B7-h3 / EGFR binding molecule and medical use thereof

Abstract

Provided are a B7-H3 binding molecule, an EGFR binding molecule, a B7-H3 / EGFR binding molecule, methods for treating cancers by using the binding molecules, and related pharmaceutical uses thereof.

Description

B7-H3 / EGFR binding molecules and medical uses thereof

[0001] The present disclosure claims priority to patent application CN202410533401.7 filed on April 29, 2024 and patent application CN202410854573.4 filed on June 27, 2024. TECHNICAL FIELD

[0002] The present disclosure relates to the field of biomedicine, in particular to B7-H3 / EGFR binding molecules, B7-H3 binding molecules, EGFR binding molecules, and methods and related pharmaceutical uses thereof for treating cancer. BACKGROUND

[0003] B7-H3, also known as B7 homolog 3, CD276 or B7RP-2, is a new member of the B7 family discovered in early 2000. Its gene was first cloned by Chapoval et al. in 2001 from a human dendritic cell cDNA library. The amino acid sequence of B7-H3 has 27% homology with B7 family proteins and has high structural similarity, and it has an impact on the function of T cells. B7-H3 is a tumor-associated antigen that is widely expressed and significantly upregulated in various malignancies, including small cell lung cancer, prostate cancer, head and neck cancer, pancreatic cancer, non-small cell lung cancer, and soft tissue sarcoma. Therefore, antibody-drug conjugates (ADC) targeting B7-H3 can specifically deliver cytotoxins to tumor cells, effectively killing tumor cells, and have the potential for tumor treatment. Several B7-H3 monoclonal antibody ADCs are currently in clinical development and have shown good results in some tumors, but there is still room for improvement in the scope of indications and efficacy.

[0004] Epidermal growth factor receptor (EGFR, ErbB-1 or HER1) is another important tumor-associated antigen. Its overexpression affects the proliferation and apoptosis of tumor cells, is highly expressed in multiple cancers, and is associated with poor prognosis in patients. However, EGFR is also expressed in multiple normal tissues, so antibodies targeting EGFR often have target-related side effects in normal tissues, limiting the dosage and efficacy of the drug.

[0005] In order to expand the population of beneficiaries, exert the synergistic effect of targeting multiple tumor-associated antigens, and improve the response rate, it is necessary to design a bispecific antibody-drug conjugate targeting two tumor-associated antigens, B7-H3 and EGFR, which are highly expressed in tumors, to improve efficacy and reduce the risk of targeting EGFR single target, and increase the therapeutic window. SUMMARY

[0006] The present disclosure provides B7-H3 binding molecules, EGFR binding molecules, B7-H3 / EGFR binding molecules, and encoding nucleic acids, vectors, host cells, pharmaceutical compositions thereof, and methods of using the same for treating cancer and related pharmaceutical uses.

[0007] B7-H3 binding molecules

[0008] The present disclosure provides B7-H3 binding molecules comprising a first binding domain that specifically binds to B7-H3.

[0009] In some embodiments, the first binding domain that specifically binds to B7-H3 comprises a heavy chain variable region (VH1) comprising HCDR1, HCDR2, and HCDR3, and / or a light chain variable region (VL1) comprising LCDR1, LCDR2, and LCDR3; wherein the sequence of the VL is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, position 92 (numbering based on the Rabat convention) is Ala or Gly, and / or position 93 (numbering based on the Rabat convention) is Arg; and the sequence of the VH is at least 90% identical to the amino acid sequence of SEQ ID NO: 4.

[0010] In some embodiments, the first binding domain that specifically binds to B7-H3 comprises a heavy chain variable region (VH1) comprising HCDR1, HCDR2, and HCDR3, and / or a light chain variable region (VL1) comprising LCDR1, LCDR2, and LCDR3; wherein the sequence of the VL is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, position 92 (numbering based on the Rabat convention) is Ala or Gly, and / or position 93 (numbering based on the Rabat convention) is Arg; and the sequence of the VH is at least 95% identical to the amino acid sequence of SEQ ID NO: 4.

[0011] In some embodiments, the first binding domain that specifically binds to B7-H3 comprises a heavy chain variable region (VH1) comprising HCDR1, HCDR2, and HCDR3, and / or a light chain variable region (VL1) comprising LCDR1, LCDR2, and LCDR3; wherein:

[0012] the HCDR1 is set forth as X1X2GMS, wherein X1 is selected from R, H, K, or S, and X2 is selected from Y or H;

[0013] HCDR2 is set forth in X3IX4SGGGSX5YYX6X7X8VKG, wherein X3 is selected from A or S, X4 is S or W, X5 is selected from I, A or K, X6 is selected from P or S, X7 is selected from D or Q, and X8 is selected from T, G or W;

[0014] HCDR3 is set forth in SEQ ID NO: 22;

[0015] LCDR1 is set forth in KAX9X 10 X 11 VNTAVA, wherein X9 is selected from P or S, X 10 is selected from Q, R or K, X 11 is selected from N, D or G;

[0016] LCDR2 is set forth in SAX 12 NX 13 YX 14 , wherein X 12 is selected from S, T or K, X 13 is selected from R, G, P or L, X 14 is selected from T, D or P; and LCDR3 is set forth in QQYX 15 X 16 X 17 X 18 T, wherein X 15 is selected from A or G, X 16 is selected from S or R, X 17 is selected from S, Q, G or H, X 18 is selected from L, I or P.

[0017] In some embodiments, the first binding domain that specifically binds B7-H3,

[0018] HCDR1 is set forth in any one of SEQ ID NOs: 20, 31, 44,

[0019] HCDR2 is set forth in any one of SEQ ID NOs: 21, 32, 36, 40, 45, 48,

[0020] HCDR3 is set forth in SEQ ID NO: 22,

[0021] LCDR1 is set forth in any one of SEQ ID NOs: 23, 33, 37, 41,

[0022] LCDR2 is set forth in any one of SEQ ID NOs: 24, 34, 38, 42, 46, 49, or

[0023] the LCDR3 is as set forth in any one of SEQ ID NOs: 26, 27, 30, 35, 39, 43, 47.

[0024] In some embodiments, the B7-H3 binding molecule comprises a first binding domain that specifically binds B7-H3; the first binding domain that specifically binds B7-H3 comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), the VH1 comprises HCDR1, HCDR2, and HCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 8, 11, 13, 15, 17, and 19; the VL1 comprises LCDR1, LCDR2, and LCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 6, 7, 9, 10, 12, 14, 16, and 18; the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0025] In some embodiments, the B7-H3 binding molecule comprises a first binding domain that specifically binds B7-H3; the first binding domain that specifically binds B7-H3 comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), the VH1 comprises HCDR1, HCDR2, and HCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 8, 11, 13, 15, 17, and 19; the VL1 comprises LCDR1, LCDR2, and LCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 6, 7, 9, 10, 12, 14, 16, and 18; the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0026] In some embodiments, the B7-H3 binding molecule comprises a first binding domain that specifically binds B7-H3; the first binding domain that specifically binds B7-H3 comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), the VH1 comprises HCDR1, HCDR2, and HCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 8, 11, 13, 15, 17, and 19; the VL1 comprises LCDR1, LCDR2, and LCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 6, 7, 9, 10, 12, 14, 16, and 18; the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0027] In some embodiments, the B7-H3 binding molecule comprises a first binding domain that specifically binds B7-H3; the first binding domain that specifically binds B7-H3 comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), the VH1 comprises HCDR1, HCDR2, and HCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 8, 11, 13, 15, 17, and 19; the VL1 comprises LCDR1, LCDR2, and LCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 6, 7, 9, 10, 12, 14, 16, and 18; the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0028] In some embodiments, the B7-H3 binding molecule comprises a first binding domain that specifically binds B7-H3; the first binding domain that specifically binds B7-H3 comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), the VH1 comprises HCDR1, HCDR2, and HCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 8, 11, 13, 15, 17, and 19; the VL1 comprises LCDR1, LCDR2, and LCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 6, 7, 9, 10, 12, 14, 16, and 18; the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0029] In some embodiments, the B7-H3 binding molecule comprises a first binding domain that specifically binds B7-H3; the first binding domain that specifically binds B7-H3 comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), the VH1 comprises HCDR1, HCDR2, and HCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 8, 11, 13, 15, 17, and 19; the VL1 comprises LCDR1, LCDR2, and LCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 6, 7, 9, 10, 12, 14, 16, and 18; the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0030] HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 13 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 12;

[0031] HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 13 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 12;

[0032] HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 15 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 14;

[0033] HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 17 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 16; or

[0034] HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 18 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 19.

[0035] In some embodiments, the B7-H3 binding molecule, HCDR1 is as set forth in any one of SEQ ID NOs: 20, 28, 31, and 44, HCDR2 is as set forth in any one of SEQ ID NOs: 21, 29, 32, 36, 40, 45, and 48; HCDR3 is as set forth in SEQ ID NO: 22; LCDR1 is as set forth in any one of SEQ ID NOs: 23, 33, 37, and 41; LCDR2 is as set forth in any one of SEQ ID NOs: 24, 34, 38, 42, 46, and 49; and LCDR3 is as set forth in any one of SEQ ID NOs: 25, 26, 27, 30, 35, 39, 43, and 47. These are CDRs defined according to the Kabat numbering system.

[0036] In some specific embodiments, the foregoing B7-H3 binding molecules, the sequences of the HCDR1, HCDR2, and HCDR3, and the LCDR1, LCDR2, and LCDR3 are as follows:

[0037] SEQ ID NOs: 20-24 and 26;

[0038] SEQ ID NOs: 20-24 and 27;

[0039] SEQ ID NOs: 20-24 and 30

[0040] SEQ ID NOs: 31, 32, 22, 33-35;

[0041] SEQ ID NOs: 31, 36, 22, 37-39;

[0042] SEQ ID NOs: 31, 40, 22, 41-43;

[0043] SEQ ID NOs: 44, 45, 22, 33, 46, 47.

[0044] SEQ ID NOs: 44, 48, 22, 33, 49, 35; or

[0045] SEQ ID NOs: 28, 29, and 22-25.

[0046] In some embodiments, the heavy chain variable region in the first binding domain that specifically binds B7-H3 in the foregoing B7-H3 binding molecules comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 8, 11, 13, 15, 17, and 19, or at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 6, 7, 9, 10, 12, 14, 16, and 18, or at least 90% identical thereto.

[0047] In some specific embodiments, the heavy chain variable region (VH1) and the light chain variable region (VL1) in the first binding domain that specifically binds B7-H3, wherein:

[0048] the VH1 is as set forth in SEQ ID NO: 4 or an amino acid sequence at least 90% identical thereto and the VL1 is as set forth in SEQ ID NO: 5 or an amino acid sequence at least 90% identical thereto;

[0049] the VH1 is as set forth in SEQ ID NO: 4 or an amino acid sequence at least 90% identical thereto and the VL1 is as set forth in SEQ ID NO: 6 or an amino acid sequence at least 90% identical thereto;

[0050] the VH1 is as set forth in SEQ ID NO: 8 or an amino acid sequence at least 90% identical thereto and the VL1 is as set forth in SEQ ID NO: 7 or an amino acid sequence at least 90% identical thereto;

[0051] the VH1 is as set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90% sequence identity thereto and the VL1 is as set forth in SEQ ID NO: 9 or an amino acid sequence having at least 90% sequence identity thereto;

[0052] the VH1 is as set forth in SEQ ID NO: 11 or an amino acid sequence having at least 90% sequence identity thereto and the VL1 is as set forth in SEQ ID NO: 10 or an amino acid sequence having at least 90% sequence identity thereto;

[0053] the VH1 is as set forth in SEQ ID NO: 13 or an amino acid sequence having at least 90% sequence identity thereto and the VL1 is as set forth in SEQ ID NO: 12 or an amino acid sequence having at least 90% sequence identity thereto;

[0054] the VH1 is as set forth in SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence identity thereto and the VL1 is as set forth in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity thereto;

[0055] the VH1 is as set forth in SEQ ID NO: 17 or an amino acid sequence having at least 90% sequence identity thereto and the VL1 is as set forth in SEQ ID NO: 16 or an amino acid sequence having at least 90% sequence identity thereto;

[0056] the VH1 is as set forth in SEQ ID NO: 19 or an amino acid sequence having at least 90% sequence identity thereto and the VL1 is as set forth in SEQ ID NO: 18 or an amino acid sequence having at least 90% sequence identity thereto.

[0057] In some embodiments, the above-mentioned amino acid mutations are substitutions, replacements, modifications, deletions, and / or additions of amino acids (e.g., conservative replacements of amino acids), which do not affect or substantially affect the function of specifically binding B7-H3.

[0058] In some embodiments, the foregoing B7-H3 binding molecules further comprise an immunoglobulin Fc region. In some embodiments, the B7-H3 binding molecules comprise a human immunoglobulin Fc region. For example, the Fc region is an Fc region of human IgGl, IgG2, or IgG4. In some embodiments, the human immunoglobulin Fc region is an Fc region of wild-type IgG or a variant thereof.

[0059] In some embodiments, the Fc region is an Fc region with increased effector function, e.g., increased antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) with increased effector function.

[0060] Exemplary IgGl Fc regions include those with the following substitutions: 239D; 239E; 239K, 241A; 262A; 264D; 264L; 264A; 264S; 265A; 265S; 265V; 296A; 296A; 301A; 332E; 239D / 332E; 239D / 330S / 332E; 239D / 330L / 332E; 298A / 333A / 334A; 247I / 339D; 247I / 339Q; 280H / 290S; 280H / 290S / 298D; 280H / 290S / 298V; 243L / 292P / 300L; 243L / 292P / 300L / 396L; 243L / 292P / 300L / 305I / 396L; 236A / 239D / 332E; 326A / 333A; 326W / 333S; 290E / 298G / 299A; 290N / 298G / 299A; 290E / 298G / 299A / 326E; or 290N / 298G / 299A / 326E; or any combination of any of the above positions. The mutations are defined according to the EU numbering system.

[0061] Exemplary IgGl Fc regions include those having the following substitutions: S239D; S239E; S239K, F241A; V262A; V264D; V264L; V264A; V264S; D265A; D265S; D265V; F296A; Y296A; R301A; I332E; S239D / I332E; S239D / A330S / I332E; S239D / A330L / I332E; S298A / D333A / K334A; P247I / A339D; P247I / A339Q; D280H / K290S; D280H / K290S / S298D; D280H / K290S / S298V; F243L / R292P / Y300L; F243L / R292P / Y300L / P396L; F243L / R292P / Y300L / V305I / P396L; G236A / S239D / I332E; K326A / E333A; K326W / E333S; K290E / S298G / T299A; K290N / S298G / T299A; K290E / S298G / T299A / K326E; or K290N / S298G / T299A / K326E, or any combination of any of the above positions. The foregoing " / " means and.

[0062] In some embodiments, the Fc region comprises an amino acid sequence as set forth in SEQ ID NOs: 51, 90, and 91.

[0063] In some embodiments, the B7-H3 binding molecule, which comprises a heavy chain and a light chain, wherein:

[0064] the heavy chain is an amino acid sequence as set forth in SEQ ID NO: 53 or having at least 90% sequence identity thereto and the light chain is an amino acid sequence as set forth in SEQ ID NO: 52 or having at least 90% sequence identity thereto;

[0065] the heavy chain is an amino acid sequence as set forth in SEQ ID NO: 63 or having at least 90% sequence identity thereto and the light chain is an amino acid sequence as set forth in SEQ ID NO: 62 or having at least 90% sequence identity thereto;

[0066] the heavy chain is an amino acid sequence as set forth in SEQ ID NO: 67 or having at least 90% sequence identity thereto and the light chain is an amino acid sequence as set forth in SEQ ID NO: 66 or having at least 90% sequence identity thereto.

[0067] In some embodiments, the B7-H3 binding molecule of the present disclosure is an anti-B7-H3 antibody.

[0068] In some embodiments, the present disclosure also provides a conjugate or fusion protein comprising the anti-B7-H3 antibody.

[0069] In some embodiments, the foregoing B7-H3 binding molecules have

[0070] (a) bind to human B7-H3 (or an epitope thereof) with a K -7 dissociation constant of < 10 D M, e.g., < 1 x 10 -7 M, e.g., < 1 x 10 -8 M, or < 1 x 10 -9 M; and / or

[0071] (b) have a higher affinity than huA3.

[0072] EGFR binding molecules

[0073] The present disclosure provides EGFR binding molecules comprising a second binding domain that specifically binds EGFR.

[0074] In some embodiments, the second binding domain that specifically binds EGFR comprises a heavy chain variable region (VH2) comprising HCDR1, HCDR2, and HCDR3 as set forth in any one of SEQ ID NOs: 71, 72, and 75; and a light chain variable region (VL2) comprising LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 69; wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0075] In some embodiments, the EGFR binding molecules have HCDR1 as set forth in SEQ ID NO: 76, HCDR2 as set forth in SEQ ID NO: 82 or 77, HCDR3 as set forth in any one of SEQ ID NOs: 78, 83, and 86, LCDR1 as set forth in SEQ ID NO: 79, LCDR2 as set forth in SEQ ID NO: 80, and LCDR3 as set forth in SEQ ID NO: 81. The CDRs are defined according to the Kabat numbering system.

[0076] In some specific embodiments, the foregoing EGFR binding molecules do not comprise a combination of VH2 and VL2 as set forth in SEQ ID NOs: 70 and 69.

[0077] In some specific embodiments, the foregoing EGFR binding molecules have the sequences of HCDR1, HCDR2, and HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in:

[0078] SEQ ID NOs: 76, 82, and 78-81;

[0079] SEQ ID NOs: 76, 77, 83, and 79-81; or

[0080] SEQ ID NOs: 76, 77, 86, and 79-81.

[0081] In some embodiments, the heavy chain variable region in the second binding domain that specifically binds EGFR in the foregoing EGFR binding molecules comprises an amino acid sequence as set forth in any one of 71, 72, and 75, or an amino acid sequence that is at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 69, or an amino acid sequence that is at least 90% identical thereto.

[0082] In some specific embodiments, the foregoing amino acid mutations are substitutions, replacements, modifications, deletions, and / or additions of amino acids (e.g., conservative replacements of amino acids), which do not affect or substantially affect the function of specifically binding EGFR.

[0083] In some embodiments, the heavy chain variable region and the light chain variable region in the second binding domain that specifically binds EGFR in the foregoing EGFR binding molecules are linked by a linker as an scFv.

[0084] In some specific embodiments, the second binding domain that specifically binds EGFR comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 96, 97, and 98, or an amino acid sequence that is at least 90% identical thereto.

[0085] In some embodiments, the foregoing EGFR binding molecules further comprise an immunoglobulin Fc region. In some embodiments, the EGFR binding molecules comprise a human immunoglobulin Fc region. For example, the Fc region is an Fc region of human IgGl, IgG2, or IgG4. In some specific embodiments, the human immunoglobulin Fc region is an Fc region of wild-type IgG or a variant thereof.

[0086] In some embodiments, the Fc region is an Fc region that increases effector function, e.g., has increased effector function for antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0087] Exemplary IgGl Fc regions include those having the following substitutions: 239D; 239E; 239K, 241A; 262A; 264D; 264L; 264A; 264S; 265A; 265S; 265V; 296A; 296A; 301A; 332E; 239D / 332E; 239D / 330S / 332E; 239D / 330L / 332E; 298A / 333A / 334A; 247I / 339D; 247I / 339Q; 280H / 290S; 280H / 290S / 298D; 280H / 290S / 298V; 243L / 292P / 300L; 243L / 292P / 300L / 396L; 243L / 292P / 300L / 305I / 396L; 236A / 239D / 332E; 326A / 333A; 326W / 333S; 290E / 298G / 299A; 290N / 298G / 299A; 290E / 298G / 299A / 326E; or 290N / 298G / 299A / 326E; or any combination of any of the above positions. The mutations are defined according to the EU numbering system.

[0088] Exemplary IgGl Fc regions include those having the following substitutions: S239D; S239E; S239K, F241A; V262A; V264D; V264L; V264A; V264S; D265A; D265S; D265V; F296A; Y296A; R301A; I332E; S239D / I332E; S239D / A330S / I332E; S239D / A330L / I332E; S298A / D333A / K334A; P247I / A339D; P247I / A339Q; D280H / K290S; D280H / K290S / S298D; D280H / K290S / S298V; F243L / R292P / Y300L; F243L / R292P / Y300L / P396L; F243L / R292P / Y300L / V305I / P396L; G236A / S239D / I332E; K326A / E333A; K326W / E333S; K290E / S298G / T299A; K290N / S298G / T299A; K290E / S298G / T299A / K326E; or K290N / S298G / T299A / K326E, or any combination of any of the above positions.

[0089] In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NOs: 51, 90, and 91.

[0090] In some embodiments, the EGFR binding molecule, which comprises a heavy chain and a light chain, wherein

[0091] the heavy chain is as set forth in SEQ ID NO: 89 or an amino acid sequence having at least 90% sequence identity thereto and the light chain is as set forth in SEQ ID NO: 87 or an amino acid sequence having at least 90% sequence identity thereto;

[0092] the heavy chain is as set forth in SEQ ID NO: 93 or an amino acid sequence having at least 90% sequence identity thereto and the light chain is as set forth in SEQ ID NO: 87 or an amino acid sequence having at least 90% sequence identity thereto.

[0093] In some embodiments, the EGFR binding molecule of the present disclosure is an anti-EGFR antibody, or a conjugate, fusion protein comprising the same.

[0094] In some embodiments, the foregoing EGFR binding molecule has an affinity weaker than Zalutumumab.

[0095] B7-H3 / EGFR binding molecules

[0096] The present disclosure provides B7-H3 / EGFR binding molecules comprising a first binding domain that specifically binds to B7-H3 and a second binding domain that specifically binds to EGFR.

[0097] In some embodiments, the B7-H3 / EGFR binding molecule, the first binding domain that specifically binds to B7-H3 comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), the VH1 comprises HCDR1, HCDR2 and HCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 8, 11, 13, 15, 17 and 19; and the VL1 comprises LCDR1, LCDR2 and LCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 5, 6, 7, 9, 10, 12, 14, 16 and 18. The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system.

[0098] In some embodiments, the VH and VL in the first binding domain comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 selected from the group consisting of:

[0099] HCDR1, HCDR2 and HCDR3 in an amino acid sequence as set forth in SEQ ID NO: 15 and LCDR1, LCDR2 and LCDR3 in an amino acid sequence as set forth in SEQ ID NO: 14;

[0100] The amino acid sequences shown in SEQ ID NO:4 contain HCDR1, HCDR2, and HCDR3, and the amino acid sequences shown in SEQ ID NO:5 contain LCDR1, LCDR2, and LCDR3; the amino acid sequences shown in SEQ ID NO:4 contain HCDR1, HCDR2, and HCDR3, and the amino acid sequences shown in SEQ ID NO:6 contain LCDR1, LCDR2, and LCDR3.

[0101] HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 8 and LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 7;

[0102] HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 4 and LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 9;

[0103] HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 11 and LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 10;

[0104] HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 13 and LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 12;

[0105] The amino acid sequences shown in SEQ ID NO: 17, such as HCDR1, HCDR2, and HCDR3, and the amino acid sequences shown in SEQ ID NO: 16, such as LCDR1, LCDR2, and LCDR3; or

[0106] HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 18 and LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 19.

[0107] In some embodiments, the first binding domain that specifically binds B7-H3 in the B7-H3 / EGFR binding molecule comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), the VH1 comprising a HCDR1, a HCDR2, and a HCDR3, and the VL1 comprising a LCDR1, a LCDR2, and a LCDR3; wherein the HCDR1 is as set forth in any one of SEQ ID NOs: 20, 28, 31, and 44, the HCDR2 is as set forth in any one of SEQ ID NOs: 21, 29, 32, 36, 40, 45, and 48; the HCDR3 is as set forth in SEQ ID NO: 22; the LCDR1 is as set forth in any one of SEQ ID NOs: 23, 33, 37, and 41; the LCDR2 is as set forth in any one of SEQ ID NOs: 24, 34, 38, 42, 46, and 49; and the LCDR3 is as set forth in any one of SEQ ID NOs: 25, 26, 27, 30, 35, 39, 43, and 47.

[0108] In some embodiments, the first binding domain that specifically binds B7-H3 in the B7-H3 / EGFR binding molecule comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), the VH1 comprising a HCDR1, a HCDR2, and a HCDR3, and the VL1 comprising a LCDR1, a LCDR2, and a LCDR3; wherein the HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and the LCDR3 have the sequences as set forth in:

[0109] SEQ ID NOs: 20-24 and 26;

[0110] SEQ ID NOs: 20-24 and 27;

[0111] SEQ ID NOs: 20-24 and 30

[0112] SEQ ID NOs: 31, 32, 22, 33-35;

[0113] SEQ ID NOs: 31, 36, 22, 37-39;

[0114] SEQ ID NOs: 31, 40, 22, 41-43;

[0115] SEQ ID NOs: 44, 45, 22, 33, 46, 47.

[0116] SEQ ID NOs: 44, 48, 22, 33, 49, 35; or

[0117] SEQ ID NOs: 28, 29, and 22-25.

[0118] In some embodiments, the B7-H3 / EGFR binding molecules, the second binding domain comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises HCDR1, HCDR2, and HCDR3 in an amino acid sequence as set forth in any one of SEQ ID NOs: 70-75; and the VL2 comprises LCDR1, LCDR2, and LCDR3 in an amino acid sequence as set forth in SEQ ID NO: 69. The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0119] In some embodiments, the B7-H3 / EGFR binding molecules, the second binding domain comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), the VH2 comprises HCDR1, HCDR2, and HCDR3, and the VL2 comprises LCDR1, LCDR2, and LCDR3; wherein the sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the second binding domain are as set forth in:

[0120] SEQ ID NO: 76-81;

[0121] SEQ ID NO: 76, 82, and 78-81;

[0122] SEQ ID NO: 76, 77, 83, and 78-81;

[0123] SEQ ID NO: 76, 84, and 78-81;

[0124] SEQ ID NO: 76, 85, and 78-81; or,

[0125] SEQ ID NO: 76, 77, 86, and 78-81.

[0126] In some embodiments, the foregoing B7-H3 / EGFR binding molecules, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the VH1 and VL1 in the first binding domain that specifically binds B7-H3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the VH2 and VL2 in the second binding domain that specifically binds EGFR are as set forth in:

[0127] SEQ ID NO: 31, 40, 22, 41, 42, 43, and SEQ ID NO: 76-81;

[0128] SEQ ID NOs: 31, 40, 22, 41, 42, 43, and SEQ ID NOs: 76, 82, 78-81;

[0129] SEQ ID NOs: 31, 40, 22, 41, 42, 43, and SEQ ID NOs: 76, 77, 86, 79-81;

[0130] SEQ ID NOs: 20, 21, 22, 23, 24, 26, and SEQ ID NOs: 76, 82, 78-81;

[0131] SEQ ID NOs: 44, 48, 22, 33, 49, 35, and SEQ ID NOs: 76, 82, 78-81; or

[0132] SEQ ID NOs: 20, 21, 22, 23, 24, 25, and SEQ ID NOs: 76, 82, 78-81.

[0133] In some embodiments, the B7-H3 / EGFR binding molecules, wherein,

[0134] the heavy chain variable region (VH1) in the first binding domain that specifically binds B7-H3 comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 8, 11, 13, 15, 17, and 19, or at least 90% identical thereto, and the light chain variable region (VL1) comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 5, 6, 7, 9, 10, 12, 14, 16, and 18, or at least 90% identical thereto; and / or,

[0135] the heavy chain variable region (VH2) in the second binding domain that specifically binds EGFR comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 70, 71, and 75, or at least 90% identical thereto, and the light chain variable region (VL2) comprises an amino acid sequence as set forth in SEQ ID NO: 69, or at least 90% identical thereto.

[0136] In some embodiments, the foregoing B7-H3 / EGFR binding molecules, the VH1 and VL1 in the first binding domain that specifically binds B7-H3, and the VH2 and VL2 in the second binding domain that specifically binds EGFR are as set forth below, respectively:

[0137] the VH1 is as set forth in SEQ ID NO: 15 or an amino acid sequence at least 90% identical thereto, the VL1 is as set forth in SEQ ID NO: 14 or an amino acid sequence at least 90% identical thereto, the VH2 is as set forth in SEQ ID NO: 71 or an amino acid sequence at least 90% identical thereto, and the VL2 is as set forth in SEQ ID NO: 69 or an amino acid sequence at least 90% identical thereto;

[0138] the VH1 is as set forth in SEQ ID NO: 15 or an amino acid sequence at least 90% identical thereto, the VL1 is as set forth in SEQ ID NO: 14 or an amino acid sequence at least 90% identical thereto, the VH2 is as set forth in SEQ ID NO: 71 or an amino acid sequence at least 90% identical thereto, and the VL2 is as set forth in SEQ ID NO: 69 or an amino acid sequence at least 90% identical thereto;

[0139] the VH1 is as set forth in SEQ ID NO: 15 or an amino acid sequence at least 90% identical thereto, the VL1 is as set forth in SEQ ID NO: 14 or an amino acid sequence at least 90% identical thereto, the VH2 is as set forth in SEQ ID NO: 75 or an amino acid sequence at least 90% identical thereto, and the VL2 is as set forth in SEQ ID NO: 69 or an amino acid sequence at least 90% identical thereto;

[0140] the VH1 is as set forth in SEQ ID NO: 4 or an amino acid sequence at least 90% identical thereto, the VL1 is as set forth in SEQ ID NO: 5 or an amino acid sequence at least 90% identical thereto, the VH2 is as set forth in SEQ ID NO: 71 or an amino acid sequence at least 90% identical thereto, and the VL2 is as set forth in SEQ ID NO: 69 or an amino acid sequence at least 90% identical thereto;

[0141] the VH1 is as set forth in SEQ ID NO: 19 or an amino acid sequence at least 90% identical thereto, the VL1 is as set forth in SEQ ID NO: 18 or an amino acid sequence at least 90% identical thereto, the VH2 is as set forth in SEQ ID NO: 71 or an amino acid sequence at least 90% identical thereto, and the VL2 is as set forth in SEQ ID NO: 69 or an amino acid sequence at least 90% identical thereto; or

[0142] the VH1 is as set forth in SEQ ID NO: 4 or an amino acid sequence at least 90% identical thereto, the VL1 is as set forth in SEQ ID NO: 3 or an amino acid sequence at least 90% identical thereto, the VH2 is as set forth in SEQ ID NO: 71 or an amino acid sequence at least 90% identical thereto, and the VL2 is as set forth in SEQ ID NO: 69 or an amino acid sequence at least 90% identical thereto.

[0143] In some embodiments, the above-mentioned amino acid mutations are substitutions, replacements, modifications, deletions and / or additions of amino acids (e.g., conservative replacements of amino acids), which do not affect or substantially affect the function of specifically binding B7-H3 and EGFR.

[0144] In some embodiments, in the second binding domain that specifically binds EGFR in the foregoing B7-H3 / EGFR binding molecules, the heavy chain variable region and the light chain variable region are linked as a scFv through a linker.

[0145] In some embodiments, the linker is as set forth in (G m S n ) h or (G m Q n ) h or (GGNGT) h (SEQ ID NO: 128) or (YGNGT) h (SEQ ID NO: 129) or (EPKSS) h (SEQ ID NO: 130), wherein each of m, n is independently selected from an integer from 1 to 8, and h is independently selected from an integer from 1 to 20; for example, (GGGGS)1(SEQ ID NO: 131), (GGGGS)2(SEQ ID NO: 132), (GGGGS)3(SEQ ID NO: 133), or (GGGGS)4(SEQ ID NO: 134).

[0146] In some embodiments, the second binding domain that specifically binds EGFR in the foregoing B7-H3 / EGFR binding molecules comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 96, 97 and 98 or an amino acid sequence at least 90% identical thereto.

[0147] In some embodiments, the foregoing B7-H3 / EGFR binding molecules, wherein the first binding domain that specifically binds B7-H3 is a Fab, and the second binding domain that specifically binds EGFR is a scFv.

[0148] In some embodiments, the foregoing B7-H3 / EGFR binding molecules further comprise an immunoglobulin Fc region. In some embodiments, the B7-H3 / EGFR binding molecules comprise a human immunoglobulin Fc region. For example, the Fc region is an Fc region of human IgGl, IgG2, or IgG4. In some particular embodiments, the human immunoglobulin Fc region is an Fc region of wild-type IgG or a variant thereof.

[0149] In some embodiments, the Fc region is an Fc region that increases effector function, e.g., increased antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) with increased effector function.

[0150] Exemplary IgGl Fc regions include those with the following substitutions: 239D; 239E; 239K, 241A; 262A; 264D; 264L; 264A; 264S; 265A; 265S; 265V; 296A; 296A; 301A; 332E; 239D / 332E; 239D / 330S / 332E; 239D / 330L / 332E; 298A / 333A / 334A; 247I / 339D; 247I / 339Q; 280H / 290S; 280H / 290S / 298D; 280H / 290S / 298V; 243L / 292P / 300L; 243L / 292P / 300L / 396L; 243L / 292P / 300L / 305I / 396L; 236A / 239D / 332E; 326A / 333A; 326W / 333S; 290E / 298G / 299A; 290N / 298G / 299A; 290E / 298G / 299A / 326E; or 290N / 298G / 299A / 326E; or any combination of the above positions. The mutations are defined according to the EU numbering system.

[0151] Exemplary IgGl Fc regions include those having the following substitutions: S239D; S239E; S239K, F241A; V262A; V264D; V264L; V264A; V264S; D265A; D265S; D265V; F296A; Y296A; R301A; I332E; S239D / I332E; S239D / A330S / I332E; S239D / A330L / I332E; S298A / D333A / K334A; P247I / A339D; P247I / A339Q; D280H / K290S; D280H / K290S / S298D; D280H / K290S / S298V; F243L / R292P / Y300L; F243L / R292P / Y300L / P396L; F243L / R292P / Y300L / V305I / P396L; G236A / S239D / I332E; K326A / E333A; K326W / E333S; K290E / S298G / T299A; K290N / S298G / T299A; K290E / S298G / T299A / K326E; or K290N / S298G / T299A / K326E, or any combination of any of the above positions.

[0152] In some embodiments, the Fc region comprises an amino acid sequence as set forth in SEQ ID NO: 51, 90, and 91.

[0153] In some embodiments, the Fc region comprises a first subunit (Fcl) and a second subunit (Fc2), a mutation that pairs the two subunits (Fcl, Fc2) of the Fc region to form a dimer, or a mutation that reduces homodimerization. In some embodiments, the Fc region comprises a knobs-into-holes mutation that facilitates association of the first subunit and the second subunit. For example, one, two, or more amino acid residues in the CH3 domain of Fcl are mutated with one or more amino acid residues having a larger side chain volume to create a protuberance (or knob) on the surface of the CH3 domain of Fcl, and one, two, or more of the amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fcl are mutated with an amino acid residue having a smaller side chain volume to create a depression (or hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fcl. In some specific embodiments, the first subunit of the Fc region comprises a knob mutation and the second subunit of the Fc region comprises a hole mutation.

[0154] The first subunit and the second subunit are intended to facilitate formation of a heterodimer, and the positions of the two are interchangeable.

[0155] In some embodiments, the Fc1 has one or more amino acid substitutions at a position selected from 354, 356, 358, and 366, and the Fc2 has one or more amino acid substitutions at a position selected from 349, 356, 358, 366, 368, and 407. In some particular embodiments, the Fc1 contains a mutation at position 366, and the Fc2 contains a mutation selected from 366, 368, and 407, or any combination thereof; in some particular embodiments, the Fc1 contains a mutation at position 354 or 356, and the Fc2 contains a mutation at position 349; in some particular embodiments, the Fc1 contains a mutation at position 354 or 356, and the Fc2 contains mutations at positions 349, 366, 368, and 407.

[0156] In some embodiments, the Fc1 has one or more amino acid substitutions selected from 354C, 356C, 356E, 358M, and 366W, and the Fc2 has one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some particular embodiments, the Fc1 contains a mutation at 366W, and the Fc2 contains a mutation selected from 366S, 368A, and 407V, or any combination thereof; in some particular embodiments, the Fc1 contains a mutation at 354C or 356C, and the Fc2 contains a mutation at 349C; or in some particular embodiments, the Fc1 contains mutations at 354C / 366W, and the Fc2 contains mutations at 349C / 366S / 368A / 407V.

[0157] In some particular embodiments, the Fc contains a C220S mutation; for example, the Fc linked to a scFv.

[0158] In some particular embodiments, the Fc1 is as set forth in SEQ ID NO: 94, and the Fc2 is as set forth in SEQ ID NO: 95.

[0159] In some particular embodiments, the Fc1 is as set forth in SEQ ID NO: 106 or 108, and the Fc2 is as set forth in SEQ ID NO: 107 or 109.

[0160] In some embodiments, the B7-H3 / EGFR binding molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein,

[0161] the first polypeptide chain comprises a heavy chain variable region that binds B7-H3, a CH1, and an Fc1;

[0162] the second polypeptide chain comprises a light chain variable region that binds B7-H3 and a light chain constant region; and

[0163] the third polypeptide chain comprises an scFv that binds EGFR and Fc2.

[0164] In some embodiments, the B7-H3 / EGFR binding molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein,

[0165] the first polypeptide chain comprises a heavy chain variable region that binds B7-H3, CH1, and Fc2;

[0166] the second polypeptide chain comprises a light chain variable region that binds B7-H3 and a light chain constant region; and

[0167] the third polypeptide chain comprises an scFv that binds EGFR and Fc1.

[0168] In some embodiments, the B7-H3 / EGFR binding molecule comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein, in order from N-terminus to C-terminus:

[0169] (I) the first polypeptide chain comprises the following structure: [VH1 of the first binding domain]-CH1-[Fc first subunit],

[0170] the second polypeptide chain comprises the following structure: [VL1 of the first binding domain]-CL;

[0171] the third polypeptide chain comprises the following structure: [VH2 of the second binding domain]-[linker]a-[VL2 of the second binding domain]-[linker]b-[Fc second subunit];

[0172] (II) the first polypeptide chain comprises the following structure: [VH1 of the first binding domain]-CH1-[Fc second subunit],

[0173] the second polypeptide chain comprises the following structure: [VL1 of the first binding domain]-CL;

[0174] the third polypeptide chain comprises the following structure: [VH2 of the second binding domain]-[linker]a-[VL2 of the second binding domain]-[linker]b-[Fc first subunit];

[0175] (III) the first polypeptide chain comprises the following structure: [VH2 of the second binding domain]-[linker]a-[VL2 of the second binding domain]-[linker]b-[Fc second subunit],

[0176] the second polypeptide chain comprises the following structure: [VL1 of the first binding domain]-CL;

[0177] the third polypeptide chain comprises the following structure: [VH1 of the first binding domain]-CH1-[Fc first subunit];

[0178] (IV) the first polypeptide chain comprises the following structure: [VH2 of the second binding domain]-[linker]a-[VL2 of the second binding domain]-[linker]b-[Fc first subunit],

[0179] the second polypeptide chain comprises the following structure: [VL1 of the first binding domain]-CL;

[0180] the third polypeptide chain comprises the following structure: [VH2 of the second binding domain]-[linker]a-[VL2 of the second binding domain]-[linker]b-[Fc two-subunit];

[0181] (V) the first polypeptide chain comprises the following structure: [VH1 of the first binding domain]-CH1-[Fc first subunit],

[0182] the second polypeptide chain comprises the following structure: [VL1 of the first binding domain]-CL;

[0183] the third polypeptide chain comprises the following structure: [VL2 of the second binding domain]-[linker]a-[VH2 of the second binding domain]-[linker]b-[Fc second subunit];

[0184] (VI) the first polypeptide chain comprises the following structure: [VH1 of the first binding domain]-CH1-[Fc second subunit],

[0185] the second polypeptide chain comprises the following structure: [VL1 of the first binding domain]-CL;

[0186] the third polypeptide chain comprises the following structure: [VL2 of the second binding domain]-[linker]a-[VH2 of the second binding domain]-[linker]b-[Fc first subunit];

[0187] wherein - represents a peptide bond, a is selected from 0 or 1, b is selected from 0 or 1; the linker is a polypeptide capable of achieving a connection function, for example, (G x S y ) n , x is selected from an integer from 1 to 6, y is selected from an integer from 0 to 4, n is selected from an integer from 1 to 6.

[0188] In some embodiments, the linker in the foregoing B7-H3 / EGFR binding molecules can be 1-20 or more amino acids, a non-functional amino acid sequence without secondary structure above. For example, the linker is a flexible linker, such as G4S (SEQ ID NO: 131), GS, GAP, (G4S)2(SEQ ID NO: 132), (G4S)3(SEQ ID NO: 133), (G4S)4(SEQ ID NO: 134), (G4S)5(SEQ ID NO: 135), ASGS (SEQ ID NO: 136), and the like.

[0189] In some embodiments, the first polypeptide chain and the second polypeptide chain of the foregoing B7-H3 / EGFR binding molecules constitute a first binding domain, and the third polypeptide chain constitutes a second binding domain.

[0190] In some embodiments, the foregoing B7-H3 / EGFR binding molecules, wherein:

[0191] the first polypeptide chain is set forth in SEQ ID NO: 99 or 110 or an amino acid sequence having at least 90% sequence identity thereto, the second polypeptide chain is set forth in SEQ ID NO: 62 or an amino acid sequence having at least 90% sequence identity thereto, and the third polypeptide chain is set forth in SEQ ID NO: 100 or 114 or an amino acid sequence having at least 90% sequence identity thereto;

[0192] the first polypeptide chain is set forth in SEQ ID NO: 99 or 115 or an amino acid sequence having at least 90% sequence identity thereto, the second polypeptide chain is set forth in SEQ ID NO: 62 or an amino acid sequence having at least 90% sequence identity thereto, and the third polypeptide chain is set forth in SEQ ID NO: 101, 111 or 116 or an amino acid sequence having at least 90% sequence identity thereto;

[0193] the first polypeptide chain is set forth in SEQ ID NO: 99 or 110 or an amino acid sequence having at least 90% sequence identity thereto, the second polypeptide chain is set forth in SEQ ID NO: 62 or an amino acid sequence having at least 90% sequence identity thereto, and the third polypeptide chain is set forth in SEQ ID NO: 102 or 113 or an amino acid sequence having at least 90% sequence identity thereto;

[0194] The first polypeptide chain is an amino acid sequence as shown in SEQ ID NO: 103 or 112 or having at least 90% sequence identity with it; the second polypeptide chain is an amino acid sequence as shown in SEQ ID NO: 52 or having at least 90% sequence identity with it; and the third polypeptide chain is an amino acid sequence as shown in SEQ ID NO: 101 or 111 or having at least 90% sequence identity with it.

[0195] The first polypeptide chain is shown in SEQ ID NO: 104 or has an amino acid sequence that has at least 90% sequence identity with it, the second polypeptide chain is shown in SEQ ID NO: 66 or has an amino acid sequence that has at least 90% sequence identity with it, and the third polypeptide chain is shown in SEQ ID NO: 101 or has an amino acid sequence that has at least 90% sequence identity with it.

[0196] The first polypeptide chain is an amino acid sequence as shown in SEQ ID NO: 105 or 117 or having at least 90% sequence identity with it, the second polypeptide chain is an amino acid sequence as shown in SEQ ID NO: 55 or having at least 90% sequence identity with it, and the third polypeptide chain is an amino acid sequence as shown in SEQ ID NO: 101 or 111 or having at least 90% sequence identity with it.

[0197] In some embodiments, the B7-H3 / EGFR binding molecule disclosed herein is an anti-B7-H3 / EGFR antibody, or a conjugate or fusion protein containing said antibody.

[0198] In some embodiments, the aforementioned B7-H3 / EGFR binding molecule has an activity selected from at least one of the following:

[0199] (a) with ≤10 -7 K D It binds to human B7-H3 or its epitopes, and has a higher affinity than huA3.

[0200] (b) with ≤10 -7 K D It binds to human EGFR or its epitopes, and has a weaker affinity than zalutumumab.

[0201] (c) Selective binding to B7-H3 and EGFR double-positive cells; for example, in Example 6, the proportion of double-positive cells in the antibody-positive cell population is more than 2, 10, 20, or 100 times that of single-positive cells.

[0202] (d) selectively blocks EGF binding to B7-H3 and EGFR double positive cells; the B7-H3 / EGFR binding molecule blocks the ability of EGF to bind double positive cells better than single positive cells, IC 50 fold or more, 4-fold or more, or 10-fold or more;

[0203] (e) selectively inhibits EGFR receptor phosphorylation in B7-H3 and EGFR double positive cells;

[0204] (f) selectively induces killing of B7-H3 and EGFR double positive cells;

[0205] (g) selectively inhibits proliferation of B7-H3 and EGFR double positive cells.

[0206] In the present disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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; "at least 90% (sequence) identity" encompasses 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.

[0207] Antibody drug conjugate

[0208] The present disclosure provides antibody drug conjugates comprising an antibody and a drug molecule. In some embodiments, the antibody comprises the first binding domain that specifically binds B7-H3 of any one of the preceding embodiments, and / or the second binding domain that specifically binds EGFR of any one of the preceding embodiments.

[0209] In some embodiments, the antibody comprises a first binding domain that specifically binds B7-H3, and a second binding domain that specifically binds EGFR.

[0210] In some embodiments, the drug molecule includes, but is not limited to, cytotoxic drugs, immunomodulators, cytostatic agents. For example, compounds, polypeptides, proteins, nucleic acids, etc. that have cell growth inhibitory, cytotoxic, and / or immunomodulatory functions. Illustratively, toxins (e.g., small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin), radioisotopes, chemotherapeutic drugs, antibiotics, and nucleolytic enzymes, among others.

[0211] In some particular embodiments, the drug molecule is a cytotoxic drug.

[0212] In some embodiments, the drug molecule is selected from MMAF or a derivative thereof, MMAE or a derivative thereof, exetecan or a derivative thereof, eribulin or a derivative thereof. In some specific embodiments, the drug molecule is exetecan or a derivative thereof.

[0213] In some embodiments, the foregoing antibody drug conjugate is of the structure shown in Formula (IV)

[0214] wherein:

[0215] -L-Y- is a linker moiety;

[0216] n is 1 to 10, n is a decimal or an integer;

[0217] Ab is an antibody, e.g., any of the foregoing anti-B7-H3 / EGFR antibodies, anti-B7-H3 antibodies, or anti-EGFR antibodies of the present disclosure.

[0218] In some embodiments, Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-;

[0219] R a and R b are the same or different and each is independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, or a heterocyclyl group; or, R a and R b together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclyl group;

[0220] R 1selected from hydrogen atom, alkyl, halo, haloalkyl, deuterium-substituted alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl;

[0221] R 2 selected from hydrogen atom, alkyl, halo, haloalkyl, deuterium-substituted alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl;

[0222] or, R 1 and R 2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

[0223] or, R a and R 2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group;

[0224] m is an integer from 0 to 4.

[0225] In some embodiments, -L- is a linker unit that is -L 1 -L 2 -L 3 -L 4

[0226] L 1 -(succinimid-3-yl-N)-W-C(O)-, -CH2-C(O)-NR 3 -W-C(O)- or -C(O)-W-C(O)-, wherein W is selected from C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, cycloalkyl, and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halo, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterium-substituted alkyl, alkoxy, and cycloalkyl;

[0227] L 2 selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)-, or a chemical bond, wherein p 1 is an integer from 1 to 20;

[0228] L 3 ​a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterium-alkyl, alkoxy, and cycloalkyl;

[0229] L 4 selected from -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 , -C(O)NR 5 (CH2) t - or a chemical bond, wherein t is an integer from 1 to 6;

[0230] R 3 , R 4 and R 5 are the same or different and each is independently selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterium-alkyl group, and a hydroxyalkyl group;

[0231] R 6 and R 7 are the same or different and each is independently selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterium-alkyl group, and a hydroxyalkyl group.

[0232] In some embodiments, the antibody drug conjugate of Formula (IV) as in any one of the preceding embodiments,

[0233] wherein:

[0234] Y is -O-(CR a R b )m-CR 1 R 2 -C(O)-;

[0235] R a and R b are the same or different and each is independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C 1-6 alkyl group, a C 1-6 haloalkyl group, a C 1-6 deuterium-alkyl group, a C 1-6 alkoxy group, a hydroxyl group, and a C 1-6 hydroxyalkyl group;

[0236] R 1 is selected from the group consisting of a hydrogen atom, a C 1-6 alkyl group, a C 3-6 cycloalkyl C 1-6 alkyl group, or a C 3-6 cycloalkyl group;

[0237] R 2 is selected from the group consisting of a hydrogen atom, a halogen, a C 1-6 haloalkyl group, a C1-6 deuterated alkyl, C 3-6 cycloalkyl and C 3-6 cycloalkyl C 1-6 alkyl;

[0238] or, R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl;

[0239] or, R a and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl; m is 0 or 1 ;

[0240] In some embodiments, the antibody drug conjugate of Formula (IV) as described in any of the preceding embodiments,

[0241] wherein:

[0242] Y is -O-(CR a R b )m-CR 1 R 2 -C(O)-;

[0243] R a and R b are the same or different and each is independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, and C 1-6 alkyl;

[0244] R 1 is C 3-6 cycloalkyl C 1-6 alkyl or C 3-6 cycloalkyl;

[0245] R 2 is selected from the group consisting of a hydrogen atom, C 1-6 haloalkyl and C 3-6 cycloalkyl;

[0246] or, R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl; m is 0 or 1.

[0247] In some embodiments, the antibody drug conjugate of Formula (IV) as described in any of the preceding embodiments,

[0248] wherein Y is selected from:

[0249] wherein the O end of Y is connected to the linker unit L.

[0250] In some embodiments, the antibody drug conjugate of Formula (IV) is

[0251] wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4

[0252] L 1 is selected from -(succinimid-3-yl-N)-W-C(O)-, -CH2-C(O)-NR 3 -W-C(O)- and -C(O)-W-C(O)-, wherein W is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl and straight chain heteroalkyl of 1 to 8 atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O and S, wherein said C 1-8 alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterium- substituted alkyl, alkoxy and cycloalkyl;

[0253] L 2 is selected from -NR 4 (CH2CH2O)pCH2CH2C(O)-, -NR 4 (CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)- or a chemical bond, wherein p is an integer from 1 to 20;

[0254] L 3 is a peptide residue consisting of 2 to 7 amino acid residues, wherein said amino acid residues are selected from the group consisting of amino acid residues formed from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and are optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterium-substituted alkyl, alkoxy and cycloalkyl;

[0255] L 4 is selected from -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 , -C(O)NR 5 (CH2) t - and a chemical bond, wherein t is an integer from 1 to 6;

[0256] R 3 , R 4 and R 5 ​They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl, deuteralkyl and hydroxyalkyl;

[0257] R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups.

[0258] In some implementations, such as the antibody-drug conjugate represented by formula (IV) as described in the previous one,

[0259] Among them, the connector unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0260] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, where W is selected from C. 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl and straight-chain heteroalkyl groups with 1 to 8 chain atoms, wherein the heteroalkyl group comprises 1 to 3 heteroatoms selected from N, O and S, wherein the C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently and optionally further substituted by one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0261] L 2 Selected from -NR 4 (CH2CH2O)pCH2CH2C(O)-、-NR 4 (CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)-, or chemical bonds, where p is an integer from 1 to 20;

[0262] L 3 It is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0263] L 4 Selected from -NR 5 (CR 6 R 7 )t -C(O)NR 5 -C(O)NR 5 (CH2) t - and a bond, wherein t is an integer from 1 to 6, non-limiting examples being 1, 2, 3, 4, 5 and 6;

[0264] R 3 , R 4 and R 5 are the same or different and each is independently selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group and a hydroxyalkyl group;

[0265] R 6 and R 7 are the same or different and each is independently selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group and a hydroxyalkyl group.

[0266] In some embodiments, the antibody drug conjugate of Formula (IV) as described in any of the preceding embodiments,

[0267] wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0268] L 1 is s 1 is an integer from 2 to 8, non-limiting examples being 2, 3, 4, 5, 6, 7 and 8;

[0269] L 2 is a bond;

[0270] L 3 is a tetrapeptide residue; preferably, L 3 is a tetrapeptide residue of GGFG;

[0271] L 4 is -NR 5 (CR 6 R 7 )t-, R 5 , R 6 or R 7 are the same or different and each is independently a hydrogen atom or an alkyl group, and t is 1 or 2;

[0272] wherein the L 1 end is attached to Ab, and the L 4 end is attached to Y.

[0273] In some embodiments, the antibody-drug conjugate as shown in formula (IV) of any of the preceding embodiments, wherein n is 1 to 10, for example 1 to 8, 2 to 8, 2 to 7, 2 to 4, 3 to 8, 3 to 7, 3 to 6, 4 to 7, or 4 to 6, and n is a decimal or an integer. In some embodiments, n is 1 to 8, and n is a decimal or an integer. In some embodiments, n is 3 to 7, and n is a decimal or an integer. In some embodiments, n is 4 to 6, and n is a decimal or an integer. In some embodiments, n is an average of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10. In some implementations, n is an average of approximately 4.1, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4.5, approximately 4.6, 4.7, approximately 4.8, approximately 4.9, approximately 5, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, or approximately 6.

[0274] In some implementations, the aforementioned antibody-drug conjugate has the structure shown in formula (II).

[0275] in:

[0276] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight-chain heteroalkyl with 1 to 8 atoms, wherein the heteroalkyl comprises 1 to 3 heteroatoms selected from N, O or S, wherein the C 1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently and optionally further substituted by one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0277] L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, p 1 Integers from 1 to 20;

[0278] L 3 It is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0279] R 1 Selected from hydrogen atoms, halogens, cycloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups;

[0280] R 2 is selected from a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group;

[0281] or, R 1 and R 2 together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclyl group;

[0282] R 4 and R 5 are the same or different and each is independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0283] R 6 and R 7 are the same or different and each is independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0284] m is an integer from 0 to 4;

[0285] n is an integer or a decimal number from 1 to 10;

[0286] Ab is an antibody, such as any of the foregoing anti-B7-H3 / EGFR antibodies, anti-B7-H3 antibodies, or anti-EGFR antibodies of the present disclosure.

[0287] In some embodiments, the antibody drug conjugate of Formula (II) as in any of the foregoing,

[0288] wherein:

[0289] said -L-Y- is as shown in the following structure:

[0290] s 1 is an integer from 2 to 8;

[0291] L 2 , L 3 , R 1 , R 2 , R 5 , R 6 , R 7 , and m are as defined in the foregoing Formula (II).

[0292] In some embodiments, the foregoing antibody drug conjugate is of the structure of Formula (III):

[0293] wherein:

[0294] s 1 is an integer from 2 to 8;

[0295] R 1 , R2 , R 5 ~ R 7 , m is as defined above for formula (II);

[0296] n is 1 to 10, n is an integer or a decimal number;

[0297] Ab is an antibody, e.g., an anti-B7-H3 / EGFR antibody, an anti-B7-H3 antibody, or an anti-EGFR antibody.

[0298] In some embodiments, the antibody drug conjugate of general formula (IV) as in any of the preceding embodiments, wherein -L- is:

[0299] In some embodiments, the antibody drug conjugate of general formula (IV) as in any of the preceding embodiments, wherein -L-Y- is optionally selected from:

[0300] In some embodiments, wherein -L-Y- is optionally selected from:

[0301] In some embodiments, wherein -L-Y- is:

[0302] In some embodiments, wherein -L-Y- is:

[0303] In some embodiments, the antibody drug conjugate is of the structure of formula (V-1) or (V-2):

[0304] the Ab is an anti-B7-H3 / EGFR antibody, n is 1 to 10, n is an integer or a decimal number;

[0305] wherein the anti-B7-H3 / EGFR antibody comprises a first binding domain that specifically binds B7-H3 and a second binding domain that specifically binds EGFR;

[0306] the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of VH1 and VL1 in the first binding domain that specifically binds B7-H3 and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of VH2 and VL2 in the second binding domain that specifically binds EGFR are as follows, respectively:

[0307] SEQ ID NOs: 31, 40, 22, 41, 42, 43, and SEQ ID NOs: 76-81;

[0308] SEQ ID NO: 31, 40, 22, 41, 42, 43 and SEQ ID NO: 76, 82, 78-81;

[0309] SEQ ID NO: 31, 40, 22, 41, 42, 43 and SEQ ID NO: 76, 77, 86, 79-81;

[0310] SEQ ID NO: 20, 21, 22, 23, 24, 26 and SEQ ID NO: 76, 82, 78-81;

[0311] SEQ ID NO: 44, 48, 22, 33, 49, 35 and SEQ ID NO: 76, 82, 78-81; or

[0312] SEQ ID NO: 20, 21, 22, 23, 24, 25 and SEQ ID NO: 76, 82, 78-81.

[0313] In some implementations, antibody-drug conjugates of formula (V-1) or (V-2) as described in the preceding one,

[0314] The heavy chain variable region of the first binding domain specifically binding to B7-H3 comprises an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 4, 8, 11, 13, 15, 17, and 19; the light chain variable region comprises an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 3, 5, 6, 7, 9, 10, 12, 14, 16, and 18; and / or,

[0315] The heavy chain variable region in the second binding domain that specifically binds to EGFR contains an amino acid sequence as shown in any of 70, 71, and 75 or having at least 90% identity with it, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO: 69 or having at least 90% identity with it.

[0316] Preferably, VH1 and VL1 in the first binding domain that specifically binds to B7-H3, and VH2 and VL2 in the second binding domain that specifically binds to EGFR, are respectively as follows:

[0317] SEQ ID NO: 15, 14, 70 and 69;

[0318] SEQ ID NO: 15, 14, 71 and 69;

[0319] SEQ ID NO: 15, 14, 75 and 69;

[0320] SEQ ID NOs: 4, 5, 71, and 69;

[0321] SEQ ID NOs: 19, 18, 71, and 69; or

[0322] SEQ ID NOs: 4, 3, 71, and 69.

[0323] In some embodiments, the antibody drug conjugate of Formula (V-1) or (V-2) as in any one of the preceding embodiments,

[0324] the Ab is an anti-B7-H3 / EGFR antibody comprising a polypeptide chain combination of:

[0325] 1) a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 99 or 110, or having at least 90% sequence identity thereto, a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 62, or having at least 90% sequence identity thereto, and a third polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 100 or 114, or having at least 90% sequence identity thereto,

[0326] 2) a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 99, 110, or 115, or having at least 90% sequence identity thereto, a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 62, or having at least 90% sequence identity thereto, and a third polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 101, 111, or 116, or having at least 90% sequence identity thereto,

[0327] 3) a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 99 or 110, or having at least 90% sequence identity thereto, a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 62, or having at least 90% sequence identity thereto, and a third polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 102 or 113, or having at least 90% sequence identity thereto,

[0328] 4) a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 103 or 112, or having at least 90% sequence identity thereto, a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 52, or having at least 90% sequence identity thereto, and a third polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 101 or 111, or having at least 90% sequence identity thereto,

[0329] 5) a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 105 or 117, or having at least 90% sequence identity thereto, a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 55, or having at least 90% sequence identity thereto, a third polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 101 or 111, or having at least 90% sequence identity thereto, or

[0330] 6) a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 104, or having at least 90% sequence identity thereto, a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 66, or having at least 90% sequence identity thereto, a third polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 101, or having at least 90% sequence identity thereto.

[0331] In some embodiments, the antibody drug conjugate of general formula (V-2) as described in any of the preceding embodiments has the following structure:

[0332] The present disclosure also provides a method of preparing the aforementioned antibody drug conjugate, comprising the following steps:

[0333] conjugating the aforementioned Ab to the drug molecule;

[0334] Optionally, purifying the antibody drug conjugate.

[0335] In some embodiments, the method of preparing the antibody drug conjugate of formula (II) comprises the following steps:

[0336] conjugating the reduced Ab to a compound of general formula (La-Y-D) to obtain a compound of general formula (II);

[0337] wherein Ab, W, L2, L3, R1, R2, R5-R7, m and n are as defined in formula (II).

[0338] Polynucleotides and vectors

[0339] The present disclosure provides polynucleotides encoding the B7-H3 binding molecules, EGFR binding molecules, or B7-H3 / EGFR binding molecules of the present disclosure. The polynucleotides of the present disclosure can be RNA, DNA, or cDNA. According to some embodiments of the present disclosure, the polynucleotides of the present disclosure are substantially isolated polynucleotides.

[0340] A polynucleotide of the present disclosure can also be in the form of, can be present in, and / or can be part of a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector can be, inter alia, an expression vector, i.e., a vector that can provide for expression of a B7-H3 binding molecule, EGFR binding molecule, B7-H3 / EGFR binding molecule in vitro and / or in vivo, i.e., in a suitable host cell, host organism, and / or expression system. Such an expression vector typically comprises at least one polynucleotide of the present disclosure operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). Selection of such elements and their sequences for expression in a particular host is a matter of choice to one of skill in the art. Control elements and other elements useful or necessary for expression of a B7-H3 binding molecule, EGFR binding molecule, B7-H3 / EGFR binding molecule of the present disclosure, e.g., promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, reporter genes.

[0341] A polynucleotide of the present disclosure can be prepared or obtained by known means, e.g., by automated DNA synthesis and / or recombinant DNA techniques, based on information of the amino acid sequence of a polypeptide of the present disclosure, and / or can be isolated from a suitable natural source.

[0342] Host Cells

[0343] The present disclosure provides a recombinant host cell that expresses or is capable of expressing one or more B7-H3 binding molecules, EGFR binding molecules, B7-H3 / EGFR binding molecules of the present disclosure and / or contains a nucleic acid or vector of the present disclosure. In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0344] Bacterial cells include, for example, cells of Gram-negative bacterial strains, such as Escherichia coli strains, Proteus strains, and Pseudomonas strains, and Gram-positive bacterial strains, such as Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains.

[0345] Fungal cells, for example, include cells of Trichoderma, Neurospora, and Aspergillus species; or cells of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula species.

[0346] Mammalian cells, for example, include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0347] However, the present disclosure can also use amphibian cells, insect cells, plant cells, and any other cells used in the art for expression of heterologous proteins.

[0348] The cells of the present disclosure are not capable of developing into a complete plant or animal individual.

[0349] Production or manufacturing methods

[0350] The present disclosure provides methods of making a B7-H3 binding molecule, EGFR binding molecule, or B7-H3 / EGFR binding molecule of the present disclosure, which generally comprise the steps of:

[0351] - culturing a host cell of the present disclosure under conditions that allow expression of a binding protein of the present disclosure; and

[0352] - recovering the binding protein expressed by the host cell from the culture; and

[0353] - optionally, including further purification and / or modification of the binding protein of the present disclosure.

[0354] A B7-H3 binding molecule, EGFR binding molecule, or B7-H3 / EGFR binding molecule of the present disclosure can be produced intracellularly (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies) in a cell as described above, then isolated from the host cell and optionally further purified; or it can be produced extracellularly (e.g., in the culture medium of a host cell in culture), then isolated from the culture medium and optionally further purified.

[0355] Methods and reagents for recombinant production of polypeptides, e.g., particular suitable expression vectors, transformation or transfection methods, selectable markers, methods for inducing expression of proteins, culture conditions, etc., are known in the art. Similarly, techniques for isolating and purifying the binding proteins or antibodies of the present disclosure are well known to those of ordinary skill in the art. Methods for producing and purifying antibodies are well known in the art and can be found in, e.g., Current Protocols in Immunology (Chapters 5-8 and 15). The engineered antibodies of the present disclosure can also be produced and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. The expression vectors can be stably transfected into cells. Mammalian expression systems result in glycosylation of the antibodies, particularly in the highly conserved N-terminus of the Fc region. Stable clones expressing antibodies that specifically bind to human-derived antigens are selected. Positive clones are expanded in serum-free media in bioreactors to produce antibodies. The culture fluid in which the antibodies are secreted can be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble aggregates and multimers can also be removed using conventional methods, such as size exclusion, ion exchange. The resulting product is immediately frozen, e.g., at -70°C, or lyophilized.

[0356] However, the B7-H3 binding molecules, EGFR binding molecules, or B7-H3 / EGFR binding molecules of the present disclosure can also be obtained by other methods known in the art for producing proteins, e.g., chemical synthesis, including solid phase or liquid phase synthesis.

[0357] Compositions / pharmaceutical compositions

[0358] The present disclosure provides compositions comprising the foregoing B7-H3 binding molecules, EGFR binding molecules, or B7-H3 / EGFR binding molecules.

[0359] For example, pharmaceutical compositions are provided containing a therapeutically, palliatively, or prophylactically effective amount of a B7-H3 binding molecule, EGFR binding molecule, or B7-H3 / EGFR binding molecule as described above and at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0360] In some embodiments, the pharmaceutical compositions can contain 0.01 to 99% by weight of the B7-H3 binding molecules, EGFR binding molecules, or B7-H3 / EGFR binding molecules in unit dose or the amount of B7-H3 binding molecules, EGFR binding molecules, or B7-H3 / EGFR binding molecules in a unit dose of the pharmaceutical composition is 0.1-2000 mg, in some embodiments, 1-1000 mg.

[0361] In some embodiments, articles of manufacture or kits are provided that contain at least one container, each independently comprising a foregoing B7-H3 binding molecule, EGFR binding molecule, or B7-H3 / EGFR binding molecule. Optionally, the kits comprise a container and a label. The container, for example, a bottle, a syringe, and a test tube. The container holds a composition that is effective for treating a condition. The label is on or associated with the container. The label indicates that the composition is used for treating the selected condition. The composition comprises a foregoing B7-H3 binding molecule, EGFR binding molecule, or B7-H3 / EGFR binding molecule.

[0362] Methods of treating disease and pharmaceutical uses

[0363] The present disclosure provides methods of using a foregoing B7-H3 binding molecule, EGFR binding molecule, B7-H3 / EGFR binding molecule, polynucleotide, composition (including pharmaceutical composition) for treating, ameliorating, preventing, diagnosing a disease or condition.

[0364] In some embodiments, methods of ameliorating, alleviating, treating, or preventing a disease are provided, comprising administering to a subject an ameliorating, alleviating, treating, or preventing effective amount of a foregoing B7-H3 binding molecule, EGFR binding molecule, B7-H3 / EGFR binding molecule, polynucleotide, or composition (including pharmaceutical composition).

[0365] In some embodiments, uses of a B7-H3 binding molecule, EGFR binding molecule, or B7-H3 / EGFR binding molecule of the present disclosure in the manufacture of a medicament for ameliorating, alleviating, treating, or preventing a disease are provided.

[0366] In some embodiments, the foregoing disease is a cancer.

[0367] In some embodiments, the cancer is B7-H3 and EGFR double positive; for example, the proportion of double positive co-expression is greater than 10%, or greater than 15% (e.g., % obtained from the method of Example 17 for detecting EGFR protein expression and B7-H3 protein expression).

[0368] In some embodiments, the cancer is head and neck cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer.

[0369] In some embodiments, methods are provided as shown below, comprising administering to a subject in need thereof an effective amount of a foregoing B7-H3 / EGFR binding molecule, polynucleotide, or composition (including pharmaceutical composition):

[0370] (1) a method of selectively binding to B7-H3 and EGFR double positive cells;

[0371] For example, the ratio of the double-positive cell population to the single-positive cell population in the antibody-positive cell population in Example 6 is more than 2-fold, more than 10-fold, more than 20-fold, or more than 100-fold; for example, the detection method shown in Example 6;

[0372] (2) selectively blocking the binding of EGF to B7-H3 and EGFR double-positive cells;

[0373] The B7-H3 / EGFR binding molecule has a blocking ability on the double-positive cells that is superior to the blocking ability on the single-positive cells, for example, IC 50 fold, more than 4-fold, or more than 10-fold; for example, the detection method shown in Example 7;

[0374] (3) selectively inhibiting the phosphorylation of EGFR receptors in B7-H3 and EGFR double-positive cells; for example, the detection method shown in Example 8;

[0375] (4) selectively killing B7-H3 and EGFR double-positive cells; for example, the detection method shown in Example 10 or the detection method shown in Example 14;

[0376] (5) selectively inhibiting the proliferation of B7-H3 and EGFR double-positive cells.

[0377] Detection and kits

[0378] The present disclosure provides detection uses of B7-H3 binding molecules, EGFR binding molecules, B7-H3 / EGFR binding molecules, polynucleotides, compositions. The present disclosure also provides methods, systems, or devices for detecting B7-H3 and / or EGFR in vivo or in vitro, which comprise treating a sample with the aforementioned binding proteins, polynucleotides, compositions of the present disclosure.

[0379] In some embodiments, the in vitro detection method, system, or device may, for example, comprise:

[0380] (1) contacting the sample with a B7-H3 binding molecule, an EGFR binding molecule, a B7-H3 / EGFR binding molecule, a composition;

[0381] (2) detecting the complex formed between the aforementioned binding protein and the sample; and / or

[0382] (3) contacting a reference sample (e.g., a control sample) with the binding protein; and

[0383] (4) determining the degree of complex formation by comparison with the reference sample. Changes in complex formation (e.g., statistically significant changes) in the sample or subject as compared to the control sample or subject in the subject indicate the presence of B7-H3 and / or EGFR in the sample.

[0384] In other embodiments, an in vivo detection method, system or device can comprise:

[0385] (1) administering to the subject a binding protein, polynucleotide described above; and

[0386] (2) detecting the formation of a complex between the binding protein described above and the subject.

[0387] The detection can comprise determining the location or time of complex formation. The binding protein described above can be labeled with a detectable substance, and detection of the label can be used to detect the substance (e.g., B7-H3 and / or EGFR) that can bind to the protein. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. Complex formation of the binding protein with B7-H3 and / or EGFR can be detected by measuring or visualizing the substance bound to or unbound from B7-H3 and / or EGFR. Conventional detection assays can be used, e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or immunohistochemistry of tissue. For detection purposes, the binding proteins, polynucleotides of the disclosure can be labeled with a fluorophore chromophore. In some embodiments, diagnostic reagents comprising the binding proteins described above are also provided, as are related diagnostic uses.

[0388] In some embodiments, kits comprising the binding proteins, polynucleotides described above are also provided, and can further comprise instructions for diagnostic use. The kits can also contain at least one additional reagent, such as a label or additional diagnostic agent. For in vivo use, the binding proteins can be formulated as a pharmaceutical composition. BRIEF DESCRIPTION OF DRAWINGS

[0389] FIG. 1A is the EGFR, B7-H3 expression level on the surface of human tumor cells evaluated by the number of antibody binding. FIG. 1B to FIG. 1E are the surface EGFR and B7-H3 expression of NCI-H1975, SK-MES-1, FaDu, NCI-H1568 parental tumor cell lines and corresponding B7-H3 knockout cells, respectively, detected by fluorescence value on flow cytometry using PE or APC fluorescently labeled antibodies.

[0390] FIG. 2A to FIG. 2E are the selective binding of B01, B02, B03 bispecific antibodies to SK-MES-1 cells that express both human EGFR and B7-H3. Cetuximab and IgG1 isotype as controls.

[0391] FIG. 3A-B show the competitive blocking effect of Cetuximab and IgGl isotype control on EGF binding to EGFR in double positive cell H1975.wt and B7-H3 knock out EGFR single positive cell H1975.bko, and in double positive cell FaDu.wt and B7-H3 knock out EGFR single positive cell FaDu.bko. No EGF treatment is the control without EGF.

[0392] FIG. 4A-L show the selectivity of bispecific antibodies to block EGF binding to EGFR in NCI-H1975 and FaDu cells expressing both human EGFR and B7-H3, and B7-H3 knock out human tumor cell lines H1975.bko and FaDu.bko are EGFR single positive control cells. FIG. 4A-F show the competitive blocking effect of B01, B02, B03, B04, B05, B06 on EGF binding to EGFR in double positive cell H1975.wt and single positive cell H1975.bko. FIG. 4G-L show the competitive blocking effect of B01, B02, B03, B04, B05, B06 on EGF binding to EGFR in double positive cell FaDu.wt and single positive cell FaDu.bko.

[0393] FIG. 5A-B show the selectivity of bispecific antibodies to block EGFR phosphorylation in SK-MES-1 cells, using EGFR and B7-H3 double positive SK-MES-1.wt and B7-H3 knock out EGFR single positive SK-MES-1 cell lines as comparison. Cells were incubated with antibodies and then stimulated with EGF, and protein and phosphorylation levels were detected by western blot. FIG. 5A shows the EGFR phosphorylation results in double positive SK-MES-1.wt cells, and FIG. 5B shows the EGFR phosphorylation results in single positive SK-MES-1.bko cells.

[0394] FIG. 6 shows the ADCC killing results of four mAbs Zmab, Zmab.DE, Zmab.scFv.DE and IgGl isotype control on NCI-H1975 cell line. IgGl isotype is wild type IgGl and serves as negative control.

[0395] Figures 7A-7H are the ADCC killing of NCI-H1975 cells co-expressing human EGFR and B7-H3 by PBMC selectively induced by bispecific antibodies. B7-H3 knock-out human tumor cell line NCI-H1975.bko is EGFR single positive control cell. Figures 7A-7H show the ADCC killing effect of B01, B02, B03, B04, B05, B06 and Cetuximab and IgGl isotype control in double positive cell H1975.wt and single positive cell H1975.bko, respectively.

[0396] Figures 8A-8H are the ADCC killing of FaDu cells co-expressing human EGFR and B7-H3 by PBMC selectively induced by bispecific antibodies. B7-H3 knock-out human tumor cell line FaDu.bko is EGFR single positive control cell. Figures 8A-8H show the ADCC killing effect of B01, B02, B03, B04, B05, B06 and Cetuximab and IgGl isotype control in double positive cell FaDu.wt and single positive cell FaDu.bko, respectively.

[0397] Figure 9A is the selective efficacy results of anti-EGFR / B7-H3 bispecific antibodies B02, B04, B05, B06 in mice subcutaneous xenograft model of double positive EGFR and B7-H3 FaDu.wt cells, single positive EGFR FaDu.bko cells, respectively.

[0398] Figure 9B is the corresponding mouse body weight graph.

[0399] Figures 10A-10E are the cell killing activity of anti-EGFR / B7-H3 bispecific antibody drug conjugates ADC-1, ADC-2, ADC-3, ADC-4, ADC-5 in SK-MES-1.wt double positive cells and SK-MES-1.bko single positive cells, respectively.

[0400] Figures 11A-11B are the selective killing of double positive cells expressing human EGFR and B7-H3 by anti-EGFR / B7-H3 bispecific antibody drug conjugate ADC-7. Figure 11A is the wild type (wt) and EGFR single positive (bko) of NCI-H1568, Figure 11B is the wild type (wt) and EGFR single positive (bko) of FaDu, which are obtained by knocking out B7-H3 from the above cells to get EGFR single positive cell line (bko). ADC-8-1 is the control.

[0401] Figure 12 is the result of killing activity of anti-EGFR / B7-H3 bispecific antibody drug conjugate ADC-7 on antigen-negative tumor cell NCI-H69. IgG1 isotype control ADC (IgG1-9) was used as negative control, ADC-8-1 and DS-7300 as single-targeting ADC controls.

[0402] Figures 13A-13F are the results of immunohistochemical staining to detect EGFR, B7-H3 expression in head and neck cancer, non-small cell lung cancer, and esophageal cancer tumor chip samples. Figure 13A: EGFR immunohistochemical staining of head and neck cancer tumor chip. Figure 13B: B7-H3 immunohistochemical staining of head and neck cancer tumor chip. Figure 13C: EGFR immunohistochemical staining of non-small cell lung cancer tumor chip. Figure 13D: B7-H3 immunohistochemical staining of non-small cell lung cancer tumor chip. Figure 13E: EGFR immunohistochemical staining of esophageal cancer tumor chip. Figure 13F: B7-H3 immunohistochemical staining of esophageal cancer tumor chip. DETAILED DESCRIPTION

[0403] TERMINOLOGY

[0404] To facilitate a better understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless specifically defined in the present disclosure, all other technical and scientific terms used in the present disclosure have the meanings commonly understood by one of ordinary skill in the art in the field of the present disclosure.

[0405] “EGFR” and “HER1” are used interchangeably and include variants, isoforms, species homologs of human EGFR, and analogs having at least one common epitope with EGFR.

[0406] “B7-H3” and “CD276” are used interchangeably and include variants, isoforms, species homologs of human B7-H3, and analogs having at least one common epitope with B7-H3.

[0407] “Binding to EGFR” or “binding to B7-H3” means the ability to interact with an antigen (EGFR or B7-H3) or an epitope thereof, which can be of human origin.

[0408] “Binding molecule” encompasses any molecule capable of specifically binding to an antigen or an epitope thereof, including but not limited to an antibody or conjugate thereof as defined in the present disclosure. The “binding molecule” of the present disclosure can comprise a heavy chain variable region (VH) and a light chain variable region (VL), such as Fab, Fab’, F(ab’)2, single chain antibody (scFv), or IgG antibody.

[0409] "Antibody" encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antibody fragments (or antigen binding fragments, or antigen binding portions), so long as they exhibit the desired antigen-binding activity. An antibody can refer to an immunoglobulin, which is a tetrameric molecule consisting of two heavy chains and two light chains connected by disulfide bonds. The heavy chains of an immunoglobulin have the same amino acid composition and order, but the antigenic properties of the immunoglobulin are different. Accordingly, the immunoglobulin can be classified into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, γ, α, and ε, respectively. The same class of Ig can be further divided into subclasses, e.g., IgG can be divided into IgG1, IgG2, IgG3, and IgG4, based on the differences in the amino acid composition and the number and location of disulfide bonds in the hinge region of the heavy chain. The light chains are classified into κ or λ chains by the constant region.

[0410] The sequences of the heavy and light chains of an antibody near the N-terminus are highly variable, forming the variable region (V region), while the sequences further from the N-terminus are relatively stable, forming the constant region (C region). The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity-determining region (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of three CDR regions and four FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0411] "Antigen binding fragment" encompasses single chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope binding fragments of any of the above (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3) 209-217).

[0412] Methods of producing and making these antigen binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Fab-Fv formats were first disclosed in WO2009 / 040562, and the disulfide stabilized version Fab-dsFv was first disclosed in WO2010 / 035012. The antigen binding fragments of the present disclosure also include the Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170 and WO2005 / 003171. Multivalent antibodies can comprise multispecificity, e.g. bispecificity, or can be monospecific (see, for example, WO92 / 22583 and WO05 / 113605), one example of the latter being the Tri-Fab (or TFM) described in WO 92 / 22583.

[0413] For the determination or definition of CDRs, a definitive delineation of CDRs and identification of residues of the binding site can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be achieved by any of a variety of techniques known to those of skill in the art, e.g., X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definition, conformational definition.

[0414] The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the location of certain structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83). The AbM numbering system uses a suite of computer programs produced by Oxford Molecular Group that model antibody structures (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies", Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from the primary sequence (see Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on analysis of complex crystal structures available (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In conformational definitions, the location of CDRs can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). Still other CDR boundary definitions can not strictly follow one of the above methods, but still overlap at least a portion of the Kabat CDRs. The boundaries of the CDRs can be shortened or lengthened in light of

[0415] Typically, "specific binding" refers to the binding of a protein to an epitope on an antigen. The B7-H3 binding molecule, EGFR binding molecule, and B7-H3 / EGFR binding molecule disclosed herein were measured to be 10 in Biacore, KinExA, or Fortibio assays. -7 Up to 10 -10 moles per liter (M), or 10 -8 Up to 10 -10 M, or 10 -9 Up to 10 -10 M, or a lower dissociation constant (K) D It binds to the antigen to be bound (i.e., B7-H3 or EGFR) or its epitopes. Any antigen greater than 10... -4 M of K D Values ​​are generally considered to indicate nonspecific binding. Specific binding of a binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays as described in this disclosure.

[0416] "Conservative substitution" refers to the substitution with another amino acid residue that has properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. Furthermore, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Additionally, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when amino acid residues in the group exhibiting similar properties as described above are substituted, it will not show a specific change in properties.

[0417] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same nucleotide or amino acid monomer—for example, if every position in two DNA molecules is occupied by the same nucleotide—then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of homology is obtained by aligning the two sequences.

[0418] "Nucleic acid" or "polynucleotide" are used interchangeably herein to refer to a molecule of DNA or RNA, either single- or double-stranded, and in the case of single-stranded, its complementary sequence, e.g., double-stranded DNA. A nucleic acid is "operably linked" when it is functionally related to another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0419] A "host cell" includes an individual cell or cell culture that can be or has been a recipient for polynucleotide insert(s). Host cells include progeny of the original recipient cell, and the progeny can not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected and / or transformed in vivo with a polynucleotide of the present disclosure. "Cell," "cell line," and "cell culture" can be used interchangeably and all such designations include descendants of the original cell strain. It is also understood that all descendants can not be identical in DNA content to the original parent cell due to deliberate or inadvertent mutation.

[0420] The term "internalization" refers to the transport of a moiety from the exterior of a cell to the interior. An internalized moiety can be located in an intracellular compartment. An "internalized" or "internalizing" antigen or antibody refers to an antigen or antibody that is capable of being transported from the exterior to the interior of a target cell. It is generally understood by those skilled in the art that the process of internalization by a cell generally refers to the transmembrane movement of a cell surface molecule from the cell surface to the interior of the cell. Following internalization, the endosome can be transported to a lysosome for degradation or recycled to the cell surface. The rate of internalization of a given cell surface molecule provides a measure of the kinetics of movement of that molecule across the plasma membrane from the cell surface to the interior of the cell. The internalization activity or rate of internalization of an antigen and antibody can be monitored and / or measured by a variety of techniques known in the art, including acid dissociation (Li N. et al., Methods Mol. Biol., 457:305-17, 2008) and toxin killing assays (Pahara J. et al. Exp Cell Res., 316:2237-50, 2010; and Mazor et al., J. Immunol. Methods, 321:41-59, 2007). A number of antibody labeling techniques, dyes, and kits for antibody labeling useful for quantifying and monitoring internalization are commercially available (e.g., pHrodo iFL antibody labeling method, reagents, and kits sold by Thermo Fisher Scientific).

[0421] The term "antibody drug conjugate" "antibody drug conjugate" (ADC) refers to a conjugate of an antibody with a drug molecule having a target effector activity. Among them, the antibody can be coupled with the drug directly or through a linking unit.

[0422] The drug molecule is not particularly limited as long as it has a substituent or a partial structure that can be linked to the linker structure. Depending on the conjugated drug, the antibody drug conjugate can be used for various purposes. Examples of such drugs can include substances having anti-tumor activity, substances effective against blood diseases, substances effective against autoimmune diseases, anti-inflammatory substances, antimicrobial substances, antifungal substances, antiparasitic substances, antiviral substances, and antinarcotic substances.

[0423] The term "drug loading" refers to the average number of cytotoxic drugs loaded on each ligand in the ADC, which can also be expressed as the ratio of the amount of drug to the amount of antibody, and the drug loading can range from 1 to 20, preferably 1 to 10, cytotoxic drugs (D) per antibody (Ab). In embodiments of the present disclosure, the drug loading is denoted as n, and exemplary values are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the average value of values between any two values. The number of drug molecules per ADC molecule after the conjugation reaction can be identified by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, monoclonal antibody size variant assay (CE-SDS), and HPLC profiling.

[0424] While the drug to antibody ratio has an exact value for a particular conjugate molecule (e.g., n in Formula (I) or (IV)), it will be understood that when used to describe a sample containing many molecules, the value will often be an average due to some degree of heterogeneity typically associated with the conjugation step. The average loading of an immunoconjugate sample is referred to herein as the drug to antibody ratio or “DAR”. In some embodiments, the DAR is between about 1 and about 10, e.g., 1-8, and typically about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0. Embodiments include immunoconjugates wherein the DAR is about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.4, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0. In some embodiments, a DAR of ‘about x’ means that the measured value of the DAR is within 20% of x (e.g., within 20%, 15%, 10%, 50% of x).

[0425] Methods for detecting DAR, e.g., extrapolating DAR values from LC-MS data of reduced and deglycosylated samples. LC / MS allows quantification of the average number of payload (drug moiety) molecules attached to the antibody in an ADC. HPLC separates the antibody into light and heavy chains, and also separates the heavy (HC) and light (LC) chains according to the number of linker-payload groups per chain. Mass spectrometry data enables identification of component species in the mixture, e.g., LC, LC+1, LC+2, HC, HC+1, HC+2, etc. From the average loading of the LC and HC chains, the average DAR of the ADC can be calculated. The DAR of a given immunoconjugate sample represents the average number of drug (payload) molecules attached to a tetrameric antibody containing two light chains and two heavy chains. Methods for detecting DAR, e.g., in WO2018142322.

[0426] The term "camptothecin drug" refers to camptothecin and its derivatives having cytotoxic properties, non-limitingly selected from 10-hydroxy camptothecin, 7-ethyl-10- hydroxy camptothecin, topotecan, exatecan, irinotecan or 9-nitro-10-hydroxy camptothecin and its derivatives or pharmaceutically acceptable salts thereof.

[0427] The term "alkyl" refers to saturated aliphatic hydrocarbon groups which are straight-chain or branched-chain groups containing 1 to 20 carbon atoms, preferably alkyl groups containing 1 to 12 carbon atoms, more preferably alkyl groups containing 1 to 10 carbon atoms, most preferably alkyl groups containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched isomers thereof, and the like. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available point of attachment, preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxo.

[0428] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein alkyl is as defined above.

[0429] The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon radical having 2 residues derived by removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, which is a straight chain or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, more preferably containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylidene (-CH(CH3)-), 1,2-ethylidene (-CH2CH2)-, 1,1-propylidene (-CH(CH2CH3)-), 1,2-propylidene (-CH2CH(CH3)-), 1,3-propylidene (-CH2CH2CH2-), 1,4-butylidene (-CH2CH2CH2CH2-), and 1,5-butylidene (-CH2CH2CH2CH2CH2-), and the like. The alkylene group can be substituted or unsubstituted, when substituted, the substituents can be substituted at any available point of attachment, which are preferably substituted with one or more substituents independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocyclyloxy, cycloalkylthio, heterocyclylthio, and oxo.

[0430] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl are as defined above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. The alkoxy group can be optionally substituted or unsubstituted, when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocyclyloxy, cycloalkylthio, heterocyclylthio.

[0431] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring comprising 3 to 20 carbon atoms, preferably comprising 3 to 12 carbon atoms, more preferably comprising 3 to 10 carbon atoms, most preferably comprising 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spirocyclic, fused, and bridged cycloalkyl groups.

[0432] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, comprising 3 to 20 ring atoms, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O) mring members are carbon. Preferably, the heteroatom containing ring has 3 to 12 ring members, of which 1 to 4 are heteroatoms; more preferably, the cycloheteroalkyl ring has 3 to 10 ring members. Non-limiting examples of monocyclic cycloheteroalkyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic cycloheteroalkyl groups include spiro, fused, and bridged cycloheteroalkyl groups.

[0433] The term "spirocycloheteroalkyl" refers to a polycyclic cycloheteroalkyl group of 5 to 20 members sharing one atom (referred to as a spiro atom) between single rings, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), with the remaining ring atoms being carbon. It can contain one or more double bonds, but no ring has a fully conjugated pi-electron system. Preferably, it is 6 to 14 membered, more preferably 7 to 10 membered. Spirocycloheteroalkyl groups are classified as mono-, bi-, or polycyclic, preferably mono- and bicyclic, based on the number of rings sharing a spiro atom. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monocyclic spirocycloheteroalkyl group. Non-limiting examples of spirocycloheteroalkyl groups include: m The term "spirocycloheteroalkyl" refers to a polycyclic cycloheteroalkyl group of 5 to 20 members sharing one atom (referred to as a spiro atom) between single rings, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), with the remaining ring atoms being carbon. It can contain one or more double bonds, but no ring has a fully conjugated pi-electron system. Preferably, it is 6 to 14 membered, more preferably 7 to 10 membered. Spirocycloheteroalkyl groups are classified as mono-, bi-, or polycyclic, preferably mono- and bicyclic, based on the number of rings sharing a spiro atom. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monocyclic spirocycloheteroalkyl group. Non-limiting examples of spirocycloheteroalkyl groups include:

[0434] The term "fused cycloheteroalkyl" refers to a polycyclic cycloheteroalkyl group of 5 to 20 members, each ring in the system sharing an adjacent pair of atoms with other rings in the system, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), with the remaining ring atoms being carbon. It can contain one or more double bonds, but no ring has a fully conjugated pi-electron system. Preferably, it is 6 to 14 membered, more preferably 7 to 10 membered. Fused cycloheteroalkyl groups are classified as bicyclic, tricyclic, tetracyclic, or polycyclic, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused cycloheteroalkyl groups. Non-limiting examples of fused cycloheteroalkyl groups include: m The term "fused cycloheteroalkyl" refers to a polycyclic cycloheteroalkyl group of 5 to 20 members, each ring in the system sharing an adjacent pair of atoms with other rings in the system, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), with the remaining ring atoms being carbon. It can contain one or more double bonds, but no ring has a fully conjugated pi-electron system. Preferably, it is 6 to 14 membered, more preferably 7 to 10 membered. Fused cycloheteroalkyl groups are classified as bicyclic, tricyclic, tetracyclic, or polycyclic, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused cycloheteroalkyl groups. Non-limiting examples of fused cycloheteroalkyl groups include:

[0435] The term "bridged cycloheteroalkyl" refers to a polycyclic cycloheteroalkyl group of 5 to 14 members, any two rings sharing two non-adjacent atoms, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), with the remaining ring atoms being carbon. It can contain one or more double bonds, but no ring has a fully conjugated pi-electron system. Preferably, it is 6 to 14 membered, more preferably 7 to 10 membered. Bridged cycloheteroalkyl groups are classified as bicyclic, tricyclic, tetracyclic, or polycyclic, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloheteroalkyl groups include: m The term "bridged cycloheteroalkyl" refers to a polycyclic cycloheteroalkyl group of 5 to 14 members, any two rings sharing two non-adjacent atoms, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O)m (where m is an integer from 0 to 2), with the remaining ring atoms being carbon. It can contain one or more double bonds, but no ring has a fully conjugated pi-electron system. Preferably, it is 6 to 14 membered, more preferably 7 to 10 membered. Bridged cycloheteroalkyl groups are classified as bicyclic, tricyclic, tetracyclic, or polycyclic, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloheteroalkyl groups include:

[0436] The heterocyclyl ring can be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring that is attached to the parent structure is a heterocyclyl ring, non-limiting examples of which include:

[0437] and the like.

[0438] The heterocyclyl group can be optionally substituted or unsubstituted, and when substituted, the substituent groups are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.

[0439] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (that is, rings which share adjacent pairs of carbon atoms) ring having a conjugated pi-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, preferably phenyl. The aryl ring can be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring that is attached to the parent structure is an aryl ring, non-limiting examples of which include:

[0440] The aryl group can be substituted or unsubstituted, and when substituted, the substituent groups are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0441] The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5- to 10-membered, more preferably 5- or 6-membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring can be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring that is attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:

[0442] The heteroaryl group can be optionally substituted or unsubstituted, and when substituted, the substituent groups are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0443] The term "amino protecting group" is used to protect an amino group from reactions occurring at other sites of the molecule, with the amino group remaining intact. Non-limiting examples include 9-fluorenylmethyloxycarbonyl, t-butyloxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl, among others. These groups can optionally be substituted with 1-3 substituents selected from halo, alkoxy, or nitro. Preferably, the amino protecting group is 9-fluorenylmethyloxycarbonyl.

[0444] The term "cycloalkylalkyl" refers to an alkyl group substituted with one or more cycloalkyl groups, preferably one cycloalkyl group, wherein alkyl is as defined above, and wherein cycloalkyl is as defined above.

[0445] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0446] The term "deuteroalkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

[0447] The term "hydroxy" refers to an -OH group.

[0448] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0449] The term "amino" refers to -NH2.

[0450] The term "nitro" refers to -NO2.

[0451] The term "amide" refers to -C(O)N(alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.

[0452] The term "carboxylate" refers to -C(O)O(alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.

[0453] The present disclosure also includes various deuterated forms of the compounds of Formula (IV). Each available hydrogen atom attached to a carbon atom can be independently replaced with a deuterium atom. One skilled in the art would be able to synthesize deuterated forms of the compounds of Formula (IV) by reference to the relevant literature. Commercially available deuterated starting materials can be used in the preparation of deuterated forms of the compounds of Formula (IV), or they can be synthesized using conventional techniques employing deuterated reagents, including but not limited to deuterated borane, trideuteroborane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide, and deuterated methyl iodide, among others.

[0454] "Substituted" means that one or more hydrogen atoms, preferably up to five, more preferably one to three, of the group are independently of each other replaced with the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, which can be determined (experimentally or theoretically) by the person skilled in the art without undue effort, as possible or impossible. For example, an amino group with a free hydrogen or a hydroxyl group can be unstable when bound to a carbon atom with an unsaturated (e.g. olefinic) bond.

[0455] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blockade. "Inhibit growth" (e.g. in reference to a cell) is intended to include any measurable decrease in cell growth.

[0456] "Pro liferative disorder" refers to a condition associated with some degree of abnormal cell proliferation. In one embodiment, a proliferative disorder refers to cancer. "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer," "cancerous," "proliferative disorder," and "tumor" are not mutually exclusive when referred to in the present disclosure.

[0457] "Administer," "administering" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with the animal, human, subject, cell, tissue, organ or biological fluid, e.g. therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Treatment of a cell includes contact of the agent with the cell, as well as contact of the agent with a fluid which is in contact with the cell. "Administer," "administering" and "treatment" also mean treatment by an agent, diagnostic, binding composition or by another cell in vitro and ex vivo, e.g. a cell. When applied to a human, veterinary or research subject, it refers to therapeutic treatment, prophylaxis or preventative measures, research and diagnostic applications.

[0458] "Treatment" means administering an oral or topical therapeutic agent, such as a pharmaceutical composition comprising any of the binding proteins of this disclosure or thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases, and for whom the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in a treated subject or population in an amount that effectively relieves symptoms of one or more diseases, whether by inducing regression of such symptoms or inhibiting their progression to any clinically measurable degree. The amount of therapeutic agent that effectively relieves symptoms of any specific disease (also referred to as the "therapeuticly effective amount") can vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease in a particular subject, they should reduce the symptoms of the target disease in a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0459] "Effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount used on a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. Effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity. Subjects in this disclosure may be animal or human subjects.

[0460] "Optional" or "optionally" means that the event or circumstance described below may, but does not necessarily, occur, and the description includes the possibility that the event or circumstance may or may not occur. "And / or" should be interpreted as specifically disclosing that each of the two specified features or components has or does not have the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" in this disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc., should be understood to have an inclusive meaning rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to."

[0461] In this disclosure, "subject" and "patient" refer to mammals, especially primates, and particularly humans.

[0462] The ordinal numbers "first", "second", "third", "1", "2", "3", and the like (such as "first subunit", "Fc2", "third chain", "linker 2") in the present disclosure are only used to distinguish different features, elements, components or steps, and are not intended to limit the number, order, level.

[0463] Examples

[0464] The following examples are used to further describe the present disclosure, but these examples do not limit the scope of the present disclosure.

[0465] The experimental methods in the embodiments or test examples of the present disclosure, which are not specified with specific conditions, are generally according to the conventional conditions, or according to the conditions suggested by the manufacturers of raw materials or commodities. See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, Ausubel et al., Greene Publishing Associates, Inc., Wiley Interscience, NY. The reagents, which are not specified with specific sources, are the conventional reagents purchased on the market.

[0466] Example 1. Affinity maturation of anti-B7-H3 antibodies based on phage display technology

[0467] Example 1-1. Screening with antigen and parent anti-B7-H3 antibody sequence

[0468] Table 1-1. Screening with B7-H3 antigen information

[0469] The sequences of human B7-H3 4Ig protein and human B7-H3 2Ig protein are as follows (SEQ ID NO: 1 and SEQ ID NO: 2).

[0470] > Human B7-H3 4Ig amino acid sequence

[0471] > Human B7-H3 2Ig amino acid sequence

[0472] Example 1-2. Construction and screening of affinity maturation mutation library

[0473] The VH and VL sequences of B7-H3 antibody (huA3) are as follows:

[0474] The huA3 sequence is analyzed and homologous modeling is performed, based on structural analysis, amino acid mutation sites are designed for 6 CDR regions of heavy chain and light chain and key framework regions, and degenerate base primers containing different amino acid mutation sites are synthesized. The fragment amplification and splicing are completed by PCR method, the enzyme digestion sites are introduced, and then the phage display vector is connected. The connection product is transformed into E. coli TG1 competent cells (Lucigen) by electroporation. A small amount of bacterial liquid is diluted and plated, and the plate colonies are sequenced to verify the library insertion rate and diversity. The remaining bacterial liquid is prepared into a recombinant phage library for screening according to the conventional method.

[0475] Liquid phase screening is performed by using streptavidin magnetic beads, biotin-labeled human B7-H3 4Ig or B7-H3 2Ig antigen is incubated and combined with magnetic beads, and the recombinant phage library is added for liquid phase screening. The eluted phage after screening is used to infect fresh activated TG1 bacterial liquid, and after plating, single colonies are picked for activity identification, sequencing and sequence analysis, and after periplasmic cavity expression calibration, 96 groups of antibody sequences with strong binding activity to human B7-H3 4Ig or B7-H3 2Ig antigen are obtained.

[0476] The corresponding CDR and framework region sequences of all obtained light and heavy chain mutation clones containing mutation sites are summarized, and new degenerate primers are designed. The fragment amplification and splicing are completed by PCR method, the enzyme digestion sites are introduced, and then the phage display vector is connected, and the construction of the combined library and the preparation of the recombinant phage library for screening are completed.

[0477] Liquid phase screening is performed by using streptavidin magnetic beads, biotin-labeled human B7-H3 4Ig or B7-H3 2Ig antigen is incubated and combined with magnetic beads, and the recombinant phage library is added for liquid phase screening. The eluted phage after screening is used to infect fresh activated TG1 bacterial liquid, and after plating, single colonies are picked for activity identification, sequencing and sequence analysis, and after periplasmic cavity expression calibration, 96 groups of antibody sequences with strong binding activity to human B7-H3 4Ig or B7-H3 2Ig antigen are obtained.

[0478] Examples 1-3. Construction and screening of affinity maturation mutation library

[0479] F2-A4, F2-F6, F3-D3, F5-B9, 15-C8, 16-B4, 17-D2, 18-A10 and 18-E4 with high affinity are selected. The variable region sequences are as follows:

[0480] The CDR sequences of the Kabat numbering system are as follows:

[0481] Table 1-2

[0482] Antibodies F2-A4, F2-F6, F3-D3, F5-B9, 15-C8, 16-B4, 17-D2, 18-A10, 18-E4 and huA3 sequences were linked to human IgGl heavy chain constant region (CH1 and IgGl Fc) and human kappa light chain constant region, respectively, and expressed and purified by routine recombinant protein techniques after construction of full-length sequences.

[0483] >Human kappa light chain constant region

[0484] >CH1

[0485] >IgGl Fc

[0486] >F2-A4 LC

[0487] >F2-A4 HC

[0488] >F2-F6 LC

[0489] >F2-F6 HC

[0490] >F3-D3 LC

[0491] >F3-D3 HC

[0492] >F5-B9 LC

[0493] >F5-B9 HC

[0494] >15-C8 LC

[0495] >15-C8 HC

[0496] >16-B4 LC

[0497] >16-B4 HC

[0498] >17-D2 LC

[0499] >17-D2 HC

[0500] >18-A10 LC

[0501] >18-A10 HC

[0502] >18-E4 LC

[0503] >18-E4 HC

[0504] >huA3 LC

[0505] >huA3 HC

[0506] Examples 1-4. Affinity Assay

[0507] The affinity of antibodies to antigen B7-H3 2lg or B7-H3 4lg was determined using the surface plasmon resonance (SPR) method. Briefly, using a commercial kit (GE Human Antibody Capture Kit, type 2 (29234600, GE)), a standard amine coupling method was used to covalently attach an anti-human IgG (Fc) polyclonal antibody to a CM5 (GE) chip, which was then used to capture the various purified antibodies to be tested to the stationary phase. Different concentration gradients of B7-H3 2lg or B7-H3 4lg protein diluted in the same buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, pH 7.4) were injected before and after each cycle, and after injection, the regeneration reagent (3 M MgCl2) provided in the kit was used for regeneration. The antigen-antibody binding kinetics were tracked for 2 minutes and the dissociation kinetics were tracked for 4-5 minutes. The resulting data were analyzed using the BIAevaluation software from GE using a 1:1 (Langmuir) binding model. The binding rate ka, dissociation rate kd, and affinity KD values determined in this way.

[0508] The results are shown in Table 2-1. The affinity of the obtained anti-B7-H3 antibodies to the antigen is better than that of huA3. Through sequence alignment analysis, the mutation of amino acid site 92 and / or 93 (numbered based on Kabat rules) of the light chain variable region CDR3 to Ala and Arg, respectively, is crucial for the enhancement of affinity. The affinities of F2-F6 (S92A mutation), F5-B9 (S92G, S93R mutation), and F2-A4 (S92A, S93R mutation) are about 10 times, about 15 times, and about 27 times, respectively, higher than that of the parent huA3. The high-affinity enriched sequences screened from the combinatorial library all contain S92A, S93R mutations (for example: 15-C8, 16-B4, 17-D2, 18-A10, and 18-E4)

[0509] Table 2-1. Affinity of antibodies after two rounds of affinity maturation

[0510] Example 2. Point mutation affinity improvement of anti-EGFR antibodies

[0511] Example 2-1. Screening with antigen

[0512] The screened antigen is an EGFR protein (ACRO Biosystems, EGR-H5222). The full-length sequence of the human EGFR protein is as follows (SEQ ID NO: 68).

[0513] > Human EGFR amino acid sequence

[0514] Example 2-2. Anti-EGFR antibody improvement

[0515] The antibody Zalutumumab was mutated and improved, with a target affinity range of 1E -09 to 5E -07 M, the selected amino acid is the side chain of the amino acid in the CDR region of the VH chain (based on Kabat rules) and the amino acid in the range thereof, which is mutated to an amino acid with similar or different physicochemical properties, and the potential PTM site of the light and heavy chain CDR region is mutated, in order to improve the drugability of the antibody.

[0516] Among them, Zalutumumab, Zmab.v1, Zmab.v27, Zmab.v18, Zmab.v19, Zmab.v6, the light chain variable region sequence and the heavy chain variable region sequence are as follows.

[0517] > Zalutumumab VH

[0518] > Zalutumumab VL ​

[0519] Zmab.vl VH

[0520] Zmab.vl VL is identical to Zalutumumab VL

[0521] Zmab.v6 VH

[0522] Zmab.v6 VL is identical to Zalutumumab VL

[0523] Zmab.vl 8 VH

[0524] Zmab.vl 8 VL is identical to Zalutumumab VL

[0525] Zmab.vl 9 VH

[0526] Zmab.vl 9 VL is identical to Zalutumumab VL

[0527] Zmab.v27 VH

[0528] Zmab.v27 VL is identical to Zalutumumab VL.

[0529] CDR sequences in the Kabat numbering system are as follows:

[0530] Table 2-2

[0531] The obtained heavy chain variable region and light chain variable region are connected with human IgGl heavy chain constant region and human kappa light chain constant region respectively, and after constructing full-length sequence, the antibody is expressed by conventional recombinant protein technology.

[0532] Zalutumumab HC

[0533] Zalutumumab LC

[0534] Zmab.vl HC

[0535] Zmab.vl LC is identical to Zalutumumab LC

[0536] Zmab.v27 HC

[0537] Zmab.v27 LC is identical to Zalutumumab LC.

[0538] Example 2-3. Affinity determination

[0539] Antibody antigen affinity was determined using a bio-layer interferometry (BLI) method using cell culture supernatant expressing mutant antibodies. First, the AHC biosensor was immersed in buffer (0.02% PBST) for 10 minutes for equilibration, then immersed in a solidified solution containing a known concentration of EGFR antigen (ACRO Biosystems, EGR-H5222) to a signal value of 1 nm, then the antigen-solidified sensor was immersed in buffer for baseline equilibration for 2 minutes, then the biosensor with solidified antigen of known concentration was immersed in a sample solution containing the antibody to be tested for 2 minutes, and finally the sensor combined with the antibody to be tested was immersed in buffer for dissociation for 10 minutes. By real-time monitoring of the thickness of the biological membrane layer of the biosensor during the experiment, the kinetic constant of the sample to be tested can be obtained.

[0540] As shown in Table 3, the point mutation anti-EGFR antibody affinity, Zmab.v1, Zmab.v6 and Zmab.v27 are weaker than Zalutumumab, while Zmab.v18, Zmab.v19 affinity is comparable to Zalutumumab. Among them, Zmab.v6 and Zmab.v27 are mutated at the same position of HCDR3, but the affinity is quite different.

[0541] Table 3. Comparison of affinity of point mutation anti-EGFR antibody and Zalutumumab parent

[0542] Example 3. Design and preparation of anti-B7-H3 / EGFR bispecific antibody

[0543] The variable region part of the anti-B7-H3 antibody huA3, F2-A4, 17-D2, 18-E4 with representative affinity, and the variable region part of the anti-EGFR antibody Zalutumumab, Zmab.V1, Zmab.V27 were selected respectively, and the Knobs-into-Holes (KiH) technology was used to construct bispecific antibodies.

[0544] The cDNA fragments were synthesized according to the gene sequences of each antibody light and heavy chains, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20) as shown in Table 4. The VH and VL of the anti-B7-H3 antibody were cloned into the plasmid vector containing the heavy chain and light chain constant regions, respectively, and the anti-B7-H3 antibody heavy chain constant region contained S354C, T366W, S239D, I332E mutations (SEQ ID NO: 94). The VL and VH of the anti-EGFR antibody were connected as scFv through a (GGGGS)3 linker, cloned into the plasmid vector containing human IgG1 Fc, and the human IgG1 Fc sequence contained C220S, Y349C, T366S, L368A, Y407V, S239D, I332E mutations (SEQ ID NO: 95), to construct bispecific antibodies B01, B02, B03, B04, B05, B06.

[0545] Table 4. Anti-B7-H3 / EGFR bispecific antibodies

[0546] >CH1

[0547] >IgG1 Fc S354C, T366W, S239D, I332E mutations

[0548] >IgG1 Fc C220S, Y349C, T366S, L368A, Y407V, S239D, I332E

[0549] >Human kappa light chain constant region is identical to SEQ ID NO: 50.

[0550] >Zalutumumab scFv

[0551] >Zmab.v1 scFv

[0552] >Zmab.v27 scFv

[0553] The full-length sequences of bispecific antibodies B01, B02, B03, B04, B05, B06 are as follows (underlined for Fc):

[0554] >B01 B7-H3 HC

[0555] B01 B7-H3 LC is identical to 17-D2 LC (SEQ ID NO: 62)

[0556] B01 EGFR HC

[0557] B02 B7-H3 HC is identical to B01 B7-H3 HC (SEQ ID NO: 99)

[0558] B02 B7-H3 LC is identical to 17-D2 LC (SEQ ID NO: 62)

[0559] B02 EGFR HC

[0560] B03 B7-H3 HC is identical to B01 B7-H3 HC (SEQ ID NO: 99)

[0561] B03 B7-H3 LC is identical to 17-D2 LC (SEQ ID NO: 62)

[0562] B03 EGFR HC

[0563] B04 B7-H3 HC

[0564] B04 B7-H3 LC is identical to F2-A4 LC (SEQ ID NO: 52)

[0565] B04 EGFR HC is identical to B02 EGFR HC (SEQ ID NO: 101)

[0566] B05 B7-H3 HC

[0567] B05 B7-H3 LC is identical to 18-E4 LC (SEQ ID NO: 66)

[0568] B05 EGFR HC is identical to B02 EGFR HC (SEQ ID NO: 101)

[0569] B06 B7-H3 HC

[0570] B06 B7-H3 LC is identical to huA3 LC (SEQ ID NO: 55)

[0571] >B06 EGFR HC is identical to B02 EGFR HC (SEQ ID NO: 101).

[0572] Bispecific antibodies with a purity greater than 90% by SEC-HPLC were obtained through conventional recombinant protein expression and purification techniques.

[0573] Example 4. Binding affinity of bispecific antibodies to antigens

[0574] Example 4-1. With EGFR antigen protein

[0575] The affinity of the bispecific antibody for the EGFR antigen was determined using the biomembrane interference (BLI) method described in Example 3. The results in Table 5 show that the bispecific antibody can bind to the EGFR antigen, and the affinity for EGFR is B01 > B02 > B03.

[0576] Table 5. Affinity of anti-B7-H3 / EGFR bispecific antibodies to EGFR antigen protein

[0577] Example 4-2. With double-positive tumor cells expressing human EGFR and B7-H3

[0578] Collect NCI-H1975 and FaDu cells, and seed 2 × 10⁶ cells per well in a 96-well plate. 5 Cells were centrifuged at 600g for 5 minutes, the supernatant was discarded, and 100 μL of the test antibody or control Cetuximab or IgG1 isotype control was added. The cells were incubated at 4°C for 0.5 hours. After centrifugation to remove the supernatant, the cells were washed twice with 200 μL of washing buffer (PBS + 2% FBS). 100 μL of 1:500 diluted anti-human IgG secondary antibody labeled with Alexa Fluor 488 (Jackson Immuno Research, 209-545-098) was added, and the cells were incubated at 4°C for 0.5 hours. After centrifugation to remove the supernatant, the cells were washed twice with 200 μL of washing buffer (PBS + 2% FBS). The cells were resuspended in 100 μL of washing buffer (PBS + 2% FBS) and analyzed by flow cytometry (BD FACSLyric). The median fluorescence value at each concentration was calculated using a four-parameter nonlinear logistic regression model (GraphPad Prism). EC5 50 .

[0579] The results in Table 6 show that all the bispecific antibodies have a strong binding ability to cells expressing EGFR and B7-H3 on their surface.

[0580] Table 6. Results of binding assays of anti-B7-H3 / EGFR bispecific antibodies to tumor cells expressing human EGFR and B7-H3.

[0581] Example 5. EGFR and B7-H3 double positive cell receptor expression detection and preparation of EGFR single positive cell line derivation

[0582] The following cell lines were grown to 90% confluence in complete medium with the respective medium components, then digested with TrypLE (Gibco, Cat# 12605028) and harvested.

[0583] Human non-small cell lung adenocarcinoma cell NCI-H1975 (ATCC, Cat# CRL-5908), medium RPMI1640 + 10% FBS + 1% P / S

[0584] Human non-small cell lung squamous carcinoma cell SK-MES-1 (Chinese Academy of Sciences Cell Bank, Cat# SCSP-5010), medium MEM + 10% FBS + 1% non-essential amino acids + 1% glutamine + 1% sodium pyruvate + 1% P / S

[0585] Human non-small cell lung adenocarcinoma cell NCI-H1568 (Chinese Academy of Sciences Cell Bank, Cat# SCSP-5072), medium RPMI1640 + 10% FBS + 1% P / S

[0586] Human head and neck cancer cell FaDu (Chinese Academy of Sciences Cell Bank, Cat# TCHu132), medium MEM + 10% FBS + 1% sodium pyruvate + 1% P / S

[0587] Human pharyngeal head cancer cell Detroit 562 (Wuhan Ponsay Life Science Co., Ltd., Cat# CL-0330), medium MEM + 10% FBS + 1% non-essential amino acids + 1% glutamine + 1% P / S.

[0588] Human tumor cells Detroit 562, SK-MES-1, FaDu, NCI-H1568 were collected. 2x10 5 Each 1.5 mL EP tube was added with 2x10 TM Simply The antibody binding capacity of each cell was obtained after quantification of the anti-Mouse IgG standard (Bangs Laboratory, Cat# 815), and the results are shown in Figure 1A.

[0589] Human tumor cells NCI-H1975, SK-MES-1, FaDu, NCI-H1568 cells were collected. Using CRISPR technology, SK-MES-1, NCI-H1975, FaDu, NCI-H1568 derivative cell lines with B7-H3 gene knockout were prepared, which were used as safety evaluation cell lines to calculate the killing selectivity window with double positive cells.

[0590] 2x10 5 Cells were placed in 1.5 mL EP tubes. Centrifugation at 300g for 5 minutes, and the supernatant was removed. 100 μL of 1:100 diluted PE anti-human CD276 (B7-H3) antibody (Biolegend, 331606) or 1:100 diluted PE anti-EGFR antibody (Abeam, Ab130738) solution was added to each tube, and incubated at 4°C for 0.5 hours. The supernatant was removed by centrifugation, and the cells were washed twice with 200 μL of washing solution (PBS+2% FBS). The cells were resuspended with 100 μL of PBS, and detected by flow cytometry (BD FACSLyric). Flow Jo software was used for data analysis to obtain fluorescence intensity values.

[0591] 2x10 5 Cells were placed in 1.5 mL EP tubes. Centrifugation at 300g for 5 minutes, and the supernatant was removed. 100 μL of 1:100 diluted PE anti-human CD276 (B7-H3) antibody (Biolegend, 331606) or 1:100 diluted PE anti-EGFR antibody (Abeam, Ab130738) solution was added to each tube, and incubated at 4°C for 0.5 hours. The supernatant was removed by centrifugation, and the cells were washed twice with 200 μL of washing solution (PBS+2% FBS). The cells were resuspended with 100 μL of PBS, and detected by flow cytometry (BD FACSLyric). Flow Jo software was used for data analysis to obtain fluorescence intensity values.

[0592] Results are shown in FIG. IB, FIG. 1C, FIG. ID, FIG. IE. NCI-H1975, SK-MES-1, FaDu, NCI-H1568 parental cells are all EGFR, B7-H3 double positive cells (hereinafter also referred to as H1975.wt, SK-MES-1.wt, FaDu.wt, NCI-H1568.wt), while after CRISPR knockout, the derived cells are all EGFR positive, B7-H3 negative cells (hereinafter also referred to as H1975.bko, SK-MES-1.bko, FaDu.bko, NCI-H1568.bko).

[0593] Example 6. Selective binding of bispecific antibodies against B7-H3 / EGFR to double positive cells

[0594] SK-MES-1.wt cells were collected and labeled with CellTrace TM Far Red Cell Proliferation Kit (Invitrogen, C34564). 1 x 10 5 SK-MES-1.bko and labeled SK-MES-1.wt were seeded into 96-well plates at the same number, centrifuged at 600g for 5 minutes, and the supernatant was removed. 100 μL of the test bispecific antibody or control drug Cetuximab or IgG1 isotype control was added, and incubated at 4°C for 0.5 hours. After centrifugation to remove the supernatant, the cells were washed twice with 200 μL of washing solution (PBS + 2% FBS), and 100 μL of 1:500 diluted anti-human IgG secondary antibody labeled with Alexa Fluor 488 (Jackson Immuno Research, 209-545-098) was added, and incubated at 4°C for 0.5 hours. After centrifugation to remove the supernatant, the cells were washed twice with 200 μL of washing solution (PBS + 2% FBS). The cells were resuspended with 100 μL of washing solution (PBS + 2% FBS), and the FITC and APC fluorescence channels were detected by flow cytometry (BD FACSLyric). Flow Jo software was used for data analysis to obtain the proportion of SK-MES-1.wt and SK-MES-1.bko in the antibody positive population.

[0595] In the case of the same number of cells, the theoretical proportion of the two cells in the non-selective antibody positive population was 50% each. The results in FIG. 2A to FIG. 2E and Table 7 show that the bispecific antibody selectively binds to double positive cells, and the selectivity is B01 < B02 < B03, while Cetuximab does not have selectivity.

[0596] Table 7. Selective binding of anti-B7-H3 / EGFR bispecific antibodies to cells expressing human EGFR and B7-H3 Table 7. Selective binding of anti-B7-H3 / EGFR bispecific antibodies to cells expressing human EGFR and B7-H3

[0597] Example 7. Anti-B7-H3 / EGFR bispecific antibodies selectively block EGF binding to double positive cells

[0598] Both EGF and bispecific antibodies can bind to EGFR antigen expressed on human tumor cells. After co-incubation, the ability of bispecific antibodies to competitively block EGF binding to EGFR can be indirectly reflected by detecting the amount of EGF. The selectivity of bispecific antibodies can be reflected in the same cell lines with and without knock-out of B7-H3.

[0599] First, the control anti-EGFR mAb Cetuximab was tested for blocking difference in the same cell lines with and without knock-out of B7-H3. 2x10 5 H1975.wt or H1975.bko or Fadu.wt or Fadu.bko cells were plated in 96-well plates per well. After 5 min centrifugation at 600g, the supernatant was removed and 50 μΐ of Cetuximab was added, followed by 50 μΐ of EGF-his (ACRO, EGF-H52H3) labeled with EZ-Link Sulfo-NHS-LC-Biotin kit (Thermo Scientific, A39257). The cells were incubated at 4°C for 0.5 h. After centrifugation to remove the supernatant, the cells were washed twice with 200 μΐ of wash buffer (PBS + 2% FBS) and 100 μΐ of 1:1000 diluted Alexa Fluor 633 Streptavidin conjugate (Invitrogen, S21375) was added. The cells were incubated at 4°C for 0.5 h. After centrifugation to remove the supernatant, the cells were washed twice with 200 μΐ of wash buffer (PBS + 2% FBS). The cells were resuspended with 100 μΐ of wash buffer (PBS + 2% FBS) and analyzed by flow cytometry (BD FACSLyric). The data were normalized and fitted with a four-parameter nonlinear logistic regression curve in Graphpad Prism. The results are shown in Figure 3 and Table 8.

[0600] Table 8. Anti-EGFR antibody blocking of EGF binding on NCI-H1975 and FaDu cells

[0601] (NA represents no IC 50 )

[0602] The same method was used to test bispecific antibodies B01, B02, B03, B04, B05, B06 for blocking difference in the same cell lines with and without knock-out of B7-H3. H1975.wt or H1975.bko or Fadu.wt or Fadu.bko cells were used. The results are shown in Figure 4 and Table 9.

[0603] As can be seen from Figure 4A to Figure 4L, in two human tumor cell lines, the bispecific antibodies B01, B02, B03, B04, B05, B06 all blocked EGF binding to both positive and EGFR single positive cells; the ability of the bispecific antibodies to block EGF binding to double positive cells (IC 50 ) was superior to the ability to block single positive cells, and the difference was large.

[0604] The results of Table 8 and Table 9 show that the bispecific antibodies B01, B02, B04, B05, B06 selectively blocked EGF binding to double positive cells, with a clear selectivity window (> 3-fold); the control anti-EGFR antibody Cetuximab blocked EGF binding to double positive cells or to single positive cells with no significant difference (0.5-2-fold).

[0605] Table 9. Anti-B7-H3 / EGFR bispecific antibodies selectively block EGF binding on NCI-H1975 and FaDu cells

[0606] (NA represents that the IC 50 )

[0607] Example 8. Anti-B7-H3 / EGFR bispecific antibodies selectively inhibit EGFR receptor phosphorylation

[0608] In a 12-well plate, 2x10 5SK-MES-1.wt or SK-MES-1.bko cells, the next day, serum-free culture was performed overnight, on the third day, 2 nM and 20 nM bi-specific or control drug Cetuximab was added, incubated for 1 h in 37 °C incubator, then 50 ng / ml of EGF-his (ACRO, EGF-H52H3) was added per well, incubated for 15 min in 37 °C incubator, then collected for sample preparation with RIPA lysis buffer (Thermo Scientific, 89900), without adding antibody, EGF was added as a positive control, without adding antibody and without adding EGF as a negative control (NC). 12-well precast gel (Invitrogen, NP0322BOX) was used, 16 μl was loaded per sample, 150 V gel running for 45 min, then transferred to membrane using a transfer kit (BIO-RAD, 1704156) at 25 V, 25 mA for 10 min. Blocking was performed using 5% BSA (PBS + 0.05% Tween 20), incubated for 1 h at room temperature with shaking. Antibodies were diluted with 1% BSA, p-EGFR Tyr1068 (Cell Signaling Technology, 2234) was diluted at 1:200, EGFR (Cell Signaling Technology, 4267S) was diluted at 1:200, beta-Actin (Cell Signaling Technology, 4970) was diluted at 1:500, and incubated overnight at 4 °C. Washing was performed 3 times with PBST, each time for 10 min with shaking. HRP-labeled anti-rabbit IgG secondary antibody (Jackson immuno, 111-035-144) was diluted at 1:5000 with 1% BSA, incubated for 1 h at room temperature with shaking, then washed 3 times with PBST, each time for 10 min with shaking. After adding developing solution (Millipore, WBKLS0500), exposure was performed using a gel imager (Bio-rad ChemiDoc MP) and the image was captured.

[0609] The results are shown in Figures 5A-5B, Cetuximab inhibited EGFR phosphorylation in both double-positive and EGFR single-positive cells without distinction, while B01, B02, B04, B05, and B06 selectively inhibited double-positive cells, and B03 had weaker inhibitory ability.

[0610] Example 9. ADCC effect of anti-EGFR antibodies

[0611] The Fc fragment of anti-EGFR antibody introduces amino acid mutation sites S239D and I332E, further enhancing the ADCC effect. Based on the sequence of Zalutumumab, the parent Zmab (HC SEQ ID NO: 121 and LC SEQ ID NO: 87), Zmab-DE (HC SEQ ID NO: 122 and LC SEQ ID NO: 87) and Zmab-scFv-DE (HC SEQ ID NO: 123) were constructed respectively. The specific sequences are as follows:

[0612] >Zmab HC

[0613] >Zmab LC is identical to Zalutumumab LC (SEQ ID NO: 87)

[0614] >Zmab-DE HC

[0615] >Zmab-scFv-DE

[0616] >IgG1 constant region D356E and L358M

[0617] >IgG1 constant region S239D I332E D356E L358M

[0618] After mixing the antibody with effector cells healthy human PBMC (Shanghai Aobio Technology Co., Ltd., T0071) and target cells NCI-H1975, the level of cell death was detected. Briefly, the target cells were washed once with culture medium, and the target cells were adjusted to about 1x10 6 / mL. Add BATDA (Perkin Elmer, cat.no.C136-100) 4 μL in the culture medium, incubate at 37°C for 10 minutes. Wash the cells 3-5 times. Resuspend the cells carefully, and adjust the concentration to about 20x10 4 / mL. Add 50 μL (total 1x10 4 NCI-H1975 cells per well in a 96-well plate. Add 100 μL (total 2.5x10 5Incubate at 37°C for 3 hours. Set up the maximum killing activity well, add 10 μΐ of lysis solution to each well, and set up the spontaneous release well. Use DELFIA EuTDA Cytotoxicity Reagents (PerkinElmer, #AD0116) to measure the killing activity. First, centrifuge at 500g for 5 minutes, and transfer 20 μΐ of supernatant to a flat-bottom plate. Add 200 μΐ of Eu-solution reaction substrate, shake at 250 rpm per minute for 15 minutes, and measure the fluorescence value using time-resolved fluorescence (TRF).

[0619] Calculate the killing activity according to the following formula: Killing activity (%) = [(ER-SR) / (MR-SR)]x100;

[0620] wherein ER is the fluorescence value of the experimental group after adding Eu-solution, SR is the fluorescence value of the spontaneous release control group after adding Eu-solution, and MR is the fluorescence value of the maximum killing activity well after adding Eu-solution.

[0621] The experimental results, as shown in Table 10A and Figure 6, show that all the tested anti-EGFR antibodies can significantly activate NK cells to kill H1975 cells, and the introduction of DE mutations in the Fc domain has stronger killing function, and the scFv mode of antibody further enhances the ADCC effect.

[0622] Table 10A. ADCC cell killing of NCI-H1975 induced by anti-EGFR antibodies

[0623] Example 10. Selective ADCC killing of anti-B7-H3 / EGFR bispecific antibodies

[0624] The bispecific antibody can exert ADCC killing effect by specifically binding to the tumor cell surface EGFR and B7-H3 antigens.

[0625] 1. NCI-H1975 cells

[0626] Wash the target cells once with the culture medium, and adjust the target cells to about 2x10 5 / mL. Add 50 μΐ (total 1x10 4PBMC (Shanghai Aobio Biotech Co., Ltd., T0071) was centrifuged at 400g for 10 min, and the cells were resuspended with medium. PBMC was added, and 30 nM gradient dilution of the test bispecific antibody or control Cetuximab or IgG1 isotype control was added, and incubated at 37°C for 3 hours. The maximum killing activity well was set, 10 μL of lysis solution was added to each well, and the spontaneous release well was set. DELFIA EuTDA Cytotoxicity Reagents (PerkinElmer, #AD0116) was used to measure the killing activity. The fluorescence value was measured using time-resolved fluorescence (TRF).

[0627] The killing activity was calculated according to the following formula: Killing activity (%) = [(ER-SR) / (MR-SR)]x100;

[0628] wherein ER is the fluorescence value of the experimental group after adding Eu-solution, SR is the fluorescence value of the spontaneous release control group after adding Eu-solution, and MR is the fluorescence value of the maximum killing activity well after adding Eu-solution.

[0629] The experimental results, as shown in Table 10B and FIG. 7, show that all the detected bispecific antibodies can significantly activate NK cells to kill H1975 cells; compared with EGFR single-positive H1975.bko cells, bispecific antibodies B01, B02, B03, B04, B05 and B06 have a significant selective induction of killing effect on double-positive H1975.wt cells.

[0630] Table 10B. ADCC killing of NCI-H1975 cells induced by antibodies

[0631] 2. The target cells were washed once with culture medium, and the target cells were adjusted to about 2x10 5 / mL. 50 μL (total 1x10 4 cells were added to each well of different 96-well plates. Healthy human PBMC (Shanghai Aobio Biotech Co., Ltd., T0071) was centrifuged at 400g for 10 min, and the cells were resuspended with medium. PBMC was added, and 30 nM gradient dilution of the test bispecific antibody or control Cetuximab or IgG1 isotype control was added, and incubated at 37°C for 3 hours. The maximum killing activity well was set, 10 μL of lysis solution was added to each well, and the spontaneous release well was set. Cytotoxicity Assay Kit PLUS (LDH) (Sigma, 4744934001) was used to measure the killing activity, and the absorbance at 490 nm wavelength was measured.

[0632] The killing activity was calculated according to the following formula: Killing activity (%) = [(ER-NR) / (MR-SR)]x100;

[0633] Wherein, ER is the absorbance of the experimental group after the reaction is terminated, NR is the absorbance of the non-drug treatment group, MR is the fluorescence value of the maximum killing activity hole after the reaction is terminated, and SR is the absorbance of the spontaneous release control group.

[0634] The experimental results, as shown in Table 11 and FIG. 8, show that all the detected bispecific antibodies can significantly activate NK cells and kill FaDu cells; compared with EGFR single-positive FaDu.bko cells, bispecific antibodies B02, B03, B04, B05 and B06 have a significant selective induction of killing effect in double-positive FaDu.wt cells.

[0635] Table 11. ADCC killing of FaDu cells induced by antibodies

[0636] Example 11. Pharmacodynamic experiment of anti-B7-H3 / EGFR bispecific antibody in a tumor-bearing mouse model in vivo

[0637] In this experiment, the parental cell FaDu.wt (EGFR and B7-H3 double-positive cell) and the B7-H3 knockout derivative cell FaDu.bko (EGFR single-positive cell) were inoculated into BALB / c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) to determine the selective killing effect of anti-EGFR / B7-H3 bispecific antibodies.

[0638] FaDu.wt cells and FaDu.bko cells were routinely subcultured using MEM medium (10% FBS). When the predicted number of cells reached the required amount, the cells were trypsinized and centrifuged to collect the cells, which were dispersed in PBS to a concentration of 5x10 7 / mouse) was inoculated into the left lower limb subcutaneous area of each BALB / c nude mouse, and the next day, 0.1 ml of FaDu.wt cell suspension (i.e., 5x10 6 / mouse) was inoculated into the right lower limb subcutaneous area of the same mouse. When the average tumor volume of FaDu.bko and FaDu.wt reached 116 mm 6 / mouse) was inoculated into the right lower limb subcutaneous area of the same mouse. When the average tumor volume of FaDu.bko and FaDu.wt reached 116 mm 3The mice were randomly divided into groups (8 mice per group) and the first day of grouping was the day of grouping. The mice were administered the antibodies, positive control antibodies, and negative control antibodies (see Table 12 for the administered antibodies and concentrations) on days 1, 4, 8, 11, 15, and 18 after grouping, and the tumor volume and body weight of the mice were measured twice per week. The monitoring was performed until the end of day 22 after grouping. The tumor inhibition rate (TGI) on day 22 after grouping was calculated according to the following formula: TGI = (1 - (tumor volume of the treatment group - initial tumor volume of the treatment group) / (tumor volume of the PBS control group - initial tumor volume of the PBS control group)) x 100%.

[0639] The maximum length (L) and maximum width (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: V = L x W 2 / 2. The body weight was measured using an electronic balance. During the entire study, the mice were euthanized when the tumor volume of the mice was > 2000 mm 3 or the body weight of the mice decreased by > 20%.

[0640] The tumor inhibition rate results are shown in FIG. 9A and Table 13: the tumor size was measured on day 22 after grouping, and the inhibition rate was calculated. In the FaDu.wt model, the tumor inhibition rates of B02, B04, B05, and B06 were 92%, 105%, 105%, and 104%, respectively, compared with the PBS control group. In the FaDu.bko model, the tumor inhibition rates of antibodies B02, B04, B05, and B06 were 72%, 98%, 68%, and 94%, respectively, compared with the PBS control group. This indicates that the bispecific antibodies B02 and B05 with strong B7-H3 ends and weak EGFR ends have a better selective window. The body weight of the mice was also monitored, and the results are shown in FIG. 9B: in the administered mouse groups, no significant weight loss was observed.

[0641] Table 12. Grouping and administration scheme for the mouse experiment

[0642] Table 13. Tumor inhibition rate on day 22 after grouping

[0643] Example 12. Modification of anti-B7-H3 / EGFR bispecific antibodies

[0644] According to the gene sequences of the light chains and heavy chains of the antibodies, the cDNA fragments were synthesized and inserted into pcDNA3.1 expression vectors (Life Technologies Cat. No. V790-20), as shown in Table 14.

[0645] After the humanized heavy chain and light chain expression vectors were transfected into 293F cells, the culture supernatant was collected, and the bispecific antibodies with a SEC HPLC purity of > 90% were obtained after Protein A affinity purification.

[0646] Table 14. Anti-B7-H3 / EGFR bispecific antibodies

[0647] wherein the two subunits of the Fc region of B12, B14 and B16 comprise S354C, T366W mutations (SEQ ID NO: 106) and C220S, Y349C, T366S, L368A, Y407V mutations (SEQ ID NO: 107), respectively;

[0648] wherein the two subunits of the Fc region of B13 and B11 comprise S354C, T366W mutations (SEQ ID NO: 106) and C220S, Y349C, T366S, L368A, Y407V mutations, and removal of C-terminal lysine (SEQ ID NO: 109), respectively;

[0649] wherein the two subunits of the Fc region of B22 comprise S354C, T366W mutations, and removal of C-terminal lysine (SEQ ID NO: 108) and C220S, Y349C, T366S, L368A, Y407V mutations, and removal of C-terminal lysine (SEQ ID NO: 109), respectively.

[0650] > Human IgGl Fc S354C, T366W mutations

[0651] > Human IgGl Fc C220S, Y349C, T366S, L368A, Y407V mutations

[0652] > Human IgGl Fc S354C, T366W mutations, removal of C-terminal K

[0653] > Human IgGl Fc C220S, Y349C, T366S, L368A, Y407V mutations, removal of C-terminal K

[0654] The full length sequences of bispecific antibodies B12, B14, B16, B13, B11, B22 are as follows:

[0655] > B12 B7-H3 HC

[0656] > B12 B7-H3 LC is identical to 17-D2 LC (SEQ ID NO: 62)

[0657] > B12 EGFR HC

[0658] B14 B7-H3 HC is identical to B12 B7-H3 HC (SEQ ID NO: 110)

[0659] B14 B7-H3 LC is identical to F2-A4 LC (SEQ ID NO: 52)

[0660] B14 EGFR HC is identical to B12 EGFR HC (SEQ ID NO: 111)

[0661] B16 B7-H3 HC is identical to B14 B7-H3 HC (SEQ ID NO: 112)

[0662] B16 B7-H3 LC is identical to huA3 LC (SEQ ID NO: 55)

[0663] B16 EGFR HC is identical to B12 EGFR HC (SEQ ID NO: 111)

[0664] B13 B7-H3 HC is identical to B12 B7-H3 HC (SEQ ID NO: 110)

[0665] B13 B7-H3 LC is identical to 17-D2 LC (SEQ ID NO: 62)

[0666] B13 EGFR HC

[0667] B11 B7-H3 HC is identical to B12 B7-H3 HC (SEQ ID NO: 110)

[0668] B11 B7-H3 LC is identical to 17-D2 LC (SEQ ID NO: 62)

[0669] B11 EGFR HC

[0670] B22 B7-H3 HC

[0671] B22 B7-H3 LC is identical to 17-D2 LC (SEQ ID NO: 62)

[0672] B22 EGFR HC

[0673] The sequences of control antibodies Cetuximab and Ifinatamab are as follows:

[0674] >Cetuximab HC

[0675] >Cetuximab LC

[0676] >Ifinatamab HC

[0677] >Ifinatamab LC

[0678] Example 13. Preparation of antibody drug conjugate

[0679] Example 13-1. Preparation of compound

[0680] 1.1 Preparation of compound 1

[0681] N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]glycylglycyl-L-phenylalanyl-N- [(2-{[(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15- hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino}-2- oxoethoxy)methyl]glycinamide Compound 1

[0682] Compound 1 was synthesized by the method provided in the reference patent “Example 58, page 163 of the specification of CN104755494A”.

[0683] 1.2 Preparation of compound 9-A and compound 9-B

[0684] N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14- tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A

[0685] Compound 9-A was synthesized by the method provided in the reference patent “Example 9, page 58 of the specification of CN112512591A”.

[0686] Example 13-2. Preparation of anti-B7-H3 / EGFR antibody drug conjugate

[0687] The antibody drug conjugate (ADC) was prepared using Compound 1 or Compound 9-A. The method is as follows:

[0688] 1. Preparation of antibody drug conjugate ADC-6

[0689] In a centrifuge tube, B22 (500 μg, 49.26 μL), PBS pH = 7.2 aqueous buffer solution (117.4 μL) and prepared aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (1.5 mM, 10.76 μL, 4.0 eq) were added respectively, and placed in a 37 °C thermostat, and shaken for 3 hours at 37 °C, and the reaction was stopped. After taking out, a DMSO (3 μL) solution of Compound 9-A (65 μg, 15 eq) was added, and placed in a 25 °C thermostat, and shaken for 3 hours, and the reaction was stopped. ADC-6 was purified by centrifugal desalting column, and stored at 4 °C. Sampling was sent for analysis, and the SEC purity was 97.9%, and the average DAR detected by mass spectrometry was 3.15.

[0690] 2. Preparation of antibody drug conjugate ADC-7

[0691] In a centrifuge tube, B22 (500 μg, 105.3 μL), PBS pH = 7.2 (61.4 μL) and aqueous solution of tris (2-carboxyethyl) phosphine (TCEP) (10 mM, 8.06 μL, 20 eq) were added respectively, and incubated at 37 °C for 3 hours. After taking out, a DMSO (3 μL) solution of Compound 9-A (65 μg, 15 eq) was added, and shaken at 25 °C for 3 hours. ADC-7 (489 μg, yield 97.8%) was purified by centrifugal desalting column, and stored at 4 °C. Sampling was sent for analysis, and the SEC purity was 97.0%, and the average DAR detected by mass spectrometry was 5.50.

[0692] 3. Preparation of antibody drug conjugate ADC-1, ADC-2, ADC-3, ADC-4, ADC-5

[0693] B12, or B14, or B16, or B13, or B11 were coupled with Compound 1 respectively, and the preparation method of ADC-7 was referred to, to obtain antibody drug conjugates ADC-1, ADC-2, ADC-3, ADC-4, ADC-5, and the average DAR values after mass spectrometry detection are shown in Table 15.

[0694] Finally, the bispecific ADCs shown in Table 15 were obtained for the next pharmacodynamic test.

[0695] Table 15. Anti-B7-H3 / EGFR ADC

[0696] Example 13-3. Preparation of anti-EGFR antibody drug conjugate and anti-B7H3 antibody drug conjugate

[0697] As shown in Table 16, Cetuximab (HC SEQ ID NO: 117 and LC SEQ ID NO: 118) and Ifinatamab (HC SEQ ID NO: 119 and LC SEQ ID NO: 120) expression plasmid vectors were constructed based on the published sequences, respectively. After expression and purification by conventional recombinant protein technology, monospecific antibodies with SEC HPLC purity greater than 95% were obtained.

[0698] Preparation of antibody drug conjugate (ADC). The method is as follows:

[0699] 1. Preparation of antibody drug conjugate ADC-8-1

[0700] Cetuximab and Compound 1 were conjugated according to the method provided in reference patent "Example 7 Procedure 1 of the specification of CN105829346B", and ADC-8-1 was purified by centrifugal desalting column and stored at 4°C. Sampling was sent for analysis, SEC = 96.14%, and mass spectrometry detection showed an average DAR of 8.01.

[0701] 2. Preparation of antibody drug conjugate DS-7300

[0702] Take 540.9 μL of 7.58 mg / mL antibody solution (4.1 mg, 50 mM sodium phosphate buffer, pH 7.4) into a 1.5 mL centrifuge tube, and add 202.7 μL of 50 mM sodium phosphate buffer, pH 7.4, to the centrifuge tube. Then add 76.4 μL of 1 mmol / L TCEP solution (corresponding to 2.82 equivalent ratio), control the reaction concentration of the antibody to be 5 mg / mL, and incubate at 22°C in a metal bath with shaking for 2 hours.

[0703] After the reaction time is reached, add 65.47 μL of DMSO (control the total content of DMSO to be 10% v / v) and 25.64 μL of 10 mg / mL Compound 1 solution (corresponding to 10 equivalents, the small molecule is dissolved in DMSO) to the antibody solution, and incubate at 22°C in a metal bath with shaking for 2 hours.

[0704] Amicon Ultr-4 mL, 30 kDa ultrafiltration centrifuge tubes were used to replace the sample buffer with 10 mM histidine (pH 5.5) buffer and remove the excess drug linker intermediates from the reaction mixture. The sample solution was added to the ultrafiltration centrifuge tubes and centrifuged at 4000 rpm for 20 min, repeated 8 times to obtain the purified sample. The sample was taken for analysis, SEC = 99.37%, average DAR = 3.90 by RP-HPLC detection.

[0705] Table 16. Anti-EGFR ADC and anti-B7H3 ADC

[0706] Example 13-4. Affinity of anti-B7-H3 / EGFR antibody drug conjugate

[0707] The affinity of bispecific antibody drug conjugate ADC-7 to B7-H3 and EGFR antigen proteins was detected by surface plasmon resonance (SPR) method.

[0708] The bispecific antibody drug conjugate to be tested was used as ligand, and the antigen protein was used as analyte. Briefly, using the commercial kit GE Human Antibody Capture Kit, type 2 (manufacturer GE, item number 29234600), the anti-human IgG (Fc) polyclonal antibody was covalently linked to the CM5 (GE) chip using the standard amino coupling method, and then this antibody was used to capture the antibody drug conjugate ADC-7 to be tested to the stationary phase. Different concentration gradients of antigen proteins diluted in the same buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20, pH 7.4) were injected before and after each cycle. The Biacore T200 instrument was used to track the antigen-antibody binding kinetics for 2 minutes and the dissociation kinetics for 4-5 minutes. The BIAevaluation software of GE was used to analyze the data with a 1:1 (Langmuir) binding model, and the association rate ka, dissociation rate kdand affinity K D values were determined in this way.

[0709] The results are shown in Table 17, ADC-7 binds to both human B7-H3 and EGFR antigens, and the affinity to B7-H3 is higher than that to EGFR.

[0710] Table 17. Affinity of anti-B7-H3 / EGFR ADC to antigen B7-H3 and EGFR

[0711] Example 14. Selective killing of anti-B7-H3 / EGFR antibody drug conjugate

[0712] Example 14-1. Selective killing of double positive cells by different anti-B7-H3 / EGFR ADCs

[0713] The in vitro activity and selectivity window of the bispecific ADC molecules ADC-1, ADC-2, ADC-3, ADC-4, ADC-5 were evaluated using the SK-MES-1 double positive cell line co-expressing B7-H3 and EGFR (SK-MES-1.wt), and the SK-MES-1 derived cell line with B7-H3 knocked out (SK-MES-1.bko).

[0714] 1000 SK-MES-1 WT or KO cells were seeded in 96-well plates, respectively, and incubated with culture medium and ADC-1, ADC-2, ADC-3, ADC-4, ADC-5 diluted in gradient. The plates were incubated at 37°C, 5% CO2 for 5 days. The number of viable cells was detected using Cell Viability Detection Kit (Suzhou Yingze Biological, Cat# EZB-CV1) and Multifunctional Enzyme Labeling Instrument SpectraMax Pro. The bioluminescence values at each concentration were plotted and fitted to calculate IC 50 . The results are shown in Figures 10A-10E and Table 18.

[0715] The results showed that ADC-1 and ADC-5 had the strongest in vitro killing activity, and ADC-1 had a greater selectivity window for killing double positive cells.

[0716] Table 18. Killing activity of anti-B7-H3 / EGFR ADCs

[0717] (NA means that the IC 50 was not obtained).

[0718] Example 14-2. Selective killing of double positive cells by anti-B7-H3 / EGFR ADC ADC-7

[0719] In this experiment, human tumor cell lines NCI-H1568, FaDu (double positive, named wt, the expression abundance of B7-H3 and EGFR on the cell surface is shown in Figure 1, the ratio is labeled in Figure 4), and each cell after knocking out B7-H3 to obtain single EGFR target cell line (single positive, named bko), and EGFR and B7-H3 double negative human small cell lung cancer cell line NCI-H69 were further evaluated for the selective killing effect of ADC-7 on various B7-H3 and EGFR double positive cells.

[0720] Example 14. Anti-B7-H3 / EGFR ADCs’ killing activity 2000 NCI-H1568 cells, or 2000 FaDu cells, or 5000 NCI-H69 cells were seeded in 96-well plates, respectively, with culture medium and the test sample diluted in gradient. The cells were incubated in a 37 °C, 5% CO2 incubator for 5-6 days. The number of viable cells was detected using Cell Viability Detection Kit (Suzhou Yingze Biological, Catalog No. EZB-CV1) and Multifunctional Enzyme Labeling Instrument SpectraMax Pro. The bioluminescence values at each concentration were plotted and fitted with IC50 using a four-parameter nonlinear logistic regression function model (GraphPad Prism). 50 .

[0721] As shown in FIGs. 11A-11B and Table 19, the results showed that ADC-7 had better dual-positive cell selectivity than ADC-8-1 in the example models where B7-H3 was expressed at a similar ratio to EGFR or B7-H3 was expressed lower than EGFR.

[0722] As shown in FIG. 12 and Table 20, ADC-7 did not produce cytotoxicity to cells negative for target antigen, which was superior to the maximum killing of the isotype control ADC IgG1-9 (loaded with toxin Compound 9-A).

[0723] Table 19. Killing activity of anti-B7-H3 / EGFR ADC-7

[0724] Table 20. Killing activity of anti-B7-H3 / EGFR ADC-7

[0725] Example 15. Binding and endocytosis of anti-B7-H3 / EGFR bispecific antibodies

[0726] The binding and endocytosis ability of anti-B7-H3 / EGFR bispecific antibody B22 to FaDu cells was evaluated. B22, Ifinatamab and Cetuximab solutions were configured with pre-cooled complete culture medium and added to FaDu cells, respectively, at 10 6Cells 100 μΐ, final antibody concentration 50 nM, incubate on ice for 1 hour. After incubation, cells were washed twice with pre-cooled complete medium, centrifuge at 300 x g for 3 min, discard supernatant. Each sample was resuspended with 100 μΐ of medium and immediately aliquoted into two 1.5 mL EP tubes. One of them was centrifuged at 300 x g for 3 min at 4°C, then 250 μΐ of 4% PFA was added to fix at room temperature for 15 min. After centrifugation and washing to 50 μΐ of FACS buffer (PBS, 2% FBS), it was kept on ice, which was the T0 sample. The other was incubated in a 37°C incubator for 3 hours, then centrifuged at 300 x g for 3 min, 250 μΐ of 4% PFA was added to fix at room temperature for 15 min. After centrifugation and washing to 50 μΐ of FACS buffer, it was the T3 sample. 50 μΐ of Alexa Fluor 488 labeled anti-human IgG secondary antibody (Jackson Immuno Research, 209-545-098) diluted 250 times with FACS buffer was added to both T0 and T3 samples. After incubation on ice for 30 min, they were washed twice with 1 ml of FACS buffer, the supernatant was discarded, 100 μΐ of FACS buffer was added, and the fluorescence was detected by flow cytometry (BD FACSLyric) and the median value of fluorescence intensity (MFI) was calculated. The MFI of the T0 sample represents the amount of antibody binding to the cells, and the difference between the MFI of T0 and T3 relative to the MFI of T0 is the antibody endocytosis rate.

[0727] The results are shown in Table 21. The cell binding amount and antibody endocytosis rate of anti-B7-H3 / EGFR bispecific antibody B22 on double-positive cells are better than those of anti-B7-H3 monoclonal antibody Ifinatamab and anti-EGFR monoclonal antibody Cetuximab.

[0728] Table 21. Binding and endocytosis of anti-B7-H3 / EGFR bispecific antibodies

[0729] Example 16. DAR value of anti-B7-H3 / EGFR antibody drug conjugate

[0730] This experiment compares the freeze-thaw stability of two bispecific antibody drug conjugates ADC-6 and ADC-7 with different toxin-antibody conjugation ratios (DAR).

[0731] The bispecific antibody conjugate molecules were tested for freeze-thaw stability by repeated freeze-thawing at -80°C for 3 times. The samples before and after freeze-thawing were tested for changes in the degree of molecular aggregation by SEC-HPLC using an Agilent 1260 Infinity II HPLC system and a chromatographic column SRT-C SEC-300 (Sedex, 235300-7830) with a buffer of 1X PBS (pH 7.4) at a flow rate of 1 mL / min. The percentage of monomeric molecules in the main peak before and after freeze-thawing was calculated by UV signal. The results showed that both ADC-6 and ADC-7 had excellent stability, with the main peak percentage being 100% before and after freeze-thawing, and there was no significant difference in the stability of the two ADCs.

[0732] Example 17. Detection of EGFR, B7-H3 expression in head and neck cancer, non-small cell lung cancer, esophageal cancer samples by immunohistochemistry

[0733] The EGFR protein expression in tumor samples from head and neck cancer, lung cancer and esophageal cancer patients was tested by standard immunohistochemistry method.

[0734] Briefly, the head and neck cancer tumor chip (No. HN810001), non-small cell lung cancer tumor chip (No. R215Lu01) and esophageal cancer tumor chip (No. D880101) purchased from Zhongke Guanghua were dewaxed, rehydrated, and treated with antigen retrieval solution (ServiceBio, Cat. No. G1202) in a microwave oven for 10 minutes. Then the chips were treated with 3% hydrogen peroxide solution to inactivate endogenous peroxidase activity. Subsequently, 5% bovine serum albumin (Shenguo Biotech, A60033200100) was used to inhibit non-specific binding, and then the monoclonal mouse anti-EGFR antibody (Thermofisher, Cat. No. MA5-13269) was used to incubate each tumor chip overnight. The next day, the ABC three-step method was used for color development. Specifically, the chip was incubated with biotin-labeled rabbit anti-mouse secondary antibody (Thermofisher, Cat. No. PA1-28567) at room temperature for 1 hour, then the ABC solution was prepared according to the kit (Thermofisher, Cat. No. 32050) requirements and incubated at room temperature for 30 minutes. Subsequently, DAB (ServiceBio, Cat. No. G1212) was used for color development to the desired intensity, and then terminated, dehydrated and mounted. The formed chip was observed using a bright field microscope (Olympus), or scanned (see Figure 13A, Figure 13C, Figure 13E).

[0735] B7-H3 protein expression in tumor samples from head and neck cancer, lung cancer and esophageal cancer patients was tested by standard immunohistochemistry method. Briefly, head and neck cancer tumor chips (No. HN810001), non-small cell lung cancer tumor chips (No. R215Lu01) and esophageal cancer tumor chips (No. D880101) purchased from Zhongke Guanghua were dewaxed, rehydrated and treated with antigen retrieval solution (Service Bio, Cat No. G1202) in a microwave oven for 10 minutes. Then the chips were treated with 3% hydrogen peroxide solution to inactivate endogenous peroxidase activity. Subsequently, 5% bovine serum albumin (Shenguo Bio, A60033200100) was used to inhibit non-specific binding, and then the monoclonal rabbit anti-B7-H3 antibody (Cell Signaling Technology, Cat No. 14058) was incubated with each tumor chip overnight. The next day, the ABC three-step method was used for color development. Specifically, the chips were incubated with biotin-labeled goat anti-rabbit secondary antibody (Thermofisher, Cat No. A16100) at room temperature for 1 hour, and then the ABC solution was prepared according to the kit (Thermofisher, Cat No. 32050) requirements and incubated at room temperature for 30 minutes. Subsequently, DAB (Service Bio, Cat No. G1212) was used for color development to the desired intensity, and then terminated, dehydrated and mounted. The formed chips were observed using a bright field microscope (Olympus), or scanned (see FIG. 13B, FIG. 13D, FIG. 13F).

[0736] The results showed that EGFR and B7-H3 had 53%, 18%, 22% double positive co-expression in head and neck cancer, non-small cell lung cancer, esophageal cancer tumors, respectively.

[0737] Example 18. Pharmacodynamic experiment of anti-B7-H3 / EGFR antibody drug conjugate in mouse model

[0738] 1. Pharmacodynamic comparison of anti-B7-H3 / EGFR ADC on NCI-H1568 non-small cell lung cancer tumor cell-bearing mouse model in vivo

[0739] In this experiment, NCI-H1568 cells (Chinese Academy of Sciences Cell Bank, Cat No. SCSP-5072) were inoculated into BALB / c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) to determine the anti-tumor effect of anti-B7-H3 / EGFR ADC.

[0740] NCI-H1568 cells were routinely subcultured using RPMI1640 medium (10% FBS). When the predicted cell number reached the required amount, the cells were trypsinized and centrifuged to collect the cells, which were dispersed in PBS to a concentration of 5×10 7 Each mouse was subcutaneously inoculated with 0.1 ml of NCI-H1568 cell suspension (i.e. 5×106 BALB / c nude mice were randomly divided into groups (7 mice per group) when the average tumor volume reached about 200 mm 3 The first day was recorded on the day of grouping. The drugs were administered by IV tail vein on days 1, 8, and 15, with a dose of 6 mg / kg each. The tumor volume and body weight of the mice were measured twice a week. The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: V = L x W 2 The body weight was measured using an electronic balance. During the entire study, when the tumor volume of the mice was >2000 mm 3 or the body weight of the mice decreased by >20%, the mice were euthanized. The tumor inhibition rate (TGI) was calculated according to the following formula: TGI = (1 - (tumor volume of the treatment group - initial tumor volume of the treatment group) / (tumor volume of the PBS control group - initial tumor volume of the PBS control group)) x 100%.

[0741] The results of the tumor inhibition rate showed that the anti-B7-H3 / EGFR ADC had a significant inhibitory effect on the mouse tumor cell model of NCI-H1568.

[0742] 2. Pharmacodynamic experiment of anti-B7-H3 / EGFR ADC on FaDu head and neck cancer tumor cell tumor-bearing mouse model

[0743] In this experiment, FaDu cells (Chinese Academy of Sciences Cell Bank, TCHu132) were used to inoculate BALB / c nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) to determine the anti-tumor effect of anti-B7-H3 / EGFR ADC in a head and neck cancer model.

[0744] FaDu cells were routinely subcultured using MEM medium (10% FBS). When the predicted cell number reached the required amount, the cells were trypsinized and collected by centrifugation, and the cells were dispersed in PBS to a concentration of 4.5 x 107 / ml. Each mouse was subcutaneously inoculated with 0.1 ml of the FaDu cell suspension (i.e. 4.5 x 106 / mouse) into the right front limb dorsal lower region of a BALB / c nude mouse. When the average tumor volume of the mice reached about 200 mm3, the mice were randomly divided into groups (6 mice per group), and the day of grouping was recorded as day 1. The mice were administered by IV tail vein on days 1 and 15, and the tumor volume and body weight of the mice were measured twice a week. The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated according to the following formula: V = L x W2 / 2. The body weight was measured using an electronic balance. During the entire study, when the tumor volume of a mouse was >2000 mm3or the body weight of a mouse decreased by >20%, the mouse was euthanized. The tumor inhibition rate (TGI) was calculated according to the following formula: TGI = (1- (tumor volume of treatment group - initial tumor volume of treatment group) / (tumor volume of PBS control group - initial tumor volume of PBS control group)) x 100%.

[0745] The tumor inhibition rate results showed that the anti-B7-H3 / EGFR ADC had a significant inhibitory effect on the mouse tumor model of head and neck cancer cells FaDu, and in the administered mouse group, no significant weight loss was found.

Claims

1. A B7-H3 binding molecule comprising a domain that specifically binds B7-H3; the domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 selected from the group consisting of: HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 15 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 14; HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 4 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 5; HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 4 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 6; HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 8 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 7; HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 4 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 9; HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 11 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 10; HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 13 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 12; HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 17 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 16; and HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 18 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 19; Preferably, the HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 are selected from the group consisting of combinations of: SEQ ID NO: 31, 40, 22, 41-43; SEQ ID NO: 20-24 and 26; SEQ ID NO: 20-24 and 27; SEQ ID NO: 20-24 and 30 SEQ ID NO: 31, 32, 22, 33-35; SEQ ID NO: 31, 36, 22, 37-39; SEQ ID NO: 44, 45, 22, 33, 46, 47; SEQ ID NO: 44, 48, 22, 33, 49, 35; or SEQ ID NO: 28, 29, and 22-25.

2. A B7-H3 binding molecule comprising a domain that specifically binds B7-H3; the domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein, the VL has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, and LCDR3 in the VL is Ala or Gly at position 92 (numbering based on the Kabat rule) and Arg at position 93 (numbering based on the Kabat rule); preferably, the VH has a sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

4.

3. A B7-H3 binding molecule comprising a domain that specifically binds B7-H3; the domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises a HCDR1, a HCDR2, and a HCDR3, the VL comprises a LCDR1, a LCDR2, and a LCDR3; wherein: the HCDR1 is as set forth in X1X2GMS, wherein X1 is selected from R, H, K, or S, and X2 is selected from Y or H; the HCDR2 is as set forth in X3IX4SGGGSX5YYX6X7X8VKG (SEQ ID NO: 124), wherein X3 is selected from A or S, X4 is S or W, X5 is selected from I, A, or K, X6 is selected from P or S, X7 is selected from D or Q, and X8 is selected from T, G, or W; the HCDR3 is as set forth in SEQ ID NO: 22; the LCDR1 is as KAX9X 10 X 11 VNTAVA (SEQ ID NO: 125), wherein X9 is selected from P or S, X 10 is selected from Q, R, or K, X 11 is selected from N, D, or G; the LCDR2 is as set forth in SAX 12 NX 13 YX 14 (SEQ ID NO: 126), wherein X 12 is selected from S, T, or K, X 13 is selected from R, G, P, or L, X 14 is selected from T, D, or P; the LCDR3 is as set forth in QQYX 15 X 16 X 17 X 18 T (SEQ ID NO: 127), wherein X 15 is selected from A or G, X 16 is selected from S or R, X 17 is selected from S, Q, G, or H, X 18 is selected from L, I, or P; preferably, the sequences of the HCDR1, HCDR2, and HCDR3, and the LCDR1, LCDR2, and LCDR3 are selected from the combinations of: SEQ ID NO: 31, 40, 22, 41-43; SEQ ID NO: 20-24 and 26; SEQ ID NO: 20-24 and 27; SEQ ID NO: 20-24 and 30 SEQ ID NO: 31, 32, 22, 33-35; SEQ ID NO: 31, 36, 22, 37-39; SEQ ID NO: 44, 45, 22, 33, 46, 47; and SEQ ID NO: 44, 48, 22, 33, 49, 35.

4. The B7-H3 binding molecule of any one of claims 1 to 3, wherein, the heavy chain variable region in the domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 8, 11, 13, 15, 17, and 19, or is at least 80% or 90% identical thereto, the light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 6, 7, 9, 10, 12, 14, 16, and 18, or is at least 80% or 90% identical thereto. Preferably, the heavy chain variable region, light chain variable region is selected from the combination of amino acid sequences of: SEQ ID NO: 15 and 14; SEQ ID NO: 4 and 5; SEQ ID NO: 4 and 6; SEQ ID NO: 8 and 7; SEQ ID NO: 4 and 9; SEQ ID NO: 11 and 10; SEQ ID NO: 13 and 12; SEQ ID NO: 17 and 16; SEQ ID NO: 19 and 18; Preferably, the B7-H3 binding molecule is an anti-B7-H3 antibody.

5. The B7-H3 binding molecule of any one of claims 1 to 4, further comprising an immunoglobulin Fc region; Preferably, the Fc region is an Fc region of IgGl, IgG2, IgG3 or IgG4.

6. A B7-H3 binding molecule, comprising a domain that specifically binds to B7-H3; the binding domain comprises a heavy chain and a light chain, wherein: the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 53 or at least 90% identical thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 52 or at least 90% identical thereto; the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 63 or at least 90% identical thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 62 or at least 90% identical thereto; or the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 67 or at least 90% identical thereto, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 66 or at least 90% identical thereto.

7. An EGFR binding molecule, comprising a domain that specifically binds to EGFR; the domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises HCDR1, HCDR2 and HCDR3 in any one of the amino acid sequences as set forth in SEQ ID NOs: 71, 72 and 75; the VL comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence as set forth in SEQ ID NO: 69; the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system.

8. An EGFR binding molecule, comprising a domain that specifically binds to EGFR; the domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprises HCDR1, HCDR2 and HCDR3, the VL comprises LCDR1, LCDR2 and LCDR3; wherein, HCDR1 is as set forth in SEQ ID NO: 76, HCDR2 is as set forth in SEQ ID NO: 82 or 77, HCDR3 is as set forth in any one of SEQ ID NOs: 78, 83 and 86, LCDR1 is as set forth in SEQ ID NO: 79, LCDR2 is as set forth in SEQ ID NO: 80, LCDR3 is as set forth in SEQ ID NO: 81; and the domain does not comprise a combination of VH and VL as set forth in SEQ ID NOs: 70 and 69; Preferably, the sequences of HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3 are selected from the combinations of: SEQ ID NOs: 76, 82 and 78-81; SEQ ID NOs: 76, 77, 83 and 79-81; or SEQ ID NOs: 76, 77, 86 and 79-81.

9. The EGFR binding molecule of claim 7 or 8, wherein, the heavy chain variable region in the domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 71, 72 and 75, or an amino acid sequence with at least 90% identity thereto, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 69, or an amino acid sequence with at least 90% identity thereto; Preferably, the heavy chain variable region and the light chain variable region are linked as a scFv by a linker; More preferably, the domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 96, 97 and 98, or an amino acid sequence with at least 90% identity thereto; Preferably, the EGFR binding molecule is an anti-EGFR antibody.

10. The EGFR binding molecule of any one of claims 7 to 9, further comprising an immunoglobulin Fc region; Preferably, the Fc region is an Fc region of IgGl, IgG2, IgG3 or IgG4; More preferably, the Fc region comprises an ADCC enhancing mutation; further preferably, the ADCC enhancing mutation is selected from 239D and 332E.

11. A B7-H3 / EGFR binding molecule, comprising: a first binding domain that specifically binds to B7-H3; and a second binding domain that specifically binds to EGFR; the first binding domain that specifically binds to B7-H3 comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), the VH and VL comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 selected from: HCDR1, HCDR2 and HCDR3 in an amino acid sequence as set forth in SEQ ID NO: 15 and LCDR1, LCDR2 and LCDR3 in an amino acid sequence as set forth in SEQ ID NO: 14; HCDR1, HCDR2 and HCDR3 in an amino acid sequence as set forth in SEQ ID NO: 4 and LCDR1, LCDR2 and LCDR3 in an amino acid sequence as set forth in SEQ ID NO: 5; HCDR1, HCDR2 and HCDR3 in an amino acid sequence as set forth in SEQ ID NO: 4 and LCDR1, LCDR2 and LCDR3 in an amino acid sequence as set forth in SEQ ID NO: 6; HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 8 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 7; HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 4 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 9; HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 11 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 10; HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 13 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 12; HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 17 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 16; and HCDR1, HCDR2, and HCDR3 in the amino acid sequence of SEQ ID NO: 18 and LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 19; or the first binding domain comprises a heavy chain variable region (VH1) comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL1) comprising LCDR1, LCDR2, and LCDR3; wherein, HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of the first binding domain have the sequences of: SEQ ID NOs: 31, 40, 22, 41-43; SEQ ID NOs: 20-24 and 26; SEQ ID NOs: 20-24 and 27; SEQ ID NOs: 20-24 and 30; SEQ ID NOs: 31, 32, 22, 33-35; SEQ ID NOs: 31, 36, 22, 37-39; SEQ ID NOs: 44, 45, 22, 33, 46, 47; SEQ ID NOs: 44, 48, 22, 33, 49, 35; or SEQ ID NOs: 28, 29, and 22-25.

12. The B7-H3 / EGFR binding molecule of claim 11, wherein, the second binding domain comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein, the VH2 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence of any one of SEQ ID NOs: 70-75; the VL2 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 69; the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; or, the second binding domain comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein, the VH2 comprises HCDR1, HCDR2, and HCDR3 in the amino acid sequence of any one of SEQ ID NOs: 70-75; the VL2 comprises LCDR1, LCDR2, and LCDR3 in the amino acid sequence of SEQ ID NO: 69; the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; or, the second binding domain comprises a heavy chain variable region (VH2) comprising HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL2) comprising LCDR1, LCDR2, and LCDR3; wherein the sequences of HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 of the second binding domain are selected from the following combinations: SEQ ID NO: 76, 82, and 78-81; SEQ ID NO: 76-81; SEQ ID NO: 76, 77, 83, and 79-81; SEQ ID NO: 76, 84, and 78-81; SEQ ID NO: 76, 85, and 78-81; or SEQ ID NO: 76, 77, 86, and 79-81.

13. The B7-H3 / EGFR binding molecule of claim 11 or 12, wherein, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of VH1 and VL1 in the first binding domain, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of VH2 and VL2 in the second binding domain, respectively, are as follows: SEQ ID NO: 31, 40, 22, 41, 42, 43, and SEQ ID NO: 76-81; SEQ ID NO: 31, 40, 22, 41, 42, 43, and SEQ ID NO: 76, 82, 78-81; SEQ ID NO: 31, 40, 22, 41, 42, 43, and SEQ ID NO: 76, 77, 86, 79-81; SEQ ID NO: 20, 21, 22, 23, 24, 26, and SEQ ID NO: 76, 82, 78-81; SEQ ID NO: 44, 48, 22, 33, 49, 35, and SEQ ID NO: 76, 82, 78-81; or SEQ ID NO: 20, 21, 22, 23, 24, 25, and SEQ ID NO: 76, 82, 78-81.

14. The B7-H3 / EGFR binding molecule of any one of claims 11 to 13, wherein, the heavy chain variable region of the first binding domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 4, 8, 11, 13, 15, 17, and 19, or at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 3, 5, 6, 7, 9, 10, 12, 14, 16, and 18, or at least 90% identical thereto; and / or, the heavy chain variable region of the second binding domain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 70, 71, and 75, or at least 90% identical thereto, and the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 69, or at least 90% identical thereto; Preferably, VH1 and VL1 in the first binding domain, and VH2 and VL2 in the second binding domain are respectively as shown below: SEQ ID NO: 15, 14, 70 and 69; SEQ ID NO: 15, 14, 71 and 69; SEQ ID NO: 15, 14, 75 and 69; SEQ ID NO: 4, 5, 71 and 69; SEQ ID NO: 19, 18, 71 and 69; or SEQ ID NO: 4, 3, 71 and 69; Preferably, the B7-H3 / EGFR binding molecule is an anti-B7-H3 / EGFR antibody.

15. The B7-H3 / EGFR binding molecule of any one of claims 11 to 14, wherein the VH2 and the VL2 are linked as a scFv by a linker; Preferably, the connector is selected from (G m S n ) h 、(G m Q n ) h (GGNGT) h (YGNGT) h (EPKSS) h As shown, where, m, n are each independently selected from an integer of 1-8, and h is independently selected from an integer of 1-20; More preferably, the second binding domain comprises an amino acid sequence as shown in any one of SEQ ID NO: 96, 97 and 98, or an amino acid sequence with at least 90% identity thereto.

16. The B7-H3 / EGFR binding molecule of any one of claims 11 to 15, further comprising an immunoglobulin Fc region; Preferably, the Fc region is an Fc region of IgG1, IgG2, IgG3 or IgG4; More preferably, the Fc region comprises an ADCC enhancing mutation; further preferably, the ADCC enhancing mutation is selected from 239D and 332E.

17. The B7-H3 / EGFR binding molecule of claim 16, wherein, the Fc region comprises a first subunit and a second subunit, the first subunit and the second subunit comprise a knob-into-hole mutation; Preferably, the first subunit comprises a knob mutation, and the second subunit comprises a hole mutation; Preferably, the first subunit comprises a mutation at position 366, and the second subunit comprises a mutation selected from the group consisting of 366, 368 and 407, or any combination thereof; or the first subunit comprises a mutation at position 354 or 356, and the second subunit comprises a mutation at position 349; or the first subunit comprises mutations at positions 354 and 366, and the second subunit comprises mutations at positions 349, 366, 368 and 407; More preferably, the first subunit comprises a mutation 366W, and the second subunit comprises a mutation selected from the group consisting of 366S, 368A and 407V, or any combination thereof; the first subunit comprises a mutation 354C or 356C, and the second subunit comprises a mutation 349C; or the first subunit comprises mutations 354C and 366W, and the second subunit comprises mutations 349C, 366S, 368A and 407V.

18. The B7-H3 / EGFR binding molecule of any one of claims 11 to 17, comprising: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein, in order from N-terminus to C-terminus: (I) the first polypeptide chain comprises the following structure: [VH1 of the first binding domain]-CH1-[Fc first subunit], the second polypeptide chain comprises the structure: [VL1 of the first binding domain] - CL; the third polypeptide chain comprises the structure: [VL2 of the second binding domain] - [linker]a- [VH2 of the second binding domain] - [linker]b- [Fc second subunit]; (I) the first polypeptide chain comprises the structure: [VH1 of the first binding domain] - CH1 - [Fc second subunit], the second polypeptide chain comprises the structure: [VL1 of the first binding domain] - CL; the third polypeptide chain comprises the structure: [VL2 of the second binding domain] - [linker]a- [VH2 of the second binding domain] - [linker]b- [Fc first subunit]; (II) the first polypeptide chain comprises the structure: [VH1 of the first binding domain] - CH1 - [Fc second subunit], the second polypeptide chain comprises the structure: [VL1 of the first binding domain] - CL; the third polypeptide chain comprises the structure: [VL2 of the second binding domain] - [linker]a- [VH2 of the second binding domain] - [linker]b- [Fc first subunit]; (III) the first polypeptide chain comprises the structure: [VH1 of the first binding domain] - CH1 - [Fc first subunit], the second polypeptide chain comprises the structure: [VL1 of the first binding domain] - CL; the third polypeptide chain comprises the structure: [VL2 of the second binding domain] - [linker]a- [VH2 of the second binding domain] - [linker]b- [Fc second subunit]; (IV) the first polypeptide chain comprises the structure: [VH1 of the first binding domain] - CH1 - [Fc second subunit], The linker is a polypeptide capable of performing a linking function, preferably (G x S y ) n , x is selected from an integer from 1-6, y is selected from an integer from 0-4, n is selected from an integer from 1-6. the second polypeptide chain comprises the structure: [VL1 of the first binding domain] - CL; the third polypeptide chain comprises the structure: [VL2 of the second binding domain] - [linker]a- [VH2 of the second binding domain] - [linker]b- [Fc first subunit]; wherein - denotes a peptide bond, a is selected from 0 or 1, and b is selected from 0 or 1; 19. The B7-H3 / EGFR binding molecule of claim 18, comprising a combination of polypeptide chains selected from: 1) a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 99 or 110, or having at least 90% sequence identity thereto, a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 62, or having at least 90% sequence identity thereto, and a third polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 100 or 114, or having at least 90% sequence identity thereto, 2) a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 99, 110, or 115, or having at least 90% sequence identity thereto, a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 62, or having at least 90% sequence identity thereto, and a third polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 101, 111, or 116, or having at least 90% sequence identity thereto, 3) a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 99 or 110, or having at least 90% sequence identity thereto, a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 62, or having at least 90% sequence identity thereto, and a third polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 102 or 113, or having at least 90% sequence identity thereto, 4) a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 103 or 112, or having at least 90% sequence identity thereto, a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 52, or having at least 90% sequence identity thereto, and a third polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 101 or 111, or having at least 90% sequence identity thereto, 5) a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 105 or 117, or having at least 90% sequence identity thereto, a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 55, or having at least 90% sequence identity thereto, and a third polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 101 or 111, or having at least 90% sequence identity thereto, or 6) a first polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 104, or having at least 90% sequence identity thereto, a second polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 66, or having at least 90% sequence identity thereto, and a third polypeptide chain comprising an amino acid sequence as set forth in SEQ ID NO: 101, or having at least 90% sequence identity thereto.

20. A B7-H3 / EGFR binding molecule comprising: a first binding domain that specifically binds to B7-H3; and a second binding domain that specifically binds to EGFR; wherein the first binding domain is a Fab and the second binding domain is a scFv; preferably, the first binding domain is a B7-H3 binding molecule as defined in any one of claims 1 to 6, preferably, the second binding domain is an EGFR binding molecule as defined in any one of claims 7 to 10.

21. An antibody drug conjugate comprising: an antibody (Ab); and a drug molecule, the Ab comprising a first binding domain that specifically binds to B7-H3 and / or a second binding domain that specifically binds to EGFR, wherein the first binding domain and the second binding domain are respectively a first binding domain and a second binding domain as defined in any one of claims 11 to 20; preferably, the drug molecule is a cytotoxic drug; more preferably, the cytotoxic drug is selected from MMAE or a derivative thereof, exatecan or a derivative thereof, eribulin or a derivative thereof.

22. The antibody drug conjugate of claim 21, wherein the drug molecule is selected from MMAE or a derivative thereof, exatecan or a derivative thereof, eribulin or a derivative thereof.

23. The antibody drug conjugate of claim 22, which is of the structure shown in formula (IV): wherein: Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- or -S-(CR a R b ) m -CR 1 R 2 -C(O)-; R a and R b are the same or different and each is independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxy group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, or a heterocyclyl group; or, R a and R b together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclyl group; R 1 is selected from a hydrogen atom, an alkyl group, a halogen, a haloalkyl group, a deuterium- substituted alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group; R 2 is selected from a hydrogen atom, an alkyl group, a halogen, a haloalkyl group, a deuterium- substituted alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclyl group, an aryl group, or a heteroaryl group; or R 1 and R 2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl; or R a and R 2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl; m is an integer from 0 to 4; n is an integer or a decimal number from 1 to 10; -L- is a linker unit; Preferably, -L- is -L 1 -L 2 -L 3 -L 4 -, wherein: L 1 -(succinimid-3-yl-N)-W-C(O)-, -CH2-C(O)-NR 3 -W-C(O)- or -C(O)-W-C(O)-, wherein W is selected from C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, C 1-8 alkyl, L 2 -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)- or a chemical bond, wherein p 1 is an integer from 1 to 20; L 3 a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterium-alkyl, alkoxy, and cycloalkyl; L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 -C(O)NR 5 (CH2) t - or chemical bond, where t is an integer from 1 to 6; R 3 , R 4 , and R 5 are the same or different and each is independently selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; R 6 and R 7 are the same or different and each is independently selected from the group consisting of a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

24. The antibody drug conjugate of claim 23, wherein: Y is -O-(CR a R b )m-CR 1 R 2 -C(O)-; R a and R b are the same or different and each is independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, a C 1-6 alkyl group, a C 1-6 haloalkyl group, a C 1-6 deuterated alkyl group, a C 1-6 alkoxy group, a hydroxyl group, and a C 1-6 hydroxyalkyl group; R 1 selected from a hydrogen atom, C 1-6 alkyl, C 3-6 cycloalkyl C 1-6 alkyl or C 3-6 cycloalkyl; R 2 selected from a hydrogen atom, a halogen, C 1-6 haloalkyl, C 1-6 deuteroalkyl, C 3-6 cycloalkyl and C 3-6 cycloalkyl C 1-6 alkyl; or R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl; or R a and R 2 together with the carbon atom to which they are attached form C 3-6 cycloalkyl; m is 0 or 1 ; Preferably, Y is -O-(CR a R b )m-CR 1 R 2 -C(O)-; R a and R b are the same or different and each is independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, and a C 1-6 alkyl group; R 1 is C 3-6 cycloalkyl C 1-6 alkyl or C 3-6 cycloalkyl; R 2 selected from a hydrogen atom, C 1-6 haloalkyl and C 3-6 cycloalkyl; or R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl; m is 0 or 1; and / or, Linker unit -L- is -L 1 -L 2 -L 3 -L 4 - L 1 -(succinimid-3-yl-N)-W-C(O)-, wherein W is C 1-8 alkyl or C 1-8 alkyl-C 3-8 cycloalkyl; L 2 -NR 4 (CH2CH2O)p 1 CH2C(O)- or a chemical bond, wherein p 1 is an integer from 1 to 20; L 3 is a peptide residue consisting of 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid; L 4 selected from -NR 5 (CR 6 R 7 ) t - wherein t is an integer from 1 to 6; R 4 selected from a hydrogen atom, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 deuteroalkyl and C 1-6 hydroxyalkyl; R 5 selected from a hydrogen atom, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 deuterated alkyl and C 1-6 hydroxyalkyl; R 6 and R 7 are the same or different and each is independently selected from the group consisting of a hydrogen atom, a C 1-6 alkyl group, a C 1-6 haloalkyl group, a C 1-6 deuteroalkyl group, and a C 1-6 hydroxyalkyl group; Preferably, the linker unit -L- is -L 1 -L 2 -L 3 -L 4 - L 1 for s 1 is an integer from 2 to 8; L 2 is a chemical bond; L 3 tetrapeptide residue of the form GGFG; L 4 -NR 5 (CR 6 R 7 ) t -,R 5 is a hydrogen atom or a C 1-6 alkyl group, R 6 and R 7 are the same or different and each independently a hydrogen atom or a C 1-6 alkyl group, and t is 1 or 2.

25. The antibody drug conjugate of any one of claims 22 to 24, which is of the structure shown in formula (III): wherein: s 1 is an integer from 2 to 8; preferably 5; m is 0 or 1; R 1 is C 3-6 cycloalkyl C 1-6 alkyl or C 3-6 cycloalkyl; R 2 selected from a hydrogen atom, C 1-6 haloalkyl and C 3-6 cycloalkyl; or R 1 and R 2 together with the carbon atom to which they are attached form C 3-6 cycloalkyl; R 5 selected from a hydrogen atom, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 deuterated alkyl and C 1-6 hydroxyalkyl; R 6 and R 7 are the same or different and each is independently selected from the group consisting of a hydrogen atom, a C 1-6 alkyl group, a C 1-6 haloalkyl group, a C 1-6 deuteroalkyl group, and a C 1-6 hydroxyalkyl group.

26. The antibody drug conjugate of any one of claims 22 to 25, wherein -L-Y- is optionally selected from: Preferably 27. A method of preparing the antibody drug conjugate of any one of claims 21 to 26, comprising the step of conjugating the Ab to the drug molecule.

28. A polynucleotide encoding: The B7-H3 binding molecule of any one of claims 1 to 6, The EGFR binding molecule of any one of claims 7 to 10, or The B7-H3 / EGFR binding molecule of any one of claims 11 to 20.

29. A vector comprising or expressing the polynucleotide of claim 28.

30. A host cell containing or expressing the polynucleotide of claim 28 or the vector of claim 29.

31. A method of making a B7-H3 binding molecule, an EGFR binding molecule, or a B7-H3 / EGFR binding molecule, comprising: expressing the polynucleotide of claim 28 or the vector of claim 29 in the host cell of claim 30, and isolating the expressed B7-H3 binding molecule, EGFR binding molecule, or B7-H3 / EGFR binding molecule from the host cell; optionally, further comprising a step of purifying the B7-H3 binding molecule, EGFR binding molecule, or B7-H3 / EGFR binding molecule.

32. A pharmaceutical composition comprising: the B7-H3 / EGFR binding molecule of any one of claims 11 to 20 or the antibody drug conjugate of any one of claims 21 to 26, and, at least one pharmaceutically acceptable excipient, diluent, or carrier.

33. A method of treating cancer, comprising a step of (1) or (2) as follows: (1) administering to a subject in need thereof a therapeutically effective amount of the B7-H3 / EGFR binding molecule of any one of claims 11 to 20, the antibody drug conjugate of any one of claims 21 to 26, or the pharmaceutical composition of claim 32; or (2) administering to a subject in need thereof a therapeutically effective amount of the B7-H3 binding molecule of any one of claims 1 to 6, the EGFR binding molecule of any one of claims 7 to 10, the polynucleotide of claim 28, or the vector of claim 29; preferably, the cancer is B7-H3 and EGFR double positive.

34. The method of claim 33, wherein the cancer is selected from the group consisting of head and neck cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer.

35. Use of (1) or (2) as follows: (1) the B7-H3 / EGFR binding molecule of any one of claims 11 to 20, the antibody drug conjugate of any one of claims 21 to 26, or the pharmaceutical composition of claim 32 for the manufacture of a medicament for treating cancer; or (2) the B7-H3 binding molecule of any one of claims 1 to 6, the EGFR binding molecule of any one of claims 7 to 10, the polynucleotide of claim 28, or the vector of claim 29 for the manufacture of a medicament for treating cancer; preferably, the cancer is B7-H3 and EGFR double positive.

36. The use of claim 35, wherein the cancer is selected from the group consisting of head and neck cancer, non-small cell lung cancer, esophageal cancer.

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