Trop2 / MUC1 binding molecule and medical use

Abstract

Provided are a Trop2 / MUC1 binding molecule and a medical use. Specifically provided are an anti-Trop2 / MUC1 bispecific antibody, an anti-Trop2 / MUC1 antibody-drug conjugate, a pharmaceutical composition containing the antibody or the conjugate, a method for treating cancer, and a pharmaceutical use.

Description

Trop2 / MUC1 binding molecules and their pharmaceutical applications Technical Field

[0001] This disclosure relates to Trop2 / MUC1 binding molecules, such as bispecific antibodies against Trop2 / MUC1, anti-Trop2 / MUC1 antibody-drug conjugates, and their use as drugs for the treatment of cancer. Background Technology

[0002] Trop2 (Trophoblast cell-surface antigen 2) is a single-pass transmembrane glycoprotein with a molecular weight of 36 kDa, encoded by the tumor-associated calcium signal transducer 2 (TACSTD2) gene. Trop2 is overexpressed in various cancer cells, such as non-small cell lung cancer (NSCLC), breast cancer, urothelial carcinoma, and gastrointestinal tumors, and is negatively correlated with prognosis. Trop2 participates in the regulation of multiple growth signaling pathways, such as MAPK and PI3K / AKT, playing a crucial role in tumor development. The high expression and biological functions of Trop2 in tumors make it a suitable drug target. Currently, drug development targeting Trop2 is underway, exploring various mechanisms. Trodelvy, an antibody-drug conjugate (ADC) drug linked to SN38, has been approved for triple-negative breast cancer (TNBC) and urothelial carcinoma. Trodelvy significantly improves patients' objective response rate (ORR) and prolongs overall survival (OS), demonstrating the clinical efficacy of Trop2 ADC drugs. However, due to the high expression of Trop2 in normal oral cells, Trodelvy results in a high rate of stomatitis as a side effect in clinical practice. Therefore, the development of drugs that can effectively reduce the clinical toxicity of Trop2 ADCs will bring clinical benefits to patients.

[0003] MUC1 is a single-transmembrane glycoprotein. In normal tissues, MUC1 is highly glycosylated extracellularly, forming a tight barrier on the surface of epithelial cells to protect them from extreme environmental influences. In tumor cells, MUC1 exhibits a hypoglycosylated phenotype and displays tumor cell-specific glycopeptide epitopes. MUC1 mediates intracellular signaling functions, plays an important role in tumor development, and is highly expressed in various epithelial tumor cells.

[0004] This disclosure provides an anti-MUC1 antibody with higher binding and endocytic activity, and provides a target for Trop2 / MUC1 binding molecules, enhancing selectivity for tumors. Summary of the Invention

[0005] This disclosure relates to Trop2 binding molecules, MUC1 binding molecules, Trop2 / MUC1 binding molecules and their encoded nucleic acids, vectors, host cells, pharmaceutical compositions, and methods for treating cancer and related pharmaceutical uses.

[0006] Trop2 binding molecules

[0007] This disclosure provides a Trop2-binding molecule comprising a first binding domain that specifically binds to Trop2.

[0008] In some embodiments, the first binding domain that specifically binds Trop2 in the Trop2-binding molecule comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO: 1 or 50; and the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO: 2 or 3; wherein the CDR is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0009] In some embodiments, the first binding domain of the Trop2 binding molecule comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2, and HCDR3, and the VL1 comprises LCDR1, LCDR2, and LCDR3; wherein the VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 48, 9, and 10, respectively; and the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 11, 47, and 13, respectively.

[0010] The HCDR1 is shown as X2YGMN (SEQ ID NO: 48), where X2 is N or E;

[0011] The LCDR2 is shown as SASX1RYT (SEQ ID NO: 47), where X1 is Y or T.

[0012] In some specific implementations, the sequences of HCDR1, HCDR2, and HCDR3 of the first binding domain, as well as LCDR1, LCDR2, and LCDR3, are as shown in SEQ ID NO: 8-13 or SEQ ID NO: 8-11, 14, and 13. These are CDRs defined according to the Kabat numbering system.

[0013] In some embodiments, in the aforementioned Trop2 binding molecule, the heavy chain variable region of the first binding domain that specifically binds Trop2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 1 or 50, and the light chain variable region contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 2 or 3.

[0014] In some specific implementations, in the first structural domain,

[0015] VH1 contains an amino acid sequence as shown in SEQ ID NO:1 or having at least 90% sequence identity with it, and VL1 contains an amino acid sequence as shown in SEQ ID NO:2 or having at least 90% sequence identity with it.

[0016] VH1 contains an amino acid sequence as shown in SEQ ID NO:1 or having at least 90% sequence identity with it, and VL1 contains an amino acid sequence as shown in SEQ ID NO:3 or having at least 90% sequence identity with it; or

[0017] VH1 comprises an amino acid sequence as shown in SEQ ID NO:50 or having at least 90% sequence identity with it, and VL1 comprises an amino acid sequence as shown in SEQ ID NO:2 or having at least 90% sequence identity with it.

[0018] In some specific implementations, the above-mentioned amino acid mutations are amino acid substitutions, replacements, modifications, deletions, and / or additions (e.g., conserved substitutions of amino acids), and the mutations do not affect or substantially do not affect the function of specifically binding to Trop2.

[0019] In some embodiments, the aforementioned Trop2 binding molecule further comprises an immunoglobulin Fc region. In some embodiments, the Trop2 binding molecule comprises a human immunoglobulin Fc region. For example, the Fc region is the Fc region of human IgG1, IgG2, or IgG4. In some specific embodiments, the human immunoglobulin Fc region is the Fc region of wild-type IgG or a variant thereof.

[0020] In some implementations, the Fc region is an Fc region that enhances effector function, for example, enhancing antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), and / or complement-dependent cytotoxicity (CDC) with enhanced effector function.

[0021] An exemplary IgG1 Fc region includes substitutions having the following: 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; 2 80H / 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.

[0022] An exemplary IgG1 Fc region includes substitutions having the following: 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; D 280H / 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 the above positions.

[0023] In some embodiments, the Trop2 binding molecule comprises a heavy chain and a light chain, wherein:

[0024] The heavy chain is an amino acid sequence as shown in SEQ ID NO:43 or having at least 90% sequence identity with it, and the light chain is an amino acid sequence as shown in SEQ ID NO:44 or having at least 90% sequence identity with it.

[0025] In some embodiments, the Trop2 binding molecule disclosed herein is an anti-Trop2 antibody, or a conjugate or fusion protein containing said antibody.

[0026] In some embodiments, the aforementioned Trop2 binding molecule has a concentration of ≤10 -7 K D Values ​​associated with human Trop2 or its epitopes, for example, ≤1×10 -7 M.

[0027] MUC1 binding molecules

[0028] This disclosure provides a MUC1 binding molecule comprising a second binding domain that specifically binds to MUC1.

[0029] In some embodiments, the second binding domain of the MUC1 binding molecule comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein VH2 comprises HCDR1, HCDR2, and HCDR3, and VL2 comprises LCDR1, LCDR2, and LCDR3; wherein HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 respectively comprise amino acid sequences as shown in SEQ ID NO:15, 49, and 17-20;

[0030] Wherein, HCDR2 is as shown in EIRLKSNNYX3THYAESVKG (SEQ ID NO: 49), and X3 in HCDR2 is T, G, D, N or P.

[0031] In some embodiments, the second binding domain of the MUC1 binding molecule comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein VH2 comprises HCDR1, HCDR2, and HCDR3, and VL2 comprises LCDR1, LCDR2, and LCDR3; wherein HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 respectively comprise amino acid sequences as shown in SEQ ID NO:15, 67, and 17-20;

[0032] Wherein, HCDR2 is as shown in EIRLKSNNYX3THYAESVKG (SEQ ID NO: 67), and X3 in HCDR2 is an amino acid other than T (Thr).

[0033] In some specific implementations, the sequences of HCDR1, HCDR2, and HCDR3 of the second binding domain, LCDR1, LCDR2, and LCDR3 are as follows: as shown in SEQ ID NO:15-20 or SEQ ID NO:15, 22, 17-20.

[0034] It is a CDR defined according to the Kabat numbering system.

[0035] In some embodiments, the second binding domain of the MUC1 binding molecule comprises a heavy chain variable region (VH2) and a light chain variable region (VL2), wherein the VH2 comprises HCDR1, HCDR2, and HCDR3, and the VL2 comprises LCDR1, LCDR2, and LCDR3; wherein HCDR1, HCDR2, and HCDR3, as well as LCDR1, LCDR2, and LCDR3, are as shown in SEQ ID NO:25-30, respectively.

[0036] In some embodiments, in the aforementioned MUC1 binding molecule, the heavy chain variable region in the second binding domain that specifically binds MUC1 contains an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO:4, 6, 7 and 23, and the light chain variable region contains an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO:5 or 24.

[0037] In some specific implementations, in the second structural domain,

[0038] VH2 contains an amino acid sequence as shown in SEQ ID NO:4 or having at least 90% sequence identity with it, and VL2 contains an amino acid sequence as shown in SEQ ID NO:5 or having at least 90% sequence identity with it;

[0039] VH2 contains an amino acid sequence as shown in SEQ ID NO:6 or having at least 90% sequence identity with it, and VL2 contains an amino acid sequence as shown in SEQ ID NO:5 or having at least 90% sequence identity with it;

[0040] VH2 contains an amino acid sequence as shown in SEQ ID NO:7 or having at least 90% sequence identity with it, and VL2 contains an amino acid sequence as shown in SEQ ID NO:5 or having at least 90% sequence identity with it;

[0041] VH2 contains an amino acid sequence as shown in SEQ ID NO:23 or having at least 90% sequence identity with it, and VL2 contains an amino acid sequence as shown in SEQ ID NO:24 or having at least 90% sequence identity with it.

[0042] In some specific implementations, the above-mentioned amino acid mutations are amino acid substitutions, replacements, modifications, deletions and / or additions (e.g., conserved substitutions of amino acids), and the mutations do not affect or substantially do not affect the function of specifically binding to MUC1.

[0043] In some embodiments, the aforementioned MUC1 binding molecule further comprises an immunoglobulin Fc region. In some embodiments, the MUC1 binding molecule comprises a human immunoglobulin Fc region. For example, the Fc region is the Fc region of human IgG1, IgG2, or IgG4. In some specific embodiments, the human immunoglobulin Fc region is the Fc region of wild-type IgG or a variant thereof.

[0044] In some implementations, the Fc region is an Fc region that enhances effector function, for example, enhancing antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), and / or complement-dependent cytotoxicity (CDC) with enhanced effector function.

[0045] An exemplary IgG1 Fc region includes substitutions having the following: 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; 2 80H / 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.

[0046] An exemplary IgG1 Fc region includes substitutions having the following: 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; D 280H / 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 the above positions.

[0047] In some embodiments, the MUC1 binding molecule disclosed herein is an anti-MUC1 antibody, or a conjugate or fusion protein containing said antibody.

[0048] In some embodiments, the aforementioned MUC1 binding molecule has a concentration of ≤10 -7 K D Values ​​associated with human MUC1 or its epitopes, for example, ≤1×10 -7 M, ≤1×10 -8 M, or ≤1×10 -9 M.

[0049] In some specific implementation schemes, the aforementioned MUC1 binding molecule has a higher affinity than antibody P, and the detection method is as described in Example 4.

[0050] Trop2 / MUC1 binding molecules

[0051] This disclosure provides a Trop2 / MUC1 binding molecule comprising a first binding domain that specifically binds Trop2 and a second binding domain that specifically binds MUC1.

[0052] This disclosure provides a Trop2 / MUC1 binding molecule comprising a first binding domain that specifically binds Trop2 and a second binding domain that specifically binds to the N-terminus of MUC1.

[0053] In some embodiments, the second binding domain specifically binds to a glycosylated polypeptide at the N-terminus of MUC1, such as APPAHGVTSAPDTRPAPGSTAPPAHGVTSA (SEQ ID NO: 65). In some specific embodiments, the glycosylated polypeptide is shown as APPAHGVTSAPDT(GalNAc)RPAPGSTAPPAHGVTSA (SEQ ID NO: 64).

[0054] In some implementations, the second binding domain at the N-terminus of the MUC1-specific binding site competes for binding with antibody P or PT59G.

[0055] In some implementations, the second binding domain at the N-terminus of the MUC1 that specifically binds to the same epitope as antibody P or PT59G.

[0056] This disclosure provides a Trop2 / MUC1 binding molecule comprising a first binding domain that specifically binds Trop2 and a second binding domain that specifically binds to the C-terminus of MUC1.

[0057] In some embodiments, the second binding domain at the C-terminus of the specifically binding MUC1 specifically binds the polypeptide shown in SEQ ID NO:66.

[0058] In some implementations, the second binding domain of the C-terminus of the specifically binding MUC1 competes for binding with SDP02313.

[0059] In some implementations, the second binding domain at the C-terminus of the specifically binding MUC1 binds to the same epitope as SDP02313.

[0060] In some embodiments, in the Trop2 / MUC1 binding molecule, each binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0061] In some embodiments, in the Trop2 / MUC1 binding molecule, the first binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO: 1 or 50; and the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO: 2 or 3. The CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0062] In some embodiments, in the Trop2 / MUC1 binding molecule, the first binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2, and HCDR3, and the VL1 comprises LCDR1, LCDR2, and LCDR3; wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO: 48, 9, and 10, respectively; and LCDR1, LCDR2, and LCDR3 comprise amino acid sequences as shown in SEQ ID NO: 11, 47, and 13, respectively.

[0063] The HCDR1 is shown as X2YGMN (SEQ ID NO: 48), where X2 is N or E;

[0064] The LCDR2 is shown as SASX1RYT (SEQ ID NO: 47), where X1 is Y or T.

[0065] In some specific embodiments, the HCDR1, HCDR2, and HCDR3 of the first binding domain, as well as LCDR1, LCDR2, and LCDR3, respectively comprise amino acid sequences as shown in SEQ ID NO: 8-13 or SEQ ID NO: 8-11, 14, and 13. These are CDRs defined according to the Kabat numbering system.

[0066] In some embodiments, in the Trop2 / MUC1 binding molecule, 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, and the VL2 comprises LCDR1, LCDR2, and LCDR3; wherein the HCDR1, HCDR2, and HCDR3 of the second binding domain, and LCDR1, LCDR2, and LCDR3, respectively comprise amino acid sequences as shown in SEQ ID NO: 15, 49, and 17-20;

[0067] The HCDR2 is shown as EIRLKSNNYX3THYAESVKG (SEQ ID NO: 49).

[0068] In some specific implementations, X3 in HCDR2 is T, G, D, N, or P.

[0069] In some specific embodiments, the second binding domains HCDR1, HCDR2, and HCDR3, LCDR1, LCDR2, and LCDR3 respectively contain amino acid sequences as shown in SEQ ID NO:15-20 or SEQ ID NO:15, 22, 17-20.

[0070] It is a CDR defined according to the Kabat numbering system.

[0071] In some embodiments, in the Trop2 / MUC1 binding molecule, 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, and the VL2 comprises LCDR1, LCDR2, and LCDR3; wherein the HCDR1, HCDR2, and HCDR3 of the second binding domain, as well as LCDR1, LCDR2, and LCDR3, each comprise the amino acid sequences shown in SEQ ID NO:25-30.

[0072] In some embodiments, in the Trop2 / MUC1 binding molecule, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of VH1 and VL1 in the first binding domain that specifically binds Trop2 respectively contain amino acid sequences as shown in SEQ ID NO: 48, 9, 10, 11, 47, and 13, respectively; and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of VH2 and VL2 in the second binding domain that specifically binds MUC1 respectively contain amino acid sequences as shown in SEQ ID NO: 15, 49, and 17-20, respectively.

[0073] In some specific embodiments, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of VH1 and VL1 in the first binding domain that specifically binds to Trop2, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of VH2 and VL2 in the second binding domain that specifically binds to MUC1, respectively contain the following:

[0074] The amino acid sequences shown in SEQ ID NO: 8-13 and SEQ ID NO: 15-20;

[0075] The amino acid sequences shown in SEQ ID NO: 8-13 and SEQ ID NO: 15, 22, 17-20;

[0076] The amino acid sequences shown in SEQ ID NO: 8-13 and SEQ ID NO: 25-30;

[0077] The amino acid sequences shown in SEQ ID NO: 8-11, 14 and 13, and SEQ ID NO: 15-20;

[0078] The amino acid sequences shown in SEQ ID NO: 8-11, 14 and 13, and SEQ ID NO: 15, 22, 17-20;

[0079] The amino acid sequences shown in SEQ ID NO: 8-11, 14 and 13, and SEQ ID NO: 25-30.

[0080] In some embodiments, in the aforementioned Trop2 / MUC1 binding molecule, the heavy chain variable region (VH1) of the first binding domain specifically binding Trop2 comprises an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 1 or 50, and the light chain variable region (VL1) comprises an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 2 or 3; and / or,

[0081] The heavy chain variable region (VH2) in the second binding domain that specifically binds to MUC1 contains an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 4, 6, 7 and 23, and the light chain variable region (VL2) contains an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 5 or 24.

[0082] In some specific embodiments, the VH1 and VL1 in the first binding domain that specifically binds Trop2, and the VH2 and VL2 in the second binding domain that specifically binds MUC1, are respectively as follows:

[0083] The VH1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 1, the VL1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 2, the VH2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 4, and the VL2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 5.

[0084] The VH1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 1, the VL1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 2, the VH2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 6, and the VL2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 5.

[0085] The VH1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 1, the VL1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 2, the VH2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 7, and the VL2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 5.

[0086] The VH1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 1, the VL1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 2, the VH2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 23, and the VL2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 24.

[0087] The VH1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 1, the VL1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 3, the VH2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 4, and the VL2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 5.

[0088] The VH1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 1, the VL1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 3, the VH2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 6, and the VL2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 5.

[0089] The VH1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 1, the VL1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 3, the VH2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 7, and the VL2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 5.

[0090] The VH1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 1, the VL1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 3, the VH2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 23, and the VL2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 24.

[0091] The VH1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 50, the VL1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 2, the VH2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 4, and the VL2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 5.

[0092] The VH1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 50, the VL1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 2, the VH2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 6, and the VL2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 5.

[0093] The VH1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 50, the VL1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 2, the VH2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 7, and the VL2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 5.

[0094] The VH1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 50, the VL1 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 2, the VH2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 23, and the VL2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 24.

[0095] In some specific implementations, the above-mentioned amino acid mutations are amino acid substitutions, replacements, modifications, deletions, and / or additions (e.g., conserved substitutions of amino acids), which do not affect or substantially do not affect the function of specifically binding Trop2 and MUC1.

[0096] In some embodiments, the aforementioned Trop2 / MUC1 binding molecule further comprises an immunoglobulin Fc region. In some embodiments, the Trop2 / MUC1 binding molecule comprises a human immunoglobulin Fc region. For example, the Fc region is the Fc region of human IgG1, IgG2, or IgG4. In some specific embodiments, the human immunoglobulin Fc region is the Fc region of wild-type IgG or a variant thereof.

[0097] In some implementations, the Fc region is an Fc region that enhances effector function, for example, enhancing antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), and / or complement-dependent cytotoxicity (CDC) with enhanced effector function.

[0098] An exemplary IgG1 Fc region includes substitutions having the following: 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; 2 80H / 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.

[0099] An exemplary IgG1 Fc region includes substitutions having the following: 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; D 280H / 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 the above positions.

[0100] In some embodiments, the Fc region comprises a first subunit (Fc1) and a second subunit (Fc2), and mutations are introduced that cause the two subunits (Fc1, Fc2) of the Fc region to pair and form a dimer, or mutations that reduce homodimerization. In some embodiments, the Fc region comprises knocks-in-hole mutations that promote the union of the first and second subunits. For example, within the CH3 / CH3 interface, one, two, or more amino acid residues in the CH3 domain of Fc1 are mutated with one or more amino acid residues having a larger side chain volume, thereby creating a bump (or pestle, knob) on the surface of the CH3 domain of Fc1; one, two, or more amino acid residues in the CH3 domain of Fc2 that interact with the CH3 domain of Fc1 are mutated with amino acid residues having a smaller side chain volume, thereby creating a depression (or hole, hole) on the surface of the CH3 domain of Fc2 that interacts with the CH3 domain of Fc1. In some specific embodiments, the first subunit of the Fc region comprises a knock mutation, and the second subunit of the Fc region comprises a hole mutation.

[0101] In some embodiments, Fc1 has one or more amino acid substitutions selected from positions 354, 356, 358, and 366, and Fc2 has one or more amino acid substitutions selected from positions 349, 356, 358, 366, 368, and 407. In some specific embodiments, Fc1 contains a mutation at position 366, and Fc2 contains a mutation selected from positions 366, 368, and 407, or any combination thereof; in some specific embodiments, Fc1 contains a mutation at position 354 or 356, and Fc2 contains a mutation at position 349; in some specific embodiments, Fc1 contains a mutation at position 354 or 356, and Fc2 contains mutations at positions 349, 366, 368, and 407.

[0102] In some embodiments, Fc1 has one or more amino acid substitutions selected from 354C, 356C, 356E, 358M, and 366W, and Fc2 has one or more amino acid substitutions selected from 349C, 356E, 358M, 366S, 368A, and 407V. In some specific embodiments, Fc1 contains a 366W mutation, and Fc2 contains a mutation selected from 366S, 368A, and 407V, or any combination thereof; in some specific embodiments, Fc1 contains a 354C or 356C mutation, and Fc2 contains a 349C mutation; or in some specific embodiments, Fc1 contains a 354C / 366W mutation, and Fc2 contains a 349C / 366S / 368A / 407V mutation.

[0103] In some specific implementations, Fc1 is as shown in SEQ ID NO:42.

[0104] In some embodiments, the Trop2 / MUC1 binding molecule contains a linker.

[0105] In some implementations, the connector such as (G) m S n ) h Or (GGNGT) h (SEQ ID NO: 51) or (YGNGT) h (SEQ ID NO: 52) or (EPKSS) h The amino acid sequence shown in (SEQ ID NO: 53) is wherein m and n are each independently selected from integers 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), and h is independently selected from integers 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

[0106] In some implementations, the connector is (G x S) y The connector, wherein x is selected from an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5), and y is selected from an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In some embodiments, the connector is selected from G4S (SEQ ID NO: 54), GS, GAP, (G4S)2 (SEQ ID NO: 55), (G4S)3 (SEQ ID NO: 56), (G4S)4 (SEQ ID NO: 57), (G4S)5 (SEQ ID NO: 58), and ASGS.

[0107] In some embodiments, the Trop2 / MUC1 binding molecule comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain; wherein

[0108] The first heavy chain has the following structure from the N end to the C end: [VH1]-[connector 1]-[Obscurin-O chain]-[connector 3]-[first subunit of Fc region].

[0109] The first light chain has the following structure from the N end to the C end: [VL1]-[connector 2]-[Titin-T chain],

[0110] The second chain has the following structure from the N-terminus to the C-terminus: [VH2]-[CH1]-[the second subunit of the Fc region], and

[0111] The second light chain has the following structure from the N-terminus to the C-terminus: [VL2]-[CL];

[0112] or,

[0113] The first heavy chain has the following structure from the N-terminus to the C-terminus: [VH2]-[connector 1]-[Obscurin-O chain]-[connector 3]-[first subunit of the Fc region].

[0114] The first light chain has the following structure from the N end to the C end: [VL2]-[connector 2]-[Titin-T chain],

[0115] The second chain has the following structure from the N-terminus to the C-terminus: [VH1]-[CH1]-[the second subunit of the Fc region], and

[0116] The second light chain has the following structure from the N-terminus to the C-terminus: [VL1]-[CL];

[0117] Wherein, - represents a peptide bond, and the linker 1, linker 2 and linker 3 may be the same or different, and may exist independently or not exist.

[0118] In some specific implementations, the amino acid sequences of linker 1 and linker 2 are GGGGS, and linker 3 is absent.

[0119] In some implementations, the Titin-T chain is as shown in SEQ ID NO: 59.

[0120] In some embodiments, the Titin-T chain is a variant of SEQ ID NO: 59, the variant being selected from one or more sites having amino acid residue substitutions at positions 3, 8, 11, 13, 20, 22, 25, 26, 39, 40, 42, 45, 47, 49, 56, 58, 60, 64, 66, 70, 75, 77, 79, 81, 82, 83, and 84. Exemplarily, substitutions are made at one or more amino acid residues selected from 3W, 8C, 11I, 13L, 20C, 22M / 22C, 25S, 26C, 39T, 40S, 42K, 45S, 47E, 49G, 56S, 58E, 60S, 64T, 66S / 66K, 70R, 75V, 77S, 79T, 81R, 82M, 83D, and 84L. In some specific embodiments, the Titin-T chain is as shown in SEQ ID NO: 38 or has an amino acid sequence that has at least 80% sequence identity with it.

[0121] In some implementations, the Obscurin-O chain is as shown in SEQ ID NO: 60.

[0122] In some embodiments, the Obscurin-O chain is a variant of SEQ ID NO: 60, which has amino acid residue substitutions at one or more sites selected from positions 2, 3, 7, 9, 11, 12, 13, 14, 17, 20, 22, 25, 30, 32, 34, 36, 41, 42, 44, 45, 48, 53, 58, 62, 66, 67, 69, 76, 82, 88, 89, 92, 93, 94, and 97. Exemplarily, one or more amino acid residues selected from 2E, 3C, 7K / 7R, 9C, 11L, 12S, 13Y / 13S, 14T, 17E, 20L, 22M / 22S, 25S, 30D, 32P / 32F, 34E, 36T, 41K, 42L, 44I, 45T, 48V, 53L, 58V, 62E / 62K / 62H, 66C, 67Q / 67T, 69S, 76S, 82H, 88C, 89L, 92E, 93C, 94G, and 97G are substituted. In some specific embodiments, the Obscurin-O chain is an amino acid sequence as shown in SEQ ID NO: 37 or having at least 80% sequence identity with it.

[0123] In some embodiments, the Trop2 / MUC1 binding molecule comprises a combination of polypeptide chains:

[0124] 1) A first heavy chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 33, a first light chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 34, a second heavy chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 31, and a second light chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 32.

[0125] or

[0126] 2) A first heavy chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 33, a first light chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 34, a second heavy chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 35, and a second light chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 36.

[0127] In some embodiments, this disclosure provides a Trop2 / MUC1 binding molecule comprising a first binding domain that specifically binds Trop2 and a second binding domain that specifically binds MUC1; its endocytic activity in Trop2 and MUC1 dual-positive cells is greater than that of the Trop2 binding molecule.

[0128] In some specific embodiments, this disclosure provides a Trop2 / MUC1 binding molecule comprising a first binding domain that specifically binds Trop2 and a second binding domain that specifically binds the C-terminus of MUC1; its endocytic activity in Trop2 and MUC1 dual-positive cells is greater than that of a binding molecule that binds only Trop2 and greater than that of a binding molecule that binds only the C-terminus of MUC1. For example, refer to the detection method in Example 10.

[0129] In some specific embodiments, the Trop2 binding molecule comprises only binding molecules that specifically bind to the first binding domain of Trop2.

[0130] In some embodiments, the aforementioned Trop2 / MUC1 binding molecule has an activity selected from at least one of the following:

[0131] (a) with ≤10 -7 K DThe value binds to human Trop2 or its epitope, and has a weaker affinity than S1920; refer to the detection method in Example 5 or 7;

[0132] (b) with ≤10 -7 The KD value binds to human MUC1 or its epitope, and the affinity is superior to that of antibody P; refer to the detection method in Example 5 or 7;

[0133] (c) Selective binding to Trop2 and MUC1 double-positive cells; refer to the detection method in Example 6 or 9;

[0134] For example, the number of molecules binding in Trop2 and MUC1 double-positive cells is greater than that binding to Trop2, or in EC... 50 The value is significantly smaller than that of binding molecules that contain only the first binding domain that specifically binds Trop2;

[0135] (d) Selective killing of Trop2 and MUC1 double-positive cells; refer to the detection method in Example 11;

[0136] For example, it is more effective at killing Trop2 and MUC1 double-positive cells than binding molecules that contain only the first binding domain that specifically binds Trop2.

[0137] (e) Selective inhibition of B7-H3 and EGFR double-positive cell proliferation; refer to the detection method in Example 12.

[0138] In some embodiments, the Trop2 / MUC1 binding molecule disclosed herein is an anti-Trop2 / MUC1 antibody, or a conjugate or fusion protein containing said antibody.

[0139] In some specific embodiments, the Trop2 / MUC1 binding molecule disclosed herein is an anti-Trop2 / MUC1 bispecific antibody, or a conjugate or fusion protein containing the bispecific antibody.

[0140] In this 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.

[0141] Antibody-drug conjugates

[0142] This disclosure provides antibody-drug conjugates comprising an antibody and a drug molecule. In some embodiments, the antibody comprises a first binding domain specifically binding to Trop2 of any of the foregoing embodiments, and / or a second binding domain specifically binding to MUC1 of any of the foregoing embodiments.

[0143] In some embodiments, the antibody comprises a first binding domain that specifically binds to Trop2 and a second binding domain that specifically binds to MUC1.

[0144] In some embodiments, the drug molecule includes, but is not limited to, cytotoxic drugs, immunomodulators, and cell inhibitors. For example, compounds, peptides, proteins, nucleic acids, etc., that have cell growth inhibition, cytotoxicity, and / or immunomodulatory functions. Exemplary examples include toxins (such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin), radioactive isotopes, chemotherapeutic drugs, antibiotics, and ribolysins, etc.

[0145] In some specific implementations, the drug molecule is a cytotoxic drug.

[0146] In some specific embodiments, the drug molecule is selected from MMAF or its derivatives, MMAE or its derivatives, ezetidine or its derivatives, and eribulin or its derivatives. In some specific embodiments, the effector molecule is ezetidine or its derivatives.

[0147] In some embodiments, the antibody-drug conjugate has the structure shown in formula (I):

[0148] in,

[0149] -LY- is the connecting part;

[0150] D represents a drug molecule;

[0151] n is between 1 and 10, and n is a decimal or an integer;

[0152] Ab stands for antibody, such as anti-Trop2 / MUC1 antibody, anti-Trop2 antibody, or anti-MUC1 antibody.

[0153] In some implementation schemes, D is a camptothecin-based drug; for example, ixotecan or its derivatives.

[0154] In some implementations, the aforementioned antibody-drug conjugate has the structure shown in formula (IV).

[0155] in:

[0156] -LY- is the connecting part;

[0157] n is between 1 and 10, and n is a decimal or an integer;

[0158] Ab stands for antibody, such as anti-Trop2 / MUC1 antibody, anti-Trop2 antibody, or anti-MUC1 antibody.

[0159] In some implementations, 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)-;

[0160] R a and R b They may be the same or different, and each is independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxyl, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, or heterocyclic groups; or, R a and R b Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0161] R 1 Selected from hydrogen atoms, alkyl groups, halogens, haloalkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups;

[0162] R 2 Selected from hydrogen atoms, alkyl groups, halogens, haloalkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups;

[0163] Or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0164] Or, R a and R 2 Together with the carbon atom attached to it, it forms a cycloalkyl or heterocyclic group;

[0165] m is an integer from 0 to 4.

[0166] In some implementations, -L- represents the connector unit, which is -L 1 -L 2 -L 3 -L 4 -,

[0167] L 1 It is -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, where 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 with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, alkoxy and cycloalkyl;

[0168] 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, where p 1 Integers from 1 to 20;

[0169] L 3 It is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids may optionally be further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, alkoxy and cycloalkyl;

[0170] 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;

[0171] R 3 R 4 and R 5 They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl and hydroxyalkyl;

[0172] 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, and hydroxyalkyl groups.

[0173] In some implementations, such as the antibody-drug conjugate represented by formula (I) as described in the previous one,

[0174] in:

[0175] Y is -O-(CR) a R b m-CR 1 R 2 -C(O)-;

[0176] R a and R b They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 alkoxy, hydroxy and C 1-6 Hydroxyalkyl;

[0177] R 1 Selected from hydrogen atoms, C 1-6 Alkyl, C 3-6 cycloalkyl C 1-6 Alkyl or C 3-6 cycloalkyl;

[0178] R 2 Selected from hydrogen atoms, halogens, C 1-6 Haloalkyl, C 3-6 cycloalkyl and C 3-6 cycloalkyl C 1-6 alkyl;

[0179] Or, R 1 and R 2 Together with the carbon atoms it is attached to, they form C 3-6 cycloalkyl;

[0180] Or, R a and R 2 Together with the carbon atom it is attached to, they form C 3-6 cycloalkyl; m is 0 or 1;

[0181] Some specific implementation schemes, such as the antibody-drug conjugates represented by formula (I) as described in the previous one,

[0182] in:

[0183] Y is -O-(CR) a R b m-CR 1 R2 -C(O)-;

[0184] R a and R b They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 alkyl;

[0185] R 1 C 3-6 cycloalkyl C 1-6 Alkyl or C 3-6 cycloalkyl;

[0186] R 2 Selected from hydrogen atoms, C 1-6 Halogenated alkyl and C 3-6 cycloalkyl;

[0187] Or, R 1 and R 2 Together with the carbon atoms it is attached to, they form C 3-6 Cycloalkyl; m is 0 or 1.

[0188] In some implementations, such as the antibody-drug conjugate represented by formula (I) as described in the previous one,

[0189] Wherein, Y is selected from:

[0190] The O end of Y is connected to the connector unit L.

[0191] In some implementations, such as the antibody-drug conjugate represented by formula (I) as described in the previous one,

[0192] Among them, the connector unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0193] 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 of 1 to 8 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 with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, alkoxy and cycloalkyl;

[0194] L 2Selected 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;

[0195] 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, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, alkoxy and cycloalkyl.

[0196] L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 -C(O)NR 5 (CH2) t - and chemical bonds, where t is an integer from 1 to 6;

[0197] R 3 R 4 and R 5 They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl and hydroxyalkyl;

[0198] 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, and hydroxyalkyl groups.

[0199] In some implementations, such as the antibody-drug conjugate represented by formula (I) as described in the previous one,

[0200] Among them, the connector unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0201] 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 C1-8 Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently and optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, alkoxy and cycloalkyl;

[0202] 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;

[0203] 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, alkoxy and cycloalkyl;

[0204] L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 -C(O)NR 5 (CH2) t - and chemical bonds, where t is an integer from 1 to 6, and non-limiting examples are 1, 2, 3, 4, 5 and 6;

[0205] R 3 R 4 and R 5 They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl and hydroxyalkyl;

[0206] 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, and hydroxyalkyl groups.

[0207] In some implementations, such as the antibody-drug conjugate represented by formula (I) as described in the previous one,

[0208] Among them, the connector unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0209] L 1 for s 1 The integers are from 2 to 8, with non-limiting examples being 2, 3, 4, 5, 6, 7, and 8;

[0210] L 2 Chemical bond;

[0211] L 3 It is a tetrapeptide residue; preferably, L 3 It is a tetrapeptide residue of GGFG;

[0212] L 4 For -NR 5 (CR 6 R 7 )t-,R 5 R 6 Or R 7 They may be the same or different, and each is independently a hydrogen atom or an alkyl group, with t being 1 or 2;

[0213] The L mentioned therein 1 The terminal is connected to Ab, L 4 The end is connected to Y.

[0214] In some embodiments, the antibody-drug conjugate as shown in formula (I) 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.

[0215] In some implementations, the aforementioned antibody-drug conjugate has the structure shown in formula (II).

[0216] in:

[0217] 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 1-8Alkyl, cycloalkyl and straight-chain heteroalkyl are each independently and optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, alkoxy and cycloalkyl;

[0218] 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;

[0219] L 3 It is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids may optionally be further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, alkoxy and cycloalkyl;

[0220] R 1 Selected from hydrogen atoms, halogens, cycloalkyl, cycloalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups;

[0221] R 2 Selected from hydrogen atoms, halogens, haloalkyl groups, cycloalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups;

[0222] Or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0223] R 4 and R 5 They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl and hydroxyalkyl;

[0224] 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, and hydroxyalkyl groups;

[0225] m is an integer from 0 to 4;

[0226] n is between 1 and 10, and n can be an integer or a decimal;

[0227] Ab stands for antibody, such as anti-Trop2 / MUC1 antibody, anti-Trop2 antibody, or anti-MUC1 antibody.

[0228] In some implementations, such as the antibody-drug conjugate represented by formula (II) as described in the preceding one,

[0229] in:

[0230] The -LY- structure is as follows:

[0231] s 1 Integers between 2 and 8;

[0232] L 2 L 3 R 1 R 2 R 5 R 6 R 7 And m are as defined in equation (II) above.

[0233] In some implementations, the aforementioned antibody-drug conjugate has the structure shown in formula (III):

[0234] in:

[0235] s 1 Integers between 2 and 8;

[0236] R 1 R 2 R 5 ~R 7 m is as defined in equation (II) above;

[0237] n is between 1 and 10, and n can be an integer or a decimal;

[0238] Ab stands for antibody, such as anti-Trop2 / MUC1 antibody, anti-Trop2 antibody, or anti-MUC1 antibody.

[0239] In some implementations, the antibody-drug conjugates of formula (I) or (IV) as described in any of the preceding embodiments, wherein -L- is:

[0240] In some implementations, antibody-drug conjugates of formula (I) or (IV) as described in any of the preceding embodiments, wherein -LY- is optionally derived from:

[0241] In some implementation schemes, -LY- is optional from:

[0242] In some implementations, -LY- means:

[0243] In some implementations, -LY- means:

[0244] In some embodiments, the antibody-drug conjugate has a structure as shown in formula (V-1) or (V-2):

[0245] or

[0246] The Ab is an anti-Trop2 / MUC1 antibody, and n is from 1 to 10, where n is an integer or a decimal.

[0247] The anti-Trop2 / MUC1 antibody contains a first binding domain that specifically binds to Trop2 and a second binding domain that specifically binds to MUC1.

[0248] The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of VH1 and VL1 in the first binding domain that specifically binds to Trop2, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of VH2 and VL2 in the second binding domain that specifically binds to MUC1 are respectively shown below:

[0249] As shown in SEQ ID NO: 8-13 and SEQ ID NO: 15-20;

[0250] As shown in SEQ ID NO: 8-13 and SEQ ID NO: 15, 22, 17-20;

[0251] As shown in SEQ ID NO: 8-13 and SEQ ID NO: 25-30;

[0252] As shown in SEQ ID NO: 8-11, 14 and 13 and SEQ ID NO: 15-20;

[0253] As shown in SEQ ID NO: 8-11, 14 and 13 and SEQ ID NO: 15, 22, 17-20; or

[0254] As shown in SEQ ID NO: 8-11, 14 and 13 and SEQ ID NO: 25-30.

[0255] In some implementations, antibody-drug conjugates of formula (V-1) or (V-2) as described in the preceding one,

[0256] The heavy chain variable region in the first binding domain that specifically binds to Trop2 contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 1 or 50, and the light chain variable region contains an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 2 or 3; and / or,

[0257] The heavy chain variable region in the second binding domain that specifically binds to MUC1 contains an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 4, 6, 7 and 23, and the light chain variable region contains an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 5 or 24.

[0258] Preferably, VH1 and VL1 in the first binding domain that specifically binds to Trop2, and VH2 and VL2 in the second binding domain that specifically binds to MUC1, are respectively as follows:

[0259] SEQ ID NO: 1, 2, 4 and 5;

[0260] SEQ ID NO: 1, 2, 6 and 5;

[0261] SEQ ID NO: 1, 2, 7 and 5;

[0262] SEQ ID NO: 1, 2, 23 and 24;

[0263] SEQ ID NO: 1, 3, 4 and 5;

[0264] SEQ ID NO: 1, 3, 6 and 5;

[0265] SEQ ID NO: 1, 3, 7 and 5;

[0266] SEQ ID NO: 1, 3, 23 and 24;

[0267] SEQ ID NO: 50, 2, 4 and 5;

[0268] SEQ ID NO: 50, 2, 6 and 5;

[0269] SEQ ID NO: 50, 2, 7 and 5; or

[0270] SEQ ID NO: 50, 2, 23 and 24.

[0271] In some embodiments, the antibody-drug conjugate represented by formula (V-1) or (V-2) as described in any of the preceding embodiments, wherein the Ab is an anti-Trop2 / MUC1 antibody comprising a combination of polypeptide chains as follows:

[0272] 1) A first heavy chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 33, a first light chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 34, a second heavy chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 31, and a second light chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 32.

[0273] or

[0274] 2) A first heavy chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 33, a first light chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 34, a second heavy chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 35, and a second light chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 36.

[0275] In some implementations, the antibody-drug conjugates of general formula (V-2) as described in the preceding one have the following structure:

[0276] This disclosure also provides a method for preparing the antibody-drug conjugate shown in formula (I) as described above, comprising the following steps:

[0277] The aforementioned Ab was coupled with (LYD) to obtain the antibody-drug conjugate shown in formula (I);

[0278] Optionally, the antibody-drug conjugate shown in formula (I) is purified.

[0279] In some embodiments, a method for preparing an antibody-drug conjugate as shown in formula (II) includes the following steps:

[0280] After reduction of Ab, it undergoes a coupling reaction with general formula (La-YD) to give the compound shown in general formula (II);

[0281] Among them, Ab, W, L2, L3, R1, R2, R5~R7, m and n are defined as in equation (II).

[0282] Polynucleotides and carriers

[0283] This disclosure provides polynucleotides encoding the Trop2-binding molecule, MUC1-binding molecule, or Trop2 / MUC1-binding molecule of this disclosure. The polynucleotides of this disclosure may be RNA, DNA, or cDNA. According to some embodiments of this disclosure, the polynucleotides of this disclosure are substantially isolated polynucleotides.

[0284] The polynucleotides of this disclosure may also be in vector form, present in a vector, and / or part of a vector, such as a plasmid, sticky-terminal plasmid, YAC, or viral vector. The vector may be, in particular, an expression vector, providing an in vitro and / or in vivo expression (i.e., in a suitable host cell, host organism, and / or expression system) of the Trop2 binding molecule, the MUC1 binding molecule, or the Trop2 / MUC1 binding molecule. This expression vector typically contains at least one polynucleotide of this disclosure operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selection of these elements and their sequences for expression in a particular host is common knowledge to those skilled in the art. Regulatory elements and other elements useful or necessary for the expression of the Trop2 binding molecule, the MUC1 binding molecule, or the Trop2 / MUC1 binding molecule of this disclosure include, for example, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.

[0285] The polynucleotides disclosed herein can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on information about the amino acid sequence of the polypeptides disclosed herein, and / or can be isolated from suitable natural sources.

[0286] host cells

[0287] This disclosure provides recombinant host cells that express or are capable of expressing one or more of the Trop2 binding molecules, MUC1 binding molecules, Trop2 / MUC1 binding molecules of this disclosure, and / or containing nucleic acids or vectors of this disclosure. In some embodiments, the host cells are bacterial cells, fungal cells, or mammalian cells.

[0288] Bacterial cells include, for example, cells of Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0289] Fungal cells include, for example, cells of species from the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells of species from the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichia pastoris* and *Pichia methanolica*), and *Hansenula*.

[0290] Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0291] However, this disclosure may also use amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0292] The cells disclosed herein cannot develop into complete plant or animal individuals.

[0293] Production or preparation method

[0294] This disclosure provides a method for preparing the Trop2-binding molecule, MUC1-binding molecule, or Trop2 / MUC1-binding molecule of this disclosure, said method generally comprising the following steps:

[0295] - Culture the host cells of this disclosure under conditions that allow expression of the binding proteins of this disclosure; and

[0296] -Recover the binding protein expressed by the host cells from the culture; and

[0297] -Optionally, this may include further purification and / or modification of the binding protein disclosed herein.

[0298] The Trop2-binding molecule, MUC1-binding molecule, or Trop2 / MUC1-binding molecule of this disclosure may be produced intracellularly in cells as described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), followed by isolation from the host cell and optionally further purification; or may be produced extracellularly (e.g., in a culture medium for culturing host cells), followed by isolation from the culture medium and optionally further purification.

[0299] Methods and reagents for recombinantly generating peptides, such as specific expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, isolation and purification techniques suitable for manufacturing the binding proteins or antibodies of this disclosure are well known to those skilled in the art. Methods for producing and purifying antibodies are well known and available in the prior art, such as Cold Spring Harbor's Guide to Laboratory Antibody Techniques (Chapters 5-8 and 15). The engineered antibodies of this disclosure can also be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Expression vectors can stably transfect cells. Mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product must be frozen immediately, such as at -70°C, or freeze-dried.

[0300] However, the Trop2-binding molecules, MUC1-binding molecules, or Trop2 / MUC1-binding molecules disclosed herein can also be obtained by other protein-generating methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

[0301] This disclosure provides a method for preparing Trop2 / MUC1 binding molecules, the method comprising:

[0302] (a) Determine the binding activity of candidate molecules with Trop2 peptide;

[0303] (b) Determine the binding activity of candidate molecules with MUC1 peptides;

[0304] (c) Select candidate molecules that have Trop2 binding activity and MUC1 binding activity, i.e., the Trop2 / MUC1 binding molecules;

[0305] In some specific implementations, the method is selected from the group consisting of:

[0306] A) The method comprises:

[0307] i) Provide the Trop2 peptide for contact with candidate molecules;

[0308] ii) Provide the MUC1 peptide for contact with the candidate molecule;

[0309] iii) Determine whether the candidate molecule has Trop2 binding activity and MUC1 binding activity as an indicator of whether the candidate molecule is the Trop2 / MUC1 binding molecule;

[0310] B) The method includes:

[0311] i) Provide a cell containing an exogenous recombinant nucleic acid encoding a human Trop2 peptide, wherein the cell expresses the human Trop2 peptide and is contacted with a candidate molecule;

[0312] ii) Provide a cell containing an exogenous recombinant nucleic acid encoding a human MUC1 peptide, wherein the cell expresses the human MUC1 peptide and is contacted with a candidate molecule;

[0313] iii) Determine whether the candidate molecule has Trop2 binding activity and MUC1 binding activity as an indicator of whether the candidate molecule is the Trop2 / MUC1 binding molecule;

[0314] C) The method described herein includes

[0315] i) Provide cells containing exogenous recombinant nucleic acids encoding human Trop2 or MUC1 peptides, wherein the cells express the human Trop2 or MUC1 peptides; and contact with candidate molecules;

[0316] ii) Provide another peptide from Trop2 or MUC1 to contact the candidate molecule;

[0317] iii) Determine whether a candidate molecule has Trop2 binding activity and MUC1 binding activity as an indicator of whether the candidate molecule is the Trop2 / MUC1 binding molecule.

[0318] Composition / Pharmaceutical Composition

[0319] This disclosure provides compositions comprising the aforementioned Trop2-binding molecule, MUC1-binding molecule, or Trop2 / MUC1-binding molecule.

[0320] For example, a pharmaceutical composition is provided which contains a therapeutically effective amount of the Trop2-binding molecule, MUC1-binding molecule, or Trop2 / MUC1-binding molecule as described above, and at least one pharmaceutically acceptable excipient, diluent, or carrier.

[0321] In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of Trop2-binding molecules, MUC1-binding molecules, or Trop2 / MUC1-binding molecules per unit dose, or the amount of Trop2-binding molecules, MUC1-binding molecules, or Trop2 / MUC1-binding molecules per unit dose of the pharmaceutical composition may be 0.1-2000 mg, and in some specific embodiments, 1-1000 mg.

[0322] In some embodiments, a product or kit is provided containing at least one container, each independently containing the aforementioned Trop2-binding molecule, MUC1-binding molecule, or Trop2 / MUC1-binding molecule. Optionally, the kit includes a container and a label. The container is, for example, a bottle, syringe, or test tube. The container holds a composition effective for treating a condition. A label on or attached to the container indicates that the composition is used to treat the selected condition. The composition contains the aforementioned Trop2-binding molecule, MUC1-binding molecule, or Trop2 / MUC1-binding molecule.

[0323] Methods of Treating Diseases and Pharmaceutical Uses This disclosure provides methods for treating, alleviating, preventing, and diagnosing diseases or conditions using the aforementioned Trop2-binding molecules, MUC1-binding molecules, Trop2 / MUC1-binding molecules, polynucleotides, or compositions (including pharmaceutical compositions). Some embodiments provide methods for improving, alleviating, treating, or preventing a disease, including administering to a subject an effective amount of the aforementioned Trop2-binding molecule, MUC1-binding molecule, Trop2 / MUC1-binding molecule, polynucleotide, or composition (including pharmaceutical compositions) for improving, alleviating, treating, or preventing the disease.

[0324] In some embodiments, the use of the Trop2-binding molecule, MUC1-binding molecule, or Trop2 / MUC1-binding molecule of this disclosure in the preparation of a medicament for improving, alleviating, treating, or preventing disease is provided.

[0325] In some implementation schemes, the aforementioned disease is cancer.

[0326] In some implementations, the cancer is double-positive for Trop2 and MUC1.

[0327] In some implementations, the cancer is breast cancer or lung cancer (e.g., non-small cell lung cancer).

[0328] Some implementations provide a method, as shown below, comprising administering an effective amount of the aforementioned Trop2 / MUC1 binding molecule, polynucleotide, or composition (including pharmaceutical compositions) to a subject in need:

[0329] (1) A method for selectively binding Trop2 and MUC1 double-positive cells;

[0330] (2) A method for selectively killing Trop2 and MUC1 double-positive cells;

[0331] (3) Methods to increase the endocytic activity of the Trop2 binding domain;

[0332] (4) A method to selectively inhibit the proliferation of Trop2 and MUC1 double-positive cells.

[0333] In some specific embodiments, a method for increasing the endocytic activity of the Trop2 binding domain is provided, the method comprising treating cells with a Trop2 / MUC1 binding molecule, said Trop2 / MUC1 binding molecule comprising a second domain that specifically binds a second binding domain of MUC1; the endocytic activity of said second domain in Trop2 and MUC1 dual-positive cells is greater than that of the Trop2 binding molecule.

[0334] Tests and kits

[0335] This disclosure provides the use of Trop2 binding molecules, MUC1 binding molecules, Trop2 / MUC1 binding molecules, polynucleotides, and compositions for detection. This disclosure also provides methods, systems, or apparatus for in vivo or in vitro detection of Trop2 and / or MUC1, comprising treating a sample with the aforementioned binding proteins, polynucleotides, or compositions of this disclosure.

[0336] In some implementations, the in vitro detection method, system, or device may include, for example:

[0337] (1) Contact the sample with Trop2 binding molecules, MUC1 binding molecules, Trop2 / MUC1 binding molecules, polynucleotides, and compositions;

[0338] (2) Detect the complexes formed between the aforementioned binding proteins, polynucleotides, and samples; and / or

[0339] (3) Contact the reference sample (e.g., control sample) with the binding protein and nucleic acid; and

[0340] (4) Determine the extent of complex formation by comparison with a reference sample. A change in complex formation in a sample or subject compared to a control sample or subject (e.g., a statistically significant change) indicates the presence of Trop2 and / or MUC1 in the sample.

[0341] In other embodiments, the in vivo detection method, system, or apparatus may include:

[0342] (1) Administer the aforementioned binding protein and polynucleotide to the subject; and

[0343] (2) Detect the formation of complexes between the aforementioned binding proteins, polynucleotides and the subject.

[0344] Detection may include determining the location or time of complex formation. The aforementioned binding proteins and polynucleotides are labeled with a detectable substance, and the detection of the label enables the detection of substances capable of binding proteins and polynucleotides (e.g., Trop2 and / or MUC1). Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. The formation of complexes between binding proteins / polynucleotides and Trop2 and / or MUC1 can be detected by measuring or visualizing the substances that bind to or do not bind to Trop2 and / or MUC1. Conventional detection assays can be used, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry. For detection purposes, the binding proteins and polynucleotides of this disclosure may be labeled with fluorophores or chromophores. In some embodiments, diagnostic reagents comprising the aforementioned polynucleotides and binding proteins are also provided, along with related diagnostic uses.

[0345] In some embodiments, a kit is also provided comprising the aforementioned binding protein, polynucleotide, and may also include diagnostic instructions for use. The kit may also contain at least one additional reagent, such as a marker or additional diagnostic agent. For in vivo use, the binding protein may be formulated as a pharmaceutical composition. Attached Figure Description

[0346] Figure 1 shows the expression of Trop2 and MUC1 in oral cancer-adjacent tissues as evaluated by IHC.

[0347] Figure 2 is a schematic diagram of the anti-Trop2 / MUC1 antibody-drug conjugate.

[0348] Figure 3 shows the binding of the anti-Trop2 / MUC1 bispecific antibody to MDA-MB-453 cells as detected by FACS; S1920 and SDP02321 are controls.

[0349] Figure 4 shows the binding of the anti-Trop2 / MUC1 bispecific antibody to cells T47D and MDA-MB-453 as detected by FACS; S1920 is the control.

[0350] Figures 5A and 5B are the natural mass spectra of the bispecific antibody ADC; Figure 5A is the natural mass spectrum of SDA008, and Figure 5B is the natural mass spectrum of SDA012.

[0351] Figure 6 shows the binding of the bispecific antibody ADC to A431 cells as detected by FACS, with SDA021 serving as the control.

[0352] Figures 7A and 7B show the endocytic activity of the bispecific antibody ADC in T47D and NCI-H441 cells.

[0353] Figures 8A and 8B show the cell-killing activity of the bispecific antibody ADC; Figure 8A shows cell A431, Figure 8B shows cell T47D, and SDA021 is the control.

[0354] Figures 9A and 9B show the antitumor activity of the bispecific antibody ADC in the T47D and NCI-H1650 xenograft models. Detailed Implementation

[0355] Terminology Definition

[0356] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined in this disclosure, all other technical and scientific terms used in this disclosure shall have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0357] “Trop2”, “Trop2”, and “Trop-2” are used interchangeably and include variants, isotypes, species homologs of human Trop-2, and analogs that share at least one common epitope with Trop2.

[0358] "MUC1" and "MUC-1" are used interchangeably and include variants, isotypes, species homologs of human Trop-2, and analogs that share at least one epitope with MUC1. MUC1 is a high-molecular-weight transmembrane protein composed of two subunits: the N-terminus (MUC1-N) and the C-terminus (NUC1-C). MUC1-N and NUC1-C are linked non-covalently, forming a heterodimeric complex in the cell membrane. MUC1-N comprises a 20-amino-acid variable-number tandem repeat (VNTR) domain, with the number of VNTR repeats varying from 20 to 120 in different individuals. These repeat sequences are rich in serine, threonine, and proline residues, allowing for heavy O-glycosylation. MUC1-C is the C-terminal domain, containing an extracellular domain (ECD), a transmembrane domain (TMD), and a cytoplasmic domain.

[0359] "Binding molecule" encompasses any molecule capable of specifically binding to an antigen or its epitopes, including but not limited to antibodies or their conjugates as defined in this disclosure. The "binding molecule" of this disclosure may contain a heavy chain variable region (VH) and a light chain variable region (VL), such as Fab, Fab', F(ab')2, a single-chain antibody (scFv), or an IgG antibody.

[0360] The term "antibody" encompasses various 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), as long as they exhibit the desired antigen-binding activity. Antibodies can refer to immunoglobulins, which are tetrapeptide chains composed of two heavy chains and two light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, different subclasses can be distinguished; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ chains or λ chains based on differences in their constant regions. Each of the five types of Ig can have either a κ chain or a λ chain.

[0361] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are known as the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0362] "Antigen-binding fragments" encompass 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, VL, or VHH), scFv, bivalent, trivalent, or quadrivalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3): 209-217).

[0363] Methods for generating and preparing these antigen-binding fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv form was first disclosed in WO2009 / 040562, and its disulfide-stabilized form, Fab-dsFv, was first disclosed in WO2010 / 035012. The antigen-binding fragments disclosed herein also include Fab and Fab' fragments described in WO2005 / 003169, WO2005 / 003170, and WO2005 / 003171. Multivalent antibodies may contain multispecificity, such as bispecificity, or may be monospecific (see, for example, WO92 / 22583 and WO05 / 113605), an example of the latter being Tri-Fab (or TFM) described in WO 92 / 22583.

[0364] The determination or definition of a contact ligand (CDR) can be achieved by resolving the structure of the antibody and / or the structure of the antibody-ligand complex, thus enabling a definitive characterization of the CDR and identification of the residues at the binding site. This can be accomplished using any of the various techniques known to those skilled in the art, such as 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 definitions, and conformational definitions.

[0365] The Kabat numbering system is the standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat system, but it takes into account the location of certain structural loop regions (see, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses a computer program integration suite produced by the Oxford Molecular Group to model antibody structures (see, for example, Martin et al., 1989, ProcNatl Acad Sci (USA), 86: 9268-9272; "AbMTM, 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 a de novo approach to model the tertiary structure of antibodies from basic sequences (see those described by Samudrala et al., 1999, “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach” in PROTEINS, Structural, Function and Genetics Suppl., 3:194-198). Contact definitions are based on the analysis of available complex crystal structures (see, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In conformational definitions, the position of the CDR can be identified as a residue that contributes enthalpy to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). Other CDR boundary definitions may not strictly follow one of the above methods but still overlap with at least a portion of the Kabat CDR. CDR boundaries may be shortened or lengthened based on predictions or experimental results that a particular residue or group of residues does not significantly affect antigen binding. As used in this disclosure, a CDR may refer to a CDR defined by any method (including combinations of methods) known in the art. The correspondences between various numbering systems are well known to those skilled in the art.

[0366] 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.

[0367] "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.

[0368] "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.

[0369] The terms “nucleic acid” or “polynucleotide” are used interchangeably herein to refer to any single-stranded or double-stranded DNA or RNA molecule, and in the case of single-stranded molecules, its complementary sequence, such as double-stranded DNA. Nucleic acids are “operably linked” when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to said coding sequence.

[0370] "Host cell" includes individual cells or cell cultures that may be, or have been, recipients of vectors for incorporating polynucleotide inserts. Host cell includes progeny of a single host cell, and progeny are not necessarily identical to the parent cell (in morphology or genomic DNA complementation) due to natural, accidental, or intentional mutations. Host cell includes cells transfected and / or transformed in vivo with the polynucleotides of this disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such names include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in DNA content. The term includes mutant progeny that have the same function or biological activity as the cells screened in the initial transformation.

[0371] The term "internalization" refers to the transport of a portion of a cell from its exterior to its interior. The internalized portion may reside within intracellular compartments. "Internalized" or "internalized" antigens or antibodies are those 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 cell internalization typically refers to the transmembrane movement of cell surface molecules from the cell surface to the cell interior. After internalization, endosomes can be transported to lysosomes for degradation or recycled back to the cell surface. The rate of internalization of a given cell surface molecule provides a measurement of the kinetics of the molecule's movement from the cell surface across the plasma membrane to the cell interior. The internalization activity or rate of antigens and antibodies 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 toxicity assays (Pahara J. et al., Exp Cell Res., 316:2237–50, 2010; and Mazor et al., J. Immunol. Methods, 321:41–59, 2007). Many antibody labeling techniques, dyes, and kits for antibody labeling are commercially available for quantifying and monitoring internalization (e.g., the pHrodo iFL antibody labeling method, reagents, and kits sold by Thermo Fisher Scientific).

[0372] The terms "antibody-drug conjugate" or "antibody-drug conjugate" (ADC) refer to the linking of an antibody to a drug molecule with a target effector activity. The antibody can be directly conjugated to the drug or via a linker unit.

[0373] There are no particular restrictions on drug molecules, as long as they have substituents or parts of the structure that can be attached to the linker structure. Depending on the conjugated drug, antibody-drug conjugates can be used for a variety of purposes. Examples of such drugs can include substances with antitumor activity, substances effective against blood disorders, substances effective against autoimmune diseases, anti-inflammatory substances, antimicrobial substances, antifungal substances, antiparasitic substances, antiviral substances, and antianesthetic substances.

[0374] The term "drug loading" refers to the average amount of cytotoxic drug loaded onto each ligand in an ADC, and can also be expressed as the ratio of drug amount to antibody amount. Drug loading can range from 1 to 20 cytotoxic drugs (D) linked to each antibody (Ab), or from 1 to 10 cytotoxic drugs (D). In embodiments of this disclosure, drug loading is expressed as n, exemplarily 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the average of any two values. The average amount of drug per ADC molecule after the coupling reaction can be characterized using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, CE-SDS (monoclonal antibody molecular size variant assay), and HPLC.

[0375] While the drug-to-antibody ratio has a precise value for a particular conjugate molecule (e.g., n in formula (I)), it should be understood that when used to describe samples containing many molecules, this value will often be an average, due to a degree of non-uniformity typically associated with the conjugation step. The average load 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, for example, 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. The embodiments include DARs of approximately 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, and 4. Immunoconjugates of approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.4, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0. In some embodiments, 'approximately x' DAR means that the measured value of DAR is within 20% of x.

[0376] Methods for detecting DAR include, for example, extrapolating DAR values ​​from LC-MS data of reduced and deglycosylated samples. LC / MS allows quantification of the average number of drug-load molecules linked to the antibody in an ADC. HPLC separates antibodies into light and heavy chains, and further separates heavy chains (HC) and light chains (LC) based on the number of linker-load groups on each chain. Mass spectrometry data can identify the types of components in a mixture, such as LC, LC+1, LC+2, HC, HC+1, HC+2, etc. The average DAR of the ADC can be calculated based on the average loading of the LC and HC chains. The DAR of a given immunoconjugate sample represents the average number of drug (load) molecules linked to a tetrameric antibody containing two light chains and two heavy chains. For example, the DAR detection method in WO2018142322.

[0377] The term "camptothecin class of drugs" refers to camptothecin and its derivatives, which are cytotoxic, and are not limited to 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin, topotecan, ixotecan, irinotecan or 9-nitro-10-hydroxycamptothecin and its derivatives or pharmaceutically acceptable salts.

[0378] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, such as an alkyl group containing 1 to 12 carbon atoms, an alkyl group containing 1 to 10 carbon atoms, or an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-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 their various branched isomers, etc. For example, it is a lower alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-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, etc. Alkyl groups can be substituted or unsubstituted. When substituted, the substituents can be substituted at any usable connection point. The substituents can be one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0379] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein the alkyl group is as defined above.

[0380] The term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It is a straight-chain or branched group containing 1 to 20 carbon atoms, for example, alkylene containing 1 to 12 carbon atoms, or alkylene containing 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). The alkylene group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent can be independently selected from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0381] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent can be one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0382] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, for example, a cycloalkyl ring containing 3 to 20 carbon atoms, 3 to 12 carbon atoms, 3 to 10 carbon atoms, or 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0383] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m(where m is an integer from 0 to 2) heteroatoms, but excluding the ring portions of -OO-, -OS-, or -SS-, with the remaining ring atoms being carbon. For example, it may contain 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; or contain 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups.

[0384] The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group consisting of 5 to 20 member monocyclic rings sharing a single atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O). m (Where m is an integer from 0 to 2) heteroatoms, with the remaining ring atoms being carbon. It may contain one or more double bonds, but no ring has a fully conjugated π-electron system. For example, it can be 6 to 14-membered, or 7 to 10-membered. Spiroheterocyclic groups are classified into monospirocyclic, bispirocyclic, or multispirocyclic groups based on the number of shared spiroatoms between rings. Non-limiting examples are 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospirocyclic groups. Non-limiting examples of spirocyclic groups include:

[0385] The term "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with the other rings in the system. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are selected from nitrogen, oxygen, or S(O). m (Where m is an integer from 0 to 2) heteroatoms, with the remaining ring atoms being carbon. For example, they can be 6 to 14 members, or 7 to 10 members. Depending on the number of rings, they can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic groups. Non-limiting examples are 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

[0386] The term "bridged heterocyclic group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly bonded atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are selected from nitrogen, oxygen, or S(O). m Bridged heterocyclic groups consist of heteroatoms (where m is an integer from 0 to 2) and the remaining ring atoms are carbon. For example, they can be 6 to 14-membered or 7 to 10-membered. Based on the number of rings, they can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic groups. Non-limiting examples of bridged heterocyclic groups include:

[0387] The heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include: wait.

[0388] The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituent can be one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0389] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, with non-limiting examples being 6- to 10-membered groups, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, with non-limiting examples including:

[0390] The aryl group can be substituted or unsubstituted. When substituted, the substituent can be one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0391] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group can be 5 to 10-membered, with non-limiting examples being 5- or 6-membered, such as furanyl, thiophene, pyridyl, pyrrole, N-alkylpyrrole, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, with non-limiting examples including:

[0392] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent can be one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0393] The term "amino protecting group" is used to protect the amino group by a group that is easily removed, so that the amino group remains unchanged when other parts of the molecule react. Non-limiting examples include 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl, etc. These groups may optionally be substituted with 1 to 3 substituents selected from halogens, alkoxy groups, or nitro groups. For example, the amino protecting group is 9-fluorenylmethoxycarbonyl.

[0394] The term "cycloalkylalkyl" refers to an alkyl group that is substituted by one or more cycloalkyl groups, for example, substituted by one cycloalkyl group, wherein the alkyl group is as defined above, and the cycloalkyl group is as defined above.

[0395] The term "halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0396] The term "hydroxyl group" refers to the -OH group.

[0397] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0398] The term "amino" refers to -NH2.

[0399] The term "nitro" refers to -NO2.

[0400] The term "amide group" refers to -C(O)N(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0401] The term "carboxylic acid ester group" refers to -C(O)O (alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0402] "Substituted" refers to one or more hydrogen atoms in a group, for example, up to five, or one to three hydrogen atoms, that are independently replaced by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated bond (such as an alkene).

[0403] "Inhibit" or "block" are used interchangeably and cover both partial and complete inhibition / blockage. "Inhibit growth" (e.g., involving cells) is intended to include any measurable reduction in cell growth.

[0404] "Proliferative disease" refers to a condition associated with a certain degree of abnormal cell proliferation. In one implementation, a proliferative disease refers to cancer. "Tumor" refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. "Cancer," "cancerous," "proliferative disease," and "tumor" are not mutually exclusive when used in this disclosure.

[0405] "Giving," "applying," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, 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, such as in therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and a cell, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cell. "Giving," "applying," and "treatment" also mean treatment, such as of cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to humans, veterinary, or research subjects, it refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0406] "Treatment" means administering, either internally or externally, a 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 immune disorders, 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 the 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.

[0407] "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.

[0408] "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."

[0409] In this disclosure, "subject" and "patient" refer to mammals, especially primates, and particularly humans.

[0410] Example

[0411] The following embodiments are used to further describe this disclosure, but these embodiments are not intended to limit the scope of this disclosure.

[0412] Experimental methods not specifically described in the embodiments or test examples of this disclosure are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; Methods in Modern Molecular Biology, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents not specifically named are commercially available, conventional reagents.

[0413] Example 1. Co-expression of Trop2 and MUC1

[0414] IHC analysis of Trop2 and MUC1 was performed on formalin-fixed paraffin-embedded PDX models and clinical tumor tissue samples of various tumor types to explore the co-expression ratio of Trop2 and MUC1 in different tumor types.

[0415] Tumor microarrays (TMAs); including patient-derived tumor xenograft (PDX) model TMAs and patient clinical tumor TMAs, were sourced from commercial sources. PDX models or tumor tissues derived from formalin-fixed and paraffin-embedded (FFPE) materials were purchased from Crown Bioscience (Suzhou) Co., Ltd. and Guilin Yaoli Biotechnology Co., Ltd., respectively.

[0416] IHC detection of Trop2 and MUC1 was performed according to the following protocol. In short, tissue samples were fixed, processed, and embedded in paraffin to prepare tissue sections. The sections were incubated at 65°C for 2 hours on a slide oven and allowed to cool to room temperature (RT) before further processing. After routine deparaffining and rehydration of the tissue samples, antigen retrieval was performed to reverse protein cross-linking that occurred after formalin fixation. Antigen retrieval was achieved by protein hydrolysis of the tissue in a protease solution or by applying heat to the tissue sections in an aqueous medium (commonly referred to as the HIER process), and detection was performed using Bond Polymer Refine Detection (Leica, catalog number DS9800). Treatment was performed at 100°C with pH 9.0 EDTA antigen retrieval solution (Sangon Biotech) for 20 minutes, followed by treatment with 3-4% hydrogen peroxide for 5 minutes. Subsequently, the antibody was incubated with either monoclonal Trop2 antibody (Abcam, EPR20043) or MUC1 antibody (self-made, variable region sequence as shown in SEQ ID NO:4 and 5), with the MUC1 IHC requiring further incubation with rabbit anti-mouse IgG. After incubation with anti-rabbit poly-HRP-IgG for 30 minutes, DAB staining was performed, followed by counterstaining with hematoxylin.

[0417] Trop2 and MUC1 showed moderate to high co-expression in PDX models and clinical samples of breast cancer and non-small cell lung cancer, as detailed in Table 1. Previous literature (Yamashita, Tomoko, et al. Biochemical and Biophysical Research Communications 516.1 (2019)) indicates that MUC1 and Trop-2 have similar distributions in tumor tissues.

[0418] Table 1. Co-expression of Trop2 and MUC1 in clinical cancer patient samples and PDX samples

[0419] Example 2. IHC staining of Trop2 and MUC1 expression in normal oral tissues

[0420] Three normal oral tissue samples were selected for Trop2 and MUC1 IHC staining (method as in Example 1). The results are shown in Figure 1. Trop2 was expressed at a intensity of 3+ in all three oral tissue samples, but MUC1 was negative in two samples and expressed at an intensity of 1+ in one sample. In buccal A5 tissue where MUC1 was expressed at 1+, MUC1 and Trop2 showed non-co-positive expression. The differential expression of Trop2 and MUC1 in the oral cavity suggests that the addition of MUC1 may reduce the side effects caused by Trop2 antibody-drug conjugate therapy.

[0421] Example 3. Design and preparation of anti-Trop2 antibody modification

[0422] The variable region at the 2-terminus of Trop is selected from antibody S1920, and its heavy chain variable region (VH) and light chain variable region (VL) sequences are as follows:

[0423] Anti-Trop2 antibody S1920 VH

[0424] Anti-Trop2 antibody S1920 VL

[0425] To reduce the affinity of anti-Trop2 antibodies S1920 (SEQ ID NO: 1 and 2), we designed two sets of mutations, N31E and Y53T. Antibody S1920 N31E is a mutation with N to E at position 31 of VH, while antibody S1920 Y53T is a mutation with Y to T at position 53 of VL.

[0426] The variable region sequence of antibody S1920 Y53T is as follows:

[0427] Anti-Trop2 antibody S1920 Y53T VH

[0428] Anti-Trop2 antibody S1920 Y53T VL

[0429] Anti-Trop2 antibody S1920 N31E VH

[0430] Anti-Trop2 antibodies were constructed. The nucleotide sequence encoding antibody VH was cloned into a pTT5 vector containing the constant region of the human IgG1 heavy chain, and the nucleotide sequence encoding antibody VL was cloned into a pTT5 vector containing the constant region of the human kappa light chain. These were then transfected into ExpiCHO cells. After 8 days, the cells were centrifuged to remove the cells, and the cell culture medium was collected and filtered. The harvested cell culture medium was purified using a protein A affinity column. The bound antibody was eluted with citric acid, and the medium was desalted and replaced with PBS to obtain the target antibody.

[0431] Further analysis was conducted to determine the binding kinetics of antibodies S1920 N31E and S1920 Y53T. The experimental protocol involved diluting antibodies S1920, S1920 N31E, and S1920 Y53T to 1 μg / mL using HBS-EP+ buffer at a flow rate of 10 μL / min, capturing antibodies up to 200 RU. His-tagged human Trop2 antigen (purchased from Acro, catalog number TR2-H5223) was diluted with HBS-EP+ buffer at specific concentration gradients of 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM. The flow rate for sample analysis was set to 30 μL / min. The binding time was 120 s, and the dissociation time was 600 s. Regeneration was then performed using pH 1.5 Gly-HCl buffer as the regeneration buffer, with a regeneration flow rate of 30 μL / min and a regeneration time of 30 s. The response signal was plotted with analysis time on the x-axis and response value on the y-axis. The obtained data were fitted using BIAcore 8K analysis software, employing a 1:1 Langmuir binding model to determine the binding rate constant (Ka), dissociation rate constant (Kd), and dissociation equilibrium constant (KD), among other kinetic constants. Table 2 shows that the binding activity of antibody S1920 Y53T to Trop2 was significantly reduced.

[0432] Table 2. Binding activity of antibody S1920 before and after mutation

[0433] Example 4. Design and preparation of anti-MUC1 antibody

[0434] 1) Anti-MUC1 antibody P

[0435] Antibody P specifically binds to the N-terminal variable number tandem repeat (VNTR) sequence of the MUC1 antigen. The heavy chain variable region (VH) and light chain variable region (VL) sequences are as follows:

[0436] Anti-MUC1 antibody P VH

[0437] Anti-MUC1 antibody P VL

[0438] To enhance antibody affinity, we designed mutations at position 59 (corresponding to position 56 of the Kabat number) of the variable region of the antibody P heavy chain, namely T59D, T59P, T59N, and T59G, and verified their binding ability with the antigen.

[0439] Antibody binding activity was assessed by detecting the fluorescence signal of antibodies bound to the surface of MUC1 single-positive NCI-H1650 cells, and the binding strength was evaluated based on the intensity of the fluorescence signal. Specifically, serially diluted antibodies were incubated with 1E5 cells at 4°C for 1 hour. Excess antibody was washed away, and murine Alexa Flour 647-labeled anti-human Fc antibody was added. The cells were incubated at 4°C for 30 minutes, and after washing away excess antibody, the cells were resuspended in 200 μL of 1% BSA / PBS buffer. The fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer.

[0440] Table 3 shows that the binding activity of modified antibodies PT59G, PT59D, PT59N, and PT59P was increased.

[0441] Table 3. Binding activity of antibody P on the surface of NCI-H1650 cells before and after mutation.

[0442] The binding activity of antibody P T59G to VNTR-Galnac glycopeptide was detected using ELISA, with antibody P serving as a control. The VNTR-Galnac glycopeptide sequence is as follows:

[0443] APPAHGVTSAPDT(GalNAc)RPAPGSTAPPAHGVTSA (SEQ ID NO:64). Specifically, 1 μg / ml glycopeptide was coated onto an ELISA plate (Corning, catalog number 3590) and incubated overnight at 4°C; washed once with PBST buffer, and the supernatant was discarded. Blocked for 1 hour at room temperature with 1% BSA / PBS buffer. Washed three times with PBST buffer, and the supernatant was discarded; serially diluted antibodies were added and incubated for 1 hour at room temperature. Washed three times with PBST buffer, and the supernatant was discarded; HRP anti-human Fc secondary antibody was added and incubated for 1 hour at room temperature; washed six times with PBST buffer; and TMB chromogenic solution was added for color development. The OD450 was read using a microplate reader. The EC50 was obtained by four-parameter fitting using GraphPad Prism. 50 .

[0444] The results in Table 4 show that the binding activity of antibody PT59G is superior to that of antibody P.

[0445] Table 4. T59G protein-level binding activity

[0446] The variable region sequences of antibodies PT59D and PT59G are as follows:

[0447] Anti-MUC1 antibody P T59D VH

[0448] Anti-MUC1 antibody P T59D VL

[0449] Anti-MUC1 antibody P T59G VH

[0450] Anti-MUC1 antibody P T59G VL

[0451] The CDR (Kabat numbering rule) for antibodies is as follows:

[0452] 2) Anti-MUC1 antibody SDP02313

[0453] The MUC1 protein extracellular domain sequence with a His tag (abbreviated: MUC1-CL-His, as shown in SEQ ID NO:61) was used as an immunogen and detection reagent.

[0454] SEQ ID NO: 61

[0455] Note: The underlined part is the 6×His tag, and the bold part is the extracellular domain of the MUC1 protein.

[0456] After obtaining the hybridoma, antibody screening yielded multiple MUC1-C antibodies. The variable region of antibody M4 obtained from the hybridoma is as follows:

[0457] >M4 heavy chain variable region sequence (M4 mVH):

[0458] SEQ ID NO: 62

[0459] >M4 light chain variable region sequence (M4 mVL):

[0460] SEQ ID NO: 63

[0461] The humanized light chain templates for murine antibody M4 were IGKV1-39*01 / IGKV6-21*02 / IGKV3-11*01 and IGKJ4*01, and the humanized heavy chain templates were IGHV1-46*01 and IGHJ6*01. The CDRs of murine antibody M4 were transplanted into their respective human templates, and then the FR amino acids of the humanized antibodies were reverse-mutated. The variable regions of the resulting humanized antibody SDP02313 (M4-H1L1) are as follows:

[0462] Anti-MUC1 antibody SDP02313 VH

[0463] Anti-MUC1 antibody SDP02313 VL

[0464] The CDR (Kabat numbering rule) for antibodies is as follows:

[0465] The affinity assay for anti-CMUC1 antibodies using Biacore was performed as follows: Antibody molecules were captured using a Protein A biosensor chip, followed by a flow of a specific concentration of MUC1-CL-His antigen molecules onto the chip surface. The injection was continuous for 180 seconds, followed by natural dissociation for 600 seconds. The reaction signal was monitored in real-time using a Biacore 8K instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated using 10 mM Glycine-HCl (pH 1.5). The Kinetics 1:1 binding model was used for data fitting.

[0466] Table 5 shows that SDP02313 binds to the C-terminus of MUC1.

[0467] Table 5. Affinity of antibody SDP02313

[0468] Example 5. Anti-Trop2 / MUC1 Bispecific Antibody

[0469] 1) Design and preparation

[0470] A 1+1 bispecific antibody was constructed using a Hot-Ig design (as shown in Figure 2). This involved replacing one arm of the antibody, CH1 and CL, with Obscurin-O and Titin-T chains, respectively, to resolve light chain mismatches. A knock-in-hole design was used to resolve heavy chain mismatches.

[0471] Construct a bispecific antibody with the following structure:

[0472] The first heavy chain, from N-terminus to C-terminus, consists of: anti-Trop2 antibody heavy chain variable region - GGGGS - [Obscurin-O chain] - [IgG1 Fc1].

[0473] The first light chain, from N-terminus to C-terminus, consists of: anti-Trop2 antibody light chain variable region - GGGGS - [Titin-T chain].

[0474] The second heavy chain, from N-terminus to C-terminus, consists of: the anti-MUC1 antibody heavy chain variable region -[CH1]-[IgG1Fc2], and...

[0475] The second light chain, from the N-terminus to the C-terminus, consists of: the variable region of the anti-MUC1 antibody light chain - [CL];

[0476] Where - represents a peptide bond, Obscurin-O chain is shown in SEQ ID NO:37, Titin-T chain is shown in SEQ ID NO:38, CH1 is shown in SEQ ID NO:39, Cκ is shown in SEQ ID NO:40, IgG1 Fc1 is shown in SEQ ID NO:42; IgG1 Fc2 is shown in SEQ ID NO:41.

[0477] The bispecific antibody SDP02311 has the Trop2 terminus of the antibody S1920 Y53T variable region and the MUC1 terminus of the antibody PT59G variable region. The bispecific antibody SDP02314 has the Trop2 terminus of the antibody S1920 Y53T variable region and the MUC1 terminus of the antibody SDP02313 variable region. The heavy chain (H) and light chain (L) sequences of these two bispecific antibodies are shown below:

[0478] >SDP02311 H1

[0479] >SDP02311 L1

[0480] >SDP02311 H2

[0481] >SDP02311 L2

[0482] SDP02314 H1 is identical to SEQ ID NO:33;

[0483] >SDP02314 L1 is identical to SEQ ID NO:34.

[0484] >SDP02314 H2

[0485] >SDP02314 L2

[0486] In the above heavy chain sequences, the italicized part represents the Fc constant region of IgG1, the underlined wavy part represents the Obscurin-O chain, and the underlined part represents CH1; in the light chain sequences, the underlined part represents Cκ, the underlined wavy part represents the Titin-T chain; the italicized and bold part represents the GGGGS (SEQ ID NO:69) linker.

[0487] Obscurin-O chain

[0488] Titin-T chain

[0489] CH1

[0490] >Cκ

[0491] >IgG1 Fc2(Y349C / T366S / L368A / Y407V)

[0492] >IgG1 Fc1 (S354C / T366W)

[0493] 2) Control antibody

[0494] The control antibody S1920 is an anti-Trop2 antibody, and its full-length sequence is as follows: >Antibody S1920 H

[0495] Antibody S1920 L

[0496] The variable regions of antibodies S1920 and S1920 Y53T were respectively conjugated with isotype (hen egg lysozyme, HEL, not binding to MUC1 or Trop2) antibodies to construct 1+1 bispecific antibodies (as shown in Figure 2) to obtain monovalent anti-Trop2 antibodies, with corresponding antibody names SDP02252 (S1920-iso) and SDP02321 (S1920Y53T-iso).

[0497] Antibody-cell binding activity was evaluated by detecting binding to Trop2 single-positive MDA-MB-453 cells. Specifically, serially diluted antibodies SDP02252 and SDP02321 were incubated with 1E5 cells at 4°C for 1 hour. Excess antibody was washed away, and murine Alexa Flour 647-labeled anti-human Fc antibody was added. The cells were incubated at 4°C for 30 minutes, and after washing away excess antibody, the cells were resuspended in 200 μL of 1% BSA / PBS buffer. The fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer.

[0498] Table 6 shows the EC50 of the modified antibody SDP02321. 50 The value increases significantly, while the maximum value of MFI decreases significantly.

[0499] Table 6. Binding activity of antibody S1920 on the surface of MDA-MB-453 cells before and after mutation.

[0500] The full-length sequence of antibody SDP02321 (anti-Trop2 monovalent antibody) is as follows:

[0501] SDP02321 H1 is identical to SEQ ID NO:33;

[0502] >SDP02321 L1 is identical to SEQ ID NO:34.

[0503] Antibody SDP02321 H2

[0504] Antibody SDP02321 L2

[0505] 3) Antibody preparation

[0506] The nucleotide sequence encoding the antibody was cloned into the pTT5 vector and transfected into ExpiCHO cells. Four strands were expressed in the same host cells. After 8 days, the cells were centrifuged, collected, and the cell culture medium was filtered. The harvested cell culture medium was purified using a Protein A affinity column (MabSelect SuRe, GE). The bound antibody was eluted with glycine, and the elution buffer was neutralized with 1M Tris before desalting. The target antibody was obtained by detection.

[0507] 4) Detection of antigen-binding activity of anti-Trop2 / MUC1 bispecific antibodies

[0508] Surface plasmon resonance (SPR) was used to detect the binding activity of the anti-Trop2 / MUC1 bispecific antibody to the antigen. SPR detection was performed using a BIAcore 8K (Cityva) system. The protein A sensor chip and related reagents were purchased from Cytiva.

[0509] Experimental Methods: SDP02311 and SDP02314 were diluted to 1 μg / mL with HBS-EP+ buffer at a flow rate of 10 μL / min to capture antibodies up to 200 RU. His-tagged human Trop2 antigen (purchased from Acro, catalog number TR2-H5223), cyno Trop2 antigen (purchased from Acro, catalog number TR2-R52H3), human CMUC1 antigen (purchased from Acro, catalog number MU1-H52H5), cyno CMUC1 antigen (purchased from Acro, catalog number MU1-C52H5), and human NMUC1 antigen (purchased from Acro, catalog number MU1-H52H7) were diluted with HBS-EP+ buffer at specific concentration gradients of 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM. The flow rate was set to 30 μL / min for sample analysis. The binding time was 120 s, and the dissociation time was 600 s. Regeneration was then performed using pH 1.5 Gly-HCl buffer at a flow rate of 30 μL / min for 30 s. The response signal was plotted with analysis time on the x-axis and response value on the y-axis. The obtained data were fitted using BIAcore 8K analysis software. The binding model of SDP02311 with human NMUC1 antigen was used, while the 1:1 Langmuir binding model was used for the others. The binding rate constant (K0) was determined. a ), dissociation rate constant (K) d and dissociation equilibrium constant (K) D Dynamic constants such as )

[0510] Experimental Results: Table 7 shows that the anti-Trop2 / MUC1 antibody SDP02311 can bind to the N-terminus of human Trop2 antigen, monkey Trop2 antigen, and human MUC1. The anti-Trop2 / MUC1 antibody SDP02314 can bind to the C-terminus of human Trop2 antigen, monkey Trop2 antigen, human MUC1, and monkey MUC1.

[0511] Table 7. Binding kinetic parameters of anti-Trop2 / MUC1 antibody

[0512] Example 6. Selective binding of anti-Trop2 / MUC1 bispecific antibody

[0513] 1) The expression of MUC1 N-terminus, MUC1 C-terminus and Trop2 was detected in various tumor cell lines using MUC1 antibody and Trop2 antibody.

[0514] Antibody binding activity is assessed by detecting the fluorescence signal of the antibody bound to the cell surface; the intensity of the fluorescence signal evaluates the binding strength. Specifically, an appropriate concentration of the detection antibody is mixed with 1x10... 5 Cells were incubated at 4°C for 1 hour, excess antibody was washed away, and mouse Alexa Flour 647-labeled anti-human Fc antibody was added. The cells were incubated at 4°C for 30 minutes, excess antibody was washed away, and the cells were resuspended in 200 μL of 1% BSA / PBS buffer. Cell surface fluorescence signals were read using a Thermo Attune NxT flow cytometer. Expression analysis of MUC1 and Trop2 in common cells was performed separately using Graphpad.

[0515] The results showed that tumor cell lines T47D, NCI-1650, and NCI-H441 highly expressed both MUC1 and Trop2; MDA-MB-453 expressed low Trop2 and no MUC1; and A431 expressed high Trop2 and very low MUC1.

[0516] 2) Anti-Trop2 / MUC1 bispecific antibody binds to Trop2 single-positive cells

[0517] The binding of anti-Trop2 / MUC1 bispecific antibodies SDP02311 and SDP02314 to Trop2-positive MDA-MB-453 tumor cells was measured by flow cytometry, with anti-Trop-2 antibodies S1920 and SDP02321 serving as controls.

[0518] Experimental method: Serially diluted antibody molecules and control molecules were mixed with 1x 10... 5 Cells were incubated at 4°C for 1 hour, excess antibody was washed away, and mouse-derived Alexa Flour 647-labeled anti-human Fc antibody was added. After incubation at 4°C for 30 minutes, excess antibody was washed away, and the cells were resuspended in 200 μL of 1% BSA / PBS buffer. The fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer.

[0519] Experimental results: As shown in Figure 3 and Table 8, the anti-Trop2 / MUC1 bispecific antibodies SDP02311 and SDP02314 can bind to Trop2 single-positive cells; and their affinity is comparable to that of SDP02321 (S1920 Y53T-iso), maintaining monovalent binding activity; but weaker than that of S1920.

[0520] Table 8. Binding activity of Trop2-related antibody molecules on MDA-MB-453 cells

[0521] 3) Anti-Trop2 / MUC1 bispecific antibody binds to Trop2 and MUC1 double-positive cells

[0522] The selectivity of dual-target antibodies was evaluated by analyzing the binding of an antibody-cell mixture. Specifically, the cell mixture consisted of the T47D tumor cell line, which highly expresses both MUC1 and Trop2, and the MDA-MB-453 cell line, which expresses low levels of Trop2 and lacks MUC1 expression. For flow cytometry analysis, to distinguish between the two cell types, the double-positive T47D cells were labeled with CFSE (catalog number V12883, Thermo Fisher) and diluted 1000-fold according to the manufacturer's instructions. The stained double-positive T47D cells and unstained MDA-MB-453 cells were mixed at a ratio of 1:50. 2.5E5 cells per well were seeded into 96-well round-bottom plates and incubated with different concentrations of antibody at 4°C for 1 hour. The cells were washed twice with 1% BSA / PBS buffer, and then incubated with Alexa 647-labeled anti-human IgG Fc secondary antibody at 4°C in the dark for 30 minutes. After washing twice with buffer, the cells were resuspended in 200 μL. Fluorescence signals on the cell surface were read using a Thermo Attune NxT flow cytometer. Approximately 1E5 cells were collected from each sample for data analysis.

[0523] As shown in Figure 4, compared with the control S1920, the results showed that the anti-Trop2 / MUC1 bispecific antibodies SDP02311 and SDA02314 selectively bound to double-positive T47D cells.

[0524] Example 7. Design and preparation of anti-Trop2 / MUC1 antibody-drug conjugates (ADCs)

[0525] 1) Preparation of compounds

[0526] 1.1 Preparation of Compound 1

[0527] Compound 1 was synthesized according to the method provided in Example 58 on page 163 of the specification of patent CN104755494A.

[0528] 1.2 Preparation of Compound 9A

[0529] Compound 9A, as described in Example 9 of patent WO2020063676 A1, has the following structure:

[0530] 2) Anti-Trop2 / MUC1 antibody-drug conjugates

[0531] 2.1 Antibody-drug conjugates (ADCs) with the following structures were prepared, as shown in Figure 2. In SDA008, the Ab is SDP02311, and in SDA012, the Ab is SDP02314.

[0532] Preparation of anti-Trop2 / MUC1 antibody-drug conjugates for DAR6:

[0533] Add a 10 mM solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) (10 mM, 1173 μL, 11.7 μmol, 10.0 eq) to a 30 mM histidine-acetic acid buffer (pH 5.5, containing 2.5 mM EDTA) containing antibody SDP02311 (11.3 mg / mL, 15.0 mL, 1.17 μmol), and place the mixture in a temperature-controlled shaker at 35 °C for 2 hours. Then cool the reaction mixture to 25 °C. Add a dimethyl sulfoxide solution of the prepared compound 9-A (10 mg, 9.38 μmol, 8.0 eq) to the above reaction mixture, and place the mixture in a temperature-controlled shaker at 25 °C for 1 hour. Finally, the reaction solution was desalted and buffered using a desalting column (HiPrep 26 / 10, GE) (buffer: 30 mM histidine-acetic acid + 8% sucrose, pH 5.5, flow rate 10 mL / min) to obtain a solution of SDA008 (10.6 mg / mL, 15.6 mL), with a yield of 97%, and stored at 4 °C. The target DAR was 6, and the drug loading was calculated by natural mass spectrometry, with an actual average DAR of 5.75. As shown in Figure 5A, the DAR6 ADC content was 90.1%, exhibiting high homogeneity and containing only small amounts of DAR2 and DAR4 byproducts.

[0534] SDP02314 was conjugated with the aforementioned compound 9-A, and prepared according to the method for SDA008, yielding SDA012. The target DAR was 6, and natural mass spectrometry analysis (Figure 5B) showed an average DAR value of 5.99. DAR6 accounted for 98.6%, DAR4 for 0.7%, and DAR2 and DAR8 were undetectable. This indicates that the anti-Trop2 / MUC1 antibody-drug conjugate (ADC) of this structure exhibits high DAR6 content and low proportions of other DAR values, demonstrating high homogeneity.

[0535] 2.2 Preparation of antibody-drug conjugates (ADCs) with the following structures: SDA001 and SDA002 were prepared according to the preparation method described in step 1.4 above. Specifically, the Ab in SDA001 is SDP02311, and the Ab in SDA002 is SDP02314, with a target DAR value of 6.

[0536] 3) Detection of anti-Trop2 / MUC1 ADC binding to antigen

[0537] Surface plasmon resonance (SPR) was used to detect the binding activity of the anti-Trop2 / MUC1 bispecific antibody to the antigen.

[0538] Experimental Methods: Antibody-drug conjugates SDA008 and SDA012 were diluted to 1 μg / mL with HBS-EP+ buffer at a flow rate of 10 μL / min to capture antibodies up to 200 RU. His-tagged human Trop2 antigen (purchased from Acro, catalog number TR2-H5223), cyno Trop2 antigen (purchased from Acro, catalog number TR2-R52H3), human CMUC1 antigen (purchased from Acro, catalog number MU1-H52H5), cyno CMUC1 antigen (purchased from Acro, catalog number MU1-C52H5), and human NMUC1 antigen (purchased from Acro, catalog number MU1-H52H7) were diluted with HBS-EP+ buffer at specific concentration gradients of 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM. The flow rate was set to 30 μL / min for sample analysis. The binding time was 120 s, and the dissociation time was 600 s. Regeneration was then performed using pH 1.5 Gly-HCl buffer at a flow rate of 30 μL / min for 30 s. The response signal was plotted with analysis time on the x-axis and response value on the y-axis. The obtained data were fitted using BIAcore 8K analysis software. The SDA008 antigen and human NMUC1 antigen were bound using a Steady-state affinity model, while the others were bound using a 1:1 Langmuir model. The binding rate constant (K008) was determined. a ), dissociation rate constant (K) d and dissociation equilibrium constant (K) D Dynamic constants such as )

[0539] Experimental Results: Table 9 shows that the anti-Trop2 / MUC1 antibody-drug conjugate SDA008 can bind to the N-terminus of human Trop2 antigen, monkey Trop2 antigen, and human MUC1 antigen. The anti-Trop2 / MUC1 antibody-drug conjugate SDA012 can bind to the C-terminus of human Trop2 antigen, monkey Trop2 antigen, human MUC1 antigen, and monkey MUC1 antigen. Furthermore, its binding activity is comparable to that of anti-Trop2 / MUC1 antibodies SDP02311 and SDP02314.

[0540] Table 9. Binding kinetic parameters of anti-Trop2 / MUC1 antibody-drug conjugates

[0541] Example 8. Preparation of anti-Trop2 ADC (control ADC)

[0542] 1) The structural formula of the antibody-drug conjugate SDA021 for anti-Trop2 antibody S1920 is shown in the figure below:

[0543] Preparation method of SDA021:

[0544] Add 10 mM (86.2 μL, 862 nmol, 2.5 eq) of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) aqueous solution to antibody S1920 (10.0 mg / mL, 5.0 mL, 345 nmol) in 30 mM histidine-acetic acid buffer (pH 5.5, containing 2.5 mM EDTA). Place the mixture in a temperature-controlled shaker and shake at 37 °C for 3 hours. Then cool the reaction mixture to 25 °C. Add a dimethyl sulfoxide solution of compound 9-A (2.96 mg, 2.76 μmol, 8.0 eq) to the above reaction mixture and shake at 25 °C for 3 hours. Finally, the reaction solution was desalted and replaced using a desalting column (HiPrep 26 / 10, GE) (buffer: 30mM histidine-acetic acid + 8% sucrose, pH 5.5, flow rate 10mL / min) to obtain a solution of SDA021. The drug loading was calculated by RP-HPLC, and the measured DAR was 4.0.

[0545] 2) The structural formula of the anti-Trop2 monovalent antibody-drug conjugate SDA013 is shown below:

[0546] SDP02321 was coupled with the aforementioned compound 9-A, and prepared according to the method for SDA008, to obtain SDA013. The average DAR value was 5.73 as determined by HIC-HPLC.

[0547] 3) The structural formula of the C-terminal antibody-drug conjugate SDA2313 against MUC1 is shown below:

[0548] SDP02321 was coupled with the aforementioned compound 9-A, and SDA2313 was obtained by referring to the preparation method of SDA021. The target DAR value is 4.

[0549] Example 9. Selective binding of anti-Trop2 / MUC1 ADC

[0550] The binding of anti-Trop2 / MUC1 ADCs to T47D and NCI-H441 cells (Trop2 and MUC1 double-positive cells) and A431 cells (Trop2 single-positive cells) was detected. Anti-Trop2 antibody-drug conjugates SDA013 and SDA021 served as controls.

[0551] Experimental method: Serially diluted antibody molecules and control molecules were mixed with 1x10... 5 Cells were incubated at 4°C for 1 hour, excess antibody was washed away, and mouse-derived Alexa Flour 647-labeled anti-human Fc antibody was added. After incubation at 4°C for 30 minutes, excess antibody was washed away, and the cells were resuspended in 200 μL of 1% BSA / PBS buffer. The fluorescence signal on the cell surface was read using a Thermo Attune NxT flow cytometer.

[0552] As shown in Figure 6 and Table 10, the binding amounts of anti-Trop2 / MUC1 ADCs SDA008 and SDA012 and EC in Trop2 single-positive cells were... 50 Both were weaker than the anti-Trop2 ADC SDA021; in Trop2 and MUC1 double-positive cells, the binding of SDA008 and SDA012 was significantly increased. This indicates that the dual anti-ADC may reduce the binding of Trop2 to single-positive normal tissues and enhance the selective binding to double-positive cells.

[0553] Table 10. Binding activities of Trop2-related ADC molecules on A431, T47D, and NCI-H441

[0554] Example 10. Endocytotic activity of anti-Trop2 / MUC1 ADC in Trop2 and MUC1 dual-positive cells

[0555] The endocytic activity of anti-Trop2 / MUC1 ADC in Trop2 and MUC1 double-positive cells (T47D and NCI-H441) was detected, with anti-Trop-2 ADC SDA013 and anti-MUC-C ADC SDA2313 serving as controls.

[0556] Experimental method: Zenon TM pHrodoTM The iFL IgG indicator reagent (Invitrogen, catalog number Z25612) labels a Fab fragment that binds to the Fc region of intact IgG antibodies, forming a labeled complex within 5 minutes. When the complex enters the cells via endocytosis, fluorescence increases dramatically with increasing acidity in the surrounding environment. The fluorescence intensity can be recorded in real-time using an Incucyte instrument, allowing for the assessment of antibody internalization. Specifically, cells are plated at 10,000 cells per well, with 50 μL per well. After cell attachment, 50 μL of the labeled complex is added to each well. The labeled complex is prepared in the same centrifuge tube: 80 nM chimeric antibody, 240 nM pHrodo, mixed, and incubated at room temperature for 5 minutes. The tube is then placed in the Incucyte instrument, and the reading conditions are set according to the instrument's operating procedures. After reading, the data is analyzed using the instrument's built-in analysis software.

[0557] Experimental results: As shown in Figure 7A, the endocytic activity of anti-Trop2 / MUC1 ADC SDA008 in double-positive cells was better than that of SDA012, but both SDA008 and SDA012 were stronger than SDA013; As shown in Figure 7B, the endocytic activity of anti-Trop2 / MUC1 ADC SDA012 in double-positive cells was better than that of SDA021, SDA013 and SDA2313.

[0558] Example 11. Killing activity against Trop2 / MUC1 ADC

[0559] The killing ability of Trop2 / MUC1 bispecific antibody ADC molecules on Trop2-positive A431 cells and T47D cells was verified by in vitro cell killing assays. A431 cells and T47D cells were cultured in DMEM and 1640 medium containing 10% FBS, respectively, and passaged 2-3 times per week at a passage ratio of 1:3 or 1:6. Before passage, the medium was aspirated, and the cells were digested with 2 ml of 0.25% trypsin at 37°C for 2 minutes. Fresh medium was then added to stop the digestion, and the cells were resuspended. 50 μL of the cell suspension was added to each 96-well cell culture plate, and the plates were filled with 2 × 10⁻⁶ cells. 3 Cells / well, 10% FBS 1640 medium, 96-well plate with only PBS buffer added to the periphery. Incubate the plate overnight in an incubator (37°C, 5% CO2).

[0560] The next day, the ADC to be tested was prepared to a final concentration of 200 nM and serially diluted 1:3 with complete culture medium for 9 wells, with an additional 0 concentration. 50 μl of each solution was added to the cell culture plate and incubated for 6 days (37°C, 5% CO2). In a 96-well cell culture plate, 100 μL of CellTiter-Glo reagent was added to each well and incubated at room temperature in the dark for 10-15 min. Chemiluminescence signal values ​​were read using a PE Envision instrument, and data were processed using GraphPad software. A graph was plotted with ADC or toxin concentration on the X-axis and cell killing rate on the Y-axis.

[0561] As shown in Figure 8B and Table 11, in Trop2 and MUC1 double-positive cells T47D, the killing ability of anti-Trop2 / MUC1 ADC SDA008 and SDA012 was better than that of SDA013; as shown in Figure 8A, in Trop2 single-positive cells A431, the killing ability of anti-Trop2 / MUC1 ADC SDA008 and SDA012 was comparable to that of SDA013.

[0562] Table 11. Killing activity against Trop2 / MUC1 ADC

[0563] Example 12. Evaluation of anti-Trop2 / MUC1 ADC in a mouse anti-tumor model

[0564] 1) T47D xenograft tumor model:

[0565] Experimental Balb / C Nude mice, female, 6-8 weeks old, 18-22g. Housing environment: SPF grade. 0.2ml (1X10) 7 100 T47D cells (with a 1:1 matrix gel volume ratio) were seeded. The average tumor volume reached approximately 100-150 mm. 3 Grouping and administration began on the same day. Animals were grouped according to tumor volume and body weight deviation being less than 30%, with 6 animals in each group. Intraperitoneal injection began on the day of grouping (D0), administered once a week for a total of 4 times. Tumor volume and body weight were measured twice weekly, and data were recorded. The dosage was 1 MPk or 0.3 mg / kg.

[0566] Tumor volume V = 1 / 2 × a × b 2 Where a and b represent length and width, respectively. Relative tumor proliferation rate T / C (%) = (T - T0) / (C - C0) × 100, where T and C are the tumor volumes of the treatment and control groups at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. Tumor inhibition rate TGI (%) = 1 - T / C (%). As mentioned above, the human IgG control antibody was used as a negative control. TGI represents the maximum tumor growth inhibition during the experiment.

[0567] The results of the T47D tumor model experiment showed, as shown in Figure 9A and Table 12, that SDA012 and SDA008 significantly inhibited tumor growth at a dose of 1 mg / kg, and the TGI of SDA012 and SDA008 was superior to that of SDA013. Mice showed normal weight gain after treatment, with no toxicity and good tolerability.

[0568] Table 12. Anti-Trop2 / MUC1 ADC inhibitory effect on mouse xenograft (T47D)

[0569] (**** indicates P-value ≤ 0.0001, ** indicates P-value ≤ 0.05, and * indicates P-value ≤ 0.1. Significant differences were analyzed using two-way ANOVA.)

[0570] 2) NCI-1650 xenograft tumor model:

[0571] Experimental NCG mice, female, 6-8 weeks old, 18-22g. Housing environment: SPF grade. 0.2ml (5 x 10 ml) of the solution was used. 5 (Number of NCI-1650 cells, plus 1:1 matrix gel volume ratio) were seeded. The average tumor volume reached approximately 300 mm². 3 Grouping and administration began on the same day. Animals were grouped according to tumor volume and body weight deviation being less than 30%, with 6 animals in each group. Intraperitoneal injection began on the day of grouping (D0), administered once a week for a total of 4 times. Tumor volume and body weight were measured and recorded twice weekly. The dosage was 3 MPa.

[0572] The results of the NCI-1650 tumor model experiment, as shown in Figure 9B and Table 13, indicated that SDA012 and SDA008 significantly inhibited tumor growth at a dose of 3 mg / kg. SDA012, at a dose of 3 mg / kg, showed significantly better efficacy than SDA013, suggesting that the efficacy of the Trop2 / MUC1 bispecific antibody ADC molecule depends on the MUC1 target. Mice showed normal weight gain after treatment, with no toxicity and good tolerability.

[0573] Table 13. Anti-Trop2 / MUC1 ADC inhibitory effect on mouse xenograft (NCI-H1650)

[0574] (*Represents V.SSDA013)

Claims

1. A Trop2 / MUC1 binding molecule comprising a first binding domain specifically binding Trop2 and a second binding domain specifically binding MUC1; the first binding domain comprising a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein the VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO: 1 or 50; wherein the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO: 2 or 3; wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system; or The first binding domain comprises a heavy chain variable region (VH1) and a light chain variable region (VL1), wherein VH1 comprises HCDR1, HCDR2, and HCDR3, and VL1 comprises LCDR1, LCDR2, and LCDR3; wherein, The VH1 includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 48, 9 and 10 respectively; the VL1 includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 11, 47 and 13 respectively; The VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 8, 9, and 10, respectively; the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 11, 14, and 13, respectively; or The VH1 includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 8, 9 and 10 respectively; the VL1 includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 11, 12 and 13 respectively.

2. The Trop2 / MUC1 binding molecule as claimed in claim 1, wherein the second binding domain comprises a heavy chain variable region (VH2) and a light chain variable region (VL2). (a) The VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequence of SEQ ID NO: 23; the VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequence of SEQ ID NO: 24; or (b) The VH2 comprises HCDR1, HCDR2 and HCDR3 in the amino acid sequence shown in SEQ ID NO: 7, 4 or 6; the VL2 comprises LCDR1, LCDR2 and LCDR3 in the amino acid sequence shown in SEQ ID NO: 5; The CDR is 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 VH2 comprises HCDR1, HCDR2, and HCDR3, and VL2 comprises LCDR1, LCDR2, and LCDR3; wherein: (i) The VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 25, 26, and 27, respectively; the VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 28, 29, and 30, respectively; or (ii) The VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 15, 49, and 17, respectively; the VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 18, 19, and 20, respectively; or Preferably, (ii-1) The VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 15, 22, and 17, respectively; the VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 18, 19, and 20, respectively; or (ii-2) The VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 15, 16, and 17, respectively; the VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 18, 19, and 20, respectively; or (ii-3) The VH2 comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, 21 and 17 respectively; the VL2 comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 18, 19 and 20 respectively.

3. The Trop2 / MUC1 binding molecule as described in claim 1 or 2, wherein, The VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 48, 9, and 10, respectively; the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 11, 47, and 13, respectively; and the VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 25, 26, and 27, respectively; and the VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 28, 29, and 30, respectively; or The VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 48, 9, and 10, respectively; the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 11, 47, and 13, respectively; the VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 15, 49, and 17, respectively; and the VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 18, 19, and 20, respectively. Preferably, The VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 8, 9, and 10, respectively; the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 11, 14, and 13, respectively; the VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 25, 26, and 27, respectively; and the VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 28, 29, and 30, respectively. The VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 8, 9, and 10, respectively; the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 11, 14, and 13, respectively; the VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 15, 22, and 17, respectively; and the VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 18, 19, and 20, respectively. The VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 8, 9, and 10, respectively; the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 11, 14, and 13, respectively; the VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 15, 16, and 17, respectively; and the VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 18, 19, and 20, respectively. The VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 8, 9, and 10, respectively; the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 11, 12, and 13, respectively; the VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 25, 26, and 27, respectively; and the VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 28, 29, and 30, respectively. The VH1 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 8, 9, and 10, respectively; the VL1 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 11, 12, and 13, respectively; the VH2 comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 15, 22, and 17, respectively; and the VL2 comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 18, 19, and 20, respectively. VH1 includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 8, 9 and 10 respectively; VL1 includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 11, 12 and 13 respectively; VH2 includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, 16 and 17 respectively; and VL2 includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 18, 19 and 20.

4. The Trop2 / MUC1 binding molecule as described in any one of claims 1 to 3, wherein, The VH1 comprises an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 1 or 50, and the VL1 comprises an amino acid sequence as shown in or having at least 90% identity with SEQ ID NO: 2 or 3; and / or, The VH2 comprises an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 4, 6, 7 and 23, and the VL2 comprises an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 5 or 24. Preferably, The VH1 contains the amino acid sequence shown in SEQ ID NO: 1; the VL1 contains the amino acid sequence shown in SEQ ID NO: 3; the VH2 contains the amino acid sequence shown in SEQ ID NO: 23; and the VL1 contains the amino acid sequence shown in SEQ ID NO: 24; The VH1 contains the amino acid sequence shown in SEQ ID NO: 1; the VL1 contains the amino acid sequence shown in SEQ ID NO: 3; the VH2 contains the amino acid sequence shown in SEQ ID NO: 7; and the VL1 contains the amino acid sequence shown in SEQ ID NO: 5; The VH1 contains the amino acid sequence shown in SEQ ID NO: 1; the VL1 contains the amino acid sequence shown in SEQ ID NO: 3; the VH2 contains the amino acid sequence shown in SEQ ID NO: 6; and the VL1 contains the amino acid sequence shown in SEQ ID NO: 5; The VH1 contains the amino acid sequence shown in SEQ ID NO: 1; the VL1 contains the amino acid sequence shown in SEQ ID NO: 3; the VH2 contains the amino acid sequence shown in SEQ ID NO: 4; and the VL1 contains the amino acid sequence shown in SEQ ID NO: 5; The VH1 contains the amino acid sequence shown in SEQ ID NO: 1; the VL1 contains the amino acid sequence shown in SEQ ID NO: 2; the VH2 contains the amino acid sequence shown in SEQ ID NO: 23; and the VL1 contains the amino acid sequence shown in SEQ ID NO: 24; The VH1 contains the amino acid sequence shown in SEQ ID NO: 1; the VL1 contains the amino acid sequence shown in SEQ ID NO: 2; the VH2 contains the amino acid sequence shown in SEQ ID NO: 7; and the VL1 contains the amino acid sequence shown in SEQ ID NO: 5; The VH1 contains the amino acid sequence shown in SEQ ID NO: 1; the VL1 contains the amino acid sequence shown in SEQ ID NO: 2; the VH2 contains the amino acid sequence shown in SEQ ID NO: 6; and the VL1 contains the amino acid sequence shown in SEQ ID NO: 5; The VH1 contains the amino acid sequence shown in SEQ ID NO: 1; the VL1 contains the amino acid sequence shown in SEQ ID NO: 2; the VH2 contains the amino acid sequence shown in SEQ ID NO: 4; and the VL1 contains the amino acid sequence shown in SEQ ID NO: 5; The VH1 contains the amino acid sequence shown in SEQ ID NO: 50; the VL1 contains the amino acid sequence shown in SEQ ID NO: 2; the VH2 contains the amino acid sequence shown in SEQ ID NO: 23; and the VL1 contains the amino acid sequence shown in SEQ ID NO: 24; The VH1 contains the amino acid sequence shown in SEQ ID NO: 50; the VL1 contains the amino acid sequence shown in SEQ ID NO: 2; the VH2 contains the amino acid sequence shown in SEQ ID NO: 7; and the VL1 contains the amino acid sequence shown in SEQ ID NO: 5; The VH1 contains the amino acid sequence shown in SEQ ID NO: 50; the VL1 contains the amino acid sequence shown in SEQ ID NO: 2; the VH2 contains the amino acid sequence shown in SEQ ID NO: 6; and the VL1 contains the amino acid sequence shown in SEQ ID NO: 5; or The VH1 contains the amino acid sequence shown in SEQ ID NO: 50; the VL1 contains the amino acid sequence shown in SEQ ID NO: 2; the VH2 contains the amino acid sequence shown in SEQ ID NO: 4; and the VL1 contains the amino acid sequence shown in SEQ ID NO: 5; Preferably, the Trop2 / MUC1 binding molecule is an anti-Trop2 / MUC1 antibody, more preferably an anti-Trop2 / MUC1 bispecific antibody.

5. The Trop2 / MUC1 binding molecule as described in any one of claims 1 to 4, further comprising an immunoglobulin Fc region; Preferably, the Fc region is the Fc region of IgG1, IgG2, IgG3 or IgG4; More preferably, the Fc region contains ADCC-enhancing mutations, such as 239D and 332E, wherein the amino acid positions are numbered according to the Eu numbering rules.

6. The Trop2 / MUC1 binding molecule as described in claim 5, wherein, The Fc region comprises a first subunit and a second subunit, the first and second subunits containing knobs-into-holes mutations; Preferably, the first subunit of the Fc region contains a knob mutation, and the second subunit of the Fc region contains a hole mutation; Preferably, The first subunit of the Fc region contains a mutation at position 366, and the second subunit contains a mutation selected from positions 366, 368, and 407, or any combination thereof. The first subunit of the Fc region contains a mutation at position 354 or 356, and the second subunit contains a mutation at position 349; or... The first subunit of the Fc region contains a mutation at position 354 or 366, and the second subunit contains a mutation at position 349, 366, 368, or 407. More preferably, the first subunit of the Fc region contains the 366W mutation, and the second subunit contains a mutation selected from 366S, 368A and 407V or any combination thereof; The first subunit of the Fc region contains a 354C or 356C mutation, and the second subunit contains a 349C mutation; or The first subunit of the Fc region contains the 354C / 366W mutation, and the second subunit contains the 349C / 366S / 368A / 407V mutation. The positions of the mutated amino acids are determined according to the Eu numbering system.

7. The Trop2 / MUC1 binding molecule as described in any one of claims 1 to 6, comprising a first heavy chain, a first light chain, a second heavy chain, and a second light chain; wherein, From N-end to C-end: The first heavy chain has the following structure: [VH1]-[connector 1]-[Obscurin-O chain]-[connector 3]-[first subunit of Fc region]. The first light chain has the following structure: [VL1]-[connector 2]-[Titin-T chain], The second heavy chain has the following structure: [VH2]-[CH1]-[second subunit of the Fc region], and The second light chain has the following structure: [VL2]-[CL]; Where - represents a peptide bond, The Obscurin-O chain contains the amino acid sequence shown in SEQ ID NO:

37. The Titin-T chain contains the amino acid sequence shown in SEQ ID NO:

38. The connector 1, connector 2 and connector 3 may be the same or different, and may exist independently or not exist. Preferably, the amino acid sequences of linker 1 and linker 2 are GGGGS, and linker 3 is absent.

8. The Trop2 / MUC1 binding molecule of claim 7, comprising a combination of polypeptide chains as follows: 1) A first heavy chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 33, a first light chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 34, a second heavy chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 31, and a second light chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO:

32. or 2) A first heavy chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 33, a first light chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 34, a second heavy chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO: 35, and a second light chain comprising an amino acid sequence as shown in or having at least 90% sequence identity with SEQ ID NO:

36.

9. A Trop2 / MUC1 binding molecule comprising a first binding domain that specifically binds Trop2 and a second binding domain that specifically binds MUC1; Preferably, its endocytic activity in Trop2 and MUC1 dual-positive cells is greater than that of the Trop2-binding molecule.

10. A method for preparing Trop2 / MUC1 binding molecules, the method comprising: (a) Determine the binding activity of candidate molecules with Trop2 peptide; (b) Determine the binding activity of candidate molecules with MUC1 peptides; (c) Select candidate molecules that have Trop2 binding activity and MUC1 binding activity, i.e., the Trop2 / MUC1 binding molecules.

11. An antibody-drug conjugate comprising an antibody and a drug molecule, wherein the antibody comprises a first binding domain that specifically binds to Trop2 and / or a second binding domain that specifically binds to MUC1, wherein the first binding domain and the second binding domain are, respectively, the first binding domain and the second binding domain of any one of claims 1 to 8. Preferably, the drug molecule is a cytotoxic drug; more preferably, the drug molecule is a camptothecin-type drug, such as eczema or its derivatives.

12. The antibody-drug conjugate according to claim 11, having the structure shown in formula (IV): in: 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 They may be the same or different, and each is independently selected from hydrogen, halogen, alkyl, haloalkyl, alkoxy, hydroxyl, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, or heterocyclic groups; or, R a and R b Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups; R 1 Selected from hydrogen atoms, alkyl groups, halogens, haloalkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups; R 2 Selected from hydrogen atoms, alkyl groups, halogens, haloalkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, or heteroaryl groups; Or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups; Or, R a and R 2 Together with the carbon atom attached to it, it forms a cycloalkyl or heterocyclic group; m is an integer from 0 to 4; n is between 1 and 10, and can be an integer or a decimal; -L- represents the connector unit; Preferably, L is -L 1 -L 2 -L 3 -L 4 -,in: L 1 It is -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, where 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 with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, alkoxy and cycloalkyl; 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, where p 1 Integers from 1 to 20; L 3 It is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids may optionally be further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, 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 They may be the same or different, and each is independently selected from hydrogen atoms, alkyl, haloalkyl and hydroxyalkyl; 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, and hydroxyalkyl groups; n is between 1 and 10, and n is a decimal or an integer; Ab stands for antibody.

13. The antibody-drug conjugate according to claim 12, wherein: Y is -O-(CR a R b )m-CR 1 R 2 -C(O)-; R a and R b They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 alkoxy, hydroxy and C 1-6 Hydroxyalkyl; R 1 Selected from hydrogen atoms, C 1-6 Alkyl, C 3-6 cycloalkyl C 1-6 Alkyl or C 3-6 cycloalkyl; R 2 Selected from hydrogen atoms, halogens, C 1-6 Haloalkyl, C 3-6 cycloalkyl and C 3-6 cycloalkyl C 1-6 alkyl; Or, R 1 and R 2 Together with the carbon atoms it is attached to, they form C 3-6 cycloalkyl; Or, R a and R 2 Together with the carbon atom it is attached to, they 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 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 alkyl; R 1 C 3-6 cycloalkyl C 1-6 Alkyl or C 3-6 cycloalkyl; R 2 Selected from hydrogen atoms, C 1-6 Halogenated alkyl groups and C 3-6 cycloalkyl; Or, R 1 and R 2 Together with the carbon atoms it is attached to, they form C 3-6 cycloalkyl; m is 0 or 1; And / or, Connector unit -L- is -L 1 -L 2 -L 3 -L 4 -, L 1 It is -(succinimide-3-yl-N)-WC(O)-, where W is C 1-8 Alkyl or C 1-8 Alkyl-C 3-8 cycloalkyl; L 2 For -NR 4 (CH2CH2O)p 1 CH2C(O)- or chemical bond, where p 1 Integers from 1 to 20; L 3 It is a peptide residue composed 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 -, where t is an integer from 1 to 6; R 4 Selected from hydrogen atoms, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups and C 1-6 Hydroxyalkyl; R 5 Selected from hydrogen atoms, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups and C 1-6 Hydroxyalkyl; R 6 and R 7 Whether the atoms are the same or different, and each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, C 1-6 Halogenated alkyl groups and C 1-6 Hydroxyalkyl; Preferably, the connector unit -L- is -L 1 -L 2 -L 3 -L 4 -, L 1 for s 1 Integers between 2 and 8; L 2 It is a chemical bond; L 3 It is a tetrapeptide residue of GGFG; L 4 For -NR 5 (CR 6 R 7 ) t -, R 5 It is a hydrogen atom or a carbon atom. 1-6 Alkyl, R 6 and R 7 They may be the same or different, and each is independently a hydrogen atom or a carbon atom. 1-6 Alkyl group, where t is 1 or 2.

14. The antibody-drug conjugate according to any one of claims 12 to 13, having the structure shown in formula (III): in: s 1 Integers from 2 to 8; preferably 5; m is 0 or 1; R 1 C 3-6 cycloalkyl C 1-6 Alkyl or C 3-6 cycloalkyl; R 2 Selected from hydrogen atoms, C 1-6 Halogenated alkyl groups and C 3-6 cycloalkyl; Or, R 1 and R 2 Together with the carbon atoms it is attached to, they form C 3-6 cycloalkyl; R 5 Selected from hydrogen atoms, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups and C 1-6 Hydroxyalkyl; R 6 and R 7 Whether the atoms are the same or different, and each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, C 1-6 Halogenated alkyl groups and C 1-6 Hydroxyalkyl.

15. The antibody-drug conjugate according to any one of claims 12 to 14, wherein -LY- is optionally selected from: Preferred 16. Antibody-drug conjugates, with the following structure: in: n is between 1 and 10, and n is a decimal or an integer; Ab is an antibody containing a first binding domain that specifically binds to Trop2 and / or a second binding domain that specifically binds to MUC1, wherein the first binding domain and the second binding domain are the first binding domain and the second binding domain of any one of claims 1 to 8, respectively.

17. A method for preparing an antibody-drug conjugate as described in any one of claims 12 to 16, comprising the step of conjugating the antibody with the drug molecule.

18. A polynucleotide encoding the Trop2 / MUC1 binding molecule as described in any one of claims 1 to 9.

19. A vector comprising or expressing the polynucleotide of claim 18.

20. A host cell containing or expressing the polynucleotide of claim 18 or the vector of claim 19.

21. A method for preparing Trop2 / MUC1 binding molecules, including: The expression of the polynucleotide of claim 18 or the vector of claim 19 in the host cell of claim 20, and the Trop2 / MUC1 binding molecule isolated and expressed from the host cell; Optionally, the process further includes a step of purifying the Trop2 / MUC1 binding molecule.

22. A pharmaceutical composition comprising the Trop2 / MUC1 binding molecule of any one of claims 1 to 9 or the antibody-drug conjugate of any one of claims 11 to 16, and at least one pharmaceutically acceptable excipient, diluent or carrier.

23. Use of the Trop2 / MUC1 binding molecule according to any one of claims 1 to 9, the antibody-drug conjugate according to any one of claims 11 to 16, the polynucleotide according to claim 18, the carrier according to claim 19, or the pharmaceutical composition according to claim 22 for the preparation of a medicament for treating cancer; Preferably, the cancer is double-positive for Trop2 and MUC1; Preferably, the cancer is breast cancer or lung cancer (e.g., non-small cell lung cancer).

24. Methods for treating cancer, including Administering a therapeutically effective amount of the Trop2 / MUC1 binding molecule of any one of claims 1 to 9, the antibody-drug conjugate of any one of claims 11 to 16, the polynucleotide of claim 18, the carrier of claim 19, or the pharmaceutical composition of claim 22 to a subject in need. Preferably, the cancer is double-positive for Trop2 and MUC1; Preferably, the cancer is breast cancer or lung cancer (e.g., non-small cell lung cancer).

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