Bispecific antigen binding molecules that bind to TROP2 and / or PRLR, conjugates and uses thereof

Abstract

The present invention provides a bispecific antibody (bsAb) comprising a bispecific antigen-binding molecule that specifically binds to a first antigen-binding domain of human TROP2 and a second antigen-binding domain of human PRLR, and an ADC thereof. The described bispecific antigen binding molecules and ADCs thereof are useful for inducing immune responses against TROP2 expressed tumors.

Description

Technical Field

[0001] The present invention relates to bispecific antigen binding molecules that bind to TROP2 and / or PRLR and uses thereof. The present invention also relates to antibody-drug conjugates, which comprise bispecific antigen binding molecules that bind to TROP2 and / or PRLR and therapeutic agents (e.g., cytotoxic drugs). Background Art

[0002] Human trophoblast cell surface antigen 2 (TROP2) is a cell surface glycoprotein encoded by the TACSTD2 gene. A large number of clinical studies and literature reports have shown that TROP2 is overexpressed in a variety of epithelial cancers, including gastric cancer, lung cancer, colorectal cancer, ovarian cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, and esophageal cancer (Tsujikawa et al. (1999) Nat

[0003] Genet.21:420–423; Fong et al. (2008) Mod path.21:186–191; Muhlmann et al. (2009) J Clin Path.62:152–158; and Wu et al. (2013) Head & Neck.35:1373–1378). In contrast, TROP2 is rarely expressed or not expressed in normal adult tissues (Cubas et al. (2009) Biochimica et Biophysica Acta.1796:309–314; Zhang et al. (1997)

[0004] Science.276:1268–1272), and only a small amount of expression is found in cells in the epithelial area, and the expression level is also lower than that in cancer, indicating that TROP2 is related to tumor formation. Overexpression of TROP2 in tumor tissue is closely related to the poor prognosis of patients (Wang et al. (2008) Mol Cancer Ther 7:280-285) and the metastasis of cancer cells, and also affects the overall survival rate of patients. Therefore, TROP2 has become a striking target in molecular targeted therapy of tumors.

[0005] Prolactin is a polypeptide growth hormone that exerts its activity by interacting with the prolactin receptor (PRLR). PRLR is a single transmembrane receptor belonging to the class I cytokine receptor superfamily. The combination of prolactin and PRLR leads to dimerization and intracellular signal transduction of the receptor. The signal transduction through PRLR is associated with various processes, such as mammary gland development, lactation, reproduction and immunoregulation. In addition, high levels of PRLR expression have been detected in breast, prostate and other tumor types. Blocking of PRLR signal transduction has been suggested as a means for treating breast cancer and prostate cancer. (See Damiano et al. (2013) Clin. Cancer Res. 19 (7): 1644-1650).

[0006] Antibody-drug conjugates (ADCs) are a class of effective therapeutic constructs, generally composed of three parts: antibodies or antibody-like ligands, cytotoxic drugs, and linkers that couple ligands and drugs. It allows targeted delivery of small molecule drugs to target cells such as cancer cells. Due to the targeting function, these compounds show significantly higher therapeutic indexes compared to the same systemically delivered agents. Since ADCs have the potential to be used as effective anticancer agents that reduce systemic toxicity, they can be used alone or as adjuvant therapy to reduce tumor load. After ADCs bind antigens with the help of the targeting of antibodies, they need to enter the interior of cells through endocytosis to release toxic drugs to kill target cells. The endocytosis efficiency of ADCs is closely related to their activity, which directly affects the aggregation of drugs in tumor cells and the subsequent killing of target cells. Therefore, it is necessary to obtain ADCs with greater internalization efficiency, so that they have better pharmacokinetic properties, higher activity, and improved drug efficacy, thereby improving the therapeutic efficacy of various diseases such as inducing immune responses to cancers expressed by TROP2 or PRLR. Summary of the invention

[0007] A first aspect of the present invention provides a bispecific antigen-binding molecule, which comprises a first antigen-binding domain that binds to human TROP2 and a second antigen-binding domain that binds to human PRLR.

[0008] In some embodiments, the first antigen binding domain binds to human cells expressing human TROP2.

[0009] In some embodiments, the second antigen binding domain binds to human cells expressing human PRLR.

[0010] In some embodiments, each of the first antigen binding domain and the second antigen binding domain is a fully human antigen binding domain.

[0011] In some embodiments, the bispecific antigen binding molecule is a bispecific antibody.

[0012] In some embodiments, the bispecific antigen-binding molecule is a bispecific antigen-binding fragment.

[0013] In some embodiments, the bispecific antigen binding molecule binds to human TROP2 or / and human PRLR expressed on cells.

[0014] In some embodiments, the first antigen binding domain comprises a heavy chain variable domain VH1 and a light chain variable domain VL1, wherein the VH1 comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2 and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; wherein the VL1 comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5 and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

[0015] In some embodiments, the first antigen binding domain comprises a heavy chain variable domain with an amino acid sequence of SEQ ID NO:7 and / or a light chain variable domain with an amino acid sequence of SEQ ID NO:8.

[0016] In some embodiments, the second antigen-binding domain comprises a heavy chain variable domain VH2 and a light chain variable domain VL2, wherein the VH2 comprises a complementarity determining region H′CDR1, H′CDR2 and H′CDR3, wherein the H′CDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H′CDR2 comprises the amino acid sequence shown in SEQ ID NO:10 and the H′CDR3 comprises the amino acid sequence shown in SEQ ID NO:11; wherein the VL2 comprises a complementarity determining region L′CDR1, L′CDR2 and L′CDR3, wherein the L′CDR1 comprises the amino acid sequence shown in SEQ ID NO:12, the L′CDR2 comprises

[0017] The amino acid sequence shown in SEQ ID NO:13 and L'CDR3 include the amino acid sequence shown in SEQ ID NO:14.

[0018] In some embodiments, the second antigen binding domain comprises a heavy chain variable domain with an amino acid sequence of SEQ ID NO:15 and / or a light chain variable domain with an amino acid sequence of SEQ ID NO:16.

[0019] In some embodiments, the second antigen binding domain comprises a second light chain, comprising a second light chain variable domain (VL2), wherein VL2 is operably linked to a first pairing domain (X1), and a second heavy chain, comprising a second heavy chain variable domain (VH2), wherein VH2 is operably linked to a second pairing domain (X2), wherein X1 and X2 can form a leucine zipper structure.

[0020] In some embodiments, the first antigen binding domain comprises a first light chain and a first heavy chain, wherein the first light chain comprises a first light chain variable domain (VL1), wherein VL1 is operably connected to a third pairing domain (X3), and the first heavy chain comprises a first heavy chain variable domain (VH1), wherein VH1 is operably connected to a fourth pairing domain (X4), and X3 and X4 can form a leucine zipper structure.

[0021] In some embodiments, the first antigen binding domain further comprises a first dimerization domain, the second antigen binding domain further comprises a second dimerization domain, and the first dimerization domain and the second dimerization domain are combined.

[0022] In some embodiments, the first dimerization domain and the second dimerization domain are not identical and associate in a manner that prevents homodimerization and / or favors heterodimerization.

[0023] In some embodiments, the first dimerization domain and the second dimerization domain are combined by a means selected from the group consisting of knob-into-hole, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or increased flexibility.

[0024] In some embodiments, the C-terminus of the first dimerization domain is operably connected to the N-terminus of the fifth pairing domain (X5), and the C-terminus of the second dimerization domain is operably connected to the N-terminus of the sixth pairing domain (X6), wherein the X5 and X6 can form a dimer, and preferably the X5 and X6 can form a leucine zipper structure.

[0025] In some embodiments, X1 comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 domain or a C / EBP domain; and X2 correspondingly comprises a binding domain that forms a leucine zipper structure with X1.

[0026] In some embodiments, X2 comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 domain or a C / EBP domain; and X1 correspondingly comprises a binding domain that forms a leucine zipper structure with X2.

[0027] In some embodiments, X1 comprises a Jun domain and X2 comprises a Fos domain.

[0028] In some embodiments, X1 comprises a Fos domain and X2 comprises a Jun domain.

[0029] In some embodiments, X1 comprises a Jun domain and X2 comprises a FosW domain.

[0030] In some embodiments, X1 comprises a FosW domain and X2 comprises a Jun domain.

[0031] In some embodiments, X1 comprises a Myc domain and X2 comprises a Max domain.

[0032] In some embodiments, X1 comprises a Max domain and X2 comprises a Myc domain.

[0033] In some embodiments, X1 comprises a WinzipA2 domain and X2 comprises a WinzipB1 domain.

[0034] In some embodiments, X1 comprises a WinzipB1 domain and X2 comprises a WinzipA2 domain.

[0035] In some embodiments, X1 comprises an ACID-p1 domain and X2 comprises a BASE-p1 domain.

[0036] In some embodiments, X1 comprises a BASE-p1 domain and X2 comprises an ACID-p1 domain.

[0037] In some embodiments, X1 comprises a GCN4 domain and X2 comprises a C / EBP domain.

[0038] In some embodiments, X1 comprises a C / EBP domain and X2 comprises a GCN4 domain.

[0039] In some embodiments, X3 comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 domain or a C / EBP domain; and X4 correspondingly comprises a binding domain that forms a leucine zipper structure with X3.

[0040] In some embodiments, X4 comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 domain or a C / EBP domain; and X3 correspondingly comprises a binding domain that forms a leucine zipper structure with X4.

[0041] In some embodiments, X3 comprises a Jun domain and X4 comprises a Fos domain.

[0042] In some embodiments, X3 comprises a Fos domain and X4 comprises a Jun domain.

[0043] In some embodiments, X3 comprises a Jun domain and X4 comprises a FosW domain.

[0044] In some embodiments, X3 comprises a FosW domain and X4 comprises a Jun domain.

[0045] In some embodiments, X3 comprises a Myc domain and X4 comprises a Max domain.

[0046] In some embodiments, X3 comprises a Max domain and X4 comprises a Myc domain.

[0047] In some embodiments, X3 comprises a WinzipA2 domain and X4 comprises a WinzipB1 domain.

[0048] In some embodiments, X3 comprises a WinzipB1 domain and X4 comprises a WinzipA2 domain.

[0049] In some embodiments, X3 comprises an ACID-p1 domain and X4 comprises a BASE-p1 domain.

[0050] In some embodiments, X3 comprises a BASE-p1 domain and X4 comprises an ACID-p1 domain.

[0051] In some embodiments, X3 comprises a GCN4 domain and X4 comprises a C / EBP domain.

[0052] In some embodiments, X3 comprises a C / EBP domain and X4 comprises a GCN4 domain.

[0053] In some embodiments, X5 comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 domain or a C / EBP domain; and X6 correspondingly comprises a domain that forms a leucine zipper structure with X5.

[0054] In some embodiments, X6 comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 domain or a C / EBP domain; and X5 correspondingly comprises a domain that forms a leucine zipper structure with X6.

[0055] In some embodiments, X5 comprises a Jun domain and X6 comprises a Fos domain.

[0056] In some embodiments, X5 comprises a Fos domain and X6 comprises a Jun domain.

[0057] In some embodiments, X5 comprises a Jun domain and X6 comprises a FosW domain.

[0058] In some embodiments, X5 comprises a FosW domain and X6 comprises a Jun domain.

[0059] In some embodiments, X5 comprises a Myc domain and X6 comprises a Max domain.

[0060] In some embodiments, X5 comprises a Max domain and X6 comprises a Myc domain.

[0061] In some embodiments, X5 comprises a WinzipA2 domain and X6 comprises a WinzipB1 domain.

[0062] In some embodiments, X5 comprises a WinzipB1 domain and X6 comprises a WinzipA2 domain.

[0063] In some embodiments, X5 comprises an ACID-p1 domain and X6 comprises a BASE-p1 domain.

[0064] In some embodiments, X5 comprises a BASE-p1 domain and X6 comprises an ACID-p1 domain.

[0065] In some embodiments, X5 comprises a GCN4 domain and X6 comprises a C / EBP domain.

[0066] In some embodiments, X5 comprises a C / EBP domain and X6 comprises a GCN4 domain.

[0067] In some embodiments, the Jun domain is a sequence as set forth in SEQ ID NO:17, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:17; and / or, the Fos domain is a sequence as set forth in SEQ ID NO:18, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:18; and / or, the FosW domain is a sequence as set forth in SEQ ID NO:19, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:19; and / or, the Max domain is a sequence as set forth in SEQ ID NO:20, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:20; and / or, the Myc domain is a sequence as set forth in SEQ ID NO:21, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:21. NO:21 has a sequence with at least 80%, 85%, 90%, 95%, or 99% identity; and / or,

[0068] The WinzipA2 domain is a sequence as shown in SEQ ID NO:22, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:22; and / or, the WinzipB1 domain is a sequence as shown in SEQ ID NO:23, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:23; and / or, the ACID-p1 domain is a sequence as shown in SEQ ID NO:24, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:24; and / or, the BASE-p1 domain is a sequence as shown in SEQ ID NO:25, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:25; and / or, the GCN4 domain is a sequence as shown in SEQ ID NO:26, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: NO:26 has a sequence with at least 80%, 85%, 90%, 95%, or 99% identity; and / or,

[0069] The C / EBP domain is the sequence set forth in SEQ ID NO:27, or a sequence at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:27.

[0070] In some embodiments, the VL1 is operably connected to X3, preferably, the VL1 is operably connected to X3 through a first linker L1; the VH1 is operably connected to X4, preferably, the VH1 is operably connected to X4 through a second linker L2; and / or, the VL2 is operably connected to X1, preferably, the VL2 is operably connected to X1 through a third linker L3; the VH2 is operably connected to X2, preferably, the VH2 is operably connected to X2 through a fourth linker L4; and / or, the C-terminus of the first dimerization domain is connected to X5, preferably, the C-terminus of the first dimerization domain is connected to X5 through a fifth linker L5; the C-terminus of the second dimerization domain is connected to X6, preferably, the C-terminus of the second dimerization domain is connected to X6 through a sixth linker L6, and L1-L6 are each independently present or absent.

[0071] In some embodiments, when L1-L6 are optionally present, L1-L6 are each independently selected from the following polypeptide fragments: (GxS)y, (TxG)y, and (SxG)y, wherein x is selected from an integer of 1, 2, 3, 4, or 5, and y is selected from an integer of 1, 2, 3, 4, 5, or 6.

[0072] In some embodiments, L1-L6 are each independently selected from the following polypeptide fragments: GGS, GGGGS, TGGGG, and SGGGG, more preferably, L1-L6 are each independently connected to a cleavage site peptide via their C-terminus, and the cleavage site peptide is selected from factoryXa, thrombin, enterokinase or SUMO.

[0073] In some embodiments, the first antigen binding domain comprises a heavy chain having an amino acid sequence of SEQ ID NO: 29 and a light chain having an amino acid sequence of SEQ ID NO: 28; and / or

[0074] The second antigen binding domain comprises a heavy chain with an amino acid sequence of SEQ ID NO: 31 and a light chain with an amino acid sequence of SEQ ID NO: 30.

[0075] The second aspect of the present invention provides an isolated polynucleotide encoding the bispecific antigen binding molecule described in any one of the first aspect; preferably, the polynucleotide is single-stranded DNA, double-stranded DNA or single-stranded RNA.

[0076] The third aspect of the present invention provides an isolated vector comprising the polynucleotide described in the second aspect.

[0077] The fourth aspect of the present invention provides a host cell comprising the polynucleotide described in the second aspect or the vector described in the third aspect.

[0078] In some embodiments, the host cell is a prokaryotic cell, preferably Escherichia coli.

[0079] In some embodiments, the host cell is a eukaryotic cell, preferably, the cell is selected from one of a CHO cell, a COS cell, a Sp2 / 0 cell or a yeast cell.

[0080] The fifth aspect of the present invention provides an antibody-drug conjugate (ADC), wherein the antibody-drug conjugate comprises the bispecific antigen binding molecule according to any one of the first aspects and a cytotoxic drug.

[0081] In some embodiments, the bispecific antigen binding molecule and the cytotoxic drug are covalently linked via a linker.

[0082] In some embodiments, the cytotoxic drug is selected from the group consisting of auristatins, maytansinoids, calicheamicin, duocarmycins, anthracyclines, pyrrolobenzodiazepine dimers, amatoxin, camptothecin derivatives, tubulysin, tomaymycin, or dolastatin derivatives.

[0083] In some embodiments, the cytotoxic drug is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoid DM1, maytansinoid DM4, camptothecin SN-38, isotecan, or a derivative thereof.

[0084] In some embodiments, the linker is one or more of MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropionyl), val-cit (valine-citrulline), val-ala (valine-alanine), ala-phe (alanine-phenylalanine), phe-lys (phenylalanine-lysine), a dipeptide site in a protease-cleavable linker, PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio) pentanoate), SMCC (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate), and SIAB (N-succinimidyl (4-iodo-acetyl) aminobenzoate). Preferably, the linker is MC.

[0085] The sixth aspect of the present invention provides a pharmaceutical composition, which contains a therapeutically effective amount of the bispecific antigen binding molecule according to any one of the first aspect or the antibody-drug conjugate (ADC) according to any one of the fifth aspect and a pharmaceutically acceptable carrier or diluent.

[0086] The seventh aspect of the present invention provides the use of the bispecific antigen binding molecule described in any one of the first aspect, the antibody-drug conjugate (ADC) described in any one of the fifth aspect, or the pharmaceutical composition described in any one of the sixth aspect in the preparation of a medicament for treating a disease or condition associated with the expression of TROP2 and / or PRLR.

[0087] In some embodiments, the disease or disorder is cancer.

[0088] Technical effects of the present invention:

[0089] The bispecific antibodies and antibody-drug conjugates (ADCs) thereof according to the present invention are particularly useful for targeting cells expressing TROP2 and / or PRLR, and have higher internalization efficiency than monoclonal antibodies; the anti-TROP2 / PRLR bispecific antibody-drug conjugate has a higher affinity for the TROP2 antigen than the anti-TROP2 monoclonal antibody-drug conjugate, and the anti-TROP2 / PRLR bispecific antibody-drug conjugate also has a higher affinity for PRLR; and the anti-TROP2 / PRLR bispecific antibody-drug conjugate has a higher cell killing activity against the TROP2-positive tumor cell line MDA-MB-468 than the anti-TROP2 monoclonal antibody-drug conjugate, for example, the anti-TROP2 / PRLR-MC-MMAF ADC is twice as effective as the anti-TROP2-MC-MMAF ADC in killing TROP2-positive MDA-MB-468 cells.

[0090] In addition, the bispecific antibody of the present invention obtained by conventional preparation methods has a higher purity, and the anti-TROP2 / PRLR bispecific antibody of the present invention can effectively prevent mismatching and assembly between light chain-heavy chain and heavy chain-heavy chain.

[0091] definition

[0092] The "antibodies" described in the present invention include antibodies or antigen-binding fragments thereof that have been modified on the basis of immunoglobulins while retaining the ability to bind to antigens; including monospecific antibodies, multispecific antibodies (such as bispecific antibodies); and also monovalent antibodies or multivalent antibodies. A natural complete antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region (VH) and the first, second and third constant regions (CH1, CH2, CH3, respectively), while each light chain consists of a variable region (VL) and a constant region (CL). The antibody is "Y"-shaped, and the backbone of the "Y"-shaped structure is composed of the second and third constant regions of the two heavy chains, which are bound by disulfide bonds. Each arm of the "Y"-shaped structure contains the variable region and the first constant region of one of the heavy chains, which are bound to the variable region and constant region of the light chain. The variable regions of the light chain and the heavy chain are responsible for the binding of the antigen. The variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the light (L) chain contain LCDR1, LCDR2, LCDR3, and the CDRs of the heavy (H) chain contain HCDR1, HCDR2, HCDR3). Among them, the three CDRs are separated by a lateral continuous part called the framework region (FR), which is more highly conserved than the CDR and forms a scaffold to support the hypervariable loops. VH and VL each contain 4 FRs, and the CDRs and FRs are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains do not participate in antigen binding, but have a variety of effector functions. Antibodies can be divided into several categories based on the amino acid sequence of the heavy chain constant region. The heavy chains of mammalian antibodies can be divided into α, δ, ε, γ and μ, and the light chains of mammalian antibodies can be divided into λ or κ. Depending on whether they contain α, δ, ε, γ and μ heavy chains, antibodies can be divided into five major categories or isotypes: IgA, IgD, IgE, IgG and IgM. Several major antibody classifications can also be divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain). In addition to the above-mentioned antibodies with complete constant regions, the antibodies of the present invention also include antibodies with incomplete constant regions, such as only CH1, CH1 and CH2, CH1 and CH3, CH2 and CH3, etc. In summary, the term "antibody" used in the present invention includes but is not limited to any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific antibody that can bind to a specific antigen, and all of them that show the desired antigen-binding activity are included in the present invention.

[0093] According to the number of different target antigens or different antigen epitopes bound by the antibody, it is divided into monospecific antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, ..., and multispecific antibodies (binding to two or more different target antigens or different antigen epitopes). For example, the term "bispecific antibody" refers to an antibody that can specifically bind to two different antigens or two different antigen epitopes of the same antigen. The prior art has disclosed bispecific antibodies of various structures; according to the integrity of the IgG molecule, it can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies; according to the number and configuration of the antigen binding region, it can be divided into bispecific antibodies of divalent, trivalent, tetravalent or more valence; according to whether the structure is symmetrical on the left and right, it can be divided into symmetrical bispecific antibodies and asymmetrical bispecific antibodies. The term "antigen" or "antigen molecule" when used herein can be used interchangeably and refers to all molecules that can be specifically bound by an antibody. A bivalent bispecific antibody specifically binds to a first antigen and a second antigen, wherein the first antigen and the second antigen can be two different antigens, or different epitopes of the same antigen. The term "antigen" or "antigenic molecule" as used herein includes, for example, proteins, different epitopes on proteins (which are different antigens within the meaning of the present invention), polysaccharides, and the like.

[0094] The term "fully human" refers to an antibody or antigen-binding fragment thereof or an immunoglobulin domain that comprises an amino acid sequence encoded by a DNA derived from a human sequence over the entire length of each polypeptide of the antibody or antigen-binding fragment thereof or immunoglobulin domain. In some cases, the fully human sequence is derived from a protein endogenous to humans. In other cases, a fully human protein or protein sequence comprises a chimeric sequence in which each component sequence is derived from a human sequence. Although not bound by any theory, chimeric proteins or chimeric sequences are typically designed to minimize the formation of immunogenic epitopes in the junctions of the component sequences, for example, compared to any wild-type human immunoglobulin region or domain.

[0095] The terms "specific binding" and "selective binding" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 The KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Generally, the antibodies disclosed in the present invention bind with an affinity (KD) of less than about 10 -7 M, for example, less than about 10 -8 M or 10 -9 The dissociation equilibrium constant (KD) of M binding to antigen.

[0096] The term "Fab fragment" refers to a fragment capable of binding to an antigen that can be produced by enzymatic treatment of an antibody, and comprises a light chain VL-CL and a heavy chain CH-CH1 structure.

[0097] The term "Fc region" or "Fc fragment" refers to the part of an antibody consisting of a CH2 constant domain and / or a CH3 constant domain of a first heavy chain bound to a CH2 constant domain and / or a CH3 constant domain of a second heavy chain via a disulfide bond. The Fc of an antibody is responsible for a variety of different effector functions, such as ADCC and CDC, but does not participate in the binding of an antigen.

[0098] The term "electrostatic interaction" is a non-covalent interaction and plays an important role in protein folding, stability, flexibility and function, including ionic interactions, hydrogen bonds and halogen bonding. Electrostatic interactions can be formed in polypeptides, such as between Lys and Asp, between Lys and Glu, between Glu and Arg, or between Glu, Trp on the first chain and Arg, Val or Thr on the second chain.

[0099] The term "hydrophilic interaction" refers to molecules with polar groups that have a strong affinity for water and can form transient bonds with water through hydrogen bonds.

[0100] The term "hydrophobic interaction" refers to a phenomenon in which hydrophobic groups gather close to each other to avoid water.

[0101] Any bispecific antibody pattern or technology can be used to prepare the bispecific antigen binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen binding specificity to produce a bispecific antigen binding molecule. Specific exemplary bispecific patterns that can be used in the context of the present invention include, but are not limited to, for example, scFv-based or double antibody bispecific patterns, IgG-scFv fusions, dual variable domains (DVD)-Ig, quadromas, knobs-in-hole structures, common light chains (e.g., common light chains with knobs-in-hole structures, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG, and Mab 2 bispecific patterns (for a review of the aforementioned patterns, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein).

[0102] In the bispecific antibody of the present invention, the leucine zipper structure pair sequence contained in the light chain and heavy chain of the first antigen binding part and / or the second antigen binding part can effectively reduce the mismatch between the light chain and the heavy chain, and help the light chain and the heavy chain to form a heterodimer; further, the knob-in-hole structure KIH, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, and the method of increasing flexibility or a combination of the above methods between the heavy chain of the first antigen binding part and the heavy chain of the second antigen binding part effectively reduce the mismatch between the heavy chains, especially the use of KIH and leucine zipper structure at the same time makes the anti-mismatch effect between the heavy chains better. The above antibody structure can ensure that the antigen binding sequence is expressed and assembled with minimal or almost no mismatch.

[0103] The term "leucine zipper" structure refers to a structural motif that occurs in DNA-binding proteins and other proteins. Leucine zippers are formed when the hydrophobic faces of two alpha-helices (often containing leucine residues) from the same or different polypeptide chains interact to form a circle-to-circle dimer structure. Specifically, leucine zippers are composed of stretched amino acids, with the seventh amino acid of every seven amino acids being leucine, which is a hydrophobic amino acid, arranged on one side of the alpha helix, and all charged amino acid residues arranged on the other side. When two protein molecules are arranged in parallel, the leucines interact with each other to form dimers, forming a "zipper" structure. Therefore, leucine zipper domains are peptides that promote the oligomerization of proteins in which leucine zipper domains are present. Known leucine zippers include naturally occurring peptides and their derivatives that are dimerizing or trimerizing. The leucine zipper structure pair can be selected from: Fos protein / Jun protein, FosW protein / Jun protein, Myc protein / Max protein, WinzipA / WinzipB1, ACID-p1 protein / BASE-p1 protein, GCN4 protein / GCN4 domain, C / EBP protein / C / EBP protein and functional variants of the above proteins / domains. The leucine zipper structure pair described herein can be selected from any of the above groups but is not limited thereto, and any sequence that can form the leucine zipper structure can be applied to the present invention.The Jun domain is a sequence as set forth in SEQ ID NO:17, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:17; and / or the Fos domain is a sequence as set forth in SEQ ID NO:18, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:18; and / or the FosW domain is a sequence as set forth in SEQ ID NO:19, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:19; and / or the Max domain is a sequence as set forth in SEQ ID NO:20, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:20; and / or the Myc domain is a sequence as set forth in SEQ ID NO:21, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:21. NO:21 has at least 80%, 85%, 90%, 95%, or 99% identity; and / or the WinzipA2 domain is a sequence as shown in SEQ ID NO:22, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:22; and / or the WinzipB1 domain is a sequence as shown in SEQ ID NO:23, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:23; and / or the ACID-p1 domain is a sequence as shown in SEQ ID NO:24, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:24; and / or the BASE-p1 domain is a sequence as shown in SEQ ID NO:25, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:25. NO:25 has a sequence with at least 80%, 85%, 90%, 95%, or 99% identity; and / or the GCN4 domain is a sequence as shown in SEQ ID NO:26, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:26; and / or the C / EBP domain is a sequence as shown in SEQ ID NO:27, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:27.

[0104] The term "operably linked" or "operably connected" refers to two or more biological sequences being connected in a manner that causes them to function in a desired manner, regardless of whether a connector (also called a linker, a linker, a connecting sequence) is present. When in the case of a polypeptide, the term indicates that the polypeptide sequences are connected in a manner that allows the connected product to have a desired biological function, and a connector may or may not be present between the two sequences. For example, an antibody variable region can be operably linked to a constant region to form a stable product with antigen binding activity. The term can also be used for polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, an enhancer, a silencer sequence, etc.), the term indicates that the polynucleotide sequence is connected in a manner that allows the polypeptide to be regulated by the polynucleotide for expression.

[0105] The term "linker" or "spacer" refers to a border or boundary region where two polypeptide sequences are fused or combined. For example, a linker domain may comprise at least a portion of a C-terminal fragment of a first fusion polypeptide fused to at least a portion of an N-terminal fragment of a second fusion polypeptide, with or without additional linkers in between. For example, a linker may be rich in glycine and proline residues, such as a linker having a single or repeated sequence of threonine / serine composition, GGS, GGGGS, TGGGG, SGGGG, or tandem repeats thereof (e.g., 2, 3, or more repeats).

[0106] The term "mismatch" refers to the interaction or association of two or more homologous or heterologous polypeptides to form an undesirable dimer or multi-specific pairing. "Not prone to mismatch" means that, for example, when polypeptides A1, B2 and B2 are co-expressed, if the expression amount of the desired A1-B1 dimer is greater than the amount of the A1-B2 dimer, it is considered that preferential pairing occurs between A1-B1, that is, mismatch is not likely to occur between A1-B2.

[0107] In some embodiments, in bi / multi-specific antibodies, mispairing is not easy to occur between VL1 of the first light chain and VH2 of the second heavy chain, and / or mispairing is not easy to occur between VH1 of the first heavy chain and VL2 of the second light chain, but VH1-VL1 are preferentially paired, and VH2-VL2 are preferentially paired.

[0108] The term "dimerization domain" refers to domains that are capable of promoting association with each other to form dimers.

[0109] In some embodiments, a first dimerization domain can be associated with a second dimerization domain.

[0110] The association can be by binding or linking or bonding in any suitable manner; for example, by a linker, a disulfide bond, a hydrogen bond, an electrostatic interaction, a salt bridge, or a hydrophobic-hydrophilic interaction, or a combination thereof.

[0111] Exemplary dimerization domains include, but are not limited to, antibody hinge regions, antibody CH2 domains, antibody

[0112] CH3 domains, and / or other suitable protein monomers capable of dimerizing and interacting with each other or

[0113] Polypeptide. The hinge region, CH2 constant domain, CH3 constant domain can be derived from any antibody isotype, such as IgG1, IgG2, IgG3 and IgG4.

[0114] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single or double-stranded form. Unless explicitly limited, the term encompasses polynucleotides containing known natural nucleotide analogs that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific polynucleotide sequence also implies its conservatively modified variants (e.g., degenerate codon substitutions), alleles, homologous genes, SNPs, and complementary sequences, as well as the sequences explicitly indicated.

[0115] The terms "homology" and "identity" refer to sequence similarity between multiple polynucleotides or between two polypeptides. When it refers to a nucleic acid or a fragment thereof, it means that when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide substitutions, insertions or deletions, the sequence has nucleotide sequence identity in at least about 80%, more preferably at least about 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotide bases as calculated using any of the following sequence identity calculation programs such as FASTA, BLAST or Gap. When applied to polypeptides, it means that when optimally aligned using programs such as Gap or BESTFIT with default gap weights, two peptide sequences have at least 80% sequence identity, more preferably at least 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and the difference between non-identical residue positions can be amino acid substitutions, deletions or insertions, and more preferably, the difference between non-identical residue positions is conservative amino acid substitutions.

[0116] The term "vector" used in the term "isolated vector" of the present application refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted and the protein can be expressed. Generally, its structure also includes appropriate regulatory sequences. The vector can be used to transform, transduce or transfect host cells so that the genetic elements it carries are expressed in the host cells. The vector may contain a variety of elements that control expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, including but not limited to viral particles, liposomes or protein shells. For example, the vector includes: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), bacteriophages such as lambda phages or M13 phages, and animal viruses, etc. Animal virus species used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). In some embodiments, the vector system includes mammals, bacteria, yeast systems, etc., and includes plasmids such as but not limited to pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., as well as other laboratory and commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). Vectors containing the polynucleotide sequences described herein can be introduced into host cells for cloning or gene expression, and the term "host cell" used in this application refers to cells into which exogenous polynucleotides and / or vectors are introduced. Host cells suitable for cloning or expressing the DNA in the vectors described herein are the prokaryotic cells, animal cells, plant cells, and fungal cells described above.

[0117] Antibody-drug conjugate (ADC) refers to connecting an antibody or antigen-binding fragment to a biologically active toxic drug through a stable linking unit (also referred to herein as a linker). Toxic drugs include any drugs that are harmful to the growth, vitality or reproduction of cells. The antibody or antigen-binding fragment of the present invention delivers these cytotoxic drugs to target cells. Examples of suitable cytotoxic drugs for forming ADC are known in the art. The term "linker" or "linking unit" refers to a chemical structure fragment or bond that is connected to an antibody or antigen-binding molecule fragment at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug.

[0118] The terms "include", "comprising", "having", "containing" or "involving" and other variations thereof herein are inclusive or open-ended and do not exclude other unlisted elements or method steps. Those skilled in the art will appreciate that the above terms such as "comprising" encompass the meaning of "consisting of".

[0119] In the present invention, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle that is administered together with the active ingredient and is suitable for contact with the tissues of humans and / or other animals within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic reaction or other problems or complications corresponding to a reasonable benefit / risk ratio. The term "active ingredient", "therapeutic agent", "active substance" or "active agent" refers to a chemical entity that can effectively treat one or more symptoms of a target disease or condition.

[0120] As used herein, the term "effective amount" (e.g., "therapeutically effective amount" or "prophylactically effective amount") refers to the amount of active ingredient that, after administration, will achieve the desired effect to some extent, such as alleviating one or more symptoms of the condition being treated or preventing the occurrence of the condition or its symptoms.

[0121] As used herein, unless otherwise indicated, the term "treat," ...

[0122] The disorder or condition or one or more symptoms of the disorder or condition may be prevented. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1 :Schematic diagram of the structure of the bispecific antibody against TROP2 / PRLR

[0124] Figure 2 : Anti-TROP2 / PRLR bispecific antibody SEC (purity) test results

[0125] Figure 3 : Anti-TROP2 monoclonal antibody SEC (purity) test results

[0126] Figure 4 :HIC-HPLC detection results of anti-TROP2 monoclonal antibody

[0127] Figure 5 :HIC-HPLC test results of anti-TROP2-MC-MMAF ADC

[0128] Figure 6 :HIC-HPLC detection results of anti-TROP2 / PRLR bsAb

[0129] Figure 7 :HIC-HPLC test results of anti-TROP2 / PRLR-MC-MMAF ADC

[0130] Figure 8 :The results of endocytic activity detection of anti-TROP2-MC-MMAF ADC and anti-TROP2 / PRLR-MC-MMAF ADC on TROP2-positive tumor cell line MDA-MB-468 DETAILED DESCRIPTION

[0131] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0132] Example 1.

[0133] Anti-TROP2 / anti-PRLR bispecific antibody sequence design

[0134] The exemplary bispecific antibody of the present invention comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain targets a first antigen TROP2, and the second antigen binding domain targets a second antigen PRLR. The bispecific antibody targeting TROP2 / PRLR is named anti-TROP2 / PRLR bsAb and is designed as follows:

[0135] The C-terminus of the second light chain variable domain VL2 (sequence as shown in SEQ ID NO: 16) of the antibody targeting PRLR is connected to the N-terminus of the Fos domain (sequence as shown in SEQ ID NO: 18) via the third linker L3 (GGGGS), and the C-terminus of the second heavy chain variable domain VH2 (sequence as shown in SEQ ID NO: 15) of the antibody targeting PRLR is connected to the N-terminus of the Jun domain (sequence as shown in SEQ ID NO: 17) via the fourth linker L4 (GGGGS); the first light chain variable domain VL1 sequence of the antibody targeting TROP2 is shown in SEQ ID NO: 8 and the heavy chain variable domain VH1 sequence is shown in SEQ ID NO: 7. The second light chain sequence is shown in SEQ ID NO:28, the second heavy chain sequence is shown in SEQ ID NO:29, the first light chain sequence is shown in SEQ ID NO:30, the first heavy chain sequence is shown in SEQ ID NO:31, and the amino acids T366S-L368A-Y407V (Hole) on the first heavy chain sequence and the amino acid T366W (Knob) on the second heavy chain sequence form a KIH (knob-into-hole) structure.

[0136] Comparative Example 1. Anti-TROP2 monoclonal antibody sequence design

[0137] The monoclonal antibody targeting TROP2 is named anti-TROP2 mAb, the light chain amino acid sequence is SEQ ID NO:32, and the heavy chain amino acid sequence is SEQ ID NO:33.

[0138] Example 2. Construction of anti-TROP2 / PRLR bsAb and anti-TROP2 mAb

[0139] Construction of anti-TROP2 / PRLR bsAb plasmid vector

[0140] 1. Gene Synthesis

[0141] The sequences for expressing the anti-TROP2 / PRLR bispecific antibody and anti-TROP2 monoclonal antibody in Example 1 were constructed and, after codon optimization, submitted to the gene synthesis company Nanjing GenScript for full gene synthesis.

[0142] 2. Construction of pCGS3 expression vector

[0143] For anti-TROP2 / PRLR bispecific antibodies:

[0144] Using the seamless cloning method, the heavy chain and light chain of the first antigen binding part were cloned into the multiple cloning site of the pCGS3 vector, and the heavy chain and light chain of the second antigen binding part were also cloned into the multiple cloning site of the pCGS3 vector, wherein the heavy chain was cloned into the BstBI and PacI restriction sites, and the light chain was cloned into the HindIII and XhoI restriction sites.

[0145] The specific method is as follows:

[0146] The enzyme digestion components in Table 1 were mixed evenly, and the resulting reaction solution was incubated in a metal bath at 37° C. for 3 h; the enzyme digestion products were recovered using an agarose gel DNA recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0147] Table 1 Heavy chain double enzyme digestion system

[0148] Components Volume (μL) <![CDATA[Buffer (Cutsmart TM )]]> 5 BstBI (NEB, USA) 1 PacI (NEB, USA) 1 template 10 <![CDATA[Double distilled water (ddH 2 O)]]> Top up to 50

[0149] T4 DNA ligase (NEB, USA) was used to connect the DNA recovery product with the pCGS3 vector digestion recovery product. The specific ligation reaction system is shown in Table 2. The ligation reaction components were mixed, and the mixed reaction solution was placed in a 16°C metal bath overnight.

[0150] Table 2 Ligation reaction system

[0151] Components Volume (μL) 10*T4 Buffer (NEB, USA) 2 Vector fragment 2 Gene fragment 7 <![CDATA[Double distilled water (ddH 2 O)]]> Top up to 20

[0152] Transformation and inoculation were performed as follows: 5 μL of the above ligation product was added to 50 μL Trans5α competent cells (Beijing Quanshijin Biotechnology Co., Ltd.), mixed and placed on ice for 30 min, then placed in a 42°C constant temperature water bath for heat shock for 90 s, then placed on ice for 1 min, and finally added 250 μL LB medium, incubated at 37°C, 220 rpm for 1 h, and 100 μL was applied to ampicillin. + (Amp + LB culture plates (Luria-Bertani medium, broth medium) were prepared from Tiangen Biochemical Technology (Beijing) Co., Ltd. and cultured at 37°C overnight.

[0153] Pick a single clone from each LB culture plate and place it in a 1.5 mL EP tube. Add 300 μL of Amp + LB medium was used, and cultured at 37°C and 220 rpm for 8 hours; electrophoresis was performed after PCR of cultured bacteria, and the bacterial solution with the correct band was sequenced. Plasmid DNA was extracted according to the following steps: After shaking and amplifying the correct bacterial solution, plasmid DNA was extracted using a plasmid extraction kit (Beijing Quanshijin Biotechnology Co., Ltd.).

[0154] Continue to perform light chain double enzyme digestion and ligation, wherein the light chain double enzyme digestion system is shown in Table 3:

[0155] Table 3 Light chain double enzyme digestion system

[0156] Components Volume (μL) <![CDATA[Buffer (Cutsmart TM )]]> 5 HindIII (NEB, USA) 1 XhoI (NEB, USA) 1 template 10 <![CDATA[Double distilled water (ddH 2 O)]]> Top up to 50

[0157] The remaining steps are the same as above.

[0158] The constructed plasmid was extracted with an endotoxin-free plasmid extraction kit (DP117, Tiangen Biochemical Technology Co., Ltd.) and named anti-TROP2 / PRLR bsAb. The concentration and purity of the plasmid DNA were determined using a NanoDrop UV spectrophotometer (Thermo Fisher Scientific, USA) and set aside.

[0159] 2.2 The anti-TROP2 monoclonal antibody sequence was constructed into the pCGS3 expression vector according to the same method as above.

[0160] Example 3. Expression of anti-TROP2 / PRLR bispecific antibodies and anti-TROP2 monoclonal antibodies

[0161] The anti-TROP2 / PRLR bispecific antibody and anti-TROP2 monoclonal antibody were expressed according to the following steps:

[0162] HEK-293f cells (Zhuhai Kairui Biotechnology Co., Ltd.) were cultured with KOP293 cell culture medium (Zhuhai Kairui, K03252) at 37°C and 5% CO 2 The shaker was set at 100-130 rpm and the humidity was controlled at above 75%. The day before transfection, 293f cells in logarithmic growth phase and in good growth condition were passaged to 2×10 6 indivual

[0163] / mL, shaker (110 rpm, 37°C, 5% CO 2 ) were cultured overnight and transfected the next day. Before transfection, TA-293 (293 cell suspension chemical transfection reagent, Zhuhai Kairui Biotechnology Co., Ltd.) and KPM (serum-free cell transfection buffer, Zhuhai Kairui Biotechnology Co., Ltd.) were preheated at room temperature. The cell density and viability were measured. The density was 4×10 6 / mL, and it can be used only when the activity rate is greater than 98%.

[0164] Perform transfection according to the instructions of the KOP293 transient transfection protein expression system: prepare two 15ml sterile centrifuge tubes, add 5ml KPM and 100μg sterile plasmid DNA to one of them, and gently blow to mix. Take another centrifuge tube, add 5ml KPM and 500μl TA-293 transfection reagent, and gently blow to mix; transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and gently blow to mix; incubate the plasmid-vector complex at room temperature for 10 minutes; remove the cells from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and return to the CO 2 The cells were shaken and cultured in a constant temperature shaker. 24 hours after transfection, 600 μl of 293 cell protein expression enhancer (KE-293, Zhuhai Kairui Biotechnology Co., Ltd.) and transient transfection nutrient additive (KT-Feed 50×, Zhuhai Kairui Biotechnology Co., Ltd.) were added to increase the expression of the product; on the 5th day after transfection, the cell supernatant was collected.

[0165] Example 4. Purification of anti-TROP2 / PRLR bispecific antibodies and anti-TROP2 monoclonal antibodies

[0166] The expression supernatants of the anti-TROP2 / PRLR bispecific antibody and the anti-TROP2 monoclonal antibody were purified by the following steps: filtration with a 0.45uM filter membrane, and the antibody with the Fc domain was obtained from the expression supernatant using a Protein A affinity chromatography column. The Protein A affinity chromatography column buffer was prepared: the equilibrium buffer was 9.5mM sodium dihydrogen phosphate, 40.5mM sodium dihydrogen phosphate, pH 7.4; the elution buffer was 0.1M glycine, pH 3.0. The eluted antibody was replaced with PBS buffer (phosphate buffer). The purified anti-TROP2 / PRLR bispecific antibody and anti-TROP2 monoclonal antibody were obtained respectively.

[0167] Test Example 1. Purity detection of anti-TROP2 / PRLR bispecific antibody and anti-TROP2 monoclonal antibody

[0168] The purified anti-TROP2 / PRLR bispecific antibody and anti-TROP2 monoclonal antibody were detected by high performance liquid chromatography (SEC-HPLC): prepare the mobile phase (50mM PBS (phosphate buffer) + 150mM NaCl, pH6.8), filter with a 0.45um filter membrane, and ultrasonically degas; filter the ultrafiltered protein with a syringe filter; insert the inner tube into the glass vial, add 200μL of the sample to be tested; put the sample into the HPLC sample slot, open the HPLC working software, flush the system with the equilibrium buffer, observe the baseline change, and when the system has reached equilibrium, set the program and start testing the sample. Detection conditions: flow rate 0.5mL / min; time 30min, injection volume 20μL, column temperature 25℃, detection wavelengths 214nm and 280nm respectively; isocratic elution; after the sample is tested, the results are analyzed.

[0169] from Figure 2 , Figure 3 The results show that the purity of the anti-TROP2 / PRLR bispecific antibody is above 94%, and the purity of the anti-TROP2 monoclonal antibody is above 96%. As can be seen from the above results, the purity of the antibodies obtained in the present invention is relatively high, and the anti-TROP2 / PRLR bispecific antibody obtained in the present invention effectively prevents mispairing and assembly between light chain-heavy chain and heavy chain-heavy chain.

[0170] Example 5. Preparation and characterization of ADC drugs of anti-TROP2 / PRLR bispecific antibodies

[0171] The ADC drug of anti-TROP2 / PRLR bispecific antibody (referred to as anti-TROP2 / PRLR-MC-MMAF ADC) and the ADC drug of anti-TROP2 antibody (referred to as anti-TROP2-MC-MMAF ADC) were prepared, wherein the linker used was maleimidocaproyl (MC), the small molecule toxic agent was monomethyl auristatin peptide F (MMAF), and the structure of Linker-MMAF (purchased from: CSN PHARM, catalog number: CSN 18491-002) was as follows:

[0172]

[0173] The specific method is as follows:

[0174] Solution preparation: dilute 0.5M EDTA (ethylenediaminetetraacetic acid) with PBS (phosphate buffered saline) to obtain 1mM EDTA-PBS solution; dilute 0.5M TCEP (Tris-2-carboxyethyl-phosphine) (purchased from Thermo Fisher, catalog number: 77720) with PBS to 5mM; prepare 10% DMSO (dimethyl sulfoxide) solution, take 100uL DMSO and mix with 900uL EDTA-PBS solution; dilute the antibody to a concentration of 10mg / mL.

[0175] Antibody reduction: Use TCEP solution to reduce and break disulfide bonds, where antibody:TCEP=1:8 (molar ratio), react on a shaker at 37°C for 2h, and place on ice after the reaction is completed.

[0176] Use the prepared DMSO solution to dissolve Linker-MMAF, add Linker-MMAF at a ratio of antibody: Linker = 1:10 (molar ratio) to make the final concentration of DMSO in the system 10%, react at room temperature for 2 hours, and perform coupling. After the reaction, dialyze the coupled protein in PBS and perform ultrafiltration to remove uncoupled small molecule drugs, thereby obtaining anti-TROP2 / PRLR-MC-MMAF ADC and anti-TROP2-MC-MMAF ADC, respectively.

[0177] Test Example 2:

[0178] The DAR value (drug to antibody ratio) was determined by spectrophotometer (UV method). The linker payload loading value (drug to antibody ratio; DAR value) was determined by UV method according to Hamblett et al. (American Association for Cancer Research. 20041015; 10(20):7063-70). The details are as follows:

[0179] 1. Take 100 μL of ADC sample and add it to a 96-well UV detection plate (corning3635). Measure the absorbance of the sample at 280 nm and 365 nm, which are recorded as A280 and A365 respectively.

[0180] 2. The total absorbance value is equal to the sum of the absorbance values ​​of the drug and the antibody:

[0181]

[0182]

[0183] (1) is the extinction coefficient of the drug at 280 nm; Cdrug is the drug concentration; is the extinction coefficient of the antibody at 280 nm; C mAb is the antibody concentration and l is the path length through the well containing the analyte (1 cm).

[0184] (2) is the extinction coefficient of the drug at 365 nm; C drug is the drug concentration; is the extinction coefficient of the antibody at 365 nm; C mAb is the antibody concentration and l is the path length through the well containing the analyte (1 cm).

[0185] 3. The above two equations can be used to obtain the concentrations of antibodies and drugs respectively.

[0186]

[0187]

[0188] 4. Use C drug Divide by C mAb The average drug-antibody conjugation ratio (DAR) was calculated as the number of moles of drug divided by the number of moles of antibody:

[0189] DAR=C drug / C mAb

[0190] The results of DAR value detection by absorbance spectrophotometry are as follows:

[0191] Table 4 DAR values ​​detected by absorbance method

[0192] ADC Name DAR value Anti-TROP2 / PRLR-MC-MMAF ADC 4.48 Anti-TROP2-MC-MMAF ADC 8

[0193] The absorbance values ​​were measured at 280 nm and 365 nm, and the ratio of small molecule drugs to antibodies in the obtained anti-TROP2 / PRLR-MC-MMAF ADC sample was DAR=4.48, and the ratio of small molecule drugs to antibodies in the anti-TROP2 mAb–MC-MMAF sample was DAR=8.

[0194] Experimental Example 3: Analysis of drug conjugation rate of ADC by hydrophobic interaction chromatography HIC-HPLC method

[0195] Waters e2695 / LC-20AT high performance liquid chromatograph (purchased from Waters) and TSK-GEL Butyl-NPR chromatographic column (4.6 mm×35 mm, purchased from TOSOH) were used to analyze anti-TROP2 / PRLR-MC-MMAF ADC, anti-TROP2-MC-MMAF ADC, anti-TROP2 / PRLR bsAb and anti-TROP2 mAb. Mobile phase A: 20 mM phosphate buffer (pH=6.8), 1.5 M ammonium sulfate; Mobile phase B: 75% (v / v) 20 mM phosphate buffer (pH=6.8), 25% (v / v) isopropanol; Mobile phase gradient during the experiment is shown in Table 5, detection wavelength: 280 nm; Column temperature: 25°C; Flow rate: 0.5 ml / min; Injection volume: about 10 μg.

[0196] Table 5 HIC-HPLC elution gradient conditions

[0197]

[0198]

[0199] The results are as follows Figure 4-7 As shown, Figure 4 , 5 , 6, and 7 are the result graphs of anti-TROP2 mAb, anti-TROP2-MC-MMAF ADC, anti-TROP2 / PRLR bsAb, and anti-TROP2 / PRLR-MC-MMAF ADC, respectively, indicating that anti-TROP2 mAb and TROP2 / PRLRbsAb were successfully coupled to the corresponding small molecule drugs, which is consistent with the results of DAR value detection by absorbance method.

[0200] Experimental Example 4: Affinity of the antibodies and ADCs of the present invention for TROP2 and PRLR antigens

[0201] Surface plasmon resonance biosensor was used to measure the affinity of anti-TROP2 / PRLR bsAb, anti-TROP2 / PRLR-MC-MMAF ADC, anti-TROP2 mAb and anti-TROP2-MC-MMAF ADC to TROP2 and PRLR, respectively. Affinity analysis was analyzed by SPR, and the instrument used was Biacore, T200 (purchased from GE Healthcare); mobile phase (GE, BR-1006-69). Unless otherwise stated, all reagents and materials can be purchased from GE.

[0202] (1) Instruments and reagents

[0203] Table 6 Instruments and reagents

[0204]

[0205] (2) Experimental procedures

[0206] According to the method described in the instructions of the human Fab capture kit (Cat.#28-9583-25, GE), the human Fab capture molecule was covalently coupled to the CM5 biosensor chip (Cat.#BR-1000-12, GE), thereby affinity capturing the antibody to be tested. The antigen used was human TROP2-his (Cat.#10428-H08H-100, Sino biological) antigen or human PRLR antigen (Cat. No.: PRR-H52Ha, purchased from: Biopsies); anti-TROP2 / PRLR bsAb, anti-TROP2 / PRLR-MC-MMAF, anti-TROP2 mAb and anti-TROP2-MC-MMAF ADC were analytes, and their affinity with TROP2 and / or PRLR was detected. After the analyte was gradiently diluted (100nM, 50nM, 25nM, 10nM, 5nM), the following operations were performed:

[0207] 1) Capture antibody: Capture antibody for 60 s at a flow rate of 10 μl / min.

[0208] 2) Analysis: After the analyte was serially diluted (100 nM, 50 nM, 25 nM, 10 nM, 5 nM), it flowed in at a flow rate of 30 μl / min, bound to the antigen for 120 s, and then dissociated at a flow rate of 30 μl / min for 900 s.

[0209] 3) Regeneration: Use Glycine-HCl, pH 3.0 solution (GE, BR-1003-57) for regeneration at a flow rate of 30 μl / min for 30 seconds.

[0210] 4) Process results: Wait for the experiment to end and open the analysis software to process the results.

[0211] (3) Experimental results are shown in Table 7

[0212] Table 7 Affinity of anti-TROP2 / PRLR bsAb, anti-TROP2 / PRLR-MC-MMAF ADC, anti-TROP2 mAb and anti-TROP2-MC-MMAF ADC to TROP2 and / or PRLR

[0213]

[0214]

[0215] The results showed that the anti-TROP2 / PRLR bispecific antibody obtained by the present invention had a higher affinity to TROP2 than the anti-TROP2 monoclonal antibody; and the anti-TROP2 / PRLR-MC-MMAF ADC had a higher affinity than the anti-TROP2–MC-MMAF.

[0216] In addition, anti-TROP2 / PRLR-MC-MMAF ADC also has a high affinity for PRLR, so anti-TROP2 / PRLR-MC-MMAF ADC can be used to target and bind to tumor cells that are positive for TROP2 or PRLR expression.

[0217] This also shows that the present invention can obtain correctly assembled bispecific antibodies and effectively prevent mismatching.

[0218] Experimental Example 4: Endocytic activity detection of anti-TROP2 / PRLR-MC-MMAF ADC and anti-TROP2-MC-MMAF ADC

[0219] The steps for detecting the endocytic activity of anti-TROP2 / PRLR-MC-MMAF ADC and anti-TROP2-MC-MMAF ADC are as follows:

[0220] 1. Harvesting cells: MAD-MB-468 (human breast cancer cells, purchased from ATCC, catalog number #HTB-132) are adherent cells. Trypsin was used to digest the cells so that they fell off the culture dish and the cell suspension was placed at the bottom of a centrifuge tube. Trypan blue staining was used to determine the cell viability, and the cells were centrifuged at 4°C and 300g for 5 minutes to precipitate the cells. FACS staining buffer was added to wash the cells once. The cells were centrifuged again and resuspended in an appropriate volume of FACS staining buffer to a cell concentration of 2×10 6 cells / mL.

[0221] 2. Prepare cells for staining: Add cell suspension at 50uL / well (1×10 5 cell /

[0222] The cells were plated in 96-well U-bottom cell plates, and a set of parallel experiments was set for each sample.

[0223] 3. Antibody binding to cells: Use FACS staining buffer to dilute the primary antibody to a concentration of 20ug / mL. Take 50uL of the primary antibody dilution and add it to the 96-well U-bottom cell plate, mix gently and place on ice for 60 minutes.

[0224] 4. Wash unbound antibodies: Add 200uL FACS staining buffer to wash the cells, centrifuge at 4°C, 300g for 5min, discard the supernatant, and repeat the wash twice.

[0225] 5. Incubate at 37°C to internalize bound antibodies: Add 30uL FACS to each well to resuspend cells and incubate at 37°C for 30min, 60min, and 90min to internalize antibodies bound to the cell surface. At each time point, use another sample incubated at 4°C for the same time as a negative control without internalization. At each time point, move the corresponding 37°C and 4°C incubated samples to ice and add 170uL ice FACS to terminate internalization. Centrifuge at 4°C, 300g for 5min to precipitate cells.

[0226] 6. Use fluorescently labeled secondary antibodies to label antibodies bound to cell surface: add secondary antibodies at a ratio of 0.5:100, 100uL / well. Incubate at 4℃ in dark for 30min.

[0227] 7. Wash unbound secondary antibody: Add 200uL FACS staining buffer to wash cells and remove unbound antibody. Centrifuge at 4°C, 300g for 5min to pellet cells and discard supernatant. Repeat washing twice. Resuspend the stained cells in 100uL ice-cold PBS.

[0228] 8. Detect the fluorescence intensity of the cell surface by flow cytometry: After the cells are washed, analyze them by flow cytometry as soon as possible.

[0229] 9. Calculation of internalization level of cell surface-bound antibody: calculated by the mean fluorescence intensity (MFI) reduction level of samples incubated at 37°C relative to samples incubated at 4°C, the formula is as follows: (a) % MFI at time point tx = MFI of samples incubated at 37°C × 100 / MFI of samples incubated at 4°C;

[0230] (b) Percent internalization at tx time point = 100 - % MFI at tx time point

[0231] Draw a bar graph based on the calculated internalization rate. Figure 8 As shown in the figure, anti-TROP2 / PRLR-MC-MMAF ADC has a higher internalization rate than anti-TROP2-MC-MMAF ADC, which is increased by about 15% to 20%, indicating that the anti-TROP2 / PRLR antibody portion can more effectively drag ADC into lysosomes and improve the internalization rate.

[0232] Test Example 5: Cell activity experiment

[0233] The TROP2-positive MDA-MB-468 cell line was selected to detect the cell killing activity of anti-TROP2 / PRLR-MC-MMAF ADC and anti-TROP2-MC-MMAF ADC as follows:

[0234] 1. Instruments and Reagents

[0235] Table 8 Instruments and reagents

[0236]

[0237]

[0238] 2. Experimental Procedure

[0239] 1) Plating & Culture: MDA-MB-468 cells were cultured at 2×10 5 Cells / 100 μL were inoculated into 96-well plates, 200 μL PBS was added to the outermost circle (to reduce the volatilization of the culture medium), and cultured at 37°C and 5% CO2 for 24 h;

[0240] 2) Culture: Take out the 96-well flat-bottom plate, dry the medium in the 96-well plate, add 100 μL of ADC sample diluted with medium, and incubate at 37°C, 5% CO 2 Process for 72 hours.

[0241] 3. Detection and data processing

[0242] 1) Take out the 96-well plate and add Cell Titer-Glo Reagent at room temperature, 100 μL / well;

[0243] 2) Oscillate on a plate shaker at room temperature in the dark for 30 min;

[0244] 3) Use the microplate reader (CEY0017) to read the value under Luminescence and record it;

[0245] 4) Graphpad Prism 5.0 was used to analyze and organize the data. The results of the cell killing activity test of the purified anti-TROP2 mAb-MC–MMAF and anti-TROP2 / PRLR-MC-MMAF ADC on the TROP2-positive tumor cell line MDA-MB-468 are shown in Table 9:

[0246] Table 9 Cytotoxic activity of anti-TROP2-MC-MMAF ADC and anti-TROP2 / PRLR-MC-MMAF ADC against TROP2-positive tumor cell line MDA-MB-468

[0247]

[0248]

[0249] The results showed that the anti-TROP2-MC-MMAF ADC and anti-TROP2 / PRLR-MC-MMAF ADC of the present invention had a high killing effect on TROP2-positive MDA-MB-468 cells; however, the anti-TROP2 / PRLR-MC-MMAF ADC had a higher killing effect, which was twice that of the anti-TROP2-MC-MMAF ADC.

[0250] Unless otherwise specified, the raw materials and reagents used in the specific embodiments of the present invention can be obtained from commercial sources.

[0251] The present invention has been described by the above embodiments, but it should be understood that the above embodiments are only for the purpose of example and description, and are not intended to limit the present invention to the scope of the described embodiments. In addition, it can be understood by those skilled in the art that the present invention is not limited to the above embodiments, and more variations and modifications can be made according to the teachings of the present invention, and these variations and modifications all fall within the scope of the protection claimed by the present invention. The protection scope of the present invention is defined by the attached claims and their equivalents.

Claims

1. A bispecific antigen-binding molecule comprising a first antigen-binding domain that binds to human TROP2 and a second antigen-binding domain that binds to human PRLR. 2 . The bispecific antigen binding molecule of claim 1 , wherein the first antigen binding domain binds to human cells expressing human TROP2.

3. according to the bispecific antigen binding molecule described in claim 1 or claim 2, wherein said second antigen binding domain is in conjunction with the human cell of expressing people PRLR.

4. The bispecific antigen binding molecule of any one of claims 1-3, wherein each of the first antigen binding domain and the second antigen binding domain is a fully human antigen binding domain. 5 . The bispecific antigen-binding molecule according to claim 1 , which is a bispecific antibody or a bispecific antigen-binding fragment.

6. A bispecific antigen binding molecule according to any one of claims 1-5, wherein the bispecific antigen binding molecule binds to human TROP2 or / and human PRLR expressed on a cell.

7. A bispecific antigen-binding molecule according to any one of claims 1 to 6, wherein the first antigen-binding domain that specifically binds to human TROP2 comprises a heavy chain variable domain VH1 and a light chain variable domain VL1, wherein the VH1 comprises complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2 and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; wherein the VL1 comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:5 and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

6.

8. The bispecific antigen-binding molecule according to claim 7, wherein the first antigen-binding domain comprises a heavy chain variable domain having an amino acid sequence of SEQ ID NO: 7 and / or a light chain variable domain having an amino acid sequence of SEQ ID NO:

8.

9. The bispecific antigen-binding molecule according to any one of claims 1 to 8, wherein the second antigen-binding domain comprises a heavy chain variable domain VH2 and a light chain variable domain VL2, wherein the VH2 comprises complementarity determining regions H′CDR1, H′CDR2 and H′CDR3, wherein the H′CDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the H′CDR2 comprises the amino acid sequence shown in SEQ ID NO:10 and the H′CDR3 comprises the amino acid sequence shown in SEQ ID NO:11; wherein the VL2 comprises complementarity determining regions L′CDR1, L′CDR2 and L′CDR3, wherein the L′CDR1 comprises the amino acid sequence shown in SEQ ID NO:12, the L′CDR2 comprises the amino acid sequence shown in SEQ ID NO:13 and the L′CDR3 comprises the amino acid sequence shown in SEQ ID NO:

14.

10. The bispecific antigen binding molecule of claim 9, wherein the second antigen binding domain comprises a heavy chain variable domain with an amino acid sequence of SEQ ID NO: 15 and / or a light chain variable domain with an amino acid sequence of SEQ ID NO:

16.

11. The bispecific antigen binding molecule according to any one of claims 1 to 10, wherein the second antigen binding domain comprises a second light chain and a second heavy chain, the second light chain comprises a second light chain variable domain (VL2), the VL2 is operably linked to the first pairing domain (X1), the second heavy chain comprises a second heavy chain variable domain (VH2), the VH2 is operably linked to the second pairing domain (X2), wherein, X1 and X2 can form a leucine zipper structure; And / or the first antigen-binding domain comprises a first light chain and a first heavy chain, the first light chain comprises a first light chain variable domain (VL1), the VL1 is operably connected to a third pairing domain (X3), the first heavy chain comprises a first heavy chain variable domain (VH1), the VH1 is operably connected to a fourth pairing domain (X4), and the X3 and X4 can form a leucine zipper structure.

12. The bispecific antigen-binding molecule according to claim 11, wherein The first heavy chain further comprises a first dimerization domain, and the second heavy chain further comprises a second dimerization domain, and the first dimerization domain and the second dimerization domain are combined.

13. The bispecific antigen binding molecule of claim 12, wherein the first dimerization domain and the second dimerization domain are not identical and bind in a manner that prevents homodimerization and / or favors heterodimerization; Preferably, the binding mode of the first dimerization domain and the second dimerization domain is selected from: knob-into-hole structure, hydrophobic interaction, electrostatic interaction, hydrophilic interaction, or a mode of increasing flexibility.

14. The bispecific antigen binding molecule according to claim 13, wherein the C-terminus of the first dimerization domain is operably connected to the N-terminus of the fifth pairing domain (X5), and the C-terminus of the second dimerization domain is operably connected to the N-terminus of the sixth pairing domain (X6), wherein the X5 and X6 can form a dimer, and preferably the X5 and X6 can form a leucine zipper structure.

15. The bispecific antigen-binding molecule of claim 14, wherein: X5 comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 domain or a C / EBP domain; X6 correspondingly comprises a domain that forms a leucine zipper structure with X5; or, X6 comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 domain or a C / EBP domain; X5 correspondingly comprises a domain that forms a leucine zipper structure with X6; Preferably, X5 comprises a Jun domain, and X6 comprises a Fos domain; X5 contains the Fos domain, and X6 contains the Jun domain; X5 contains the Jun domain, and X6 contains the FosW domain; X5 contains the FosW domain, and X6 contains the Jun domain; X5 contains the Myc domain, and X6 contains the Max domain; X5 contains the Max domain, and X6 contains the Myc domain; X5 contains the WinzipA2 domain, and X6 contains the WinzipB1 domain; X5 contains the WinzipB1 domain, and X6 contains the WinzipA2 domain; X5 contains the ACID-p1 domain, and X6 contains the BASE-p1 domain; X5 contains the BASE-p1 domain, and X6 contains the ACID-p1 domain; X5 contains the GCN4 domain, and X6 contains the C / EBP domain; or, X5 contains a C / EBP domain and X6 contains a GCN4 domain; More preferably, the Jun domain is a sequence as shown in SEQ ID NO: 17, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 17; and / or the Fos domain is a sequence as shown in SEQ ID NO: 18, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 18; and / or the FosW domain is a sequence as shown in SEQ ID NO: 19, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 19; and / or the Max domain is a sequence as shown in SEQ ID NO: 20, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 20; and / or the Myc domain is a sequence as shown in SEQ ID NO: 21, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: NO:21 has at least 80%, 85%, 90%, 95%, or 99% identity; and / or, the WinzipA2 domain is a sequence as shown in SEQ ID NO:22, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:22; and / or, the WinzipB1 domain is a sequence as shown in SEQ ID NO:23, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:23; and / or, the ACID-p1 domain is a sequence as shown in SEQ ID NO:24, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:24; and / or, the BASE-p1 domain is a sequence as shown in SEQ ID NO:25, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:

25. NO:25 has a sequence with at least 80%, 85%, 90%, 95%, or 99% identity; and / or, the GCN4 domain is a sequence as shown in SEQ ID NO:26, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:26; and / or, the C / EBP domain is a sequence as shown in SEQ ID NO:27, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:

27.

16. The bispecific antigen binding molecule according to any one of claims 11 to 15, wherein X1 comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 domain or a C / EBP domain; X2 correspondingly comprises a binding domain that forms a leucine zipper structure with X1; or, X2 comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 domain or a C / EBP domain; X1 correspondingly comprises a binding domain that forms a leucine zipper structure with X2; Preferably, X1 comprises a Jun domain, and X2 comprises a Fos domain; X1 contains the Fos domain, and X2 contains the Jun domain; X1 contains the Jun domain, and X2 contains the FosW domain; X1 contains the FosW domain, and X2 contains the Jun domain; X1 contains the Myc domain, and X2 contains the Max domain; X1 contains the Max domain, and X2 contains the Myc domain; X1 contains the WinzipA2 domain, and X2 contains the WinzipB1 domain; X1 contains the WinzipB1 domain, and X2 contains the WinzipA2 domain; X1 contains the ACID-p1 domain, and X2 contains the BASE-p1 domain; X1 contains the BASE-p1 domain, and X2 contains the ACID-p1 domain; X1 contains the GCN4 domain, and X2 contains the C / EBP domain; or, X1 contains a C / EBP domain and X2 contains a GCN4 domain; More preferably, the Jun domain is a sequence as shown in SEQ ID NO: 17, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 17; and / or the Fos domain is a sequence as shown in SEQ ID NO: 18, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 18; and / or the FosW domain is a sequence as shown in SEQ ID NO: 19, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 19; and / or the Max domain is a sequence as shown in SEQ ID NO: 20, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 20; and / or the Myc domain is a sequence as shown in SEQ ID NO: 21, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: NO:21 has at least 80%, 85%, 90%, 95%, or 99% identity; and / or, the WinzipA2 domain is a sequence as shown in SEQ ID NO:22, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:22; and / or, the WinzipB1 domain is a sequence as shown in SEQ ID NO:23, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:23; and / or, the ACID-p1 domain is a sequence as shown in SEQ ID NO:24, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:24; and / or, the BASE-p1 domain is a sequence as shown in SEQ ID NO:25, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:

25. NO:25 has a sequence with at least 80%, 85%, 90%, 95%, or 99% identity; and / or, the GCN4 domain is a sequence as shown in SEQ ID NO:26, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:26; and / or, the C / EBP domain is a sequence as shown in SEQ ID NO:27, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:

27.

17. The bispecific antigen binding molecule according to any one of claims 11 to 16, wherein X3 comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 domain or a C / EBP domain; X4 correspondingly comprises a binding domain that forms a leucine zipper structure with X3; or, X4 comprises a Jun domain, a Fos domain, a FosW domain, a Myc domain, a Max domain, a WinzipA2 domain, a WinzipB1 domain, an ACID-p1 domain, a BASE-p1 domain, a GCN4 domain or a C / EBP domain; X3 correspondingly comprises a binding domain that forms a leucine zipper structure with X4; Preferably, X3 comprises a Jun domain, and X4 comprises a Fos domain; X3 contains the Fos domain, and X4 contains the Jun domain; X3 contains the Jun domain, and X4 contains the FosW domain; X3 contains the FosW domain, and X4 contains the Jun domain; X3 contains the Myc domain, and X4 contains the Max domain; X3 contains the Max domain, and X4 contains the Myc domain; X3 contains the WinzipA2 domain, and X4 contains the WinzipB1 domain; X3 contains the WinzipB1 domain, and X4 contains the WinzipA2 domain; X3 contains the ACID-p1 domain, and X4 contains the BASE-p1 domain; X3 contains the BASE-p1 domain, and X4 contains the ACID-p1 domain; X3 contains the GCN4 domain, and X4 contains the C / EBP domain; or, X3 contains a C / EBP domain and X4 contains a GCN4 domain; More preferably, the Jun domain is a sequence as shown in SEQ ID NO: 17, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 17; and / or the Fos domain is a sequence as shown in SEQ ID NO: 18, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 18; and / or the FosW domain is a sequence as shown in SEQ ID NO: 19, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 19; and / or the Max domain is a sequence as shown in SEQ ID NO: 20, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: 20; and / or the Myc domain is a sequence as shown in SEQ ID NO: 21, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO: NO:21 has at least 80%, 85%, 90%, 95%, or 99% identity; and / or, the WinzipA2 domain is a sequence as shown in SEQ ID NO:22, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:22; and / or, the WinzipB1 domain is a sequence as shown in SEQ ID NO:23, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:23; and / or, the ACID-p1 domain is a sequence as shown in SEQ ID NO:24, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:24; and / or, the BASE-p1 domain is a sequence as shown in SEQ ID NO:25, or a sequence having at least 80%, 85%, 90%, 95%, or 99% identity with SEQ ID NO:

25. NO:25 has a sequence with at least 80%, 85%, 90%, 95%, or 99% identity; and / or, the GCN4 domain is a sequence as shown in SEQ ID NO:26, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:26; and / or, the C / EBP domain is a sequence as shown in SEQ ID NO:27, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity to SEQ ID NO:

27.

18. The bispecific antigen binding molecule according to any one of claims 11 to 17, wherein the VL1 is operably connected to X3, preferably, the VL1 is operably connected to X3 via a first connector L1; the VH1 is operably connected to X4, preferably, the VH1 is operably connected to X4 via a second connector L2; and / or the VL2 is operably connected to X1, preferably, the VL2 is operably connected to X1 via a third connector L3; the VH2 is operably connected to X2, preferably, the VH2 is operably connected to X2 via a fourth connector L4; and / or , the C-terminus of the first dimerization domain is connected to X5, preferably, the C-terminus of the first dimerization domain is connected to X5 via a fifth connector L5; the C-terminus of the second dimerization domain is connected to X6, preferably, the C-terminus of the second dimerization domain is connected to X6 via a sixth connector L6; L1-L6 are each independently present or absent, and when L1-L6 are optionally present, L1-L6 are each independently selected from the following polypeptide fragments: (GxS)y, (TxG)y, and (SxG)y, wherein x is selected from an integer of 1, 2, 3, 4, or 5, and y is selected from an integer of 1, 2, 3, 4, 5, or 6; Preferably, L1-L6 are each independently selected from the following polypeptide fragments: GGS, GGGGS, TGGGG, and SGGGG, more preferably, L1-L6 are each independently connected to a cleavage site peptide via their C-termini, and the cleavage site peptide is selected from factory Xa, thrombin, enterokinase or SUMO.

19. The bispecific antigen binding molecule according to claims 1-18, wherein the first antigen binding domain comprises a heavy chain having an amino acid sequence of SEQ ID NO: 29 and a light chain having an amino acid sequence of SEQ ID NO: 28; and / or The second antigen binding domain comprises a heavy chain with an amino acid sequence of SEQ ID NO: 31 and a light chain with an amino acid sequence of SEQ ID NO:

30.

20. An isolated polynucleotide encoding the bispecific antigen binding molecule of any one of claims 1-19; preferably, the polynucleotide is single-stranded DNA, double-stranded DNA or single-stranded RNA.

21. An isolated vector comprising the polynucleotide of claim 20.

22. A host cell comprising the isolated polynucleotide of claim 20, or the isolated vector of claim 21.

23. The host cell according to claim 22, which is a prokaryotic cell, preferably Escherichia coli.

24. The host cell according to claim 22, wherein the host cell is a eukaryotic cell, preferably, the cell is selected from one of a CHO cell, a COS cell, a Sp2 / 0 cell or a yeast cell.

25. An antibody-drug conjugate (ADC), comprising the bispecific antigen binding molecule according to any one of claims 1-19 and a cytotoxic drug.

26. The antibody-drug conjugate (ADC) of claim 25, wherein the bispecific antigen binding molecule and the cytotoxic drug are covalently linked via a linker.

27. The antibody-drug conjugate (ADC) of claim 26, wherein the cytotoxic drug is selected from the group consisting of auristatins, maytansinoids, calicheamicin, duocarmycins, anthracyclines, pyrrolobenzodiazepine dimers, amatoxin, camptothecin derivatives, tubulysin, tomaymycin, or dolastatin derivatives; preferably, the cytotoxic drug is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansinoid DM1, maytansinoid DM4, camptothecin SN-38, exitecan or a derivative thereof.

28. The antibody-drug conjugate (ADC) according to claim 27, wherein the linker is one or more of MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropionyl), val-cit (valine-citrulline), val-ala (valine-alanine), ala-phe (alanine-phenylalanine), phe-lys (phenylalanine-lysine), a dipeptide site in a protease-cleavable linker, PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio) pentanoate), SMCC (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate), and SIAB (N-succinimidyl (4-iodo-acetyl) aminobenzoate), preferably, the linker is MC.

29. A pharmaceutical composition comprising a therapeutically effective amount of the bispecific antigen binding molecule of any one of claims 1 to 19 or the antibody-drug conjugate (ADC) of any one of claims 25 to 28 and a pharmaceutically acceptable carrier or diluent.

30. Use of the bispecific antigen binding molecule of any one of claims 1-19, the antibody-drug conjugate (ADC) of any one of claims 25-28, or the pharmaceutical composition of claim 29 in the preparation of a medicament for treating a disease or condition associated with the expression of TROP2 or PRLR.

31. The use according to claim 30, wherein the disease or disorder is cancer.

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