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the first aspect, and a second nucleotide sequence encoding a light chain or light chain variable region of an antibody or antigen-binding fragment thereof of the first aspect, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules.

[0122] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a first polypeptide chain of the bispecific antibody of the third aspect and a second nucleotide sequence encoding a second polypeptide chain of the bispecific antibody of the third aspect, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different isolated nucleic acid molecules.

[0123] In a fifth aspect, the present disclosure provides a vector comprising the nucleic acid molecule described above. In certain embodiments, the vector is a cloning vector or an expression vector.

[0124] In a sixth aspect, the present disclosure provides a host cell comprising the nucleic acid molecule or vector described above.Such host cells include, but are not limited to, prokaryotic cells, such as bacterial cells (e.g., E.coli cells), and eukaryotic cells, such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0125] In another aspect, the present disclosure provides a method for preparing an antibody or antigen-binding fragment thereof according to the first aspect, a single domain antibody according to the second aspect, or a multispecific antibody according to the third aspect, comprising culturing a host cell as described above under conditions allowing protein expression, and harvesting the antibody or antigen-binding fragment thereof, single domain antibody, or multispecific antibody from the culture of the cultured host cells.

[0126] Pharmaceutical Compositions The antibodies (also referred to as active ingredients) disclosed herein can be incorporated into pharmaceutical compositions suitable for administration.

[0127] Accordingly, in a seventh aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first aspect, a single domain antibody or antigen-binding fragment thereof according to the second aspect, a multispecific antibody according to the third aspect, an isolated nucleic acid molecule according to the fourth aspect, a vector according to the fifth aspect, or a host cell according to the sixth aspect, and a pharma- ceutically acceptable carrier and / or excipient.

[0128] In certain embodiments, the pharmaceutical composition may further comprise an additional pharma- ceutical active agent, e.g., an anti-neoplastic agent. In certain embodiments, the additional pharma- ceutical active agent and the antibody or antigen-binding fragment thereof, single domain antibody or antigen-binding fragment thereof, multispecific antibody, isolated nucleic acid molecule, vector, or host cell of the present disclosure are provided separately or as components of the same composition.

[0129] therapeutic use In an eighth aspect, the present disclosure relates to a method for preventing and / or treating a disease comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof, the isolated nucleic acid molecule, the vector or host cell encoding the antibody or antigen-binding fragment thereof, or a pharmaceutical composition containing same, according to the first aspect. The present disclosure also relates to the use of the antibody or antigen-binding fragment thereof, the isolated nucleic acid molecule, the vector or host cell encoding the antibody or antigen-binding fragment thereof, or a pharmaceutical composition containing same, according to the first aspect, for preventing and / or treating a disease or in the manufacture of a medicament for preventing and / or treating a disease.

[0130] The antibody or antigen-binding fragment thereof described in the first aspect of the present disclosure can be used in the treatment of any disease, so long as the effector mechanisms of cytotoxic T cells are required in the treatment of the disease.

[0131] In certain embodiments, the disease is a tumor, an inflammatory disease, or an autoimmune disease.

[0132] In certain embodiments, the tumor is a solid tumor or a hematological tumor, such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, colorectal cancer, squamous cell carcinoma of the head and neck, pancreatic cancer, testicular cancer, bile duct cancer, colon cancer, fallopian tube cancer, malignant melanoma, soft tissue cancer (e.g., synovial sarcoma), indolent or aggressive forms of B-cell lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, or acute lymphoblastic leukemia.

[0133] In certain embodiments, the treatment of a disease involves the effector mechanisms of T cells (e.g., cytotoxic T cells). In certain embodiments, antibodies or antigen-binding fragments thereof are used to enhance the ability of T cells (e.g., cytotoxic T cells) to treat or prevent a disease.

[0134] In a ninth aspect, the present disclosure relates to a method for preventing and / or treating a tumor comprising administering to a subject in need thereof a single domain antibody or antigen-binding fragment thereof, an isolated nucleic acid molecule, a vector or host cell encoding the antibody or antigen-binding fragment thereof, or a pharmaceutical composition containing same, according to the second aspect. The present disclosure also relates to the use of a single domain antibody or antigen-binding fragment thereof, an isolated nucleic acid molecule, a vector or host cell encoding the antibody or antigen-binding fragment thereof, or a pharmaceutical composition containing same, according to the second aspect for preventing and / or treating a tumor or in the manufacture of a medicament for preventing and / or treating a tumor.

[0135] In certain embodiments, the tumor is an MSLN-positive tumor.

[0136] In certain embodiments, the tumor is a solid tumor or a hematological tumor, such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, colorectal cancer, squamous cell carcinoma of the head and neck, pancreatic cancer, testicular cancer, bile duct cancer, colon cancer, fallopian tube cancer, malignant melanoma, soft tissue cancer (e.g., synovial sarcoma), indolent or aggressive forms of B-cell lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, or acute lymphoblastic leukemia.

[0137] In certain embodiments, the tumor is selected from a solid tumor, e.g., mesothelioma, ovarian cancer, pancreatic cancer, breast cancer, bile duct cancer, colon cancer, gastric cancer, fallopian tube cancer, lung cancer, or colorectal cancer.

[0138] In certain embodiments, the tumor is selected from a hematological tumor, e.g., acute myeloid leukemia.

[0139] In a tenth aspect, the present disclosure relates to a method for preventing and / or treating a disease comprising administering to a subject in need thereof a multispecific antibody, an isolated nucleic acid molecule, a vector or a host cell encoding the multispecific antibody, or a pharmaceutical composition containing same, according to the third aspect. The disclosure also relates to the use of a multispecific antibody, an isolated nucleic acid molecule, a vector or a host cell encoding the multispecific antibody, or a pharmaceutical composition containing same, according to the third aspect, for preventing and / or treating a disease or in the manufacture of a medicament for preventing and / or treating a disease.

[0140] The multispecific antibody described in the third embodiment of the present disclosure can be used in the treatment of any disease, as long as the effector mechanism of cytotoxic T cells is required in the treatment of the disease.For example, when the multispecific antibody of the present disclosure comprises a CD3 binding arm for T cell recruitment and a tumor targeting arm specific to tumor-associated antigen (TAA), it brings T cells into close contact with target tumor cells, locally activates T cells, and then destroys target cells by perforin and granzyme released from the toxic granules of T cells.For example, when the multispecific antibody of the present disclosure comprises a CD3 binding arm for T cell recruitment and a targeting arm specific to immune checkpoint molecules, it activates T cells in the tumor microenvironment, and then kills tumor cells.

[0141] In certain embodiments, the disease is a tumor, an inflammatory disease, or an autoimmune disease.

[0142] In certain embodiments, the tumor is a solid tumor or a hematological tumor, such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, colorectal cancer, squamous cell carcinoma of the head and neck, pancreatic cancer, testicular cancer, bile duct cancer, colon cancer, fallopian tube cancer, malignant melanoma, soft tissue cancer (e.g., synovial sarcoma), indolent or aggressive forms of B-cell lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, or acute lymphoblastic leukemia.

[0143] In certain embodiments, the treatment of a disease involves the effector mechanisms of T cells (e.g., cytotoxic T cells). In certain embodiments, multispecific antibodies are used to recruit T cells around target cells, activate the T cells, and induce T cell-dependent cytotoxicity (TDCC), thereby effectively killing the target cells to treat or prevent the disease.

[0144] In certain embodiments, the tumor is an MSLN-positive tumor, e.g., a solid tumor, e.g., mesothelioma, ovarian cancer, pancreatic cancer, breast cancer, bile duct cancer, colon cancer, gastric cancer, fallopian tube cancer, lung cancer, or colorectal cancer; e.g., a hematological tumor, e.g., acute myeloid leukemia.

[0145] In certain embodiments, the tumor is a CD19-positive tumor, e.g., a B-cell malignancy, e.g., a lymphoma or leukemia.

[0146] In certain embodiments, the tumor is a CD20 positive tumor, e.g., a B cell malignancy, e.g., a lymphoma or leukemia.

[0147] In certain embodiments, the tumor is a Trop2-positive tumor, e.g., a solid tumor, e.g., breast cancer.

[0148] In certain embodiments, the tumor is a Her2-positive tumor, e.g., a solid tumor, e.g., a lung cancer.

[0149] In certain embodiments, the tumor is a claudin 18.2-positive tumor, e.g., a solid tumor, e.g., gastric cancer.

[0150] In any one of the eighth to tenth aspects of the present disclosure, the antibody or antigen-binding fragment thereof, single domain antibody or antigen-binding fragment thereof, multispecific antibody, or pharmaceutical composition comprising same described in the present disclosure may be formulated into any dosage form known in the medical field.

[0151] In any one of the eighth to tenth aspects of the present disclosure, the antibody or antigen-binding fragment thereof, single domain antibody or antigen-binding fragment thereof, multispecific antibody, or pharmaceutical composition containing same described in the present disclosure can be administered by any suitable method known in the art.

[0152] In any one of the eighth to tenth aspects of the present disclosure, the antibody or antigen-binding fragment thereof, single domain antibody or antigen-binding fragment thereof, multispecific antibody, or pharmaceutical composition comprising same described in the present disclosure can be formulated into a unit dosage form for ease of administration.

[0153] In any one of the eighth to tenth aspects of the present disclosure, the antibody or antigen-binding fragment thereof, single domain antibody or antigen-binding fragment thereof, multispecific antibody, or pharmaceutical composition comprising same described in the present disclosure may be administered alone or in combination with an additional pharma- ceutical active agent (e.g., an anti-neoplastic agent) or an additional treatment (e.g., an anti-neoplastic treatment).

[0154] In any of the eighth to tenth aspects of the present disclosure, the subject may be a mammal, for example a human.

[0155] Definition of Terms In this disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the laboratory procedures of virology, biochemistry, and immunology used herein are routine procedures that are widely used in the corresponding fields. Meanwhile, in order to better understand this disclosure, the definitions and explanations of relevant terms are provided below.

[0156] When the terms "eg," "such as," "for example," "comprise," "contain," or variations thereof are used herein, these terms are not to be construed as limiting terms, but instead are to be interpreted as meaning "but not limited to" or "without limitation."

[0157] Unless otherwise indicated herein or clearly contradicted by context, in the context described in this disclosure (and particularly in the context of the claims which follow), "a" and "an," as well as "the," and similar referents, are to be construed to encompass the singular and plural.

[0158] As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, which can define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and heavy chains also contain a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in binding the antibody to the antigen, but exhibits various effector functions, such as mediating the interaction of the immunoglobulin with host tissues or factors, including binding various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into highly variable regions called complementarity determining regions (CDRs) interrupted by more conserved regions called framework regions (FRs). H and V LEach of the heavy and light chains consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair respectively form an antigen-binding site. The assignment of amino acids to regions or domains may be according to the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0159] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are involved in antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2 and CDR3. The exact boundaries of these CDRs can be determined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al. al., Dev. Comparat. Immunol. 27:55-77, 2003), the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989), Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), or according to the Contact numbering system (MacCallum, RM, Martin, ACR, & Thornton, JM (1996). Antibody-antigen Interactions: Contact Analysis and Binding Site Topography. Journal of Molecular Biology, 262(5), 732-745.). For a given antibody, one of skill in the art will readily identify the CDRs defined by each numbering system.Moreover, the correspondence between different numbering systems is well known to those of skill in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0160] In the present disclosure, the CDRs contained in the antibody or antigen-binding fragment thereof of the present disclosure can be determined according to various numbering systems known in the art, such as Kabat, Chothia, IMGT, AbM or Contact numbering systems.In certain embodiments, the anti-CD3 antibody or antigen-binding fragment thereof of the present disclosure contains the CDRs determined by Kabat numbering system.In certain embodiments, the anti-MSLN single domain antibody or antigen-binding fragment thereof of the present disclosure contains the CDRs determined by Kabat, Chothia, IMGT, AbM or Contact numbering system.

[0161] As used herein, the term "framework region" or "FR" residues refers to amino acid residues in an antibody variable region other than the CDR residues defined above.

[0162] The term "antibody" is not limited to any particular method of producing an antibody. It includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0163] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be obtained by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and such polypeptides that contain at least a portion of an antibody sufficient to confer specificity to the polypeptide with antigen-binding ability.

[0164] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Among them, the "full-length heavy chain" refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain from the N-terminus to the C-terminus, and if the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain, if necessary. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 from the N-terminus to the C-terminus. The "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) from the N-terminus to the C-terminus. The two pairs of full-length antibody chains are linked to each other by a disulfide bond between CL and CH1, and a disulfide bond between the HRs of the two full-length heavy chains. A full-length antibody of the present disclosure contains two antigen-binding sites, each formed by a VH and VL pair, which specifically recognize / bind to the same antigen.

[0165] As used herein, the term "single domain antibody (sdAb)" has the meaning normally understood by those skilled in the art and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), usually derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a nanobody is composed of four framework regions and three complementarity determining regions, with the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A single domain antibody can be truncated at the N- or C-terminus to contain only a portion of FR1 and / or FR4, or to lack one or two of its framework regions, so long as it substantially retains the ability to specifically bind to an antigen. Single domain antibodies are also called nanobodies, the two being used interchangeably. As used herein, the term "antigen-binding fragment" of a single domain antibody refers to a polypeptide comprising a fragment of a single domain antibody that retains the ability to specifically bind to the same antigen that the single domain antibody binds and / or competes with the single domain antibody for specific binding to the antigen. Antigen-binding fragments of the single domain antibodies of the present disclosure can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of the single domain antibodies of the present disclosure. In some embodiments, compared to a full-length single domain antibody, an "antigen-binding fragment" of a single domain antibody can be truncated at the N- or C-terminus to contain only a portion of FR1 and / or FR4 or to lack one or two framework regions, so long as it substantially retains the ability to specifically bind to an antigen.

[0166] As used herein, the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains, the term "F(ab')2 fragment" refers to an antibody fragment consisting of two Fab fragments connected by a disulfide bridge on the hinge region, and the term "Fab' fragment" refers to a fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab')2 fragment and consisting of an intact light chain and a heavy chain Fd fragment (consisting of the VH and CH1 domains).

[0167] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. In general, the six CDRs are considered to confer antigen-binding specificity to the antibody. However, further variable regions (e.g., Fd fragments containing only three antigen-specific CDRs) can recognize and bind antigen, but the affinity may be lower than that of the intact binding site.

[0168] As used herein, the term "Fc domain" or "Fc region" refers to a portion of the heavy chain constant region, including CH2 and CH3. The Fc fragment of an antibody has many different functions, but does not participate in antigen binding. The "effector functions" mediated by the Fc region include Fc receptor binding; C1q binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation. In some embodiments, the Fc region includes a hinge, a CH2, and a CH3. When the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype, for example, IgG1, IgG2, IgG3, or IgG4.

[0169] The Fc domain may comprise either a native Fc region or a variant Fc region. A native Fc region comprises an amino acid sequence that corresponds to the amino acid sequence of an Fc region found in nature, for example, a native sequence human Fc region comprises a native sequence human IgG1 Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; a native sequence human IgG4 Fc region, and naturally occurring variants thereof. A variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification. In some embodiments, a variant Fc region may have an altered effector function (e.g., Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function) compared to a native Fc region.

[0170] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, where the VL and the VH are connected via a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. A suitable linker in the prior art consists of a repeated GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)2 can be used, but variants thereof can also be used. In some cases, a disulfide bond may also be present between the VH and VL of the scFv.

[0171] As used herein, the term "diabody" refers to an antibody whose VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, which causes the domains to pair with complementary domains on another chain, resulting in two antigen-binding moieties (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0172] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen.

[0173] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the present disclosure) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques, or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of an antibody are screened for specificity in the same manner as intact antibodies.

[0174] As used herein, when the term "antibody" is mentioned, unless the context clearly dictates otherwise, this includes intact antibodies as well as antigen-binding fragments of antibodies.

[0175] As used herein, the term "germline antibody gene" or "germline antibody gene segment" refers to an immunoglobulin coding sequence present in the genome of an organism, which has not undergone the maturation process that results in gene rearrangements and mutations for the expression of a specific immunoglobulin. In the present disclosure, the expression "heavy chain germline gene" refers to a germline antibody gene or gene fragment that encodes an immunoglobulin heavy chain, which includes V genes (variable), D genes (diversity), J genes (joining), and C genes (constant); similarly, the expression "light chain germline gene" refers to a germline antibody gene or gene fragment that encodes an immunoglobulin light chain, which includes V genes (variable), J genes (joining), and C genes (constant). In the present disclosure, the amino acid sequence encoded by a germline antibody gene or germline antibody gene fragment is also referred to as a "germline sequence". Germline antibody genes or germline antibody gene fragments, and their corresponding germline sequences, are well known to those skilled in the art and can be obtained or queried from specialized databases (e.g., IMGT, UNSWIg, NCBI, or VBASE2). In general, germline antibody genes are considered to be more likely than mature antibody genes to retain important amino acid sequence structures unique to individuals within a species, and therefore less likely to be considered foreign when used therapeutically in that species.

[0176] As used herein, the term "identity" is used to refer to the sequence match between two polypeptides or two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into the first amino acid sequence or nucleic acid sequence for best alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are identical if the positions in the first sequence are occupied by the same amino acid residues or nucleotides at the corresponding positions in the second sequence. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical overlapping positions / total number of positions×100%). In certain embodiments, both sequences are sequences of the same length.

[0177] The determination of percent identity between two sequences can also be achieved using mathematical algorithms.A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, which is improved by Karlin and Altschul, 1993, Proc. Natl.. Acad. Sci. USA 90:5873-5877.Such an algorithm is integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0178] As used herein, the term "variant" in the context of a polypeptide (including a polypeptide) also refers to a polypeptide or peptide that contains an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not limited to, a polypeptide may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be generated by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Furthermore, a variant has a similar, identical, or improved function relative to the polypeptide or peptide from which it is derived. In certain embodiments, a variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived.

[0179] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. The strength or affinity of a specific binding interaction is determined by the equilibrium dissociation constant (K D In this disclosure, "K D The term "equilibrium constant" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0180] The specific binding properties between two molecules can be determined using methods known in the art. One approach involves measuring the rate at which the antigen-binding site / antigen complex forms and dissociates. Both the "association rate constant" (ka or k) and the "dissociation rate constant" (k or k) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of k / k is the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990; 59:439-473). D , k and k values ​​can be measured by any effective method. In certain embodiments, dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). Alternatively, dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0181] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. If the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements of the carrier can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages, such as lambda phages or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40). Vectors can contain a variety of expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may contain an origin of replication site.

[0182] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell, such as E. coli or Bacillus subtilis, a fungal cell, such as a yeast cell or Aspergillus, an insect cell, such as an S2 Drosophila cell or Sf9, or an animal cell, such as a fibroblast cell, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK 293 cell or a human cell.

[0183] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the expected properties of a protein / polypeptide that contains an amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue that has a similar side chain, for example, an amino acid residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, includes the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues that have similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. In addition, amino acid residues can be divided into categories defined by physical and functional properties as needed, e.g., alcohol-containing residues (S and T), aliphatic residues (I, L, V, and M), cycloalkenyl-related residues (F, H, W, and Y), hydrophobic residues (A, C, F, G, H, I, L, M, R, T, V, W, and Y), negatively charged residues (D and E), polar residues (C, D, E, H, K, N, Q, R, S, and T), positively charged residues (H, K, and R), small residues (A, C, D, G, N, P, S, T, and V), very small residues (A, G, and S), corner-forming residues (A, C, D, E, G, H, K, N, Q, R, S, P, and T), flexible residues (Q, T, K, S, G, P, D, E, and R).Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0184] The twenty conventional amino acids included herein are written according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this disclosure, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in this disclosure, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0185] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, diluents, osmotic pressure maintaining agents, absorption retarding agents, and preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween®-80. Ionic strength enhancing agents include, but are not limited to, sodium chloride. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and the like. Absorption retarding agents include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in pharmaceuticals, and include, but are not limited to, sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or their degradation products (e.g., lactalbumin hydrolysate), etc.

[0186] As used herein, the term "prevention" refers to a method performed to prevent or delay the appearance of a disease or condition or symptom in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical result. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of disease, stabilization (i.e., not worsening) of a disease state, whether detectable or undetectable, delaying or slowing the progression of disease, relieving or alleviating a disease state, and alleviation of symptoms (whether partial or complete). In addition, "treatment" can also refer to prolonging survival compared to expected survival in the absence of treatment.

[0187] As used herein, the term "subject" refers to a mammal, e.g., a primate mammal, e.g., a human. In certain embodiments, the subject (e.g., a human) has or is at risk for a tumor (e.g., an MSLN-expressing tumor), an inflammatory disease, or an autoimmune disease.

[0188] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease refers to an amount sufficient to prevent, suppress or delay the appearance of a disease (e.g., a tumor, an inflammatory disease, or an autoimmune disease), and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease or its complications in a patient who already has the disease. The determination of such an effective amount is well within the capabilities of a person skilled in the art. For example, an effective amount for therapeutic use depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the overall condition of the patient, such as age, weight and sex, the manner in which the drug is administered, and other treatments administered in conjunction, etc.

[0189] Beneficial Effects of the Present Disclosure The present disclosure provides novel CD3 antibodies with reduced cytokine release, which show their ability to moderately activate T cells. The present disclosure also provides multispecific antibodies that target CD3 and additional antigens (e.g., tumor-associated antigens and / or immune checkpoint molecules), such as bispecific antibodies that target CD3 and MSLN. The bispecific antibodies can bind to CD3 present on T cells and specific target antigens on tumor cells to promote the targeting and recruitment of T cells to tumor cells, thereby inducing the tumor-specific (MHC-independent) killing activity of cytotoxic T cells, significantly reducing the release of cellular factors, and resulting in significantly improved safety. Thus, the antibodies of the present disclosure have significant clinical value.

[0190] The embodiments of the present disclosure are described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the drawings and examples are only used to explain the present disclosure and do not limit the scope of the present disclosure. Various objects and advantageous aspects of the present disclosure will become apparent to those skilled in the art from the following detailed description of the accompanying drawings and examples.

[0191] Sequence information A description of the sequences encompassed in this application is provided in the table below. [Table 1-1] [Table 1-2] [Table 1-3]

[0192] Unless otherwise specified, the molecular biology experiments and immunoassays used in this disclosure are carried out essentially by reference to the methods described by J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Compiled Experimental Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995, and restriction enzymes are used according to the conditions recommended by the product manufacturers. Those skilled in the art will recognize that this example is for describing the present disclosure by way of example, and is not intended to limit the scope of the present disclosure. EXAMPLES

[0193] Example 1 Construction of CD3 monoclonal antibody In this example, mutations were introduced into the heavy and light chain variable regions of a CD3 monoclonal antibody to establish an scFv phage mutation library. The heavy chain variable regions of the constructed CD3 monoclonal antibody were named VH1 to VH46, which had the amino acid sequences shown in SEQ ID NOs: 1 to 46, respectively. The light chain variable regions of the constructed CD3 monoclonal antibody were named VL-1 to VL-2, which had the amino acid sequences shown in SEQ ID NOs: 47 and 48, respectively. The CDR information of each variable region is shown in Tables 1 and 2. The constructed VH1 to VH46 were paired with VL-1, respectively, to establish an scFv phage mutation library and the constructed CD3 monoclonal antibody. The specific structure of the CD3 monoclonal antibody is an IgG antibody, and the VH of the CD3 monoclonal antibody is connected to an IgG1 mutant heavy chain constant region (SEQ ID NO: 164) to form a structure shown as [VH]-[CH], and the VL-1 of the CD3 monoclonal antibody is connected to a light chain constant region (SEQ ID NO: 165) to form a structure shown as [VL]-[CL]. For convenience of description, the CD3 monoclonal antibody containing VH1 and VL-1 is named M1, the CD3 monoclonal antibody containing VH2 and VL-1 is named M2, and thus, a total of 46 CD3 monoclonal antibodies are named M1 to M46. Coating with CD3εγ (company: acrobiosystems, catalog number: CDG-H5253), adding packaged phages, incubating at room temperature for 1 hour, washing 10 times, patting dry, then eluting the phages, infecting TG1 competent cells overnight, collecting the phages, repeating the previous procedure twice, picking single colonies, subjecting them to IPTG induction, and expressing overnight. Positive clones were determined by ELISA. [Table 2-1] [Table 2-2]

[0194] Example 2 Purity determination of CD3 monoclonal antibody 100μl of each of CD3 monoclonal antibodies M1-M46 was transferred into a 1.5ml centrifuge tube and centrifuged at 12000rpm for 5 minutes, then 90μl of sample was taken and transferred into a liquid phase injection bottle and loaded into the machine (HPLC, Shimadzu, 2030C) for detection. The results were shown in Table 3. From Table 3, it can be seen that the SEC purity values ​​were all higher than 90%, and the in vitro efficacy experiment could be continued. [Table 3]

[0195] Example 3 Affinity detection of CD3 monoclonal antibody In this example, the binding activity of CD3 monoclonal antibodies to Jurkat cells (which naturally express human CD3) (company: ATCC; catalog number: TIB-152) and cynomolgus monkey HSC-F cells (which express monkey CD3) (company: JCRB Cell Bank; catalog number: JCRB1164) was examined.

[0196] (1) Preparation of cells: Jurkat and HSC-F cells were each diluted to 1 × 10 with PBS. 7 The concentration was adjusted to 50 cells / ml, and 50 μl of cell suspension was prepared for each sample.

[0197] (2) Preparation of antibody: Using PBS, the antibody was adjusted to a final concentration of 2 μg / ml, and then a 3-fold gradient dilution was performed to obtain 8 gradients. 50 μl of antibody was prepared for each well.

[0198] (3) Cell and antibody incubation: 50 μl of cell suspension was taken, i.e., the total number of cells was 5 × 10 5 The final concentration of the antibody was 2 μg / ml starting concentration, 3-fold gradient dilutions were performed in 8 wells, and incubation was performed at 4° C. for 1 hour.

[0199] (4) After centrifugation at 500 g for 5 minutes, the supernatant was gently removed and washing was performed twice by adding 200 μl of PBS.

[0200] (5) Addition of secondary antibody: Fluorescent secondary antibody diluted in PBS (used at 1:100) was added and incubated at 4°C for 30 minutes.

[0201] (6) The fluorescent secondary antibody was washed off by centrifugation at 500 g for 5 min, the supernatant was gently removed, and washing was performed once by adding 200 μl of PBS, followed by upflow detection.

[0202] Two positive control antibodies were used in this experiment: for control antibody OKT3, its VH amino acid sequence is shown in SEQ ID NO: 166 and its VL amino acid sequence is shown in SEQ ID NO: 167, while for control antibody 1, its VH amino acid sequence is shown in SEQ ID NO: 168 and its VL amino acid sequence is shown in SEQ ID NO: 169.

[0203] The affinity detection results are shown in Table 4. [Table 4-1] [Table 4-2]

[0204] Example 4 Dynamic affinity determination of CD3 monoclonal antibodies Three concentrations of antibody, 4, 2, and 1 μg / mL, were immobilized on the HC200M chip containing anti-human IgGFc secondary antibody (Xantec, Cat. No.: HC200M), and the antibody binding amount was controlled to approximately 1000 RU. After the baseline was stabilized, serially diluted human CD3εγ (company: acrobiosystems, Cat. No.: CDG-H52W5) recombinant antigen (starting from 592.5 nM, 3-fold dilution, 8 gradient) was flowed through the chip at a flow rate of 1000 μL / min, with an association time of 8 min and a dissociation time of 20 min. Kinetic constants were obtained by fitting using the 1:1 binding model of Carterra's Kinetics software.

[0205] The results are shown in Table 5. Table 5 demonstrates that the CD3 monoclonal antibody of the present disclosure has affinity for the human CD3εγ antigen. [Table 5-1] [Table 5-2]

[0206] Example 5 Construction of human MSLN stably transfected cell lines A construct containing the full-length sequence of human MSLN (huMSLN) (cDNA purchased from Sino biological, HG13128-UT) was cloned into a lentiviral vector. The constructed lentiviral plasmid and packaging plasmid were co-transfected into HEK293T cells, and the cell supernatant was collected at 48 hours and 72 hours, respectively. The supernatant was sequentially added to MC38 cells (Nanjing Kebai, Catalog No.: CBP60825), and 4 μg / ml puromycin was added after 24 hours for screening. Single cells with high expression of huMSLN were sorted by cell sorter (Sony, LE-SH800SBP), and finally, stably transfected monoclonal cells MC38 / MSLN with high expression of huMSLN were obtained.

[0207] Example 6 Alpaca Immunity Anti-huMSLN nanobody was generated by immunizing alpaca. Alpaca immunization was commissioned to Apak Biotechnology Co., Ltd., which included immunizing two alpacas. The immunizing antigen was hFc-tagged human MSLN recombinant protein (huMSLN-hFc, Kaika Biotech, MSL-HM280). For the first immunization, subcutaneous immunization was performed by mixing Freund's complete adjuvant (CFA) and huMSLN-hFc, and for the remaining booster immunizations, subcutaneous immunization was performed three times by mixing Freund's incomplete adjuvant (IFA) and huMSLN-hFc.

[0208] Example 7 Establishment of a phage antibody library After the fourth immunization of the alpaca, 50 ml of peripheral blood was taken and the alpaca PBMC was isolated according to the instructions for lymphocyte separation solution (Haoyang Biotech, LTS1077). Total RNA was then extracted using RNAiso Plus (takara, 9109) reagent, and 5 μg of RNA was transcribed to obtain cDNA by referring to the instructions of PrimeScript™ II 1st Strand cDNA synthesis kit. The obtained cDNA was used as a template to amplify the heavy chain variable region (VHH) fragment, which was then ligated with the digested vector and electrotransduced into TG1 competent cells. One alpaca bacterial library was 2.14 × 10 9 The other alpaca bacterial library had a size of 7.8×10 9 had a size of

[0209] Example 8 MSLN single domain antibody screening The two constructed alpaca libraries were subjected to both cell and protein screening. After 3 or 4 rounds of cell screening and 2 rounds of protein screening, the plasmids of the screened bacterial libraries were extracted and subjected to PCR amplification of VHH fragments, ligated into an expression vector, clones were selected, and VHH secretory expression was induced.

[0210] Coating with antigens of human MSLN-his (huMSLN-his, Kaiji Biotech, MSL-HM18D), monkey MSLN-his (cynoMSLN-his, Kaiji Biotech, MSL-CM180), and mouse MSLN-his (muMSLN-his, Kaiji Biotech, MSL-MM180) was performed, in which the antigens were diluted to 2 μg / ml in antigen coating solution (carbonate buffer with pH 9.6), then added to the enzyme plate at 50 μl / well, and coating was performed overnight at 2-8°C; the next day, washing was performed three times with PBST, and 2% BSA was added at 37°C for blocking every 2 hours; the induced expression supernatant was added to each well at 100 μl / well, and incubated at 37°C for 1 hour; washing was performed three times with PBST, and then incubated with HRP-labeled anti-DYKDDDDK antibody (Wuhan Sanying Biotech, HRP-66008) was added at 50 μl / well and incubated at 37° C. for 1 hour; washed 3 times with PBST, then TMB was added for development and reading.

[0211] Obtained MSLN single domain antibody with good cross-reactivity against human, monkey and mouse, named 3C6.Sequenced antibody, sequence analysis was performed using CLC software, and clones with unique sequences were derived, expressed and purified.The VHH amino acid sequence of 3C6 is shown in SEQ ID NO:149, and its CDR sequence is shown in Table 6. [Table 6]

[0212] Example 9 Induction, expression and purification of MSLN single domain antibodies The stored glycerol bacteria were taken and activated overnight in 2YT medium (containing Amp) at 37°C and 250 rpm, and the next day inoculated into fresh medium at a ratio of 1:100 to reach an OD 600 Then, induction medium was added for induction and cultured overnight (30°C, 250 rpm). After induction, the supernatant was collected by centrifugation and purified using Ni (NTA type) affinity chromatography medium gravity column to obtain MSLN single domain antibody.

[0213] Example 10 Affinity detection of MSLN single domain antibodies ELISA affinity determination: Human MSLN-his (huMSLN-his, Kaika Biotech, MSL-HM18D), monkey MSLN-his (cynoMSLN-his, Kaika Biotech, MSL-CM180), and mouse MSLN-his (muMSLN-his, Kaika Biotech, MSL-MM180) were coated overnight at 4°C. After blocking with 2% BSA for 2 hours, different dilutions of single domain antibodies were added to each well (for antibodies, starting from 100 nM, 3-fold dilution, 8 concentrations) and incubated for 1 hour; HRP-labeled anti-DYKDDDDK antibody (Wuhan Sanying Biotech, HRP-66008) was added, and after color development with TMB solution, 2 M hydrochloric acid was used for stopping, and the absorbance at 450 nm was read. EC 50 were counted using Graphpad Prism software and the affinity results are shown in Table 7. [Table 7]

[0214] Example 11 Affinity determination of anti-MSLN single domain antibodies at the cellular level 50 μl of MC38 / MSLN cells (2×10 5 ECs were taken from the wells (100 μl) and added to a 96-well V-shaped plate, then 50 μl of serially diluted antibodies were added to each well (for antibodies, starting from 300 nM, 3-fold dilution, 8 concentrations) and incubated on ice for 1 hour. After washing off the primary antibody, diluted Alexa Fluro488-labeled anti-DYKDDDDK antibody (Wuhan Mitaka Biotech, HRP-66008) was added and incubated on ice for 1 hour, washed 4 times, and then resuspended in each well with 200 μl of PBS, and then detection was performed in a flow cytometer. 50 were counted using Graphpad Prism software, and the cell affinity results were shown in Table 8. [Table 8]

[0215] Example 12 Determination of the dynamic affinity of MSLN single domain antibodies Anti-DYKDDDDK antibody was bound to HC30M chip by amino coupling, and then 2 μg / ml of MSLN single domain antibody was captured. After the baseline was stabilized, serial dilutions of huMSLN-Fc (Kaika Biotech, MSL-HM280) and cynoMSLN-Fc (Kaika Biotech, MSL-CM280) (starting from 100 nM, 3-fold gradient dilution, total 8 concentrations) were run over the chip from low to high concentration with 8 minutes of association time and 20 minutes of dissociation time. Kinetic constants were obtained by fitting with Carteria software. The results of dynamic affinity between MSLN single domain antibody and MSLN protein are shown in Table 9. [Table 9]

[0216] Example 13 Expression and purification of CD3-MSLN bispecific antibody 1. Construction of CD3-MSLN bispecific antibody The structure of the CD3-MSLN bispecific antibody in this example is shown in Figure 1, where the humanized CD3 antibody has two arms (heavy and light chain pairing), and the C-terminus of the mutant IgG1 is connected to the MSLN single domain antibody (VHH antibody) via (GGGGS)2 to form the CD3-MSLN bispecific antibody (bivalent). Specifically, the VH of the humanized CD3 antibody obtained in Example 1 is connected to the IgG1 mutant heavy chain constant region (SEQ ID NO: 164), and the C-terminus of the heavy chain constant region is further connected to the above-mentioned 3C6 antibody through the linker (GGGGS)2 to form a first polypeptide chain having a structure shown as [VH]-[CH]-[L]-[VHH]; the VL-1 of the humanized CD3 antibody obtained in Example 1 is connected to the light chain constant region (SEQ ID NO: 165) to form a second polypeptide chain having a structure shown as [VL]-[CL]. The nucleic acid sequences encoding the above polypeptide chains were constructed into the PTT5 plasmid vector (Ubao Biotech, lot: VT2202) and sufficient plasmid was extracted for further use.

[0217] 2. Expression and purification of CD3-MSLN bispecific antibodies The day before transfection (D-1), cells were diluted in culture medium to 2 × 10 6 On the day of transfection (D0), cells were harvested and counted (cell viability should be ≥95%) and the cell density was adjusted to 4.0 × 10 6The antibody fragment plasmids were mixed at a ratio of heavy chain:light chain=1:1, then mixed with PEI and co-transfected into CHO-S cells. After transfection was completed, the cells were transferred to an 8% CO2 incubator and cultured at 37°C and 120 rpm. One day after transfection (D1), the cells were supplemented with pre-warmed CHOgro complete culture medium to 1 / 5 the volume of the expression system, cooled to 32°C, and continuously cultured. Two, four, and six days after transfection, the cells were supplemented with Advanced CHO Feed1 to 8%, 5%, and 5%, respectively, and cell viability was monitored daily starting from D7. When the viability was <80%, the cells were harvested; centrifuged, the supernatant was collected, and purified using a Protein A column for later use. The purity was shown in Table 10. For ease of description, the bispecific antibody containing VH1 has been designated B1, the bispecific antibody containing VH2 has been designated B2, and so on. [Table 10]

[0218] Example 14 Determination of binding activity of CD3-MSLN bispecific antibodies 1. The binding activity of the CD3-MSLN bispecific antibody to Jurkat (which naturally expresses human CD3) and cynomolgus monkey HSC-F cells (company: JCRB Cell Bank; catalog number: JCRB1164) was examined, where two positive control antibodies were used.

[0219] The bispecific antibody structures of positive control antibody OKT3-3C6 and control antibody 1-3C6 were constructed with reference to "CD3-MSLN bispecific antibody" in Example 13, where for OKT3-3C6, the VH sequence of the CD3 antibody had the amino acid sequence shown in SEQ ID NO: 166, the VL sequence of the CD3 antibody had the amino acid sequence shown in SEQ ID NO: 167, and the VHH was the 3C6 antibody of MSLN; while for control antibody 1-3C6, the VH sequence of the CD3 antibody had the amino acid sequence shown in SEQ ID NO: 168, the VL sequence of the CD3 antibody had the amino acid sequence shown in SEQ ID NO: 169, and the VHH was the 3C6 antibody of MSLN.

[0220] The experimental method was as follows: (1) Cell preparation: Jurkat and HSC-F cells were diluted to 1 × 10 with PBS. 7 The concentration was adjusted to 50 cells / ml, and 50 μl of the cell suspension was prepared and added to each sample. (2) Preparation of antibody: Using PBS, the antibody was adjusted to a final concentration of 2 μg / ml, then a 3-fold gradient dilution was performed to obtain 8 gradients, and 50 μl of antibody was added to each well. (3) Cell and antibody incubation: 50 μl of cell suspension was taken, i.e., the total number of cells was 5 × 10 5 The final concentration of the antibody was 2 μg / ml starting concentration, 3-fold gradient dilutions were performed in 8 wells, and incubation was performed at 4° C. for 1 hour. (4) After centrifugation at 500 g for 5 minutes, the supernatant was gently removed and washing was performed twice by adding 200 μl of PBS. (5) Addition of secondary antibody: Fluorescent secondary antibody diluted in PBS (used at 1:100) was added and incubated at 4°C for 30 minutes. (6) The fluorescent secondary antibody was rinsed and centrifuged at 500 g for 5 min, then the supernatant was gently removed and washed once by adding 200 μl of PBS, followed by upflow detection.

[0221] The results are shown in Table 11. The results showed that most of the CD3-MSLN bispecific antibodies of the present disclosure had the same order of magnitude of binding activity to human CD3-expressing cells and cynomolgus monkey CD3-expressing cells. [Table 11-1] [Table 11-2]

[0222] 2. Binding activity of CD3-MSLN bispecific antibodies to MC38-MSLN cells (1) Construction of MC38-MSLN cell line: The full-length DNA sequence of MSLN (NCBI genbank: accession number AAH09272) was synthesized into pLVX-IRES-Puro vector (Ubao Biotech, catalog number: VT1464); the lentivirus was packaged and infected into MC38 cells; after screening under pressure, monoclonal selection was performed to obtain a monoclonal cell line that highly expressed MSLN.

[0223] (2) Cell preparation: MC38-MSLN cells were diluted to 1 × 10 with PBS. 7 The concentration was adjusted to 50 cells / ml, and 50 μl of the cell suspension was prepared and added to each sample.

[0224] (3) Preparation of antibody: Using PBS, the antibody was adjusted to a final concentration of 2 μg / ml, and a 3-fold gradient dilution was performed to obtain 8 gradients. 50 μl of antibody was added to each well.

[0225] (4) Cell and antibody incubation: 50 μl of cell suspension was taken, i.e., the total number of cells was 5 × 10 5 The final antibody concentration was 2 μg / ml starting concentration, 3-fold gradient dilutions were performed in 8 wells, and incubation was at 4° C. for 1 hour.

[0226] (5) After centrifugation at 500 g for 5 minutes, the supernatant was gently removed and washing was performed twice by adding 200 μl of PBS.

[0227] (6) Addition of secondary antibody: Fluorescent secondary antibody diluted in PBS (used at 1:100) was added and incubated at 4°C for 30 minutes.

[0228] (7) The fluorescent secondary antibody was washed, centrifuged at 500 g for 5 min, the supernatant was gently removed, and washed once by adding 200 μl of PBS, followed by upflow detection.

[0229] The results are shown in Figure 2. The results demonstrated that the CD3-MSLN bispecific antibody of the present disclosure has good binding activity to MSLN-expressing cells.

[0230] 3. Detection of CD3-MSLN Bispecific Antibody Affinity (1) Affinity of CD3-MSLN Bispecific Antibody to CD3εγ Recombinant Antigen Three concentrations of the antibody, 4, 2, and 1 μg / mL, were immobilized on the HC200M chip containing the anti-human IgG Fc secondary antibody, and the antibody binding amount was controlled to approximately 1000 RU. After the baseline was stabilized, serially diluted human CD3εγ (company: acrobiosystems, catalog number: CDG-H52W5) recombinant antigen (starting from 592.5 nM, 3-fold dilution, 8 gradient) was flowed through the chip at a flow rate of 1000 μL / min, with an association time of 8 minutes and a dissociation time of 20 minutes. The kinetic constants were obtained by fitting with the 1:1 model of Carterra's Kinetics software. The affinity determination results are shown in Table 12 below, and the dynamic affinity diagrams are shown in Figures 3A-3D. The results show that the affinity of the CD3-MSLN bispecific antibody of the present disclosure for the human CD3εγ recombinant antigen was 10 -8 ~10 -10 It was shown that it is within the range of M. [Table 12-1] [Table 12-2]

[0231] (2) Affinity of CD3-MSLN bispecific antibodies to MSLN recombinant antigens Three concentrations of antibodies, 4, 2, and 1 μg / mL, were immobilized on the HC200M chip containing anti-human IgG Fc secondary antibody, and the antibody binding amount was controlled to approximately 1000 RU. After the baseline was stabilized, serially diluted human MSLN recombinant antigen (company: acrobiosystems, catalog number: MSN-H8223) (starting from 592.5 nM, 3-fold dilution, 8 gradient) was flowed through the chip at a flow rate of 1000 μL / min, with an association time of 8 minutes and a dissociation time of 20 minutes. The kinetic constants were obtained by fitting with the 1:1 model of Carterra's Kinetics software. The affinity determination results of the exemplary bispecific antibodies are shown in Table 13, and the dynamic affinity diagram is shown in FIG. 4. The results showed that all of the CD3-MSLN bispecific antibodies of the present disclosure have good binding activity to MSLN. [Table 13]

[0232] Example 15 Jurkat-TIGIT-luc cell stability experiment 1. Antibody dilution Using 1640+10% FBS culture medium, B8, B13, B22, B33, B37 and B43 antibodies were each diluted to 10 nM, a 2-fold gradient dilution was performed to obtain a total of 11 gradients, and 50 μl of diluted antibody was added to each well.

[0233] 2. Addition of Jurkat-TIGIT-luc cells Jurkat-TIGIT-luc (Nanjing Kebai, Cat. No.: CBP74020) cells in the logarithmic phase of growth were taken and cultured at 6 × 10 in 1640 + 10% FBS. 5The cells were adjusted to 3x10 cells / ml and added to wells containing the antibodies described above, where 50 μl of cells were added to each well (3x10 4 cells / well).

[0234] 3. Incubation The above cell and antibody mixture was gently mixed and incubated at 37°C and 5% CO2 for 5-6 hours.

[0235] 4. Loading and Detection Chemiluminescent substrate was added at 20 μl / well and then loaded onto the machine for detection.

[0236] The above steps 1-4 were repeated four times to obtain data from four repeated experiments of antibody activation of Jurkat-TIGIT-luc cells to verify the stability of Jurkat-TIGIT-luc cells. The detection results of activated Jurkat-TIGIT-luc cells were shown in Figure 5A-5F. The results showed that there was no significant difference in the results of the four repeated experiments of Jurkat-TIGIT-luc cells activated by B8, B13, B22, B33, B37 and B43 antibodies, indicating that Jurkat-TIGIT-luc cells were stable. Therefore, the Jurkat-TIGIT-luc cell line was used to carry out subsequent T cell activation signaling pathway activation experiments.

[0237] Example 16 Activation of T cell activation signaling pathways by CD3-MSLN bispecific antibodies Antibodies were diluted to 2μg / ml in 1640+10%FBS, where a 3-fold gradient dilution was performed to obtain a total of 12 gradients, and 50μl of diluted antibodies was added to each well. For the group without tumor cells, Jurkat-TIGIT-luc cells (Nanjing Kebai, Cat. No.: CBP74020) in the logarithmic phase of growth were taken and diluted to 6×10 in 1640+10%FBS. 5 The cells were adjusted to 3 × 10 cells / ml and 50 μl of cells were added to each well (3 × 10 4The plates were incubated at 37 °C for 5-6 h at 37 °C (100 cc / well), mixed gently, and incubated at 37 °C and 5% CO for 5-6 h, then chemiluminescent substrate was added at 20 μl / well and then loaded onto the machine for detection.

[0238] For the group with tumor cells, Jurkat-TIGIT-luc cells in the logarithmic phase of growth were cultured at 1.2 × 10 6 The cells were adjusted to 3 × 10 cells / ml and 25 μl of cells were added to each well (3 × 10 4 cells / well); digest the MC38 / MSLN cells constructed in Example 5, and culture the digested MC38 / MSLN cells in 1640 + 10% FBS at 1.2 × 10 6 Adjust the concentration to 3 x 10 cells / ml and add 25 μl of cells to each well, mix gently and add 3 x 10 4 10 cells / well were achieved and inoculated for 5-6 hours at 37°C and 5% CO2, chemiluminescent substrate was added at 20 μl / well and loaded onto the machine for detection.

[0239] The measurement results of the activation of human CD3-expressing T cells (i.e., Jurkat-TIGIT-luc) are shown in Figures 6A-6F and Table 14. The results showed that when tumor cells were not added, the activation of human CD3-expressing T cells by the CD3-MSLN bispecific antibodies of the present disclosure was weaker than that of the control antibody 1-3C6, and furthermore, no activated Jurkat / NFAT / LUC cells were detected, but they could moderately activate Jurkat-TIGIT-luc cells after tumor cells were added. [Table 14]

[0240] (Example 17) TDCC mediated by CD3-MSLN bispecific antibody (1) Resuscitation of PBMCs: 1. Prepare a 15 ml centrifuge tube and add 5 ml of inactivated complete culture medium. 2. Frozen PBMCs were thawed in a 37°C water bath, 5ml of cell solution was taken and added vertically to the tube, mixed thoroughly and centrifuged at 360g for 10 minutes. 3. The supernatant was discarded, 3 ml of inactivated complete culture medium was added and the cells were counted. 4. Increase cell concentration to 1 x 10 in inactivated complete culture medium 6 The concentration was adjusted to 10 cells / ml.

[0241] (2) Detection of cell proliferation (Cell trace violet) 1. MC38 / MSLN tumor cells were digested and centrifuged at 360 g for 5 min. 2. Count the cells and measure 2 x 10 6 Individual cells were harvested and centrifuged at 360 g for 5 minutes, and the supernatant was discarded. 3. 2 μl of cell trace (Invitrogen C34571) was resuspended using 1 ml of serum free 1640 to form a diluted staining solution. 4. The cells were then resuspended in 0.5 ml of staining solution and incubated in the dark at 37° C. for 30 minutes. 5. Fluorescence intensity is detected by flow cytometry, where the fluorescence intensity is 10% of that of the negative sample. 2 It was more than double. 6. The reaction was terminated by adding 5-10 ml of 1640 complete medium. 7. Centrifugation was carried out at 360g for 5 minutes. 8. Discard the supernatant, add serum-free medium, resuspend the cells, count the cells, and confirm the cell concentration is 5×10 4 The concentration was adjusted to 10 cells / ml. 9. Using a flat-bottom 96-well plate, inoculate tumor cells at 5 × 10 4 cells / ml, and PBMC cells at 1 × 10 6 The cells were diluted to 100 μl / ml and 100 μl of cell suspension of each type of cell was added each time for a total of 200 μl per well. 10. The final serum concentration was 5% and the culture was continued for 48 hours. 11. A new 96 well plate was used to dilute the antibodies and then the diluted antibodies were added. 12. Centrifugation was carried out at 360 g for 5 minutes, the supernatant was discarded, and the cells were digested with trypsin, followed by terminating the reaction. 13. Cells were stained, subjected to flow cytometry, and data received.

[0242] The results are shown in Figures 7A to 7I. The results indicate that the CD3-MSLN bispecific antibody of the present disclosure can exert a T cell-mediated tumor cell killing (TDCC) effect on tumor cells expressing the tumor antigen MSLN, which may be because the CD3-MSLN bispecific antibody of the present disclosure specifically binds to tumor cells and T cells expressing the tumor antigen MSLN, recruits T cells to the periphery of tumor cells, activates T cells, and induces TDCC, thereby effectively killing tumor cells.

[0243] (Example 18) TDCC mediated by CD3-CD19 bispecific antibodies In this example, a bispecific antibody was constructed based on the CD3 antibody and CD19 antibody of the present disclosure, which is a CD3-CD19 bispecific antibody, named 5Y2-175, and the TDCC activity mediated by 5Y2-175 was investigated. 5Y2-175 was prepared by referring to the method of constructing a bispecific antibody in Example 13, and the schematic diagram of its structure is shown in Figure 8, and its specific structure was as follows: VH of the CD3 monoclonal antibody (M33) obtained in Example 1 CD3 is ligated to an IgG1 mutant heavy chain constant region (SEQ ID NO: 164), and the C-terminus of the heavy chain constant region is linked to the VH of the CD19 antibody described above through the linker (GGGGS)2. CD19 (SEQ ID NO: 170) and VL CD19 (SEQ ID NO: 171) and VH CD19 and VL CD19 are connected through (GGGGS)2, and [VH CD3 ]-[CH]-[L]-[VH CD19 ]-[L]-[VL CD19] is formed; the VL of the humanized CD3 antibody (M33) obtained in Example 1 is CD3 is ligated to the light chain constant region (SEQ ID NO: 165) to form [VL CD3 A second polypeptide chain was formed having the structure shown as ]-[CL].

[0244] 1. Preparation of Raji-luc cells A sequence containing Luc (NCBI:GenBank:MF062157.1) was constructed and cloned into a lentiviral vector (plvx-IRES-puro, psPAX2, pMD2G). The constructed lentiviral plasmid and packaging plasmid were co-infected into HEK293T cells, the cell supernatant was collected after 48 hours and added to Raji cells (ATCC, CCL-86), and 4 μg / ml puromycin was added at 24 hours for screening. Single cells expressing Luc were sorted by cell sorter (Sony, LESH800SBP), and finally stable Raji-luc cells were obtained.

[0245] 2. Preparation of PBMCs Frozen PBMC cells were resuscitated, centrifuged at 500g for 3 min, the supernatant was removed, washed once with 1640+10% FBS (inactivated); then centrifuged at 500g for 3 min, the supernatant was removed, resuspended in an appropriate volume of 1640+10% FBS (inactivated), counted, diluted to the corresponding density, and added 25 μl per well to the well plate, so that the total number of PBMC cells was 1.25×10 5 The cells were cultured at 100x the volume of each well.

[0246] 3. Plating of Raji-luc cells Take 25 μl of Raji-luc cells and inoculate 1.25 × 10 cells into the PBMC well plate. 4 One cell / well was added.

[0247] 4. Dilution of CD3-CD19 double antibody CD3-CD19 bispecific antibody was taken and adjusted to a concentration of 2 nM in culture medium and diluted 3-fold to obtain 9 gradients; 50 μl of diluted antibody was taken and added to the cells including PBMC cells and Raji-luc cells.

[0248] 5. Incubation Incubation was carried out for 2 days in a 37° C., 5% CO 2 incubator.

[0249] 6. Read The well plates were removed, 50 μl of luciferase substrate was added, and the data was read and analyzed mechanically.

[0250] The experimental results are shown in FIG. 9, which demonstrated that the CD3-CD19 bispecific antibody could exert a T cell-mediated tumor cell killing (TDCC) effect against CD19-expressing lymphoma cells.

[0251] (Example 19) TDCC mediated by CD3-CD20 bispecific antibodies In this example, a bispecific antibody was constructed based on the CD3 antibody and CD20 antibody of the present disclosure, which is a CD3-CD20 bispecific antibody, named 5Y2-176, and the TDCC activity mediated by the CD3-CD20 bispecific antibody was examined. 5Y2-176 was constructed by referring to the CD3-CD19 bispecific antibody of Example 18, in which the VH amino acid sequence of CD20 is shown in SEQ ID NO: 172, and the VL amino acid sequence of CD20 is shown in SEQ ID NO: 173.

[0252] 1. Preparation of PBMCs Frozen PBMC cells were resuscitated, centrifuged at 500g for 3 min to remove the supernatant, washed once with 1640+10% FBS (inactivated), then centrifuged at 500g for 3 min to remove the supernatant, resuspended in an appropriate volume of 1640+10% FBS (inactivated), counted, diluted to the corresponding density, and added 25 μl per well to the well plate, resulting in a total number of PBMC cells of 1.25×10 5The cells were cultured at 100x the volume of each well.

[0253] 2. Plating of Raji-luc cells 25 μl of Raji-luc cells prepared according to the method for preparing Raji-luc cells in Example 18 was taken and placed in the PBMC well plate described above at 1.25×10 4 One cell / well was added.

[0254] 3. Dilution of CD3-CD20 double antibody CD3-CD20 antibody was taken and adjusted to have a concentration of 2 nM in culture medium, and 3-fold dilutions were performed to obtain 9 gradients; 50 μl of diluted antibody was added to the cells including PBMC cells and Raji-luc cells.

[0255] 4. Incubation Incubation was carried out for 2 days in a 37° C., 5% CO 2 incubator.

[0256] 5. Read The well plates were removed, 50 μl of luciferase substrate was added, and the data was read and analyzed mechanically.

[0257] The experimental results are shown in FIG. 10, which demonstrated that the CD3-CD20 bispecific antibody could exert a T cell-mediated tumor cell killing (TDCC) effect against CD20-expressing lymphoma cells.

[0258] (Example 20) TDCC mediated by CD3-trop2 bispecific antibodies In this example, a bispecific antibody was constructed based on the CD3 and trop2 antibodies of the present disclosure, which is a CD3-trop2 bispecific antibody, named 5Y2-174, and the TDCC activity mediated by the CD3-trop2 bispecific antibody was examined. The 5Y2-174 bispecific antibody was prepared according to the method for constructing a bispecific antibody in Example 13, and the humanized CD3 antibody involved in its specific structure was M33, and the VHH amino acid sequence of trop2 was shown in SEQ ID NO:174.

[0259] 1. Construction of HCC1806-luc cells: A sequence containing Luc gene was constructed and cloned into lentiviral vector (plvx-IRES-puro, psPAX2, pMD2G), the constructed lentiviral plasmid and packaging plasmid were co-infected into HEK293T cells, the cell supernatant was collected at 48 hours and added into HCC1806 cells (Nanjing Kebai, CBP60373), and 4μg / ml puromycin was added at 24 hours for screening. Luc-expressing single cells were sorted by cell sorter (Sony, LESH800SBP), and stable HCC1806-luc cells were finally obtained.

[0260] 2. Preparation of PBMCs Frozen PBMC cells were resuscitated, centrifuged at 500g for 3 min to remove the supernatant, washed once with 1640+10% FBS (inactivated), then centrifuged at 500g for 3 min to remove the supernatant, resuspended in an appropriate volume of 1640+10% FBS (inactivated), counted, diluted to the corresponding density, and added 25 μl per well to the well plate, resulting in a total number of PBMC cells of 1.25×10 5 The cells were cultured at 100x the volume of each well.

[0261] 3. Plating of HCC1806-luc cells Take 25 μl of the above HCC1806-luc cells and add 1.25 x 10 cells to the above PBMC well plate. 4 One cell / well was added.

[0262] 4. Dilution of CD3-Trop2 double antibodies CD3-trop2 double antibody was taken and adjusted to have a concentration of 1 nM in culture medium, and 3-fold dilutions were performed to obtain a 10 gradient; 50 μl of diluted antibody was added to cells including PBMC cells and HCC1806-luc cells.

[0263] 5. Incubation Incubation was carried out for 2 days in a 37° C., 5% CO 2 incubator.

[0264] 6. Read The well plates were removed, 50 μl of luciferase substrate was added, and the data was read and analyzed mechanically.

[0265] The experimental results are shown in FIG. 11, which demonstrated that the CD3-trop2 bispecific antibody can exert T cell-mediated tumor cell killing (TDCC) effect against trop2-expressing human breast squamous cell carcinoma cells.

[0266] Example 21 CD3-Her2 bispecific antibody-mediated TDCC In this example, a bispecific antibody was constructed based on the CD3 antibody and Her2 antibody of the present disclosure, which is a CD3-Her2 bispecific antibody, named 5Y2-177, and the TDCC activity mediated by the CD3-Her2 bispecific antibody was examined. The 5Y2-177 bispecific antibody was prepared according to the method for constructing a bispecific antibody in Example 13. The humanized CD3 antibody involved in its specific structure is M33, and the VHH amino acid sequence of Her2 is shown in SEQ ID NO:175.

[0267] 1. Construction of Calu-1-luc cells: A sequence containing Luc gene was constructed and cloned into lentiviral vector (plvx-IRES-puro, psPAX2, pMD2G). The constructed lentiviral plasmid and packaging plasmid were co-infected into HEK293T cells, the cell supernatant was collected at 48 hours, and added to Calu-1 cells (Nanjing Kebai, CBP60085), and 4μg / ml puromycin was added at 24 hours for screening. Single cells expressing Luc were sorted by cell sorter (Sony, LESH800SBP), and finally stable Calu-1-luc cells were obtained.

[0268] 2. Preparation of PBMCs Frozen PBMC cells were resuscitated, centrifuged at 500g for 3 min to remove the supernatant, washed once with 1640+10% FBS (inactivated), then centrifuged at 500g for 3 min to remove the supernatant, suspended in an appropriate volume of 1640+10% FBS (inactivated), counted, diluted to the corresponding density, and added 25 μl per well to the well plate, resulting in a total number of PBMC cells of 1.25×10 5 The cells were cultured at 100x the volume of each well.

[0269] 3. Plating Calu-1-luc cells Take 25 μl of the Calu-1-luc cells and inoculate 1.25 x 10 cells into the PBMC well plate. 4 One cell / well was added.

[0270] 4. Dilution of CD3-Her2 double antibody CD3-Her2 was taken and adjusted to have a concentration of 30 nM in culture medium and 3-fold dilutions were performed to obtain an 11 gradient; 50 μl of the diluted antibody was added to the cells including PBMC cells and Calu-1-luc cells.

[0271] 5. Incubation Incubation was carried out for 2 days in a 37° C., 5% CO 2 incubator.

[0272] 6. Read The well plates were removed, 50 μl of luciferase substrate was added, and the data was read and analyzed mechanically.

[0273] The experimental results are shown in FIG. 12, which demonstrated that the CD3-Her2 bispecific antibody could exert T cell-mediated tumor cell killing (TDCC) effect on Her2-expressing human lung cancer cells.

[0274] Example 22 TDCC mediated by CD3-claudin 18.2 bispecific antibody In this example, a bispecific antibody was constructed based on the CD3 antibody and claudin18.2 antibody of the present disclosure, which is a CD3-claudin18.2 bispecific antibody, named 5Y2-178, and the TDCC activity mediated by the CD3-claudin18.2 bispecific antibody was examined. The 5Y2-178 bispecific antibody was prepared according to the method for constructing a bispecific antibody in Example 13. The humanized CD3 antibody involved in the specific structure is M33, and the VHH amino acid sequence of claudin18.2 is shown in SEQ ID NO:176.

[0275] 1. Construction of NUGC4-luc cells A sequence containing Luc gene was constructed and cloned into lentiviral vector (plvx-IRES-puro, psPAX2, pMD2G). The constructed lentiviral plasmid and packaging plasmid were co-infected into HEK293T cells, the cell supernatant was collected at 48 hours, and added to NUGC4 cells (Nanjing Kebai, CBP60493), and 4μg / ml puromycin was added at 24 hours for screening. Single cells expressing Luc were sorted by cell sorter (Sony, LESH800SBP), and finally stable NUGC4-lucc cells were obtained.

[0276] 2. Preparation of PBMCs Frozen PBMC cells were resuscitated, centrifuged at 500g for 3 min to remove the supernatant, washed once with 1640+10% FBS (inactivated), then centrifuged at 500g for 3 min to remove the supernatant, resuspended in an appropriate volume of 1640+10% FBS (inactivated), counted, diluted to the corresponding density, and added 25 μl per well to the well plate, resulting in a total number of PBMC cells of 1.25×10 5 The cells were cultured at 100x the volume of each well.

[0277] 3. Plating of NUGC4-luc cells Take 25 μl of the above NUGC4-luc cells and add 1.25 x 10 cells to the above PBMC well plate. 4 One cell / well was added.

[0278] 4. Dilution of CD3-Claudin 18.2 Double Antibody CD3-claudin 18.2 double antibody was taken and adjusted to have a concentration of 100 nM in culture medium, and a 3-fold dilution was performed to obtain a 12-gradient; 50 μl of the diluted antibody was added to the cells, including PBMC cells and NUGC4-luc cells.

[0279] 5. Incubation Incubation was carried out for 2 days in a 37° C., 5% CO 2 incubator.

[0280] 6. Read The well plates were removed, 50 μl of luciferase substrate was added, and the data was read and analyzed mechanically.

[0281] The experimental results are shown in FIG. 13, which demonstrated that the CD3-claudin18.2 bispecific antibody could exert a T cell-mediated tumor cell killing (TDCC) effect on claudin18.2-expressing human gastric cancer cells.

[0282] (Example 23) Cytokine experiments with CD3-MSLN bispecific antibodies Bispecific antibodies were adjusted to have a concentration of 1 nM and 3-fold dilutions were performed to obtain 9 gradients, diluted bispecific antibodies were added (50 μl / well), and no bispecific antibody was added to the last well; PBMC cells were added at 2×10 6 The cells were adjusted to have a density of 100 cells / ml, 50 μl / well; the culture was carried out at 37°C and 5% CO2 for 48 hours; 30 μl of supernatant was taken at 24 hours and 48 hours, respectively, and the IL2 and INF-γ contents in the supernatant were detected by HTRF kits (cisbio 62HIL02PEH, 62HIFNGPEH).

[0283] The IL2 detection results are shown in Figures 14A-14I, and the INF-γ detection results are shown in Figures 15A-15I. The results showed that co-incubation of the CD3-MSLN bispecific antibody of the present disclosure with PBMCs produced significantly lower IL2 and INF-γ compared to the positive control OKT3-3C6, and the CD3-MSLN bispecific antibody of the present disclosure has good safety.

[0284] Although the specific embodiments of the present disclosure have been described in detail, it is understood that those skilled in the art can make various modifications and changes to the details based on all the teachings disclosed, and these modifications are within the scope of protection of the present disclosure. The full scope of the present disclosure is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to CD3, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL); the VH comprises an HCDR1 comprising the sequence set forth in SEQ ID NO: 107, an HCDR2 comprising the sequence set forth in SEQ ID NO: 108, and X １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ X １０ WX １１ X １２ X １３ (SEQ ID NO: 112) (wherein X １ is A, H or P; X ２ is A, E, G, H, K, Q or S; X ３ is D, N or R; X ４ is F or P; X ５ is G, K, L, P, Q, R, S, V, W or Y; X ６ is M, N, Q or R; X ７ is G, N, S or T; X ８ is A, Q, R or Y; X ９ is G, I or V; X １０ is N or S; X １１ is F or W; X １２ is A, E, K or Q; X １３ is H, L, M, S, or Y); with the proviso that the HCDR3 is not set forth in SEQ ID NO: 90; and The VL comprises an LCDR1 comprising the sequence set forth in SEQ ID NO: 137, an LCDR2 comprising the sequence set forth in SEQ ID NO: 138, and an LCDR3 comprising the sequence set forth in SEQ ID NO:

139. An antibody or antigen-binding fragment thereof.

2. the HCDR3 is X １ X ２ X ３ FX ４ NX ５ YX ６ SWFAX ７ (SEQ ID NO: 148) (wherein X １ is H or P; X ２ is G, E, or A; X ３ is N or R; X ４ is G, K, S, or P; X ５ is T, S, or N; X ６ is V or G; X ７ is M, Y, S, or L; Or, the HCDR3 comprises a sequence set forth in any one of SEQ ID NOs: 79, 56, 61, 69, 82, and 87; The antibody or antigen-binding fragment thereof according to claim 1.

3. The HCDR3 is HGNFGNX ７ YVSWFAX １３ (SEQ ID NO: 133) (wherein X ７ is T or N, and X １３ is M or S); or the HCDR3 comprises the sequence set forth in any one of SEQ ID NOs: 79 and 56; The antibody or antigen-binding fragment thereof according to claim 1.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises an HCDR1 comprising the sequence set forth in SEQ ID NO: 107, an HCDR2 comprising the sequence set forth in SEQ ID NO: 108, and an HCDR3 comprising the sequence set forth in any one of SEQ ID NOs: 79, 49-78, and 80-89. (i) the antibody or antigen-binding fragment thereof further comprises a framework region derived from a human immunoglobulin; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region contained in an amino acid sequence encoded by a gene derived from the human heavy chain germline, and / or a light chain framework region contained in an amino acid sequence encoded by a gene derived from the human light chain germline; or (ii) the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, and diabody; The antibody or antigen-binding fragment thereof according to claim 1.

6. the VH comprises HFR1, HFR2, HFR3 and HFR4; the HFR1 comprises the sequence set forth in SEQ ID NO: 109; the HFR2 comprises the sequence set forth in SEQ ID NO: 110; The HFR3 is VKX １ RFTISRDDSKSX ２ LYLQMNX ３ LKTEDTAX ４ YYCVR (SEQ ID NO: 136) (wherein X １ is G or D, and X ２ is I or S, and X ３ is N or S, and X ４ is M or V; the HFR4 comprises the sequence set forth in SEQ ID NO: 111; Preferably, the HFR3 comprises a sequence as set forth in any one of SEQ ID NOs: 91 to 106. The antibody or antigen-binding fragment thereof according to claim 1.

7. The VH is (1) an amino acid sequence shown in any one of SEQ ID NOs: 33, 1 to 32, and 34 to 46, or a variant thereof; (2) an amino acid sequence set forth in any one of SEQ ID NOs: 33, 8, 13, 22, 37, and 43, or a variant thereof; or (3) An amino acid sequence shown in any one of SEQ ID NOs: 33 and 8 or a variant thereof. Including; The variant according to any one of (1) to (3) has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and / or the VL comprises the amino acid sequence set forth in SEQ ID NO: 47 or 48 or a variant thereof, and the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; The antibody or antigen-binding fragment thereof according to claim 1.

8. - a VH comprising a sequence as set forth in any one of SEQ ID NOs: 33, 1 to 32, and 34 to 46 or a variant thereof, and a VL comprising a sequence as set forth in SEQ ID NO: 47 or a variant thereof; or - VH comprising the sequence shown in any one of SEQ ID NOs: 33, 1 to 32, and 34 to 46 or a variant thereof, and VL comprising the sequence shown in SEQ ID NO: 48 or a variant thereof. Including; The variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. The antibody or antigen-binding fragment thereof according to claim 1.

9. A single domain antibody or antigen-binding fragment thereof capable of specifically binding to MSLN, wherein the single domain antibody or antigen-binding fragment thereof comprises: (1) the following CDRs as defined by the IMGT numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 150, CDR2 comprising the sequence set forth in SEQ ID NO: 151, and CDR3 comprising the sequence set forth in SEQ ID NO: 152; (2) the following CDRs as defined by the Kabat numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 153, CDR2 comprising the sequence set forth in SEQ ID NO: 154, and CDR3 comprising the sequence set forth in SEQ ID NO: 155; (3) The following CDRs as defined by the AbM numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 156, CDR2 comprising the sequence set forth in SEQ ID NO: 157, and CDR3 comprising the sequence set forth in SEQ ID NO: 155; (4) the following CDRs as defined by the Chothia numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 158, CDR2 comprising the sequence set forth in SEQ ID NO: 159, and CDR3 comprising the sequence set forth in SEQ ID NO: 155; or (5) The following CDRs as defined by the Contact numbering system: CDR1 comprising the sequence set forth in SEQ ID NO: 160, CDR2 comprising the sequence set forth in SEQ ID NO: 161, and CDR3 comprising the sequence set forth in SEQ ID NO:

162. Including, A single domain antibody or antigen-binding fragment thereof.

10. 10. The single domain antibody or antigen-binding fragment thereof according to claim 9, comprising the sequence set forth in SEQ ID NO: 149 or a variant thereof, wherein said variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions compared thereto; preferably, said substitutions are conservative substitutions.

11. 9. A multispecific antibody comprising a CD3-targeting antigen-binding domain and at least one antigen-binding domain targeting an additional antigen, wherein the CD3-targeting antigen-binding domain is selected from the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and the additional antigen is selected from a tumor-associated antigen (TAA) and / or an immune checkpoint molecule.

12. The multispecific antibody is a bispecific antibody comprising the CD3-targeting antigen-binding domain and the tumor-associated antigen-targeting antigen-binding domain; or the multispecific antibody is a trispecific antibody comprising the CD3-targeting antigen-binding domain, the tumor-associated antigen-targeting antigen-binding domain, and the immune checkpoint molecule-targeting antigen-binding domain; The multispecific antibody of claim 11.

13. (I) to (VI): (I) the multispecific antibody comprises the CD3-targeting antigen-binding domain and the MSLN-targeting antigen-binding domain, and the MSLN-targeting antigen-binding domain is selected from the single domain antibody or antigen-binding fragment thereof of claim 9 or 10; or (II) the multispecific antibody comprises the CD3-targeting antigen-binding domain and the CD19-targeting antigen-binding domain, wherein the CD19-targeting antigen-binding domain comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 171 or a variant thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 170 or a variant thereof; or (III) the multispecific antibody comprises the CD3-targeting antigen-binding domain and the CD20-targeting antigen-binding domain, wherein the CD20-targeting antigen-binding domain comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the sequence set forth in SEQ ID NO: 173 or a variant thereof, and the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 172 or a variant thereof; or (IV) the multispecific antibody comprises the CD3-targeting antigen-binding domain and a TROP2-targeting antigen-binding domain, wherein the TROP2-targeting antigen-binding domain comprises a VHH, and the VHH comprises the sequence set forth in SEQ ID NO: 174 or a variant thereof; or (V) the multispecific antibody comprises the CD3-targeting antigen-binding domain and a HER2-targeting antigen-binding domain, wherein the HER2-targeting antigen-binding domain comprises a VHH, and the VHH comprises the sequence set forth in SEQ ID NO: 175 or a variant thereof; or (VI) The multispecific antibody comprises the CD3-targeting antigen-binding domain and a claudin 18.2-targeting antigen-binding domain, wherein the claudin 18.2-targeting antigen-binding domain comprises a VHH, and the VHH comprises the sequence set forth in SEQ ID NO: 176 or a variant thereof.

12. The multispecific antibody of claim 11, characterized by one of the following:

14. 11. A multispecific antibody comprising the single domain antibody or antigen-binding fragment thereof according to claim 9 or 10, wherein the multispecific antibody is preferably a bispecific, trispecific or tetraspecific antibody.

15. (i) An antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or a heavy chain variable region and / or a light chain variable region thereof; (ii) a single domain antibody or antigen-binding fragment thereof according to any one of claims 9 to 10; (iii) a multispecific antibody, or polypeptide chains thereof, comprising a CD3-targeting antigen-binding domain and at least one antigen-binding domain targeting an additional antigen, wherein the CD3-targeting antigen-binding domain is the antibody or antigen-binding fragment of (i), and the additional antigen is selected from a tumor-associated antigen (TAA) and / or an immune checkpoint molecule; or (iv) A multispecific antibody, or a polypeptide chain thereof, wherein the multispecific antibody comprises a single domain antibody or antigen-binding fragment thereof of (ii). An isolated nucleic acid molecule encoding

16. A vector comprising the nucleic acid molecule of claim 15.

17. 16. A host cell comprising the nucleic acid molecule of claim 15 or a vector comprising said nucleic acid molecule.

18. 20. A method for preparing an antibody or antigen-binding fragment thereof, a single domain antibody or antigen-binding fragment thereof, or a multispecific antibody, comprising culturing the host cell of claim 17 under conditions that allow protein expression, and harvesting the antibody or antigen-binding fragment thereof, the single domain antibody or antigen-binding fragment thereof, or the multispecific antibody from a culture of the cultured host cells.

19. (i) to (vii): (i) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; (ii) a single domain antibody or antigen-binding fragment thereof according to any one of claims 9 to 10, or (iii) a multispecific antibody comprising a CD3-targeting antigen-binding domain and at least one antigen-binding domain targeting an additional antigen, wherein the CD3-targeting antigen-binding domain is the antibody or antigen-binding fragment thereof of (i), and the additional antigen is selected from a tumor-associated antigen (TAA) and / or an immune checkpoint molecule; (iv) a multispecific antibody comprising the single domain antibody of (ii) or an antigen-binding fragment thereof; (v) an isolated nucleic acid molecule encoding any one of (i) to (iv); (vi) a vector comprising the isolated nucleic acid molecule of (v); or (vii) a host cell comprising the isolated nucleic acid molecule of (v), or the vector of (vi). and a pharmaceutically acceptable carrier and / or excipient.

20. (i) to (viii) in the manufacture of a medicament for preventing and / or treating a disease: (i) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; (ii) a single domain antibody or antigen-binding fragment thereof according to any one of claims 9 to 10, or (iii) a multispecific antibody comprising a CD3-targeting antigen-binding domain and at least one antigen-binding domain targeting an additional antigen, wherein the CD3-targeting antigen-binding domain is the antibody or antigen-binding fragment thereof of (i), and the additional antigen is selected from a tumor-associated antigen (TAA) and / or an immune checkpoint molecule; (iv) a multispecific antibody comprising the single domain antibody of (ii) or an antigen-binding fragment thereof; (v) an isolated nucleic acid molecule encoding any one of (i) to (iv); (vi) a vector comprising the isolated nucleic acid molecule of (v); (vii) a host cell comprising the isolated nucleic acid molecule of (v), or the vector of (vi); or (viii) A pharmaceutical composition comprising one of (i)-(vii) and a pharmaceutically acceptable carrier and / or excipient. The use of one of Preferably, the disease is a tumor, an inflammatory disease or an autoimmune disease; preferably, the tumor is selected from the group consisting of mesothelioma, ovarian cancer, pancreatic cancer, breast cancer, bile duct cancer, colon cancer, gastric cancer, fallopian tube cancer, lung cancer, acute myeloid leukemia and colorectal cancer.

21. (i) to (viii) for preventing and / or treating a disease: (i) the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8; (ii) a single domain antibody or antigen-binding fragment thereof according to any one of claims 9 to 10, or (iii) a multispecific antibody comprising a CD3-targeting antigen-binding domain and at least one antigen-binding domain targeting an additional antigen, wherein the CD3-targeting antigen-binding domain is the antibody or antigen-binding fragment thereof of (i), and the additional antigen is selected from a tumor-associated antigen (TAA) and / or an immune checkpoint molecule; (iv) a multispecific antibody comprising the single domain antibody of (ii) or an antigen-binding fragment thereof; (v) an isolated nucleic acid molecule encoding any one of (i) to (iv); (vi) a vector comprising the isolated nucleic acid molecule of (v); (vii) a host cell comprising the isolated nucleic acid molecule of (v), or the vector of (vi); or (viii) A pharmaceutical composition comprising one of (i)-(vii) and a pharmaceutically acceptable carrier and / or excipient. A composition comprising one of: Preferably, the disease is a tumor, an inflammatory disease or an autoimmune disease; preferably, the tumor is selected from the group consisting of mesothelioma, ovarian cancer, pancreatic cancer, breast cancer, bile duct cancer, colon cancer, gastric cancer, fallopian tube cancer, lung cancer, acute myeloid leukemia and colorectal cancer.

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