Nothing Special   »   [go: up one dir, main page]

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

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
seq
sequence shown
antibody
antigen
binding fragment
Prior art date
Application number
PCT/CN2022/142626
Other languages
English (en)
French (fr)
Inventor
胡彦
郭三友
滕毓敏
韦涛
丁兆
Original Assignee
四川汇宇制药股份有限公司
四川汇宇海玥医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 四川汇宇制药股份有限公司, 四川汇宇海玥医药科技有限公司 filed Critical 四川汇宇制药股份有限公司
Priority to EP22914863.0A priority Critical patent/EP4458861A1/en
Priority to CN202280085681.9A priority patent/CN118591561A/zh
Publication of WO2023125611A1 publication Critical patent/WO2023125611A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

一种靶向 CD3的抗体或其抗原结合片段，其具备适度的激活T细胞的能力，可有效降低细胞因子释放。所述靶向 CD3 和另外的抗原(例如肿瘤相关抗原和/或免疫检查点分子)的多特异性抗体，该多特异性抗体在确保能够高效且特异性杀伤靶细胞的同时，可显著降低细胞因子释放，具有明显提高的安全性。还涉及所述靶向 CD3 的抗体或其抗原结合片段、多特异性抗体或包含它们的组合物在疾病治疗中的用途。

Description

靶向CD3的抗体及多特异性抗体及其用途 技术领域
本发明涉及靶向CD3的抗体或其抗原结合片段，其具备适度激活T细胞的能力，可有效降低细胞因子释放。本发明还涉及靶向CD3和另外的抗原(例如肿瘤相关抗原和/或免疫检查点分子)的多特异性抗体，该多特异性抗体能够高效且特异性杀伤靶细胞，同时可显著降低细胞因子释放，具有明显提高的安全性。本发明还涉及所述靶向CD3的抗体或其抗原结合片段、多特异性抗体或包含它们的组合物在疾病治疗中的用途。
背景技术
CD3是一种蛋白复合物和T细胞共受体，主要表达在T细胞上，参与激活细胞毒性T细胞(CD8+幼稚T细胞)和T辅助细胞(CD4+幼稚T细胞)。CD3由一条CD3γ链、一条CD3δ链和两条CD3ε链组成。这些链与T细胞受体(T-cell receptor，TCR)和ζ链(zeta链)结合，在T淋巴细胞中产生激活信号。目前处于临床阶段的或者已上市药物的CD3抗体主要来源于人源化的CD3抗体(OKT3/UCHT1/L2K/TR66等)，然而它们亲和力相对较高，因而容易导致T细胞的过度激活，释放出大量细胞因子，从而造成细胞因子风暴综合症，对人体产生严重副作用，严重时甚至会威胁生命安全。为应对细胞因子风暴综合症(CRS)风险，给药剂量一般非常低，这导致治疗窗口很窄，不利于病人获益。
因此，本领域存在开发新的CD3抗体和双特异性抗体的需求，以提供适度激活T细胞的能力，降低细胞因子风暴综合症发生的概率，从而提高安全性，以利于疾病治疗，尤其是癌症治疗。
发明概述
本申请的发明人经过大量的研究，筛选获得了新的CD3抗体，这些抗体具有降低的细胞因子释放，表明其能够适度激活T细胞。本申请的发明人还筛选获得了新的MSLN单域抗体，所述单域抗体与MSLN具有良好的结合活性。基于此，进一步提供了靶向CD3和另外的抗原(例如MSLN、CD19、CD20、Trop2、Her2或Caludin18.2)的多特异性抗体，这些抗体表现出增强的肿瘤细胞定位并引发有效的T细胞活化，从而特异性杀伤肿瘤细胞，且具有显著降低的细胞因子释放，因而具有明显提高的安全性。由此提供 了以下方面。
在第一方面，本发明涉及能够特异性结合CD3的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中，所述VH包含：包含SEQ ID NO:107所示序列的HCDR1、包含SEQ ID NO:108所示序列的HCDR2以及包含X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10WX 11X 12X 13(SEQ ID NO:112)所示序列的HCDR3；其中X 1为A，H或P；X 2为A、E、G、H、K、Q或S；X 3为D，N或R；X 4为F或P；X 5为G、K、L、P、Q、R、S、V、W或Y；X 6为M，N，Q或R；X 7为G、N、S或T；X 8为A、Q、R或Y；X 9为G、I或V；X 10为N或S；X 11为F或W；X 12为A、E、K或Q；X 13为H、L、M、S或Y；并且条件是所述HCDR3不是SEQ ID NO:90所示。
在第二方面，本发明涉及能够特异性结合MSLN的单域抗体或其抗原结合片段，所述单域抗体或其抗原结合片段包含：
(1)IMGT编号系统定义的下述CDRs：包含SEQ ID NO:150所示序列或其变体的CDR1，包含SEQ ID NO:151所示序列或其变体的CDR2，以及包含SEQ ID NO:152所示序列或其变体的CDR3；
(2)Kabat编号系统定义的下述CDRs：包含SEQ ID NO:153所示序列或其变体的CDR1，包含SEQ ID NO:154所示序列或其变体的CDR2，以及包含SEQ ID NO:155所示序列或其变体的CDR3；
(3)AbM编号系统定义的下述CDRs：包含SEQ ID NO:156所示序列或其变体的CDR1，包含SEQ ID NO:157所示序列或其变体的CDR2，以及包含SEQ ID NO:155所示序列或其变体的CDR3；
(4)Chothia编号系统定义的下述CDRs：包含SEQ ID NO:158所示序列或其变体的CDR1，包含SEQ ID NO:159所示序列或其变体的CDR2，以及包含SEQ ID NO:155所示序列或其变体的CDR3；或，
(5)Contact编号系统定义的下述CDRs：包含SEQ ID NO:160所示序列或其变体的CDR1，包含SEQ ID NO:161所示序列或其变体的CDR2，以及包含SEQ ID NO:162所示序列或其变体的CDR3；
其中，(1)-(5)任一项中所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加；优选地，所述置换为保守置换；
优选地，所述单域抗体或其抗原结合片段包含：
(1)IMGT编号系统定义的下述CDRs：包含SEQ ID NO:150所示序列的CDR1， 包含SEQ ID NO:151所示序列的CDR2，以及包含SEQ ID NO:152所示序列的CDR3；
(2)Kabat编号系统定义的下述CDRs：包含SEQ ID NO:153所示序列的CDR1，包含SEQ ID NO:154所示序列的CDR2，以及包含SEQ ID NO:155所示序列的CDR3；
(3)AbM编号系统定义的下述CDRs：包含SEQ ID NO:156所示序列的CDR1，包含SEQ ID NO:157所示序列的CDR2，以及包含SEQ ID NO:155所示序列的CDR3；
(4)Chothia编号系统定义的下述CDRs：包含SEQ ID NO:158所示序列的CDR1，包含SEQ ID NO:159所示序列的CDR2，以及包含SEQ ID NO:155所示序列的CDR3；或，
(5)Contact编号系统定义的下述CDRs：包含SEQ ID NO:160所示序列的CDR1，包含SEQ ID NO:161所示序列的CDR2，以及包含SEQ ID NO:162所示序列的CDR3。
在第三方面，本发明涉及多特异性抗体，其包含靶向CD3的抗原结合结构域和至少一个靶向其他抗原的抗原结合结构域，所述靶向CD3的抗原结合结构域选自第一方面所述的抗体或其抗原结合片段，所述其他抗原选自肿瘤相关抗原(TAA)和/或免疫检查点分子。
在第四方面，本发明涉及分离的核酸分子，其编码：
-第一方面所述的抗体或其抗原结合片段、或其重链可变区和/或轻链可变区；
-第二方面所述的单域抗体或其抗原结合片段；或，
-第三方面所述的多特异性抗体，或其多肽链。
在第五方面，本发明涉及载体，其包含第四方面所述的核酸分子。
在第六方面，本发明涉及宿主细胞，其包含第四方面所述的核酸分子或第五方面所述的载体。
在第七方面，本发明涉及药物组合物，其包含第一方面所述的抗体或其抗原结合片段、第二方面所述的单域抗体或其抗原结合片段、第三方面所述的多特异性抗体、第四方面所述的分离的核酸分子、第五方面所述的载体、或第六方面所述的宿主细胞，以及药学上可接受的载体和/或赋形剂。
在第八方面，本发明涉及第一方面所述的抗体或其抗原结合片段、第二方面所述的单域抗体或其抗原结合片段、第三方面所述的多特异性抗体、第四方面所述的分离的核酸分子、第五方面所述的载体、或第六方面所述的宿主细胞、或第七方面所述的药物组合物，用于预防和/或治疗疾病的用途，或在制备用于预防和/或治疗疾病的药物中的用途。
在第九方面，本发明涉及用于预防和/或治疗疾病的方法，其包括向有此需要的受试者施用第一方面所述的抗体或其抗原结合片段、第二方面所述的单域抗体或其抗原结合 片段、第三方面所述的多特异性抗体、第四方面所述的分离的核酸分子、第五方面所述的载体、或第六方面所述的宿主细胞、或第七方面所述的药物组合物。
附图说明
图1显示了实施例13中CD3-MSLN双特异性抗体的结构示意图。
图2显示了实施例14中CD3-MSLN双特异性抗体对MC38-MSLN细胞结合活性的测定结果。
图3A-图3D显示了实施例14中CD3-MSLN双特异性抗体对人CD3εγ重组抗原的动态亲和力测定结果。
图4显示了实施例14中CD3-MSLN双特异性抗体对MSLN重组抗原的动态亲和力测定结果。
图5A-图5F显示了实施例15中Jurkat-TIGIT-luc细胞稳定性实验检测结果。
图6A-图6F显示了实施例16中CD3-MSLN双特异性抗体对T细胞活化信号通路的激活测定结果。
图7A-图7I显示了实施例17中CD3-MSLN双特异性抗体介导的TDCC测定结果。
图8显示了实施例18中CD3-CD19双特异性抗体的结构示意图。
图9显示了实施例18中CD3-CD19双特异性抗体介导的TDCC测定结果。
图10显示了实施例19中CD3-CD20双特异性抗体介导的TDCC测定结果。
图11显示了实施例20中CD3-trop2双特异性抗体介导的TDCC测定结果。
图12显示了实施例21中CD3-Her2双特异性抗体介导的TDCC测定结果。
图13显示了实施例22中CD3-Caludin18.2双特异性抗体介导的TDCC测定结果。
图14A-图14I显示了实施例23中CD3-MSLN双特异性抗体介导的IL-2释放水平检测结果。
图15A-图15I显示了实施例23中CD3-MSLN双特异性抗体介导的INF-γ释放水平检测结果。
发明详述
本发明提供了新的CD3抗体，具体提供了以下方面。
CD3抗体
在第一方面，本发明涉及能够特异性结合CD3的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中，所述VH包含： 包含SEQ ID NO:107所示序列的HCDR1、包含SEQ ID NO:108所示序列的HCDR2以及包含X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10WX 11X 12X 13(SEQ ID NO:112)所示序列的HCDR3；其中X 1为A，H或P；X 2为A、E、G、H、K、Q或S；X 3为D，N或R；X 4为F或P；X 5为G、K、L、P、Q、R、S、V、W或Y；X 6为M，N，Q或R；X 7为G、N、S或T；X 8为A、Q、R或Y；X 9为G、I或V；X 10为N或S；X 11为F或W；X 12为A、E、K或Q；X 13为H、L、M、S或Y；并且条件是所述HCDR3不是SEQ ID NO:90所示。
在某些实施方案中，所述VL包含：包含SEQ ID NO:137所示序列的LCDR1、包含SEQ ID NO:138所示序列的LCDR2以及包含SEQ ID NO:139所示序列的LCDR3。
在某些实施方案中，所述HCDR3不是SEQ ID NOs:60、64、77、89中任一项所示。
在某些实施方案中，所述HCDR3包含：X 1X 2X 3FX 4NX 5YX 6SWFAX 7(SEQ ID NO:148)所示的序列，其中，X 1是H或P；X 2是G、E、或A；X 3是N或R；X 4是G、K、S，或P；X 5是T、S、或N；X 6是V或G；X 7是M、Y、S、或L。在某些实施方案中，所述HCDR3包含SEQ ID NOs:56、61、69、79、82、87任一项所示的序列。
在某些实施方案中，所述HCDR3包含：
(1)HX 2NFGNSYVSWFAY(SEQ ID NO:113)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为H；
(2)HGNFX 5NSYVSWFAY(SEQ ID NO:114)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为K；
(3)HGNFGNSX 8VSWFAY(SEQ ID NO:115)所示的序列，其中，X 8是A、Q或R，优选为R；或，
(4)HGNFGNSYVSWFX 12Y(SEQ ID NO:116)所示的序列，其中，X 12是E、K或Q。
在某些实施方案中，所述HCDR3包含SEQ ID NOs:75、53、72、51、57、64任一项所示的序列。
在某些实施方案中，所述HCDR3包含：
(1)X 1X 2NFGNSYVSWFAY(SEQ ID NO:117)所示的序列，其中，X 1是A或P，优选为A；X 2是A、E、H、K、Q或S，优选为K；优选地，X 1为A，X 2为K；
(2)X 1GNFGNSYVX 10WFAY(SEQ ID NO:118)所示的序列，其中，X 1是A或P，优选为A；X 10是N；优选地，X 1为A，X 10为N；或，
(3)X 1GNFGNSYVSWFX 12Y(SEQ ID NO:119)所示的序列，其中，X 1是A或P， 优选为A；X 12是E、K或Q，优选为E或Q；优选地，X 1为A，X 12为E或Q；。
在某些实施方案中，所述HCDR3包含SEQ ID NOs:50、66、55、85任一项所示的序列。
在某些实施方案中，所述HCDR3包含：
(1)X 1X 2NFGNSYVSWFAY(SEQ ID NO:117)所示的序列，其中，X 1是A或P，优选为A；X 2是A、E、H、K、Q或S，优选为K；优选地，X 1为A，X 2为K；
(2)HX 2X 3FGNSYVSWFAY(SEQ ID NO:120)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为E；X 3是D或R，优选为R；优选地，X 2为E，X 3为R；
(3)HX 2NFX 5NSYVSWFAY(SEQ ID NO:121)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为A或S；X 5是K、L、P、Q、R、S、V、W或Y，优选为L、P、S、Q、V或R；优选地，X 2为A或S，X 5为L、P、S、Q、V或R；优选地，X 2和X 5分别为A/L、S/P、S/L、A/S、S/Q、S/V或S/R；
(4)HX 2NFGX 6SYVSWFAY(SEQ ID NO:122)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 6是M、Q或R，优选为R；优选地，X 2为S，X 6为R；
(5)HX 2NFGNX 7YVSWFAY(SEQ ID NO:123)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 7是G、N或T，优选为T或G；优选地，X 2为S，X 7为T或G；
(6)HX 2NFGNSX 8VSWFAY(SEQ ID NO:124)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 8是A、Q或R，优选为R；优选地，X 2为S，X 8为R；
(7)HX 2NFGNSYVSWX 11AY(SEQ ID NO:125)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 11为W；优选地，X 2为S，X 11为W；
(8)HX 2NFGNSYVSWFX 12Y(SEQ ID NO:126)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 12是E、K或Q，优选为E或Q；优选地，X 2为S或Q，X 12为E或Q；优选地，X 2和X 12分别为S/E、Q/Q或S/Q；或，
(9)HX 2NFGNSYVSWFAX 13(SEQ ID NO:127)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为A；X 13是H、L、M或S，优选为S；优选地，X 2为A，X 13为S。
在某些实施方案中，所述HCDR3包含SEQ ID NOs:50、69、49、58、59、60、70、82、86、71、68、77、78、81、62、84、89、74任一项所示的序列。
在某些实施方案中，所述HCDR3包含：
(1)HX 2NFX 5NSYVSWFAY(SEQ ID NO:121)所示的序列，其中，X 2是A、E、 H、K、Q或S，优选为A或S；X 5是K、L、P、Q、R、S、V、W或Y，优选为L、P、S、Q、V或R；优选地，X 2为A或S，X 5为L、P、S、Q、V或R；优选地，X 2和X 5分别为A/L、S/P、S/L、A/S、S/Q、S/V或S/R；
(2)HGNFX 5X 6SYVSWFAY(SEQ ID NO:129)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为P或K；X 6是M、Q或R，优选为M或Q；优选地，X 5为P或K，X 6为M或Q；优选地，X 5和X 6分别为P/M或K/Q；
(3)HGNFX 5NSYX 9SWFAY(SEQ ID NO:130)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为K或Q；X 9是G或I；优选地，X 5为K或Q，X 9是G或I；优选地，X 5和X 9分别为K/G或Q/I；
(4)HGNFX 5NSYVSWFX 12Y(SEQ ID NO:131)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为Q或L；X 12是E、K或Q，优选为Q；优选地，X 5为Q或L，X 12为Q；或，
(5)HGNFX 5NSYVSWFAX 13(SEQ ID NO:132)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为Y或W；X 13是H、L、M或S，优选为H或S；优选地，X 5为Y或W，X 13为H或S；优选地，X 5和X 13分别为Y/H或W/S。
在某些实施方案中，所述HCDR3包含SEQ ID NOs:49、58、59、60、70、82、86、76、80、61、88、52、83、54、65任一项所示的序列。
在某些实施方案中，所述HCDR3包含：
(1)X 1GNFGNSYVSWFX 12Y(SEQ ID NO:119)所示的序列，其中，X 1是A或P，优选为A；X 12是E、K或Q，优选为Q；优选地，X 1为A，X 12为Q或E；
(2)HX 2NFGNSYVSWFX 12Y(SEQ ID NO:126)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 12是E、K或Q，优选为E或Q；优选地，X 2为S，X 12为E或Q；优选地，X 2和X 12分别为S/E、Q/Q或S/Q；
(3)HGNFX 5NSYVSWFX 12Y(SEQ ID NO:131)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为Q或L；X 12是E、K或Q，优选为Q；优选地，X 5为Q或L，X 12为Q；或，
(4)HGNFGNSX 8VSWFX 12Y(SEQ ID NO:134)所示的序列，其中，X 8是A、Q或R，优选为A或Q；X 12是E、K或Q，优选为Q；优选地，X 8为A或Q，X 12为Q。
在某些实施方案中，所述HCDR3包含SEQ ID NOs:55、85、62、84、89、52、83、67、73任一项所示的序列。
在某些实施方案中，所述HCDR3包含：
(1)HX 2NFGNSYVSWFAX 13(SEQ ID NO:127)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为A；X 13是H、L、M或S，优选为S；优选地，X 2为A，X 13为S；
(2)HGNFX 5NSYVSWFAX 13(SEQ ID NO:132)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为Y或W；X 13是H、L、M或S，优选为H或S；优选地，X 5为Y或W，X 13为H或S；优选地，X 5和X 13分别为Y/H或W/S；或，
(3)HGNFGNX 7YVSWFAX 13(SEQ ID NO:133)所示的序列，其中，X 7是G、N或T，优选为T或N；X 13是H、L、M或S，优选为M或S；优选地，X 7为T或N，X 13为M或S；优选地，X 7和X 13分别为T/M或N/S。
在某些实施方案中，所述HCDR3包含SEQ ID NOs:74、54、65、56、79任一项所示的序列。
在某些实施方案中，所述HCDR3包含：
(1)HX 2X 3FGNSYVSWFAY(SEQ ID NO:120)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为E；X 3是D或R，优选为R；优选地，X 2为E，X 3为R；或，
(2)HGX 3X 4GNSYVSWFAY(SEQ ID NO:128)所示的序列，其中，X 3是D或R，优选为D；X 4是P；优选地，X 3为D，X 4是P。
在某些实施方案中，所述HCDR3包含SEQ ID NOs:69、63任一项所示的序列。
在某些实施方案中，所述HCDR3包含：X 1GNFX 5NSYVSWFAX 13(SEQ ID NO:135)所示的序列，其中，X 1是A或P，优选为P；X 5是K、L、P、Q、R、S、V、W或Y，优选为P；X 13是H、L、M或S，优选为L；优选地，X 1为P，X 5为P，X 13为L。
在某些实施方案中，所述HCDR3包含SEQ ID NO:87所示的序列。
在某些实施方案中，所述VH包含：包含SEQ ID NO:107所示序列的HCDR1、包含SEQ ID NO:108所示序列的HCDR2以及包含SEQ ID NOs:49-89任一项所示序列的HCDR3；并且，所述VL包含：包含SEQ ID NO:137所示序列的LCDR1、包含SEQ ID NO:138所示序列的LCDR2以及包含SEQ ID NO:139所示序列的LCDR3。优选地，条件是所述HCDR3不是SEQ ID NOs:60、64、77、89中任一项所示。
在某些实施方案中，所述抗体或其抗原结合片段进一步包含源自人免疫球蛋白的框架区。
在某些实施方案中，所述抗体或其抗原结合片段包含源自人胚系抗体基因所编码的氨基酸序列中所包含的框架区。在某些实施方案中，所述抗体或其抗原结合片段包含源 自人重链胚系基因所编码的氨基酸序列中所包含的重链框架区，和/或源自人轻链胚系基因所编码的氨基酸序列中所包含的轻链框架区。
在某些实施方案中，所述VH包含HFR1、HFR2、HFR3和HFR4，其中：
所述HFR1包含SEQ ID NO:109所示的序列；
所述HFR2包含SEQ ID NO:110所示的序列；
所述HFR3包含VKX 1RFTISRDDSKSX 2LYLQMNX 3LKTEDTAX 4YYCVR(SEQ ID N O:136)所示的序列；其中，X 1为G或D，X 2为I或S，X 3为N或S，X 4为M或V；
所述HFR4包含SEQ ID NO:111所示的序列。
在某些实施方案中，所述HFR3包含SEQ ID NOs:91-106任一项所示的序列。
在某些实施方案中，所述VH包含：SEQ ID NOs:1-46任一项所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述VH包含：SEQ ID NOs:8、13、22、33、37、43任一项所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述VH包含：SEQ ID NOs:3、5、9、16、20、25、28任一项所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述VH包含：SEQ ID NOs:2、7、18、40任一项所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述VH包含：SEQ ID NOs:2、22、1、10、11、12、23、36、37、41、24、21、30、32、35、14、31、39、45、27、46任一项所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述VH包含：SEQ ID NOs:1、10、11、12、23、36、37、41、29、34、13、44、4、38、42、6、17任一项所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述VH包含：SEQ ID NOs:7、40、14、31、39、45、4、38、42、19、26任一项所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述VH包含：SEQ ID NOs:27、46、6、17、8、33任一项所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述VH包含：SEQ ID NOs:22或15所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述VH包含：SEQ ID NO:43所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述VH包含：SEQ ID NOs:1-11、13-15、17-19、21-29、31-35、37-44、46任一项所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述VL包含LFR1、LFR2、LFR3和LFR4，其中：
所述LFR1包含如SEQ ID NO:140所示的序列；
所述LFR2包含如WX 1QQTPGQAX 2RX 3LIX 4(SEQ ID NO:144)所示的序列；其中， X 1为V或Y，X 2为F或P，X 3为G或T，X 4为G或Y；
所述LFR3包含如GVPARFSGSX 4X 5GX 6KAALTITGAQADDESX 7YFCA(SEQ ID N O:147)所示的序列；其中，X 4为L或I，X 5为L或I，X 6为D或N，X 7为I或D；
所述LFR4包含如SEQ ID NO:141所示的序列。
在某些实施方案中，所述LFR2包含SEQ ID NO:142或143所示的序列。
在某些实施方案中，所述LFR3包含SEQ ID NO:145或146所示的序列。
在某些实施方案中，所述VL包含：SEQ ID NO:140所示的LFR1、SEQ ID NO:142所示的LFR2、SEQ ID NO:145所示的LFR3、SEQ ID NO:141所示的LFR4。
在某些实施方案中，所述VL包含：SEQ ID NO:140所示的LFR1、SEQ ID NO:143所示的LFR2、SEQ ID NO:146所示的LFR3、SEQ ID NO:141所示的LFR4。
在某些实施方案中，所述VL包含：SEQ ID NO:47或48所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含SEQ ID NOs:1-46任一项所示序列或其变体的VH，和包含SEQ ID NO:47所示序列或其变体的VL；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含SEQ ID NOs:1-46任一项所示序列或其变体的VH，和包含SEQ ID NO:48所示序列或其变体的VL；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含SEQ ID NOs:1-11、13-15、17-19、21-29、31-35、37-44、46任一项所示序列或其变体的VH，和包含SEQ ID NO:47所示序列或其变体的VL；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含SEQ ID NOs:1-11、13-15、17-19、21-29、31-35、37-44、46任一项所示序列或其变体的VH，和包含SEQ ID NO:48所示序列或其变体的VL；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含SEQ ID NOs:8、13、22、33、37、43任一项所示序列或其变体的VH，和包含SEQ ID NO:47所示序列或其变体的VL；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含SEQ ID NOs:8、13、22、33、37、43任一项所示序列或其变体的VH，和包含SEQ ID NO:48所示序列或其变体的VL；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
在某些实施方案中，所述VH包含：HCDR1～HCDR3和HFR1～HFR4，其具备以下特征：
项目1：所述HCDR1包含SEQ ID NO:107所示的序列；
项目2：所述HCDR2包含SEQ ID NO:108所示的序列；
项目3：所述HCDR3包含选自下列的序列:
(3-1)X 1X 2NFGNSYVSWFAY(SEQ ID NO:117)所示的序列，其中，X 1是A或P，优选为A；X 2是A、E、H、K、Q或S，优选为K；优选地，X 1为A，X 2为K；
(3-2)HX 2X 3FGNSYVSWFAY(SEQ ID NO:120)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为E；X 3是D或R，优选为R；优选地，X 2为E，X 3为R；
(3-3)HX 2NFX 5NSYVSWFAY(SEQ ID NO:121)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为A或S；X 5是K、L、P、Q、R、S、V、W或Y，优选为L、P、S、Q、V或R；优选地，X 2为A或S，X 5为L、P、S、Q、V或R；优选地，X 2和X 5分别为A/L、S/P、S/L、A/S、S/Q、S/V或S/R；
(3-4)HX 2NFGX 6SYVSWFAY(SEQ ID NO:122)所示的序列，其中，X 2是A、E、 H、K、Q或S，优选为S；X 6是M、Q或R，优选为R；优选地，X 2为S，X 6为R；
(3-5)HX 2NFGNX 7YVSWFAY(SEQ ID NO:123)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 7是G，N或T，优选为T或G；优选地，X 2为S，X 7为T或G；
(3-6)HX 2NFGNSX 8VSWFAY(SEQ ID NO:124)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 8是A、Q或R，优选为R；优选地，X 2为S，X 8为R；
(3-7)HX 2NFGNSYVSWX 11AY(SEQ ID NO:125)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 11为W；优选地，X 2为S，X 11为W；
(3-8)HX 2NFGNSYVSWFX 12Y(SEQ ID NO:126)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 12是E、K或Q，优选为E或Q；优选地，X 2为S，X 12为E或Q；优选地，X 2和X 12分别为S/E、Q/Q或S/Q；或，
(3-9)HX 2NFGNSYVSWFAX 13(SEQ ID NO:127)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为A；X 13是H、L、M或S，优选为S；优选地，X 2为A，X 13为S；
项目4：所述HFR1包含SEQ ID NO:109所示的序列；
项目5：所述HFR2包含SEQ ID NO:110所示的序列；
项目6：所述HFR3包含VKX 1RFTISRDDSKSX 2LYLQMNX 3LKTEDTAX 4YYCVR(SEQ ID NO:136)所示的序列；其中，X 1为G或D，X 2为I或S，X 3为N或S，X 4为M或V；
项目7：所述HFR4包含SEQ ID NO:111所示的序列；
优选地，所述VL具备以下特征：项目8：所述VL包含SEQ ID NO:47所示的序列；或者，项目9：所述VL包含SEQ ID NO:48所示的序列。
在某些实施方案中，所述VH具备以下特征：所述项目1、项目2、项目3、项目4、项目5、项目6’、项目7，其中，项目6’：所述HFR3包含SEQ ID NOs:91-106任一项所示的序列。所述VL优选地具备以下特征：项目8或项目9。
在某些实施方案中，所述VH具备以下特征：所述项目1、项目2、项目3’、项目4、项目5、项目6、项目7，其中，项目3’：所述HCDR3包含SEQ ID NOs:50、69、49、58、59、60、70、82、86、71、68、77、78、81、62、84、89、74任一项所示的序列。所述VL优选地具备以下特征：项目8或项目9。
在某些实施方案中，所述VH具备以下特征：所述项目1、项目2、项目3’、项目4、项目5、项目6’、项目7。所述VL优选地具备以下特征：项目8或项目9。
在某些实施方案中，所述VH包含：SEQ ID NOs:2、22、1、10、11、12、23、36、37、41、24、21、30、32、35、14、31、39、45、27、46任一项所示的氨基酸序列。所述VL优选地包含SEQ ID NO:47或48所示的序列，例如SEQ ID NO:47所示的序列。
在某些实施方案中，所述VH包含：HCDR1～HCDR3和HFR1～HFR4，其具备以下特征：
项目11：所述HCDR1包含SEQ ID NO:107所示的序列；
项目12：所述HCDR2包含SEQ ID NO:108所示的序列；
项目13：所述HCDR3包含选自下列的序列:
(13-1)HX 2NFX 5NSYVSWFAY(SEQ ID NO:121)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为A或S；X 5是K、L、P、Q、R、S、V、W或Y，优选为L、P、S、Q、V或R；优选地，X 2为A或S，X 5为L、P、S、Q、V或R；优选地，X 2和X 5分别为A/L、S/P、S/L、A/S、S/Q、S/V或S/R；
(13-2)HGNFX 5X 6SYVSWFAY(SEQ ID NO:129)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为P或K；X 6是M、Q或R，优选为M或Q；优选地，X 5为P或K，X 6为M或Q；优选地，X 5和X 6分别为P/M或K/Q；
(13-3)HGNFX 5NSYX 9SWFAY(SEQ ID NO:130)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为K或Q；X 9是G或I；优选地，X 5为K或Q，X 9是G或I；优选地，X 5和X 9分别为K/G或Q/I；
(13-4)HGNFX 5NSYVSWFX 12Y(SEQ ID NO:131)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为Q或L；X 12是E、K或Q，优选为Q；优选地，X 5为Q或L，X 12为Q；或，
(13-5)HGNFX 5NSYVSWFAX 13(SEQ ID NO:132)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为Y或W；X 13是H、L、M或S，优选为H或S；优选地，X 5为Y或W，X 13为H或S；优选地，X 5和X 13分别为Y/H或W/S；
项目14：所述HFR1包含SEQ ID NO:109所示的序列；
项目15：所述HFR2包含SEQ ID NO:110所示的序列；
项目16：所述HFR3包含VKX 1RFTISRDDSKSX 2LYLQMNX 3LKTEDTAX 4YYCVR(SEQ ID NO:136)所示的序列；其中，X 1为G或D，X 2为I或S，X 3为N或S，X 4为M或V；
项目17：所述HFR4包含SEQ ID NO:111所示的序列；
优选地，所述VL具备以下特征：项目18：所述VL包含SEQ ID NO:47所示的序列；或者，项目19：所述VL包含SEQ ID NO:48所示的序列。
在某些实施方案中，所述VH具备以下特征：所述项目11、项目12、项目13、项目14、项目15、项目16’、项目17，其中，项目16’：所述HFR3包含SEQ ID NOs:91-106任一项所示的序列。所述VL优选地具备以下特征：项目18或项目19。
在某些实施方案中，所述VH具备以下特征：所述项目11、项目12、项目13’、项目14、项目15、项目16、项目17，其中，项目13’：所述HCDR3包含SEQ ID NOs:49、58、59、60、70、82、86、76、80、61、88、52、83、54、65任一项所示的序列。所述VL优选地具备以下特征：项目18或项目19。
在某些实施方案中，所述VH具备以下特征：所述项目11、项目12、项目13’、项目14、项目15、项目16’、项目17。所述VL优选地具备以下特征：项目18或项目19。
在某些实施方案中，所述VH包含：SEQ ID NOs:1、10、11、12、23、36、37、41、29、34、13、44、4、38、42、6、17任一项所示的氨基酸序列。所述VL优选地包含SEQ ID NO:47或48所示的序列，例如SEQ ID NO:47所示的序列。
在某些实施方案中，所述抗体或其抗原结合片段进一步包含源自人免疫球蛋白的恒定区。
在某些实施方案中，所述抗体或其抗原结合片段的重链包含源自人免疫球蛋白的重链恒定区。在某些实施方案中，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区。
在某些实施方案中，所述抗体或其抗原结合片段的轻链包含源自人免疫球蛋白的轻链恒定区。在某些实施方案中，所述轻链恒定区是κ轻链恒定区。在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:165所示的轻链恒定区(CL)。
在某些实施方案中，所述抗体或其抗原结合片段所包含的Fc结构域是天然Fc区，其包含与自然界中发现的Fc区的氨基酸序列一致的氨基酸序列。天然Fc区可具有效应子功能。示例性“效应子功能”包括与Fc受体结合；Clq结合和补体依赖性细胞毒性(CDC)；抗体依赖性细胞介导的细胞毒性(ADCC)；抗体依赖性细胞吞噬作用(ADCP)；对细胞表面受体(例如B细胞受体)的下调；和B细胞活化等。
在某些实施方案中，所述抗体或其抗原结合片段所包含的Fc结构域也可以是变异Fc区，其与天然Fc区相比可以包含一个或多个(例如1-10个，例如1-5个)氨基酸突变或化学修饰以改变本发明抗体的下列中的一个或更多个特性：Fc受体结合、抗体糖 基化、半胱氨酸残基的数目、效应细胞功能或补体功能等。可以通过将天然Fc区中的至少一个氨基酸残基替换为不同残基或化学修饰，产生功能改变，例如，改变抗体对效应子配体(如FcR或补体C1q)的亲和力，从而改变效应子功能(例如降低或增强)。
在某些实施方案中，所述抗体或其抗原结合片段包含突变的或化学修饰的Fc区，其与野生型Fc区相比具有降低或增强的抗体依赖性细胞毒性作用(ADCC)、降低或增强的抗体依赖性细胞吞噬作用(ADCP)和/或降低或增强的补体依赖性细胞毒性作用(CDC)。获得上述具有改变(例如降低)的效应子功能的方法是本领域已知的，例如可以在重链恒定区引入选自下列的一个或多个(例如1个、2个、3个或4个)突变：根据EU编号的L234A、L235A、G237A、K322A。在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:164所示的重链恒定区(CH)。
在某些实施方案中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、双抗体(diabody)。
在某些实施方案中，所述抗体是IgG抗体，例如IgG1，IgG2，IgG3或IgG4抗体。
在某些实施方案中，所述抗体或其抗原结合片段特异性结合CD3(例如人和/或食蟹猴CD3)的ε和/或γ链。
在某些实施方案中，当通过表面等离子体共振术(SPR)测定时，所述抗体或其抗原结合片段以不低于6×10 -10M、不低于6.5×10 -10M、不低于7×10 -10M、不低于7.5×10 -10M、不低于8×10 -10M、不低于8.5×10 -10M、不低于9×10 -10M、不低于9.5×10 -10M、不低于1×10 -9M、不低于1.5×10 -9M、不低于2×10 -9M、不低于2.5×10 -9M、不低于3×10 -9M、不低于3.5×10 -9M、不低于4×10 -9M、不低于4.5×10 -9M、不低于5×10 -9M、不低于5.5×10 - 9M、不低于6×10 -9M、不低于6.5×10 -9M、不低于7×10 -9M、不低于7.5×10 -9M、不低于8×10 -9M、不低于8.5×10 -9M、不低于9×10 -9M、不低于9.5×10 -9M、不低于1×10 -8M、不低于1.5×10 -8M、或不低于2×10 -8M的K D结合CD3(例如，人和/或食蟹猴CD3，例如ε和/或γ链)。在某些实施方案中，当通过SPR测定时，所述抗体或其抗原结合片段以不高于10 -7M，例如不高于9.5×10 -8M、不高于9×10 -8M、不高于8.5×10 -8M、不高于8×10 -8M、不高于7.5×10 -8M、不高于7×10 -8M、不高于6.5×10 -8M、不高于6×10 -8M、不高于5.5×10 -8M、不高于5×10 -8M、不高于4.5×10 -8M、不高于4×10 -8M、不高于3.5×10 - 8M、不高于3×10 -8M、不高于2.5×10 -8M或不高于2×10 -8M的K D结合CD3(例如，人和/或食蟹猴CD3，例如ε和/或γ链)。在某些实施方案中，当通过SPR测定时，所述抗体或其抗原结合片段以10 -8-10 -10M的K D结合CD3(例如，人和/或食蟹猴CD3，例如ε 和/或γ链)。
在某些实施方案中，当通过流式细胞技术测定时，所述抗体或其抗原结合片段以不低于0.05nM、不低于0.1nM、不低于0.2nM、不低于0.3nM、不低于0.4nM、不低于0.5nM、不低于0.6nM、不低于0.7nM、不低于0.8nM、不低于0.9nM、不低于1nM、不低于1.5nM、不低于2nM、不低于2.5nM、不低于3nM、不低于3.5nM、不低于4nM、不低于4.5nM、不低于5nM、不低于5.5nM、不低于6nM、不低于6.5nM、不低于7nM、不低于7.5nM、不低于8nM、不低于8.5nM、不低于9nM的EC 50结合CD3(例如，人和/或食蟹猴CD3，例如表达CD3的细胞)。在某些实施方案中，当通过流式细胞技术测定时，所述抗体或其抗原结合片段以不高于20nM，例如不高于15nM、不高于10nM、不高于9.5nM、不高于9nM、不高于8.5nM、不高于8nM、不高于7.5nM、不高于7nM、不高于6.5nM、不高于6nM、不高于5.5nM、不高于5nM、不高于4.5nM、不高于4nM、不高于3.5nM、不高于3nM、不高于2.5nM、不高于2nM、不高于1.5nM或不高于1nM的EC 50结合CD3(例如，人和/或食蟹猴CD3，例如表达CD3的细胞)。
MSLN单域抗体
在第二方面，本发明涉及能够特异性结合MSLN(例如人MSLN)的单域抗体或其抗原结合片段。单域抗体典型地由4个框架区(FRs)和3个互补决定区(CDRs)组成，称为FR1、CDR1、FR2、CDR2、FR3、CDR3及FR4，其抗原结合片段包含该单域抗体的至少一部分，该部分足以赋予该片段特异性结合抗原(例如MSLN)的能力。单域抗体可在N端或C端处截短以使其仅包含部分FR1和/或FR4，或缺少那些骨架区中的一个或两个，只要其实质上保持抗原结合和特异性即可。
在某些实施方案中，本发明的单域抗体或其抗原结合片段包含IMGT编号系统定义的下述CDRs：包含SEQ ID NO:150所示序列或其变体的CDR1，包含SEQ ID NO:151所示序列或其变体的CDR2，以及包含SEQ ID NO:152所示序列或其变体的CDR3；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)。优选地，所述置换为保守置换。在某些实施方案中，所述单域抗体或其抗原结合片段包含IMGT编号系统定义的下述CDRs：包含SEQ ID NO:150所示序列的CDR1，包含SEQ ID NO:151所示序列的CDR2，以及包含SEQ ID NO:152所示序列的CDR3。
在某些实施方案中，本发明的单域抗体或其抗原结合片段包含Kabat编号系统定义的下述CDRs：包含SEQ ID NO:153所示序列或其变体的CDR1，包含SEQ ID NO:154所 示序列或其变体的CDR2，以及包含SEQ ID NO:155所示序列或其变体的CDR3；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)。优选地，所述置换为保守置换。在某些实施方案中，所述单域抗体或其抗原结合片段包含Kabat编号系统定义的下述CDRs：包含SEQ ID NO:153所示序列的CDR1，包含SEQ ID NO:154所示序列的CDR2，以及包含SEQ ID NO:155所示序列的CDR3。
在某些实施方案中，本发明的单域抗体或其抗原结合片段包含AbM编号系统定义的下述CDRs：包含SEQ ID NO:156所示序列或其变体的CDR1，包含SEQ ID NO:157所示序列或其变体的CDR2，以及包含SEQ ID NO:155所示序列或其变体的CDR3；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)。优选地，所述置换为保守置换。在某些实施方案中，所述单域抗体或其抗原结合片段包含AbM编号系统定义的下述CDRs：包含SEQ ID NO:156所示序列的CDR1，包含SEQ ID NO:157所示序列的CDR2，以及包含SEQ ID NO:155所示序列的CDR3。
在某些实施方案中，本发明的单域抗体或其抗原结合片段包含Chothia编号系统定义的下述CDRs：包含SEQ ID NO:158所示序列或其变体的CDR1，包含SEQ ID NO:159所示序列或其变体的CDR2，以及包含SEQ ID NO:155所示序列或其变体的CDR3；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)。优选地，所述置换为保守置换。在某些实施方案中，所述单域抗体或其抗原结合片段包含Chothia编号系统定义的下述CDRs：包含SEQ ID NO:158所示序列的CDR1，包含SEQ ID NO:159所示序列的CDR2，以及包含SEQ ID NO:155所示序列的CDR3。
在某些实施方案中，本发明的单域抗体或其抗原结合片段包含Contact编号系统定义的下述CDRs：包含SEQ ID NO:160所示序列或其变体的CDR1，包含SEQ ID NO:161所示序列或其变体的CDR2，以及包含SEQ ID NO:162所示序列或其变体的CDR3；其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)。优选地，所述置换为保守置换。在某些实施方案中，所述单域抗体或其抗原结合片段包含Contact编号系统定义的下述CDRs：包含SEQ ID NO:160所示序列的CDR1，包含SEQ ID NO:161所示序列的CDR2，以及包含SEQ ID NO:162所示序列的CDR3。
在某些实施方案中，所述单域抗体或其抗原结合片段包含SEQ ID NO:149所示的序列或其变体，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸置换、缺失或添加)。在某些实施方案中，所述置换为保守置换。
在某些实施方案中，本发明的单域抗体或其抗原结合片段可以是人源化的VHH，即其中一或多个骨架区已基本上由人类骨架区替换的VHH。在某些实施方案中，所述单域抗体或或其抗原结合片段进一步包含人免疫球蛋白的重链框架区(例如，人重链胚系抗体基因所编码的氨基酸序列中所包含的重链框架区)，所述重链框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至驼源残基的回复突变。
在某些实施方案中，当通过SPR测定时，所述单域抗体或其抗原结合片段以不高于10 -8M，不高于9.5×10 -9M、9×10 -9M、不高于8.5×10 -9M、不高于8×10 -9M、不高于7.5×10 -9M、不高于7×10 -9M、不高于6.5×10 -9M、不高于6×10 -9M、不高于5.5×10 -9M、不高于5×10 -9M、不高于4.5×10 -9M、不高于4×10 -9M、不高于3.5×10 -9M、不高于3×10 -9M、不高于2.5×10 -9M、不高于2×10 -9M、不高于1.5×10 -9M、不高于10 -9M、不高于9.5×10 - 10M、不高于9×10 -10M、不高于8.5×10 -10M、不高于8×10 -10M、不高于7.5×10 -10M、不高于7×10 -10M的K D结合MSLN(例如，人MSLN)。
在某些实施方案中，当通过流式细胞技术测定时，所述抗体或其抗原结合片段以小于约100nM、10nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM或更小的EC 50结合MSLN(例如，人MSLN，例如表达MSLN的细胞)。
多特异性抗体
本发明第一方面的抗体或其抗原结合片段和/或第二方面的单域抗体或其抗原结合片段可用于形成多特异性抗体。多特异性抗体是对至少两种(例如两种、三种或四种)不同抗原具有结合特异性的抗体，从而能够结合至少两个不同的结合位点和/或靶分子。
因此，在第三方面，本发明涉及多特异性抗体。
多特异性抗体1
本发明提供了多特异性抗体1，其包含靶向CD3的抗原结合结构域和至少一个靶向 其他抗原的抗原结合结构域，所述靶向CD3的抗原结合结构域选自第一方面所述的抗体或其抗原结合片段，所述其他抗原选自肿瘤相关抗原(TAA)和/或免疫检查点分子。
在某些实施方案中，所述多特异性抗体是双特异性抗体，其包含第一方面的靶向CD3的抗原结合结构域和靶向所述肿瘤相关抗原的抗原结合结构域。当双特异性抗体包含结合T细胞特异性靶(例如CD3)的第一抗原结合区(如第一方面所述的抗体)和结合肿瘤特异性靶的第二抗原结合区时，该双特异性抗体可通过结合T细胞上存在的CD3以及肿瘤细胞上的特异性靶抗原促进T细胞对肿瘤细胞的靶向和募集，诱导肿瘤特异(不依赖于MHC)细胞毒性T细胞杀伤活性。
在某些实施方案中，所述多特异性抗体是三特异性抗体，其包含第一方面的靶向CD3的抗原结合结构域、靶向所述肿瘤相关抗原的抗原结合结构域、以及靶向所述免疫检查点分子的抗原结合结构域。
本领域技术人员应理解，本领域已知的双特异性抗体结构形式均可以用于本发明。在某些实施方案中，本发明的双特异性抗体可以是：(i)具有包含不同抗原结合区的两个臂的单一抗体，(ii)例如经由通过额外肽接头串联连接的两个scFv，对于两种不同表位具有特异性的单链抗体；(iii)双重可变结构域抗体(DVD-Ig TM)，其中每条轻链和重链含有通过短肽连接串联的两个可变结构域；(iv)化学连接的双特异性(Fab’)2片段；(v)TandAb，其为两个单链双抗体的融合物，导致对于靶抗原各自具有两个结合位点的四价双特异性抗体；(vi)柔性体(flexibody)，其为scFv与双抗体的组合，导致多价分子；(vii)所谓的“对接锁定(dock and lock)”分子，基于蛋白质激酶A中的“二聚化和对接结构域”，当应用于Fab时，其可以获得由与不同Fab片段连接的两个相同Fab片段组成的三价双特异性结合蛋白；(viii)所谓的蝎形分子(Scorpion molecule)，其包含例如融合至人Fab臂的两个末端的两个scFv；和(ix)双抗体。
在某些实施方案中，所述靶向CD3的抗原结合结构域是全长抗体、Fv片段、Fab片段、F(ab’)2片段或scFv。在某些实施方案中，所述多特异性抗体所包含的其他抗原结合结构域(例如靶向肿瘤相关抗原的抗原结合结构域，靶向免疫检查点分子的抗原结合结构域)各自独立地选自全长抗体、Fv片段、Fab片段、F(ab’)2片段、scFv或VHH。
在某些实施方案中，所述多特异性抗体所包含的各个抗原结合结构域通过肽接头连接。在某些实施方案中，所述靶向肿瘤相关抗原或免疫检查点分子的抗原结合结构域任选地通过接头连接在所述靶向CD3的抗原结合结构域的重链的N末端和/或C末端，和/或连接在所述靶向CD3的抗原结合结构域的轻链的N末端和/或C末端。在某些实施方案 中，所述靶向CD3的抗原结合结构域包含：至少一条重链和至少一条轻链，并且所述靶向肿瘤相关抗原或免疫检查点分子的抗原结合结构域与所述重链连接；或者所述靶向CD3的抗原结合结构域包含：两条相同的重链和两条相同的轻链，并且所述靶向肿瘤相关抗原或免疫检查点分子的抗原结合结构域与两条重链连接。
在某些实施方案中，所述肿瘤相关抗原选自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、GloboH、GM3、hTERT、LMP2、p53、PAP、PAX3、PAX5、PCTA-1、PLAC1、PRLR、Ras、SART-3、TRP-1、TRP-2、CD22、CD30、FOLR1、或其任意组合；
在某些优选的实施方案中，所述肿瘤相关抗原选自MSLN、CD19、CD20、TROP2、HER2或Caludin18.2。
在某些实施方案中，所述免疫检查点分子选自PD-1、PD-L1、PD-L2CTLA-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、或其任意组合。
在某些实施方案中，所述多特异性抗体包含第一多肽链和第二多肽链，其中所述第一多肽链包含第一方面所述的抗体的重链以及与其N或C端连接的特异性结合肿瘤相关抗原(如MSLN、CD19、CD20、Trop2、Her2或Caludin18.2)的抗原结合结构域；所述第二多肽链包含第一方面所述的抗体的轻链。
在某些实施方案中，所述多特异性抗体为靶向CD3/MSLN的双特异性抗体，其包含特异性结合CD3的抗原结合结构域和特异性结合MSLN的抗原结合结构域，所述靶向CD3的抗原结合结构域选自第一方面所述的抗体或其抗原结合片段；所述靶向MSLN的抗原结合结构域选自第二方面所述的单域抗体或其抗原结合片段。
在某些实施方案中，所述靶向CD3/MSLN的双特异性抗体包含第一多肽链和第二多肽链，其中，所述第一多肽链包含如下所示的结构：[VH]-[CH]-[L]-[VHH]，其中，[VH]选自第一方面中所提供的重链可变区序列(例如SEQ ID NOs:1-46任一项所示序列)，[CH]选自第一方面中所提供的重链恒定区序列(例如SEQ ID NO:164所示序列)， [L]为肽接头(例如包含一个或多个甘氨酸和/或一个或多个丝氨酸的肽接头，例如SEQ ID NO:163所示序列)，[VHH]选自第二方面中所提供的单域抗体序列(例如SEQ ID NO:149所示序列)；所述第二多肽链包含如下所示的结构：[VL]-[CL]，其中，[VL]选自第一方面中所提供的轻链可变区序列(例如SEQ ID NO:47或48所示序列)，[CL]选自第一方面中所提供的轻链恒定区序列(例如SEQ ID NO:165所示序列)。
在某些实施方案中，当通过SPR测定时，本发明所述的靶向CD3和MSLN的双特异性抗体以不低于6×10 -10M、不低于6.5×10 -10M、不低于7×10 -10M、不低于7.5×10 -10M、不低于8×10 -10M、不低于8.5×10 -10M、不低于9×10 -10M、不低于9.5×10 -10M、不低于1×10 -9M、不低于1.5×10 -9M、不低于2×10 -9M、不低于2.5×10 -9M、不低于3×10 -9M、不低于3.5×10 -9M、不低于4×10 -9M、不低于4.5×10 -9M、不低于5×10 -9M、不低于5.5×10 - 9M、不低于6×10 -9M、不低于6.5×10 -9M、不低于7×10 -9M、不低于7.5×10 -9M、不低于8×10 -9M、不低于8.5×10 -9M、不低于9×10 -9M、不低于9.5×10 -9M、不低于1×10 -8M、不低于1.5×10 -8M、或不低于2×10 -8M的K D结合CD3(例如，人和/或食蟹猴CD3，例如ε和/或γ链)。在某些实施方案中，当通过SPR测定时，本发明所述的靶向CD3和MSLN的双特异性抗体以不高于10 -7M，例如不高于9.5×10 -8M、不高于9×10 -8M、不高于8.5×10 -8M、不高于8×10 -8M、不高于7.5×10 -8M、不高于7×10 -8M、不高于6.5×10 -8M、不高于6×10 -8M、不高于5.5×10 -8M、不高于5×10 -8M、不高于4.5×10 -8M、不高于4×10 - 8M、不高于3.5×10 -8M、不高于3×10 -8M、不高于2.5×10 -8M或不高于2×10 -8M的K D结合CD3(例如，人和/或食蟹猴CD3，例如ε和/或γ链)。在某些实施方案中，当通过SPR测定时，所述双特异性抗体以10 -8-10 -10M的K D结合CD3(例如，人和/或食蟹猴CD3，例如ε和/或γ链)。
在某些实施方案中，当通过流式细胞技术测定时，本发明所述的靶向CD3和MSLN的双特异性抗体以不低于0.05nM、不低于0.1nM、不低于0.2nM、不低于0.3nM、不低于0.4nM、不低于0.5nM、不低于0.6nM、不低于0.7nM、不低于0.8nM、不低于0.9nM、不低于1nM、不低于1.5nM、不低于2nM、不低于2.5nM、不低于3nM、不低于3.5nM、不低于4nM、不低于4.5nM、不低于5nM、不低于5.5nM、不低于6nM、不低于6.5nM、不低于7nM、不低于7.5nM、不低于8nM、不低于8.5nM、不低于9nM的EC 50结合CD3(例如，人和/或食蟹猴CD3，例如表达CD3的细胞)。在某些实施方案中，当通过流式细胞技术测定时，本发明所述的靶向CD3和MSLN的双特异性抗体以不高于20nM，例如不高于15nM，不高于10nM，不高于9.5nM，不高于9nM，不高于8.5nM， 不高于8nM，不高于7.5nM，不高于7nM，不高于6.5nM，不高于6nM，不高于5.5nM，不高于5nM，不高于4.5nM，不高于4nM，不高于3.5nM，不高于3nM，不高于2.5nM，不高于2nM，不高于1.5nM或不高于1nM的EC 50结合CD3(例如，人和/或食蟹猴CD3，例如表达CD3的细胞)。
在某些实施方案中，当通过SPR测定时，本发明所述的靶向CD3和MSLN的双特异性抗体以不高于10 -8M，不高于9.5×10 -9M、9×10 -9M、不高于8.5×10 -9M、不高于8×10 - 9M、不高于7.5×10 -9M、不高于7×10 -9M、不高于6.5×10 -9M、不高于6×10 -9M、不高于5.5×10 -9M、不高于5×10 -9M、不高于4.5×10 -9M、不高于4×10 -9M、不高于3.5×10 -9M、不高于3×10 -9M、不高于2.5×10 -9M、不高于2×10 -9M、不高于1.5×10 -9M、不高于10 -9M、不高于9.5×10 -10M、不高于9×10 -10M、不高于8.5×10 -10M、不高于8×10 -10M、不高于7.5×10 -10M、不高于7×10 -10M的K D结合MSLN(例如，人MSLN)。
在某些实施方案中，当通过流式细胞技术测定时，本发明所述的靶向CD3和MSLN的双特异性抗体以小于约100nM、10nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM或更小的EC 50结合MSLN(例如，人MSLN，例如表达MSLN的细胞)。
在某些实施方案中，所述多特异性抗体为靶向CD3/CD19的双特异性抗体，其包含特异性结合CD3的抗原结合结构域和特异性结合CD19的抗原结合结构域，所述靶向CD3的抗原结合结构域选自第一方面所述的抗体或其抗原结合片段；所述靶向CD19的抗原结合结构域包含轻链可变区和重链可变区，所述轻链可变区包含SEQ ID NO:171所示的序列或其变体，所述重链可变区包含SEQ ID NO:170所示的序列或其变体。
在某些实施方案中，所述多特异性抗体为靶向CD3/CD20的双特异性抗体，其包含靶向CD3的抗原结合结构域和特异性结合CD20的抗原结合结构域，所述靶向CD3的抗原结合结构域选自第一方面所述的抗体或其抗原结合片段；所述靶向CD20的抗原结合结构域包含轻链可变区和重链可变区，所述轻链可变区包含SEQ ID NO:173所示的序列或其变体，所述重链可变区包含SEQ ID NO:172所示的序列或其变体。
在某些实施方案中，所述多特异性抗体为靶向CD3/trop2的双特异性抗体，其包含靶向CD3的抗原结合结构域和特异性结合trop2的抗原结合结构域，所述靶向CD3的抗原结合结构域选自第一方面所述的抗体或其抗原结合片段；所述靶向trop2的抗原结合结构域为VHH，其包含SEQ ID NO:174所示的序列或其变体。
在某些实施方案中，所述多特异性抗体为靶向CD3/Her2的双特异性抗体，其包含靶 向CD3的抗原结合结构域和特异性结合Her2的抗原结合结构域，所述靶向CD3的抗原结合结构域选自第一方面所述的抗体或其抗原结合片段；所述靶向Her2的抗原结合结构域为VHH，其包含SEQ ID NO:175所示的序列或其变体。
在某些实施方案中，所述多特异性抗体为靶向CD3/Caludin18.2的双特异性抗体，其包含靶向CD3的抗原结合结构域和特异性结合Caludin18.2的抗原结合结构域，所述靶向CD3的抗原结合结构域选自第一方面所述的抗体或其抗原结合片段；所述靶向Caludin18.2的抗原结合结构域为VHH，其包含SEQ ID NO:176所示的序列或其变体。
多特异性抗体2
本发明还提供多特异性抗体2，其包含第二方面所述的单域抗体或其抗原结合片段；在某些优选的实施方案中，所述多特异性抗体为双特异性抗体、三特异性抗体或四特异性抗体。
抗体的制备
本发明第一方面抗体、第二方面单域抗体或第三方面多特异性抗体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码它们的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体、单域抗体或多特异性抗体。
在第四方面，本发明提供了分离的核酸分子，其编码：
-第一方面所述的抗体或其抗原结合片段、或其重链可变区和/或轻链可变区；
-第二方面所述的单域抗体或其抗原结合片段；或，
-第三方面所述的多特异性抗体1或多特异性抗体2，或其多肽链。
在某些实施方案中，所述分离的核酸分子包含编码第一方面所述的抗体或其抗原结合片段的重链或重链可变区的第一核苷酸序列和编码其轻链或轻链可变区的第二核苷酸序列，其中所述第一核苷酸序列和所述第二核苷酸序列存在于相同或不同的分离的核酸分子上。
在某些实施方案中，所述分离的核酸分子包含编码第三方面中所述的双特异性抗体的第一多肽链的第一核苷酸序列和编码其第二多肽链的第二核苷酸序列，其中所述第一核苷酸序列和所述第二核苷酸序列存在于相同或不同的分离的核酸分子上。
在第五方面，本发明提供了载体，其包含如上所述的核酸分子。在某些实施方案中，所述载体为克隆载体或表达载体。
在第六方面，本发明提供了宿主细胞，其包含如上所述的核酸分子或载体。此类宿主细胞包括但不限于，原核细胞例如细菌细胞(如大肠杆菌细胞)，以及真核细胞例如真菌细胞(例如酵母细胞)，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。
在另一方面，本发明提供了制备第一方面所述的抗体或其抗原结合片段、第二方面所述的单域抗体或第三方面所述的多特异性抗体的方法，其包括，在允许蛋白表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中收集所述抗体或其抗原结合片段、单域抗体或多特异性抗体。
药物组合物
本文所公开的抗体(也称为活性成分)可以被整合到适合于施用的药物组合物中。
因此，在第七方面，本发明涉及药物组合物，其包含第一方面所述的抗体或其抗原结合片段、第二方面所述的单域抗体或其抗原结合片段、第三方面所述的多特异性抗体、第四方面所述的分离的核酸分子、第五方面所述的载体、或第六方面所述的宿主细胞，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，所述药物组合物还可以包含另外的药学活性剂，例如抗肿瘤剂。在某些实施方案中，所述另外的药学活性剂与本发明的抗体或其抗原结合片段、单域抗体或其抗原结合片段、多特异性抗体、分离的核酸分子、载体或第宿主细胞分开提供，或作为同一组合物的组分提供。
治疗用途
在第八方面，本发明涉及用于预防和/或治疗疾病的方法，其包括向有此需要的受试者施用第一方面所述的抗体或其抗原结合片段、编码所述抗体或其抗原结合片段的分离的核酸分子、载体或宿主细胞、或包含它们的药物组合物。本发明还涉及第一方面所述的抗体或其抗原结合片段、编码所述抗体或其抗原结合片段的分离的核酸分子、载体或宿主细胞、或包含它们的药物组合物，用于预防和/或治疗疾病的用途，或在制备用于预防和/或治疗疾病的药物中的用途。
本发明第一方面所述的抗体或其抗原结合片段可以用于任何疾病的治疗中，只要所述疾病的治疗中需要细胞毒性T细胞的效应机理。
在某些实施方案中，所述疾病是肿瘤、炎性疾病或自身免疫性疾病。
在某些实施方案中，所述肿瘤是实体瘤或血液肿瘤，例如胃癌、肺癌、卵巢癌、食管癌、胰腺癌、宫颈癌、间皮瘤、乳腺癌、前列腺癌、膀胱癌、卵巢癌、结直肠癌、头 颈部鳞状细胞癌、胰腺癌、睾丸癌、胆管癌、大肠癌、输卵管癌、恶性黑色素瘤、软组织癌(例如滑膜肉瘤)、B细胞淋巴瘤的无痛或侵袭性形式、慢性淋巴性白血病、急性髓性白血病或急性淋巴细胞白血病。
在某些实施方案中，所述疾病的治疗涉及T细胞(例如细胞毒性T细胞)的效应机理。在某些实施方案中，所述抗体或其抗原结合片段用于增强T细胞(例如细胞毒性T细胞)活性以治疗或预防所述疾病。
在第九方面，本发明涉及用于预防和/或治疗肿瘤的方法，其包括向有此需要的受试者施用第二方面所述的单域抗体或其抗原结合片段、编码所述抗体或其抗原结合片段的分离的核酸分子、载体或宿主细胞、或包含它们的药物组合物。本发明还涉及第二方面所述的单域抗体或其抗原结合片段、编码所述抗体或其抗原结合片段的分离的核酸分子、载体或宿主细胞、或包含它们的药物组合物，用于预防和/或治疗肿瘤的用途，或在制备用于预防和/或治疗疾病的肿瘤的药物中的用途。
在某些实施方案中，所述肿瘤为MSLN阳性的肿瘤。
在某些实施方案中，所述肿瘤是实体瘤或血液肿瘤，例如胃癌、肺癌、卵巢癌、食管癌、胰腺癌、宫颈癌、间皮瘤、乳腺癌、前列腺癌、膀胱癌、卵巢癌、结直肠癌、头颈部鳞状细胞癌、胰腺癌、睾丸癌、胆管癌、大肠癌、输卵管癌、恶性黑色素瘤、软组织癌(例如滑膜肉瘤)、B细胞淋巴瘤的无痛或侵袭性形式、慢性淋巴性白血病、急性髓性白血病或急性淋巴细胞白血病。
在某些实施方案中，所述肿瘤选自实体瘤，例如间皮瘤、卵巢癌、胰腺癌、乳腺癌、胆管癌、大肠癌、胃癌、输卵管癌、肺癌或结直肠癌。
在某些实施方案中，所述肿瘤选自血液肿瘤，例如急性髓性白血病。
在第十方面，本发明涉及用于预防和/或治疗疾病的方法，其包括向有此需要的受试者施用第三方面所述的多特异性抗体、编码所述多特异性抗体的分离的核酸分子、载体或宿主细胞、或包含它们的药物组合物。本发明还涉及第三方面所述的多特异性抗体、编码所述多特异性抗体的分离的核酸分子、载体或宿主细胞、或包含它们的药物组合物，用于预防和/或治疗疾病的用途，或在制备用于预防和/或治疗疾病的药物中的用途。
本发明第三方面所述的多特异性抗体可以用于任何疾病的治疗中，只要所述疾病的治疗中需要细胞毒性T细胞的效应机理。例如，当本发明的多特异性抗体包括用于T细胞募集的CD3结合臂和对肿瘤相关抗原(TAA)有特异性的肿瘤靶向臂时，其使T细胞与靶肿瘤细胞紧密接触，局部激活T细胞，随后由T细胞毒性颗粒释放的穿孔素和颗粒酶 破坏靶细胞。例如，当本发明的多特异性抗体包括用于T细胞募集的CD3结合臂和对免疫检查点分子有特异性的靶向臂时，其使肿瘤微环境中的T细胞激活，随后杀伤肿瘤细胞。
在某些实施方案中，所述疾病是肿瘤、炎性疾病或自身免疫性疾病。
在某些实施方案中，所述肿瘤是实体瘤或血液肿瘤，例如胃癌、肺癌、卵巢癌、食管癌、胰腺癌、宫颈癌、间皮瘤、乳腺癌、前列腺癌、膀胱癌、卵巢癌、结直肠癌、头颈部鳞状细胞癌、胰腺癌、睾丸癌、胆管癌、大肠癌、输卵管癌、恶性黑色素瘤、软组织癌(例如滑膜肉瘤)、B细胞淋巴瘤的无痛或侵袭性形式、慢性淋巴性白血病、急性髓性白血病或急性淋巴细胞白血病。
在某些实施方案中，所述疾病的治疗涉及T细胞(例如细胞毒性T细胞)的效应机理。在某些实施方案中，所述多特异性抗体用于将T细胞招募至靶细胞周围，激活T细胞，诱导T细胞介导的细胞杀伤(TDCC)作用，从而有效杀伤靶细胞，以治疗或预防所述疾病。
在某些实施方案中，所述肿瘤为MSLN阳性的肿瘤，例如为实体瘤，例如间皮瘤、卵巢癌、胰腺癌、乳腺癌、胆管癌、大肠癌、胃癌、输卵管癌、肺癌或结直肠癌；例如为血液肿瘤，例如急性髓性白血病。
在某些实施方案中，所述肿瘤为CD19阳性的肿瘤，例如B细胞恶性肿瘤，例如淋巴瘤或白血病。
在某些实施方案中，所述肿瘤为CD20阳性的肿瘤，例如B细胞恶性肿瘤，例如淋巴瘤或白血病。
在某些实施方案中，所述肿瘤为Trop2阳性的肿瘤，例如为实体瘤，例如乳腺癌。
在某些实施方案中，所述肿瘤为Her2阳性的肿瘤，例如为实体瘤，例如肺癌。
在某些实施方案中，所述肿瘤为Caludin18.2阳性的肿瘤，例如为实体瘤，例如胃癌。
在本发明的第八到第十方面中的任一方面中，本发明的抗体或其抗原结合片段、单域抗体或其抗原结合片段、多特异性抗体或包含它们的药物组合物可以配制成医学领域已知的任何剂型。
在本发明的第八到第十方面中的任一方面中，本发明的抗体或其抗原结合片段、单域抗体或其抗原结合片段、多特异性抗体或包含它们的药物组合物可以通过本领域已知的任何合适的方法来施用。
在本发明的第八到第十方面中的任一方面中，本发明的抗体或其抗原结合片段、单 域抗体或其抗原结合片段、多特异性抗体或包含它们的药物组合物可以以剂量单位形式配制以易于施用。
在本发明的第八到第十方面中的任一方面中，本发明的抗体或其抗原结合片段、单域抗体或其抗原结合片段、多特异性抗体或包含它们的药物组合物可以单独施用，也可以与另外的药学活性剂(例如抗肿瘤剂)或另外的疗法(例如抗肿瘤疗法)联合施用。
在本发明的第八到第十方面中的任一方面中，所述受试者可以是哺乳动物，例如人。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的病毒学、生物化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
当本文使用术语“例如”、“如”、“诸如”、“包括”、“包含”或其变体时，这些术语将不被认为是限制性术语，而将被解释为表示“但不限于”或“不限于”。
除非本文另外指明或根据上下文明显矛盾，否则术语“一个”和“一种”以及“该”和类似指称物在描述本发明的上下文中(尤其在以下权利要求的上下文中)应被解释成覆盖单数和复数。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为框架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat，Sequences of Proteins of Immunological  Interest(National Institutes of Health，Bethesda，Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDR，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，Md.，1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)、IMGT编号系统(Lefranc et al.，Dev.Comparat.Immunol.27:55-77，2003)、AbM编号系统(Martin ACR，Cheetham JC，Rees AR(1989)Modelling antibody hypervariable loops:A combined algorithm.Proc Natl Acad Sci USA 86:9268–9272)或Contact编号系统(MacCallum，R.M.，Martin，A.C.R.，&Thornton，J.M.(1996).Antibody-antigen Interactions:Contact Analysis and Binding Site Topography.Journal of Molecular Biology，262(5)，732-745.)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.，Dev.Comparat.Immunol.27:55-77，2003)。
在本发明中，本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定，例如通过Kabat、Chothia、IMGT、AbM或Contact编号系统确定。在某些实施方案中，本发明所涉及的抗CD3抗体或其抗原结合片段含有的CDR通过Kabat编号系统确定。在某些实施方案中，本发明所涉及的抗MSLN单域抗体或其抗原结合片段含有的CDR通过Kabat、Chothia、IMGT、AbM或Contact编号系统确定。
如本文中所使用的，术语“框架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology， Ch.7(Paul，W.，ed.，第2版，Raven Press，N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’) 2、Fd、Fv、互补决定区(CDR)片段、scFv、双抗体(diabody)、单域抗体(single domain antibody)、嵌合抗体、线性抗体(linear antibody)、纳米抗体(技术来自Domantis)、probody和包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分的多肽。
如本文中所使用的，术语“全长抗体”意指，由两条“全长重链”和两条“全长轻链”组成的抗体。其中，“全长重链”是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域。优选地，“全长重链”是在N端到C端方向上由VH、CH1、HR、CH2和CH3组成的多肽链。“全长轻链”是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链。两对全长抗体链通过在CL和CH1之间的二硫键和两条全长重链的HR之间的二硫键连接在一起。全长抗体包含分别由VH和VL对形成的两个抗原结合部位，这两个抗原结合部位特异性识别/结合相同的抗原。
如本文中所使用的，术语“单域抗体(single-domain antibody，sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，通常来源于重链抗体(例如骆驼科动物抗体或鲨鱼抗体)的可变区。典型地，纳米抗体由4个框架区和3个互补性决定区组成，具有FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的结构。单域抗体可在N端或C端处截短以使其仅包含部分FR1和/或FR4，或缺少那些骨架区中的一个或两个，只要其实质上保持抗原结合和特异性即可。单域抗体也称为纳米抗体(nanobody)，两者可互换使用。在本文中，术语单域抗体的“抗原结合片段”是指包含单域抗体的片段的多肽，其保持特异性结合单域抗体所结合的相同抗原的能力，和/或与单域抗体竞争对抗原的特异性结合。可通过重组DNA技术或通过本发明单域抗体的酶促或化学断裂产生本发明单域抗体的抗原结合片段。在一些实施方案中，所述单域抗体的“抗原结合片段”与全长单域抗体相比可在N端或C端处截短以使其仅包含部分FR1和/或FR4，或缺少那些骨架区中的一个或两个，只要其实质上保持抗原结合和特异性即可。
如本文中所使用的，术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’) 2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段 的抗体片段；术语“Fab’片段”意指还原连接F(ab’) 2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
如本文中所使用的，术语“Fc结构域”或“Fc区”意指，包含CH2和CH3的重链恒定区的一部分。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。由Fc区介导的“效应子功能”包括Fc受体结合；Clq结合和补体依赖性细胞毒性(CDC)；抗体依赖性细胞介导的细胞毒性(ADCC)；噬菌作用；对细胞表面受体(例如B细胞受体)的下调；和B细胞活化等。在一些实施方案中，Fc区包含铰链、CH2和CH3。当Fc区包含铰链时，铰链调节两个含Fc的多肽之间的二聚作用。Fc区可为任何抗体重链恒定区同型，例如IgG1、IgG2、IgG3或IgG4。
Fc结构域既可以包括天然Fc区，也可以包括变异Fc区。天然Fc区包含与自然界中发现的Fc区的氨基酸序列一致的氨基酸序列，例如天然序列人类Fc区包括天然序列人类IgG1Fc区(非A和A同种异型)；天然序列人类IgG2Fc区；天然序列人类IgG3Fc区；及天然序列人类IgG4Fc区，以及其天然存在的变异体。变异Fc区包含因至少一个氨基酸修饰而与天然序列Fc区的氨基酸序列不同的氨基酸序列。在一些实施方案中，变异Fc区可具备相比于天然Fc区改变的效应子功能(例如Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能)。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 2的接头，但也可使用其变体。在一些情况下，scFv的VH与VL之间还可以存在二硫键。
如本文中所使用的，术语“双抗体”意指，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.等 人，Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人，Structure2:1121-1123(1994))。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“胚系抗体基因(germline antibody gene)”或“胚系抗体基因片段(germline antibody gene segment)”是指，存在于生物体的基因组中的编码免疫球蛋白的序列，其没有经历过能够导致表达特异性免疫球蛋白的遗传学重排及突变的成熟过程。在本发明中，表述“重链胚系基因”是指，编码免疫球蛋白重链的胚系抗体基因或基因片段，其包括V基因(variable)、D基因(diversity)、J基因(joining)和C基因(constant)；类似地，表述“轻链胚系基因”是指，编码免疫球蛋白轻链的胚系抗体基因或基因片段，其包括V基因(variable)、J基因(joining)和C基因(constant)。在本发明中，由所述胚系抗体基因或胚系抗体基因片段所编码的氨基酸序列也称为“胚系序列(germline sequence)”。胚系抗体基因或胚系抗体基因片段及其相应的胚系序列是本领域技术人员熟知的，并且可从专业数据库(例如，IMGT、UNSWIg、NCBI或VBASE2)获得或查询。通常认为，种系抗体基因比成熟抗体基因更有可能保留在物种中个体特有的关键氨基酸序列结构，因此当在该物种中用于治疗时，被认为是外源的可能性相对较小。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。为了测定两个氨基酸序列或两个核酸序列的百分比同一性，为了最佳比较目的将序列进行比对(例如，可在第一氨基酸序列或核酸序列中引入缺口以与第二氨基酸或核酸序列最佳比对)。然后比较对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置被与第二序列中的对应位置相同的氨基酸残基或核苷酸占据时，则分子在该位置上是同一的。两个序列之间的百分比同一性是由序列所共享的同一性位置的数目的函数(即，百分比同一性＝同一重叠位置的数目/位置的总数×100％)。在某些实施方案中，两个序列长度相同。
两个序列之间的百分比同一性的测定还可使用数学算法来实现。用于两个序列的比较的数学算法的一个非限制性实例是Karlin和Altschul的算法，1990，Proc.Natl.Acad.Sci.U.S.A.87:2264-2268，如同Karlin和Altschul，1993，Proc.Natl.Acad.Sci.U.S.A.90:5873-5877中改进的。将这样的算法整合至Altschul等人，1990，J.Mol.Biol.215:403的NBLAST和XBLAST程序中。
如本文中所使用的，术语“变体”，在多肽的情境中(包括多肽)也指包含已通过引入氨基酸残基置换、缺失或添加改变的氨基酸序列的多肽或肽。在某些情况下，术语“变体”还指已被修饰(即，通过将任何类型的分子共价连接至多肽或肽)的多肽或肽。例如，但非限制性地，多肽可以被修饰，例如通过糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知保护/封闭基团进行的衍生化、蛋白水解切割、连接至细胞配体或其它蛋白质等。衍生多肽或肽可使用本领域技术人员已知的技术通过化学修饰来产生，所述技术包括但不限于特异性化学切割、乙酰化、甲酰化、衣霉素的代谢合成等。此外，变体具有与其所源自的多肽或肽相似、相同或改善的功能。在某些实施方案中，所述变体与其所源自的序列相比具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(K D)表示。在本发明中，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M，Nature，1993，361:186-187)。kdis/kon的比率等于解离常数K D(参见Davies等人，Annual Rev Biochem，1990；59:439-473)。可用任何有效的方法测量K D、kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数，还可用生物发光干涉测量法或Kinexa来测量解离常数。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞 中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体，例如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞、CHO细胞、COS细胞、NSO细胞、HeLa细胞、BHK细胞、HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。此外，氨基酸残基还可以分成通过可选物理和功能特性限定的类别，例如，含醇基残基(S和T)、脂肪族残基(I、L、V和M)、环烯基相关残基(F、H、W和Y)、疏水性残基(A、C、F、G、H、I、L、M、R、T、V、W和Y)、带负电的残基(D和E)、极性残基(C、D、E、H、K、N、Q、R、S和T)、带正电的残基(H、K和R)、小残基(A、C、D、G、N、P、S、T和V)，极小残基(A、G和S)、涉及转角形成的残基(A、C、D、E、G、H、K、N、Q、R、S、P和T)、柔性残基(Q、T、K、S、G、P、D、E和R)。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人 Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition，E.S.Golub and D.R.Gren，Eds.，Sinauer Associates，Sunderland，Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR，19th ed.Pennsylvania:Mack Publishing Company，1995)，并且包括但不限于：pH调节剂、表面活性剂、佐剂、离子强度增强剂、稀释剂、维持渗透压的试剂、延迟吸收的试剂、防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子、阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水、水性缓冲液(如缓冲盐水)、醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞、2-苯氧乙醇、对羟苯甲酸酯、三氯叔丁醇、苯酚、山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠、明胶、SPGA、糖类(如山梨醇、甘露醇、淀粉、蔗糖、乳糖、葡聚糖、或葡萄糖)、氨基酸(如谷氨酸、甘氨酸)、蛋白质(如干燥乳清、白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括(但不限于)减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方案中，所述受试者(例如人)患有肿瘤(例如表达MSLN的肿瘤)、炎性 疾病或自身免疫病，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤、炎性疾病或自身免疫病)有效量是指，足以预防，阻止，或延迟疾病(例如，肿瘤、炎性疾病或自身免疫病)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
本发明提供了新的CD3抗体，其具有降低的细胞因子释放，表明其具有适度激活T细胞的能力。本发明还提供了靶向CD3和另外的抗原(例如肿瘤相关抗原和/或免疫检查点分子)的多特异性抗体，例如靶向CD3和MSLN的双特异性抗体，该双特异性抗体可通过结合T细胞上存在的CD3以及肿瘤细胞上的特异性靶抗原促进T细胞对肿瘤细胞的靶向和募集，诱导肿瘤特异(不依赖于MHC)细胞毒性T细胞杀伤活性，且具有显著降低的细胞因子释放，因而具有明显提高的安全性。因此，本发明的抗体具有重要的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
序列信息
本申请涉及的序列的描述提供于下表中。
表1：序列信息
Figure PCTCN2022142626-appb-000001
Figure PCTCN2022142626-appb-000002
Figure PCTCN2022142626-appb-000003
Figure PCTCN2022142626-appb-000004
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：构建CD3单克隆抗体
本实施例在CD3单克隆抗体的重链可变区和轻链可变区引入突变，建立scFv噬菌体突变库。所构建的CD3单克隆抗体的重链可变区命名为VH1～VH46，并且分别具有SEQ  ID NOs:1-46所示的氨基酸序列，所构建的CD3单克隆抗体的轻链可变区命名为VL-1～VL-2，并且分别具有SEQ ID NOs:47和48所示的氨基酸序列，各可变区的CDR信息如表1和表2中所示。将所构建的VH1～VH46分别和VL-1进行配对，建立scFv噬菌体突变库，构建CD3单克隆抗体。CD3单克隆抗体的具体结构为IgG抗体，将CD3单克隆抗体的VH连接至IgG1突变型重链恒定区(SEQ ID NO:164)，形成具有[VH]-[CH]所示的结构；将CD3单克隆抗体的VL-1连接至轻链恒定区(SEQ ID NO:165)，形成具有[VL]-[CL]所示的结构。为行文方便，包含VH1和VL-1的CD3单克隆抗体成为M1，包含VH2和VL-1的CD3单克隆抗体成为M2，以此类推，共计46条CD3单克隆抗体命名为M1～M46。包被CD3εγ(公司：acrobiosystems货号:CDG-H5253)，加入包装好的噬菌体，常温孵育1h，洗涤10次，拍干后，洗脱噬菌体，将洗脱的噬菌体侵染TG1感受态细胞，过夜后收取噬菌体；重复前面操作2次；挑选单菌落，IPTG诱导过夜表达。通过ELISA方法确定阳性克隆。
表2：CD3单抗的CDR序列
Figure PCTCN2022142626-appb-000005
Figure PCTCN2022142626-appb-000006
Figure PCTCN2022142626-appb-000007
实施例2：CD3单抗的纯度测定
将CD3单抗M1至M46分别转移100μl转移至1.5ml离心管中，12000rpm离心5min，然后取90μl样品转移至液相进样瓶中，上机(HPLC，岛津，2030C)进行检测。结果如表3，由表3可知，SEC纯度大于90％，可以继续进行体外药效实验。
表3：CD3单抗的纯度检测结果
抗体名称 M1 M2 M3 M4 M5 M6 M7 M8 M9 M10
SEC％ 95.5 97.1 93.5 98.2 96.1 95.2 95.3 98.9 92.7 94.6
抗体名称 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20
SEC％ 99.2 92.8 96.2 97.2 95.1 95.4 95.1 96.5 92.9 96.4
抗体名称 M21 M22 M23 M24 M25 M26 M27 M28 M29 M30
SEC％ 97.3 96.2 96.7 95.2 95.6 95.4 95.8 97.5 94.2 93.7
抗体名称 M31 M32 M33 M34 M35 M36 M37 M38 M39 M40
SEC％ 95.8 93.7 94.8 96.1 96.2 96.7 95.1 97.5 94.3 97.1
抗体名称 M41 M42 M43 M44 M45 M46        
SEC％ 96.7 94.2 93.8 97.6 95.5 94.8        
实施例3：CD3单抗亲和力检测
本实施例考察CD3单抗与Jurkat细胞(天然表达人CD3)(公司：ATCC；货号：TIB-152)和食蟹猴HSC-F细胞(表达猴CD3)(公司：JCRB Cell Bank；货号：JCRB1164)的结合活性。
①.准备细胞：将Jurkat和HSC-F细胞用PBS分别调整成至1*10 7个细胞/ml，准备每个样品加入50μl的细胞悬液。
②.准备抗体：用PBS将抗体调整成终浓度为2μg/ml，然后做3倍梯度稀释，做8个梯度。每孔50μl的抗体。
③.细胞和抗体孵育：取50μl的细胞悬液，细胞总量即为5*10 5个细胞/孔。抗体终浓度即为2μg/ml的起始浓度，三倍梯度稀释8个孔，4℃，孵育1h。
④.500g，5min，轻轻去掉上清，加入200μl PBS洗2次。
⑤.加入二抗：加入用PBS稀释好的荧光二抗(1:100使用)，4℃，30min。
⑥.洗涤荧光二抗，500g，5min，轻轻去掉上清，加入200μl PBS洗1次后，上流式检测。
本实验中使用两株阳性对照抗体，对照抗体OKT3的VH氨基酸序列如SEQ ID NO:166所示；VL的氨基酸序列如SEQ ID NO:167所示；对照抗体1的VH氨基酸序列如SEQ ID NO:168所示；VL的氨基酸序列如SEQ ID NO:169所示。
亲和力检测结果如表4。
表4：CD3单抗的亲和力检测结果
Figure PCTCN2022142626-appb-000008
Figure PCTCN2022142626-appb-000009
实施例4：CD3单抗的动态亲和力测定
将4、2、1μg/mL三种浓度的抗体固定在含有anti-human IgG Fc二抗的HC200M芯片上(Xantec货号HC200M)，控制抗体结合量约1000RU。基线平稳后，将梯度稀释的人CD3εγ(公司：acrobiosystems货号:CDG-H52W5)重组抗原(从592.5nM起，3倍稀释8个梯度)，以1000μL/min的流速流过芯片，结合时间8min，解离时间20min。用Carterra自带的Kinetics软件1：1结合模型，拟合得到动力学常数。
结果如表5，由表5可知，本发明的CD3单抗与人的CD3εγ抗原具有亲和力。
表5：CD3单抗的动态亲和力测定结果
Figure PCTCN2022142626-appb-000010
Figure PCTCN2022142626-appb-000011
实施例5：人MSLN稳转细胞株的构建
构建含有人MSLN(huMSLN)全长序列(cDNA购自Sino biological，HG13128-UT)克隆到慢病毒载体上，将构建好的慢病毒质粒和包装质粒共转染HEK293T细胞，分别收集48h和72h细胞上清，先后加入到MC38细胞(南京科佰，货号CBP60825)，24h后加入4μg/ml嘌呤霉素进行筛选。通过细胞分选仪(Sony，LE-SH800SBP)分选高表达huMSLN的单细胞，最后得到高表达huMSLN的稳转细胞单克隆MC38/MSLN。
实施例6：羊驼免疫
抗huMSLN纳米抗体通过免疫羊驼产生。羊驼免疫委托阿帕克生物技术有限公司完成，即免疫两只羊驼。免疫抗原为hFc标签的人MSLN重组蛋白(huMSLN-hFc，恺佧生物，MSL-HM280)。首免用弗氏完全佐剂(CFA)与huMSLN-hFc混合后皮下免疫，其余加强免疫用弗氏不完全佐剂(IFA)与huMSLN-hFc混合后皮下免疫三次。
实施例7：建立噬菌体抗体库
羊驼四免之后取50ml外周血，按照淋巴细胞分离液(灏洋生物，LTS1077)使用说明分离羊驼PBMC。然后用RNAiso Plus(takara，9109)试剂提取总RNA，参见PrimeScript TMII 1st Strand cDNA Synthesis Kit，说明书，共转录5μg RNA，获得的cDNA。用获得的cDNA作为模版扩增重链可变区(VHH)片段，然后与酶切后的载体进行连接，并电转TG1感受态细胞。一只羊驼细菌库容2.14×10 9，另一只羊驼细菌库容7.8×10 9。
实施例8：MSLN单域抗体筛选
构建好的两只羊驼文库，均同时采用细胞和蛋白淘选。经过细胞三轮或四轮淘选，蛋白两轮淘选后，提取淘选后细菌文库质粒，PCR扩增VHH片段，连接到表达载体上，再挑克隆诱导VHH分泌表达。
包被抗原人MSLN-his(huMSLN-his，恺佧生物，MSL-HM18D)、猴MSLN-his(cynoMSLN-his，恺佧生物，MSL-CM180)、鼠MSLN-his(muMSLN-his，恺佧生物，MSL-MM180)，将抗原包被液(pH9.6的碳酸盐缓冲液)将抗原稀释至2μg/ml，然后分加到酶标板中，50μl/孔，2～8℃包被过夜；次日用PBST洗涤三次，加入2％BSA 37℃封闭2h；每孔加入诱导表达上清，100μl/孔，37℃孵育1h；用PBST洗涤三次，加入HRP标记的anti-DYKDDDDK抗体(武汉三鹰生物HRP-66008)，50μl/孔，37℃孵育1h；用PBST洗涤三次，加入TMB显色并读数。
获得一个人、猴和鼠交叉良好的MSLN单域抗体，命名为3C6。抗体送测序，用CLC软件进行序列分析，将unique序列的克隆诱导表达并纯化。3C6的VHH氨基酸序列如SEQ ID NO:149所示，其CDR序列如表6所示。
表6：3C6的CDR序列如下所示：
Figure PCTCN2022142626-appb-000012
Figure PCTCN2022142626-appb-000013
实施例9：MSLN单域抗体诱导表达和纯化
取保存的甘油菌在2YT培养基(含Amp)，37℃，250rpm活化过夜，次日按照1:100接种到新鲜培养基中培养(37℃，250rpm)至OD600＝0.6左右，加入诱导培养基诱导培养过夜(30℃，250rpm)。诱导后的菌液离心收集上清，用Ni(NTA型)亲和层析介质重力柱纯化，获得MSLN单域抗体。
实施例10：MSLN单域抗体亲和力检测
ELISA亲和力测定：包被人MSLN-his(huMSLN-his，恺佧生物，MSL-HM18D)、猴MSLN-his(cynoMSLN-his，恺佧生物，MSL-CM180)、鼠MSLN-his(muMSLN-his，恺佧生物，MSL-MM180)，4℃包被过夜。2％BSA封闭2h后，每孔加入不同稀释倍数的单域抗体(抗体从100nM起，3倍稀释8个浓度)，孵育1h；加入HRP标记的anti-DYKDDDDK抗体(武汉三鹰生物，HRP-66008)，TMB溶液显色后，用2M盐酸终止，并读450nm处的吸光度。用Graphpad Prism软件计数EC 50，亲和力结果见表7。
表7：MSLN单域抗体与MSLN蛋白的结合
抗体名称 huMSLN(nM) cynoMSLN(nM) muMSLN(nM)
3C6 0.463 0.7877 28.64
实施例11：细胞水平抗MSLN单域抗体亲和力测定
取50μl实施例5获得的MC38/MSLN细胞(2×10 5个细胞)加入到96孔V型板中，每孔再加入50μl梯度稀释的抗体(抗体从300nM起，3倍稀释8个浓度)，冰上孵育1h。洗去一抗后，加入稀释好的Alexa Fluro488标记的anti-DYKDDDDK抗体(武汉三鹰生物HRP-66008)，冰上孵育1h后，4洗涤后每孔重悬200μl PBS后，上流式仪检测。用Graphpad Prism软件计数EC 50，细胞亲和力结果见表8。
表8：MSLN单域抗体与MSLN稳转细胞的结合
Figure PCTCN2022142626-appb-000014
Figure PCTCN2022142626-appb-000015
实施例12：MSLN单域抗体动态亲和力的测定
anti-DYKDDDDK抗体通过氨基偶联结合到HC30M芯片上，然后捕获2μg/ml的MSLN单域抗体，基线平稳后，将梯度稀释好的huMSLN-Fc(恺佧生物，MSL-HM280)和cynoMSLN-Fc(恺佧生物，MSL-CM280)(从100nM开始，3倍梯度稀释，共8个浓度)分别从低浓度到高浓度流过芯片，结合时间8min，接力时间20min。用Carteria软件拟合得到动力学常数。MSLN单域抗体与MSLN蛋白的动态亲和力的结果见表9。
表9：MSLN单域抗体与MSLN蛋白的动态亲和力
Figure PCTCN2022142626-appb-000016
实施例13：CD3-MSLN双特异性抗体的表达和纯化
1、CD3-MSLN双特异性抗体的构建
本实施例的CD3-MSLN双特异性抗体的结构如图1所示，其中，人源化CD3抗体双臂(重轻链配对)，IgG1突变型C端以(GGGGS) 2连接MSLN单域抗体(VHH抗体)形成CD3-MSLN双特异性抗体(双价)。具体地，将实施例1中获得的人源化CD3抗体的VH连接至IgG1突变型重链恒定区(SEQ ID NO:164)，并进一步重链恒定区的C端通过连接子(GGGGS) 2连接上述3C6抗体，形成具有[VH]-[CH]-[L]-[VHH]所示结构的第一多肽链；将实施例1中获得的人源化CD3抗体的VL-1连接轻链恒定区(SEQ ID NO:165)，形成具有[VL]-[CL]所示结构的第二多肽链。将编码上述多肽链的核酸序列构建至PTT5质粒载体上(优宝生物，lot:VT2202)，分别提取足够的质粒备用。
2、CD3-MSLN双特异性抗体的表达和纯化
转染前一天(D1)，将细胞密度用培养基稀释至2×10 6个细胞/ml，转染当天(D0)，取细胞计数(细胞活率应≥95％)，将细胞密度调整为4.0×10 6个细胞/ml，将抗体片段的质粒，按重链：轻链1:1的比率混合，与PEI混合，共同转染CHO-S细胞，转染完成将细胞转移到37℃、120rpm、8％CO 2培养箱中培养，转染后第一天(D1)，按照表达体积的1/5补加预热CHOgro完全培养基培养基，降温至32℃继续培养，转染后第2、4、6天分别按照8％、5％、5％补加Advanced CHO Feed1，从D7开始每天监测细胞活率，活率<80％时收获细胞；离心收上清用protein A柱子纯化备用。纯度如表 10。为行文方便，包含VH1的双特异性抗体成为B1，包含VH2的双特异性抗体成为B2，以此类推。
表10：双特异性抗体纯度检测结果
抗体名称 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
抗体名称 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
抗体名称 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
抗体名称 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
抗体名称 B41 B42 B43 B44 B45 B46        
SEC％ 94.9 96.3 91.7 95.1 93.8 96.4        
实施例14：CD3-MSLN双特异性抗体的结合活性测定
1、考察CD3-MSLN双特异性抗体与Jurkat(天然表达人CD3)和食蟹猴HSC-F细胞(公司：JCRB Cell Bank；货号：JCRB1164)的结合活性，其中，使用两株阳性对照抗体：
阳性对照抗体OKT3-3C6及对照抗体1-3C6的双抗结构参照实施例13中的“CD3-MSLN双特异性抗体”进行构建。其中，OKT3-3C6的CD3抗体的VH序列具有SEQ ID NO:166所示的氨基酸序列，CD3抗体的VL序列具有SEQ ID NO:167所示的氨基酸序列，VHH是MSLN的3C6抗体；对照抗体1-3C6的CD3抗体的VH序列具有SEQ ID NO:168所示的氨基酸序列，CD3抗体的VL序列具有SEQ ID NO:169所示的氨基酸序列，VHH是MSLN的3C6抗体。
实验方法如下：
①.准备细胞：将Jurkat和HSC-F细胞用PBS调整成至1*10 7个细胞/ml，准备每个样品加入50μl的细胞悬液。
②.准备抗体：用PBS将抗体调整成终浓度为2μg/ml，然后做3倍梯度稀释，做8个梯度。每孔50μl的抗体。
③.细胞和抗体孵育：取50μl的细胞悬液，细胞总量即为5*10 5个细胞/孔。抗体终浓度即为2μg/ml的起始浓度，三倍梯度稀释8个孔，4℃，孵育1h。
④.500g，5min，轻轻去掉上清，加入200μl PBS洗2次。
⑤.加入二抗：加入用PBS稀释好的荧光二抗(1:100使用)，4℃，30min。
⑥.洗涤荧光二抗，500g，5min，轻轻去掉上清，加入200μl PBS洗1次后，上流式检测。
结果如表11所示。结果显示，本发明的CD3-MSLN双特异性抗体大部分在针对表达人CD3的细胞的结合活性和针对表达食蟹猴CD3的细胞的结合活性方面处于同一个数量级。
表11：双特异性抗体表达CD3细胞的结合活性测定
Figure PCTCN2022142626-appb-000017
Figure PCTCN2022142626-appb-000018
2、CD3-MSLN双特异性抗体与MC38-MSLN细胞的结合活性
①MC38-MSLN细胞系构建：合成MSLN(NCBI genbank:Accession#AAH09272)全长DNA序列至pLVX-IRES-Puro载体中(优宝生物货号：VT1464)；包装慢病毒并感染MC38细胞，加压筛选后单克隆分选获得高表达MSLN的单克隆细胞系。
②准备细胞：将MC38-MSLN细胞用PBS调整成至1*10 7个细胞/ml，准备每个样品加入50μl的细胞悬液。
③准备抗体：用PBS将抗体调整成终浓度为2μg/ml，然后做3倍梯度稀释，做8个梯度。每孔50μl的抗体。
④细胞和抗体孵育：取50μl的细胞悬液，细胞总量即为5*10 5个细胞/孔。抗体终浓度即为2μg/ml的起始浓度，三倍梯度稀释8个孔，4℃，孵育1h。
⑤.500g，5min，轻轻去掉上清，加入200μl PBS洗2次。
⑥.加入二抗：加入用PBS稀释好的荧光二抗(1:100使用)，4℃，30min。
⑦.洗涤荧光二抗，500g，5min，轻轻去掉上清，加入200μl PBS洗1次后，上流式检测。
结果如图2所示，结果显示，本发明的CD3-MSLN双特异性抗体对表达MSLN细胞具有良好的结合活性。
3、CD3-MSLN双特异性抗体的亲和力检测
①.CD3-MSLN双特异性抗体与CD3εγ重组抗原的亲和力
将4、2、1μg/mL三种浓度的抗体固定在含有anti-human IgG Fc二抗的HC200M芯片上，控制抗体结合量约1000RU。基线平稳后，将梯度稀释的人CD3εγ(公司：acrobiosystems货号:CDG-H52W5)重组抗原(从592.5nM起，3倍稀释8个梯度)，以1000μL/min的流速流过芯片，结合时间8min，解离时间20min。用Carterra自带的Kinetics软件1：1结合模型，拟合得到动力学常数。亲和力测定结果见下表12，动态亲和力图如图3A-3D所示。结果显示，本发明的CD3-MSLN双特异性抗体对人CD3εγ重组抗原的亲和力在10 -8-10 -10M的范围内。
表12：动态亲和力测定结果
Figure PCTCN2022142626-appb-000019
Figure PCTCN2022142626-appb-000020
②.CD3-MSLN双特异性抗体与MSLN重组抗原的亲和力
将4、2、1μg/m L三种浓度的抗体固定在含有anti-human IgG Fc二抗的HC200M芯片上，控制抗体结合量约1000RU。基线平稳后，将梯度稀释的人MSLN重组抗原(公司：acrobiosystems货号:MSN-H8223)(从592.5nM起，3倍稀释8个梯度)，以1000μL/min的流速流过芯片，结合时间8min，解离时间20min。用Carterra自带的Kinetics软件1：1结合模型，拟合得到动力学常数。示例性的双特异性抗体亲和力测定结果如表13，动态亲和力图如图4所示。结果显示，本发明的CD3-MSLN双特异性抗体均对MSLN具有良好的结合活性。
表13：动态亲和力测定结果
Figure PCTCN2022142626-appb-000021
实施例15：Jurkat-TIGIT-luc细胞稳定性实验
1.抗体稀释
用1640+10％FBS培养基分别将B8、B13、B22、B33、B37和B43稀释抗体至10nM，2倍梯度稀释，共11个梯度，每孔添加50μl稀释的抗体；
2.加入Jurkat-TIGIT-luc细胞
取生长对数期Jurkat-TIGIT-luc(南京科佰，货号CBP74020)细胞，用1640+10％FBS调整成6*10 5个细胞/ml，加入至含有上述抗体的孔中，每孔加入50μl细 胞(3*10 4个细胞/孔)；
3.孵育
将上述细胞和抗体的混合物轻轻混匀后，放置37℃、5％CO 2孵育5-6h；
4.上机检测
加入20μl/孔的化学发光底物后上机检测；
重复4次上述1-4的步骤，得到4次抗体激活Jurkat-TIGIT-luc细胞的实验数据，从而验证Jurkat-TIGIT-luc细胞的稳定性。激活Jurkat-TIGIT-luc细胞的测定结果如图5A-5F所示。结果显示，B8、B13、B22、B33、B37和B43抗体激活Jurkat-TIGIT-luc细胞的4次重复实验结果无显著差异，表明Jurkat-TIGIT-luc细胞稳定。因此，用Jurkat-TIGIT-luc细胞系进行后续的T细胞活化信号通路的激活实验。
实施例16：CD3-MSLN双特异性抗体对T细胞活化信号通路的激活
用1640+10％FBS稀释抗体至2μg/ml，三倍梯度稀释，共12个梯度，每孔添加50μl稀释的抗体；不加肿瘤组：取生长对数期Jurkat-TIGIT-luc(南京科佰，货号CBP74020)细胞，用1640+10％FBS调整成6*10 5个细胞/ml，每孔加入50μl细胞(3*10 4个细胞/孔)，轻轻混匀后，放置37℃、5％CO 2孵育5-6h，加入20μl/孔的化学发光底物后上机检测；
加入肿瘤组：取生长对数期Jurkat-TIGIT-luc细胞，用1640+10％FBS调整成1.2*10 6个细胞/ml，每孔加入25μl细胞(3*10 4个细胞/孔)；消化实施例5构建的MC38/MSLN细胞，用1640+10％FBS调整消化后的MC38/MSLN细胞至1.2*10 6个细胞/ml，每孔加入25μl细胞，3*10 4个细胞/孔轻轻混匀后，放置37℃、5％CO 2孵育5-6h，加入20μl/孔的化学发光底物后上机检测。
激活表达人CD3的T细胞(即Jurkat-TIGIT-luc)的测定结果如图6A-6F及表14所示。结果显示，在没有加入肿瘤细胞时，本发明的CD3-MSLN双特异性抗体对表达人CD3的T细胞的激活与对照抗体1-3C6相比较弱，甚至没有检测到激活Jurkat/NFAT/LUC细胞，但是在加入肿瘤细胞后均能适度激活Jurkat-TIGIT-luc细胞。
表14：CD3-MSLN双特异性抗体对T细胞活化信号通路的激活
Figure PCTCN2022142626-appb-000022
Figure PCTCN2022142626-appb-000023
实施例17：CD3-MSLN双特异性抗体介导的TDCC
(一)PBMC复苏：
1、准备一个15ml离心管，加入5ml灭活的完全培养基。
2、将冻存的PBMC 37℃水浴融化，吸取5ml细胞溶液，竖直加入，混匀后，360g离心10min。
3、弃掉上清，加入3ml灭活的完全培养基。计数。
4、使用灭活完全培养基调整细胞浓度使细胞浓度到达1×10 6个细胞/ml。
(二)细胞增殖检测(Cell trace violet)
1、将MC38/MSLN肿瘤细胞消化离心，360g离心5min。
2、计数，取2×10 6个细胞离心，360g离心5min，舍弃上清。
3、使用1ml无血清1640重悬2μl cell trace(Invitrogen C34571)，形成稀释的染液。
4、再用0.5ml染液重悬细胞，37℃避光培养30min。
5、通过流式检测荧光强度，荧光强度是阴性样本10 2倍以上。
6、加入5-10ml 1640完全培养基终止反应。
7、360g离心5min。
8、舍弃上清，加入无血清培养基重悬细胞，计数调整细胞浓度，使肿瘤细胞浓度达到5×10 4个细胞/ml。
9、使用平底96孔板，将肿瘤细胞按照5×10 4个细胞/ml、PBMC细胞按照1×10 6个细胞/ml比例稀释，每种细胞每次加入100μl细胞悬液。每孔共200μl。
10、终血清浓度为5％，培养48h。
11、使用新的96孔板稀释抗体，然后再加入稀释后的抗体。
12、360g离心5min，弃上清，用胰酶消化细胞，并终止。
13、细胞染色，流式收据数据。
结果如图7A-7I所示，结果显示，本发明的CD3-MSLN双特异性抗体能对表达肿瘤抗原MSLN的肿瘤细胞发挥T细胞介导的肿瘤细胞杀伤(TDCC)作用，这可能是 由于本发明的CD3-MSLN双特异性抗体特异性结合表达肿瘤抗原MSLN的肿瘤细胞和T细胞，将T细胞招募至肿瘤细胞周围，激活T细胞，诱导TDCC作用，从而有效杀伤肿瘤细胞。
实施例18：CD3-CD19双特异性抗体介导的TDCC
本实施例基于本发明的CD3抗体以及CD19抗体构建双特异性抗体，CD3-CD19双特异性抗体命名为5Y2-175，并考察5Y2-175介导的TDCC活性。5Y2-175参照实施例13构建双抗的方法进行制备，其结构示意图如图8所示，具体结构为：将实施例1中获得的CD3单克隆抗体(M33)的VH CD3连接至IgG1突变型重链恒定区(SEQ ID NO:164)，并进一步将重链恒定区的C端通过连接子(GGGGS) 2连接上述CD19抗体的VH CD19(SEQ ID NO:170)与VL CD19(SEQ ID NO:171)，VH CD19与VL CD19通过(GGGGS) 2连接，形成具有[VH CD3]-[CH]-[L]-[VH CD19]-[L]-[VL CD19]所示结构的第一多肽链；将实施例1中获得的人源化CD3抗体(M33)的VL CD3连接轻链恒定区(SEQ ID NO:165)，形成具有[VL CD3]-[CL]所示结构的第二多肽链。
1.Raji-luc细胞准备
构建含有Luc序列(NCBI:GenBank:MF062157.1)克隆至慢病毒载体上(plvx-IRES-puro,psPAX2,pMD2G)，将构建好的慢病毒质粒和包装质粒共染HEK293T细胞，收集48h的细胞上清，加入到Raji细胞(ATCC，CCL-86)中，24h时候加入4μg/ml嘌呤霉素进行筛选。通过细胞分选仪(Sony，LESH800SBP)分选表达Luc的单细胞，最后得到稳定的Raji-luc细胞。
2.PBMC准备
复苏冻存的PBMC细胞，500g，3min，离心去上清，再用1640+10％FBS(灭活过)洗一次，500g，3min，离心去上清，用适量1640+10％FBS(灭活过)重悬，并计数，稀释至对应的密度后，加入孔板中，每孔加入25μl，使PBMC细胞总数1.25*10 5个细胞/孔。
3.Raji-luc细胞铺板
取25μl Raji-luc细胞加入至上述PBMC孔板中，1.25*10 4个细胞/孔；
4.CD3-CD19双抗稀释
取CD3-CD19双抗，用培养基调整浓度至2nM，3倍稀释，9个梯度；取50μl稀释好的抗体加入含有PBMC和Raji-luc细胞中；
5.孵育
37℃、5％CO 2培养箱中孵育2天；
6.读数
取出孔板，加入50μl荧光素酶底物，上机读数并分析数据；
实验结果如图9所示，CD3-CD19双特异性抗体能对表达CD19的淋巴瘤细胞发挥T细胞介导的肿瘤细胞杀伤(TDCC)作用。
实施例19：CD3-CD20双特异性抗体介导的TDCC
本实施例基于本发明的CD3抗体以及CD20抗体构建双特异性抗体，CD3-CD20双特异性抗体命名为5Y2-176，并考察该CD3-CD20双特异性抗体介导的TDCC活性。5Y2-176参照实施例18的CD3-CD19双特异性抗体进行构建，其中CD20的VH氨基酸序列如SEQ ID NO:172所示，CD20的VL氨基酸序列如SEQ ID NO:173所示。
1.PBMC准备
复苏冻存的PBMC细胞，500g，3min，离心去上清，再用1640+10％FBS(灭活过)洗一次，500g，3min，离心去上清，用适量1640+10％FBS(灭活过)重悬，并计数，稀释至对应的密度后，加入孔板中，每孔加入25μl，使PBMC细胞总数1.25*10 5个细胞/孔。
2.Raji-luc细胞铺板
取25μl参照实施例18的Raji-luc细胞制备方法制备得到的Raji-luc细胞加入至上述PBMC孔板中，1.25*10 4个细胞/孔；
3.CD3-CD20双抗稀释
取CD3-CD20抗体，用培养基调整浓度至2nM，3倍稀释，9个梯度；将稀释好的抗体取50μl加入含有PBMC和Raji-luc细胞中；
4.孵育
37℃、5％CO 2培养箱中孵育2天；
5.读数
取出孔板，加入50μl荧光素酶底物，上机读数并分析数据；
实验结果如图10所示，CD3-CD20双特异性抗体能对表达CD20的淋巴瘤细胞发挥T细胞介导的肿瘤细胞杀伤(TDCC)作用。
实施例20：CD3-trop2双特异性抗体介导的TDCC
本实施例基于本发明的CD3抗体以及trop2抗体构建双特异性抗体，CD3-trop2双特异性抗体命名为5Y2-174，并考察该CD3-trop2双特异性抗体介导的TDCC活性。5Y2-174双特异性抗体参照实施例13构建双抗的方法进行制备，其具体结构涉及的人源化CD3抗体为M33，trop2的VHH氨基酸序列如SEQ ID NO:174所示。
1.HCC1806-luc细胞构建：
构建含有Luc基因序列克隆至慢病毒载体上(plvx-IRES-puro,psPAX2,pMD2G)，将构建好的慢病毒质粒和包装质粒共染HEK293T细胞，收集48h的细胞上清，加入到HCC1806(南京科佰，CBP60373)细胞中，24h时候加入4μg/ml嘌呤霉素进行筛选。通过细胞分选仪(Sony，LESH800SBP)分选表达Luc的单细胞，最后得到稳定的HCC1806-luc细胞。
2.PBMC准备
复苏冻存的PBMC细胞，500g，3min，离心去上清，再用1640+10％FBS(灭活过)洗一次，500g，3min，离心去上清，用适量1640+10％FBS(灭活过)重悬，并计数，稀释至对应的密度后，加入孔板中，每孔加入25μl，使PBMC细胞总数1.25*10 5个细胞/孔。
3.HCC1806-luc细胞铺板
取25μl上述HCC1806-luc细胞加入至上述PBMC孔板中，1.25*10 4个细胞/孔；
4.CD3-trop2双抗稀释
取CD3-trop2双抗，用培养基调整浓度至1nM，3倍稀释，10个梯度；将稀释好的抗体取50μl加入含有PBMC和HCC1806-luc细胞中；
5.孵育
37℃、5％CO 2培养箱中孵育2天；
6.读数
取出孔板，加入50μl荧光素酶底物，上机读数并分析数据；
实验结果如图11所示，CD3-trop2双特异性抗体能对表达trop2的人乳腺鳞状癌细胞发挥T细胞介导的肿瘤细胞杀伤(TDCC)作用。
实施例21：CD3-Her2双特异性抗体介导的TDCC
本实施例基于本发明的CD3抗体以及Her2抗体构建双特异性抗体，CD3-Her2双 特异性抗体命名为5Y2-177，并考察该CD3-Her2双特异性抗体介导的TDCC活性。5Y2-177双特异性抗体参照实施例13构建双抗的方法进行制备，其具体结构涉及的人源化CD3抗体为M33，Her2的VHH氨基酸序列如SEQ ID NO:175所示。
1.Calu-1-luc细胞构建：
构建含有Luc基因序列克隆至慢病毒载体上(plvx-IRES-puro,psPAX2,pMD2G)，将构建好的慢病毒质粒和包装质粒共染HEK293T细胞，收集48h的细胞上清，加入到Calu-1(南京科佰，CBP60085)细胞中，24h时候加入4μg/ml嘌呤霉素进行筛选。通过细胞分选仪(Sony，LESH800SBP)分选表达Luc的单细胞，最后得到稳定的Calu-1-luc细胞。
2.PBMC准备
复苏冻存的PBMC细胞，500g，3min，离心去上清，再用1640+10％FBS(灭活过)洗一次，500g，3min，离心去上清，用适量1640+10％FBS(灭活过)重悬，并计数，稀释至对应的密度后，加入孔板中，每孔加入25μl，使PBMC细胞总数1.25*10 5个细胞/孔。
3.Calu-1-luc细胞铺板
取25μl上述Calu-1-luc细胞加入至上述PBMC孔板中，1.25*10 4个细胞/孔；
4.CD3-Her2双抗稀释
取CD3-Her2，用培养基调整浓度至30nM，3倍稀释，11个梯度；将稀释好的抗体取50μl加入含有PBMC和Calu-1-luc细胞中；
5.孵育
37℃、5％CO 2培养箱中孵育2天；
6.读数
取出孔板，加入50μl荧光素酶底物，上机读数并分析数据；
实验结果如图12所示，CD3-Her2双特异性抗体能对表达Her2的人肺癌细胞发挥T细胞介导的肿瘤细胞杀伤(TDCC)作用。
实施例22：CD3-Caludin18.2双特异性抗体介导的TDCC
本实施例基于本发明的CD3抗体以及Caludin18.2抗体构建双特异性抗体，CD3-Caludin18.2双特异性抗体命名为5Y2-178，并考察该CD3-Caludin18.2双特异性抗体介导的TDCC活性。5Y2-178双特异性抗体参照实施例13构建双抗的方法进行制备，其 具体结构涉及的人源化CD3抗体为M33,Caludin18.2的VHH氨基酸序列如SEQ ID NO:176所示。
1.NUGC4-luc细胞构建
构建含有Luc基因序列克隆至慢病毒载体上(plvx-IRES-puro,psPAX2,pMD2G)，将构建好的慢病毒质粒和包装质粒共染HEK293T细胞，收集48h的细胞上清，加入到NUGC4(南京科佰CBP60493)细胞中，24h时候加入4μg/ml嘌呤霉素进行筛选。通过细胞分选仪(Sony，LESH800SBP)分选表达Luc的单细胞，最后得到稳定的NUGC4-lucc细胞。
2.PBMC准备
复苏冻存的PBMC细胞，500g，3min，离心去上清，再用1640+10％FBS(灭活过)洗一次，500g，3min，离心去上清，用适量1640+10％FBS(灭活过)重悬，并计数，稀释至对应的密度后，加入孔板中，每孔加入25μl，使PBMC细胞总数1.25*10 5个细胞/孔。
3.NUGC4-luc细胞铺板
取25μl上述NUGC4-luc细胞加入至上述PBMC孔板中，1.25*10 4个细胞/孔；
4.CD3-Caludin18.2双抗稀释
取CD3-Caludin18.2双抗，用培养基调整浓度至100nM，3倍稀释，12个梯度；将稀释好的抗体取50μl加入含有PBMC和NUGC4-luc细胞中；
5.孵育
37℃、5％CO 2培养箱中孵育2天；
6.读数
取出孔板，加入50μl荧光素酶底物，上机读数并分析数据；
实验结果如图13所示，CD3-Caludin18.2双特异性抗体能对表达Caludin18.2的人胃癌细胞发挥T细胞介导的肿瘤细胞杀伤(TDCC)作用。
实施例23：CD3-MSLN双特异性抗体细胞因子实验
调整双抗浓度至1nM，3倍稀释，9个梯度，添加稀释后的双抗(50μl/孔)，最后一个孔不加双抗；调整PBMC细胞密度至2*10 6个细胞/ml，50μl/孔；37℃、5％CO 2培养48h；分别在24h和48各取30μl上清，HTRF试剂盒(cisbio 62HIL02PEH，62HIFNGPEH)分别检测上清中的IL2和INF-γ含量。
IL2检测结果如图14A-14I所示，INF-γ检测结果如图15A-15I所示。结果显示，本发明的CD3-MSLN双特异性抗体与PBMC共同孵育产生显著低于阳性对照OKT3-3C6的IL2和INF-γ，表明本发明的CD3-MSLN双特异性抗体具有良好的安全性。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (42)

  1. 能够特异性结合CD3的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中，
    所述VH包含：包含SEQ ID NO:107所示序列的HCDR1、包含SEQ ID NO:108所示序列的HCDR2以及包含X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10WX 11X 12X 13(SEQ ID NO:112)所示序列的HCDR3；其中X 1为A，H或P；X 2为A、E、G、H、K、Q或S；X 3为D、N或R；X 4为F或P；X 5为G、K、L、P、Q、R、S、V、W或Y；X 6为M、N、Q或R；X 7为G、N、S或T；X 8为A、Q、R或Y；X 9为G、I或V；X 10为N或S；X 11为F或W；X 12为A、E、K或Q；X 13为H、L、M、S或Y；并且条件是所述HCDR3不是SEQ ID NO:90所示。
  2. 权利要求1所述的抗体或其抗原结合片段，其中，所述HCDR3包含：X 1X 2X 3FX 4NX 5YX 6SWFAX 7(SEQ ID NO:148)所示的序列，其中，X 1是H或P；X 2是G、E、或A；X 3是N或R；X 4是G、K、S、或P；X 5是T、S、或N；X 6是V或G；X 7是M、Y、S、或L；
    优选地，所述HCDR3包含SEQ ID NOs:56、61、69、79、82、87任一项所示的序列。
  3. 权利要求1所述的抗体或其抗原结合片段，其中，所述HCDR3包含：
    (1)HX 2NFGNSYVSWFAY(SEQ ID NO:113)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为H；
    (2)HGNFX 5NSYVSWFAY(SEQ ID NO:114)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为K；
    (3)HGNFGNSX 8VSWFAY(SEQ ID NO:115)所示的序列，其中，X 8是A、Q或R，优选为R；或，
    (4)HGNFGNSYVSWFX 12Y(SEQ ID NO:116)所示的序列，其中，X 12是E、K或Q；
    优选地，所述HCDR3包含SEQ ID NOs:75、53、72、51、57、64任一项所示的序列。
  4. 权利要求1所述的抗体或其抗原结合片段，其中，所述HCDR3包含：
    (1)X 1X 2NFGNSYVSWFAY(SEQ ID NO:117)所示的序列，其中，X 1是A或P，优选为A；X 2是A、E、H、K、Q或S，优选为K；优选地，X 1为A，X 2为K；
    (2)X 1GNFGNSYVX 10WFAY(SEQ ID NO:118)所示的序列，其中，X 1是A或P，优选为A；X 10是N；优选地，X 1为A，X 10为N；或，
    (3)X 1GNFGNSYVSWFX 12Y(SEQ ID NO:119)所示的序列，其中，X 1是A或P，优选为A；X 12是E、K或Q，优选为E或Q；优选地，X 1为A，X 12为E或Q；
    优选地，所述HCDR3包含SEQ ID NOs:50、66、55、85任一项所示的序列。
  5. 权利要求1所述的抗体或其抗原结合片段，其中，所述HCDR3包含：
    (1)X 1X 2NFGNSYVSWFAY(SEQ ID NO:117)所示的序列，其中，X 1是A或P，优选为A；X 2是A、E、H、K、Q或S，优选为K；优选地，X 1为A，X 2为K；
    (2)HX 2X 3FGNSYVSWFAY(SEQ ID NO:120)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为E；X 3是D或R，优选为R；优选地，X 2为E，X 3为R；
    (3)HX 2NFX 5NSYVSWFAY(SEQ ID NO:121)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为A或S；X 5是K、L、P、Q、R、S、V、W或Y，优选为L、P、S、Q、V或R；优选地，X 2为A或S，X 5为L、P、S、Q、V或R；优选地，X 2和X 5分别为A/L、S/P、S/L、A/S、S/Q、S/V或S/R；
    (4)HX 2NFGX 6SYVSWFAY(SEQ ID NO:122)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 6是M、Q或R，优选为R；优选地，X 2为S，X 6为R；
    (5)HX 2NFGNX 7YVSWFAY(SEQ ID NO:123)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 7是G、N或T，优选为T或G；优选地，X 2为S，X 7为T或G；
    (6)HX 2NFGNSX 8VSWFAY(SEQ ID NO:124)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 8是A、Q或R，优选为R；优选地，X 2为S，X 8为R；
    (7)HX 2NFGNSYVSWX 11AY(SEQ ID NO:125)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 11为W；优选地，X 2为S，X 11为W；
    (8)HX 2NFGNSYVSWFX 12Y(SEQ ID NO:126)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 12是E、K或Q，优选为E或Q；优选地，X 2为S或Q，X 12为E或Q；优选地，X 2和X 12分别为S/E、Q/Q或S/Q；或，
    (9)HX 2NFGNSYVSWFAX 13(SEQ ID NO:127)所示的序列，其中，X 2是A、E、 H、K、Q或S，优选为A；X 13是H、L、M或S，优选为S；优选地，X 2为A，X 13为S；
    优选地，所述HCDR3包含SEQ ID NOs:50、69、49、58、59、60、70、82、86、71、68、77、78、81、62、84、89、74任一项所示的序列。
  6. 权利要求1所述的抗体或其抗原结合片段，其中，所述HCDR3包含：
    (1)HX 2NFX 5NSYVSWFAY(SEQ ID NO:121)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为A或S；X 5是K、L、P、Q、R、S、V、W或Y，优选为L、P、S、Q、V或R；优选地，X 2为A或S，X 5为L、P、S、Q、V或R；优选地，X 2和X 5分别为A/L、S/P、S/L、A/S、S/Q、S/V或S/R；
    (2)HGNFX 5X 6SYVSWFAY(SEQ ID NO:129)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为P或K；X 6是M、Q或R，优选为M或Q；优选地，X 5为P或K，X 6为M或Q；优选地，X 5和X 6分别为P/M或K/Q；
    (3)HGNFX 5NSYX 9SWFAY(SEQ ID NO:130)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为K或Q；X 9是G或I；优选地，X 5为K或Q，X 9是G或I；优选地，X 5和X 9分别为K/G或Q/I；
    (4)HGNFX 5NSYVSWFX 12Y(SEQ ID NO:131)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为Q或L；X 12是E、K或Q，优选为Q；优选地，X 5为Q或L，X 12为Q；或，
    (5)HGNFX 5NSYVSWFAX 13(SEQ ID NO:132)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为Y或W；X 13是H、L、M或S，优选为H或S；优选地，X 5为Y或W，X 13为H或S；优选地，X 5和X 13分别为Y/H或W/S；
    优选地，所述HCDR3包含SEQ ID NOs:49、58、59、60、70、82、86、76、80、61、88、52、83、54、65任一项所示的序列。
  7. 权利要求1所述的抗体或其抗原结合片段，其中，所述HCDR3包含：
    (1)X 1GNFGNSYVSWFX 12Y(SEQ ID NO:119)所示的序列，其中，X 1是A或P，优选为A；X 12是E、K或Q，优选为Q；优选地，X 1为A，X 12为Q或E；
    (2)HX 2NFGNSYVSWFX 12Y(SEQ ID NO:126)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为S；X 12是E、K或Q，优选为E或Q；优选地，X 2为S，X 12为E或Q；优选地，X 2和X 12分别为S/E、Q/Q或S/Q；
    (3)HGNFX 5NSYVSWFX 12Y(SEQ ID NO:131)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为Q或L；X 12是E、K或Q，优选为Q；优选地，X 5为Q或L，X 12为Q；或，
    (4)HGNFGNSX 8VSWFX 12Y(SEQ ID NO:134)所示的序列，其中，X 8是A、Q或R，优选为A或Q；X 12是E、K或Q，优选为Q；优选地，X 8为A或Q，X 12为Q；
    优选地，所述HCDR3包含SEQ ID NOs:55、85、62、84、89、52、83、67、73任一项所示的序列。
  8. 权利要求1所述的抗体或其抗原结合片段，其中，所述HCDR3包含：
    (1)HX 2NFGNSYVSWFAX 13(SEQ ID NO:127)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为A；X 13是H、L、M或S，优选为S；优选地，X 2为A，X 13为S；
    (2)HGNFX 5NSYVSWFAX 13(SEQ ID NO:132)所示的序列，其中，X 5是K、L、P、Q、R、S、V、W或Y，优选为Y或W；X 13是H、L、M或S，优选为H或S；优选地，X 5为Y或W，X 13为H或S；优选地，X 5和X 13分别为Y/H或W/S；或，
    (3)HGNFGNX 7YVSWFAX 13(SEQ ID NO:133)所示的序列，其中，X 7是G、N或T，优选为T或N；X 13是H、L、M或S，优选为M或S；优选地，X 7为T或N，X 13为M或S；优选地，X 7和X 13分别为T/M或N/S；
    优选地，所述HCDR3包含SEQ ID NOs:74、54、65、56、79任一项所示的序列。
  9. 权利要求1所述的抗体或其抗原结合片段，其中，所述HCDR3包含：
    (1)HX 2X 3FGNSYVSWFAY(SEQ ID NO:120)所示的序列，其中，X 2是A、E、H、K、Q或S，优选为E；X 3是D或R，优选为R；优选地，X 2为E，X 3为R；或，
    (2)HGX 3X 4GNSYVSWFAY(SEQ ID NO:128)所示的序列，其中，X 3是D或R，优选为D；X 4是P；优选地，X 3为D，X 4是P；
    优选地，所述HCDR3包含SEQ ID NOs:69、63任一项所示的序列。
  10. 权利要求1所述的抗体或其抗原结合片段，其中，所述HCDR3包含：X 1GNFX 5NSYVSWFAX 13(SEQ ID NO:135)所示的序列，其中，X 1是A或P，优选为P；X 5是K、L、P、Q、R、S、V、W或Y，优选为P；X 13是H、L、M或S，优选为L；优选地，X 1为P，X 5为P，X 13为L；
    优选地，所述HCDR3包含SEQ ID NO:87所示的序列。
  11. 权利要求1-10任一项所述的抗体或其抗原结合片段，条件是所述HCDR3不是SEQ ID NOs:60、64、77、89中任一项所示。
  12. 权利要求1-11任一项所述的抗体或其抗原结合片段，其中，所述VH包含：包含SEQ ID NO:107所示序列的HCDR1、包含SEQ ID NO:108所示序列的HCDR2以及包含SEQ ID NOs:49-89任一项所示序列的HCDR3；
    优选地，条件是所述HCDR3不是SEQ ID NOs:60、64、77、89中任一项所示。
  13. 权利要求1-12任一项所述的抗体或其抗原结合片段，其中，所述VL包含：包含SEQ ID NO:137所示序列的LCDR1、包含SEQ ID NO:138所示序列的LCDR2以及包含SEQ ID NO:139所示序列的LCDR3。
  14. 权利要求1-13任一项的抗体或其抗原结合片段，所述抗体或其抗原结合片段进一步包含源自人免疫球蛋白的框架区；
    优选地，所述抗体或其抗原结合片段包含源自人重链胚系基因所编码的氨基酸序列中所包含的重链框架区，和/或源自人轻链胚系基因所编码的氨基酸序列中所包含的轻链框架区。
  15. 权利要求1-14任一项的抗体或其抗原结合片段，其中，所述VH包含HFR1、HFR2、HFR3和HFR4，其中：
    所述HFR1包含SEQ ID NO:109所示的序列；
    所述HFR2包含SEQ ID NO:110所示的序列；
    所述HFR3包含VKX 1RFTISRDDSKSX 2LYLQMNX 3LKTEDTAX 4YYCVR(SEQ ID NO:136)所示的序列；其中，X 1为G或D，X 2为I或S，X 3为N或S，X 4为M或V；
    所述HFR4包含SEQ ID NO:111所示的序列；
    优选地，所述HFR3包含SEQ ID NOs:91-106任一项所示的序列。
  16. 权利要求1-15任一项的抗体或其抗原结合片段，其中，所述VH包含：
    (1)SEQ ID NOs:1-46任一项所示的氨基酸序列或其变体；
    (2)SEQ ID NOs:8、13、22、33、37、43任一项所示的氨基酸序列或其变体；
    (3)SEQ ID NOs:3、5、9、16、20、25、28任一项所示的氨基酸序列或其变体；
    (4)SEQ ID NOs:2、7、18、40任一项所示的氨基酸序列或其变体；
    (5)SEQ ID NOs:2、22、1、10、11、12、23、36、37、41、24、21、30、32、35、14、31、39、45、27、46任一项所示的氨基酸序列或其变体；
    (6)SEQ ID NOs:1、10、11、12、23、36、37、41、29、34、13、44、4、38、42、6、17任一项所示的氨基酸序列或其变体；
    (7)SEQ ID NOs:7、40、14、31、39、45、4、38、42、19、26任一项所示的氨基酸序列或其变体；
    (8)SEQ ID NOs:27、46、6、17、8、33任一项所示的氨基酸序列或其变体；
    (9)SEQ ID NOs:22、15任一项所示的氨基酸序列或其变体；
    (10)SEQ ID NO:43所示的氨基酸序列或其变体；或，
    (11)SEQ ID NOs:1-11、13-15、17-19、21-29、31-35、37-44、46任一项所示的氨基酸序列或其变体；
    其中，(1)-(11)任一项中所述的变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
  17. 权利要求1-16任一项的抗体或其抗原结合片段，其中，所述VL包含LFR1、LFR2、LFR3和LFR4，其中：
    所述LFR1包含如SEQ ID NO:140所示的序列；
    所述LFR2包含如WX 1QQTPGQAX 2RX 3LIX 4(SEQ ID NO:144)所示的序列；其中，X 1为V或Y，X 2为F或P，X 3为G或T，X 4为G或Y；
    所述LFR3包含如GVPARFSGSX 4X 5GX 6KAALTITGAQADDESX 7YFCA(SEQ ID NO:147)所示的序列；其中，X 4为L或I，X 5为L或I，X 6为D或N，X 7为I或D；
    所述LFR4包含如SEQ ID NO:141所示的序列；
    优选地，所述LFR2包含SEQ ID NO:142或143所示的序列；
    优选地，所述LFR3包含SEQ ID NO:145或146所示的序列；
    优选地，所述VL包含：SEQ ID NO:140所示的LFR1、SEQ ID NO:142所示的 LFR2、SEQ ID NO:145所示的LFR3、SEQ ID NO:141所示的LFR4；
    优选地，所述VL包含：SEQ ID NO:140所示的LFR1、SEQ ID NO:143所示的LFR2、SEQ ID NO:146所示的LFR3、SEQ ID NO:141所示的LFR4。
  18. 权利要求1-17任一项的抗体或其抗原结合片段，其中，所述VL包含：SEQ ID NO:47或48所示的氨基酸序列或其变体，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
  19. 权利要求1-18任一项的抗体或其抗原结合片段，其包含：
    -包含SEQ ID NOs:1-46任一项所示序列或其变体的VH，和包含SEQ ID NO:47所示序列或其变体的VL；或，
    -包含SEQ ID NOs:1-46任一项所示序列或其变体的VH，和包含SEQ ID NO:48所示序列或其变体的VL；
    其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加，或者具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。
  20. 权利要求1-19任一项所述的抗体或其抗原结合片段，其进一步包含源自人免疫球蛋白的恒定区；
    优选地，所述抗体或其抗原结合片段的重链包含源自人免疫球蛋白的重链恒定区；优选地，所述抗体或其抗原结合片段包含突变的或化学修饰的Fc区，其与野生型Fc区相比具有改变的效应子功能；
    优选地，所述抗体或其抗原结合片段的轻链包含源自人免疫球蛋白的轻链恒定区。
  21. 权利要求1-20任一项所述的抗体或其抗原结合片段，其中，所述抗原结合片段选自Fab、Fab’、(Fab’) 2、Fv、二硫键连接的Fv、scFv、双抗体。
  22. 能够特异性结合MSLN的单域抗体或其抗原结合片段，所述单域抗体或其抗原结 合片段包含：
    (1)IMGT编号系统定义的下述CDRs：包含SEQ ID NO:150所示序列或其变体的CDR1，包含SEQ ID NO:151所示序列或其变体的CDR2，以及包含SEQ ID NO:152所示序列或其变体的CDR3；
    (2)Kabat编号系统定义的下述CDRs：包含SEQ ID NO:153所示序列或其变体的CDR1，包含SEQ ID NO:154所示序列或其变体的CDR2，以及包含SEQ ID NO:155所示序列或其变体的CDR3；
    (3)AbM编号系统定义的下述CDRs：包含SEQ ID NO:156所示序列或其变体的CDR1，包含SEQ ID NO:157所示序列或其变体的CDR2，以及包含SEQ ID NO:155所示序列或其变体的CDR3；
    (4)Chothia编号系统定义的下述CDRs：包含SEQ ID NO:158所示序列或其变体的CDR1，包含SEQ ID NO:159所示序列或其变体的CDR2，以及包含SEQ ID NO:155所示序列或其变体的CDR3；或，
    (5)Contact编号系统定义的下述CDRs：包含SEQ ID NO:160所示序列或其变体的CDR1，包含SEQ ID NO:161所示序列或其变体的CDR2，以及包含SEQ ID NO:162所示序列或其变体的CDR3；
    其中，(1)-(5)任一项中所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加；优选地，所述置换为保守置换；
    优选地，所述单域抗体或其抗原结合片段包含：
    (1)IMGT编号系统定义的下述CDRs：包含SEQ ID NO:150所示序列的CDR1，包含SEQ ID NO:151所示序列的CDR2，以及包含SEQ ID NO:152所示序列的CDR3；
    (2)Kabat编号系统定义的下述CDRs：包含SEQ ID NO:153所示序列的CDR1，包含SEQ ID NO:154所示序列的CDR2，以及包含SEQ ID NO:155所示序列的CDR3；
    (3)AbM编号系统定义的下述CDRs：包含SEQ ID NO:156所示序列的CDR1，包含SEQ ID NO:157所示序列的CDR2，以及包含SEQ ID NO:155所示序列的CDR3；
    (4)Chothia编号系统定义的下述CDRs：包含SEQ ID NO:158所示序列的CDR1，包含SEQ ID NO:159所示序列的CDR2，以及包含SEQ ID NO:155所示序列的CDR3；或，
    (5)Contact编号系统定义的下述CDRs：包含SEQ ID NO:160所示序列的CDR1，包含SEQ ID NO:161所示序列的CDR2，以及包含SEQ ID NO:162所示序列的CDR3。
  23. 权利要求22所述的单域抗体或其抗原结合片段，其包含SEQ ID NO:149所示的序列或其变体，所述变体与其所源自的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列，或与其相比具有一个或几个氨基酸置换、缺失或添加；优选地，所述置换为保守置换。
  24. 多特异性抗体，其包含靶向CD3的抗原结合结构域和至少一个靶向其他抗原的抗原结合结构域，所述靶向CD3的抗原结合结构域选自权利要求1-21任一项所述的抗体或其抗原结合片段，所述其他抗原选自肿瘤相关抗原(TAA)和/或免疫检查点分子。
  25. 权利要求24所述的多特异性抗体，所述多特异性抗体是双特异性抗体、三特异性抗体或四特异性抗体；
    优选地，所述多特异性抗体是双特异性抗体，其包含所述靶向CD3的抗原结合结构域和靶向所述肿瘤相关抗原的抗原结合结构域；
    优选地，所述多特异性抗体是三特异性抗体，其包含所述靶向CD3的抗原结合结构域、靶向所述肿瘤相关抗原的抗原结合结构域和靶向所述免疫检查点分子的抗原结合结构域。
  26. 权利要求24或25所述的多特异性抗体，其中，所述靶向肿瘤相关抗原或免疫检查点分子的抗原结合结构域任选地通过接头连接在所述靶向CD3的抗原结合结构域的重链的N末端和/或C末端，和/或连接在所述靶向CD3的抗原结合结构域的轻链的N末端和/或C末端；
    优选地，所述靶向CD3的抗原结合结构域包含：至少一条重链和至少一条轻链，并且所述靶向肿瘤相关抗原或免疫检查点分子的抗原结合结构域与所述重链连接；或者所述靶向CD3的抗原结合结构域包含：两条相同的重链和两条相同的轻链，并且所述靶向肿瘤相关抗原或免疫检查点分子的抗原结合结构域与两条重链连接。
  27. 权利要求24-26任一项所述的多特异性抗体，所述肿瘤相关抗原选自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、GloboH、GM3、hTERT、LMP2、p53、PAP、PAX3、PAX5、PCTA-1、PLAC1、PRLR、Ras、SART-3、TRP-1、TRP-2、CD22、CD30、FOLR1、Caludin18.2或其任意组合；优选地，所述肿瘤相关抗原选自MSLN、CD19、CD20、TROP2、HER2或Caludin18.2；
    所述免疫检查点分子选自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、或其任意组合。
  28. 权利要求24-27任一项所述的多特异性抗体，其包含所述靶向CD3的抗原结合结构域和靶向MSLN的抗原结合结构域，所述靶向MSLN的抗原结合结构域选自权利要求22或23所述的单域抗体或其抗原结合片段。
  29. 权利要求24-27任一项所述的多特异性抗体，其包含所述靶向CD3的抗原结合结构域和靶向CD19的抗原结合结构域；所述靶向CD19的抗原结合结构域包含轻链可变区和重链可变区，所述轻链可变区包含SEQ ID NO:171所示的序列或其变体，所述重链可变区包含SEQ ID NO:170所示的序列或其变体。
  30. 权利要求24-27任一项所述的多特异性抗体，其包含所述靶向CD3的抗原结合结构域和靶向CD20的抗原结合结构域，所述靶向CD20的抗原结合结构域包含轻链可变区和重链可变区，所述轻链可变区包含SEQ ID NO:173所示的序列或其变体，所述重链可变区包含SEQ ID NO:172所示的序列或其变体。
  31. 权利要求24-27任一项所述的多特异性抗体，其包含所述靶向CD3的抗原结合结构域和靶向TROP2的抗原结合结构域；所述靶向TROP2的抗原结合结构域包含VHH，所述VHH包含SEQ ID NO:174所示的序列或其变体。
  32. 权利要求24-27任一项所述的多特异性抗体，其包含所述靶向CD3的抗原结合结构域和靶向HER2的抗原结合结构域；所述靶向HER2的抗原结合结构域包含VHH，所述VHH包含SEQ ID NO:175所示的序列或其变体。
  33. 权利要求24-27任一项所述的多特异性抗体，其包含所述靶向CD3的抗原结合结构域和靶向Caludin18.2的抗原结合结构域；所述靶向Caludin18.2的抗原结合结构域包含VHH，所述VHH包含SEQ ID NO:176所示的序列或其变体。
  34. 多特异性抗体，其包含权利要求22或23所述的单域抗体或其抗原结合片段；优选地，所述多特异性抗体为双特异性抗体、三特异性抗体或四特异性抗体。
  35. 分离的核酸分子，其编码：
    -权利要求1-21任一项所述的抗体或其抗原结合片段、或其重链可变区和/或轻链可变区；
    -权利要求22-23任一项所述的单域抗体或其抗原结合片段；
    -权利要求24-33任一项所述的多特异性抗体或其多肽链；或，
    -权利要求34所述的多特异性抗体或其多肽链。
  36. 载体，其包含权利要求35所述的核酸分子。
  37. 宿主细胞，其包含权利要求35所述的核酸分子或权利要求36所述的载体。
  38. 制备抗体或其抗原结合片段、单域抗体或其抗原结合片段、或多特异性抗体的方法，其包括：在允许蛋白表达的条件下，培养权利要求37所述的宿主细胞，和从所培养的宿主细胞的培养物中收集所述抗体或其抗原结合片段、所述单域抗体或其抗原结合片段、或所述多特异性抗体。
  39. 药物组合物，其包含权利要求1-21任一项所述的抗体或其抗原结合片段、权利要求22-23任一项所述的单域抗体或其抗原结合片段、权利要求24-33任一项所述的多特异 性抗体、权利要求34所述的多特异性抗体、权利要求35所述的分离的核酸分子、权利要求36所述的载体、或权利要求37所述的宿主细胞，以及药学上可接受的载体和/或赋形剂。
  40. 权利要求1-21任一项所述的抗体或其抗原结合片段、权利要求22-23任一项所述的单域抗体或其抗原结合片段、权利要求24-33任一项所述的多特异性抗体、权利要求34所述的多特异性抗体、权利要求35所述的分离的核酸分子、权利要求36所述的载体、权利要求37所述的宿主细胞、或权利要求39所述的药物组合物，在制备用于预防和/或治疗疾病的药物中的用途。
  41. 用于预防和/或治疗疾病的方法，其包括向有此需要的受试者施用有效量的权利要求1-21任一项所述的抗体或其抗原结合片段、权利要求22-23任一项所述的单域抗体或其抗原结合片段、权利要求24-33任一项所述的多特异性抗体、权利要求34所述的分离的核酸分子、权利要求35所述的载体、权利要求36所述的宿主细胞、或权利要求38所述的药物组合物。
  42. 权利要求40所述的用途或权利要求41所述的方法，其中，所述疾病为肿瘤、炎性疾病或自身免疫性疾病；优选地，所述肿瘤选自间皮瘤、卵巢癌、胰腺癌、乳腺癌、胆管癌、大肠癌、胃癌、输卵管癌、肺癌、急性髓性白血病或结直肠癌。
PCT/CN2022/142626 2021-12-29 2022-12-28 靶向cd3的抗体及多特异性抗体及其用途 WO2023125611A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP22914863.0A EP4458861A1 (en) 2021-12-29 2022-12-28 Antibody targeting cd3, multispecific antibody, and uses thereof
CN202280085681.9A CN118591561A (zh) 2021-12-29 2022-12-28 靶向cd3的抗体及多特异性抗体及其用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111639610 2021-12-29
CN202111639610.2 2021-12-29

Publications (1)

Publication Number Publication Date
WO2023125611A1 true WO2023125611A1 (zh) 2023-07-06

Family

ID=86997950

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/142626 WO2023125611A1 (zh) 2021-12-29 2022-12-28 靶向cd3的抗体及多特异性抗体及其用途

Country Status (3)

Country Link
EP (1) EP4458861A1 (zh)
CN (1) CN118591561A (zh)
WO (1) WO2023125611A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170029502A1 (en) * 2015-07-31 2017-02-02 Amgen Research (Munich) Gmbh Antibody constructs for msln and cd3
US20180327508A1 (en) * 2017-05-12 2018-11-15 Harpoon Therapeutics, Inc. Msln targeting trispecific proteins and methods of use
WO2020252907A1 (zh) * 2019-06-19 2020-12-24 北京智仁美博生物科技有限公司 抗cd3e和bcma双特异性抗体及其用途
WO2021155635A1 (zh) * 2020-02-05 2021-08-12 北京智仁美博生物科技有限公司 抗cd3和cd123双特异性抗体及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170029502A1 (en) * 2015-07-31 2017-02-02 Amgen Research (Munich) Gmbh Antibody constructs for msln and cd3
US20180327508A1 (en) * 2017-05-12 2018-11-15 Harpoon Therapeutics, Inc. Msln targeting trispecific proteins and methods of use
CN110913908A (zh) * 2017-05-12 2020-03-24 哈普恩治疗公司 靶向msln的三特异性蛋白质及使用方法
WO2020252907A1 (zh) * 2019-06-19 2020-12-24 北京智仁美博生物科技有限公司 抗cd3e和bcma双特异性抗体及其用途
WO2021155635A1 (zh) * 2020-02-05 2021-08-12 北京智仁美博生物科技有限公司 抗cd3和cd123双特异性抗体及其用途

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403
BRUMMELL ET AL., BIOCHEM., vol. 32, 1993, pages 1180 - 1187
BURKS ET AL., PROC. NATL ACAD. SET USA, vol. 94, 1997, pages 412 - 417
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 878 - 883
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
DAVIES ET AL., ANNUAL REV BIOCHEM, vol. 59, 1990, pages 439 - 473
F. M. AUSUBEL ET AL.: "Compiled Experimental Guide to Molecular Biology", 1995, JOHN WILEY & SONS, INC.
HOLLIGER P. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
J. SAMBROOK ET AL.: "Molecular Cloning: Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, NATIONAL INSTITUTES OF HEALTH
KARLINALTSCHUL, PROC. NATL. ACAD. SCI. U.S.A., vol. 87, 1990, pages 2264 - 2268
KARLINALTSCHUL, PROC. NATL.. ACAD. SCI. U.S.A., vol. 90, 1993, pages 5873 - 5877
KOBAYASHI ET AL., PROTEIN ENG., vol. 12, no. 10, 1999, pages 879 - 884
LEFRANC ET AL., DEV. COMPARAT. IMMUNOL., vol. 27, 2003, pages 55 - 77
MACCALLUM, R. M.MARTIN, A. C. R.THORNTON, J. M.: "Antibody-antigen Interactions: Contact Analysis and Binding Site Topography", JOURNAL OF MOLECULAR BIOLOGY, vol. 262, no. 5, 1996, pages 732 - 745, XP002242391, DOI: 10.1006/jmbi.1996.0548
MAHNQVIST M, NATURE, vol. 361, 1993, pages 186 - 187
MARTIN ACRCHEETHAM JCREES AR: "Modeling antibody hypervariable loops: A combined algorithm", PROC NATL ACAD SCI USA, vol. 86, 1989, pages 9268 - 9272, XP000165667, DOI: 10.1073/pnas.86.23.9268
POLJAK R. J. ET AL., STRUCTURE, vol. 2, 1994, pages 1121 - 1123
SHAO, RONGGUANG: "Monoclonal Antibody- Based Cancer Immunotherapy", ACTA PHARMACOLOGICA SINICA, NATURE PUBLISHING GROUP, GB, vol. 55, no. 6, 30 June 2020 (2020-06-30), GB , pages 1110 - 1118, XP009537351, ISSN: 1671-4083 *

Also Published As

Publication number Publication date
EP4458861A1 (en) 2024-11-06
CN118591561A (zh) 2024-09-03

Similar Documents

Publication Publication Date Title
JP6783886B2 (ja) 抗ｃｔｌａ４モノクローナル抗体またはその抗原結合断片、医薬組成物および使用
US11136413B2 (en) PDL-1 antibody, pharmaceutical composition thereof, and uses thereof
WO2018095428A1 (zh) Cd47抗体、其抗原结合片段及其医药用途
EP3728314A1 (en) Bispecific antibody for icos and pd-l1
TW202126694A (zh) 標靶cd3的抗體、雙特異性抗體及其用途
WO2018219327A1 (zh) 抗cd40抗体、其抗原结合片段及其医药用途
WO2022174813A1 (zh) 抗GPRC5DxBCMAxCD3三特异性抗体及其用途
WO2021218874A1 (zh) 一种靶向人claudin和人PDL1蛋白的双特异抗体及其应用
WO2021013142A1 (zh) 抗4-1bb抗体、其抗原结合片段及双特异性抗体
WO2018176992A1 (zh) 新型双特异性抗体及其用途
JP2023504733A (ja) 抗ｃｌｅｃ－１ａ抗体及びその抗原結合性断片
CA3235697A1 (en) Anti-mesothelin nanobodies and use thereof
WO2022143670A1 (zh) 结合trop2的抗体及其用途
WO2021228218A1 (zh) 抗cd25抗体、其抗原结合片段及其医药用途
WO2023186081A1 (zh) 抗tigit-抗pvrig双特异性抗体、其药物组合物及用途
WO2023186063A1 (zh) 抗pvrig抗体、其药物组合物及用途
CN115151572B (zh) 针对ror1的抗体及其用途
WO2023025194A1 (zh) Fap/cd40结合分子及其医药用途
WO2023040940A1 (zh) Pvrig/tigit结合蛋白联合免疫检查点抑制剂用于治疗癌症
WO2023125611A1 (zh) 靶向cd3的抗体及多特异性抗体及其用途
WO2022127066A9 (zh) 一种特异性中和辅助性T细胞TGF-β信号的双特异性抗体、其药物组合及其用途
CN117355540A (zh) 抗cd137抗体和使用方法
WO2023241656A1 (zh) 包含抗cldn18.2抗体的双特异性抗体、药物组合物及用途
WO2024131751A1 (zh) 抗体及其用途
WO2022188652A1 (zh) Ror1结合蛋白及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22914863

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202280085681.9

Country of ref document: CN

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022914863

Country of ref document: EP

Effective date: 20240729