WO2023125611A1 - 靶向cd3的抗体及多特异性抗体及其用途 - Google Patents

靶向cd3的抗体及多特异性抗体及其用途 Download PDF

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WO2023125611A1
WO2023125611A1 PCT/CN2022/142626 CN2022142626W WO2023125611A1 WO 2023125611 A1 WO2023125611 A1 WO 2023125611A1 CN 2022142626 W CN2022142626 W CN 2022142626W WO 2023125611 A1 WO2023125611 A1 WO 2023125611A1
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seq
sequence shown
antibody
antigen
binding fragment
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French (fr)
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胡彦
郭三友
滕毓敏
韦涛
丁兆
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Sichuan Huiyu Pharmaceutical Co Ltd
Sichuan Huiyu Seacross Pharmaceutical Technology Co Ltd
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Sichuan Huiyu Pharmaceutical Co Ltd
Sichuan Huiyu Seacross Pharmaceutical Technology Co Ltd
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Priority to CN202280085681.9A priority Critical patent/CN118591561A/zh
Priority to EP22914863.0A priority patent/EP4458861A1/en
Priority to JP2024538668A priority patent/JP2025503513A/ja
Priority to US18/723,721 priority patent/US20250059278A1/en
Publication of WO2023125611A1 publication Critical patent/WO2023125611A1/zh
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to an antibody targeting CD3 or an antigen-binding fragment thereof, which has the ability to moderately activate T cells and can effectively reduce the release of cytokines.
  • the invention also relates to multispecific antibodies targeting CD3 and additional antigens (such as tumor-associated antigens and/or immune checkpoint molecules), which are capable of efficiently and specifically killing target cells while significantly reducing cytokine release, with significantly improved safety.
  • the present invention also relates to the use of the CD3-targeting antibody or its antigen-binding fragment, multispecific antibody or the composition comprising them in the treatment of diseases.
  • CD3 is a protein complex and T cell co-receptor mainly expressed on T cells and involved in the activation of cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells).
  • CD3 consists of a CD3 ⁇ chain, a CD3 ⁇ chain and two CD3 ⁇ chains. These chains bind to the T-cell receptor (TCR) and the ⁇ chain (zeta chain) to generate activation signals in T lymphocytes.
  • TCR T-cell receptor
  • zeta chain zeta chain
  • CD3 antibodies currently in the clinical stage or marketed drugs are mainly derived from humanized CD3 antibodies (OKT3/UCHT1/L2K/TR66, etc.), but their affinity is relatively high, so it is easy to cause excessive activation of T cells and release
  • a large number of cytokines, resulting in cytokine storm syndrome have serious side effects on the human body, and even threaten life safety in severe cases.
  • CRS cytokine storm syndrome
  • the doses administered are generally very low, resulting in a narrow therapeutic window that is not conducive to patient benefit.
  • the inventors of the present application screened and obtained new CD3 antibodies through extensive research, and these antibodies have reduced release of cytokines, indicating that they can moderately activate T cells.
  • the inventors of the present application also screened and obtained a new MSLN single-domain antibody, which has good binding activity to MSLN. Based on this, there are further provided multispecific antibodies targeting CD3 and additional antigens (such as MSLN, CD19, CD20, Trop2, Her2 or Caludin18.2), which exhibit enhanced tumor cell localization and elicit potent T cells Activated to specifically kill tumor cells, and has significantly reduced release of cytokines, thus having significantly improved safety.
  • additional antigens such as MSLN, CD19, CD20, Trop2, Her2 or Caludin18.2
  • the present invention relates to an antibody or antigen-binding fragment thereof capable of specifically binding CD3, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein,
  • the VH comprises: HCDR1 comprising the sequence shown in SEQ ID NO:107, HCDR2 comprising the sequence shown in SEQ ID NO:108 and comprising X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 HCDR3 of the sequence shown in 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 ,
  • the present invention relates to a single domain antibody or antigen-binding fragment thereof capable of specifically binding to MSLN, said single domain antibody or antigen-binding fragment thereof comprising:
  • CDR1 comprising the sequence shown in SEQ ID NO: 150 or a variant thereof
  • CDR2 comprising the sequence shown in SEQ ID NO: 151 or a variant thereof
  • SEQ ID NO: CDR3 of the sequence shown in 152 or a variant thereof
  • CDR1 comprising the sequence shown in SEQ ID NO:153 or a variant thereof
  • CDR2 comprising the sequence shown in SEQ ID NO:154 or a variant thereof
  • SEQ ID NO: CDR3 of the sequence shown in 155 or a variant thereof
  • CDR1 comprising the sequence shown in SEQ ID NO: 156 or a variant thereof
  • CDR2 comprising the sequence shown in SEQ ID NO: 157 or a variant thereof
  • SEQ ID NO: CDR3 of the sequence shown in 155 or a variant thereof
  • CDR1 comprising the sequence shown in SEQ ID NO: 158 or a variant thereof
  • CDR2 comprising the sequence shown in SEQ ID NO: 159 or a variant thereof
  • SEQ ID NO: CDR3 of the sequence shown in 155 or a variant thereof
  • CDR1 comprising the sequence shown in SEQ ID NO: 160 or its variants
  • CDR2 comprising the sequence shown in SEQ ID NO: 161 or its variants
  • SEQ ID NO: CDR3 of the sequence shown in 162 or a variant thereof
  • the variant described in any one of (1)-(5) has one or several amino acid substitutions, deletions or additions compared with the sequence it is derived from; preferably, the substitutions are conservative substitutions;
  • the single domain antibody or antigen-binding fragment thereof comprises:
  • CDR1 comprising the sequence shown in SEQ ID NO:150
  • CDR2 comprising the sequence shown in SEQ ID NO:151
  • CDR3 comprising the sequence shown in SEQ ID NO:152;
  • CDR1 comprising the sequence shown in SEQ ID NO:153
  • CDR2 comprising the sequence shown in SEQ ID NO:154
  • CDR3 comprising the sequence shown in SEQ ID NO:155;
  • CDR1 comprising the sequence shown in SEQ ID NO:156
  • CDR2 comprising the sequence shown in SEQ ID NO:157
  • CDR3 comprising the sequence shown in SEQ ID NO:155;
  • CDR1 comprising the sequence set forth in SEQ ID NO:158
  • CDR2 comprising the sequence set forth in SEQ ID NO:159
  • CDR3 comprising the sequence set forth in SEQ ID NO:155; or
  • CDR1 including the sequence shown in SEQ ID NO:160
  • CDR2 including the sequence shown in SEQ ID NO:161
  • CDR3 including the sequence shown in SEQ ID NO:162.
  • the invention in a third aspect, relates to a multispecific antibody comprising a CD3-targeting antigen-binding domain and at least one other antigen-targeting antigen-binding domain, said CD3-targeting antigen-binding domain being selected from a first According to the antibody or antigen-binding fragment thereof, the other antigen is selected from tumor-associated antigens (TAAs) and/or immune checkpoint molecules.
  • TAAs tumor-associated antigens
  • the present invention relates to an isolated nucleic acid molecule encoding:
  • the multispecific antibody of the third aspect or a polypeptide chain thereof.
  • the present invention relates to a vector comprising the nucleic acid molecule of the fourth aspect.
  • the present invention relates to a host cell comprising the nucleic acid molecule of the fourth aspect or the vector of the fifth aspect.
  • the present invention relates to a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof described in the first aspect, the single domain antibody or antigen-binding fragment thereof described in the second aspect, the multispecific antibody described in the third aspect Antibody, the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, or the host cell of the sixth aspect, and pharmaceutically acceptable carriers and/or excipients.
  • the present invention relates to the antibody or antigen-binding fragment thereof in the first aspect, the single domain antibody or antigen-binding fragment thereof in the second aspect, the multispecific antibody in the third aspect, the fourth aspect
  • the isolated nucleic acid molecule, the vector of the fifth aspect, or the host cell of the sixth aspect, or the pharmaceutical composition of the seventh aspect are used for preventing and/or treating diseases, or in Use in the preparation of medicines for preventing and/or treating diseases.
  • the present invention relates to a method for preventing and/or treating a disease, which comprises administering the antibody or antigen-binding fragment thereof of the first aspect, the monoclonal antibody of the second aspect to a subject in need thereof.
  • a domain antibody or an antigen-binding fragment thereof, the multispecific antibody of the third aspect, the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, or the host cell of the sixth aspect, or the host cell of the sixth aspect The pharmaceutical composition described in the seventh aspect.
  • Fig. 1 shows a schematic diagram of the structure of the CD3-MSLN bispecific antibody in Example 13.
  • Fig. 2 shows the assay results of the binding activity of the CD3-MSLN bispecific antibody to MC38-MSLN cells in Example 14.
  • Fig. 3A-Fig. 3D show the results of the dynamic affinity determination of the CD3-MSLN bispecific antibody in Example 14 to the human CD3 ⁇ recombinant antigen.
  • Fig. 4 shows the results of dynamic affinity determination of CD3-MSLN bispecific antibody to MSLN recombinant antigen in Example 14.
  • 5A-5F show the test results of Jurkat-TIGIT-luc cell stability in Example 15.
  • Fig. 6A-Fig. 6F show the activation assay results of CD3-MSLN bispecific antibody on T cell activation signaling pathway in Example 16.
  • Fig. 8 shows a schematic diagram of the structure of the CD3-CD19 bispecific antibody in Example 18.
  • Figure 9 shows the results of TDCC assay mediated by the CD3-CD19 bispecific antibody in Example 18.
  • Figure 10 shows the results of TDCC assay mediated by the CD3-CD20 bispecific antibody in Example 19.
  • Figure 11 shows the results of TDCC assay mediated by the CD3-trop2 bispecific antibody in Example 20.
  • Figure 12 shows the results of TDCC assay mediated by the CD3-Her2 bispecific antibody in Example 21.
  • Figure 13 shows the results of TDCC assay mediated by the CD3-Caludin18.2 bispecific antibody in Example 22.
  • Figure 14A- Figure 14I show the detection results of IL-2 release level mediated by CD3-MSLN bispecific antibody in Example 23.
  • Figure 15A- Figure 15I show the detection results of INF- ⁇ release level mediated by CD3-MSLN bispecific antibody in Example 23.
  • the present invention provides novel CD3 antibodies, and specifically provides the following aspects.
  • the present invention relates to an antibody or antigen-binding fragment thereof capable of specifically binding CD3, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein,
  • the VH comprises: HCDR1 comprising the sequence shown in SEQ ID NO:107, HCDR2 comprising the sequence shown in SEQ ID NO:108 and comprising X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 HCDR3 of the sequence shown in 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 ,
  • the VL comprises: LCDR1 comprising the sequence shown in SEQ ID NO:137, LCDR2 comprising the sequence shown in SEQ ID NO:138, and LCDR3 comprising the sequence shown in SEQ ID NO:139.
  • the HCDR3 is not set forth in any one of SEQ ID NOs: 60, 64, 77, 89.
  • the HCDR3 comprises: 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.
  • the HCDR3 comprises the sequence shown in any one of SEQ ID NOs: 56, 61, 69, 79, 82, 87.
  • the HCDR3 comprises:
  • HX 2 NFGNSYVSWFAY SEQ ID NO: 113
  • X 2 is A, E, H, K, Q or S, preferably H;
  • HGNFX 5 NSYVSWFAY SEQ ID NO: 114
  • X 5 is K, L, P, Q, R, S, V, W or Y, preferably K;
  • the HCDR3 comprises the sequence shown in any one of SEQ ID NOs: 75, 53, 72, 51, 57, 64.
  • the HCDR3 comprises:
  • the HCDR3 comprises the sequence shown in any one of SEQ ID NOs: 50, 66, 55, 85.
  • the HCDR3 comprises:
  • HX 2 X 3 FGNSYVSWFAY SEQ ID NO: 120
  • X 2 is A, E, H, K, Q or S, preferably E
  • X 3 is D or R, preferably R
  • X 2 is E
  • X 3 is R;
  • HX 2 NFX 5 NSYVSWFAY (SEQ ID NO: 121), wherein X 2 is A, E, H, K, Q or S, preferably A or S; X 5 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 respectively A/L, S/P, S/L, A/S, S/Q, S/V or S/R;
  • HX 2 NFGNX 7 YVSWFAY SEQ ID NO: 123
  • X 2 is A, E, H, K, Q or S, preferably S
  • X 7 is G, N or T, Preferably T or G
  • X 2 is S
  • X 7 is T or G
  • HX 2 NFGNSX 8 VSWFAY (SEQ ID NO: 124), wherein X 2 is A, E, H, K, Q or S, preferably S; X 8 is A, Q or R, Preferably R; Preferably, X 2 is S, X 8 is R;
  • HX 2 NFGNSYVSWX 11 AY (SEQ ID NO: 125), wherein, X 2 is A, E, H, K, Q or S, preferably S; X 11 is W; preferably, X 2 is S, X 11 is W;
  • HX 2 NFGNSYVSWFX 12 Y (SEQ ID NO: 126), wherein X 2 is A, E, H, K, Q or S, preferably S; X 12 is E, K or Q, is preferably E or Q; preferably, X2 is S or Q, and X12 is E or Q; preferably, X2 and X12 are S/E, Q/Q or S/Q, respectively; or,
  • HX 2 NFGNSYVSWFAX 13 (SEQ ID NO: 127), wherein, X 2 is A, E, H, K, Q or S, preferably A; X 13 is H, L, M or S , preferably S; preferably, X 2 is A, and X 13 is S.
  • the HCDR3 comprises SEQ ID NOs: 50, 69, 49, 58, 59, 60, 70, 82, 86, 71, 68, 77, 78, 81, 62, 84, 89, 74 Any of the sequences shown.
  • the HCDR3 comprises:
  • HX 2 NFX 5 NSYVSWFAY (SEQ ID NO: 121), wherein X 2 is A, E, H, K, Q or S, preferably A or S; X 5 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 respectively A/L, S/P, S/L, A/S, S/Q, S/V or S/R;
  • HGNFX 5 X 6 SYVSWFAY SEQ ID NO: 129
  • X is K, L, P, Q, R, S , V, W or Y, preferably P or K
  • X 6 is M, Q or R, preferably M or Q
  • X 5 is P or K
  • X 6 is M or Q
  • X 5 and X 6 are P/M or K/Q respectively;
  • HGNFX 5 NSYVSWFX 12 Y (SEQ ID NO: 131), wherein X 5 is K, L, P, Q, R, S, V, W or Y, preferably Q or L; X 12 is E, K or Q, preferably Q; preferably, X 5 is Q or L, X 12 is Q; or,
  • HGNFX 5 NSYVSWFAX 13 (SEQ ID NO: 132), wherein X 5 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 X13 is H or S; preferably, X5 and X13 are Y/H or W/S respectively.
  • the HCDR3 comprises SEQ ID NOs: 49, 58, 59, 60, 70, 82, 86, 76, 80, 61, 88, 52, 83, 54, 65 shown in any one sequence.
  • the HCDR3 comprises:
  • HX 2 NFGNSYVSWFX 12 Y (SEQ ID NO: 126), wherein X 2 is A, E, H, K, Q or S, preferably S; X 12 is E, K or Q, Preferably E or Q; Preferably, X 2 is S, X 12 is E or Q; Preferably, X 2 and X 12 are S/E, Q/Q or S/Q respectively;
  • HGNFX 5 NSYVSWFX 12 Y (SEQ ID NO: 131), wherein X 5 is K, L, P, Q, R, S, V, W or Y, preferably Q or L; X 12 is E, K or Q, preferably Q; preferably, X 5 is Q or L, X 12 is Q; or,
  • HGNFGNSX 8 VSWFX 12 Y (SEQ ID NO: 134), wherein X 8 is A, Q or R, preferably A or Q; X 12 is E, K or Q, preferably Q; Preferably, X 8 is A or Q, and X 12 is Q.
  • the HCDR3 comprises the sequence shown in any one of SEQ ID NOs: 55, 85, 62, 84, 89, 52, 83, 67, 73.
  • the HCDR3 comprises:
  • HX 2 NFGNSYVSWFAX 13 (SEQ ID NO: 127), wherein X 2 is A, E, H, K, Q or S, preferably A; X 13 is H, L, M or S , preferably S; preferably, X 2 is A, X 13 is S;
  • HGNFX 5 NSYVSWFAX 13 (SEQ ID NO: 132), wherein X 5 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, X13 is H or S; preferably, X5 and X13 are Y/H or W/S respectively; or,
  • HGNFGNX 7 YVSWFAX 13 SEQ ID NO: 133
  • X 7 is G, N or T, preferably T or N
  • X 13 is H, L, M or S, preferably M or S
  • X 7 is T or N
  • X 13 is M or S
  • X 7 and X 13 are T/M or N/S respectively.
  • the HCDR3 comprises the sequence shown in any one of SEQ ID NOs: 74, 54, 65, 56, 79.
  • the HCDR3 comprises:
  • HX 2 X 3 FGNSYVSWFAY SEQ ID NO: 120
  • X 2 is A, E, H, K, Q or S, preferably E
  • X 3 is D or R, preferably R
  • X 2 is E and X 3 is R; or,
  • the HCDR3 comprises the sequence shown in any one of SEQ ID NOs: 69, 63.
  • the HCDR3 comprises: X 1 GNFX 5 NSYVSWFAX 13 (SEQ ID NO: 135) the sequence shown, wherein, X 1 is A or P, preferably P; X 5 is K, L, P, Q, R, S, V, W or Y, preferably P; X13 is H, L, M or S, preferably L; preferably, X1 is P, X5 is P, X13 is L .
  • the HCDR3 comprises the sequence shown in SEQ ID NO:87.
  • the VH comprises: HCDR1 comprising the sequence shown in SEQ ID NO: 107, HCDR2 comprising the sequence shown in SEQ ID NO: 108 and any one of the sequences comprising SEQ ID NOs: 49-89 and, the VL comprises: LCDR1 comprising the sequence shown in SEQ ID NO:137, LCDR2 comprising the sequence shown in SEQ ID NO:138 and LCDR3 comprising the sequence shown in SEQ ID NO:139.
  • the condition is that the HCDR3 is not shown in any one of SEQ ID NOs: 60, 64, 77, 89.
  • the antibody or antigen-binding fragment thereof further comprises a framework region derived from a human immunoglobulin.
  • the antibody or antigen-binding fragment thereof comprises a framework region derived from an amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain framework region derived from an amino acid sequence encoded by a human heavy chain germline gene, and/or a heavy chain framework region derived from a human light chain germline gene The light chain framework region contained in the encoded amino acid sequence.
  • the VH comprises HFR1, HFR2, HFR3, and HFR4, wherein:
  • the HFR1 comprises the sequence shown in SEQ ID NO: 109;
  • the HFR2 comprises the sequence shown in SEQ ID NO:110;
  • the HFR3 comprises the sequence shown in VKX 1 RFTISRDDSKSX 2 LYLQMNX 3 LKTEDTAX 4 YYCVR (SEQ ID NO: 136); wherein, X 1 is G or D, X 2 is I or S, X 3 is N or S, X 4 is M or V;
  • the HFR4 comprises the sequence shown in SEQ ID NO:111.
  • the HFR3 comprises the sequence shown in any one of SEQ ID NOs: 91-106.
  • the VH comprises: the amino acid sequence shown in any one of SEQ ID NOs: 1-46 or a variant thereof, the variant having one or more amino acids compared to the sequence from which it is derived Substitutions, deletions or additions, or having 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 VH comprises: the amino acid sequence shown in any one of SEQ ID NOs: 8, 13, 22, 33, 37, 43 or a variant thereof, the variant and the sequence from which it is derived Compared to having one or several amino acid substitutions, deletions or additions, or having 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 VH comprises: the amino acid sequence shown in any one of SEQ ID NOs: 3, 5, 9, 16, 20, 25, 28 or a variant thereof, the variant is derived from has one or several amino acid substitutions, deletions or additions, 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 VH comprises: the amino acid sequence shown in any one of SEQ ID NOs: 2, 7, 18, 40, or a variant thereof, which has a or several amino acid substitutions, deletions or additions, or have 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 VH comprises: SEQ ID NOs: 2, 22, 1, 10, 11, 12, 23, 36, 37, 41, 24, 21, 30, 32, 35, 14, 31,
  • the VH comprises: An amino acid sequence or a variant thereof having one or several amino acid substitutions, deletions or additions, or at least 90%, at least 91%, at least 92% of the sequence from which it is derived , 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 VH comprises: the amino acid sequence shown in any one of SEQ ID NOs: 7, 40, 14, 31, 39, 45, 4, 38, 42, 19, 26 or a variant thereof, Said variant has one or several amino acid substitutions, deletions or additions, or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of the sequence from which it is derived , at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the VH comprises: the amino acid sequence shown in any one of SEQ ID NOs: 27, 46, 6, 17, 8, 33 or a variant thereof, the variant and the sequence from which it is derived Compared to having one or several amino acid substitutions, deletions or additions, or having 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 VH comprises: the amino acid sequence shown in SEQ ID NOs: 22 or 15, or a variant thereof, which has one or several amino acid substitutions compared to the sequence from which it is derived, Deletion or addition, or having 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 VH comprises: the amino acid sequence shown in SEQ ID NO: 43, or a variant thereof, which has one or several amino acid substitutions, deletions, or Add, or have 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 sex.
  • the VH comprises: the amino acid sequence shown in any one of SEQ ID NOs: 1-11, 13-15, 17-19, 21-29, 31-35, 37-44, 46 or A variant thereof, which has a substitution, deletion or addition of one or several amino acids, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94% of the sequence from which it is derived , at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the VL comprises LFR1, LFR2, LFR3, and LFR4, wherein:
  • the LFR1 comprises the sequence shown in SEQ ID NO:140;
  • the LFR2 comprises the sequence shown as WX 1 QQTPGQAX 2 RX 3 LIX 4 (SEQ ID NO: 144); wherein, X 1 is V or Y, X 2 is F or P, X 3 is G or T, X 4 be G or Y;
  • the LFR3 comprises the sequence shown as GVPARFSGSX 4 X 5 GX 6 KAALTITGAQADDESX 7 YFCA (SEQ ID NO: 147); wherein, X 4 is L or I, X 5 is L or I, X 6 is D or N, X 7 is I or D;
  • the LFR4 comprises the sequence shown in SEQ ID NO:141.
  • the LFR2 comprises the sequence shown in SEQ ID NO: 142 or 143.
  • the LFR3 comprises the sequence shown in SEQ ID NO: 145 or 146.
  • the VL comprises: LFR1 shown in SEQ ID NO:140, LFR2 shown in SEQ ID NO:142, LFR3 shown in SEQ ID NO:145, LFR3 shown in SEQ ID NO:141 LFR4.
  • the VL comprises: LFR1 shown in SEQ ID NO:140, LFR2 shown in SEQ ID NO:143, LFR3 shown in SEQ ID NO:146, LFR3 shown in SEQ ID NO:141 LFR4.
  • the VL comprises: the amino acid sequence shown in SEQ ID NO: 47 or 48 or a variant thereof, the variant having one or several amino acid substitutions compared to the sequence from which it is derived, Deletion or addition, or having 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 comprises: a VH comprising a sequence shown in any one of SEQ ID NOs: 1-46 or a variant thereof, and a VH comprising a sequence shown in SEQ ID NO: 47 or a variant thereof VL of a variant; wherein said variant has one or several amino acid substitutions, deletions or additions, or 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 comprises: a VH comprising a sequence shown in any one of SEQ ID NOs: 1-46 or a variant thereof, and a VH comprising a sequence shown in SEQ ID NO: 48 or a variant thereof VL of a variant; wherein said variant has one or several amino acid substitutions, deletions or additions, or 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 comprises: comprising any one of SEQ ID NOs: 1-11, 13-15, 17-19, 21-29, 31-35, 37-44, 46 A VH of the sequence shown or a variant thereof, and a VL comprising the sequence shown in SEQ ID NO: 47 or a variant thereof; wherein the variant has one or several amino acid substitutions compared to the sequence from which it is derived, Deletion or addition, or having 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 comprises: comprising any one of SEQ ID NOs: 1-11, 13-15, 17-19, 21-29, 31-35, 37-44, 46 A VH of the sequence shown or a variant thereof, and a VL comprising the sequence shown in SEQ ID NO: 48 or a variant thereof; wherein the variant has one or several amino acid substitutions compared to the sequence from which it is derived, Deletion or addition, or having 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 comprises: VH comprising a sequence shown in any one of SEQ ID NOs: 8, 13, 22, 33, 37, 43 or a variant thereof, and comprising SEQ ID NOs: The VL of the sequence shown in NO:47 or its variant; wherein, the variant has one or several amino acid substitutions, deletions or additions compared to the sequence it is derived from, 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 comprises: VH comprising a sequence shown in any one of SEQ ID NOs: 8, 13, 22, 33, 37, 43 or a variant thereof, and comprising SEQ ID NOs: The VL of the sequence shown in NO:48 or its variant; wherein, the variant has one or several amino acid substitutions, deletions or additions compared to the sequence it is derived from, 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 VH comprises: HCDR1-HCDR3 and HFR1-HFR4, which have the following characteristics:
  • the HCDR1 comprises the sequence shown in SEQ ID NO: 107;
  • the HCDR2 comprises the sequence shown in SEQ ID NO: 108;
  • the HCDR3 comprises a sequence selected from the group consisting of:
  • HX 2 NFX 5 NSYVSWFAY (SEQ ID NO: 121), wherein X 2 is A, E, H, K, Q or S, preferably A or S; X 5 is K, L, P, Q, R, S, V, W or Y, preferably L, P, S, Q, V or R; preferably, X 2 is A or S, X 5 is L, P, S, Q , V or R; preferably, X 2 and X 5 are A/L, S/P, S/L, A/S, S/Q, S/V or S/R respectively;
  • HX 2 NFGX 6 SYVSWFAY (SEQ ID NO: 122), wherein, X 2 is A, E, H, K, Q or S, preferably S; X 6 is M, Q or R, preferably R; preferably, X 2 is S, X 6 is R;
  • HX 2 NFGNX 7 YVSWFAY SEQ ID NO: 123
  • X 2 is A, E, H, K, Q or S, preferably S
  • X 7 is G, N or T, preferably T or G
  • X 2 is S
  • X 7 is T or G
  • HX 2 NFGNSYVSWX 11 AY (SEQ ID NO: 125), wherein X 2 is A, E, H, K, Q or S, preferably S; X 11 is W; preferably , X 2 is S, X 11 is W;
  • HX 2 NFGNSYVSWFX 12 Y (SEQ ID NO: 126), wherein, X 2 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, X12 is E or Q; preferably, X2 and X12 are respectively S/E, Q/Q or S/Q; or,
  • HX 2 NFGNSYVSWFAX 13 (SEQ ID NO: 127), wherein, X 2 is A, E, H, K, Q or S, preferably A; X 13 is H, L, M Or S, preferably S; Preferably, X 2 is A, X 13 is S;
  • HFR1 comprises the sequence shown in SEQ ID NO:109;
  • HFR2 comprises the sequence shown in SEQ ID NO:110;
  • the HFR3 comprises the sequence shown by VKX 1 RFTISRDDSKSX 2 LYLQMNX 3 LKTEDTAX 4 YYCVR (SEQ ID NO: 136); wherein, X 1 is G or D, X 2 is I or S, and X 3 is N or S , X 4 is M or V;
  • HFR4 comprises the sequence shown in SEQ ID NO:111;
  • the VL has the following characteristics: 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.
  • the VH has the following characteristics: the item 1, the item 2, the item 3, the item 4, the item 5, the item 6', the item 7, wherein, the item 6': the HFR3 comprises SEQ ID NOs: the sequence shown in any one of 91-106.
  • the VL preferably has the following characteristics: Item 8 or Item 9.
  • the VH has the following characteristics: the item 1, the item 2, the item 3', the item 4, the item 5, the item 6, the item 7, wherein, the item 3': the HCDR3 comprises SEQ ID NOs: the sequence shown in any one of 50, 69, 49, 58, 59, 60, 70, 82, 86, 71, 68, 77, 78, 81, 62, 84, 89, 74.
  • the VL preferably has the following characteristics: Item 8 or Item 9.
  • said VH is characterized by said item 1, item 2, item 3', item 4, item 5, item 6', item 7.
  • the VL preferably has the following characteristics: Item 8 or Item 9.
  • the VH comprises: SEQ ID NOs: 2, 22, 1, 10, 11, 12, 23, 36, 37, 41, 24, 21, 30, 32, 35, 14, 31, The amino acid sequence shown in any one of 39, 45, 27 and 46.
  • Said VL preferably comprises the sequence shown in SEQ ID NO:47 or 48, for example the sequence shown in SEQ ID NO:47.
  • the VH comprises: HCDR1-HCDR3 and HFR1-HFR4, which have the following characteristics:
  • the HCDR1 comprises the sequence shown in SEQ ID NO: 107;
  • the HCDR2 comprises the sequence shown in SEQ ID NO: 108;
  • the HCDR3 comprises a sequence selected from the group consisting of:
  • HX 2 NFX 5 NSYVSWFAY SEQ ID NO: 121
  • X 2 is A, E, H, K, Q or S, preferably A or S
  • X 5 is K, L, P, Q, R, S, V, W or Y, preferably L, P, S, Q, V or R
  • X 2 is A or S
  • X 5 is L, P, S, Q , V or R
  • X 2 and X 5 are A/L, S/P, S/L, A/S, S/Q, S/V or S/R respectively;
  • HGNFX 5 X 6 SYVSWFAY SEQ ID NO: 129
  • X 5 is K, L, P, Q, R, S, V, W or Y, preferably P or K
  • X 6 is M, Q or R, preferably M or Q
  • X 5 is P or K
  • X 6 is M or Q
  • X 5 and X 6 are P/M or K/Q respectively ;
  • HGNFX 5 NSYX 9 SWFAY SEQ ID NO: 130
  • X 5 is K, L, P, Q, R, S, V, W or Y, preferably K or Q
  • X 9 is G or I
  • X 5 is K or Q
  • X 9 is G or I
  • X 5 and X 9 are K/G or Q/I respectively;
  • HGNFX 5 NSYVSWFX 12 Y (SEQ ID NO: 131), wherein X 5 is K, L, P, Q, R, S, V, W or Y, preferably Q or L ; X 12 is E, K or Q, preferably Q; Preferably, X 5 is Q or L, X 12 is Q; or,
  • HGNFX 5 NSYVSWFAX 13 (SEQ ID NO: 132), wherein X 5 is K, L, P, Q, R, S, V, W or Y, preferably Y or W; X13 is H, L, M or S, preferably H or S; preferably, X5 is Y or W, X13 is H or S; preferably, X5 and X13 are Y/H or W/ S;
  • HFR1 comprises the sequence shown in SEQ ID NO: 109;
  • HFR2 comprises the sequence shown in SEQ ID NO: 110;
  • the HFR3 comprises the sequence shown in VKX 1 RFTISRDDSKSX 2 LYLQMNX 3 LKTEDTAX 4 YYCVR (SEQ ID NO: 136); wherein, X 1 is G or D, X 2 is I or S, and X 3 is N or S , X 4 is M or V;
  • HFR4 comprises the sequence shown in SEQ ID NO: 111;
  • the VL has the following characteristics: 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.
  • the VH is characterized by: the item 11, the item 12, the item 13, the item 14, the item 15, the item 16', the item 17, wherein, the item 16': the HFR3 comprises SEQ ID NOs: the sequence shown in any one of 91-106.
  • the VL is preferably characterized by: Item 18 or Item 19.
  • said VH has the following characteristics: said item 11, item 12, item 13', item 14, item 15, item 16, item 17, wherein, item 13': said HCDR3 comprises SEQ ID NOs: the sequence shown in any one of 49, 58, 59, 60, 70, 82, 86, 76, 80, 61, 88, 52, 83, 54, 65.
  • the VL is preferably characterized by: Item 18 or Item 19.
  • said VH is characterized by: said item 11, item 12, item 13', item 14, item 15, item 16', item 17.
  • the VL is preferably characterized by: Item 18 or Item 19.
  • the VH comprises: Amino acid sequence shown in one item.
  • Said VL preferably comprises the sequence shown in SEQ ID NO:47 or 48, for example the sequence shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment thereof further comprises a constant region derived from a human immunoglobulin.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin.
  • the heavy chain constant region is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.
  • the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin.
  • the light chain constant region is a kappa light chain constant region.
  • the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) set forth in SEQ ID NO: 165.
  • the Fc domain comprised by the antibody or antigen-binding fragment thereof is a native Fc region comprising an amino acid sequence identical to that of an Fc region found in nature.
  • a native Fc region may have effector functions.
  • effector functions include binding to Fc receptors; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); Downregulation of cell surface receptors (eg, B cell receptors); and B cell activation, among others.
  • the Fc domain contained in the antibody or antigen-binding fragment thereof may also be a variant Fc region, which may contain one or more (for example, 1-10, for example, 1 - 5) amino acid mutations or chemical modifications to alter one or more of the following properties of the antibodies of the invention: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function or complement function wait.
  • Effector function can be altered by substituting at least one amino acid residue in the native Fc region with a different residue or by chemical modification, e.g., altering the affinity of the antibody for an effector ligand such as FcR or complement C1q (e.g. decrease or increase).
  • the antibody or antigen-binding fragment thereof comprises a mutated or chemically modified Fc region that has 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).
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP Enhanced antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • Methods for obtaining the above-mentioned effector functions with changes (such as reduction) are known in the art, for example, one or more (such as 1, 2, 3 or 4) selected from the following can be introduced into the heavy chain constant region ) mutations: L234A, L235A, G237A, K322A according to EU numbering.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 164.
  • the antigen-binding fragment is selected from Fab, Fab', (Fab') 2 , Fv, disulfide-linked Fv, scFv, diabody.
  • the antibody is an IgG antibody, such as an IgGl, IgG2, IgG3 or IgG4 antibody.
  • the antibody or antigen-binding fragment thereof specifically binds the ⁇ and/or ⁇ chains of CD3 (eg, human and/or cynomolgus CD3).
  • the antibody or antigen-binding fragment thereof has a concentration of not lower than 6 ⁇ 10 ⁇ 10 M, not lower than 6.5 ⁇ 10 ⁇ 10 M, not lower than 6.5 ⁇ 10 ⁇ 10 M, as determined by surface plasmon resonance (SPR).
  • SPR surface plasmon resonance
  • the antibody or antigen-binding fragment thereof is present at no more than 10 -7 M, such as no more than 9.5 x 10 -8 M, no more than 9 x 10 -8 M when determined by SPR , not higher than 8.5 ⁇ 10 -8 M, not higher than 8 ⁇ 10 -8 M, not higher than 7.5 ⁇ 10 -8 M, not higher than 7 ⁇ 10 -8 M, not higher than 6.5 ⁇ 10 -8 M , not higher than 6 ⁇ 10 -8 M, not higher than 5.5 ⁇ 10 -8 M, not higher than 5 ⁇ 10 -8 M, not higher than 4.5 ⁇ 10 -8 M, not higher than 4 ⁇ 10 -8 M , not higher than 3.5 ⁇ 10 -8 M , not higher than 3 ⁇ 10 -8 M, not higher than 2.5 ⁇ 10 -8 M, or not higher than 2 ⁇ 10 -8 M K D binding to CD3 (for example, human and and/or cynomolgus CD3, eg ⁇ and/or ⁇ chain).
  • CD3 for example, human and and/or cyn
  • the antibody or antigen-binding fragment thereof is present at a concentration of not less than 0.05 nM, not less than 0.1 nM, not less than 0.2 nM, not less than 0.3 nM, not less than 0.2 nM, or not less than 0.3 nM when determined by flow cytometry.
  • nM EC 50 binding to CD3 (eg, human and/or cynomolgus CD3, eg CD3 expressing cells) .
  • CD3 eg, human and/or cynomolgus CD3, eg CD3 expressing cells
  • the antibody or antigen-binding fragment thereof is present at no greater than 20 nM, such as no greater than 15 nM, no greater than 10 nM, no greater than 9.5 nM, no greater than 9nM, not higher than 8.5nM, not higher than 8nM, not higher than 7.5nM, not higher than 7nM, not higher than 6.5nM, not higher than 6nM, not higher than 5.5nM, not higher than 5nM, not higher than 4.5 nM, not higher than 4 nM, not higher than 3.5 nM, not higher than 3 nM, not higher than 2.5 nM, not higher than 2 nM, not higher than 1.5 nM, or not higher than 1 nM EC50 binding to CD3 (e.g., human and/or or cynomolgus CD3, eg CD3-expressing cells).
  • CD3 e.g., human and/or or cynomolgus CD3, eg CD3-expressing cells.
  • the invention relates to single domain antibodies or antigen-binding fragments thereof capable of specifically binding to MSLN, eg human MSLN.
  • Single domain antibodies typically consist of four framework regions (FRs) and three complementarity determining regions (CDRs), called FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, the antigen-binding fragments of which comprise the single domain antibody At least a portion, the portion is sufficient to confer on the fragment the ability to specifically bind an antigen (eg, MSLN).
  • Single domain antibodies can be truncated at the N-terminus or C-terminus so that they comprise only part of FR1 and/or FR4, or lack one or both of those framework regions, so long as they substantially retain antigen binding and specificity.
  • the single domain antibody or antigen-binding fragment thereof of the invention comprises the following CDRs defined by the IMGT numbering system: CDR1 comprising the sequence shown in SEQ ID NO: 150 or a variant thereof comprising SEQ ID NO: 151
  • the substitution is a conservative substitution.
  • the single domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the IMGT numbering system: CDR1 comprising the sequence shown in SEQ ID NO:150, CDR2 comprising the sequence shown in SEQ ID NO:151 , and a CDR3 comprising the sequence shown in SEQ ID NO:152.
  • the single domain antibody or antigen-binding fragment thereof of the invention comprises the following CDRs defined by the Kabat numbering system: CDR1 comprising the sequence shown in SEQ ID NO: 153 or a variant thereof comprising SEQ ID NO: 154
  • the substitution is a conservative substitution.
  • the single domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the Kabat numbering system: CDR1 comprising the sequence shown in SEQ ID NO: 153, CDR2 comprising the sequence shown in SEQ ID NO: 154 , and a CDR3 comprising the sequence shown in SEQ ID NO:155.
  • the single domain antibody or antigen-binding fragment thereof of the invention comprises the following CDRs defined by the AbM numbering system: CDR1 comprising the sequence shown in SEQ ID NO: 156 or a variant thereof comprising SEQ ID NO: 157
  • the substitution is a conservative substitution.
  • the single domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the AbM numbering system: CDR1 comprising the sequence shown in SEQ ID NO:156, CDR2 comprising the sequence shown in SEQ ID NO:157 , and a CDR3 comprising the sequence shown in SEQ ID NO:155.
  • the single domain antibody or antigen-binding fragment thereof of the invention comprises the following CDRs as defined by the Chothia numbering system: CDR1 comprising the sequence shown in SEQ ID NO: 158 or a variant thereof comprising SEQ ID NO: 159
  • the substitution is a conservative substitution.
  • the single domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the Chothia numbering system: CDR1 comprising the sequence shown in SEQ ID NO:158, CDR2 comprising the sequence shown in SEQ ID NO:159 , and a CDR3 comprising the sequence shown in SEQ ID NO:155.
  • the single domain antibody or antigen-binding fragment thereof of the present invention comprises the following CDRs defined by the Contact numbering system: CDR1 comprising the sequence shown in SEQ ID NO: 160 or a variant thereof comprising SEQ ID NO: 161
  • the substitution is a conservative substitution.
  • the single domain antibody or antigen-binding fragment thereof comprises the following CDRs defined by the Contact numbering system: CDR1 comprising the sequence shown in SEQ ID NO:160, CDR2 comprising the sequence shown in SEQ ID NO:161 , and a CDR3 comprising the sequence shown in SEQ ID NO:162.
  • the single domain antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 149 or a variant thereof 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 , or have one or several amino acid substitutions, deletions or additions compared thereto (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.
  • a single domain antibody or antigen-binding fragment thereof of the invention may be a humanized VHH, ie a VHH in which one or more framework regions have been substantially replaced with human framework regions.
  • 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 contained in the amino acid sequence encoded by a human heavy chain germline antibody gene).
  • framework region optionally comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ) back mutation from human residues to camel residues.
  • the single domain antibody or antigen-binding fragment thereof is present at no higher than 10 -8 M, no higher than 9.5 x 10 -9 M, 9 x 10 -9 M, no higher than 9.5 x 10 -9 M, or Higher than 8.5 ⁇ 10 -9 M, not higher than 8 ⁇ 10 -9 M, not higher than 7.5 ⁇ 10 -9 M, not higher than 7 ⁇ 10 -9 M, not higher than 6.5 ⁇ 10 -9 M, not higher Higher than 6 ⁇ 10 -9 M, not higher than 5.5 ⁇ 10 -9 M, not higher than 5 ⁇ 10 -9 M, not higher than 4.5 ⁇ 10 -9 M, not higher than 4 ⁇ 10 -9 M, not higher Higher than 3.5 ⁇ 10 -9 M, not higher than 3 ⁇ 10 -9 M, not higher than 2.5 ⁇ 10 -9 M, not higher than 2 ⁇ 10 -9 M, not higher than 1.5 ⁇ 10 -9 M, not higher Higher than 10 -9 M, not higher than 9.5 ⁇ 10 -10 M , not higher than 9 ⁇ 10 -10 M, not higher than 8.5 ⁇
  • the antibody or antigen-binding fragment thereof is present at 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 when determined by flow cytometry. , 0.3 nM, 0.2 nM, 0.1 nM or less EC 50 binding to MSLN (eg, human MSLN, eg, cells expressing MSLN).
  • MSLN eg, human MSLN, eg, cells expressing MSLN
  • the antibodies or antigen-binding fragments thereof of the first aspect of the invention and/or the single domain antibodies or antigen-binding fragments thereof of the second aspect may be used to form multispecific antibodies.
  • Multispecific antibodies are antibodies that have binding specificities for at least two (eg, two, three or four) different antigens, thereby being able to bind at least two different binding sites and/or target molecules.
  • the invention relates to multispecific antibodies.
  • the present invention provides a multispecific antibody 1, which comprises an antigen-binding domain targeting CD3 and at least one antigen-binding domain targeting other antigens, and the antigen-binding domain targeting CD3 is selected from the first aspect Antibodies or antigen-binding fragments thereof, the other antigens are selected from tumor-associated antigens (TAAs) and/or immune checkpoint molecules.
  • TAAs tumor-associated antigens
  • the multispecific antibody is a bispecific antibody comprising the CD3-targeting antigen-binding domain of the first aspect and the tumor-associated antigen-targeted antigen-binding domain.
  • the bispecific antibody comprises a first antigen-binding region (antibody as described in the first aspect) that binds a T cell-specific target (such as CD3) and a second antigen-binding region that binds a tumor-specific target
  • the bispecific Antibodies can promote the targeting and recruitment of T cells to tumor cells by binding to CD3 present on T cells and specific target antigens on tumor cells, and induce tumor-specific (MHC-independent) cytotoxic T cell killing activity.
  • the multispecific antibody is a trispecific antibody comprising the CD3-targeting antigen-binding domain of the first aspect, the tumor-associated antigen-targeted antigen-binding domain, and the The antigen binding domain of the immune checkpoint molecule.
  • a bispecific antibody of the invention may be: (i) a single antibody with two arms comprising different antigen binding regions, (ii) for example via two scFvs connected in series by an additional peptide linker, Single-chain antibodies with specificity for two different epitopes; (iii) dual variable domain antibodies (DVD-Ig TM ), in which each light and heavy chain contains two variable structures connected in tandem by a short peptide link domains; (iv) chemically linked bispecific (Fab')2 fragments; (v) TandAb, which is a fusion of two single chain diabodies, resulting in a tetravalent bispecific with two binding sites each for the target antigen specific antibodies; (vi) flexible bodies (flexibodies), which are combinations of scFv and diabodies, resulting in multivalent molecules; (vii) so-called "dock and lock" molecules,
  • the CD3-targeting antigen binding domain is a full-length antibody, Fv fragment, Fab fragment, F(ab')2 fragment, or scFv.
  • the other antigen-binding domains comprised by the multispecific antibody are each independently selected from From full length antibody, Fv fragment, Fab fragment, F(ab')2 fragment, scFv or VHH.
  • the individual antigen binding domains comprised by the multispecific antibody are linked by a peptide linker.
  • the antigen-binding domain targeting a tumor-associated antigen or an immune checkpoint molecule is optionally linked to the N-terminus of the heavy chain of the antigen-binding domain targeting CD3 and/or C-terminus, and/or the N-terminus and/or C-terminus of the light chain connected to the CD3-targeting antigen-binding domain.
  • the antigen-binding domain targeting CD3 comprises: at least one heavy chain and at least one light chain, and the antigen-binding domain targeting a tumor-associated antigen or an immune checkpoint molecule is associated with the The heavy chain is connected; or the antigen-binding domain targeting CD3 comprises: two identical heavy chains and two identical light chains, and the antigen-binding domain targeting tumor-associated antigens or immune checkpoint molecules and The two heavy chains are connected.
  • the tumor-associated antigen is selected from CD19, BCMA, EGFR, HER2, HER3, HER4, PSMA, EpCAM, EphA2, CD33, CD123, CD38, CLDN18, MSLN, TROP2, Mucin1, 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, Glo
  • the tumor-associated antigen is selected from MSLN, CD19, CD20, TROP2, HER2 or Caludin18.2.
  • the immune checkpoint molecule is selected from 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, or any combination thereof.
  • the multispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the heavy chain of the antibody described in the first aspect together with N or C An antigen-binding domain that specifically binds to a tumor-associated antigen (such as MSLN, CD19, CD20, Trop2, Her2 or Caludin18.2) connected at the end; the second polypeptide chain comprises the light chain of the antibody described in the first aspect.
  • a tumor-associated antigen such as MSLN, CD19, CD20, Trop2, Her2 or Caludin18.
  • the multispecific antibody is a bispecific antibody targeting CD3/MSLN, which comprises an antigen-binding domain that specifically binds CD3 and an antigen-binding domain that specifically binds MSLN, and the target
  • the antigen-binding domain targeting CD3 is selected from the antibody or antigen-binding fragment thereof in the first aspect; the antigen-binding domain targeting MSLN is selected from the single-domain antibody or antigen-binding fragment thereof in the second aspect.
  • the bispecific antibody targeting CD3/MSLN comprises a first polypeptide chain and a second polypeptide chain
  • the first polypeptide chain comprises the structure shown below: [VH ]-[CH]-[L]-[VHH], wherein [VH] is selected from the heavy chain variable region sequence provided in the first aspect (for example, the sequence shown in any one of SEQ ID NOs: 1-46)
  • [CH] is selected from the heavy chain constant region sequence provided in the first aspect (such as the sequence shown in SEQ ID NO: 164)
  • [L] is a peptide linker (such as comprising one or more glycine and/or one or more A serine peptide linker, such as the sequence shown in SEQ ID NO: 163)
  • [VHH] is selected from the single domain antibody sequence provided in the second aspect (such as the sequence shown in SEQ ID NO: 149);
  • the second multiple The peptide chain comprises the following structure: [VL]-[CL], wherein [VL] is selected from the light chain
  • the bispecific antibody targeting CD3 and MSLN of the present invention has a concentration of no less than 6 ⁇ 10 -10 M, no less than 6.5 ⁇ 10 -10 M, no less than 6.5 ⁇ 10 -10 M, no less than Less than 7 ⁇ 10 -10 M, no less than 7.5 ⁇ 10 -10 M, no less than 8 ⁇ 10 -10 M, no less than 8.5 ⁇ 10 -10 M, no less than 9 ⁇ 10 -10 M, no Less than 9.5 ⁇ 10 -10 M, not less than 1 ⁇ 10 -9 M, not less than 1.5 ⁇ 10 -9 M, not less than 2 ⁇ 10 -9 M, not less than 2.5 ⁇ 10 -9 M, not less than Less than 3 ⁇ 10 -9 M, not less than 3.5 ⁇ 10 -9 M, not less than 4 ⁇ 10 -9 M, not less than 4.5 ⁇ 10 -9 M, not less than 5 ⁇ 10 -9 M, no Less than 5.5 ⁇ 10 -9 M , not less than 6 ⁇ 10 -9 M, not less than 6.5 ⁇ 10
  • the bispecific antibody targeting CD3 and MSLN according to the present invention is not higher than 10 -7 M, such as not higher than 9.5 ⁇ 10 -8 M, not higher than 9.5 ⁇ 10 -8 M, when measured by SPR.
  • 10 -7 M such as not higher than 9.5 ⁇ 10 -8 M, not higher than 9.5 ⁇ 10 -8 M, when measured by SPR.
  • 9 ⁇ 10 -8 M not higher than 8.5 ⁇ 10 -8 M, not higher than 8 ⁇ 10 -8 M, not higher than 7.5 ⁇ 10 -8 M, not higher than 7 ⁇ 10 -8 M, not higher At 6.5 ⁇ 10 -8 M, not higher than 6 ⁇ 10 -8 M, not higher than 5.5 ⁇ 10 -8 M, not higher than 5 ⁇ 10 -8 M, not higher than 4.5 ⁇ 10 -8 M, not higher K D at 4 ⁇ 10 -8 M , not higher than 3.5 ⁇ 10 -8 M, not higher than 3 ⁇ 10 -8 M, not higher than 2.5 ⁇ 10 -8 M or not higher than 2 ⁇ 10 -8 M Binds CD3 (e
  • the bispecific antibody targeting CD3 and MSLN of the present invention has a concentration of not less than 0.05 nM, not less than 0.1 nM, not less than 0.2 nM, Not lower than 0.3nM, not lower than 0.4nM, not lower than 0.5nM, not lower than 0.6nM, not lower than 0.7nM, not lower than 0.8nM, not lower than 0.9nM, not lower than 1nM, not lower than 1.5nM, not lower than 2nM, not lower than 2.5nM, not lower than 3nM, not lower than 3.5nM, not lower than 4nM, not lower than 4.5nM, not lower than 5nM, not lower than 5.5nM, not lower than 6 nM, not less than 6.5 nM, not less than 7 nM, not less than 7.5 nM, not less than 8 nM, not less than 8.5 nM, not less than 9 nM EC 50 binding to CD3 (
  • the CD3- and MSLN-targeting bispecific antibody of the present invention is not higher than 20 nM, such as not higher than 15 nM, not higher than 10 nM, not higher when measured by flow cytometry.
  • the CD3- and MSLN-targeting bispecific antibody of the present invention is not higher than 20 nM, such as not higher than 15 nM, not higher than 10 nM, not higher when measured by flow cytometry.
  • At 9.5nM not higher than 9nM, not higher than 8.5nM, not higher than 8nM, not higher than 7.5nM, not higher than 7nM, not higher than 6.5nM, not higher than 6nM, not higher than 5.5nM, not higher EC50 binding at 5 nM, not higher than 4.5 nM, not higher than 4 nM, not higher than 3.5 nM, not higher than 3 nM, not higher than 2.5 nM, not higher than 2 nM, not higher than 1.5 nM or not higher than 1
  • the bispecific antibody targeting CD3 and MSLN of the present invention is not higher than 10 -8 M, not higher than 9.5 ⁇ 10 -9 M, 9 ⁇ 10 -9 M, not higher than 8.5 ⁇ 10 -9 M, not higher than 8 ⁇ 10 -9 M, not higher than 7.5 ⁇ 10 -9 M, not higher than 7 ⁇ 10 -9 M, not higher than 6.5 ⁇ 10 -9 M, not higher than 6 ⁇ 10 -9 M, not higher than 5.5 ⁇ 10 -9 M, not higher than 5 ⁇ 10 -9 M, not higher than 4.5 ⁇ 10 -9 M, not higher than 4 ⁇ 10 -9 M, not higher than 3.5 ⁇ 10 -9 M, not higher than 3 ⁇ 10 -9 M, not higher than 2.5 ⁇ 10 -9 M, not higher than 2 ⁇ 10 -9 M, not higher than 1.5 ⁇ 10 -9 M, not higher than 10 -9 M, not higher than 9.5 ⁇ 10 -10 M, not higher than 9 ⁇ 10 -10 M, not higher than 8.5 ⁇ 10 -10 M, not higher than 8 ⁇ 10
  • the bispecific antibodies targeting CD3 and MSLN described herein exhibit a concentration of less than about 100 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM when determined by flow cytometry.
  • An EC50 of nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less binds to MSLN (eg, human MSLN, eg, a cell expressing MSLN).
  • the multispecific antibody is a bispecific antibody targeting CD3/CD19, which comprises an antigen-binding domain that specifically binds CD3 and an antigen-binding domain that specifically binds CD19, and the target
  • the antigen-binding domain to CD3 is selected from the antibody or antigen-binding fragment thereof described in the first aspect;
  • the antigen-binding domain targeting CD19 comprises a light chain variable region and a heavy chain variable region, and the light chain can be
  • the variable region comprises the sequence shown in SEQ ID NO: 171 or a variant thereof
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO: 170 or a variant thereof.
  • the multispecific antibody is a bispecific antibody targeting CD3/CD20, which comprises an antigen binding domain targeting CD3 and an antigen binding domain specifically binding CD20, the targeting The antigen-binding domain of CD3 is selected from the antibody or antigen-binding fragment thereof described in the first aspect; the antigen-binding domain targeting CD20 comprises a light chain variable region and a heavy chain variable region, and the light chain variable region The region comprises the sequence shown in SEQ ID NO: 173 or a variant thereof, and the heavy chain variable region comprises the sequence shown in SEQ ID NO: 172 or a variant thereof.
  • the multispecific antibody is a bispecific antibody targeting CD3/trop2, which comprises an antigen binding domain targeting CD3 and an antigen binding domain specifically binding to trop2, the targeting The antigen-binding domain of CD3 is selected from the antibody or antigen-binding fragment thereof described in the first aspect; the antigen-binding domain targeting trop2 is VHH, which comprises the sequence shown in SEQ ID NO: 174 or variants thereof.
  • the multispecific antibody is a bispecific antibody targeting CD3/Her2, which comprises an antigen-binding domain targeting CD3 and an antigen-binding domain specifically binding to Her2, and the targeting The antigen-binding domain of CD3 is selected from the antibody or antigen-binding fragment thereof described in the first aspect; the antigen-binding domain targeting Her2 is VHH, which includes the sequence shown in SEQ ID NO: 175 or variants thereof.
  • the multispecific antibody is a bispecific antibody targeting CD3/Caludin18.2, which comprises an antigen-binding domain targeting CD3 and an antigen-binding domain that specifically binds Caludin18.2,
  • the antigen-binding domain targeting CD3 is selected from the antibody or antigen-binding fragment thereof described in the first aspect;
  • the antigen-binding domain targeting Caludin18.2 is VHH, which comprises SEQ ID NO:176 sequence or its variants.
  • the present invention also provides a multispecific antibody 2, which comprises the single domain antibody or antigen-binding fragment thereof according to the second aspect; in certain preferred embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody Antibodies or tetraspecific antibodies.
  • the antibody of the first aspect, the single domain antibody of the second aspect or the multispecific antibody of the third aspect of the present invention can be prepared by various methods known in the art, for example, by genetic engineering and recombination techniques. For example, DNA molecules encoding them are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. Then, the transfected host cells are cultured under specific conditions, and express the antibody, single domain antibody or multispecific antibody of the present invention.
  • the invention provides an isolated nucleic acid molecule encoding:
  • the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or a heavy chain variable region of the antibody or antigen-binding fragment thereof of the first aspect and a first nucleotide sequence encoding a light chain or a light chain variable region thereof.
  • the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the first polypeptide chain of the bispecific antibody described in the third aspect and a second nucleotide sequence encoding the second polypeptide chain thereof.
  • the present invention provides a vector comprising a nucleic acid molecule as described above.
  • the vector is a cloning vector or an expression vector.
  • the present invention provides a host cell comprising a nucleic acid molecule or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as E. coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells, and animal cells (such as mammalian cells, such as small mouse cells, human cells, etc.).
  • the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the first aspect, the single domain antibody of the second aspect, or the multispecific antibody of the third aspect, comprising, in Host cells as described above are cultured under conditions that allow protein expression, and the antibody or antigen-binding fragment thereof, single domain antibody or multispecific antibody is collected from the cultured host cell culture.
  • the antibodies also referred to as active ingredients
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof in the first aspect, the single domain antibody or antigen-binding fragment thereof in the second aspect, the antibody or antigen-binding fragment thereof in the third aspect,
  • the multispecific antibody the isolated nucleic acid molecule of the fourth aspect, the vector of the fifth aspect, or the host cell of the sixth aspect, and pharmaceutically acceptable carriers and/or excipients.
  • the pharmaceutical composition may also comprise additional pharmaceutically active agents, such as antineoplastic agents.
  • additional pharmaceutically active agent is separated from 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 invention. provided, or provided as a component of the same composition.
  • the present invention relates to a method for preventing and/or treating a disease, which comprises administering the antibody or antigen-binding fragment thereof of the first aspect, encoding the antibody or its antigen to a subject in need thereof An isolated nucleic acid molecule, vector or host cell, or a pharmaceutical composition comprising the same that binds the fragment.
  • the present invention also relates to the antibody or antigen-binding fragment thereof according to the first aspect, an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, a vector or a host cell, or a pharmaceutical composition comprising them, for preventing and/or Or the use of treating a disease, or the use in the preparation of a medicament for preventing and/or treating a disease.
  • the antibody or antigen-binding fragment thereof according to the first aspect of the present invention can be used in the treatment of any disease, as long as the treatment of the disease requires the effector mechanism of cytotoxic T cells.
  • the disease is a tumor, an inflammatory disease, or an autoimmune disease.
  • the tumor is a solid or 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, Indolent or aggressive in colorectal cancer, squamous cell carcinoma of the head and neck, pancreatic cancer, testicular cancer, cholangiocarcinoma, colorectal cancer, fallopian tube cancer, malignant melanoma, soft tissue cancer (eg, synovial sarcoma), B-cell lymphoma forms, chronic lymphocytic leukemia, acute myeloid leukemia, or acute lymphoblastic leukemia.
  • gastric cancer such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, Indolent or aggressive in colorectal cancer, squa
  • the treatment of the disease involves T cell (eg, cytotoxic T cell) effector mechanisms.
  • T cell eg, cytotoxic T cell
  • the antibodies or antigen-binding fragments thereof are used to enhance T cell (eg, cytotoxic T cell) activity to treat or prevent the disease.
  • the present invention relates to a method for preventing and/or treating tumors, which comprises administering the single domain antibody or antigen-binding fragment thereof described in the second aspect, encoding the antibody or Isolated nucleic acid molecules, vectors or host cells, antigen-binding fragments thereof, or pharmaceutical compositions comprising them.
  • the present invention also relates to the single domain antibody or antigen-binding fragment thereof according to the second aspect, an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, a vector or a host cell, or a pharmaceutical composition comprising them, for preventing And/or the purposes of treating tumor, or the purposes in the preparation of the medicine for preventing and/or treating the tumor of disease.
  • the tumor is a MSLN positive tumor.
  • the tumor is a solid or 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, Indolent or aggressive in colorectal cancer, squamous cell carcinoma of the head and neck, pancreatic cancer, testicular cancer, cholangiocarcinoma, colorectal cancer, fallopian tube cancer, malignant melanoma, soft tissue cancer (eg, synovial sarcoma), B-cell lymphoma forms, chronic lymphocytic leukemia, acute myeloid leukemia, or acute lymphoblastic leukemia.
  • gastric cancer such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, Indolent or aggressive in colorectal cancer, squa
  • the tumor is selected from solid tumors such as mesothelioma, ovarian cancer, pancreatic cancer, breast cancer, cholangiocarcinoma, colorectal cancer, gastric cancer, fallopian tube cancer, lung cancer or colorectal cancer.
  • solid tumors such as mesothelioma, ovarian cancer, pancreatic cancer, breast cancer, cholangiocarcinoma, colorectal cancer, gastric cancer, fallopian tube cancer, lung cancer or colorectal cancer.
  • the tumor is selected from hematological tumors, such as acute myeloid leukemia.
  • the present invention relates to a method for preventing and/or treating a disease, comprising administering to a subject in need thereof the multispecific antibody of the third aspect, an isolated antibody encoding the multispecific antibody nucleic acid molecules, vectors or host cells, or pharmaceutical compositions comprising them.
  • the present invention also relates to the multispecific antibody described in the third aspect, an isolated nucleic acid molecule encoding the multispecific antibody, a vector or a host cell, or a pharmaceutical composition containing them, for the prevention and/or treatment of diseases purposes, or in the preparation of medicines for the prevention and/or treatment of diseases.
  • the multispecific antibody described in the third aspect of the present invention can be used in the treatment of any disease, as long as the treatment of the disease requires the effector mechanism of cytotoxic T cells.
  • the multispecific antibody of the present invention includes a CD3 binding arm for T cell recruitment and a tumor targeting arm specific for a tumor-associated antigen (TAA)
  • TAA tumor-associated antigen
  • it brings T cells into close contact with target tumor cells, locally T cells are activated, followed by destruction of target cells by perforin and granzymes released by T cytotoxic granules.
  • TAA tumor-associated antigen
  • the multispecific antibody of the present invention includes a CD3 binding arm for T cell recruitment and a targeting arm specific for an immune checkpoint molecule, it activates T cells in the tumor microenvironment and subsequently kills tumor cells .
  • the disease is a tumor, an inflammatory disease, or an autoimmune disease.
  • the tumor is a solid or 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, Indolent or aggressive in colorectal cancer, squamous cell carcinoma of the head and neck, pancreatic cancer, testicular cancer, cholangiocarcinoma, colorectal cancer, fallopian tube cancer, malignant melanoma, soft tissue cancer (eg, synovial sarcoma), B-cell lymphoma forms, chronic lymphocytic leukemia, acute myeloid leukemia, or acute lymphoblastic leukemia.
  • gastric cancer such as gastric cancer, lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, prostate cancer, bladder cancer, ovarian cancer, Indolent or aggressive in colorectal cancer, squa
  • the treatment of the disease involves T cell (eg, cytotoxic T cell) effector mechanisms.
  • T cell eg, cytotoxic T cell
  • the multispecific antibody is used to recruit T cells to target cells, activate T cells, and induce T cell-mediated cell killing (TDCC), thereby effectively killing target cells, for the treatment of or prevent the disease.
  • TDCC T cell-mediated cell killing
  • the tumor is an MSLN-positive tumor, such as a solid tumor, such as mesothelioma, ovarian cancer, pancreatic cancer, breast cancer, bile duct cancer, colorectal cancer, gastric cancer, fallopian tube cancer, lung cancer, or colorectal cancer Cancer; for example, a hematological neoplasm, such as acute myeloid leukemia.
  • a solid tumor such as mesothelioma, ovarian cancer, pancreatic cancer, breast cancer, bile duct cancer, colorectal cancer, gastric cancer, fallopian tube cancer, lung cancer, or colorectal cancer Cancer
  • a hematological neoplasm such as acute myeloid leukemia.
  • the tumor is a CD19 positive tumor, eg, a B cell malignancy, eg, lymphoma or leukemia.
  • the tumor is a CD20 positive tumor, eg, a B cell malignancy, eg, lymphoma or leukemia.
  • the tumor is a Trop2 positive tumor, eg, a solid tumor, eg, breast cancer.
  • the tumor is a Her2-positive tumor, eg, a solid tumor, eg, lung cancer.
  • the tumor is a Caludin 18.2 positive tumor, eg, a solid tumor, eg, gastric cancer.
  • the antibodies or antigen-binding fragments thereof, single domain antibodies or antigen-binding fragments thereof, multispecific antibodies or pharmaceutical compositions comprising them of the present invention may be formulated as Any dosage form known in the medical art.
  • the antibodies or antigen-binding fragments thereof, single domain antibodies or antigen-binding fragments thereof, multispecific antibodies or pharmaceutical compositions comprising them of the present invention may be obtained by means of the present invention Any suitable method known in the art for administration.
  • the antibodies or antigen-binding fragments thereof, single domain antibodies or antigen-binding fragments thereof, multispecific antibodies or pharmaceutical compositions comprising them of the present invention may be dosed in Unit forms are formulated for ease of administration.
  • the antibodies or antigen-binding fragments thereof, single domain antibodies or antigen-binding fragments thereof, multispecific antibodies or pharmaceutical compositions comprising them of the present invention may be administered alone , can also be administered in combination with another pharmaceutically active agent (eg, an antineoplastic agent) or another therapy (eg, an antineoplastic therapy).
  • another pharmaceutically active agent eg, an antineoplastic agent
  • another therapy eg, an antineoplastic therapy
  • the subject may be a mammal, such as a human.
  • antibody refers to an immunoglobulin molecule, usually composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC).
  • Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain is composed 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 domains are not directly involved in antibody-antigen binding, but exhibit a variety of effector functions, such as mediating immunoglobulin interactions with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement Binding of the first component (C1q) of the system.
  • VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminus to carboxy-terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antigen binding site, respectively.
  • the allocation of amino acids in each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J.Mol.Biol.196:901- 917; Definition by Chothia et al. (1989) Nature 342:878-883.
  • CDR complementarity determining region
  • the variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2 and CDR3.
  • CDR1, CDR2 and CDR3 The precise boundaries of these CDRs can be defined 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), Chothia numbering system (Chothia & Lesk (1987) J.Mol.Biol.196:901-917; Chothia et al.
  • the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be identified according to various numbering systems known in the art, for example, Kabat, Chothia, IMGT, AbM or Contact numbering systems.
  • the anti-CD3 antibody or antigen-binding fragment thereof according to the present invention contains CDRs identified by the Kabat numbering system.
  • the CDRs contained in the anti-MSLN single domain antibody or antigen-binding fragment thereof of the present invention are determined by the Kabat, Chothia, IMGT, AbM or Contact numbering system.
  • framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • antibody is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • 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 to which the full-length antibody binds, and/or competes with the full-length antibody for Specific binding to an antigen, which is also referred to as an "antigen-binding moiety".
  • an antigen-binding moiety 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. Can be obtained by recombinant DNA techniques. or by enzymatic or chemical cleavage of intact antibodies to generate antigen-binding fragments of antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, complementarity determining region (CDR) fragments, scFv, diabody, single domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), probody, and antibodies containing enough to confer specific antigen-binding capabilities on the polypeptide at least a portion of the polypeptide.
  • full-length antibody means an antibody consisting of two “full-length heavy chains” and two “full-length light chains”.
  • “full-length heavy chain” refers to such a polypeptide chain, which consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain The CH2 domain of the constant region and the CH3 domain of the constant region of the heavy chain; and, when the full-length antibody is of the IgE isotype, optionally further includes the CH4 domain of the constant region of the heavy chain.
  • a "full-length heavy chain” is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction.
  • a "full-length light chain” is a polypeptide chain consisting, in the N-terminal to C-terminal direction, of a light chain variable region (VL) and a light chain constant region (CL).
  • VL light chain variable region
  • CL light chain constant region
  • the two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and between the HRs of the two full-length heavy chains.
  • a full-length antibody contains two antigen-binding sites formed by a VH and VL pair, respectively, which specifically recognize/bind to the same antigen.
  • Single-domain antibody has the meaning commonly understood by those skilled in the art, which refers to a single monomer variable antibody domain (such as a single heavy chain variable antibody domain) region), usually derived from the variable region of a heavy chain antibody (such as a camelid antibody or a shark antibody).
  • a Nanobody consists of 4 framework regions and 3 complementarity determining regions, having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • Single domain antibodies can be truncated at the N-terminus or C-terminus so that they comprise only part of FR1 and/or FR4, or lack one or both of those framework regions, so long as they substantially retain antigen binding and specificity.
  • Single-domain antibodies are also known as nanobodies, and the two are used interchangeably.
  • 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 to which the single domain antibody binds, and/or competes with the single domain antibody for Antigen specific binding.
  • Antigen-binding fragments of the single domain antibodies of the invention can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the single domain antibodies of the invention.
  • the "antigen-binding fragment" of the single domain antibody may be truncated at the N-terminus or C-terminus compared to the full-length single domain antibody such that it only contains part of FR1 and/or FR4, or lacks those One or both of the framework regions are sufficient as long as they substantially maintain antigen binding and specificity.
  • Fab fragment means an antibody fragment consisting of VL, VH, CL and CH1 domains; An antibody fragment of two Fab fragments linked; the term “Fab'fragment” means a fragment obtained after reduction of the disulfide bond linking the two heavy chain fragments in an F(ab') fragment, consisting of an intact light chain and heavy chain The Fd fragment of the chain (consisting of the VH and CH1 domains).
  • the term "Fv" means 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 capable of forming a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a variable region (such as the Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although perhaps with a lower affinity than the full binding site.
  • the term "Fc domain” or "Fc region” means a part of the heavy chain constant region comprising CH2 and CH3.
  • the Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding.
  • "Effector functions" mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; Downregulation of body (eg, B cell receptor); and B cell activation, etc.
  • the Fc region comprises a hinge, CH2 and CH3. When the Fc region contains a hinge, the hinge mediates dimerization between two Fc-containing polypeptides.
  • the Fc region can be of any antibody heavy chain constant region isotype, eg IgGl, IgG2, IgG3 or IgG4.
  • the Fc domain can include both a native Fc region and a variant Fc region.
  • a native Fc region comprises an amino acid sequence identical to that of an Fc region found in nature, for example, a native sequence human Fc region includes a native sequence human IgG1 Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human Fc region; an IgG3 Fc region; and a native sequence human IgG4 Fc region, and naturally occurring variants thereof.
  • a variant Fc region comprises an amino acid sequence that differs from that of a native sequence Fc region by at least one amino acid modification.
  • a variant Fc region may possess altered effector functions (e.g., Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function) compared to a native Fc region .
  • scFv refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker.
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof.
  • GGGGS amino acid sequence
  • the term "diabody” means that its VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, This forces the domain to pair with the complementary domain of another chain and creates two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).
  • Each of the above antibody fragments maintains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • Antigen-binding fragments of antibodies can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) ), and antigen-binding fragments of antibodies are screened for specificity in the same manner as for whole antibodies.
  • antibody includes not only whole antibodies but also antigen-binding fragments of antibodies.
  • germline antibody gene or “germline antibody gene segment” refers to an immunoglobulin-encoding sequence present in the genome of an organism , which have not undergone the maturation process of genetic rearrangements and mutations leading to the expression of specific immunoglobulins.
  • the expression “heavy chain germline gene” refers to the germline antibody gene or gene fragment encoding the heavy chain of immunoglobulin, which includes V gene (variable), D gene (diversity), J gene (joining) and C gene (constant); similarly, the expression “light chain germline gene” refers to a germline antibody gene or gene fragment encoding an immunoglobulin light chain, which includes V gene (variable), J gene (joining) and C gene (constant).
  • the amino acid sequence encoded by the germline antibody gene or germline antibody gene fragment is also called “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 professional databases (eg, IMGT, UNSWIg, NCBI or VBASE2). It is generally believed that germline antibody genes are more likely than mature antibody genes to retain key amino acid sequence structures unique to individuals in a species, and therefore are less likely to be considered exogenous when used for therapy in that species.
  • the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • the sequences are aligned for optimal comparison purposes (for example, gaps may be introduced in a first amino acid sequence or nucleic acid sequence to best align with a second amino acid or nucleic acid sequence).
  • Jiabi pair The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • a non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Modified from .Acad.Sci.U.S.A. 90:5873-5877. Such an algorithm was incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.
  • variant in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions or additions. In certain instances, the term “variant” also refers to a polypeptide or peptide that has been modified (ie, by covalently linking molecules of any type to the polypeptide or peptide).
  • polypeptides may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, Attachment to cellular ligands or other proteins, etc.
  • Derivative polypeptides or peptides can be produced 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, and the like.
  • a variant has a similar, identical or improved function to the polypeptide or peptide from which it is derived.
  • the 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% of the sequence from which it is derived %, at least 98%, at least 99%, or 100% sequence identity.
  • 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 can be expressed in terms of the equilibrium dissociation constant ( KD ) for that interaction.
  • KD 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.
  • the specific binding properties between two molecules can be determined using methods well known in the art.
  • One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473).
  • KD , kon and kdis values can be measured by any effective method.
  • dissociation constants can be measured in Biacore using surface plasmon resonance (SPR), and can also be measured using bioluminescence interferometry or Kinexa.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage, such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • Phage such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg
  • the term "host cell” refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • 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 substitutions for amino acid residues with amino acid residues that have similar side chains, e.g., are physically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including Substitution of residues with the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art.
  • These families include those 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), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acid, proline, phenylalanine, methionine), beta branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g.
  • 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
  • non-polar side chains such as
  • amino acid residues can also be divided into classes defined by alternative physical and functional properties, 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), minimal residues (A, G and S), residues involved in turn formation (A, C, D, E, G, H,
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient compatible with the subject and the active ingredient pharmacologically and/or physiologically, These are well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • 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 enhancers include, but are not limited to, sodium chloride.
  • Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearates and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • the stabilizing agent has the meaning generally understood by those skilled in the art, and it can stabilize the desired activity of the active ingredient in the medicine, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc.
  • prevention refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • a beneficial or desired clinical outcome includes, but is not limited to, relief of symptoms, reduction of the extent of the disease, stabilization (i.e., no longer worsening) of the disease state, delay or slowing of the progression of the disease, amelioration or palliation of the disease status, and relief of symptoms (whether partial or total), whether detectable or undetectable.
  • treating can also refer to prolonging survival as compared to expected survival if not receiving treatment.
  • the term "subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject e.g., a human
  • has, or is at risk of having, a tumor e.g., an MSLN-expressing tumor
  • an inflammatory disease e.g., an autoimmune disease.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • an effective amount for preventing a disease refers to an amount sufficient to prevent, arrest, or delay the occurrence of a disease (for example, a tumor, an inflammatory disease or an autoimmune disease); treating a disease
  • An effective amount is an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capability of those skilled in the art.
  • amounts effective for therapeutic use will depend on the severity of the disease being treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly etc.
  • the present invention provides novel CD3 antibodies with reduced cytokine release, indicating their ability to modestly activate T cells.
  • the invention also provides multispecific antibodies targeting CD3 and additional antigens (such as tumor-associated antigens and/or immune checkpoint molecules), such as bispecific antibodies targeting CD3 and MSLN, which bispecific antibodies can be obtained by Binding to CD3 present on T cells and specific target antigens on tumor cells promotes T cell targeting and recruitment of tumor cells, induces tumor-specific (MHC-independent) cytotoxic T cell killing activity, and has significantly reduced cell Factor release, thus having significantly improved safety. Therefore, the antibodies of the present invention have important clinical value.
  • the molecular biology experiment methods and immunoassay methods used in the present invention are basically with reference to J.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M.Ausubel et al., Compiled Molecular Biology Experimental Guide, 3rd Edition, John Wiley & Sons, Inc., 1995 by the method described; restriction endonucleases were used in accordance with the conditions recommended by the product manufacturer.
  • restriction endonucleases were used in accordance with the conditions recommended by the product manufacturer.
  • mutations were introduced into the heavy chain variable region and light chain variable region of the CD3 monoclonal antibody to establish a scFv phage mutant library.
  • the heavy chain variable regions of the constructed CD3 monoclonal antibodies are named VH1 ⁇ VH46, and have the amino acid sequences shown in SEQ ID NOs: 1-46 respectively, and the light chain variable regions of the constructed CD3 monoclonal antibodies are named as VL-1 ⁇ VL-2, and respectively have the aminoacid sequence shown in SEQ ID NOs:47 and 48, the CDR information of each variable region is as shown in table 1 and table 2.
  • the constructed VH1-VH46 were paired with VL-1 respectively to establish scFv phage mutation library and 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 the IgG1 mutant heavy chain constant region (SEQ ID NO: 164) to form a structure shown as [VH]-[CH];
  • the VL-1 of the CD3 monoclonal antibody is linked to the light chain constant region (SEQ ID NO: 165), forming a structure shown as [VL]-[CL].
  • M1 ⁇ M46 a total of 46 CD3 monoclonal antibodies
  • Coat CD3 ⁇ (company: acrobiosystems product number: CDG-H5253), add packaged phage, incubate at room temperature for 1 h, wash 10 times, pat dry, elute phage, and infect TG1 competent cells with the eluted phage overnight. Collect phage; repeat the previous operation twice; pick a single colony and induce overnight expression with IPTG. Positive clones were determined by ELISA method.
  • Embodiment 2 the purity determination of CD3 monoclonal antibody
  • This example investigates the interaction of CD3 monoclonal antibody with Jurkat cells (naturally expressing human CD3) (company: ATCC; product number: TIB-152) and cynomolgus monkey HSC-F cells (expressing monkey CD3) (company: JCRB Cell Bank; product number: JCRB1164) binding activity.
  • Cell preparation Adjust Jurkat and HSC-F cells to 1*10 7 cells/ml with PBS, and prepare 50 ⁇ l of cell suspension for each sample.
  • Cell and antibody incubation take 50 ⁇ l of cell suspension, the total amount of cells is 5*10 5 cells/well.
  • the final concentration of the antibody is the initial concentration of 2 ⁇ g/ml, three-fold serial dilution of 8 wells, and incubation at 4°C for 1 hour.
  • VH amino acid sequence of the control antibody OKT3 is shown in SEQ ID NO:166; the VL amino acid sequence is shown in SEQ ID NO:167; the VH amino acid sequence of the control antibody 1 is shown in SEQ ID NO Shown in: 168; The aminoacid sequence of VL is shown in SEQ ID NO: 169.
  • Antibodies at three concentrations of 4, 2, and 1 ⁇ g/mL were immobilized on the HC200M chip containing anti-human IgG Fc secondary antibody (Xantec product number HC200M), and the antibody binding amount was controlled to be about 1000RU.
  • the human CD3 ⁇ company: acrobiosystems product number: CDG-H52W5
  • recombinant antigen company: acrobiosystems product number: CDG-H52W5
  • the binding time was 8 minutes. Dissociation time 20min.
  • the kinetic constants were obtained by fitting the 1:1 binding model with the Kinetics software that comes with Carterra.
  • Example 5 Construction of human MSLN stably transfected cell lines
  • huMSLN human MSLN
  • cDNA purchased from Sino biological, HG13128-UT
  • cDNA purchased from Sino biological, HG13128-UT
  • cDNA purchased from Sino biological, HG13128-UT
  • cDNA purchased from Sino biological, HG13128-UT
  • HEK293T cells co-transfect the constructed lentiviral plasmid and packaging plasmid into HEK293T cells, and collect the cells at 48h and 72h respectively
  • the supernatant was successively added to MC38 cells (Nanjing Kebai, Cat. No. CBP60825), and 4 ⁇ g/ml puromycin was added after 24 hours for selection.
  • the single cells highly expressing huMSLN were sorted by a cell sorter (Sony, LE-SH800SBP), and finally the stably transfected cell monoclonal MC38/MSLN highly expressing huMSLN was obtained.
  • Anti-huMSLN nanobodies were produced by immunizing alpacas.
  • the immunization of alpacas was entrusted to Apak Biotechnology Co., Ltd., that is, to immunize two alpacas.
  • the immunization antigen was human MSLN recombinant protein with hFc tag (huMSLN-hFc, Kaijia Biology, MSL-HM280).
  • the first immunization was subcutaneously immunized with complete Freund's adjuvant (CFA) mixed with huMSLN-hFc, and the remaining booster immunizations were subcutaneously immunized three times with incomplete Freund's adjuvant (IFA) mixed with huMSLN-hFc.
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • RNAiso Plus takara, 9109
  • VHH heavy chain variable region
  • the two constructed alpaca libraries were both panned by cells and proteins. After three or four rounds of panning for cells and two rounds of panning for proteins, extract the panned bacterial library plasmids, amplify the VHH fragments by PCR, connect them to the expression vector, and then pick clones to induce the secretion and expression of VHH.
  • 3C6 A MSLN single-domain antibody with good crossover between human, monkey and mouse was obtained, named 3C6.
  • the antibody is sent for sequencing, sequence analysis is performed with CLC software, and the clone of the unique sequence is induced to express 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.
  • ELISA affinity determination Coating human MSLN-his (huMSLN-his, Kay Biology, MSL-HM18D), monkey MSLN-his (cynoMSLN-his, Kay Biology, MSL-CM180), mouse MSLN-his (muMSLN-his , Kaijia Biology, MSL-MM180), and coated overnight at 4°C.
  • Example 11 Affinity determination of anti-MSLN single domain antibody at cell level
  • Example 5 Take 50 ⁇ l of MC38/MSLN cells (2 ⁇ 10 5 cells) obtained in Example 5 and add them to a 96-well V-type plate, and add 50 ⁇ l of gradiently diluted antibodies to each well (antibodies start at 300 nM, 3-fold diluted to 8 concentrations) , and incubated on ice for 1h. After the primary antibody was washed away, the diluted Alexa Fluro488-labeled anti-DYKDDDDK antibody (Wuhan Sanying Biological HRP-66008) was added, incubated on ice for 1 hour, and after 4 washes, 200 ⁇ l of PBS per well was resuspended, and then detected by a flow cytometer. The EC 50 was counted by Graphpad Prism software, and the cell affinity results are shown in Table 8.
  • the anti-DYKDDDDK antibody was bound to the HC30M chip through amino coupling, and then 2 ⁇ g/ml MSLN single domain antibody was captured.
  • the serially diluted huMSLN-Fc (Kaijia Biology, MSL-HM280) and cynoMSLN-Fc (Kaijia Biology, MSL-CM280) starting from 100nM, 3-fold serial dilution, a total of 8 concentrations
  • the binding time was 8 minutes
  • the relay time was 20 minutes.
  • Kinetic constants were obtained by fitting with Carteria software.
  • the results of the dynamic affinity between the MSLN single domain antibody and the MSLN protein are shown in Table 9.
  • CD3-MSLN bispecific antibody in this example is shown in Figure 1, in which, the humanized CD3 antibody has two arms (heavy and light chain pairing), and the C-terminus of the IgG1 mutant is connected to the MSLN single domain antibody by (GGGGS) 2 (VHH antibody) forms a CD3-MSLN bispecific antibody (bivalent).
  • the VH of the humanized CD3 antibody obtained in Example 1 was linked to the IgG1 mutant heavy chain constant region (SEQ ID NO: 164), and further the C-terminal of the heavy chain constant region was passed through a linker (GGGGS) 2 Link the above 3C6 antibody to form the first polypeptide chain with the structure shown as [VH]-[CH]-[L]-[VHH]; link the VL-1 of the humanized CD3 antibody obtained in Example 1 to light chain constant region (SEQ ID NO: 165), forming a second polypeptide chain having the structure shown in [VL]-[CL].
  • the nucleic acid sequence encoding the above polypeptide chain was constructed on a PTT5 plasmid vector (Youbao Biology, lot: VT2202), and enough plasmids were extracted for use.
  • D1 One day before transfection (D1), dilute the cell density with medium to 2 ⁇ 106 cells/ml, on the day of transfection (D0), count the cells (cell viability should be ⁇ 95%), and adjust the cell density to 4.0 ⁇ 10 6 cells/ml, mix the plasmids of antibody fragments according to the ratio of heavy chain: light chain 1:1, mix with PEI, and co-transfect CHO-S cells. After transfection, transfer the cells to 37°C, Culture in 120rpm, 8% CO2 incubator. On the first day (D1) after transfection, add preheated CHOgro complete medium according to 1/5 of the expression volume, cool down to 32°C and continue culturing.
  • Antibody name B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 SEC% 93.5 100 92.9 97 95 93.5 94.5 97.45 91.1 99.4
  • Antibody name B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 SEC% 98.1 97.8 96.8 96 96.6 94.7 97.6 93.7 93.8 97.8
  • Antibody name B21 B22 B23 B24 B25 B26 B27 B28 B29 B30 SEC% 98.4 93.3 91.3 86 95.8 94.8 91 99.2 80 91.2
  • Antibody name B31 B32 B33 B34 B35 B36 B37 B38 B39 B40 SEC% 94.6 96.1 93.5 95.4 95 96.2 96.2 96.49 92.8 93.7
  • Antibody name B41 B42 B43 B44 B45 B46 the the the SEC% 94.9 96.3 91.7 95.1 93.8 96.4 the the the the the the the the the the the the the the the
  • CD3-MSLN bispecific antibody to Jurkat (naturally expressing human CD3) and cynomolgus monkey HSC-F cells (company: JCRB Cell Bank; product number: JCRB1164), among which, two positive control antibodies were used:
  • the double antibody structures of the positive control antibody OKT3-3C6 and the control antibody 1-3C6 were constructed referring to the "CD3-MSLN bispecific antibody" in Example 13.
  • the VH sequence of the CD3 antibody of OKT3-3C6 has the amino acid sequence shown in SEQ ID NO: 166
  • the VL sequence of the CD3 antibody has the amino acid sequence shown in SEQ ID NO: 167
  • the VHH is the 3C6 antibody of MSLN
  • the VH sequence of the CD3 antibody of 3C6 has the amino acid sequence shown in SEQ ID NO: 168
  • the VL sequence of the CD3 antibody has the amino acid sequence shown in SEQ ID NO: 169
  • the VHH is the 3C6 antibody of MSLN.
  • the experimental method is as follows:
  • Cell and antibody incubation take 50 ⁇ l of cell suspension, the total amount of cells is 5*10 5 cells/well.
  • the final concentration of the antibody is the initial concentration of 2 ⁇ g/ml, three-fold serial dilution of 8 wells, and incubation at 4°C for 1 hour.
  • results are shown in Table 11.
  • the results show that most of the CD3-MSLN bispecific antibodies of the present invention have the same order of magnitude in binding activity to cells expressing human CD3 and to cells expressing cynomolgus CD3.
  • MC38-MSLN cell line Synthesize the full-length DNA sequence of MSLN (NCBI genbank: Accession#AAH09272) into the pLVX-IRES-Puro vector (Youbao biological product number: VT1464); package lentivirus and infect MC38 cells, after pressurized screening Monoclonal cell lines with high expression of MSLN were obtained by monoclonal sorting.
  • 3Prepare the antibody adjust the antibody to a final concentration of 2 ⁇ g/ml with PBS, then make 3-fold serial dilutions and make 8 gradients. 50 ⁇ l of antibody per well.
  • Antibodies with three concentrations of 4, 2, and 1 ⁇ g/mL were immobilized on the HC200M chip containing anti-human IgG Fc secondary antibody, and the antibody binding amount was controlled to be about 1000RU.
  • the recombinant antigen of human CD3 ⁇ (company: acrobiosystems, article number: CDG-H52W5) diluted serially (starting from 592.5nM, 3-fold dilution and 8 gradients) was flowed through the chip at a flow rate of 1000 ⁇ L/min, and the binding time was 8min. Dissociation time 20min.
  • the kinetic constants were obtained by fitting the 1:1 binding model with the Kinetics software that comes with Carterra.
  • Antibodies with three concentrations of 4, 2, and 1 ⁇ g/mL were immobilized on the HC200M chip containing anti-human IgG Fc secondary antibody, and the antibody binding amount was controlled to be about 1000RU.
  • the gradiently diluted human MSLN recombinant antigen (company: acrobiosystems product number: MSN-H8223) (starting from 592.5nM, 3-fold diluted 8 gradients) flowed through the chip at a flow rate of 1000 ⁇ L/min, and the binding time was 8min. Dissociation time 20min.
  • the kinetic constants were obtained by fitting the 1:1 binding model with the Kinetics software that comes with Carterra.
  • Exemplary bispecific antibody affinity determination results are shown in Table 13, and the dynamic affinity diagram is shown in FIG. 4 . The results show that all the CD3-MSLN bispecific antibodies of the present invention have good binding activity to MSLN.
  • Embodiment 15 Jurkat-TIGIT-luc cell stability experiment
  • Example 16 Activation of T cell activation signaling pathway by CD3-MSLN bispecific antibody
  • Add tumor group Take Jurkat-TIGIT-luc cells in logarithmic phase of growth, adjust to 1.2*10 6 cells/ml with 1640+10% FBS, add 25 ⁇ l cells per well (3*10 4 cells/well); digest For the MC38/MSLN cells constructed in Example 5, use 1640+10% FBS to adjust the digested MC38/MSLN cells to 1.2*10 6 cells/ml, add 25 ⁇ l cells to each well, and gently add 3*10 4 cells/well After mixing, place at 37°C and 5% CO 2 to incubate for 5-6h, add 20 ⁇ l/well of chemiluminescent substrate, and then detect on the machine.
  • the assay results of activation of T cells expressing human CD3 are shown in FIGS. 6A-6F and Table 14.
  • the results showed that when no tumor cells were added, the activation of T cells expressing human CD3 by the CD3-MSLN bispecific antibody of the present invention was weaker than that of the control antibody 1-3C6, and the activation of Jurkat/NFAT/LUC cells was not even detected , but all can moderately activate Jurkat-TIGIT-luc cells after adding tumor cells.
  • Table 14 Activation of T cell activation signaling pathway by CD3-MSLN bispecific antibody
  • the fluorescence intensity was detected by flow cytometry, and the fluorescence intensity was more than 10 2 times that of the negative sample.
  • the final serum concentration was 5%, cultured for 48 hours.
  • the results are shown in Figures 7A-7I, and the results show that the CD3-MSLN bispecific antibody of the present invention can exert a T cell-mediated tumor cell killing (TDCC) effect on tumor cells expressing the tumor antigen MSLN, which may be due to this
  • TDCC tumor cell killing
  • the invented CD3-MSLN bispecific antibody specifically binds tumor cells and T cells expressing the tumor antigen MSLN, recruits T cells to the surrounding tumor cells, activates T cells, induces TDCC, and effectively kills tumor cells.
  • Example 18 CD3-CD19 bispecific antibody-mediated TDCC
  • a bispecific antibody was constructed based on the CD3 antibody and CD19 antibody of the present invention.
  • the CD3-CD19 bispecific antibody was named 5Y2-175, and the TDCC activity mediated by 5Y2-175 was investigated.
  • 5Y2-175 was prepared by referring to the method of constructing the double antibody in Example 13, and its structural diagram is shown in Figure 8.
  • the specific structure is: link the VH CD3 of the CD3 monoclonal antibody (M33) obtained in Example 1 to the IgG1 mutant
  • the heavy chain constant region (SEQ ID NO:164), and further the C-terminal of the heavy chain constant region is connected to the VH CD19 (SEQ ID NO:170) and VL CD19 (SEQ ID NO :171), VH CD19 and VL CD19 are connected through (GGGGS) 2 to form the first structure with [VH CD3 ]-[CH]-[L]-[VH CD19 ]-[L]-[VL CD19 ] Polypeptide chain;
  • the VL CD3 of the humanized CD3 antibody (M33) obtained in Example 1 was connected to the light chain constant region (SEQ ID NO: 165) to form the first having the structure shown in [VL CD3 ]-[CL] Two polypeptide chains.
  • a Luc sequence (NCBI: GenBank: MF062157.1) and clone it into a lentiviral vector (plvx-IRES-puro, psPAX2, pMD2G), co-infect HEK293T cells with the constructed lentiviral plasmid and packaging plasmid, and collect the cells for 48 hours The supernatant was added to Raji cells (ATCC, CCL-86), and 4 ⁇ g/ml puromycin was added at 24 hours for selection. Single cells expressing Luc were sorted by a cell sorter (Sony, LESH800SBP), and finally stable Raji-luc cells were obtained.
  • Resuscitate the frozen PBMC cells 500g, 3min, centrifuge to remove the supernatant, then wash once with 1640+10% FBS (inactivated), 500g, 3min, centrifuge to remove the supernatant, wash with an appropriate amount of 1640+10%FBS (inactivated) (over) resuspended, counted, diluted to the corresponding density, added to the well plate, 25 ⁇ l was added to each well, so that the total number of PBMC cells was 1.25*10 5 cells/well.
  • CD3-CD19 double antibody adjust the concentration to 2nM with medium, dilute 3 times, and use 9 gradients; take 50 ⁇ l of the diluted antibody and add it to PBMC and Raji-luc cells;
  • the CD3-CD19 bispecific antibody can exert T cell-mediated tumor cell killing (TDCC) effect on CD19-expressing lymphoma cells.
  • TDCC tumor cell killing
  • a bispecific antibody was constructed based on the CD3 antibody and CD20 antibody of the present invention.
  • the CD3-CD20 bispecific antibody was named 5Y2-176, and the TDCC activity mediated by the CD3-CD20 bispecific antibody was investigated.
  • 5Y2-176 was constructed with reference to the CD3-CD19 bispecific antibody in Example 18, wherein 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.
  • Resuscitate the frozen PBMC cells 500g, 3min, centrifuge to remove the supernatant, then wash once with 1640+10% FBS (inactivated), 500g, 3min, centrifuge to remove the supernatant, wash with an appropriate amount of 1640+10%FBS (inactivated) (over) resuspended, counted, diluted to the corresponding density, added to the well plate, 25 ⁇ l was added to each well, so that the total number of PBMC cells was 1.25*10 5 cells/well.
  • CD3-CD20 antibody adjust the concentration to 2nM with medium, dilute 3 times, and use 9 gradients; take 50 ⁇ l of the diluted antibody and add it to PBMC and Raji-luc cells;
  • the CD3-CD20 bispecific antibody can exert T cell-mediated tumor cell killing (TDCC) effect on CD20-expressing lymphoma cells.
  • TDCC tumor cell killing
  • Example 20 CD3-trop2 bispecific antibody-mediated TDCC
  • a bispecific antibody was constructed based on the CD3 antibody and trop2 antibody of the present invention.
  • the CD3-trop2 bispecific antibody was named 5Y2-174, and the TDCC activity mediated by the CD3-trop2 bispecific antibody was investigated.
  • the 5Y2-174 bispecific antibody was prepared by referring 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 trop2 is shown in SEQ ID NO:174.
  • Resuscitate the frozen PBMC cells 500g, 3min, centrifuge to remove the supernatant, then wash once with 1640+10% FBS (inactivated), 500g, 3min, centrifuge to remove the supernatant, wash with an appropriate amount of 1640+10%FBS (inactivated) (over) resuspended, counted, diluted to the corresponding density, added to the well plate, 25 ⁇ l was added to each well, so that the total number of PBMC cells was 1.25*10 5 cells/well.
  • CD3-trop2 double antibody adjust the concentration to 1nM with medium, dilute 3 times, and have 10 gradients; take 50 ⁇ l of the diluted antibody and add it to cells containing PBMC and HCC1806-luc;
  • the CD3-trop2 bispecific antibody can exert T cell-mediated tumor cell killing (TDCC) effect on trop2-expressing human breast squamous cell carcinoma cells.
  • TDCC tumor cell killing
  • Example 21 CD3-Her2 bispecific antibody-mediated TDCC
  • a bispecific antibody was constructed based on the CD3 antibody and Her2 antibody of the present invention.
  • the CD3-Her2 bispecific antibody was named 5Y2-177, and the TDCC activity mediated by the CD3-Her2 bispecific antibody was investigated.
  • the 5Y2-177 bispecific antibody was prepared by referring to the method for constructing the 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.
  • lentiviral vector plvx-IRES-puro, psPAX2, pMD2G
  • co-infect HEK293T cells with the constructed lentiviral plasmid and packaging plasmid, collect the cell supernatant for 48 hours, and add it to Calu-1 (Nanjing Kebai, CBP60085) cells were screened by adding 4 ⁇ g/ml puromycin at 24 hours.
  • Single cells expressing Luc were sorted by a cell sorter (Sony, LESH800SBP), and finally stable Calu-1-luc cells were obtained.
  • Resuscitate the frozen PBMC cells 500g, 3min, centrifuge to remove the supernatant, then wash once with 1640+10% FBS (inactivated), 500g, 3min, centrifuge to remove the supernatant, wash with an appropriate amount of 1640+10%FBS (inactivated) (over) resuspended, counted, diluted to the corresponding density, added to the well plate, 25 ⁇ l was added to each well, so that the total number of PBMC cells was 1.25*10 5 cells/well.
  • CD3-Her2 adjust the concentration to 30nM with medium, dilute 3 times, and have 11 gradients; take 50 ⁇ l of the diluted antibody and add it to cells containing PBMC and Calu-1-luc;
  • the CD3-Her2 bispecific antibody can exert T cell-mediated tumor cell killing (TDCC) effect on Her2-expressing human lung cancer cells.
  • TDCC tumor cell killing
  • Example 22 TDCC mediated by CD3-Caludin18.2 bispecific antibody
  • a bispecific antibody was constructed based on the CD3 antibody and Caludin18.2 antibody of the present invention.
  • the CD3-Caludin18.2 bispecific antibody was named 5Y2-178, and the CD3-Caludin18.2 bispecific antibody-mediated TDCC activity.
  • the 5Y2-178 bispecific antibody was prepared by referring to the method for constructing the bispecific antibody in Example 13.
  • the humanized CD3 antibody involved in its specific structure is M33, and the VHH amino acid sequence of Caludin18.2 is shown in SEQ ID NO:176.
  • lentiviral vector plvx-IRES-puro, psPAX2, pMD2G
  • co-infect HEK293T cells with the constructed lentiviral plasmid and packaging plasmid, collect the cell supernatant for 48 hours, and add it to NUGC4 (Nanjing In Kebai CBP60493) cells, 4 ⁇ g/ml puromycin was added for selection at 24 hours.
  • Single cells expressing Luc were sorted by a cell sorter (Sony, LESH800SBP), and finally stable NUGC4-lucc cells were obtained.
  • Resuscitate the frozen PBMC cells 500g, 3min, centrifuge to remove the supernatant, then wash once with 1640+10% FBS (inactivated), 500g, 3min, centrifuge to remove the supernatant, wash with an appropriate amount of 1640+10%FBS (inactivated) (over) resuspended, counted, diluted to the corresponding density, added to the well plate, 25 ⁇ l was added to each well, so that the total number of PBMC cells was 1.25*10 5 cells/well.
  • the experimental results are shown in Figure 13.
  • the CD3-Caludin18.2 bispecific antibody can exert T cell-mediated tumor cell killing (TDCC) effect on human gastric cancer cells expressing Caludin18.2.
  • TDCC tumor cell killing
  • the IL2 detection results are shown in Figures 14A-14I, and the INF- ⁇ detection results are shown in Figures 15A-15I.
  • the results showed that co-incubating the CD3-MSLN bispecific antibody of the present invention with PBMC produced significantly lower levels of IL2 and INF- ⁇ than the positive control OKT3-3C6, indicating that the CD3-MSLN bispecific antibody of the present invention has good safety.

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