TW202346362A - An anti-trop-2/cd3 dual-specific antibody - Google Patents

Abstract

The present invention relates to the field of antibody drug technology. Specifically, it involves a dual-specificity antibody against TROP-2/CD3. The anti-TROP-2/CD3 dual-specificity antibody of the present invention comprises a first antigen-binding domain D1 and a second antigen-binding domain D2, wherein D1 is an anti-TROP-2 antibody or its antigen-binding fragment, and D2 is an anti-CD3 antibody or its antigen-binding fragment. The anti-TROP-2/CD3 dual-specificity antibody of the present invention can be used to achieve T-cell-mediated immune response, particularly to effectively promote T-cell-mediated killing of tumor cells expressing TROP-2, thereby effectively inhibiting tumor growth.

Description

An anti-TROP-2/CD3 bispecific antibody

The present invention relates to the technical field of antibody drugs, specifically, to an anti-TROP-2/CD3 bispecific antibody.

Human trophoblast cell surface antigen 2 (TROP-2) is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene. TROP-2 is a single-transmembrane type I membrane protein composed of 323 amino acids, including 26 amino acids in the message peptide, 248 amino acids in the extracellular region, and 23 amino acids in the transmembrane region. 26 amino acids in the inner region. Up to now, the ligand protein of TROP-2 has not been identified, so its physiological and biochemical functions are not very clear yet. However, a large number of clinical studies and literature reports indicate that TROP-2 protein is highly expressed in various human epithelial cancers and is closely related to poor prognosis and cancer cell metastasis, including breast cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, and cervical cancer. . The US FDA has approved the TROP-2 antibody conjugate drug sacituzumab govitecan for the treatment of metastatic triple-negative breast cancer.

Cluster of differentiation 3 (CD3) is an important differentiation antigen on the surface of T cells. CD3 molecules form a TCR-CD3 complex with T cell receptor (TCR). The TCRαβ subunit recognizes extracellular signals and The extracellular signal is transmitted to CD3, and the CD3 molecule relies on the immunoreceptor tyrosine-based activation motif (ITAM) to transmit the signal into the cell. Anti-CD3 antibodies can stimulate or block T cell activation signals, eliminate effector T cells or induce the production of regulatory T cells.

Bispecific antibodies (BsAb), also known as bifunctional antibodies, are specific drugs that simultaneously target two different antigens or different epitopes of the same antigen. BsAb can act on tumor cells by immune cells and viral molecules to enhance the killing effect on target cells. It can also combine with different antigens of tumor cells at the same time to enhance its binding specificity and reduce off-target effects. Bispecific antibodies have broadened the application fields of antibody drugs and provided new research ideas for tumor immunity.

However, there is currently a lack of satisfactory bispecific antibodies against TROP-2 and CD3 in the field. Therefore, there is an urgent need in this field to develop a new bispecific antibody against TROP-2 and CD3.

In view of the above shortcomings of the prior art, the purpose of the present invention is to provide an anti-TROP-2/CD3 bispecific antibody to solve the problems in the prior art.

The purpose of the present invention is to provide a new bispecific antibody against TROP-2 and CD3. The bispecific antibody can specifically bind to TROP-2 and CD3 at the same time, thereby initiating T cell targeted killing of TROP-2 positive expression. of tumor cells. The present invention also aims to provide a polynucleotide molecule encoding the bispecific antibody; to provide an expression vector containing the molecule; to provide a host cell containing the expression vector; to provide a method for preparing the bispecific antibody; to provide a method containing the bispecific antibody. Pharmaceutical compositions of specific antibodies; immunoconjugates comprising the bispecific antibodies are provided; applications of the bispecific antibodies or pharmaceutical compositions in preparing drugs for treating cancer or tumors are provided; fusion proteins, the Methods for treating cancer or tumors with pharmaceutical compositions or immunoconjugates.

In order to achieve the above objects, the present invention provides the following technical solutions: A first aspect of the invention provides a bispecific antibody, the bispecific antibody comprising a first antigen-binding domain D1 and a second antigen-binding domain D2, where D1 is an anti-TROP-2 antibody or an antigen-binding fragment thereof , the D2 is an anti-CD3 antibody or an antigen-binding fragment thereof, and the anti-TROP-2 antibody or anti-CD3 antibody or their respective antigen-binding fragments comprise the heavy chain complementarity determining region HCDR1-3 and the light chain complementarity determining region LCDR1-3.

In another preferred embodiment, the bispecific antibody contains a monomer or a dimer or multimer formed of monomers. The dimer can be homologous or heterologous, and the monomer from N-terminus to C-terminus contains a structure selected from any of the following groups: in, VLA represents the light chain variable region of an anti-TROP-2 antibody or antigen-binding fragment thereof; VHA represents the heavy chain variable region of an anti-TROP-2 antibody or antigen-binding fragment thereof; VLB represents the light chain variable region of an anti-CD3 antibody or antigen-binding fragment thereof; VHB represents the heavy chain variable region of an anti-CD3 antibody or its antigen-binding fragment; CH represents the heavy chain constant region; CL represents the light chain constant region; L1, L2 and L3 are each independently a bond or linker; "~" represents disulfide bond or covalent bond; "-" represents peptide bond.

In another preferred embodiment, the bispecific antibody comprises a structure selected from any of the following groups: a) Homodimer formed by the monomer of structure I; b) Homodimers formed from monomers of structure II.

In another preferred embodiment, the anti-CD3 antibody or antigen-binding fragment thereof includes a heavy chain complementarity determining region HCDR1-3 and a light chain complementarity determining region LCDR1-3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9 are shown, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12 respectively; and /Or, the anti-TROP-2 antibody or antigen-binding fragment thereof includes the heavy chain complementarity determining region HCDR1-3 and the light chain complementarity determining region LCDR1-3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively as shown in SEQ ID NO. 1. SEQ ID NO. 2 and SEQ ID NO. 3 are shown. The amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 respectively.

In another preferred embodiment, the complementarity determining region contains at least one amino acid mutation.

In another preferred embodiment, the mutation is used to increase the expression level of the bispecific antibody.

In another preferred embodiment, the mutation is used to reduce the affinity of the bispecific antibody to human CD3.

In another preferred embodiment, the mutation is selected from any one or more of the following: a) Anti-CD3 antibody or its antigen-binding fragment HCDR1:X1X2AMN, b) HCDR1 of anti-TROP-2 antibody or antigen-binding fragment thereof: X3YWLG, c) LCDR2 of anti-CD3 antibody or antigen-binding fragment thereof: X4TNKRAP, Among them, X1 is not T, X2 is not Y, X3 is not I, and X4 is not G.

In another preferred example, the amino acid mutation is selected from the group consisting of X1 being G, X2 being S, H or G, X3 being D or E, and X4 being A.

In another preferred embodiment, the anti-CD3 antibody or antigen-binding fragment thereof includes a heavy chain complementarity determining region HCDR1-3 and a light chain complementarity determining region LCDR1-3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO. 15, SEQ ID NO. 8, and SEQ ID NO. 9 are shown, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12 respectively; and /Or, the anti-TROP-2 antibody or antigen-binding fragment thereof includes the heavy chain complementarity determining region HCDR1-3 and the light chain complementarity determining region LCDR1-3, wherein the amino acid sequences of HCDR1, HCDR2, and HCDR3 are respectively as shown in SEQ ID NO. 13 or SEQ ID NO. 14, SEQ ID NO. 2, SEQ ID NO. 3, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are as SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 respectively. shown.

In another preferred example, the HCDR1 of the anti-CD3 antibody or its antigen-binding fragment is X1X2AMN, where X1 is G, As shown in SEQ ID NO. 8 and SEQ ID NO. 9 respectively, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12 respectively; and/or , the HCDR1 of the anti-TROP-2 antibody or its antigen-binding fragment is X3YWLG, where X3 is D, and the amino acid sequence is shown in SEQ ID NO. 13; the amino acid sequences of HCDR2 and HCDR3 are respectively shown in SEQ ID NO. 2, SEQ ID NO. 3 is shown, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.

In another preferred example, the HCDR1 of the anti-CD3 antibody or its antigen-binding fragment is X1X2AMN, where X1 is G, As shown in SEQ ID NO. 8 and SEQ ID NO. 9 respectively, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12 respectively; and/or , the HCDR1 of the anti-TROP-2 antibody or its antigen-binding fragment is X3YWLG, where X3 is D, and the amino acid sequence is shown in SEQ ID NO. 13; the amino acid sequences of HCDR2 and HCDR3 are shown in SEQ ID NO. 2 and SEQ ID NO. 3 is shown, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.

In another preferred example, the HCDR1 of the anti-CD3 antibody or its antigen-binding fragment is X1X2AMN, where X1 is G, As shown in SEQ ID NO. 8 and SEQ ID NO. 9 respectively, the amino acid sequences of LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12 respectively; and/or , the HCDR1 of the anti-TROP-2 antibody or its antigen-binding fragment is X3YWLG, where X3 is D, and the amino acid sequence is shown in SEQ ID NO. 13; the amino acid sequences of HCDR2 and HCDR3 are shown in SEQ ID NO. 2 and SEQ ID NO. 3 is shown, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.

In another preferred example, the LCDR2 of the anti-CD3 antibody or its antigen-binding fragment is X4TNKRAP, where X4 is A, and the amino acid sequence is shown in SEQ ID NO. 19; the amino acid sequences of LCDR1 and LCDR3 are shown in SEQ ID NO. NO. 10, SEQ ID NO. 12, HCDR1, HCDR2, HCDR3 amino acid sequences are shown in SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 respectively; and/or, the anti-TROP The HCDR1 of the -2 antibody or its antigen-binding fragment is X3YWLG, where 3, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 respectively.

In another preferred embodiment, the anti-CD3 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO. 22, and the light chain variable region is as shown in SEQ ID NO. 22. The amino acid sequence of the chain variable region is shown in SEQ ID NO. 23; and/or the anti-TROP-2 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region, wherein the heavy chain can The amino acid sequence of the variable region is shown in SEQ ID NO. 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 21.

In another preferred embodiment, the variable region contains at least one amino acid mutation.

In another preferred embodiment, the variable region contains amino acid mutations selected from any one or more of the following: 1) The SEQ ID NO. 20 has an amino acid mutation at position 31; 2) The SEQ ID NO. 22 has an amino acid mutation at position 30; 3) The SEQ ID NO. 22 has an amino acid mutation at position 31; 4) The SEQ ID NO. 22 has an amino acid mutation at position 32; 5) The SEQ ID NO. 23 has an amino acid mutation at position 50.

Preferably, the variable region of the bispecific antibody contains the mutations shown in 1), 3) and 4);

Preferably, the variable region of the bispecific antibody contains the mutations shown in 1), 2), 3) and 4);

Preferably, the variable region of the bispecific antibody contains the mutations shown in 1) and 5).

In another preferred embodiment, the variable region contains amino acid mutations selected from any one or more of the following: a) The SEQ ID NO. 20 has I31D; b) The SEQ ID NO. 20 has I31E; c) The SEQ ID NO. 22 has N30S; d) The SEQ ID NO. 22 has T31G; e) The SEQ ID NO. 22 has Y32S, Y32H, and Y32G; f) The SEQ ID NO. 23 has G50A.

Preferably, the variable region of the bispecific antibody includes the T31G in d), the Y32S in e) and the I31D in a), or the variable region of the bispecific antibody includes the T31G in d), e) The Y32S in b) and the I31E mutation in b). Correspondingly, the anti-CD3 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 26, and the light chain variable region The amino acid sequence is shown in SEQ ID NO. 23; and/or the anti-TROP-2 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amine group The acid sequence is shown in SEQ ID NO. 24 and SEQ ID NO. 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 21.

Preferably, the variable region of the bispecific antibody includes the N3OS in c), the T31G in d), the Y32S in e) and the I31D mutation in a), and the variable region of the bispecific antibody includes c) the N30S in d), the T31G in d), the Y32S in e) and or the I31E mutation in b);

Preferably, the variable region of the bispecific antibody includes the T31G in d), the Y32H in e) and the I31D mutation in a), or the variable region of the bispecific antibody includes the T31G, e in d) ) the Y32H and b) the I31E mutation;

Preferably, the variable region of the bispecific antibody includes the T31G in d), the Y32G in e) and the I31D mutation in a), or the variable region of the bispecific antibody includes the T31G, e in d) ) the Y32G and b) the I31E mutation;

Preferably, the variable region of the bispecific antibody comprises the G50A in f) and the I31D mutation in a), or the variable region of the bispecific antibody comprises the G50A in f) and the I31E mutation in b).

In another preferred embodiment, the anti-TROP-2 or anti-CD3 antigen-binding fragment is selected from Fab, F(ab'), F(ab')2, Fv or single-chain Fv (scFv); the anti-TROP-2 or Anti-CD3 antibodies are IgG antibodies.

In another preferred embodiment, the anti-TROP-2 or anti-CD3 antibody is selected from chimeric antibodies (such as human-mouse chimeric antibodies), murine antibodies or humanized antibodies.

In another preferred embodiment, the anti-TROP-2 antibody is a low-endocytotic antibody, which is more conducive to sustained effects.

In another preferred embodiment, the IgG antibody includes a heavy chain constant region and a light chain constant region; more preferably, the heavy chain constant region is selected from human IgG1, human IgG2, human IgG3 or human IgG4, and the light chain constant region is selected from human IgG1, human IgG2, human IgG3 or human IgG4. From human κ (Kappa) or human λ (Lambda).

In another preferred embodiment, the IgG antibody includes the IgG1 heavy chain constant region whose amino acid sequence is shown in SEQ ID NO. 51.

In another preferred embodiment, the IgG antibody includes the human kappa (Kappa) light chain constant region whose amino acid sequence is shown in SEQ ID NO. 52.

In another preferred embodiment, the constant region of the IgG antibody contains at least one amino acid mutation.

In another preferred example, the amino acid mutation is a knob-in-hole (KIH) mutation that can promote heterodimerization of two heavy chains; more preferably, the mutation is located in the CH3 region of the constant region.

In another preferred example, the D1 is an IgG antibody, and the D2 is a scFv; more preferably, the D2 is connected to the N-terminal or C-terminal of D1, or between CH1 and CH2 of D1; further more preferably, This D2 is linked to the heavy chain of D1.

In another preferred embodiment, the D2 contains one, two, three or more anti-CD3 scFvs.

In another preferred embodiment, the scFv contains a VH-L1-VL structure or a VL-L1-VH structure from N-terminus to C-terminus.

In another preferred embodiment, the anti-TROP-2 antibody is selected from the group consisting of anti-TROP-2-VH-I31D monoclonal antibody and anti-TROP-2-VH-I31E monoclonal antibody.

In another preferred embodiment, the anti-CD3 antigen-binding fragment is selected from CD3-scFv and CD3-scFv-VH-T31G-Y32S.

The above-mentioned connection refers to direct connection through a linker or through a peptide bond.

In another preferred example, the amino acid sequences of the linkers L1, L2, and L3 are independently (G4S)n or 4G, and n is selected from 1, 2, 3, 4, 5, and 6.

In another preferred example, the amino acid sequence of the linker L1 is shown in SEQ ID NO. 27.

In another preferred example, the amino acid sequence of the linker L2 is shown in SEQ ID NO. 28.

In another preferred example, the amino acid sequence of the linker L3 is shown in SEQ ID NO. 29.

In another preferred embodiment, the bispecific antibody is a homodimer or heterodimer.

In the preferred example shown in Figure 1A, the bispecific antibody is a homodimer, including an anti-TROP-2 IgG antibody and two anti-CD3 scFv, wherein each scFv includes a variable region VH and an Variable region VL, VH and VL are connected through linker L1, and each anti-CD3 scFv is connected to between CH1 and CH2 of anti-TROP-2 immunoglobulin antibody IgG through linkers L2 and L3.

In the preferred example shown in Figure 1B, the bispecific antibody is a homodimer, including an anti-TROP-2 IgG antibody and two anti-CD3 scFv, wherein each scFv includes a variable region VH and an The variable regions VL, VH and VL are connected through linker L1, and each anti-TROP-2 scFv is connected in series with the N-terminus of the anti-TROP-2 immunoglobulin antibody IgG heavy chain through linker L2.

In another preferred embodiment, the bispecific antibody is a heterodimer, comprising an anti-TROP-2 IgG antibody and an anti-CD3 scFv, wherein the scFv includes a variable region VH and a variable region VL, VH and VL The anti-CD3 scFv is connected in series with the N-terminus of any anti-TROP-2 immunoglobulin antibody IgG heavy chain through linker L2.

In another preferred embodiment, the bispecific antibody includes a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are selected from any of the following: a) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 36, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; b) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 37, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; c) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 38, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; d) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 39, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; e) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 40, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; f) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 41, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; g) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 42, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; or, h) The amino acid sequence in a) to g) is formed by substituting, deleting or adding one or more amino acid residues, and has both anti-TROP-2 activity and anti-CD3 activity. to g) derivatized polypeptides.

In another preferred embodiment, the bispecific antibody includes an active fragment and/or derivative of the bispecific antibody, wherein the active fragment and/or derivative retains 70-100% of the bispecific antibody. (such as 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%) anti-TROP-2 activity and 70-100% anti-CD3 activity .

In another preferred embodiment, the derivative of the bispecific antibody is that the bispecific antibody undergoes one or several amino acid mutations (after amino acid deletion, insertion and/or substitution) and maintains at least 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity of the polypeptide.

In another preferred embodiment, the amino acid mutation is a conservative amino acid substitution.

In another preferred embodiment, the amino acid mutation is located in the framework region or constant region.

A second aspect of the invention provides a polynucleotide molecule encoding said bispecific antibody.

In another preferred embodiment, the polynucleotide molecule includes a polynucleotide molecule encoding the bispecific antibody heavy chain, and the nucleotide sequence is as shown in SEQ ID NO. 43, SEQ ID NO. 44 or SEQ ID NO. 45. Shown; and, the polynucleotide molecule encoding the light chain of the bispecific antibody, the nucleotide sequence is shown in SEQ ID NO. 50.

In another preferred embodiment, the polynucleotide molecule includes a polynucleotide molecule encoding the bispecific antibody heavy chain, with a nucleotide sequence such as SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48 or SEQ ID NO. 49 is shown; and, the polynucleotide molecule encoding the light chain of the bispecific antibody, the nucleotide sequence is shown in SEQ ID NO. 50.

A third aspect of the present invention provides an expression vector containing the polynucleotide molecule.

In another preferred embodiment, the expression vector is a virus or plasmid, preferably a phage or a phagemid.

In another preferred embodiment, the expression vector is selected from the following group: pcDNA3.4, pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, Preferably it is pcDNA3.4.

A fourth aspect of the present invention provides a cell containing the expression vector.

In another preferred example, the cell is selected from any of the following: COS, CHO, 293F, 293E, NSO, sf9, sf21, DH5α, BL21 (DE3) or TG1, preferably E.coli TG1, BL21 (DE3 ) cells (expressing single-chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies).

A fifth aspect of the present invention provides a method for preparing the above-mentioned bispecific antibody, which method includes the following steps: a) Cultivate the cells under expression conditions to express bispecific antibodies; b) Isolate and purify the bispecific antibody described in step a).

The sixth aspect of the present invention provides a pharmaceutical composition, which contains an effective amount of the above-mentioned bispecific antibody and one or more pharmaceutically acceptable carriers, diluents or excipients.

In another preferred embodiment, the pharmaceutical composition contains the above-mentioned bispecific antibody, acetate, trehalose, arginine hydrochloride, Tween, etc.

In another preferred embodiment, the dosage form of the pharmaceutical composition includes a gastrointestinal dosage form or a parenteral dosage form.

In another preferred embodiment, the parenteral administration dosage form includes intravitreal injection, intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection or intracavity injection. wait.

The seventh aspect of the present invention provides the use of the above-mentioned bispecific antibody or pharmaceutical composition in the preparation of drugs for cancer or tumors.

In another preferred embodiment, the cancer or tumor is a TROP-2 positive cancer or tumor.

In another preferred embodiment, the cancer or tumor is selected from: lung cancer, bone cancer, gastric cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, fallopian tube cancer, endometrial cancer, Cervical cancer, vaginal cancer, vulva cancer, rectal cancer, colon cancer, anal area cancer, breast cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethra cancer, penile cancer, Prostate cancer, pancreatic cancer, brain cancer, testicular cancer, lymphoma, transitional cell cancer, bladder cancer, kidney or ureter cancer, renal cell cancer, renal pelvis cancer, Hodgkin lymphoma, non-Hodgkin lymphoma Non-Hodgkin lymphoma, soft tissue sarcoma, pediatric solid tumors, lymphocytic lymphoma, central nervous system tumors, primary central nervous system lymphoma, spinal tumors, brainstem glioma, pituitary adenoma, Melanoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, chronic, acute leukemia, or combinations thereof.

An eighth aspect of the present invention provides an immunoconjugate, the immunoconjugate comprising: a) the bispecific antibodies described above; and b) The conjugate part is selected from any one or more of the following: detectable markers, drugs, toxins, cytokines, radionuclides or enzymes, etc.

In another preferred embodiment, the conjugate moiety is selected from: fluorescent or luminescent markers, radioactive markers, contrast agents for magnetic resonance imaging or computerized X-ray tomography, or enzymes capable of producing detectable products, Radionuclides, biotoxins or cytokines, etc.

In another preferred embodiment, the immunoconjugate includes an antibody-drug conjugate.

In another preferred embodiment, the immunoconjugate is used to prepare a pharmaceutical composition for treating tumors or cancer.

In the ninth aspect of the present invention, a method for treating cancer or tumors is provided, which method includes administering the bispecific antibody according to the first aspect of the present invention, the bispecific antibody according to the sixth aspect of the present invention to a subject in need. the pharmaceutical composition described above, or the immunoconjugate described in the eighth aspect of the present invention.

In another preferred embodiment, the method further includes combined administration with other anti-tumor drugs.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.

The positive and progressive effects of the anti-TROP-2/CD3 bispecific antibody of the present invention are: (1) It can regulate T cell immune activity by binding to T cells, and specifically bind to tumor cells expressing TROP-2, thereby inducing immune cell targets To kill TROP-2 positive tumor cells. In particular, its tumor cell killing effect and ability to stimulate immune cells to release IL-2 cytokines are better than the control anti-CD3 monoclonal antibody; (2) high specificity and good safety; (3) high expression amount; (4) Structurally stable.

After extensive and in-depth research, the inventors obtained a new bispecific antibody targeting tumor cell surface molecules TROP-2 and CD3, which can maintain the activity of the antibodies at both ends and can simultaneously bind to TROP-2 and CD3 antigens. The bispecific antibodies of the present invention can be used to achieve T cell-mediated immune responses, especially to effectively promote T cell-mediated killing of tumor cells expressing TROP-2, thereby effectively inhibiting tumors. Unexpectedly, its tumor cell killing effect and ability to stimulate immune cells to release IL-2 cytokines were better than the control anti-CD3 monoclonal antibody, and it was safe. Therefore, the bispecific antibody of the present invention can be developed as an anti-tumor drug with superior efficacy. On this basis, the present invention was completed.

Terminology

In order that this disclosure may be more easily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below unless expressly stated otherwise herein.

The term "about" may refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

In the present invention, the terms "Antibody (Ab)" and "Immunoglobulin G (IgG)" are heterotetrameric proteins with the same structural characteristics, which are composed of two identical light chains (L) and Composed of two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes (isotypes) is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by a constant domain, which consists of three domains, CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end. The light chain constant region includes a domain CL; the constant region of the light chain pairs with the CH1 domain of the heavy chain constant region, and the light chain can The variable region is paired with the variable region of the heavy chain. Constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes κ (Kappa) or λ (Lambda). The heavy and light chains of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of the antibody are covalently linked together by the inter-polypeptide disulfide formed between the hinge regions. Bonds are held together covalently.

The "immunoglobulin antibody IgG" described in the present invention is about 150kDa and consists of four peptide chains, including two identical gamma heavy chains of approximately 50kDa and two identical light chains of approximately 25kDa, thus having a tetramer structure. level structure. The two heavy chains are linked to each other by disulfide bonds and to a light chain each. The resulting tetramer has two identical halves that form a fork or Y-like shape, with each end of the fork containing an identical antigen-binding site. IgG antibodies can be divided into multiple subclasses (eg, IgG1, 2, 3, 4) based on minor differences in amino acid sequences in the constant regions of the heavy chains.

In the present invention, the term "bispecific antibody (or bispecific antibody)" refers to an antibody molecule that can specifically bind to two antigens (targets) or two epitopes at the same time. Based on symmetry, bispecific antibodies can be divided into structurally symmetric and asymmetric molecules. According to the number of binding sites, bispecific antibodies can be divided into bivalent, trivalent, tetravalent and multivalent molecules.

In the present invention, the term "monoclonal antibody (monoclonal antibody)" refers to an antibody obtained from a substantially homogeneous population, that is, the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may exist. Monoclonal antibodies target a single antigenic site with high specificity. Furthermore, unlike conventional polyclonal antibody preparations, which are typically mixtures of different antibodies directed against different antigenic determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the nature of the antibody as having been obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any special method to produce the antibody.

As used herein, the term "antigen-binding fragment" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, or a single Fv fragment, etc., which has antigen-binding activity. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fv fragments; or (iv) single chain Fv (scFv). As used herein, the expression "antigen-binding fragment" also encompasses other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tribodies , tetrabody antibodies, minibodies, nanobodies (such as monovalent nanobodies and bivalent nanobodies, etc.), small module immune drugs and shark variable IgNAR domains, etc.

In the present invention, the terms "Fab" and "Fc" mean that papain can cleave an antibody into two identical Fab segments and one Fc segment. The Fab segment consists of the VH and CH1 domains of the heavy chain of the antibody and the VL and CL domains of the light chain. The Fc segment is a crystallizable fragment (Fc), which consists of the CH2 and CH3 domains of the antibody. The Fc segment has no antigen-binding activity and is the site where the antibody interacts with effector molecules or cells. The term "F(ab')2" fragment antibody is obtained by pepsin digestion of the entire IgG antibody, removing most of the Fc region while leaving some hinge regions intact, and has two antigen-binding F( ab') part.

In the present invention, the term "scFv" or "single chain variable region fragment scFv" is a single chain antibody (single chain antibody fragment, scFv), which refers to a fusion protein containing the variable regions of the immunoglobulin heavy chain VH and light chain VL. , VH and VL are connected through a linker containing 15-25 amino acids, in which the fusion protein retains the same antigen specificity of the intact immunoglobulin.

In the present invention, the term "Fv fragment" or "Fv antibody" contains the antibody heavy chain variable region and the light chain variable region, but does not have a constant region, and is the smallest antibody fragment that has all antigen-binding sites. Typically, the Fv fragment also contains a polypeptide linker between the VH and VL domains and is capable of forming the structure required for antigen binding.

In the present invention, the term "variable" means that certain parts of the variable regions of the antibody differ in sequence, which contribute to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy and light chain variable regions. The more conserved part of the variable region is called the frame region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-sheet configuration and are connected by three CDRs forming a connecting loop. In some cases, a partial β-sheet structure can be formed. The CDRs in each chain are held closely together by the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pp. 647-669 (1991)).

As used herein, the term "framework region FR" refers to the amino acid sequences inserted between the CDRs, i.e., those portions of the light and heavy chain variable regions of an immunoglobulin that are relatively conserved among different immunoglobulins within a single species. . The light chain and heavy chain of immunoglobulins each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, and FR4-H respectively. Accordingly, the light chain variable domain may thus be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain may thus be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof. The derivative of the human antibody FR is basically the same as the naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%. Knowing the amino acid sequence of the CDR, those skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR4- H. As used herein, the term "human framework region" is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to that of a naturally occurring human antibody .

As used herein, the term "linker" refers to one or more amine groups inserted into an immunoglobulin domain that provide sufficient mobility for the light and heavy chain domains to fold into an exchange dual variable region immunoglobulin. acid residue. In the present invention, preferred linkers refer to linkers L1, L2 and L3, where L1 connects the VH and VL of the single chain antibody (scFv), and L2 and L3 connect the scFv to the CH1 and CH2 of the antibody heavy chain, Or directly connected to the heavy chain of the antibody through L2 (such as the N-terminal or C-terminal of the antibody heavy chain). Examples of suitable linkers include single glycine (Gly) or serine (Ser) residues. The identity and sequence of the amino acid residues in the linker may vary depending on the type of secondary structural elements desired to be implemented in the linker.

In the present invention, the terms "anti", "binding" and "specific binding" refer to the non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. Typically, the antibody binds the antigen with an equilibrium dissociation constant (KD) of less than about 10-7M, such as less than about 10-8M, 10-9M, 10-10M, 10-11M, or less. As used herein, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, surface plasmon resonance (SPR) is used to determine the binding affinity of the antibody to the antigen in a BIACORE instrument or ELISA is used to determine the relative affinity of the antibody to the antigen.

In the present invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody or a region of an antigen that is bound by an antibody.

bispecific antibodies

The bispecific antibody of the present invention is a bispecific antibody that can specifically bind to TROP-2 and CD3, and it contains an anti-CD3 antibody part and an anti-TROP-2 antibody part.

The bispecific antibodies of the present invention can be dimers, trimers or multimers, preferably homo- or heterodimers. The anti-CD3 or anti-TROP-2 antibody portion of the bispecific antibody of the present invention may include one or more antibodies or antigen-binding fragments thereof, preferably 1, 2, 3, 4, 5 or 6.

The bispecific antibody of the present invention also includes conservative variants thereof, which means that compared with the amino acid sequence of the bispecific antibody of the present invention, there are at most 10, preferably at most 8, and more preferably at most 5. , a polypeptide formed by replacing at most 3 amino acids with amino acids of similar or similar properties. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.

Table A initial residue representative substitution Preferred substitutions Ile (I) Asp; Glu; Leu; Val; Met Asp Thr(T) Gly; Ser; Trp; Lys Gly Tyr(Y) Ser; Cys; Val Ser

The sequence of the present invention adopts the Kabat system numbering rule.

In constructing the bispecific antibodies of the invention, issues related to the chemical and physical stability of the bispecific antibodies are also addressed, such as expressing physically stable molecules, increasing heat and salt-dependent stability, reducing aggregation, Increase solubility at high concentrations and maintain affinity for the two antigens TROP-2 and CD3 respectively.

Encoding nucleic acids and expression vectors

Another aspect of the invention provides a polynucleotide molecule encoding the bispecific antibody described above. The polynucleotides of the invention may be in DNA form or RNA form. Forms of DNA include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. Once the relevant sequence is obtained, recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

The preparation method of the polynucleotide molecule of the present invention is a conventional preparation method in the art, and preferably includes the following preparation method: obtaining the polynucleotide molecule encoding the above-mentioned monoclonal antibody through gene cloning technology such as PCR method, or artificially The polynucleotide molecule encoding the above-mentioned monoclonal antibody is obtained through full-sequence synthesis.

Those skilled in the art know that the nucleotide sequence encoding the amino acid sequence of the above-mentioned bispecific antibody can be appropriately introduced with substitutions, deletions, changes, insertions or additions to provide a polynucleotide homologue. Homologues of the polynucleotides of the present invention can be prepared by replacing, deleting or adding one or more hydroxyl groups encoding the bispecific antibody gene within the range of maintaining the activity of the antibody.

Another aspect of the invention provides an expression vector containing the above-mentioned polynucleotide molecule.

These vectors can be used to transform appropriate host cells to enable expression of the protein. The expression vector is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate Sequence expression vector. The expression vector can be a virus or a plasmid, such as an appropriate phage or phagemid. For more technical details, please refer to, for example, Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, 1989, edited by Sambrook et al. Many known techniques and protocols for nucleic acid manipulation can be found, for example, in Current Protocols in Molecular Biology, 2nd edition, edited by Ausubel et al. The expression vector of the present invention is preferably pDR1, pcDNA3.1(+), pcDNA3.4, pcDNA3.1/ZEO(+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pcDNA3. 4.

The present invention also provides a cell containing the above-mentioned expression vector.

The cells of the present invention are obtained by transforming the expression vector into host cells. The host cell can be any conventional host cell in the art, as long as it can make the above-mentioned recombinant expression vector stably replicate itself, and the carried nucleotide can be effectively expressed. The host cells include prokaryotic expression cells and eukaryotic expression cells. The host cells preferably include: COS, CHO (Chinese Hamster Ovary, Chinese Hamster Ovary), NSO, sf9, sf21, DH5α, BL21 (DE3) or TG1 , more preferably E.coli TG1, BL21 (DE3) cells (expressing single chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies). The preferred recombinant expression transformant of the present invention can be obtained by transforming the aforementioned expression vector into a host cell. The transformation method is a conventional transformation method in this field, preferably a chemical transformation method, a heat shock method or an electroporation method.

Preparation method

The cell culture method and the antibody separation and purification method of the present invention are conventional methods in this field. For specific operation methods, please refer to the corresponding cell culture technical manual and antibody separation and purification technical manual. The method for preparing anti-TROP-2/CD3 bispecific antibodies disclosed in the present invention includes: cultivating the above-mentioned cells under expression conditions to express bispecific antibodies that can specifically bind to TROP-2 and CD3; and isolating and The anti-TROP-2/CD3 bispecific antibody was purified. Using the above methods, the recombinant protein can be purified into a substantially homogeneous material.

The anti-TROP-2/CD3 bispecific antibodies disclosed in the present invention can be separated and purified by affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high-salt buffer, changing pH, etc. can be used to wash the antibody. Deconjugation of anti-TROP-2/CD3 bispecific antibodies from affinity columns. The inventor of the present invention conducted detection experiments on the obtained anti-TROP-2/CD3 bispecific antibodies. The experimental results showed that the anti-TROP-2/CD3 bispecific antibodies can bind to target cells and antigens well and have high affinity.

pharmaceutical composition

Another aspect of the invention provides a composition, preferably said composition is a pharmaceutical composition.

In the present invention, the term "pharmaceutical composition" means that the bispecific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to exert a more stable therapeutic effect. These preparations can ensure the bispecific antibodies disclosed in the present invention. It protects the conformational integrity of the amino acid core sequence of the antibody while also protecting the polyfunctional groups of the protein from degradation (including but not limited to aggregation, deamination or oxidation).

Generally, these materials may be formulated in a nontoxic, inert, and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5.0-8.0, preferably at a pH of about 6.0-8.0, although the pH may be formulated as desired It varies depending on the nature of the substance and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection). ), intracranial injection or intracavity injection. Generally, for liquid preparations, they can usually remain stable at 2-8°C for at least one year; for lyophilized preparations, they can usually remain stable at 30°C for at least six months. The bispecific antibody preparation can be suspension, water injection, freeze-drying and other preparations commonly used in the pharmaceutical field.

The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned bispecific antibody (or conjugate thereof) of the present invention and Pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The drug formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example, about 10 micrograms/kg body weight to 50 mg/kg body weight per day. In addition, the bispecific antibodies of the invention can also be used with other therapeutic agents.

When using the pharmaceutical composition, a safe and effective amount of the bispecific antibody or immunoconjugate thereof is administered to the mammal. The safe and effective dose is usually at least about 10 micrograms/kg of body weight, and in most cases does not exceed about 50 mg/kg of body weight. Preferably, the dose is about 10 micrograms/kg of body weight to 10 mg/kg of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.

Application

Another aspect of the present invention provides the use of the above-mentioned bispecific antibody that can specifically bind to TROP-2 and CD3, or the above-mentioned pharmaceutical composition in the preparation of a medicine for the treatment of cancer or tumors.

The drugs used to treat cancer or tumors referred to in the present invention refer to drugs that inhibit and/or treat tumors, which may include delaying the development of tumor-related symptoms and/or reducing the severity of these symptoms, and further include existing Cancer is accompanied by the reduction of symptoms and prevention of the occurrence of other symptoms, including reducing or preventing tumor metastasis.

Tumors targeted by the medicine of the present invention preferably include, but are not limited to: lung cancer, bone cancer, gastric cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, fallopian tube cancer, intrauterine cancer, Membrane cancer, cervical cancer, vaginal cancer, vulvar cancer, rectal cancer, colon cancer, anal cancer, breast cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, urethra cancer, Penile cancer, prostate cancer, pancreatic cancer, brain cancer, testicular cancer, lymphoma, transitional cell cancer, bladder cancer, kidney or ureter cancer, renal cell cancer, renal pelvis cancer, Hodgkin lymphoma, non- Non-Hodgkin lymphoma, soft tissue sarcoma, pediatric solid tumors, lymphocytic lymphoma, central nervous system tumors, primary central nervous system lymphoma, spinal tumors, brainstem glioma, pituitary gland adenoma, melanoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, chronic or acute leukemia, and combinations of this cancer.

When the bispecific antibody and its composition of the present invention are administered to animals, including humans, the dosage varies depending on the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. Animal experiments can be referred to Depending on the results and various circumstances, the total dose cannot exceed a certain range. Specifically, the intravenous dosage is 1-1800mg/day.

The bispecific antibodies and their compositions of the present invention can also be administered in combination with other anti-tumor drugs to achieve more effective treatment of tumors. These anti-tumor drugs include but are not limited to: 1. Cytotoxic drugs: 1) Action Drugs based on the chemical structure of nucleic acids: alkylating agents such as nitrogen mustards, nitrosoureas, and methanesulfonates; platinum compounds such as cisplatin (Cisplatin), carboplatin (Carboplatin), and oxaliplatin (Oxaliplatin), etc.; Antibiotics such as Adriamycin/Doxorubicin, Dactinomycin D, Daunorubicin, Epirubicin, Mithramycin, etc.; 2) Affect nucleic acid metabolism Drugs: dihydrofolate reductase inhibitors such as methotrexate (Methotrexate) and pemetrexed (Pemetrexed), etc.; thymidine synthase inhibitors such as fluorouracils (5-fluorouracil, capecitabine), etc.; Purine nucleoside synthase inhibitors such as 6-mercaptopurine, etc.; nucleotide reductase inhibitors such as hydroxyurea (Hydroxycarbamide), etc.; DNA polymerase inhibitors such as cytarabine (Cytosinearabinoside) and gemcitabine (Gemcitabine), etc.; 3) Drugs that act on tubulin: Docetaxel, Vincristine, Vinorelbine, podophylline, homoharringtonine, etc.; 2. Hormone drugs: antiestrogens Hormones such as Tamoxifen, Droloxifene, Exemestane, etc.; aromatase inhibitors such as Aminoglutethimide, Formestane, Letrox Letrozle, Anastrozole, etc.; anti-androgens: flutamide RH-LH agonists/antagonists: Noride, enastrone, etc.; 3. Biological response modifier drugs: these drugs Mainly by regulating the body's immune function to achieve anti-tumor effects, such as interferons (Interferon), interleukin-2 (Interleukin-2), thymosins (Thymosins), etc.; 4. Monoclonal antibody drugs: Tratuximab Trastuzumab, Rituximab, Cetuximab, Bevacizumab, etc. The bispecific antibodies and their compositions disclosed in the present invention can be used in combination with one of the above-mentioned anti-tumor drugs or a combination thereof.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying detailed conditions in the following examples usually follow conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. Suggested conditions. Unless otherwise stated, percentages and parts are by weight. When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected.

The experimental materials and sources used in the following examples as well as the preparation methods of experimental reagents are described in detail below.

Experimental materials:

Competent cells: Brand Shengong, item number B528412.

293F cells: Brand GIBCO, Cat. No. R79007.

Colo205 cells: Cell Bank of the Chinese Academy of Sciences, catalog number TCHu102.

MDA-MB-231 cells: Cell Bank of Chinese Academy of Sciences, catalog number TCHu227.

A549 cells: purchased from ATCC.

Human lung cancer cell NCI-H292: Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-582.

hPBMC: purchased from Aucells, product number FPB004F-C-MLR.

Experimental reagents:

Endotoxin-free plasmid maximal extraction kit: brand Tiangen, product number DP117.

DNA purification and recovery kit: brand Tiangen, product number DP214.

ClonExpressTM II One Step Cloning Kit: Brand Novozymes, item number C112.

PrimeSTAR® HS DNA Polymerase: Brand takara, Cat. No. R010B.

Coating solution: 1.59 grams of sodium carbonate, 2.93 grams of sodium bicarbonate, add double distilled water to adjust the volume to 1L.

PBST: PBS+0.05% Tween 20.

Tween 20: Brand Aladdin, item number T104863.

ELISA blocking solution: PBST+1%BSA.

BSA: Brand Shenggong, item number A60332.

TROP-2-HIS protein: brand Kaika, product number TRP-HM121.

CD3-FC protein: brand Kaika, product number CD3-HM205.

HRP-labeled goat anti-human FC antibody: Brand Boaolong, Cat. No. BF03031.

HRP-labeled goat anti-human Fab antibody: Brand Thermo Fisher, Cat. No. A0293.

TMB: Brand BD Biosciences, Cat. No. 555214.

Stop solution: 2M sulfuric acid solution.

0.25% Trypsin-EDTA: Brand GIBCO, Cat. No. 25200-072.

1640 complete medium: RPMI1640 medium + 10% FBS + 1% Pen Strep + 1% sodium pyruvate.

RPMI1640 culture medium: brand GIBCO, item number 22400089.

FBS: Brand GIBCO, item number 10099-141.

Pen Strep: Brand GIBCO, item number 1514022.

Sodium pyruvate: Brand GIBCO, Cat. No. 11360-070.

DMEM complete medium: DMEM high glucose medium + 10% FBS + 1% Pen Strep + 1% GLUMAX.

DMEM high sugar medium: brand GIBCO, product number 11965-092.

GlutaMAX: Brand GIBCO, Cat. No. 35050-061.

CellTiter-Glo® Luminescent Cell Viability Assay: Available from Promega, Cat. No. G7572.

Purified Mouse Anti-Human IL-2: Available from BD Pharmingen, Cat. No. 55051.

Biotin Mouse Anti-Human IL-2: Available from BD Pharmingen, Cat. No. 555040.

Recombinant Human IL-2: Purchased from BD Pharmingen, Cat. No. 554603.

Streptavidin HRP: purchased from BD Biosciences, Cat. No. 554066.

Experimental instruments:

Mastercycler nexus PCR instrument: purchased from Eppendorf.

Hitrap Mabselect Sure column: purchased from Cytiva.

HiLoad 26/600 Superdex 200 pg column: purchased from Cytiva.

SpectraMax i3x microplate reader: purchased from Molecular Devices.

SpectraMaxM5 microplate reader: purchased from Molecular Devices.

The sequence of this application is shown in the table below:

Table B. SEQ ID NO. Name sequence 1 Amino acid sequence of the complementarity determining region HCDR1 in the variable region VH of the anti-TROP-2 monoclonal antibody heavy chain ikB 2 Amino acid sequence of the complementarity determining region HCDR2 in the variable region VH of the anti-TROP-2 monoclonal antibody heavy chain NIFPGSAYINYNEKFKG 3 Amino acid sequence of the complementarity determining region HCDR3 in the variable region VH of the anti-TROP-2 monoclonal antibody heavy chain EGSNSGY 4 Amino acid sequence of the complementarity determining region LCDR1 in the variable region VL of the light chain of anti-TROP-2 monoclonal antibody KSSQSLLNSGNQQNYLA 5 Amino acid sequence of the complementarity determining region LCDR2 in the variable region VL of the anti-TROP-2 monoclonal antibody light chain GASTRES 6 Amino acid sequence of the complementarity determining region LCDR3 in the variable region VL of the anti-TROP-2 monoclonal antibody light chain QSDHIYPYT 7 Amino acid sequence of the complementarity determining region HCDR1 in the variable region VH of the anti-CD3 monoclonal antibody heavy chain TYAMN 8 Amino acid sequence of the complementarity determining region HCDR2 in the variable region VH of the anti-CD3 monoclonal antibody heavy chain RIRSKYNNYATYYADSVKD 9 Amino acid sequence of the complementarity determining region HCDR3 in the variable region VH of the anti-CD3 monoclonal antibody heavy chain HGNFGNSYVSWFAY 10 Amino acid sequence of the complementarity determining region LCDR1 in the variable region VL of the light chain of anti-CD3 monoclonal antibody RSSTGAVTTSNYAN 11 Amino acid sequence of the complementarity determining region LCDR2 in the variable region VL of the anti-CD3 monoclonal antibody light chain GTNKRAP 12 Amino acid sequence of the complementarity determining region LCDR3 in the variable region VL of the anti-CD3 monoclonal antibody light chain ALWYSNLWV 13 Amino acid sequence of the complementarity determining region HCDR1-I31D in the variable region VH of the anti-TROP-2 monoclonal antibody heavy chain DYWLG 14 Amino acid sequence of the complementarity determining region HCDR1-I31E in the variable region VH of the anti-TROP-2 monoclonal antibody heavy chain EYW 15 The amino acid sequence of the complementarity determining region HCDR1-T31G-Y32S in the variable region VH of the anti-CD3 monoclonal antibody heavy chain GSAMN 16 Amino acid sequence of the complementarity determining region HCDR1-T31G-Y32S (VH-FR-N30S) in the variable region VH of the anti-CD3 monoclonal antibody heavy chain GSAMN 17 The amino acid sequence of the complementarity determining region HCDR1-T31G-Y32H in the variable region VH of the anti-CD3 monoclonal antibody heavy chain GHAMN 18 The amino acid sequence of the complementarity determining region HCDR1-T31G-Y32G in the variable region VH of the anti-CD3 monoclonal antibody heavy chain GGAMN 19 Amino acid sequence of the complementarity determining region LCDR2-G50A in the variable region VL of the anti-CD3 monoclonal antibody heavy chain ATNKRAP 20 Anti-TROP-2 monoclonal antibody heavy chain variable region VH amino acid sequence QVQLVQSGPEVKKPGASVKVSCKASGYTFTIYWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSTSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSS twenty one Anti-TROP-2 monoclonal antibody light chain variable region VL amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQQNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQSDHIYPYTFGQGTKLEIKR twenty two Anti-CD3 monoclonal antibody heavy chain variable region VH amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS twenty three Anti-CD3 monoclonal antibody light chain variable region VL amino acid sequence QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIG G TNKRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGQGTKVEIKR twenty four Anti-TROP-2 monoclonal antibody heavy chain variable region VH-I31D amino acid sequence QVQLVQSGPEVKKPGASVKVSCKASGYTFT D YWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSTSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSS 25 Anti-TROP-2 monoclonal antibody heavy chain variable region VH-I31E amino acid sequence QVQLVQSGPEVKKPGASVKVSCKASGYTFT E YWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSTSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSS 26 Anti-CD3 monoclonal antibody heavy chain variable region VH-T31G-Y32S amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFN GS AMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 27 Amino acid sequence of peptide linker L1 GGGGSGGGGSGGGGSGGGGS 28 Amino acid sequence of peptide linker L2 GGGGSGGGGSGGGGS 29 Amino acid sequence of peptide linker L3 GGGG 30 Anti-TROP-2 monoclonal antibody heavy chain amino acid sequence QVQLVQSGPEVKKPGASVKVSCKASGYTFTIYWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 31 Anti-TROP-2 monoclonal antibody light chain amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQQNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQSDHIYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC 32 Anti-TROP-2 monoclonal antibody heavy chain HC-I31D amino acid sequence QVQLVQSGPEVKKPGASVKVSCKASGYTFTDYWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 33 Anti-TROP-2 monoclonal antibody heavy chain HC-I31E amino acid sequence QVQLVQSGPEVKKPGASVKVSCKASGYTFTEYWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 34 Amino acid sequence of variable fragment scFv of anti-CD3 monoclonal antibody EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAAL TLSGAQPEDEAEYYCALWYSNLWVFGQGTKVEIKR 35 Amino acid sequence of variable fragment scFv-VH-T31G-Y32S of anti-CD3 monoclonal antibody EVQLVESGGGLVQPGGSLRLSCAASGFTFNGSAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTL SGAQPEDEAEYYCALWYSNLWVFGQGTKVEIKR 36 Heavy chain amino acid sequence of anti-TROP-2/CD3 double antibody a QVQLVQSGPEVKKPGASVKVSCKASGYTFTDYWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIG GTNKRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGQGTKVEIKRGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 37 Heavy chain amino acid sequence of anti-TROP-2/CD3 double antibody b QVQLVQSGPEVKKPGASVKVSCKASGYTFTDYWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNGSAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWQQKPGQAPRGLIGGT NKRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGQGTKVEIKRGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 38 Heavy chain amino acid sequence of anti-TROP-2/CD3 double anti-c EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAAL TLSGAQPEDEAEYYCALWYSNLWVFGQGTKVEIKRGGGGSGGGGSGGGGSQVQLVQSGPEVKKPGASVKVSCKASGYTFTDYWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSTSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 39 Heavy chain amino acid sequence of anti-TROP-2/CD3 double antibody b ₁ QVQLVQSGPEVKKPGASVKVSCKASGYTFTDYWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSGSAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGT NKRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGQGTKVEIKRGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 40 Heavy chain amino acid sequence of anti-TROP-2/CD3 double antibody b ₂ QVQLVQSGPEVKKPGASVKVSCKASGYTFTDYWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNGHAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGT NKRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGQGTKVEIKRGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 41 Heavy chain amino acid sequence of anti-TROP-2/CD3 double antibody b ₃ QVQLVQSGPEVKKPGASVKVSCKASGYTFTDYWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNGGAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWQQKPGQAPRGLIGGT NKRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGQGTKVEIKRGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 42 Heavy chain amino acid sequence of anti-TROP-2/CD3 double antibody b ₄ QVQLVQSGPEVKKPGASVKVSCKASGYTFTDYWLGWVRQAPGQGLEWMGNIFPGSAYINYNEKFKGKVTITADTSSTAYMELSSLRSEDTAVYFCAREGSNSGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV DKKVEPKSCGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWQQKPGQAPRGLIG ATNKRAPGVPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGQGTKVEIKRGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 43 Heavy chain nucleic acid sequence of anti-TROP-2/CD3 double antibody a See sequence listing 44 Heavy chain nucleic acid sequence of anti-TROP-2/CD3 double antibody b See sequence listing 45 Heavy chain nucleic acid sequence of anti-TROP-2/CD3 double anti-c See sequence listing 46 Heavy chain nucleic acid sequence of anti-TROP-2/CD3 double antibody b ₁ See sequence listing 47 Heavy chain nucleic acid sequence of anti-TROP-2/CD3 double antibody b ₂ See sequence listing 48 Heavy chain nucleic acid sequence of anti-TROP-2/CD3 double antibody b ₃ See sequence listing 49 Heavy chain nucleic acid sequence of anti-TROP-2/CD3 double antibody b ₄ See sequence listing 50 Anti-TROP-2 monoclonal antibody, double antibody a, b, c, b ₁ , b ₂ , b ₃ and b ₄ light chain nucleic acid sequences See sequence listing 51 Human IgG1 heavy chain constant region amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 52 Amino acid sequence of human kappa chain constant region TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Example 1. Construction of anti-TROP-2/CD3 double antibody molecules

The present invention uses anti-TROP-2 monoclonal antibody IgG and anti-CD3 monoclonal antibody scFv in series to construct anti-TROP-2/CD3 bispecific antibodies a, b and c. The structures of a and b are shown in Figure 1A. The c structure is shown in Figure 1B.

Among them, the anti-TROP-2 monoclonal antibody sequence in the anti-TROP-2/CD3 bispecific antibody is derived from the KM4097HV3aLV0 humanized monoclonal antibody disclosed in the US20120237518A1 patent. The KM4097HV3aLV0 humanized monoclonal antibody serves as the anti-TROP-2 monoclonal antibody. Positive control. The CDR sequence of the anti-CD3 monoclonal antibody in the anti-TROP-2/CD3 bispecific antibody is derived from SEQ ID NO. 2 and SEQ ID NO. 4 in US8236308B2, and has been humanized. The present invention uses modified humanized SP34 as a positive control for anti-CD3 monoclonal antibodies.

The specific construction method of humanized SP34 is as follows:

(1) Obtaining the CDR region sequence of mouse anti-human CD3 monoclonal antibody mouse SP34 (mSP34)

The amino acid sequences of the heavy chain and light chain variable regions of the murine anti-human CD3 monoclonal antibody are respectively derived from SEQ ID NO. 2 and SEQ ID NO. 4 in US8236308B2.

mSP34 heavy chain variable region amino acid sequence:

EVKLLESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSA

mSP34 light chain variable region amino acid sequence:

QAVVTQESALTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL

The amino acid sequences of the heavy chain and light chain variable regions of mSP34 were analyzed, and the complementarity determining region CDR and framework region FR of mSP34 heavy chain and light chain were determined according to Kabat's rules. The amino acid sequences of the mSP34 heavy chain CDR are HCDR1: TYAMN (SEQ ID NO. 7), HCDR2: RIRSKYNNYATYYADSVKD (SEQ ID NO. 8) and HCDR3: HGNFGNSYVSWFAY (SEQ ID NO. 9), and the amino acid sequences of the light chain CDR The sequences are LCDR1: RSSTGAVTTSNYAN (SEQ ID NO. 10), LCDR2: GTNKRAP (SEQ ID NO. 11), and LCDR3: ALWYSNLWV (SEQ ID NO. 12).

(2) Humanized construction process of SP34 monoclonal antibody

At https://www.ncbi.nlm.nih.gov/igblast/, the homology between the heavy chain variable region of mSP34 and the human IgG germline sequence was compared, and IGHV3-23*04 was selected as the heavy chain CDR transplantation template. The heavy chain CDR of mSP34 was transplanted into the IGHV3-23*04 framework region, and WGQGTLVTVSS was added as the fourth framework region after HCDR3 to obtain the CDR-grafted heavy chain variable region sequence. Similarly, the homology between the light chain variable region of mSP34 and the human IgG germline sequence was compared, IGLV7-46*01 was selected as the light chain CDR transplantation model, and the light chain CDR of SP34 was transplanted into the backbone of IGLV7-46*01. region, and add FGQGTKVEIK as the fourth framework region after LCDR3 to obtain the CDR grafted light chain variable region sequence. On the basis of the CDR grafted variable region, some amino acid sites are mutated. When performing mutations, the amino acid sequence is Kabat coded, and the position of the site is indicated by the Kabat code.

Preferably, for the CDR transplanted heavy chain variable region, the S at position 49 is mutated to A, the N at position 73 is mutated to D, the A at position 93 is mutated to V, and the K at position 94 is mutated to R. For the CDR grafted light chain variable region, mutate the F at position 36 to V, the T at position 46 to G, the Y at position 49 to G, the W at position 57 to G, and the W at position 58. T mutates into V. The above-mentioned heavy chain and light chain variable regions with back mutation sites are respectively defined as humanized SP34 heavy chain and light chain variable regions (SEQ ID NO. 18 and SEQ ID NO. 19).

(3) Construction process of full-length humanized SP34 monoclonal antibody:

The synthesized humanized heavy chain variable region is connected to the coding gene of the human IgG1 heavy chain constant region (SEQ ID NO. 39) to obtain a full-length humanized heavy chain gene; the humanized light chain variable region is combined with The coding genes of the human Kappa chain constant region (SEQ ID NO. 40) were connected to obtain the full-length humanized light chain gene.

The positive control monoclonal antibody was finally obtained by referring to the expression and purification method in Example 2.

In the process of measuring the protein expression of the purified product, it was found that the expression level of the positive control anti-TROP-2 monoclonal antibody was low, about 10-20 mg/l, while the expression level of the anti-TROP-2 monoclonal antibody increased to Above 30mg/l, the expression of anti-TROP-2 monoclonal antibody protein increases to above 70mg/l after I31D mutation. Therefore, I31E or I31D mutation is used in anti-TROP-2/CD3 double antibodies to increase the expression of double antibodies. The specific mutations of TROP-2/CD3 double antibodies are as follows:

Anti-TROP-2 heavy chain variable region VH (SEQ ID NO. 20) was mutated at the amino acid I31D site to obtain anti-TROP-2 heavy chain variable region VH-I31D (SEQ ID NO. 24), thereby obtaining anti-TROP-2 heavy chain variable region VH-I31D (SEQ ID NO. 24). The TROP-2 heavy chain HC-I31D (SEQ ID NO. 32) and the light chain of the anti-TROP-2 monoclonal antibody (SEQ ID NO. 31) remained unchanged, and the anti-TROP-2-VH-I31D monoclonal antibody was obtained.

Anti-TROP-2 heavy chain variable region VH (SEQ ID NO. 20) was mutated at the amino acid I31E site to obtain anti-TROP-2 heavy chain variable region VH-I31E (SEQ ID NO. 25), thereby obtaining anti-TROP-2 heavy chain variable region VH-I31E (SEQ ID NO. 25). The TROP-2 heavy chain HC-I31E (SEQ ID NO. 33) and the light chain of the anti-TROP-2 monoclonal antibody (SEQ ID NO. 31) remained unchanged, and the anti-TROP-2-VH-I31E monoclonal antibody was obtained.

The anti-CD3 heavy chain variable region VH-T31G-Y32S (SEQ ID NO. 26) was obtained by mutating the amino acid T31G and Y32S sites of the anti-CD3 heavy chain variable region VH (SEQ ID NO. 22).

The scFv in anti-TROP-2/CD3 bispecific antibodies a and c is VH-L1-VL, which is connected to the anti-CD3 heavy chain variable region VH (SEQ ID NO. 22) through L1 (SEQ ID NO. 27). Anti-CD3 light chain variable region VL (SEQ ID NO. 23) was obtained, namely CD3-scFv (SEQ ID NO. 34).

In another embodiment, the scFv in the anti-TROP-2/CD3 bispecific antibody b is VH-T31G-Y32S-L1-VL, which is connected to the anti-CD3 heavy chain variable region VH through L1 (SEQ ID NO. 27) -T31G-Y32S (SEQ ID NO. 26) and anti-CD3 light chain variable region VL (SEQ ID NO. 23), namely CD3-scFv-VH-T31G-Y32S (SEQ ID NO. 35).

The CD3-scFv of anti-TROP-2/CD3 bispecific antibodies a and b is linked to the anti-TROP-2 heavy chain HC-131D (SEQ ID NO. . 32) Between CH1 and CH2, the heavy chain of anti-TROP-2/CD3 double antibody a (SEQ ID NO. 36) and the heavy chain of anti-TROP-2/CD3 double antibody b (SEQ ID NO. 37) are obtained, The light chain of the anti-TROP-2 mAb (SEQ ID NO. 31) remained unchanged.

The CD3-scFv of the anti-TROP-2/CD3 bispecific antibody c is connected to the N terminus of the anti-TROP-2 heavy chain HC-I31D (SEQ ID NO. 32) through L2 (SEQ ID NO. 28) to obtain anti-TROP- The heavy chain of the 2/CD3 double anti-c (SEQ ID NO. 38) and the light chain of the anti-TROP-2 monoclonal antibody (SEQ ID NO. 31) remain unchanged.

Further mutations were made to the amino acid sequence of the CD3 variable region:

Anti-CD3 heavy chain variable region VH (SEQ ID NO. 22) was mutated at the amino acid N30S, T31G and Y32S sites to obtain the anti-CD3 heavy chain variable region VH-N30S-T31G-Y32S;

The anti-CD3 heavy chain variable region VH (SEQ ID NO. 22) was mutated at the amino acid T31G and Y32H sites to obtain the anti-CD3 heavy chain variable region VH-T31G-Y32H;

The anti-CD3 heavy chain variable region VH (SEQ ID NO. 22) was mutated at the amino acid T31G and Y32G sites to obtain the anti-CD3 heavy chain variable region VH-T31G-Y32G;

Anti-CD3 light chain variable region VL-G50A was obtained by mutating the amino acid G50A site of the anti-CD3 light chain variable region VL (SEQ ID NO. 23).

The anti-CD3 light chain variable region VL (SEQ ID NO. 23) was combined with the anti-CD3 heavy chain variable region VH-N30S-T31G-Y32S, VH-T31G-Y32H and VH- through L1 (SEQ ID NO. 27), respectively. T31G-Y32G is connected to obtain VH-N30S-T31G-Y32S-L1-VL (CD3-scFv-VH-N30S-T31G-Y32S), VH-T31G-Y32H-L1-VL (CD3-scFv-VH-T31G-Y32H ) and VH-T31G-Y32G-L1-VL (CD3-scFv-VH-T31G-Y32G); combining anti-CD3 heavy chain variable region VH (SEQ ID NO. 22) with anti-CD3 heavy chain variable region VH (SEQ ID NO. 22) via L1 (SEQ ID NO. 27) CD3 light chain variable region VL-G50A was connected to obtain VH-L1-VL-G50A (CD3-scFv-VL-G50A).

CD3-scFv-VH-N30S-T31G-Y32S, CD3-scFv-VH-T31G-Y32H, CD3-scFv-VH-T31G-Y32G via L2 (SEQ ID NO. 28) and L3 (SEQ ID NO. 29) and CD3-scFv-VL-G50A were sequentially connected to the anti-TROP-2 heavy chain HC-I31D (SEQ ID NO. 32) between CH1 and CH2 to obtain anti-TROP-2/CD3 double antibodies b1, b2, b3 and b4. The heavy chain and amino acid sequences are shown in SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41 and SEQ ID NO. 42 respectively. The light chain of the anti-TROP-2 monoclonal antibody (SEQ ID NO. 31 ) remains unchanged.

Example 2. Expression and purification of anti-TROP-2/CD3 dual antibodies

The DNA fragments of the heavy chain and light chain of the anti-TROP-2/CD3 bispecific antibody were subcloned into vector pcDNA3.4 respectively, and the recombinant plasmid was extracted and co-transfected into 293F cells and/or CHO cells. After the cells are cultured for 5-7 days, the culture medium is centrifuged at high speed, filtered with a 0.22 μm filter membrane, and loaded into a Hitrap Mabselect Sure affinity chromatography column. The protein is eluted in one step with 100 mM citric acid, pH 3.5 eluent, and recovered. The target sample was dialyzed and replaced into PBS with pH 7.4, and the purified protein was detected by UPLC-SEC.

The detection results of anti-TROP-2/CD3 double antibody a, b, c, b1-b4 molecules are shown in Figure 2A, Figure 2B and Figure 2C, and Figure 2D-Figure 2G. The antibody molecule state is uniform and the monomer purity reaches above 95.

Example 3. Enzyme-linked immunosorbent assay (ELISA) to determine the affinity of dual antibodies to antigens

3.1 Detection of the affinity of anti-TROP-2/CD3 bispecific antibodies for TROP-2

Dilute the recombinant Human-TROP-2-HIS protein in the coating solution to 200ng/ml, add 100μl/well to the enzyme plate, and leave it at room temperature for 2 hours or at 4 degrees Celsius overnight. Remove the coating solution (use absorbent paper to remove residual droplets), add blocking solution at 200 μl/well to the enzyme plate, and leave it at room temperature for 1-2 hours. Remove the blocking solution (use absorbent paper to remove residual droplets), then dilute the TROP-2/CD3 bispecific antibody and anti-TROP-2 monoclonal antibody to 10 μg/ml with the blocking solution, and dilute four times to form 12 concentration gradients (the highest concentration 10μg/ml, minimum concentration 0.002ng/ml), add 100μl/well to the blocked enzyme plate in sequence, and leave it at room temperature for 1 hour. Wash the plate three times with PBST (use absorbent paper to remove residual droplets), dilute the HRP-labeled goat anti-human FC antibody 1:3000 with blocking solution, add 100 μl/well to the enzyme plate, and leave it at room temperature for 30 minutes. Wash the plate three times with PBST (use absorbent paper to remove residual droplets), add TMB at 100 μl/well, and keep it at room temperature in the dark for 5 minutes. Add stop solution at 80 μl/well to terminate the substrate color reaction, and use a microplate reader Read the OD value at 450nm, analyze the data with GraphPad, draw a graph and calculate EC50.

The test results are shown in Figure 3A. The EC50 (unit: nM) of the anti-TROP-2/CD3 bispecific antibody molecules a and c and the positive control anti-TROP-2 monoclonal antibody binding to TROP-2-HIS are 0.01649 and 0.01875 respectively. and 0.02606, indicating that the insertion of CD3-scFv, whether attached to the N-terminus of the anti-TROP-2 mAb heavy chain or inserted between CH1 and CH2 of the anti-TROP-2 mAb heavy chain, does not affect bispecificity Antibody binding ability to TROP-2 antigen.

3.2 Detection of the affinity of anti-TROP-2/CD3 bispecific antibodies for CD3

Dilute the recombinant Human-CD3-FC protein in the coating solution to 200ng/ml, add 100μl/well to the enzyme plate, and leave it at room temperature for 2 hours or at 4 degrees Celsius overnight. Remove the coating solution (use absorbent paper to remove residual droplets), add blocking solution at 200 μl/well to the enzyme plate, and leave it at room temperature for 1-2 hours. Remove the blocking solution (use absorbent paper to remove residual droplets), then dilute the anti-TROP-2/CD3 bispecific antibody and anti-CD3 monoclonal antibody to 10 μg/ml with the blocking solution, and dilute four times to form 12 concentration gradients (the highest concentration is 10 μg /ml, the lowest concentration is 0.002ng/ml), add 100μl/well to the blocked enzyme plate in sequence, and leave it at room temperature for 1 hour. Wash the plate three times with PBST (use absorbent paper to remove residual droplets), dilute the HRP-labeled goat anti-human Fab antibody 1:3000 with blocking solution, add 100 μl/well to the enzyme plate, and leave it at room temperature for 30 minutes. Wash the plate three times with PBST (use absorbent paper to remove residual droplets), add TMB at 100 μl/well, and keep it at room temperature in the dark for 5 minutes. Add stop solution at 80 μl/well to terminate the substrate color reaction, and use a microplate reader Read the OD value at 450nm, analyze the data with GraphPad, draw a graph and calculate EC50.

The test results are shown in Figure 3B. The EC50 (unit: nM) of the anti-TROP-2/CD3 bispecific antibody molecules a, b, and c and the positive control anti-CD3 monoclonal antibody binding to CD3-FC are 0.07924, 0.6071, and 0.04141 respectively. and 0.06182. It shows that CD3-scFv is inserted between CH1 and CH2 of the heavy chain of the anti-TROP-2 monoclonal antibody, which slightly reduces the binding ability of the bispecific antibody to CD3 antigen and is effective against the variable region of the CD3 heavy chain on the basis of the bispecific antibody a. After the amino acid of VH is mutated, the binding ability of the bispecific antibody to the CD3 antigen is significantly reduced.

At the same time, compare the differences in binding affinity between anti-TROP-2/CD3 double antibodies a, b, b1, b2, b3 and b4 and CD3. The experimental steps and reaction conditions are the same as above. The test results are shown in Figure 3C. The EC50 (unit: nM) of the anti-TROP-2/CD3 duplex a, b, b1, b2, b3, b4 and the positive control anti-CD3 monoclonal antibody binding to the antigen CD3-FC are 0.0532 respectively. , 0.3208, 0.0939, 0.1431, 5.4670, 0.1273 and 0.0229. This shows that after mutating the variable region of the anti-CD3 heavy chain or light chain, the affinity of the antibody to the CD3 antigen is reduced, among which the affinity of the anti-TROP-2/CD3 dimeric b3 decreases most significantly.

Example 4. Fortibio determines the affinity of anti-TROP-2/CD3 dual antibodies for CD3 antigen

The kinetic parameters of the binding of anti-TROP-2/CD3 bispecific antibody and antigen CD3-Fc were determined using the Fortebio Octet Molecular Interaction Instrument and Octet AR2G Biosensors probe. Use 1×HBS-EP buffer to dilute the TROP-2/CD3 double antibody concentration to 10nM, 2-fold dilution, and set up 7 concentration gradients; use 1×Octet Sodium Acetate buffer to dilute the antigen CD3-Fc concentration to 10ug/ml. After activation of the Octet AR2G Biosensors probe, the antigen-antibody binding and dissociation rates are measured.

The kinetic parameters of the combination of anti-TROP-2/CD3 double antibodies and CD3-Fc are shown in Table 1, and the kinetic characteristic parameter map is shown in Figure 4. The results show that anti-TROP-2/CD3 double antibodies a, b1 and b4 have higher affinity to CD3-Fc, anti-TROP-2/CD3 double antibodies b have weaker affinity to CD3-Fc, and anti-TROP-2/CD3 Double anti-b3 has the weakest affinity with CD3-Fc.

Table 1 Kinetic parameters of anti-TROP-2/CD3 dual antibody binding to CD3-Fc antigen Sample name Ka(1/Ms) Kd(1/s) KD(M) Anti-TROP-2/CD3 double antibody a 9.52E+05 1.96E-04 2.06E-10 Anti-TROP-2/CD3 double antibody b 1.92E+06 2.32E-03 1.21E-09 Anti-TROP-2/CD3 double antibody b ₁ 3.55E+06 1.06E-03 2.98E-10 Anti-TROP-2/CD3 double anti-b ₂ 1.57E+06 6.96E-04 4.42E-10 Anti-TROP-2/CD3 double anti-b ₃ 4.65E+04 2.66E-03 5.73E-08 Anti-TROP-2/CD3 double antibody b ₄ 1.13E+06 3.12E-04 2.76E-10 KD is the affinity constant; ka is the antigen-antibody binding rate; kd is the antigen-antibody dissociation rate; KD=kd/ka.

Example 5. Experiment on killing tumor cells by hPBMC under the action of double antibodies

Take Colo205 cells (human colon cancer cells), MDA-MB-231 cells (human breast cancer cells) and A549 cells (human lung adenocarcinoma cells) growing in the logarithmic phase, and dilute the cells with complete culture medium to 1×105 cells/ml. Add 100 μl per well into a white transparent 96-well plate. No cells are added to the edge wells of the 96-well plate. Only complete culture medium is added for edge sealing. Place it in a 37°C and 5% _CO2 incubator for overnight culture.

Take fresh human peripheral blood mononuclear cells (hPBMC), dilute the cells with complete culture medium to 1.2×106 cells/ml, add 50 μl per well to a white transparent 96-well plate, and set to add only hPBMC. Cells were in control wells without tumor cells.

The concentration and dilution factor of adding antibodies to different tumor cells are different: dilute the anti-TROP-2/CD3 double antibody b with complete culture medium to 1 μg/ml, and dilute it 6 times to form 10 concentration gradients (the highest concentration is 1 μg/ml, the lowest concentration is 9.92× 10-8 μg/ml), add 50 μl per well to a white transparent 96-well plate containing Colo205 cells and hPBMC cells, place it in a 37°C and 5% CO ₂ incubator for 48 hours; dilute the anti-TROP- 2/CD3 double antibody b to 20 μg/ml, 4-fold dilution to form 10 concentration gradients (the highest concentration is 20 μg/ml, the lowest concentration is 7.63×10-5 μg/ml), and 50 μl per well is added to the solution containing MDA-MB-231 Cells and hPBMC cells, white transparent 96-well plates containing A549 cells and hPBMC cells were placed in a 37°C and 5% CO ₂ incubator for 48 hours. Before detection, the bottom of the white transparent 96-well plate needs to be sealed with white cardboard to ensure that the bottom of the plate is light-proof. Aspirate 100 μl of supernatant from each well, and the remaining 100 μl of culture medium containing cells is added to The CellTiter-Glo® Luminescent Cell Viability Assay. CellTiter-Glo®Reagent, 100μl per well, react in the dark for 10 minutes, then use SoftMax 6.3 software for computer reading and detection.

In order to study the synergistic effect of the two targets in anti-TROP-2/CD3 dual antibodies, tumor cells with different expression levels of TROP-2 protein were selected. Both Colo205 cells and MDA-MB-231 cells can express TROP-2 protein, and The expression of TROP-2 in Colo205 cells was higher than that in MDA-MB-231 cells, and A549 cells hardly expressed TROP-2 protein. The test results are shown in Figure 5A. The anti-TROP-2/CD3 double antibody b has the most obvious killing effect on Colo205 cells, and also has a certain effect on MDA-MB-231 cells. It also has a certain effect on A549 cells that do not express TROP-2 protein. There is no killing effect, indicating that anti-TROP-2/CD3 dual antibodies can be positioned on target cells to ensure that normal human tissues will not be killed by the T cell killing effect induced by anti-TROP-2/CD3 dual antibodies.

To verify the difference in the killing of tumor cells by anti-TROP-2/CD3 dual antibodies a and b and anti-CD3 monoclonal antibodies, Colo205 cells with relatively high TROP-2 expression were selected as target cells. The experimental steps and reaction conditions were the same as above. The experiment The results are shown in Figure 5B. The anti-TROP-2/CD3 double antibody b after T31G and Y32S site mutations has a lower killing effect on target cells than the anti-TROP-2/CD3 double antibody a, but both are higher than the anti-CD3 single antibody. anti.

To further verify the killing effect of anti-TROP-2/CD3 dual antibodies on tumor cells after mutating the variable region of the anti-CD3 heavy chain or light chain, Colo205 cells with relatively high TROP-2 expression were selected as target cells. The experimental steps were the same as The reaction conditions are the same as above. The test results are shown in Figure 5C. The killing effect of anti-TROP-2/CD3 double antibodies b1, b2 and b4 on Colo205 cells is more obvious, and the killing effect of anti-TROP-2/CD3 double antibodies b is intermediate. The killing effect of anti-TROP-2/CD3 double anti-b3 is relatively weak.

Example 6. Cytokine release experiment under the action of double antibodies

Take the Colo205 cells growing in the logarithmic phase, dilute the cells to 1×105 cells/ml with complete medium, and add 100 μl per well into a white transparent 96-well plate. No cells are added to the edge wells of the 96-well plate, and only complete medium is added for edge sealing. Process and place in a 37°C 5% _CO2 incubator overnight.

Take fresh hPBMC cells, dilute the cells with complete culture medium to 1.2×106 cells/ml, and add 50 μl per well into a white transparent 96-well plate. At the same time, set up a control well with only hPBMC cells and no tumor cells.

Dilute anti-CD3 monoclonal antibody and anti-TROP-2/CD3 double antibody to 1 μg/ml with complete culture medium, and dilute 6 times to form 10 concentration gradients (the highest concentration is 1 μg/ml, the lowest concentration is 9.92×10-8 μg/ml). Add 50 μl per well to a white transparent 96-well plate containing Colo205 cells and hPBMC cells, and place it in a 37°C and 5% _CO2 incubator for 48 hours.

The supernatant was collected, and the contents of IL-2, INF-γ and TNF-α in the supernatant were detected by ELISA. Dilute Purified Mouse Anti-Human IL-2, Purified Mouse Anti-Human INF-γ and Purified Mouse Anti-Human TNF-α with coating solution to 4μg/ml respectively, and add 50μl/well to a 96-well enzyme plate. Incubate overnight in a 4°C refrigerator. Remove the coating solution, remove residual droplets with absorbent paper, add blocking solution (PBST containing 1% BSA) at 200 μl/well, and leave at room temperature for 2 hours. Remove the blocking solution, use absorbent paper to remove residual droplets, and use the blocking solution to dilute Recombinant Human IL-2 and Recombinant Human TNF-α to 50ng ng/ml respectively, and perform 3-fold gradient dilution to form 12 concentration gradients for making a standard curve. ; Dilute Recombinant Human INF-γ with blocking solution to 100ng/ml, and dilute it 3 times to form 12 concentration gradients, which are used to prepare a standard curve; dilute the supernatant 3 times with blocking solution and add it to 96 wells at 100 μl/well. Place in the enzyme plate at room temperature for 1 hour. Wash the microtiter plate three times with PBST, and dilute Biotin Mouse Anti-Human IL-2, Biotin Mouse Anti-Human INF-γ and Biotin Mouse Anti-Human TNF-α with blocking solution at a ratio of 1:1000 to 100 μl/well. Add to 96-well microplate and place at room temperature for 1 hour. Wash the ELISA plate three times with PBST, dilute Streptavidin HRP with blocking solution at a ratio of 1:1000, add 100 μl/well to the 96-well ELISA plate, and leave it at room temperature for 1 hour. Wash the microplate plate with PBST three times, add TMB at 100 μl/well, and place it at room temperature in the dark for 5 minutes. Add stop solution at 70 μl/well. After terminating the substrate color reaction, immediately use a microplate reader to read at a wavelength of 450 nm. Take the OD value of each well, use SoftMax 6.4 software to draw a standard curve, convert the concentration of IL-2 cytokine in each well, and use GraphPad Prism6 to plot and analyze the data.

Compare the contents of the cytokines IL-2, INF-γ and TNF-α released by immune cells during the process of killing tumor cells by anti-TROP-2/CD3 dual antibodies a and b and anti-CD3 monoclonal antibodies in collaboration with hPBMC. The experimental results are as follows As shown in Figures 6A, 6B and 6C, during the killing of tumor cells by hPBMC, anti-TROP-2/CD3 double antibody b can stimulate immune cells to release fewer cytokines, including IL-2, compared with double antibody a. , INF-γ and TNF-α. At the same time, compared with Figure 5B, it is found that anti-CD3 monoclonal antibodies with almost no killing effect will also stimulate immune cells to release small amounts of cytokines such as INF-γ and TNF-α when the concentration accumulates to a certain amount, indicating that anti-TROP-2/ The killing effect of CD3 double antibodies on tumor cells and the impact on the autoimmune system is better than that of anti-CD3 monoclonal antibodies, and the killing effect of anti-TROP-2/CD3 double antibodies b is better than that of anti-CD3 monoclonal antibodies, but it releases relatively less Cytokines.

After comparing the anti-CD3 heavy chain or light chain variable region mutations, anti-TROP-2/CD3 dual antibodies cooperated with hPBMC to kill tumor cells, stimulating immune cells to release the content of cytokine INF-γ. The experimental results are shown in Figure 6D showed that the anti-TROP-2/CD3 double antibodies b1, b2 and b4 stimulated the highest content of cytokine INF-γ, the anti-TROP-2/CD3 double antibody b stimulated the cytokine INF-γ content was relatively small, and the anti-TROP- The 2/CD3 double antibody b3 hardly stimulated immune cells to release cytokines. At the same time, compared with Figure 5C, it was found that the killing effect of the anti-TROP-2/CD3 double antibody on tumor cells was proportional to the content of stimulated cytokines.

The concentration of anti-TROP-2/CD3 double antibody b that induces the release of IL-2 cytokines is much higher than the concentration that causes tumor cell killing.

Example 7. Anti-tumor effect of anti-TROP-2/CD3 dual antibodies on NCI-H292 xenograft tumor model

7.1 Anti-tumor effect of anti-TROP-2/CD3 double antibody b on NCI-H292 transplanted tumor model

Human peripheral blood mononuclear cells hPBMC were used to reconstruct the human immune system in NOG mice, and a human lung cancer NCI-H292 subcutaneous transplant tumor model was established in these mice. The specific implementation steps are as follows:

Collect NCI-H292 cells (human non-small cell lung cancer) cultured in vitro, adjust the cell suspension concentration to 1×108 cells/ml, and mix with Matrigel in an equal ratio of 1:1. Resuscitate hPBMC in vitro and resuspend hPBMC cells in PBS. Adjust the concentration of hPBMC suspension to 1×107 cells/ml. Mix the mixed tumor cell suspension and hPBMC suspension at a ratio of 1:1. Under sterile conditions, 200 μl of cell mixture suspension was inoculated subcutaneously on the right upper back of NOG mice. When the subcutaneous tumor grows to 150mm3, the mice are randomly divided into 3 groups according to the tumor volume, with 8 mice in each group, including: blank control group, only injected with PBS as a control; anti-TROP-2/CD3 bispecific antibody b The two dose groups of 1 mg/kg and 5 mg/kg were administered by intraperitoneal injection three times a week for four consecutive weeks. During the entire experiment, the diameter of the transplanted tumors was measured twice a week, and the weight of the mice was measured at the same time. The growth curve of the tumors in each group over time and the weight curve of the mice were drawn.

The experimental results are shown in Figures 7A and 7B. On the NCI-H292 cell mixed hPBMC transplanted tumor model, the inhibitory effect of anti-TROP-2/CD3 dual anti-b on tumors was significantly higher than that of the control group, and 1mg/kg and 5mg/kg There is no significant difference between the two dose groups, indicating that a dose of 1 mg/kg can exert a better inhibitory effect; the weight of mice did not decrease significantly after several weeks of continuous administration, indicating that TROP-2/CD3 dual anti-b It has less toxic side effects on mice. The results showed that in this transplanted tumor model, anti-TROP-2/CD3 double anti-b can significantly inhibit tumor growth without obvious toxic side effects on mice.

7.2 Comparison of the anti-tumor effects of different anti-TROP-2/CD3 dual antibodies on the NCI-H292 transplanted tumor model

Human peripheral blood mononuclear cells hPBMC were used to reconstruct the human immune system in NOG mice, and a human lung cancer NCI-H292 subcutaneous transplant tumor model was established in these mice. The specific implementation steps are as follows:

Human non-small cell lung cancer NCI-H292 cells cultured in vitro were collected, the cell suspension concentration was adjusted to 1×108 cells/ml, and mixed with Matrigel in an equal ratio of 1:1. Resuscitate hPBMC in vitro and resuspend hPBMC cells in PBS. Adjust the concentration of hPBMC suspension to 1×107 cells/ml. Mix the mixed tumor cell suspension and hPBMC suspension at a ratio of 1:1. The right upper back of the NOG mouse was shaved, and 200 μl of mixed cell suspension was inoculated subcutaneously into the right upper back of the NOG mouse under sterile conditions. When the average tumor volume of subcutaneous tumors is approximately 200mm3, the mice are randomly divided into 5 groups, with 8 mice in each group, including: blank control group, only injected with PBS as a control; anti-TROP-2/CD3 bispecific antibody a , b, b2 and b3 dose group of 1mg/kg, administered intraperitoneally twice a week for four consecutive weeks. During the entire experiment, the diameter of the transplanted tumors was measured twice a week, and the growth curve of the tumors in each group over time was drawn with the number of days of administration as the abscissa and the tumor volume as the ordinate.

The experimental results are shown in Figure 7C. In the NCI-H292 cell mixed hPBMC transplant tumor model, different anti-TROP-2/CD3 dual antibodies demonstrated strong anti-tumor activity in vivo at a dose of 1 mg/kg. The inhibitory effects were significantly higher than those of the control group; when comparing the anti-tumor activities of anti-TROP-2/CD3 dual antibodies, at the same dose of 1 mg/kg, anti-TROP-2/CD3 dual antibody a had the strongest anti-tumor activity. The anti-tumor activity of anti-TROP-2/CD3 dual antibodies b2 is second, and the anti-tumor activity of anti-TROP-2/CD3 dual antibodies b and b3 is slightly weaker than b2, but in general, the anti-tumor activity of each TROP-2/CD3 dual antibody is There was no significant difference in activity, and the anti-tumor activities were all above 75% on day 17.

Collect mouse blood samples 16 hours after the first dose, separate and obtain serum, and detect the cytokine content in mouse serum under the action of anti-TROP-2/CD3 double antibodies a, b and b3, using Bio-plex Pro Mouse Cytokine Grp 1 Panel 23-Plex kit was used to process serum samples, and the Bio-PlexTM 200 system platform was used to detect the samples to obtain the contents of 23 cytokines in mouse serum. The experimental results are shown in Figure 7D. Except for IL-1b and IL-12(P40), the levels of cytokines stimulated by anti-TROP-2/CD3 double antibodies a are higher than those of anti-TROP-2/CD3 double antibodies b and b3; anti-TROP-2/CD3 double antibodies Compared with b and b3, the anti-TROP-2/CD3 double antibody b stimulates IL-1a, IL-6, G-CSF and MIP-1b cytokines to a higher level, but has no significant difference in other cytokines, indicating that After the anti-TROP-2/CD3 double anti-a mutation reduces its affinity, it can significantly reduce the production of cytokines in animals while maintaining its anti-tumor activity.

Example 8. Stability study of anti-TROP-2/CD3 double antibody a

This experiment can be used to evaluate protein stability in the presence of excipients, revealing important information needed to develop optimal formulations.

Anti-TROP-2/CD3 double antibody a was stored in a buffer of 20mM acetate + 6% trehalose + 1% arginine hydrochloride + 0.1% Tween 80 with a pH of 5.0, and was cultured at 25°C and 37°C respectively. After 28 days in the box, the HPLC-SEC results are shown in Figure 8A and Figure 8B.

The results show that the structure of the anti-TROP-2/CD3 double antibody a is relatively stable, and its purity can still remain above 95% after being placed at a high temperature of 37°C for up to 28 days.

Discuss

It can be seen from the above experiments that the bispecific antibody provided by the present invention can bind to TROP-2 and CD3 at the same time, regulate the immune activity of T cells by binding to CD3, and specifically kill tumor cells expressing TROP-2 protein. The results in Figures 3B and 3C show that the ability of the bispecific antibody to bind CD3 is reduced compared with its monoclonal antibody, but the results in Figures 5A and 5B show that the bispecific antibody's killing effect on tumor cells is significantly increased compared with its monoclonal antibody, and the tumor cells The higher the expression level of TROP-2, the stronger the killing effect of the bispecific antibody on tumor cells. Comparing the results of PBMC killing of tumor cells and their cytokine release, it was found that the stronger the bispecific antibody's killing effect on cells, the more TH1 and TH2 cytokines were released, which was positively correlated. Comparing the killing of target cells by different mutations of the bispecific antibody It was found that the killing effect of bispecific antibody b is relatively strong, and at the same time it stimulates less release of cytokines and has a larger medication window. In the NCI-H292 transplanted tumor model, the stronger the bispecific antibody's inhibitory activity against tumor cells, the more TH1 and TH2 cytokines were released, which was also positively correlated. From the perspective of in vivo cellular immunity experiments, it was once again proven that anti-TROP- 2/CD3 double anti-b anti-tumor killing effect and safety. This shows that the bispecific antibody has a reduced affinity for CD3, which is more helpful for T cells to exert immune effects. It also has the function of targeting the TROP-2 protein and can specifically bind and kill tumor cells expressing TROP-2.

All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the patent scope of the appended claims of this application.

The above examples are for illustrating the disclosed embodiments of the present invention and are not to be construed as limitations of the present invention. In addition, various modifications and variations in methods of the invention set forth herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

Figure 1A shows a schematic structural diagram of anti-TROP-2/CD3 double antibodies a and b.

Figure 1B shows a schematic structural diagram of anti-TROP-2/CD3 bis-antibody c.

Figure 2A shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody a.

Figure 2B shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody b.

Figure 2C shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody c.

Figure 2D shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody b1.

Figure 2E shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody b2.

Figure 2F shows the UPLC detection pattern of anti-TROP-2/CD3 double anti-b3.

Figure 2G shows the UPLC detection pattern of anti-TROP-2/CD3 double antibody b4.

Figure 3A shows the ELISA detection of the binding of anti-TROP-2/CD3 double antibodies a and c to TROP-2.

Figure 3B shows the ELISA detection of the binding of anti-TROP-2/CD3 double antibodies a, b and c to CD3.

Figure 3C shows the ELISA detection of the binding of different mutations of anti-TROP-2/CD3 dual antibodies to CD3.

Figure 4 shows the kinetic characteristic parameter map of anti-TROP-2/CD3 double antibody.

Figure 5A shows the killing effect of hPBMC on different target cells under the action of anti-TROP-2/CD3 double antibody b.

Figure 5B shows the killing effect of hPBMC on Colo205 cells under the action of anti-TROP-2/CD3 double antibodies.

Figure 5C shows the killing effect of hPBMC on Colo205 cells under the action of different mutations of anti-TROP-2/CD3 double antibodies.

Figure 6A shows the IL-2 content released by hPBMC during the killing of target cells under the action of anti-TROP-2/CD3 double antibodies.

Figure 6B shows the IFN-γ content released during the killing of target cells by hPBMC under the action of anti-TROP-2/CD3 double antibodies.

Figure 6C shows the content of TNF-α released during the killing of target cells by hPBMC under the action of anti-TROP-2/CD3 double antibodies.

Figure 6D shows the IFN-γ content released by hPBMC during the killing of target cells under the action of different mutations of anti-TROP-2/CD3 double antibodies.

Figure 7A shows the anti-tumor effect of anti-TROP-2/CD3 dual antibody b on the NCI-H292 transplanted tumor model.

Figure 7B shows the toxic side effects of anti-TROP-2/CD3 double antibody b on mice in the NCI-H292 transplanted tumor model.

Figure 7C shows the anti-tumor effects of anti-TROP-2/CD3 dual antibodies with different mutations on the NCI-H292 transplanted tumor model.

Figure 7D shows the cytokine content in mouse serum under the action of anti-TROP-2/CD3 double antibodies.

Figure 8A shows the HPLC detection pattern of anti-TROP-2/CD3 double antibody a treated at 25°C for 28 days.

Figure 8B shows the HPLC detection pattern of anti-TROP-2/CD3 double antibody a treated at 37°C for 28 days.

TW202346362A_112118587_SEQL.xml

Claims (15)

A bispecific antibody, wherein the bispecific antibody includes: first antigen-binding domain D1 and second antigen-binding domain D2, The first antigen-binding domain D1 is an anti-TROP-2 antibody or an antigen-binding fragment thereof, The second antigen-binding domain D2 is an anti-CD3 antibody or an antigen-binding fragment thereof, The anti-TROP-2 antibody or anti-CD3 antibody or antigen-binding fragment of each comprises a heavy chain complementarity determining region HCDR1-3 and a light chain complementarity determining region LCDR1-3. The bispecific antibody according to claim 1, wherein the HCDR1-3 and LCDR1-3 amino acid sequences of the anti-TROP-2 antibody or its antigen-binding fragment are as follows: SEQ ID NO. 1-3 and SEQ ID NO, respectively 4-6; and/or, the HCDR1-3 and LCDR1-3 amino acid sequences of the anti-CD3 antibody or its antigen-binding fragment are as shown in SEQ ID NO. 7-9 and SEQ ID NO. 10-12 respectively. Show; Preferably, the complementarity determining region contains at least one amino acid mutation; Wherein, the mutation is selected from any one or more of the following: a) Anti-CD3 antibody or its antigen-binding fragment HCDR1:X1X2AMN, b) HCDR1 of anti-TROP-2 antibody or antigen-binding fragment thereof: X3YWLG, c) LCDR2 of anti-CD3 antibody or antigen-binding fragment thereof: X4TNKRAP, The amino acid mutation is selected from the group in which X1 is not T, X2 is not Y, X3 is not I, and X4 is not G; Preferably, the amino acid mutation is selected from the group consisting of X1 being G, X2 being S, H or G, X3 being D or E, and X4 being A. The bispecific antibody as described in claim 2, wherein the bispecific antibody is selected from any of the following: 1) The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the anti-CD3 antibody or its antigen-binding fragment are shown in SEQ ID NO. 15, SEQ ID NO. 8, and SEQ ID NO. 9 respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 are as follows: The acid sequences are shown in SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12 respectively; And/or, the HCDR1, HCDR2, and HCDR3 amino acid sequences of the anti-TROP-2 antibody or its antigen-binding fragment are respectively SEQ ID NO. 13 or SEQ ID NO. 14, SEQ ID NO. 2, and SEQ ID NO. 3. As shown, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 respectively; 2) The HCDR1 of the anti-CD3 antibody or its antigen-binding fragment is X1X2AMN, where X1 is G, X2 is S, and the amino acid sequence is as shown in SEQ ID NO. 16; the amino acid sequences of HCDR2 and HCDR3 are as shown in SEQ ID NO. . 8. SEQ ID NO. 9 is shown, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12 respectively; And/or, the HCDR1 of the anti-TROP-2 antibody or its antigen-binding fragment is X3YWLG, where X3 is D, and the amino acid sequence is as shown in SEQ ID NO. 13; the amino acid sequences of HCDR2 and HCDR3 are as shown in SEQ ID NO. . 2. SEQ ID NO. 3 is shown, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 respectively; 3) The HCDR1 of the anti-CD3 antibody or its antigen-binding fragment is X1X2AMN, where X1 is G, X2 is H, and the amino acid sequence is as shown in SEQ ID NO. 17; the amino acid sequences of HCDR2 and HCDR3 are as shown in SEQ ID NO. . 8. SEQ ID NO. 9 is shown, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12 respectively; And/or, the HCDR1 of the anti-TROP-2 antibody or its antigen-binding fragment is X3YWLG, where X3 is D, and the amino acid sequence is as shown in SEQ ID NO. 13; the amino acid sequences of HCDR2 and HCDR3 are as shown in SEQ ID NO. . 2. SEQ ID NO. 3 is shown, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 respectively; 4) The HCDR1 of the anti-CD3 antibody or its antigen-binding fragment is X1X2AMN, where X1 is G, X2 is G, and the amino acid sequence is as shown in SEQ ID NO. 18; the amino acid sequences of HCDR2 and HCDR3 are as shown in SEQ ID NO. . 8. SEQ ID NO. 9 is shown, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12 respectively; And/or, the HCDR1 of the anti-TROP-2 antibody or its antigen-binding fragment is X3YWLG, where X3 is D, and the amino acid sequence is as shown in SEQ ID NO. 13; the amino acid sequences of HCDR2 and HCDR3 are as shown in SEQ ID NO. . 2. SEQ ID NO. 3 is shown, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 respectively; 5) The LCDR2 of the anti-CD3 antibody or its antigen-binding fragment is X4TNKRAP, where ID NO. 12 is shown, and the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9 respectively; And/or, the HCDR1 of the anti-TROP-2 antibody or its antigen-binding fragment is X3YWLG, where X3 is D, and the amino acid sequence is as shown in SEQ ID NO. 13; the amino acid sequences of HCDR2 and HCDR3 are as shown in SEQ ID NO. . 2. As shown in SEQ ID NO. 3, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 respectively. The bispecific antibody of claim 1, wherein the anti-CD3 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is such as SEQ ID As shown in NO. 22, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO. 23; and/or, the anti-TROP-2 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region. Region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 20, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 21; Preferably, the variable region contains at least one amino acid mutation; more preferably, the amino acid mutation is selected from any one or more of the following: 1) The SEQ ID NO. 20 has an amino acid mutation at position 31; 2) The SEQ ID NO. 22 has an amino acid mutation at position 30; 3) The SEQ ID NO. 22 has an amino acid mutation at position 31; 4) The SEQ ID NO. 22 has an amino acid mutation at position 32; 5) The SEQ ID NO. 23 has an amino acid mutation at position 50; Preferably, the variable region of the bispecific antibody contains the mutations shown in 1), 3) and 4); Preferably, the variable region of the bispecific antibody contains the mutations shown in 1), 2), 3) and 4); Preferably, the variable region of the bispecific antibody contains the mutations shown in 1) and 5); Wherein, the variable region contains amino acid mutations selected from any one or more of the following: a) The SEQ ID NO. 20 has the amino acid mutation I31D; b) The SEQ ID NO. 20 has the amino acid mutation I31E; c) The SEQ ID NO. 22 has an amino acid mutation of N30S; d) The SEQ ID NO. 22 has the amino acid mutation of T31G; e) The SEQ ID NO. 22 has the amino acid mutation of Y32S, Y32H or Y32G; f) The SEQ ID NO. 23 has the amino acid mutation of G50A; Preferably, the variable region of the bispecific antibody includes the T31G in d), the Y32S in e) and the I31D mutation in a), or the variable region of the bispecific antibody includes the T31G, e in d) ) the Y32S and b) the I31E mutation; Preferably, the variable region of the bispecific antibody includes the N30S in c), the T31G in d), the Y32S in e) and the I31D mutation in a), or the variable region of the bispecific antibody includes c The N30S in ), the T31G in d), the Y32S in e) and the I31E mutation in b); Preferably, the variable region of the bispecific antibody includes the T31G in d), the Y32H in e) and the I31D mutation in a), or the variable region of the bispecific antibody includes the T31G, e in d) ) the Y32H and b) the I31E mutation; Preferably, the variable region of the bispecific antibody includes the T31G in d), the Y32G in e) and the I31D mutation in a), or the variable region of the bispecific antibody includes the T31G, e in d) ) the Y32G and b) the I31E mutation; Preferably, the variable region of the bispecific antibody comprises the G50A in f) and the I31D mutation in a), or the variable region of the bispecific antibody comprises the G50A in f) and the I31E mutation in b). The bispecific antibody of claim 4, wherein the anti-CD3 antibody or antigen-binding fragment thereof contains a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as follows SEQ ID NO. 26 is shown, and the light chain variable region amino acid sequence is shown in SEQ ID NO. 23; and/or the anti-TROP-2 antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain. Variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 24 or SEQ ID NO. 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 21. The bispecific antibody of claim 1, wherein the anti-TROP-2 or anti-CD3 antigen-binding fragment is selected from Fab, F(ab'), F(ab')2, Fv or scFv; preferably, the The anti-TROP-2 or anti-CD3 antibody is an IgG antibody; wherein, the D1 is an IgG antibody, and the D2 is a scFv; preferably, the D2 is connected to the N-terminal or C-terminal of D1, or between CH1 and CH2 of D1 ;More preferably, the D2 is linked to the heavy chain of D1. The bispecific antibody of claim 1, wherein the bispecific antibody includes a heavy chain and a light chain, and the amino acid sequences of the heavy chain and light chain are selected from any of the following: a) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 36, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; b) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 37, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; c) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 38, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; d) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 39, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; e) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 40, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; f) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 41, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; g) The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 42, and the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO. 31; or, h) The amino acid sequence in a) to g) is formed by substituting, deleting or adding one or more amino acid residues, and has both anti-TROP-2 activity and anti-CD3 activity. to g) derivatized polypeptides. A polynucleotide molecule, wherein the polynucleotide molecule encodes the bispecific antibody according to any one of claims 1 to 7. An expression vector, wherein the expression vector contains the polynucleotide molecule as described in claim 8. A cell, wherein the cell contains the expression vector according to claim 9. A method for preparing a bispecific antibody as described in any one of claims 1 to 7, wherein the preparation method includes the following steps: a) Cultivate the cells as described in claim 10 under expression conditions to express the bispecific antibody; b) Isolate and purify the bispecific antibody described in step a). A pharmaceutical composition, wherein the pharmaceutical composition contains an effective amount of the bispecific antibody as described in any one of claims 1 to 7 and one or more pharmaceutically acceptable carriers or excipients. Use of the bispecific antibody as described in any one of claims 1 to 7 or the pharmaceutical composition as described in claim 12 in the preparation of medicaments for cancer or tumors; preferably, the cancer or tumor is TROP-2 Positive cancer or tumor. The use as described in claim 13, wherein the cancer or tumor is selected from: lung cancer, bone cancer, gastric cancer, pancreatic cancer, prostate cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, fallopian tube cancer, uterine cancer Endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, rectal cancer, colon cancer, anal cancer, breast cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, urethra cancer, Penile cancer, prostate cancer, pancreatic cancer, brain cancer, testicular cancer, lymphoma, transitional cell cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell cancer, renal pelvis cancer, Hodgkin lymphoma, non- Non-Hodgkin lymphoma, soft tissue sarcoma, pediatric solid tumors, lymphocytic lymphoma, central nervous system tumors, primary central nervous system lymphoma, spinal tumors, brainstem glioma, pituitary gland adenoma, melanoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, chronic, acute leukemia, or combinations thereof. An immunoconjugate, wherein the immunoconjugate contains: a) a bispecific antibody as described in any one of claims 1 to 7; and b) Any one or more of the following conjugates: detectable markers, drugs, toxins, cytokines, radionuclides or enzymes.