Targeting Trop2 and HER2 bispecific antibody as well as preparation method and application thereof

Abstract

Provided are a novel bispecific antigen-binding antibody, an antigen-binding fragment thereof, and a Trop2 * HER2 bispecific antibody drug conjugate (Trop2 * HER2 Abs ADC) which comprise a Trop2-binding arm and an HER2-binding arm, are capable of simultaneously specifically binding to HER2 and Trop2, have a high binding affinity to HER2 and Trop2, can be endocytosed by cells expressing HER2 and / or Trop2, and can be used as an antigen-binding fragment of a Trop2 * HER2 bispecific antibody drug conjugate. Endocytosis of the two binding arms L has a synergistic effect; the Trop2 * HER2 Abs ADC can inhibit the growth of tumor cells expressing HER2 and / or Trop2 in vitro and in vivo, and the bispecific antibody can be used for preventing and treating human diseases related to HER2 and / or Trop2, such as cancers.

Description

Technical Field

[0001] This invention relates to an antibody that specifically binds to Trop2, an antigen-binding fragment, a bispecific antibody that binds to Trop2 and HER2, and an antibody-drug conjugate (Trop2 and HER2 bispecific ADC), as well as its preparation method and uses, particularly its use in the preparation of drugs for the diagnosis, prevention and treatment of diseases associated with HER2 and Trop2 overexpression, including tumors such as breast cancer, gastric cancer, pancreatic cancer, ovarian cancer, colorectal cancer, etc. Background Technology

[0002] HER2, a membrane-bound tyrosine kinase of the ErbB family, lacks an endogenous ligand. Through heterodimerization with other members of the ERBB family, it activates signaling pathways such as PI3K, MAPK, and JAK / STAT, promoting cell proliferation and survival. Tumors with high HER2 expression exhibit strong metastatic and invasive abilities; therefore, HER2 mutations are considered a potential driver of tumorigenesis. Current research indicates that abnormal expression of the HER2 gene exists in multiple tumor types, including breast cancer, ovarian cancer, gastric cancer, uterine cancer, cervical cancer, and biliary tract cancer. Overexpression of the HER2 gene is closely related to the severity of cancer progression; therefore, HER2 is a popular target in the field of oncology. The market size for breast cancer treatment drugs is expected to continue to expand, reaching $47.7 billion by 2029, indicating a huge market potential for HER2-targeted drugs. However, drug resistance caused by HER2 antibody therapy is a pressing issue that needs to be addressed in antibody therapy.

[0003] Anti-Trop2 ADC drugs have shown significant efficacy in the treatment of HR+ / HER2- advanced breast cancer.

[0004] Trop2, or human trophoblast cell surface glycoprotein antigen 2, also known as tumor-associated calcium signal transducer 2 (TACSTD2), is a cell surface glycoprotein encoded by the TACSTD2 gene. It plays a role in various cell signal transduction pathways and is associated with cell proliferation and differentiation. Studies have found that Trop2 overexpression promotes tumor cell growth, proliferation, and metastasis by regulating calcium signaling pathways, cyclin expression, and reducing fibronectin adhesion. Furthermore, Trop2 can interact with β-catenin in the Wnt signaling cascade, thereby affecting the transcription of nuclear oncogenes and cell proliferation. Therefore, Trop2 overexpression is closely related to the proliferation, invasion, and metastasis of cancer cells.

[0005] Trop2 is overexpressed in various malignant tumors, such as cervical cancer (89%), urothelial carcinoma (83%), papillary thyroid carcinoma (83%), breast cancer (80%), squamous cell carcinoma of the lung, endometrial carcinoma (72%), prostate cancer, colon cancer, lung adenocarcinoma, ovarian epithelial carcinoma, gastric cancer, and pancreatic cancer, with Trop2 positivity rates exceeding 55%. Therefore, Trop2 has become a potential target for oncology drug development. Currently, there are several Trop2-targeted drugs under development globally, mainly as adjuvant inhibitors (ADCs), with indications primarily for solid tumors such as NSCLC and triple-negative breast cancer. However, due to the complexity of Trop2's downstream signaling and function, the development of conventional targeted drugs faces numerous challenges. The safety profile of some products still requires improvement; for example, a significant proportion of ≥ grade 3 treatment-related adverse events (TRAEs) were observed in the SKB264, KB264, and ESG-401 studies.

[0006] For the treatment of double-positive tumors involving complex cell signaling pathways targeting both HER2 and Trop2, as well as for patients with HER2-resistant tumors, there are still unmet clinical needs regarding the efficacy and safety of existing drugs. Summary of the Invention

[0007] The purpose of this invention is to provide an anti-Trop2 antibody or its antigen-binding fragment with binding specificity and internalization activity against Trop2 in humans and / or monkeys, a bispecific antibody or its antigen-binding fragment that specifically binds to both HER2 and Trop2 target proteins, and a bispecific antibody-drug conjugate involving an anti-Trop2 / HER2 antibody or its antigen-binding fragment and a drug. This conjugate can deliver cytotoxic plasmid drugs to tumor target tissues, exhibiting a higher level of targeted cytotoxic killing effect on Trop2 and / or HER2-positive tumor cells compared to Trop2 monoclonal antibodies and HER2 monoclonal antibodies or monoclonal antibody ADCs. It can effectively inhibit tumor cell growth, while having no significant effect on body weight, and exhibiting lower toxicity and side effects compared to parental antibody ADCs. This invention can be used in the preparation of drugs for treating tumors and other diseases.

[0008] In some embodiments, the anti-Trop2 antibody of the present invention is selected from at least one of murine antibodies, chimeric antibodies, human antibodies or humanized antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, single-chain antibodies, biantibodies, triantibodies, tetraantibodies or single-domain antibodies, or nanobodies or single-domain antibodies.

[0009] On one hand, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to Trop2, comprising a heavy chain variable region A (VHA) and a light chain variable region A (VLA), wherein the VHA comprises VHA CDR1, VHA CDR2, and VHA CDR3, wherein the VHA CDR1, VHA CDR2, and VHA CDR3 each comprise an amino acid sequence that is at least 95% or 100% identical to the amino acid sequence of a selected VHA CDR1, VHA CDR2, and VHA CDR3; and

[0010] The VLA comprises VLA CDR1, VHA CDR2, and VHA CDR3, wherein VLA CDR1, VHA CDR2, and VHA CDR3 each contain an amino acid sequence that is at least 95% or 100% identical to the amino acid sequence of the selected VLA CDR1, VHA CDR2, and VHA CDR3.

[0011] In some embodiments, VHA CDR1, VHA CDR2, and VHA CDR3 each comprise an amino acid sequence having at least 95% sequence similarity to the amino acid sequences shown in SEQ ID NO:1, 2, and 3, and VLA CDR1, VLA CDR2, and VLA CDR3 each comprise an amino acid sequence having at least 95% sequence similarity to the amino acid sequences shown in SEQ ID NO:4, 5, and 6.

[0012] In some embodiments, VHA comprises VHACDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO:1-3, respectively, and VLA comprises VLA CDR1, 2, and 3 having the amino acid sequences shown in SEQ ID NO:4-6, respectively (according to the Kabat numbering scheme).

[0013] In some embodiments, the antibody or antigen-binding fragment specifically binds to human Trop2 or cynomolgus monkey Trop2.

[0014] In some embodiments, the antibody is a chimeric antibody.

[0015] In some embodiments, the antibody is a human or humanized antibody.

[0016] In some embodiments, the VHA comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in any one of SEQ ID NO:7-14.

[0017] In some embodiments, the VLA comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in any one of SEQ ID NO:15-21.

[0018] In some embodiments, the antibody that specifically binds to Trop2 further comprises a heavy chain constant region (CHA') and a light chain constant region (CLA).

[0019] In some implementations, the CHA' is a human IgG isotype.

[0020] In some embodiments, the CHA' is one of the human IgG1, IgG2, IgG3 and IgG4 isotypes.

[0021] In some preferred embodiments, CHA' is the heavy chain constant region of the human IgG4 isotype.

[0022] In some embodiments, the CLA is a type of human κ and λ light chain.

[0023] In some preferred embodiments, the CLA is a human κ-type light chain.

[0024] In some embodiments, the antibody is at least one of monoclonal antibody, humanized antibody, chimeric antibody, and bispecific antibody; the antigen-binding fragment is at least one of Fab, F(ab'), F(ab')2, Fd, single-chain antibody scFv, disulfide-linked Fv(sdFv), or single-domain antibody.

[0025] On one hand, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to Trop2, comprising a full-length heavy chain (HA') and a full-length light chain (LA), wherein the HA' comprises the VHA of the aforementioned antibody or antigen-binding fragment that binds to Trop2 and a heavy chain constant region A' (CHA'), and the LA comprises the VLA of the aforementioned antibody or antigen-binding fragment that binds to Trop2 and a light chain constant region A (CLA); wherein the HA' comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in any one of SEQ ID NO:22-29.

[0026] In some embodiments, the LA comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in any one of SEQ ID NO:30-36.

[0027] On the one hand, the present invention provides an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof that specifically binds to Trop2 as described herein.

[0028] On the one hand, the present invention provides an antigen-binding protein construct that specifically binds to Trop2.

[0029] In some embodiments, the antigen-binding protein construct is a bispecific or multispecific antibody or its antigen-binding fragment.

[0030] On the one hand, the present invention also provides a bispecific molecule that may comprise the anti-Trop2 antibody of the present invention or its antigen-binding portion, which is linked to a second functional portion (e.g., a second antibody) having a binding specificity different from that of the antibody.

[0031] In some embodiments, the bispecific molecule is a bispecific antibody.

[0032] In some embodiments, the bispecific antibody is a bispecific antibody or antigen-binding fragment thereof that targets Trop2 and another tumor cell surface target.

[0033] On one hand, the present invention provides a novel bispecific antibody that specifically binds to Trop2 and HER2, and an antigen-binding fragment comprising a first antigen-binding arm that specifically binds to Trop2 and a second antigen-binding arm that specifically binds to HER2; wherein the first antigen-binding arm and the second antigen-binding arm are both single-chain peptide chains containing a light chain variable region and a heavy chain variable region, wherein the first antigen-binding arm contains an amino acid mutation that forms a knob structure, and the second antigen-binding arm contains an amino acid mutation that forms a hole structure;

[0034] The first antigen-binding arm includes a heavy chain variable region A (VHA) and a light chain variable region A (VLA) that specifically bind to Trop2, and the second antigen-binding arm includes a heavy chain variable region B (VHB) and a light chain variable region B (VLB) that specifically bind to HER2, wherein the VHA and VLA are the VHA and VLA in the antibody or antigen-binding fragment that binds to Trop2 as described above.

[0035] In some embodiments, the first antigen-binding arm is a single-chain peptide chain with an LA-linker-HA structure formed by LA and HA being linked by a linker, and the second antigen-binding arm is a single-chain peptide chain with an LB-linker-HB structure formed by LB and HB being linked by a linker.

[0036] In some embodiments, LA, HA, LB and HB respectively comprise amino acid sequences as shown in SEQ ID NO:32, SEQ ID NO:48, SEQ ID NO:56 and SEQ ID NO:57.

[0037] In some implementations, the linker is as shown in formula (GGGGS). N The peptide chain shown is (n is any integer between 4 and 10).

[0038] On one hand, the present invention provides an antibody-drug conjugate comprising a therapeutic agent covalently bound to the following:

[0039] (a) the anti-Trop2 antibody or its antigen-binding fragment described above herein; or

[0040] (b) The bispecific antibodies or their antigen-binding fragments described above in this document.

[0041] In some embodiments, the antibody-drug conjugate comprises a novel bispecific antibody that specifically binds to Trop2 and HER2 as described above, as well as an antigen-binding fragment.

[0042] In some embodiments, the therapeutic agent is a cytotoxic agent or a cell inhibitor.

[0043] In some embodiments, the cytotoxic agent is MMAE or MMAF.

[0044] The bispecific antibody is linked to the cytotoxic agent via a linker structure.

[0045] In some embodiments, the connector structure is MC-VC-PAB.

[0046] In some embodiments, the antibody-drug conjugate has a particular advantage in that it has a higher level of ability to bind to cancer cells expressing HER2 and Trop2 than parental antibodies against Trop2 and against HER2.

[0047] In some embodiments, a particular advantage of the antibody-drug conjugate is its ability to achieve a sustained and / or complete antitumor response.

[0048] On the one hand, the present invention also provides nucleic acid molecules encoding the aforementioned anti-Trop2 antibody or its antigen-binding portion, bispecific molecules, and immunoconjugates of the present invention, as well as expression vectors that may contain such nucleic acid molecules and host cells that may contain such expression vectors.

[0049] On the one hand, the present invention also provides a method for preparing the anti-Trop2 antibody or its antigen-binding portion, bispecific molecule, or immunoconjugate of the present invention using host cells, which may include the steps of (i) culturing host cells containing the aforementioned vector in a culture medium, and (ii) isolating the target antibody molecule from the host cells or their cell cultures.

[0050] On one hand, the present invention provides a pharmaceutical composition comprising:

[0051] (i) an active ingredient, and (ii) a pharmaceutically acceptable carrier; wherein the active ingredient comprises:

[0052] The anti-Trop2 antibody described above has an antigen-binding fragment or a mutant thereof that is specific to it, or the bispecific antibody that specifically binds to Trop2 and HER2 as described above, or the antibody-drug conjugate described above.

[0053] On one hand, the present invention provides the use of the aforementioned antibody specifically binding to Trop2, its antigen-binding fragment or a mutant thereof, or the aforementioned bispecific antibody specifically binding to Trop2 and HER2, or the aforementioned bispecific antibody-drug conjugate, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating a disease; wherein the use includes:

[0054] (1) Use in the preparation of medicaments for the prevention or treatment of diseases related to Trop2 overexpression or dysfunction or tumors targeting Trop2;

[0055] (2) Use in the preparation of drugs for killing cells overexpressing Trop2 or HER2;

[0056] (4) Use in the preparation of drugs for treating Trop2 / HER2 double-positive tumors;

[0057] (5) Use in the preparation of Trop2 / HER2 bispecifically binding antibody-drug conjugates;

[0058] (6) Use in the preparation of medicaments for the treatment of HER2-resistant tumors.

[0059] On one hand, the present invention provides a method for a subject suffering from cancer, the method comprising administering a therapeutically effective amount of a composition, providing the subject with an anti-Trop2 antibody or an antigen-binding fragment thereof as described herein, an anti-Trop2 / HER2 bispecific antibody as described herein, or a Trop2 / HER2 bispecific antibody-drug conjugate as described herein, or a pharmaceutical composition.

[0060] In some implementations, the subject has a solid tumor.

[0061] In some implementations, the subjects are humans.

[0062] In some preferred embodiments, the cancer is preferably HER2 and Trop2 double positive.

[0063] In some preferred embodiments, the disease associated with Trop2 expression or dysfunction is invasive breast cancer.

[0064] In some implementations, the cancer is HER2 positive and / or Trop2 positive.

[0065] In other implementations, the cancer is characterized by low expression of HER2 and / or low expression of Trop2.

[0066] In some implementations, cancer is characterized by high / medium expression of HER2 and / or high / medium expression of Trop2.

[0067] In some embodiments, the treatment is the administration of an antibody that specifically binds to Trop2 as described above, its antigen-binding fragment or a mutant thereof, or a bispecific antibody that specifically binds to Trop2 and HER2 as described above, or a bispecific antibody-drug conjugate as described above and a combination therapy selected from one or more other treatment methods, wherein the other treatment methods are selected from immunotherapy, chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, vaccination regimens, stem cell transplantation and immunotherapy (CAR-T, CAR-NK) as adjunctive therapies, wherein the combination therapy increases the cytotoxicity of tumor cells.

[0068] On one hand, this disclosure relates to a method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, a bispecific antibody or antigen-binding protein construct as described herein, or an antibody-drug conjugate as described herein.

[0069] On one hand, the present invention provides a method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein, a bispecific antibody or antigen-binding protein construct thereof, or an antibody-drug conjugate as described herein.

[0070] In some implementations, the diseases associated with Trop2 overexpression or dysfunction are solid tumors or non-solid tumors, including but not limited to breast cancer, colorectal cancer, gastric adenocarcinoma, esophageal cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, ovarian epithelial cancer, prostate cancer, pancreatic ductal adenocarcinoma, head and neck cancer, squamous cell carcinoma, renal cell carcinoma, bladder tumor, cervical cancer, endometrial cancer, follicular thyroid cancer, and glioblastoma multiforme.

[0071] In various embodiments, HER2-positive cancers are selected from colorectal cancer (CRC), renal cell carcinoma, non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, liver cancer, head and neck cancer, and glioma. In various embodiments, the subject had previously responded to anticancer therapy but relapsed after treatment was discontinued (hereinafter referred to as "recurrent cancer"). In various embodiments, the subject has drug-resistant or refractory cancer.

[0072] The anti-Trop2 antibody of the present invention has an ELISA binding EC50 of no more than 0.39 nM to no more than 0.14 nM for recombinant HIS-Trop and an extremely high affinity of no more than 1.69E-08, or an FACS binding EC50 of no more than 10.9 nM to no more than 5.25 nM for Trop2-overexpressing cells. It has cross-binding activity with cynomolgus monkey Trop2, but no cross-binding activity with mouse Trop2. It does not bind to human EpCAM protein of the same family and has endocytic activity in Trop2-positive tumor cells.

[0073] The anti-Trop / HER2 bispecific antibody (Trop2×Her2 bsAb) of this invention exhibits an ELISA binding EC50 of approximately 0.88 nM with recombinant human Her2 protein, showing binding activity comparable to Herceptin. For recombinant human Trop, it has an ELISA binding EC50 of no more than 1.5 nM. The binding activity of Trop2×Her2 bsAb is significantly weaker than that of hu44E7-3. For the MDA-MB-468 cell line, which exhibits high Trop2 expression and low Her2 expression, the binding activity of Trop2×Her2 bsAb is weaker than that of hu44E7-3, but stronger than that of Herceptin. For the HCC1569 cell line, which exhibits low Trop2 expression and high Her2 expression, the binding activity of Trop2×Her2 bsAb is significantly stronger than that of hu44E7-3. Although the EC50 value of Trop2×Her2 bsAb is slightly higher than that of Herceptin, its maximum binding amount is also higher. For the HCC1954 cell line with high Trop2 and high Her2 expression, although the EC50 of Trop2×Her2bsAb was slightly higher than that of the two parent monoclonal antibodies, its maximum binding amount was higher than that of hu44E7-3 and Herceptin. The affinity with recombinant human Trop2-His protein was comparable to that of the parent antibody hu44E7-3, and the affinity with recombinant human Her2-His protein was comparable to that of the parent antibody Herceptin. The endocytic activity was higher than that of the parent monoclonal antibodies hu44E7-3 and Herceptin, indicating that the two antibodies have a synergistic effect in terms of endocytic activity.

[0074] The Trop / HER2 bispecific ADC of the present invention exhibits significant killing activity against tumor cell lines. In a human gastric cancer cell subcutaneous xenograft animal model, it inhibits tumor growth and causes less elevation of gamma-glutamyl transferase, alanine aminotransferase, creatine kinase, and aspartate aminotransferase than Herceptin-MMAE and hu44E7-3-MMAE. Therefore, the Trop / HER2 bispecific ADC of the present invention has better safety than the parental monoclonal antibody ADC.

[0075] Beneficial effects:

[0076] The anti-Trop2 antibody of this invention is expected to provide a targeted therapeutic antibody with broad indications, high specificity, and high affinity for the field of tumor targeted therapy. The Trop2 / HER2 bispecific antibody can further expand the types of cancer that can be treated. The Trop2 / HER2 bispecific ADC provides stronger and more precise tumor killing activity against Trop2 and / or HER2-specific tumor cells, and has good safety, which is expected to provide a new option for the unmet needs of the tumor treatment market. Attached Figure Description

[0077] Figure 1 Results of detection of human Trop2-overexpressing cell clone 1E11 (named CHO-hTrop2) (A. Flow cytometry results of FACS identification of human Trop2 expression in mixed cell pools; B. Protein expression level of human Trop2 in 1E11 monoclonal cells identified by FACS).

[0078] Figure 2 ELISA detection curves showing the binding activity of ch44E7 chimeric antibody to recombinant human Trop2 protein.

[0079] Figure 3 The binding curve of ch44E7 chimeric antibody to human Trop2 protein-expressing cell lines was detected by FACS.

[0080] Figure 4 ELISA detection of binding curves of ch44E7 chimeric antibody to recombinant Trop2 protein of other species (A. binding to recombinant cynomolgus monkey Trop2 protein; B. binding to mouse Trop2 protein).

[0081] Figure 5 ELISA detection curves showing the binding activity of ch44E7 chimeric antibody to recombinant human EpCAM protein.

[0082] Figure 6 ELISA binding curves for detecting the binding activity of humanized antibodies with recombinant human Trop2 protein.

[0083] Figure 7 Binding curves of humanized antibodies to human Trop2 protein-expressing cell lines as detected by FACS.

[0084] Figure 8 ELISA binding curves of humanized antibodies to recombinant Trop2 proteins from other species (A. binding to recombinant cynomolgus monkey Trop2 protein; B. binding to recombinant mouse Trop2 protein).

[0085] Figure 9ELISA detection curves showing the binding activity of humanized antibodies to recombinant human EpCAM protein.

[0086] Figure 10 A bar chart of FACS detection of humanized antibody endocytic activity.

[0087] Figure 11 ELISA binding curves of Trop2×Her2 bsAb and parental monoclonal antibody to recombinant expressed antigen protein (A. binding to recombinant human HER2 protein; B. binding to recombinant human Trop2 protein).

[0088] Figure 12 FACS analysis of binding activity curves of Trop2×Her2 bsAb and parent monoclonal antibody with different tumor cells (A is the binding activity curve of MDA-MB-468 cells, B is the binding activity curve of HCC1569 cells, and C is the binding activity curve of HCC1954 cells).

[0089] Figure 13 FACS detection curves for the endocytic activity of Trop2×Her2 bsAb and parental monoclonal antibody (A. Antibody endocytosis in NCI-N87 cells; B. Antibody endocytosis in HCC1954 cells).

[0090] Figure 14 .MC-VC-PAB-MMAE chemical structure.

[0091] Figure 15 Example 20 shows the killing activity curves of the Trop2×Her2 bispecific antibody-drug conjugate against different tumor cells (A is the killing activity curve of NCI-N87 cells (human gastric cancer cells), B is the killing activity curve of MDA-MB-468 cells (human breast cancer cells), and C is the killing activity curve of HCC1569 cells (human breast cancer cell line)).

[0092] Figure 16 Inhibitory activity curves of Trop2×Her2 bispecific antibody-drug conjugate against NCI-N87 subcutaneous xenografts.

[0093] Figure 17 Animal weight was measured after inhibition of NCI-N87 subcutaneous xenografts by Trop2×Her2 bispecific antibody-drug conjugate.

[0094] Figure 18Results of blood toxicity assays in animals after inhibition of NCI-N87 subcutaneous xenografts by Trop2×Her2 bispecific antibody-drug conjugate (A. Neutrophil percentage-time curve; B. Lymphocyte percentage-time curve; C. Erythrocyte density-time curve; D. Platelet density-time curve).

[0095] Figure 19 The results of biochemical marker detection in animals after inhibition of NCI-N87 subcutaneous xenografts by Trop2×Her2 bispecific antibody-drug conjugate (A. Gamma-glutamyl transferase; B. Alanine aminotransferase; C. Myosinase).

[0096] Figure 20 Schematic diagram of the pHr-hκ carrier.

[0097] Figure 21 Schematic diagram of the pHr-hIgG4 vector structure.

[0098] Figure 22 Schematic diagram of the pHr-hIgG1 vector structure. Detailed Implementation

[0099] Other features and advantages of the invention will become apparent from the following detailed description and examples, and should not be construed as limiting. All references, Genbank entries, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0100] Based on the information contained in this application, those skilled in the art can make various changes to the precise description of the invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0101] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0102] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification (including definitions) shall prevail.

[0103] antigen protein

[0104] In this article, "Trop2" refers to trophoblast cell surface antigen 2, its variants, homologs, and analogs. Tropocyte surface antigen 2 (Trop2), also known as tumor-associated calcium signaling transducer 2 (TACSTD2), is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene. It has a high structural sequence similarity to the epithelial adhesion molecule EpCAM. Studies have found that Trop2 protein is highly expressed in breast cancer, colon cancer, bladder cancer, gastric cancer, oral squamous cell carcinoma, and ovarian cancer. This protein can promote tumor cell proliferation, invasion, metastasis, and spread. Furthermore, in cancers such as breast cancer, high expression of Trop2 has also been found to be closely related to more aggressive tumors and poor clinical prognosis.

[0105] As used herein, the term "Trop2" includes human Trop2 (hTrop2), variants, subtypes, and species homologs of hTrop2, as well as analogs having at least one epitope co-occurring with hTrop2. In various embodiments, as used herein...

[0106] The hTrop2 polypeptide may comprise the amino acid sequence (SEQ ID NO:86), and its encoding nucleic acid is as shown in SEQ ID NO:87: MPLLLLLPLL WAGALAYPYD VPDYAGGGGS HTAAQDNCTC PTNKMTVCSP DGPGGRCQCRALGSGMAVDCSTLTSKCLLL KARMSAPKNA RTLVRPSEHA LVDNDGLYDP DCDPEGRFKA RQCNQTSVCWCVNSVGVRRTDKGDLSLRCD ELVRTHHILIDLRHRPTAGA FNHSDLDAEL RRLFRERYRL HPKFVAAVHYEQPTIQIELRQNTSQKAAGD VDIGDAAYYF ERDIKGESLF QGRGGLDLRV RGEPLQVERT LIYYLDEIPPKFSMKRLTAGLIAVIVVVVV ALVAGMAVLV ITNRRKSGKY KKVEIKELGE LRKEPSL (SEQ ID NO:86).

[0107] atgccactgc tgctcttgct gcctctgctt tgggctggag ctctggctta tccttacgacgtgcctgact

[0108] acgccggtgg aggcggtagc cacacggccg cgcaggacaa ctgcacgtgt cccaccaacaagatgaccgt

[0109] gtgcagcccc gacggccccg gcggccgctg ccagtgccgc gcgctgggct cgggcatggcggtcgactgc

[0110] tccacgctga cctccaagtg tctgctgctc aaggcgcgca tgagcgcccc caagaacgcccgcacgctgg

[0111] tgcggccgag tgagcacgcg ctcgtggaca acgatggcct ctacgacccc gactgcgaccccgagggccg

[0112] cttcaaggcg cgccagtgca accagacgtc ggtgtgctgg tgcgtgaact cggtgggcgtgcgcgcacg

[0113] gacaagggcg acctgagcct acgctgcgat gagctggtgc gcacccacca catcctcattgacctgcgcc

[0114] accgccccac cgccggcgcc ttcaaccact cagacctgga cgccgagctg aggcggctcttccgcgagcg

[0115] ctatcggctg caccccaagt tcgtggcggc cgtgcactac gagcagccca ccatccagatcgagctgcgg

[0116] cagaacacgt ctcagaaggc cgccggtgac gtggatatcg gcgatgccgc ctactacttcgagagggaca

[0117] tcaagggcga gtctctattc cagggccgg gcggcctgga cttgcgcgtg cgcggagaacccctgcaggt

[0118] ggagcgcacg ctcatctatt acctggacga gattccccg aagttctcca tgaagcgcctcaccgcggc

[0119] ctcatcgccg tcatcgtggt ggtcgtggtg gccctcgtcg ccggcatggc cgtcctggtgatcaccaacc

[0120] ggagaaagtc ggggaagtac aagaaggtgg agatcaagga actgggggag ttgagaaaggaaccgagctt gtaa(SEQ ID NO:87)。

[0121] In various embodiments, the Trop2 polypeptide comprises an amino acid sequence having observed, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the human Trop2 shown in SEQ ID NO: 86.

[0122] HER2 antigen

[0123] HER2, a member of the human epidermal growth factor receptor family, is aberrantly expressed in certain malignancies, including breast, gastric, and lung cancer, primarily due to HER2 genomic amplification. HER2 overexpression is associated with an increased risk of recurrence and poor prognosis in these cancers. In HER2-positive malignancies, this protein stimulates downstream RAS-RAF-ERK and PI3K-PTEN-AKT signaling pathways and plays a crucial role in cell proliferation.

[0124] As used herein, the term "HER2" includes human HER2 (hHER2), variants, subtypes, and species homologs of hHER2, as well as analogs that share at least one common epitope with hHER2. In some embodiments, the hHER2 polypeptide, as used herein, may comprise the amino acid sequence (SEQ ID NO:88):

[0125]

[0126] In some embodiments, the HER2 polypeptide comprises an amino acid sequence having observed, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the human HER2 sequence number SEQ ID NO:87.

[0127] To overcome the lack and unsatisfactory nature of existing targeted immunotherapies for tumors involving simultaneous overexpression of HER2 and Trop2, as well as HER2-resistant tumors, the inventors, through extensive and in-depth research and screening, discovered a class of anti-Trop2 antibodies and their antigen-binding fragments. These antibodies specifically recognize Trop2, bind to Trop2 with high affinity, and exhibit good tumor internalization activity. Furthermore, using this monoclonal antibody, the present invention constructs a bispecific antibody and its ADC that can simultaneously and specifically target Trop2 and HER2, and develops its application in the preparation of drugs for the prevention and treatment of cancers or tumors with abnormal expression of Trop2 and HER2 and / or HER2-resistant tumors.

[0128] Anti-Trop2 antibody

[0129] As used herein, the term "antibody" refers to any antigen-binding molecule containing at least one (e.g., one, two, three, four, five, or six) complementarity-determining regions (CDRs) as described herein (e.g., any one of three or more complementarity-determining regions). This includes three CDRs from the immunoglobulin light chain or any one of three CDRs from the immunoglobulin heavy chain and capable of specific binding.

[0130] On the one hand, the present invention provides an anti-Trop2 antibody that can specifically bind to the extracellular domain of human Trop2 and has good targeting specificity and tumor killing activity, or its antigen-binding fragment or functionally conserved mutant, which includes Trop2-specific heavy chain variable region A (VHA) and light chain variable region A (VLA).

[0131] In the following text, the variable domains of the heavy and light chains of the anti-Trop2 antibody of this invention may be referred to as "VHA" and "VLA," respectively. These domains are typically the most variable portions of the anti-Trop2 antibody (relative to other antibodies of the same type) and contain antigen-binding sites. The variable domains mediate antigen binding and define the specificity of a particular antibody for its specific antigen. The variability is concentrated in three segments called hypervariable regions (HVRs) (present in both the light and heavy chain variable domains), namely VHA CDR1, VHA CDR2, and VHA CDR3 in the heavy chain variable domain and VLA CDR1, VLA CDR2, and VLA CDR3 in the light chain variable domain. The highly conserved portions of the variable domains are called backbone regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4, respectively), and the structure of the light chain variable domain is as follows:

[0132] The structure of the heavy chain variable region is VLAFR1-VLACDR1-VLAFR2-VLACDR2-VLAFR3-VLACDR3-VLAFR4, and VHAFR1-VHACDR1-VHAFR2-VHACDR2-VHAFR3-VHACDR3-VHAFR4. There are several labeling schemes for the variable region of an antibody, including Chothia, Kabat, IMGT, and Contact. The exemplary anti-Trop2 antibody in this article uses the Kabat labeling scheme.

[0133] In the anti-Trop2 antibody of the present invention, paired VHA and VLA together form the Trop2 antigen binding site.

[0134] In some embodiments, the anti-Trop2 antibody of the present invention may be at least one selected from murine antibodies, chimeric antibodies, human antibodies or humanized antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, single-chain antibodies, biantibodies, triantibodies, tetraantibodies or single-domain antibodies, or nanobodies or single-domain antibodies.

[0135] Preferably, the anti-Trop2 antibody of the present invention is a monoclonal antibody. Furthermore, the anti-Trop2 antibody of the present invention can be, for example, a murine, chimeric, or human monoclonal antibody, preferably a humanized antibody.

[0136] Anti-Trop2 monoclonal antibody

[0137] In the context of this invention, "monoclonal antibody that specifically binds to Trop2 protein" and "monoclonal antibody against Trop2 protein" are used interchangeably and both refer to monoclonal antibodies that specifically bind to Trop2 protein (including human Trop2). "Specific binding" is a well-known term in the art. A molecule exhibits "specific binding" if it reacts more frequently, more rapidly, for a longer duration, and / or has a greater affinity for a particular target antigen or epitope than it reacts with other target antigens or epitopes. "Specific binding," or "preferential binding," does not necessarily require (although may include) exclusive binding.

[0138] On one hand, the present invention provides an anti-Trop2 antibody or its antigen-binding portion thereof, which comprises a heavy chain variable region A (VHA) and a light chain variable region A (VLA), wherein the VHA comprises VHA CDR1, VHA CDR2, and VHA CDR3, and the VLA comprises VLA CDR1, VLA CDR2, and VLA CDR3.

[0139] Technicians familiar with the field of antibodies know how to generate novel antibodies that bind to human Trop2.

[0140] In some preferred embodiments, the anti-Trop2 antibody of the present invention is a murine monoclonal antibody. The murine anti-Trop2 monoclonal antibody can be prepared using methods well-known and commonly used by those skilled in the art, such as any method selected from hybridoma method, phage display method, yeast display method, recombinant DNA method, single-cell screening, or single-cell sequencing method.

[0141] In some embodiments, the method of the present invention for generating monoclonal antibodies that specifically bind to Trop2 can be a hybridoma method, comprising the steps of administering an effective amount of an immunogenic composition containing Trop2 to mice to stimulate a detectable immune response, obtaining antibody-producing cells from the mice (e.g., cells derived from mouse spleens), fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify those producing monoclonal antibodies that specifically bind to Trop2. Once obtained, the hybridomas can be amplified in cell culture, optionally under culture conditions in which hybridoma-derived cells produce monoclonal antibodies that specifically bind to Trop2. The monoclonal antibodies can be purified from the cell culture.

[0142] For example, the present invention firstly uses hybridoma fusion technology to prepare immune spleen cells by immunizing BALB / C mice with Trop2 antigen, fuses them with SP20 myeloma cells, prepares a high-titer, high-affinity, and high-specificity mouse anti-Trop2 parent clone 44E7 by hybridoma fusion method and selects it by ELISA and FACS methods for binding activity, and further isolates monoclonal antibodies with Trop2 binding activity, named 44E7.

[0143] The amino acid sequences (SEQ ID NO) of exemplary murine monoclonal 44E7 CDRs of VHA and VLA with Trop2 binding activity of the present invention are summarized in Table 1 of Example 2.

[0144] The anti-Trop2 antibody or its antigen-binding portion of the present invention is a monoclonal antibody having the structural and chemical characteristics described below and in the following examples.

[0145] In some embodiments, the heavy chain variable region includes:

[0146] VHA CDR1, comprising the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having one or two substituted, deleted, or added amino acid residues relative to SEQ ID NO:1,

[0147] VHA CDR2, comprising the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having one or two substituted, deleted, or added amino acid residues relative to SEQ ID NO:2, and

[0148] VHA CDR3, which contains the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having one or two amino acid residues substituted, deleted or added relative to SEQ ID NO:3.

[0149] In some embodiments, the light chain variable region comprises:

[0150] VLA CDR1, comprising the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having one or two substituted, deleted, or added amino acid residues relative to SEQ ID NO:4,

[0151] VLA CDR2, comprising the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having one or two amino acid residues substituted, deleted, or added relative to SEQ ID NO:5, and

[0152] VLA CDR3, comprising the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having one or two amino acid residues substituted, deleted, or added relative to SEQ ID NO:6.

[0153] The VHA and / or VLA sequences (or CDR sequences) of the anti-Trop2 antibody of the present invention can be mixed and matched with the VHA and / or VLA sequences (or CDR sequences) of other anti-Trop2 antibodies that bind to human Trop2. Preferably, in some embodiments using immunoglobulin-like antibodies, when the VHA and VLA chains (or CDRs in such chains) are mixed and matched, the VHA from a particular VHA / VLA pair can be substituted with a structurally similar heavy chain variable region amino acid sequence. Similarly, the VLA from a particular VHA / VLA pair can be substituted with a structurally similar light chain variable region amino acid sequence.

[0154] In one embodiment, the antibody or its antigen-binding portion of the present invention may comprise:

[0155] (a) VHA, which may contain the amino acid sequences of VHA CDR1, VHA CDR2 and VHA CDR3 listed in Table 1 above; and / or

[0156] (b) VLA, which may contain the amino acid sequences of VLA CDR1, VLA CDR2 and VLA CDR3 listed in Table 1 above, or the light chain variable region of another anti-Trop2 antibody, wherein the antibody specifically binds to human Trop2 with the amino acid sequence shown in SEQ ID NO:86.

[0157] In some embodiments, the amino acid sequence of the CDR of the heavy / light chain variable region of the Trop2 antibody of the present invention is selected from one or more CDRs as shown in Table 1(a)-(f):

[0158] In another embodiment, the antibody or its antigen-binding portion of this disclosure may include VHA and / or VLA sequences comprising CDR1, CDR2, and CDR3 sequences, which are conserved modifications of one or more amino acids to the VHA and / or VLA sequences of the anti-Trop2 antibody of this disclosure. It should be understood in the art that certain conserved sequence modifications may be made that do not eliminate antigen binding.

[0159] Therefore, in one embodiment, the anti-Trop2 antibody may comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and / or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:

[0160] (a) The heavy chain variable region CDR1 sequence may contain the sequences listed in Table 1, and / or their conservative modifications; and / or

[0161] (b) The heavy chain variable region CDR2 sequence may contain the sequences listed in Table 1, and / or their conservative modifications; and / or

[0162] (c) The heavy chain variable region CDR3 sequence may contain the sequences listed in Table 1 and their conserved modifications; and / or

[0163] (d) The light chain variable regions CDR1, and / or CDR2, and / or CDR3 sequences may contain the sequences listed in Table 1 above; and / or their conservative modifications; and

[0164] (e) Antibody specifically binds to human Trop2.

[0165] As used herein, the term "conserved sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody containing an amino acid sequence. Such conserved modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this disclosure using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine), cysteine, tryptophan, nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues within the CDR region of the antibody of this disclosure can be replaced with other amino acid residues from the same side chain family, and the retained function of the altered antibody (i.e., the functions listed above) can be tested using the functional assays described herein.

[0166] In some embodiments, the murine anti-Trop2 antibody of the present invention (e.g., the murine anti-Trop2 monoclonal antibody named 44E7) comprises VHA CDR1 with the amino acid sequence DYNMH (SEQ ID NO:1) and amino acid sequence [missing information].

[0167] VHA CDR2 with amino acid sequence YIYPYNGGSAYNQKFKN (SEQ ID NO:2) and VHA CDR3 with amino acid sequence EDYGYDPGYYFDY (SEQ ID NO:3), VLA CDR1 with amino acid sequence RSSQSLVHSNGNSYLH (SEQ ID NO:4), VLA CDR2 with amino acid sequence KVSNRFS (SEQ ID NO:5) and VLA CDR6 with amino acid sequence SQSTHVWT (SEQ ID NO:6).

[0168] Furthermore, typically no more than one or two residues within the CDR region are altered. The modifications can be introduced into the antibodies disclosed herein using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0169] The amino acid sequence of the antibody complementarity-determining region (CDR) determines the specificity and affinity of the antibody. The monoclonal antibody with the above-mentioned CDR sequence described in this invention has the ability to specifically bind to Trop2 protein, including natural Trop2 protein and recombinant Trop2 protein. It can specifically recognize human Trop2 protein, cynomolgus monkey Trop2 protein, and cells and tissues expressing the corresponding Trop2 protein. It has strong versatility. ELISA and FACS detection can confirm that it has good specificity, high sensitivity, and strong affinity. It is suitable for targeted therapy of diseases with Trop2 overexpression and for immunological diagnosis and detection.

[0170] Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies.

[0171] In some preferred embodiments, the anti-Trop2 antibody of the present invention is a chimeric antibody or a humanized antibody.

[0172] In some preferred embodiments, the anti-Trop2 antibody of the present invention is, for example, a chimeric antibody.

[0173] As used herein, the term "chimeric antibody" refers to an antibody containing a sequence present in at least two different species (e.g., antibodies from two different mammalian species, such as human and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody containing a variable domain sequence (e.g., all or part of a light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and a constant sequence of a human antibody. Further examples of chimeric antibodies are described herein and are known in the art.

[0174] The encoding nucleic acid of chimeric antibodies is prepared using recombinant DNA technology.

[0175] In some exemplary embodiments, the applicant of this invention, based on the murine anti-Trop2 monoclonal antibody 44E7 obtained by the hybridoma method, linked the nucleic acids encoding the heavy chain variable region and the light chain variable region of the 44E7 murine antibody to the N-terminal signal peptide-containing human IgG4 heavy chain constant region-containing nucleic acid and the N-segment of the human light chain kappa constant region to construct expression vectors, respectively generating recombinant expression vectors containing recombinant heavy chain (HA')-containing nucleic acid and recombinant light chain (LA)-containing nucleic acid of the chimeric antibody, and simultaneously transfected host cells to express the chimeric anti-Trop2 antibody of this invention, named ch44E7.

[0176] In some exemplary embodiments, the chimeric anti-Trop2 monoclonal antibody ch44E7 of the present invention comprises a light chain variable region and a heavy chain variable region, respectively, of the amino acid sequences shown in SEQ ID NO:7 and SEQ ID NO:15 as shown in Example 3.

[0177] In some exemplary embodiments, the chimeric anti-Trop2 monoclonal antibody ch44E7 of the present invention comprises the light chain and heavy chain of the amino acid sequences shown in SEQ ID NO:22 and SEQ ID NO:30, respectively, as shown in Example 3.

[0178] In some exemplary embodiments, the nucleic acids encoding the light chain variable region and the heavy chain variable region of the chimeric anti-Trop2 monoclonal antibody ch44E7 of the present invention comprise nucleotide sequences as shown in SEQ ID NO:63 and SEQ ID NO:71, respectively.

[0179] In some preferred embodiments, the anti-Trop2 antibody or its antigen-binding portion of the present invention is humanized.

[0180] As used herein, the term "humanized antibody" refers to a non-human antibody containing a minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and a sequence derived from a human immunoglobulin. In some non-limiting instances, a humanized antibody is a human antibody (receptor antibody) in which hypervariable (e.g., CDR) region residues of the receptor antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., donor antibody) such as a mouse or rat antibody, possessing the desired specificity and affinity.

[0181] In some embodiments, the Fv framework residues of human immunoglobulins can be replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, humanized antibodies may contain residues not found in receptor or donor antibodies, and these modifications can be made to further improve antibody performance.

[0182] In some embodiments, the humanized antibody contains substantially all of at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops (CDRs) correspond to non-human (e.g., mouse) immunoglobulins, and all or substantially all of the frame regions are frame regions of human immunoglobulins. The humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically the constant region of human immunoglobulins. Humanized antibodies can be generated using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0183] In some implementations, methods for preparing humanized antibodies include, but are not limited to, CDR transplantation methods.

[0184] In some implementations, CDR transplantation can be used to modify the variable region of an antibody. Antibodies primarily interact with target antigens through amino acid residues located in the complementarity-determining regions (CDRs) of the six heavy and light chains. Therefore, the amino acid sequences within the CDRs are more diverse among individual antibodies than those outside the CDRs. Since the CDR sequence is responsible for most antibody-antigen interactions, recombinant antibodies mimicking the characteristics of a specific naturally occurring antibody can have different properties by constructing expression vectors containing CDR sequences from a specific naturally occurring antibody and transplanting them into framework sequences from different antibodies. CDR transplantation utilizes genetic engineering techniques to construct variable region modifications, such as mutating amino acid residues in the VHA and / or VLA regions, replacing the amino acid sequence of the complementarity-determining region (CDR) in the variable region of a human antibody (human VH / VL) with a murine monoclonal antibody CDR sequence. This results in humanized antibodies that possess both the specificity of murine monoclonal antibodies and maintain antibody affinity. It is an antibody humanization technology that minimizes the heterology of murine monoclonal antibodies, thereby improving one or more binding properties (e.g., affinity) of the target antibody. During CDR transplantation, FR is screened, and the human FR gene sequence that is most similar to the mouse antibody sequence is selected to make the antibody have higher activity.

[0185] In some embodiments, the preparation of VHA and VLA of the humanized anti-Trop2 antibody of the present invention may also be carried out by using the sequences of VHA / VLA of the humanized anti-Trop2 antibody generated by the aforementioned CDR transplantation and reversion mutation of the present invention as starting materials to generate humanized variable region variants (i.e., humanized VHA' and VLA') with one or two conserved amino acid modifications in one or more frame regions.

[0186] The most common approach is to select several human antibody heavy and light chains to form a framework, transplant the same CDR region into them, and finally select one or two light and heavy chain combinations for downstream applications.

[0187] The framework sequence of the humanized antibody can be obtained from public DNA databases including germline antibody gene sequences or from published references. The antibody protein sequence is compared with a compiled protein sequence database using one of the sequence similarity search methods known as Gapped BLAST, a method well-known to those skilled in the art. The preferred FR sequence in the antibody used in this invention is structurally similar to the FR sequence used in the antibody of this disclosure.

[0188] The engineered anti-Trop2 antibody of the present invention includes modifications, for example, to improve the properties of the antibody, to the framework residues within VH and / or VL.

[0189] Because certain key mouse FR region residues play a crucial role in regulating CDR structure, some amino acid residues in the human FR region can affect the conformation of the mouse CDR region. Therefore, direct CDR transplantation can lead to a decrease in the affinity or specificity of recombinant antibodies for the target antigen. Typically, such framework modifications resulting from CDR transplantation are performed to reduce the immunogenicity of the antibody.

[0190] The FR region of murine and human antibody sequences contains numerous inconsistencies, and direct CDR transplantation can lead to decreased affinity of recombinant antibodies for the target antigen. Certain key murine FR region residues play crucial roles in regulating CDR structure. After CDR transplantation, structural simulation is needed to identify amino acid residues in the FR region that affect CDR conformation, and these residues should be mutated back to murine amino acids to restore their affinity.

[0191] For example, one approach is to "back-mutate" one or more framework residues to the corresponding germline sequence of the CDR. Selecting appropriate back-mutation residues is crucial for humanization design. Back-mutation is a critical step after CDR transplantation. More specifically, antibodies that have undergone somatic mutations may contain framework residues different from the germline sequence from which the antibody originated. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody originated.

[0192] To minimize the immunogenicity of antibodies, antibody humanization can be achieved by transplanting only the specificity-determining residues into the human FR region. The overall technical approach is similar to CDR transplantation and often requires the assistance of reversal mutations.

[0193] Another type of framework modification involves mutating one or more residues within the framework region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also known as "deimmunization".

[0194] The humanized Trop2-specific antibodies of the present invention comprise amino acid modifications, such as those improving antibody properties, to framework residues within the VHA and / or VLA of the chimeric anti-Trop2 antibody of the present invention. Typically, such framework modifications can increase antibody affinity and / or decrease antibody immunogenicity. For example, one method is to “reverse mutate” one or more framework residues into a germline framework region sequence derived from a corresponding CDR, such as a mouse germline FR region. Such residues can be identified by comparing the antibody framework sequence with the germline FR region sequence of the derived antibody.

[0195] In some exemplary embodiments, the inventors employed a CDR transplantation method combined with key amino acid reverse mutation technology to humanize the variable region sequence of the murine chimeric monoclonal antibody ch44E7 against Trop2. This included first using bioinformatics analysis, through sequence alignment, to identify the antibody germline sequence most similar to the light and heavy chains of the murine monoclonal antibody ch4E7 in a known database. The three CDR sequences of VHA and VLA from the murine monoclonal antibody 44E7 were transplanted into the frame region (FR) of the human antibody, respectively. Further BLAST analysis was used to identify the homologous structure with the highest structural similarity to the variable region sequence of the murine monoclonal antibody 44E7. A three-dimensional structural model was established after sequence alignment of the antibody light and heavy chain variable regions. Key amino acids were then identified through the three-dimensional structure and reversed, resulting in the encoding nucleic acids of 7 humanized heavy chain variable region sequences and 6 humanized light chain variable region sequences. These nucleic acids were then constructed into expression vectors containing signal peptides at the N-terminus, yielding recombinant expression vectors encoding the recombinant full-length heavy chain A' (HA') and the recombinant full-length light chain (LA), respectively.

[0196] In some exemplary embodiments, the appropriate combination of light and heavy chains is then co-transfected and expressed using a combination of a full-length heavy chain (HA') expression vector and a corresponding full-length light chain (LA) expression vector to obtain a plurality of exemplary humanized anti-Trop2 antibodies of the present invention. These are humanized anti-Trop2 antibodies that have undergone conserved modification and reduced immunogenicity relative to chimeric antibodies, while having affinity for human Trop2 protein and species cross-binding characteristics comparable to or close to those of the chimeric antibody before humanization.

[0197] In some exemplary embodiments, the heavy chain variable region A (VHA) comprises the amino acid sequence of VHA as listed in any row of Table 3 of Example 9;

[0198] In some exemplary embodiments, the light chain variable region A (VLA) comprises the amino acid sequence of the VLA as listed in any row of Table 4 of Example 9, or the VLA region of another anti-Trop2 antibody that specifically binds to human Trop2.

[0199] In some embodiments, the heavy chain variable region A comprises an amino acid sequence as shown in any one of SEQ ID NO:7-14.

[0200] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:7.

[0201] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:8.

[0202] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:9.

[0203] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:10.

[0204] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:11.

[0205] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:12.

[0206] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:13.

[0207] In some embodiments, the heavy chain variable region A comprises the amino acid sequence shown in SEQ ID NO:14.

[0208] In some embodiments, the light chain variable region A comprises an amino acid sequence as shown in any one of SEQ ID NO:15-21.

[0209] In some embodiments, the light chain variable region A comprises the amino acid sequence shown in SEQ ID NO:15.

[0210] In some embodiments, the light chain variable region A comprises the amino acid sequence shown in SEQ ID NO:16.

[0211] In some embodiments, the light chain variable region A comprises the amino acid sequence shown in SEQ ID NO:17.

[0212] In some embodiments, the light chain variable region A comprises the amino acid sequence shown in SEQ ID NO:18.

[0213] In some embodiments, the light chain variable region A comprises the amino acid sequence shown in SEQ ID NO:19.

[0214] In some embodiments, the light chain variable region A comprises the amino acid sequence shown in SEQ ID NO:20.

[0215] In some embodiments, the light chain variable region A comprises the amino acid sequence shown in SEQ ID NO:21.

[0216] The VH and / or VL sequences (or CDR sequences) of other Trop2 antibodies that bind to recombinant human Trop2 can be “mixed and paired” with the VLA and / or VHA sequences (or CDR sequences) of the anti-human Trop2 antibody of the present invention.

[0217] In some embodiments, in the Trop2 antibody of the present invention, the sequence combination of VHA and VLA and the resulting anti-Trop2 antibody are shown in any row of Table 5 of Example 9.

[0218] In some embodiments, the anti-Trop2 antibody or antigen-binding fragment is a chimeric antibody.

[0219] In some embodiments, the anti-Trop2 antibody or antigen-binding fragment is a human or humanized antibody.

[0220] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VHA shown in SEQ ID NO:7 and the VLA shown in SEQ ID NO:15, exemplarily, such as a chimeric anti-Trop2 antibody named ch44E7.

[0221] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VHA shown in SEQ ID NO:8 and the VLA shown in SEQ ID NO:16, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-1.

[0222] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VHA shown in SEQ ID NO:9 and the VLA shown in SEQ ID NO:16, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-2.

[0223] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:10 and the VLA shown in SEQ ID NO:17, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-3.

[0224] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:10 and the VLA shown in SEQ ID NO:18, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-4.

[0225] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:10 and the VLA shown in SEQ ID NO:19, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-5.

[0226] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:11 and the VLA shown in SEQ ID NO:17, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-6.

[0227] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:12 and the VLA shown in SEQ ID NO:20, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-7.

[0228] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:13 and the VLA shown in SEQ ID NO:21, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-8.

[0229] In some embodiments, the anti-Trop2 antibody of the present invention comprises the VH shown in SEQ ID NO:14 and the VLA shown in SEQ ID NO:19, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-9.

[0230] The preparation of variants of the humanized anti-Trop2 antibody of the present invention involves introducing conserved modifications well known in the art into VHA and VLA. These conserved modification mutations can be amino acid substitutions, additions, or deletions, but substitution is preferred.

[0231] In some implementations, genetic modification can be performed by modifying one or more residues within one or two variable regions (i.e., VHA and / or VLA) (e.g., in one or more CDR regions and / or one or more frame regions) to improve binding affinity and / or increase similarity to naturally occurring antibodies of certain species. For example, the frame region can be modified with conserved amino acids to create the frame region of a humanized antibody. Alternatively, antibodies can be genetically modified by modifying residues in constant regions, for example, to alter the antibody's effector function.

[0232] Conservative amino acid substitution is the substitution of an amino acid residue by an amino acid residue with a similar side chain.

[0233] Those skilled in the art can learn from well-known textbooks that amino acids from families of amino acid residues with similar side chains in natural amino acids are used for conserved modification.

[0234] Through the antibody engineering and amino acid modification methods described above, variants of anti-Trop2 antibodies with novel FR sequences for VHA and VLA were generated.

[0235] In some embodiments, the present invention provides a functionally conserved mutant of the aforementioned anti-human Trop2 antibody, comprising a heavy chain variable region A' (VHA') and a light chain variable region A' (VLA'), wherein VHA' comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:7-14, or an amino acid sequence formed by altering, adding, or deleting one, two, three, four, or five amino acids in the amino acid sequence shown in any one of SEQ ID NO:7-14; wherein the VHA' has, compared to the aforementioned sequence VHA, at least one of the following: a) binding the same Trop2 antigenic epitope; b) an amino acid sequence with sequence identity greater than 85%, 90%, 95%, 98%, or 99%.

[0236] In some embodiments, the VLA' comprises an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in any of SEQ ID NO:15-21, or an amino acid sequence formed by altering, adding, or deleting one, two, three, four, or five amino acids from the amino acid sequence shown in any of SEQ ID NO:15-21, wherein the VLA' has at least one of the following conditions compared to the aforementioned sequence VLA: a) binding the same antigenic epitope; b) an amino acid sequence with sequence identity greater than 85%, 90%, 95%, 98%, or 99%.

[0237] In some embodiments, the anti-Trop2 antibody of the present invention, its antigen-binding fragment, or its mutant further comprises a constant region, wherein the constant region and the variable region constitute a full-length light chain or heavy chain, such that the heavy chain constant region and the heavy chain variable region constitute a full-length heavy chain, and the light chain variable region and the light chain constant region constitute a full-length light chain. The constant region sequence of the antibody is generally available through public searches, such as searching the IMGT online database to obtain the heavy chain constant region and light chain constant region sequences.

[0238] In some embodiments, the heavy chain constant region of the anti-Trop2 antibody of the present invention is the IgG heavy chain constant region, preferably an isotype of the human IgG heavy chain constant region.

[0239] In some embodiments, the human IgG heavy chain constant region is selected from one of the human IgG1, IgG2, IgG3 or IgG4 antibody heavy chain constant regions.

[0240] In some preferred embodiments, the heavy chain constant region of the anti-Trop2 antibody of the present invention is the heavy chain constant region of the human IgG4 antibody.

[0241] In some embodiments, the antibody light chain constant region is selected from either the human κ or λ chain light chain constant regions.

[0242] In some embodiments, the antibody light chain constant region is a human κ-type light chain constant region.

[0243] In one embodiment, the human IgG4 heavy chain constant region of the anti-Trop2 antibody of the present invention has, for example, the human IgG4 heavy chain constant region of the amino acid sequence shown in SEQ ID NO:37.

[0244] The constant region of the heavy chain contains the "Fc region" (crystallizable fragment region), also known as the "Fc domain" or simply "Fc". The Fc region refers to the C-terminal region of the antibody heavy chain, which mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or binding to the first component (C1q) of the classical complement system. In IgG antibody isotypes, the Fc region consists of two identical protein fragments from the CH2 and CH3 domains of both antibody heavy chains.

[0245] In some embodiments, the anti-Trop2 antibody of the present invention has a human κ-type light chain constant region, for example, the amino acid sequence shown in SEQ ID NO:38.

[0246] As an alternative to modifications within the framework or CDR region, the antibodies of the present invention can be genetically engineered to form a genetically modified Fc region to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or Fc receptor binding and / or antibody-dependent cytotoxicity (ADCC).

[0247] In a second aspect, the present invention provides an anti-Trop2 antibody or an antigen-binding fragment thereof or a mutant thereof, comprising a heavy chain A' (HA') and a light chain A (LA), wherein the HA comprises an amino acid sequence as shown in any one of SEQ ID NO:22-29 in Table 6 of Example 9, and the LA comprises an amino acid sequence as shown in any one of SEQ ID NO:30-36 in Table 7 of Example 9.

[0248] In some embodiments, the HA' comprises the amino acid sequence shown in SEQ ID NO:22.

[0249] In some embodiments, the HA' comprises the amino acid sequence shown in SEQ ID NO:23.

[0250] In some embodiments, the HA' comprises the amino acid sequence shown in SEQ ID NO:24.

[0251] In some embodiments, the HA' comprises the amino acid sequence shown in SEQ ID NO:25.

[0252] In some embodiments, the HA' comprises the amino acid sequence shown in SEQ ID NO:26.

[0253] In some embodiments, the HA' comprises the amino acid sequence shown in SEQ ID NO:27.

[0254] In some embodiments, the HA' comprises the amino acid sequence shown in SEQ ID NO:28.

[0255] In some embodiments, the HA' comprises the amino acid sequence shown in SEQ ID NO:29.

[0256] In some embodiments, the LA comprises the amino acid sequence shown in SEQ ID NO:30.

[0257] In some embodiments, the LA comprises the amino acid sequence shown in SEQ ID NO:31.

[0258] In some embodiments, the LA comprises the amino acid sequence shown in SEQ ID NO:32.

[0259] In some embodiments, the LA comprises the amino acid sequence shown in SEQ ID NO:33.

[0260] In some embodiments, the LA comprises the amino acid sequence shown in SEQ ID NO:34.

[0261] In some embodiments, the LA comprises the amino acid sequence shown in SEQ ID NO:35.

[0262] In some embodiments, the LA comprises the amino acid sequence shown in SEQ ID NO:36.

[0263] In some embodiments, the amino acid sequence combination of HA' and LA and the light chain in the Trop2 antibody of the present invention and the naming of the anti-Trop2 antibody are shown in any row of Table 8 of Example 9.

[0264] In some embodiments, the anti-Trop2 antibody of the present invention comprises a combination of heavy and light chains shown in any row of Table 8 of Example 9.

[0265] In some embodiments, the anti-Trop2 antibody of the present invention comprises a heavy chain A' (HA') as shown in SEQ ID NO:22 and a light chain (LA) as shown in SEQ ID NO:30, exemplarily, such as a chimeric anti-Trop2 antibody named ch44E7.

[0266] In some embodiments, the anti-Trop2 antibody of the present invention comprises heavy chain A' (HA'1) as shown in SEQ ID NO:23 and light chain A (LA2) as shown in SEQ ID NO:31, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-1.

[0267] In some embodiments, the anti-Trop2 antibody of the present invention comprises heavy chain A' (HA'4) as shown in SEQ ID NO:24 and light chain A (LA2) as shown in SEQ ID NO:31, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-2.

[0268] In some embodiments, the anti-Trop2 antibody of the present invention comprises heavy chain A' (HA'2) as shown in SEQ ID NO:25 and light chain A (LA1) as shown in SEQ ID NO:32, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-3.

[0269] In some embodiments, the anti-Trop2 antibody of the present invention comprises heavy chain A' (HA'2) as shown in SEQ ID NO:25 and light chain A (LA6) as shown in SEQ ID NO:33, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-4.

[0270] In some embodiments, the anti-Trop2 antibody of the present invention comprises heavy chain A' (HA) as shown in SEQ ID NO:25 and light chain A (LA5) as shown in SEQ ID NO:34, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-5.

[0271] In some embodiments, the anti-Trop2 antibody of the present invention comprises heavy chain A' (HA'5) as shown in SEQ ID NO:26 and light chain A (LA1) as shown in SEQ ID NO:32, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-6.

[0272] In some embodiments, the anti-Trop2 antibody of the present invention comprises heavy chain A' (HA'3) as shown in SEQ ID NO:27 and light chain A (LA3) as shown in SEQ ID NO:35, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-7.

[0273] In some embodiments, the anti-Trop2 antibody of the present invention comprises heavy chain A (HA'7) as shown in SEQ ID NO:28 and light chain A (LA4) as shown in SEQ ID NO:36, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-8.

[0274] In some embodiments, the anti-Trop2 antibody of the present invention comprises heavy chain A' (HA'6) as shown in SEQ ID NO:29 and light chain A (LA5) as shown in SEQ ID NO:34, exemplarily, such as a humanized anti-Trop2 antibody named hu44E7-9.

[0275] In some embodiments, the present invention provides a functionally conserved variant of the aforementioned anti-Trop2 antibody, comprising a functionally conserved variant of heavy chain A' (HA”) and a functionally conserved variant of light chain A (LA’), wherein HA” comprises an amino acid sequence having at least 85% sequence identity as shown in any one of SEQ ID NO:22-29, or an amino acid sequence formed by altering, adding, or deleting one, two, three, four, or five amino acids as shown in any one of SEQ ID NO:22-29; LA’ comprises an amino acid sequence having at least 85% sequence identity as shown in any one of SEQ ID NO:30-36, or an amino acid sequence formed by altering, adding, or deleting one, two, three, four, or five amino acids as shown in any one of SEQ ID NO:30-36. The HA’ and LA’ have, compared to the aforementioned sequences VHA and VLA, at least one of the following: a) binding to the same antigenic epitope; b) an amino acid sequence with sequence identity greater than 85%, 90%, 95%, 98%, or 99%.

[0276] In some embodiments, a person skilled in the art of antibodies can further obtain the human antibody against Trop2 according to the teachings of the present invention.

[0277] As used herein, the term "human antibody" refers to an antibody encoded by an endogenous nucleic acid derived from a human (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, human antibodies are generated from human collections or in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies can be generated in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are generated in bacterial or yeast cells. In some embodiments, human antibodies are generated in transgenic non-human animals (e.g., mice or rats) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0278] Trop2 binding fragment

[0279] In some embodiments, the present invention also provides an antigen-binding fragment that specifically binds to Trop2. The antibody fragment moderately retains the affinity and specificity required for a full-length antibody.

[0280] In some embodiments, the antibody or its antigen-binding fragment may be, for example, at least one of the following: a Fab fragment, a Fab' fragment, a Fab 2 fragment, an F(ab)'2 fragment, an Fv fragment, a scFv fragment and a scFv-Fc fragment, a dAb fragment, a disulfide-linked Fv (sdFv), and any polypeptide containing a binding domain homologous to the antibody binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy chain and / or light chain CDR of a complete antibody, the variable region of the heavy chain and / or light chain of a complete antibody, the full-length heavy chain or light chain of a complete antibody, or a single CDR derived from the heavy chain or light chain of a complete antibody.

[0281] For example, the Fv fragment is an antibody fragment containing a complete antigen recognition and binding site. In this configuration, the three CDRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. The six CDRs of the antibody confer antigen-binding specificity. Antigen-binding fragments can be prepared using a variety of techniques, including but not limited to hydrolyzing the complete antibody protein and expression from host cells transfected with a recombinant expression vector containing the antigen-binding fragment.

[0282] "Derivatives" and "Analogs"

[0283] The Trop2 binding fragments of the present invention, their derivatives, or analogues may be: (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing an anti-Trop2 polypeptide with another compound (e.g., a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a protein formed by fusing with a leader sequence, secretion sequence, or tag sequence such as 6His). Based on the teachings herein, these fragments, derivatives, and analogues are within the scope well known to those skilled in the art.

[0284] As is well known to those skilled in the art, in some embodiments, the affinity of the antibody for the antigen can also be increased, for example, by altering the level of glycosylation.

[0285] In some embodiments well known to those skilled in the art, a preferred class of active derivatives refers to... Tables 3 and 4 variable region Compared to other amino acid sequences, at most three, preferably at most two, and more preferably at most one amino acid are replaced by amino acids with similar or related properties to form a functionally conserved mutant polypeptide.

[0286] In this invention, “fragments,” “derivatives,” and “analytes” are polypeptides that substantially retain Trop2 binding affinity function or activity.

[0287] Multispecific antibodies that specifically bind to Trop2

[0288] On the one hand, the present invention provides a multispecific antibody that specifically binds to Trop2.

[0289] As used in this article, the term "bispecific antibody" refers to an antibody that binds to two different antigens, with epitopes located on the same antigen or different antigens.

[0290] In some embodiments, the antigen-binding protein construct may be in IgG / IgG form. Two complete IgG antibodies are assembled into a bispecific antibody by interaction at the C-terminus of the heavy chain, comprising a Trop2-specific binding moiety and a specific binding moiety of another tumor surface target protein, wherein the Trop2-specific binding moiety comprises a light chain variable region A (VLA) and a heavy chain variable region A (VHA) derived from the aforementioned humanized anti-Trop2 antibody, its antigen-binding fragment, or a mutant thereof.

[0291] In some embodiments, the bispecific antibody (e.g., a bispecific antibody) may comprise an anti-HER2 antibody or an antigen-binding fragment thereof, and an anti-Trop2 antibody or an antigen-binding fragment thereof. These bispecific antibodies, including the anti-Trop2 antibody and its antigen-binding fragment, and the anti-HER2 antibody and its antigen-binding fragment, may take various forms.

[0292] As used herein, the term "antigen-binding arm" refers to one or more protein domains (e.g., formed from amino acids from a single polypeptide or from amino acids from two or more polypeptides, e.g., the same or different polypeptides). In some embodiments, an antigen-binding arm can bind to an antigen or epitope with similar specificity and affinity to naturally occurring antibodies. An antigen-binding arm can be an antibody or a fragment thereof. One example of an antigen-binding arm is an antigen-binding arm formed from a VH-VL dimer.

[0293] In some embodiments, the antigen-binding arm is a fusion peptide single-chain structure with a ScFab-Fc structure. In some embodiments, the antigen-binding arm is a fusion peptide single-chain with a light chain variable region-linker-heavy chain variable region structure. Non-limiting examples of antigen-binding domains are described herein. Other examples of antigen-binding domains are known in the art. In some examples, the antigen-binding domain can bind a single antigen.

[0294] Anti-Trop2 / TAA bispecific antibody

[0295] Thirdly, the present invention provides a bispecific antibody against Trop2 / TAA.

[0296] An important type of bispecific antibody (BsAb) is the anti-TAA / TAA bispecific antibody, which targets two tumor antigens (TAAs). This refers to an artificial antibody that can simultaneously and specifically bind to two different tumor surface antigens or different tumor surface antigen epitopes, functioning as a bridge connecting two antigens (eptopes). The mechanisms of action of anti-TAA bispecific antibodies include: (1) simultaneously blocking two different growth signaling pathways in the carcinogenesis process, thus exerting unique or overlapping functions, interfering with / inhibiting the growth, proliferation, and survival of tumor cells; (2) simultaneously targeting different antigens or epitopes on the cell surface, enhancing their specific binding to tumor cells, and directly utilizing ADCC to kill tumor cells. Specific binding TAA / TAA bispecific antibodies are an important type of bispecific antibody, producing effects superior to dual-targeted combined therapy, and can also effectively reduce treatment costs.

[0297] Whether bispecific antibody products meet clinical efficacy and safety requirements, and whether they can be industrially produced, depends on the individual biological activities of the two antibody-binding units, the choice of affinity combination of the two protein-binding arms, and the bispecific antibody configuration formed by the selected binding arms. Different bispecific antibody structures can affect antibody efficacy. Bispecific antibody drugs also exhibit differentiated molecular characteristics due to differences in platform technology and corresponding structural design, including clinical efficacy, safety, pharmacokinetic properties, physicochemical properties, immunogenicity, and ease of clinical and commercial production.

[0298] In the following text, an exemplary TAA / TAA bispecific antibody option is a bispecific antibody that binds Trop2 and HER2.

[0299] Trop2 / HER2 bispecific antibody

[0300] On the one hand, the present invention provides a bispecific antibody that specifically binds to Trop2 and HER2, its active fragment, its preparation method, and its application.

[0301] Overexpression or amplification of HER2 not only enhances signal transduction through ligand-dependent heterodimerization but also leads to ligand-independent dimerization and aberrant signal transduction. Targeted therapies against HER2 have shown significant therapeutic effects in these "HER2-positive" tumors. Anti-HER2 therapy may develop resistance through various mechanisms, and the presence of HER2 activating mutations may also hinder effective inhibition of HER2. Trop2 is a transmembrane glycoprotein highly expressed in various tumors, such as pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, and ovarian cancer. It promotes tumor cell proliferation, invasion, metastasis, and spread, and its high expression is closely related to shortened survival and poor prognosis in cancer patients. Furthermore, in several cancers, including breast cancer, high Trop2 expression has been found to be associated with more aggressive disease and poor prognosis. HER2 and Trop2 are two tumor-associated antigens (TAAs) that have been co-expressed in various tumor types, including breast cancer, gastric cancer, colorectal cancer, bladder cancer, pancreatic cancer, and non-small cell lung cancer. This invention develops a bispecific antibody targeting HER2 and Trop2 for the treatment of cancer and other unmet medical needs.

[0302] In some embodiments, the present invention also uses the anti-Trop2 humanized antibody obtained in the foregoing aspects and another anti-HER2 antibody as parental antibodies. Through a genetic engineering method involving nucleic acid-level splicing and ligation, constructing two recombinant expression vectors respectively containing a first antigen-binding arm encoding nucleic acid with Trop2 binding specificity and a second antigen-binding arm encoding nucleic acid with HER2 binding specificity, and transiently transfecting them into the same host cell for expression, a bispecific antibody containing a Knob-mutated Trop2 binding arm and a hole-mutated HER2 binding arm is obtained. This antibody can simultaneously bind to the tumor cell surface antigens Trop2 and HER2, exhibiting higher synergistic internalization and inhibiting and killing various tumor cells.

[0303] In some embodiments, the present invention screened and tested exemplary anti-Trop2 / HER2 bispecific antibodies, which adopted the IgG1(1+1) symmetrical form.

[0304] In some embodiments, the amino acid sequences of the fragments contained in the anti-Trop2 binding arm and anti-Her2 binding arm of the exemplary anti-Trop2 / HER2 bispecific antibody of the present invention are shown in Table 13 and Table 14 of Example 15.

[0305] In some embodiments, the present invention provides a bispecific antibody against Trop2 / HER2 with a configuration of ScFab(A)-Fc(A) / ScFab(B)-Fc(B). This is a 1+1 symmetrical IgG bispecific antibody constructed using KIH technology, characterized by monovalent dual targets, ultra-stable structure, and mammalian cell production. These advantages are due to the presence of the ScFab and modified Fc portions in its two binding arms.

[0306] In some embodiments, the inventors of this application have introduced a combination of amino acid mutations constituting the Knobs-into-Holes (KIH) structure into the ScFab(A)-Fc(A) / ScFab(B)-Fc(B) configuration anti-Trop2 / HER2 bispecific antibodies of this invention. The KIH method involves replacing a large amino acid (hole) with a smaller amino acid in one heavy chain of the bsAb, and vice versa in the other heavy chain (knob), ultimately forming a heterodimer. This technology achieves a correct assembly rate of 90%-95% for the bispecific antibody heavy chains, meeting the requirements for large-scale production.

[0307] In some embodiments, the amino acid sequence fragments contained in the Trop2-specific antigen-binding arm of the ScFab(A)-Fc(A) configuration and the HER2-specific antigen-binding arm of the ScFab(B)-Fc(B) configuration of the Trop2 / HER2 bispecific antibody of the present invention are shown in Tables 13 and 14 of Example 15.

[0308] In some embodiments, in the Trop2 / HER2 bispecific antibody, the Trop2 binding arm may be a single-chain peptide chain of the ScFab(A)-Fc(A) structure composed of ScFab(A) fragments and Fc(A) fragments (containing a VHA / VLA pair forming a Trop2-specific binding site), while the HER2 binding arm may be a single-chain peptide chain of the ScFab(B)-Fc(B) structure composed of ScFab(B) fragments and Fc(B) fragments (containing a VHB / VLB pair forming a HER2-specific binding site).

[0309] In some implementations, the Trop2 binding arm portion may comprise a ScFab(A)-Fc(A)(knob) single-chain peptide, while the HER2 binding arm portion comprises a ScFab(B)-Fc(B)(hole) single-chain peptide.

[0310] In some embodiments, the anti-Trop2 / HER2 bispecific antibody of the present invention is a heterodimer assembled by the interaction of a knob single chain with a ScFab(A)-Fc(A) structure and a hole single chain with a ScFab(B)-Fc(B) structure.

[0311] In one exemplary embodiment, the Trop2 / HER2 bispecific antibody of the present invention comprises the Trop2 first antigen-binding arm as shown in SEQ ID NO:46.

[0312] In one exemplary embodiment, the second antigen-binding arm of the anti-Trop2 / HER2 bispecific antibody of the present invention comprises ScFab(B) as shown in SEQ ID NO:54.

[0313] In some implementations, heavy chain mismatch can be prevented by introducing mutations into the Fc fragment at the site involved in Fc receptor binding.

[0314] In some implementations, the Fc(A) segment contains a knob mutation consisting of amino acid site mutations S354C and T366W (both according to EU numbers).

[0315] In some implementations, the Fc(B) segment contains a hole mutation consisting of amino acid site mutations Y349C, T366S, L368A, and Y407V (all according to EU numbers).

[0316] In some embodiments, the Fc(A) segment may contain the amino acid sequence shown in SEQ ID NO:47.

[0317] In some embodiments, the Fc(B) segment may contain the amino acid sequence shown in SEQ ID NO:55.

[0318] In some exemplary embodiments, the first antigen-binding arm of the anti-Trop2 / HER2 bispecific antibody of the present invention specifically binds to Trop2, comprising ScFab(A) as shown in SEQ ID NO:46 and Fc(A) as shown in SEQ ID NO:47, with the exemplary parent antibody being hu44E7-3; the second antigen-binding arm specifically binds to HER2, comprising ScFab(B) as shown in SEQ ID NO:54 and Fc(B) as shown in SEQ ID NO:55, with the exemplary parent antibody being Herceptin; and the assembled exemplary bispecific antibody is Trop2×Her2 bsAb.

[0319] The fragment numbers contained in the Trop2 binding arm and HER2 binding arm of exemplary bispecific antibodies such as Trop2×Her2 bsAb are shown in Tables 13 and 14 of Example 15.1.

[0320] On one hand, the present invention provides a bispecific antibody against Trop2 / HER2, comprising a first antigen-binding arm specific to Trop2 binding and a second antigen-binding arm specific to HER2 binding. The first antigen-binding arm comprises a light chain variable region A (VLA), a light chain constant region A (CLA), a linker, and a heavy chain variable region A (VHA) from the anti-Trop2 antibody or its antigen-binding fragments described above, and further comprises a CH1 segment of the IgG1 heavy chain constant region and a variant of a (CH2A-CH3A) segment containing a hole structure. The second antigen-binding arm comprises a light chain variable region B (VLB) and a light chain constant region B (CLB), a linker, and a heavy chain variable region B (VHB) from the anti-HER2 antibody, and further comprises a CH1 segment of the IgG1 heavy chain constant region and a variant of a (CH2B-CH3B) segment containing a hole structure.

[0321] In some exemplary embodiments, the first antigen-binding arm comprises a VLA having the amino acid sequence shown in SEQ ID NO:17, derived from the anti-Trop2 antibody of the present invention.

[0322] In some exemplary embodiments, the first antigen-binding arm comprises a VHA having the amino acid sequence shown in SEQ ID NO:10, derived from the anti-Trop2 antibody of the present invention.

[0323] In some exemplary embodiments, the second antigen-binding arm comprises a VLB of an anti-HER2 antibody having the amino acid sequence shown in SEQ ID NO: 50.

[0324] In some exemplary embodiments, the second antigen-binding arm comprises a VHB of an anti-HER2 antibody having the amino acid sequence shown in SEQ ID NO: 52.

[0325] In some embodiments, the first antigen-binding arm of the bispecific antibody further includes a constant structural domain, such as a light chain constant region A (CHA) and a modified heavy chain constant region A (CHA), and the second antigen-binding arm further includes a constant structural domain, such as a light chain constant region B (CHB) and a heavy chain constant region B (CHB).

[0326] In some embodiments, the CHA and CHB are selected from one of the human IgG1, IgG2, IgG3 or IgG4 sequences.

[0327] In some embodiments, CHA and CHB are constant regions of the human IgG1 isotype heavy chain.

[0328] In some implementations, the CLA and CLB are human k-type or λ-type light chain constant regions.

[0329] In some implementations, the CLA and CLB are human k-type light chain constant regions.

[0330] In some exemplary embodiments, the first antigen-binding arm comprises CH1A of the IgG1 heavy chain constant region (such as the amino acid sequence shown in SEQ ID NO:62).

[0331] In some exemplary embodiments, the first antigen-binding arm may comprise VLA as shown in SEQ ID NO:17, CLA as shown in SEQ ID NO:38, VHA as shown in SEQ ID NO:10 from the humanized anti-Trop2 antibody of the present invention, and CH1A from the amino acid sequence shown in SEQ ID NO:62 from the constant region of the IgG1 heavy chain.

[0332] In some exemplary embodiments, the second antigen-binding arm comprises the light chain constant region B (CLB) of the amino acid sequence of the anti-HER2 antibody as shown in SEQ ID NO:51.

[0333] In some exemplary embodiments, the second antigen-binding arm comprises CH1B having an amino acid sequence as shown in SEQ ID NO:62.

[0334] In some exemplary embodiments, the second antigen-binding arm may include VLB as shown in SEQ ID NO:50, CLB as shown in SEQ ID NO:51, VHB as shown in SEQ ID NO:52 from the autoanti-HER2 antibody, and CH1B as shown in SEQ ID NO:62 from the constant region of the IgG1 heavy chain.

[0335] In some implementations, the (CH2A-CH3A) contained in CHA may include a knob mutation consisting of amino acid site mutations S354C and T366W.

[0336] In some implementations, CHB may contain (CH2B-CH3B) a hole mutation consisting of amino acid site mutations Y349C, T366S, L368A and Y407V.

[0337] The knob mutation composed of S354C and T366W and the hole mutation composed of Y349C, T366S, L368A and Y407V can form a KIH structure to prevent heavy chain mismatch.

[0338] In some exemplary embodiments, the first antigen-binding arm includes Trop2-specific light chain variable region A (VLA), light chain constant region A (CLA), heavy chain variable region A (VHA), and heavy chain constant region A (CHA), and the second antigen-binding arm includes HER2-specific light chain variable region B (VLB), light chain constant region B (CLB), heavy chain variable region B (VHB), and heavy chain constant region B (CHB).

[0339] In some exemplary embodiments, the CHA comprises a variant of the IgG1 heavy chain constant region with a knockb mutation, having an amino acid sequence as shown in SEQ ID NO:45.

[0340] In some exemplary embodiments, the CHB comprises a variant of the IgG1 heavy chain constant region that introduces a hole mutation, having an amino acid sequence as shown in SEQ ID NO:53.

[0341] In some exemplary embodiments, the first antigen-binding arm comprises a CLA derived from the amino acid sequence shown in SEQ ID NO:38 of the anti-Trop2 parent antibody hu44E7-3 of the present invention.

[0342] In some exemplary embodiments, the second antigen-binding arm comprises a CLB of the parent antibody Herceptin, as shown in SEQ ID NO: 51.

[0343] In some exemplary embodiments, the parent antibody of the first antigen-binding arm is hu44E7-3, which includes anti-Trop2 antibodies such as VLA as shown in SEQ ID NO:17, CLA as shown in SEQ ID NO:38, VHA as shown in SEQ ID NO:10, and CHA as shown in SEQ ID NO:45.

[0344] In some exemplary embodiments, the parent antibody of the second antigen-binding arm is Herceptin, comprising VLB as shown in SEQ ID NO:50, CLB as shown in SEQ ID NO:51, VHB as shown in SEQ ID NO:52, and CHB as shown in SEQ ID NO:53.

[0345] In some exemplary embodiments, the first antigen-binding arm comprises VLA as shown in SEQ ID NO:17, CLA as shown in SEQ ID NO:38, VHA as shown in SEQ ID NO:10, and CHA containing a combination of amino acid mutations of the knob structure as shown in SEQ ID NO:45, all from the anti-Trop2 antibody hu44E7-3, with hu44E7-3 being an exemplary parent antibody; the second antigen-binding arm comprises VLB as shown in SEQ ID NO:50, CLB as shown in SEQ ID NO:51, VHB as shown in SEQ ID NO:52, and CHB containing a combination of amino acid mutations of the hole structure as shown in SEQ ID NO:53, all from Herceptin, with Herceptin being an exemplary parent antibody.

[0346] In some exemplary embodiments, exemplary heterodimeric bispecific antibodies, such as the bispecific antibody named Trop2×HER2 Ab1, are assembled through the interaction of a first antigen-binding arm and a second antigen-binding arm.

[0347] In some embodiments, those skilled in the art may further modify the antigen-binding arm, particularly the Trop2-binding arm, of the anti-Trop2 / HER2 bispecific antibody disclosed herein to distinguish it in its amino acid sequence from that of naturally occurring binding polypeptides derived therefrom. For example, the polypeptide or amino acid sequence derived from the specified protein may be similar, for example, having a certain percentage identity with the starting sequence, such as at least 85%, 90%, 95%, 98%, or 99% identity with the starting sequence.

[0348] In some embodiments, the modification is the deletion, addition, or substitution of individual amino acids.

[0349] In some embodiments, the modification is a conservative substitution of an amino acid.

[0350] In other embodiments, the modification may be the replacement of an amino acid string with a structurally similar string that differs in the order and / or composition of its side chain family members.

[0351] Methods for conserved modification of the antibody's specific binding functional sequence can be found in well-known publications in the field of antibody engineering.

[0352] On one hand, the present invention provides an anti-Trop2 / HER2 bispecific antibody comprising a first antigen-binding arm specific to Trop2 binding and a second antigen-binding arm specific to HER2 binding. The Trop2 binding arm comprises a single-chain peptide A with a (LA-linker-HA) structure formed by a linker of a Trop2-binding specific light chain A and a heavy chain A. The HER2 binding arm comprises a single-chain peptide B with a (LB-linker-HB) structure formed by a linker of a HER2-binding specific light chain B and a heavy chain B. The HA and HB interact through a knot and hole structure to form a KIH structure, and the first and second antigen-binding arms assemble into a heterodimer via the KIH structure.

[0353] In some embodiments, the HA and the HB are selected from the same type of human IgG1, IgG2, IgG3 or IgG4.

[0354] In some implementations, the HA and HB are isotypes of human IgG1.

[0355] In some embodiments, the (CH2A-CH3A) portion of HA may include a knob mutation consisting of amino acid site mutations S354C and T366W to prevent heavy chain mismatch.

[0356] In some embodiments, the (CH2B-CH2B) portion of HB may include a hole mutation consisting of amino acid site mutations Y349C, T366S, L368A, and Y407V to prevent heavy chain mismatch.

[0357] The Trop2-specific first antigen-binding arm and the HER2-specific second antigen-binding arm interact with the knock mutation region of HA and the hole mutation region of HB to form a KIH structure, which is then assembled into a heterodimeric bispecific antibody.

[0358] Those skilled in the art of antibodies will also understand that the antibodies disclosed herein can be modified to distinguish them in their amino acid sequences from naturally occurring binding polypeptides derived therefrom. For example, a polypeptide or amino acid sequence derived from a specified protein may be similar, for example, having a certain percentage identity with the starting sequence, such as at least 85%, 90%, 95%, 98%, or 99% identity with the starting sequence.

[0359] In some embodiments, the modification is the deletion, addition, or substitution of individual amino acids.

[0360] In some embodiments, the modification is the substitution of individual amino acids with functionally conserved amino acids.

[0361] Methods for conserved modification of the antibody's specific binding functional sequence can be found in publications in the field of antibody engineering.

[0362] In some implementations, LA and LB are human k-type light chains.

[0363] In some embodiments, the first antigen-binding arm that specifically binds to Trop2 comprises a light chain (LA) of the amino acid sequence shown in SEQ ID NO:32.

[0364] In some embodiments, the first antigen-binding arm that specifically binds to Trop2 comprises a modified heavy chain A (HA) containing a knob mutation of the amino acid sequence shown in SEQ ID NO:48.

[0365] In some embodiments, the second antigen-binding arm that specifically binds to HER2 comprises a light chain (LB) of the amino acid sequence shown in SEQ ID NO:56.

[0366] In some embodiments, the second antigen-binding arm that specifically binds to HER2 comprises a modified heavy chain B (HB) containing a hole mutation of the amino acid sequence shown in SEQ ID NO:57.

[0367] In some implementations, LA and HA in the specifically bound Trop2 are linked together as a single-chain peptide A via a linker, and LB and HB in the specifically bound HER2 are linked together as a single-chain peptide B via a linker.

[0368] In some implementations, the linker is a linker peptide.

[0369] In some implementations, the length of the peptide linker can be, for example, 2 to 100.

[0370] In some embodiments, the length of each peptide linker may be at least 2 amino acids, or at least 3, 4, 5, 7, 8, 9, 10, 12, 15, 17, 20, 22 or 25 amino acids.

[0371] An exemplary connector includes multiple glycine (G) and serine (S).

[0372] In some embodiments, the connector comprises at least 50%, 60%, 70%, or 80% glycine.

[0373] In some implementations, the connector comprises at least 80% glycine.

[0374] In some implementations, shorter connectors result in more moderate T-cell binding activity.

[0375] In some embodiments, the peptide linker is (GGGGS). n The structure shown.

[0376] In some implementations, n is any positive integer from 4 to 10.

[0377] In some embodiments, the peptide linker is linker1 of the amino acid sequence (GGGGS)4 (SEQ ID NO:58) or linker2 of the amino acid sequence (GGGGS)6 (SEQ ID NO:49).

[0378] In some embodiments, the peptide linker is linker2 of the amino acid sequence shown in (GGGGS)6 (SEQ ID NO:49).

[0379] In some embodiments, the first antigen-binding arm that specifically binds to Trop2 comprises the light chain (LA) of the amino acid sequence shown in SEQ ID NO:32 and the modified heavy chain A (HA) of the amino acid sequence shown in SEQ ID NO:48, with the exemplary parental monoclonal antibody being hu44E7-3; the second antigen-binding arm that specifically binds to HER2 comprises the light chain (LB) of the amino acid sequence shown in SEQ ID NO:56 and the heavy chain (HB) of the amino acid sequence shown in SEQ ID NO:57, with the exemplary parental monoclonal antibody being Herceptin; the exemplary bispecific antibody is named Trop2×Her2 bsAb.

[0380] In some embodiments, the first antigen-binding arm that specifically binds to Trop2 contains the amino acid sequence shown in SEQ ID NO:59, with an exemplary parental monoclonal antibody being hu44E7-3; the second antigen-binding arm that specifically binds to HER2 contains the amino acid sequence shown in SEQ ID NO:60, with an exemplary parental monoclonal antibody being Herceptin; and an exemplary bispecific antibody such as Trop2×Her2bsAb is formed.

[0381] Trop2 / HER2 bispecific antibody-drug conjugate

[0382] On one hand, the present invention provides a bispecific antibody-drug conjugate against Trop2 / HER2 (Trop2×Her2 bs ADC), wherein the Trop2 / HER2 bispecific ADC comprises the aforementioned bispecific antibody portion against Trop2 / HER2.

[0383] In some embodiments of the Trop2 / HER2 bispecific ADC, the Trop2 / HER2 bispecific antibody or antigen-binding fragment is linked to the therapeutic drug via a connector structure.

[0384] In some embodiments, the therapeutic agent includes, for example, a cytotoxic agent (i.e., a cytotoxic drug), a chemotherapeutic agent, or a radioactive isotope.

[0385] In some embodiments, the cytotoxic agent includes any agent that is harmful to the growth, viability, or proliferation of tumor cells.

[0386] In some embodiments, the Trop2 / HER2 bispecific ADC of the present invention can be used for local delivery of cytotoxic agents, such as for the treatment of cancer. The Trop2 / HER2 bispecific ADC of the present invention can target the delivery of cytotoxic agents to tumors and the cells therein to generate accumulation, and is therefore particularly suitable for situations where systemic administration of these unconjugated cytotoxic agents may cause unacceptable levels of toxicity to normal cells and tumor cells to be eliminated.

[0387] In some preferred embodiments, the cytotoxic agent is a chemotherapeutic agent, such as a growth inhibitor, DNA alkylating agent, topoisomerase inhibitor, tubulin conjugate or other anticancer drug, toxin or highly reactive radioisotope.

[0388] In some preferred embodiments, the cytotoxic agent may also be a small molecule toxin and a chemotherapeutic agent.

[0389] In some embodiments, the cytotoxic agents conjugated to the anti-Trop2 / HER2 bispecific antibody of the present invention include, but are not limited to, macromolecular bacterial toxins and plant toxins, such as diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin, abrinogen, modeccin, alpha-sarcin, Clostridium difficile toxin, Pseudomonas exotoxin, botulinum toxin, bryophyll, saponin, and pokeweed toxin. Such macromolecular toxins can be conjugated to the anti-Trop2 antibody provided by the present invention using methods known in the art.

[0390] In some embodiments, the small molecule toxins in the antibody-drug conjugates (ADCs) of the present invention include, but are not limited to, tubulin inhibitors, DNA damaging agents, maytansine alkaloids, or topoisomerase I inhibitors, aristamine, succinyl benzoate, vincristine, pyrrolobenzodiazepine, paclitaxel, docetaxel, and cryptophytic acid.

[0391] In some embodiments, the cytotoxic agents include, but are not limited to, tubulolysin, camptothecin analogues, maytansine alkaloids, and arithmetic.

[0392] In some embodiments, the cytotoxic payload is a topoisomerase I inhibitor.

[0393] In some embodiments, the cytotoxic agent is camptothecin or a camptothecin analogue.

[0394] According to some implementations, the cytotoxic agent is a maytansine alkaloid, such as DM1 or DM4, or a cytomegalovirus derivative.

[0395] According to some implementations, the cytotoxic agent conjugated with the anti-Trop2 antibody is auristatin, such as MMAE, MMAF, or derivatives thereof.

[0396] In some implementations, the cytotoxic agent is MMAE.

[0397] In some embodiments, in the ADC provided by the present invention, a Trop2 / HER2 bispecific antibody or its antigen-binding fragment is conjugated to a cytotoxic agent (e.g., any cytotoxic agent disclosed above) via a linker structure, wherein the linker is any group or portion that links or bonds the antibody or antigen-binding protein of the present invention to a therapeutic portion such as a cytotoxic agent. Suitable linkers may be employed, for example, in antibody-drug conjugates and immunotoxins.

[0398] Suitable binders or linkers used to prepare the antibody-drug conjugates of the present invention are sufficiently stable to take advantage of the antibody's cyclic half-life and simultaneously capable of releasing their effective load upon antigen binding and / or antigen-mediated internalization of the conjugate. Any linker molecule or linker technology known in the art can be used to generate or construct the ADC of the present invention.

[0399] In some implementations, the connector is one of a cuttable connector and a non-cuttable connector.

[0400] In some implementations, the connector is a cuttable connector.

[0401] In some implementations, the linker is cleavable under specific physiological conditions, thereby facilitating the release of cytotoxic agents into the cell. For example, the linker may be an acid-instable linker, a peptidase-sensitive linker, a light-instable linker, a dimethyl linker, or a disulfide-containing linker.

[0402] In some embodiments, the connector includes a portion that can be cleaved by an enzyme. Exemplary enzyme-cleavable portions include, but are not limited to, peptide bonds, ester bonds, hydrazone bonds, and disulfide bonds.

[0403] In some embodiments, the linkers that can conjugate the antibody of the present invention to the therapeutic drug portion include, but are not limited to, hydrazine linkers, disulfide linkers, bifunctional linkers, dipeptide-pentapeptide linkers, glucuronide linkers, or thioether linkers.

[0404] In some embodiments, the bifunctional connector includes, but is not limited to, for example, PAB (p-aminobenzyloxycarbonyl) or its derivatives, p-aminobenzyloxycarbonyl (PABC), MC (6-maleimide hexanoyl), MP (maleimide propionyl), SPP (N-succinimide 4-(2-pyridinylthio)valerate), SMCC (N-succinimide 4-(N-maleimide methyl)cyclohexane-1-carboxylate), SIAB (N-succinimide (4-iodoacetyl)aminobenzoate), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), diazide derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bifunctional fluorinated compounds (such as 1,5-difluoro-2,4-dinitrobenzene) and their variants and combinations.

[0405] In some embodiments, the linker is a peptide linker in which one or more side chains of an amino acid are attached to a side chain group, as described below. In some embodiments, the peptide linker comprises lysine, valine, and citrulline.

[0406] In some embodiments, the peptide linker comprises lysine, valine, and alanine.

[0407] In some implementations, the peptide linker comprises valine and alanine.

[0408] In some embodiments, the peptide linker comprises a dipeptide, a tripeptide, or a tetrapeptide, such as valine-citrulline (val-cit or VC), glutamic acid-valine-citrulline (EVC), glycine-glycine-phenylalanine (GGF), or glycine-glycine-phenylalanine-glycine (GGFG).

[0409] In some embodiments, the peptide linker is valine-citrulline (val-cit or VC).

[0410] In some exemplary embodiments, in the exemplary ADC of the present invention, the connector includes a portion having the following structure: MC-VC-PAB, as shown in Formula I:

[0411]

[0412] in, A disulfide bond is a bond to an antibody or antigen-binding protein (e.g., via a lysine residue) and is Bonds with cytotoxic agents.

[0413] In some exemplary embodiments, in the exemplary ADC of the present invention, the linker-drug conjugate comprises a portion having the following structure: MC-VC-PAB-MMAE (Formula II, as shown below). Figure 14 (As shown).

[0414] Preparation of Trop2 / HER2 Bispecific ADC

[0415] The Trop2 / HER2 bispecific ADC provided by this invention can be prepared by any suitable method known in the art.

[0416] In some embodiments, the nucleophilic group of the antibody reacts first with a bifunctional linker reagent and then is linked to a cytotoxic agent, or vice versa, that is, the nucleophilic group of the cytotoxic agent reacts first with a bifunctional linker and then is linked to the antibody.

[0417] In some embodiments, the cytotoxic agent may contain (or be modified to contain) a thiol-reactive functional group that reacts with the cysteine ​​thiol group of the free cysteine ​​in the antibody provided by the present invention. Exemplary thiol-reactive functional groups include, for example, maleic anhydride, iodoacetamide, pyridyl disulfide, haloacetyl, succinimide esters (e.g., NHS, N-hydroxysuccinimide), isothiocyanates, sulfonyl chlorides, 2,6-dichlorotriazine, pentafluorophenyl esters, or aminophosphates.

[0418] In some implementations, the cytotoxic agent and antibody can be activated and reactively linked in one step to form an ADC.

[0419] In some embodiments, the cytotoxic agent is MMAE.

[0420] In some implementations, the bifunctional linker is MC-VC-PAB.

[0421] In some embodiments, the linker-drug conjugate is MC-VC-PAB-MMAE.

[0422] In some preferred embodiments, the Trop2 and HER2 bispecific ADC of the present invention is Trop2×Her2 bsAb-MC-VC-PAB-MMAE, and its preparation method includes the following steps:

[0423] First, the disulfide bonds between antibody chains were opened using TCEP reducing agent; then, the antibody was prepared by purification after a thiol coupling reaction, and further purified by gel chromatography.

[0424] In some embodiments, the molar ratio of TCEP to antibody is 2 to 10.

[0425] In some embodiments, the molar ratio of TCEP to antibody is 4 to 8.

[0426] In some embodiments, the molar ratio of TCEP to antibody is 5 to 7.

[0427] In some embodiments, the molar ratio of the MC-VC-PAB-MMAE:antibody is 10 to 18.

[0428] In some embodiments, the molar ratio of the MC-VC-PAB-MMAE:antibody is 12 to 16.

[0429] In some embodiments, the molar ratio of the MC-VC-PAB-MMAE:antibody is 14 to 16.

[0430] In some embodiments, the ratio of drug to antibody concentration (average DAR value) in the preparation method of the Trop2 / HER2 bispecific ADC of the present invention is 2-10.

[0431] In some embodiments, the ratio of drug to antibody concentration (average DAR value) in the preparation method of the Trop2 / HER2 bispecific ADC of the present invention is 3-8.

[0432] In some embodiments, the ratio of drug to antibody concentration (average DAR value) in the Trop2 / HER2 bispecific ADC of the present invention is 5-7.

[0433] In some embodiments, in the preparation method of the Trop2 / HER2 bispecific ADC of the present invention, the linking reaction of the Trop2 / HER2 bispecific antibody and MC-VC-PAB-MMAE occurs in a water bath at 25°C.

[0434] In some embodiments, the preparation method of the Trop2 / HER2 bispecific ADC of the present invention involves a linkage reaction between the Trop2 / HER2 bispecific antibody and MC-VC-PAB-MMAE in a water bath at 25°C for 3 hours.

[0435] Encoding nucleic acids

[0436] On one hand, the present invention provides isolated nucleic acid molecules encoding the aforementioned antibodies or antigen-binding fragments of the present invention. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized for a host cell used for expression. In some embodiments, the nucleic acid molecule of the present invention is operatively linked to an expression regulatory sequence.

[0437] In some embodiments, the nucleic acid composition comprises a nucleic acid encoding the aforementioned antibody or antigen-binding fragment thereof that binds to Trop2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region A (VHA) and a light chain variable region A (VLA), wherein the VHA comprises VHA CDR1, VHA CDR2 and VHA CDR3 as shown in SEQ ID NO:1-3, respectively, and the VLA comprises VLA CDR1, VLA CDR2 and VLA CDR3 as shown in SEQ ID NO:4-6, respectively.

[0438] In some embodiments, the antibody is a chimeric antibody.

[0439] In some embodiments, the nucleic acids encoding the heavy chain variable region (VH) and light chain variable region (VL) of the chimeric antibody respectively comprise SEQ ID NO:63 and SEQ ID NO:71 as described in Example 3.

[0440] In some embodiments, the antibody is a humanized antibody.

[0441] In some embodiments, the nucleic acid encoding composition comprises a nucleic acid encoding the heavy chain variable region A (VHA) of an antibody binding to Trop2 or an antigen-binding fragment thereof, as shown in any of SEQ ID NO:63-70 in Table 9 of Example 9, or a variant nucleic acid thereof with at least 85% sequence homology, wherein the VHA comprises an amino acid sequence with at least 85% identity to the amino acid sequence shown in any of SEQ ID NO:7-14 and / or comprises a nucleic acid encoding the light chain variable region A (VLA) of any of SEQ ID NO:71-77 in Table 10 of Example 9, or a variant nucleic acid thereof with at least 85% sequence homology, wherein the VLA comprises an amino acid sequence with at least 85% identity to the amino acid sequence shown in any of SEQ ID NO:15-21.

[0442] In some implementations, the VHA specifically binds to human Trop2 when paired with VLA.

[0443] In some embodiments, the nucleic acid molecule further comprises nucleic acid encoding the heavy chain constant region A' (CHA') and nucleic acid encoding the light chain constant region A (CLA).

[0444] In one aspect, the present invention also provides a composition or fusion comprising a heavy chain A' (HA') or a functionally conserved variant thereof encoding the anti-Trop2 antibody of the present invention and a light chain A' (LA') or a functionally conserved variant thereof encoding a nucleic acid, wherein the heavy chain A' (HA) comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in any one of SEQ ID NO: 22-29, and the light chain A (LA) comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in any one of SEQ ID NO: 30-36.

[0445] In some embodiments, the nucleic acid-coding compositions / fusions of the present invention include nucleic acids encoding the Trop2 binding arm and the HER2 binding arm as shown in Table 15 of Example 15, wherein the nucleic acid encoding the Trop2 binding arm includes nucleic acids encoding VLA and VHA as shown in SEQ ID NO:73 and SEQ ID NO:66, respectively, and the nucleic acid encoding the HER2 binding arm includes nucleic acids encoding VLB and VHB as shown in SEQ ID NO:84 and SEQ ID NO:85, respectively.

[0446] In some embodiments, the nucleic acid-coding compositions / fusions of the present invention include nucleic acids encoding the Trop2 binding arm and the HER2 binding arm, as shown in SEQ ID NO:82 and SEQ ID NO:83, respectively.

[0447] The nucleic acid fusions or compositions of the present invention contain encoding nucleic acids that may be present in whole cells, cell lysates, or in a partially purified or substantially pure form. The nucleic acid compositions of the present invention contain encoding nucleic acids that may, for example, be DNA or RNA and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0448] The nucleic acids of this invention can be obtained using standard molecular biology techniques.

[0449] Once DNA fragments encoding the heavy chain variable region and the light chain variable region are obtained, these fragments can be further manipulated using standard DNA recombination techniques, such as converting the variable region gene into a full-length heavy chain gene, a full-length light chain gene, a Fab fragment gene, or an scFv gene. In these operations, the DNA fragments encoding the heavy chain variable region and the light chain variable region are operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible adapter. The term "operatively linked" means that the two DNA fragments are joined together such that the amino acid sequences encoded by both fragments are within their reading frames.

[0450] For example, isolated DNA encoding the VHA region can be transformed into a full-length heavy chain gene by operatively linking the isolated VHA-coding DNA with another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3).

[0451] In some preferred embodiments, the heavy chain constant region may be the human IgG1, IgG2, IgG3, or IgG4 constant region, preferably the human IgG1 medium chain constant region. The sequence of the human IgG1 heavy chain constant region gene is known in the field, and DNA fragments including these regions can be obtained by standard PCR amplification.

[0452] For Fab fragment heavy chain genes, the DNA encoding the VH region can be operatively linked to another DNA molecule that encodes only the CH1 constant region of the heavy chain.

[0453] Isolated DNA encoding the VLA region can be converted into a full-length light chain gene by operatively linking the VLA-coding DNA to another DNA molecule encoding the light chain constant region (CLA). The sequence of the human light chain constant region gene is known in the antibody field, and DNA fragments including these regions can be obtained by standard PCR amplification.

[0454] In some preferred embodiments, the light chain constant regions (such as CLA and CLB) can be human κ and human λ constant regions, preferably human κ-type light chain constant regions.

[0455] In some preferred embodiments, to create the coding gene for scFab(A), a DNA fragment encoding VLA and a DNA fragment encoding CLA can be operatively coupled with a gene encoding a flexible linker, such as a gene encoding an amino acid sequence (Gly4-Ser). 4-6 The DNA fragments are linked and further operatively linked to an expression cassette of a promoter with DNA fragments encoding VHA and CH1; further, a DNA fragment encoding Fc(A) is added downstream of the CH1-encoding DNA, so that VLA, CLA, VLA, VHA and CH1, the hinge region and Fc(A) can be expressed as a continuous single-stranded protein, wherein the VHA and CLA regions are linked through the flexible linker to form a Trop2-specific first antigen-binding arm.

[0456] In some preferred embodiments, to create the coding gene for scFab(B), a DNA fragment encoding VLB and a DNA fragment encoding CLB can be operatively coupled with a gene encoding a flexible linker, such as a gene encoding an amino acid sequence (Gly4-Ser). 4-6The DNA fragments are ligated and further operatively linked to an expression cassette of a promoter with DNA fragments encoding VHB and CH1. Further, a DNA fragment encoding Fc(B) is added downstream of the CH1-encoding DNA, so that VLB, CLB, VHB, CH1, the hinge region, and Fc(B) can be expressed as a continuous single-stranded protein, wherein the VHB and CLB regions are connected via the flexible linker to form a HER2-specific second antigen-binding arm.

[0457] In some preferred embodiments, to create the gene encoding LA-linker-HA, the C-terminus of the DNA fragment encoding LA can be operatively linked to another fragment encoding a flexible linker, such as the amino acid sequence (Gly4-Ser)6, and the other end of the flexible linker is then linked to the N-terminus of the nucleotide sequence encoding HA, so that the LA and HA sequences are linked through the flexible linker so that HA and LA can be expressed as a continuous single-chain protein, wherein the LA and HA regions are linked through the flexible linker to form a Trop2-specific first antigen-binding arm.

[0458] In some preferred embodiments, to create the gene encoding LB-linker-HB, the C-terminus of the DNA fragment encoding LB can be operatively linked to another fragment encoding a flexible linker, such as the amino acid sequence (Gly4-Ser)6, and the other end of the flexible linker linker is then linked to the N-terminus of the nucleotide sequence encoding HB, so that the LB and HB regions are linked through the flexible linker and HB and LB can be expressed as a continuous single-chain protein, wherein the LB and HB regions are linked through the flexible linker as a HER2-specific second antigen-binding arm.

[0459] In some embodiments, the peptide linker is linker1 of the amino acid sequence (GGGGS)4 (SEQ ID NO:58) or linker2 of the amino acid sequence (GGGGS)6 (SEQ ID NO:49).

[0460] In some embodiments, the peptide linker is linker2 of the amino acid sequence shown in (GGGGS)6 (SEQ ID NO:49).

[0461] In some embodiments, the nucleic acid-encoding compositions / fusions of the present invention comprise a polynucleotide molecule as shown in SEQ ID NO:82, which encodes a fusion peptide single chain A formed by linking the full-length light chain of the Trop2-binding arm of the above-described anti-Trop2 / HER2 bispecific antibody of the present invention with a linker and a full-length heavy chain; and a polynucleotide molecule as shown in SEQ ID NO:83, which encodes a fusion peptide single chain B formed by linking the HER2-binding arm and the full-length light chain of the above-described anti-Trop2 / HER2 bispecific antibody of the present invention with a linker and a full-length heavy chain.

[0462] Recombinant vector

[0463] In one respect, the present invention also relates to recombinant vectors.

[0464] The term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, created, or isolated through recombinant means, such as antibodies expressed via a recombinant expression vector transfected into host cells; antibodies isolated from a recombinant combined human antibody library; antibodies isolated from transgenic animals (e.g., mice) carrying the human immunoglobulin gene; or antibodies prepared, expressed, created, or isolated by any means involving splicing the human immunoglobulin gene sequence with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in various embodiments, the amino acid sequences of the VH and VL regions contained in the recombinant antibody, while derived from and associated with human germline VH and VL sequences, may not be naturally present in a human antibody germline library in vivo. All these recombinant methods are well known to those skilled in the art.

[0465] In some exemplary embodiments, the recombinant vector contains the aforementioned composition or fusion of the encoded nucleic acid, and one or more regulatory sequences operatively linked to the encoded nucleic acid of the composition. The encoded nucleic acid constituting the nucleic acid composition of the present invention can be manipulated in various ways to ensure the expression of the antibody. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0466] In some preferred embodiments, the nucleic acid construct is a plasmid expression vector.

[0467] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is typically operatively linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence exhibiting transcriptional activity in the selected host cell and can be obtained from a gene encoding an extracellular or intracellular polypeptide homologous or heterologous to that host cell. The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, the untranslated region of mRNA important for translation in the host cell. The leader sequence is operatively linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in this invention.

[0468] In some preferred embodiments, the recombinant vector used in the preparation of the humanized anti-Trop2 antibody of the present invention is a pHR expression vector with a signal peptide at the N-terminus.

[0469] In some embodiments, the pHR expression vector is pHr-hκ, such as... Figure 20 As shown.

[0470] In some embodiments, the pHR expression vector is pHr-hIgG4, such as... Figure 21 As shown.

[0471] In some embodiments, the pHR expression vector is pHr-hIgG1, such as... Figure 22 As shown.

[0472] cell

[0473] The host cells suitable for introducing the nucleic acid constructs described herein can be prokaryotic cells or higher eukaryotic cells, such as mammalian cells and immune cells.

[0474] In a preferred embodiment, the host cell used for introducing the nucleic acid constructs described herein is a eukaryotic cell.

[0475] Eukaryotic cells, especially mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0476] In some preferred embodiments, preferred mammalian host cells for expressing the recombinant antibodies of the present invention include Chinese hamster ovary (CHO), NSO myeloma cells, COS cells, SP2 cells, and the GS gene expression system.

[0477] In some preferred embodiments, the host cell used for introducing the nucleic acid constructs described herein is a CHO cell.

[0478] In some exemplary embodiments, the host cell used for introducing the nucleic acid constructs described herein is an Expi CHO-S cell.

[0479] Preparation method of Trop2 antibody

[0480] In one aspect, the present invention provides a method for preparing anti-Trop2 antibodies.

[0481] In some embodiments, the anti-Trop2 antibody of the present invention is a murine antibody.

[0482] In some embodiments, the anti-Trop2 mouse antibody of the present invention is prepared and screened using somatic cell hybridization (hybridoma) technology.

[0483] For example, mice were immunized with human Trop2 protein fused with a 6×His tag at the C-terminus. Hybridomas were generated by electrofusion of spleen single cells from high-titer serum mice with SP20 myeloma cells. Functional activity was detected to screen out the parent clone 44E7 that could bind human Trop2 and to isolate single clones with Trop2 binding activity. Sequencing yielded the heavy chain variable region sequence and the light chain variable region sequence of the mouse monoclonal 44E7 of this invention.

[0484] In some embodiments, the anti-Trop2 antibody of the present invention is a chimeric antibody or a humanized antibody.

[0485] In some embodiments, the preparation of the anti-Trop2 antibody of the present invention as a chimeric antibody or humanized antibody employs genetic engineering methods, including the following steps:

[0486] First, variable region-encoding nucleic acids are designed and synthesized, and then linked to expression vectors containing corresponding constant region-encoding nucleic acids to generate recombinant expression vectors of full-length heavy and light chains, which are then transfected into host cells. Subsequently, engineered host cells are cultured, and several packets are collected and purified to obtain the target chimeric anti-Trop2 antibody or humanized anti-Trop2 antibody.

[0487] In some embodiments, the anti-Trop2 antibody of the present invention is a chimeric antibody.

[0488] Chimeric antibodies have a CDR derived from non-human antibodies, usually murine antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody.

[0489] In some embodiments, the present invention encodes a nucleic acid that is operatively linked to construct a chimeric anti-Trop2 antigen-binding fragment.

[0490] In this field, reducing the immunogenicity of antibodies can be achieved through a variety of methods, including transplanting the entire non-human variable domain into a human constant region to generate chimeric antibodies.

[0491] In some embodiments, the design of the full-length heavy chain (HA') and full-length light chain (LA) of the chimeric anti-Trop2 antibody of the present invention involves placing the variable regions of the heavy chain and light chain of a murine antibody at the N-terminus of the constant region of the heavy chain and the constant region of the light chain kappa of human IgG4, respectively, to obtain a fusion gene fragment encoding the full-length heavy chain A (HA') of the chimeric anti-Trop2 antibody and a fusion gene fragment expressing the full-length light chain (LA) of the chimeric Trop2 antibody.

[0492] In some embodiments, the chimeric antibody comprises VHA and VLA as shown in SEQ ID NO:7 and 15 of Example 3, respectively.

[0493] In some embodiments, the chimeric antibody comprises HA and LA as shown in SEQ ID NO:22 and 30 of Examples 3, respectively.

[0494] In some embodiments, the anti-Trop2 antibody of the present invention is a humanized antibody.

[0495] In some embodiments, the preparation of the humanized anti-Trop2 antibody of the present invention uses techniques recognized in the art to modify the Trop2 binding peptide, its variants or derivatives to reduce its immunogenicity.

[0496] In this art, reducing the immunogenicity of an antibody can be achieved by a variety of methods, including (a) transplanting the entire non-human variable domain into a human constant region to produce a chimeric antibody; (b) transplanting at least a portion of one or more non-human complementarity-determining regions (CDRs) into a human framework and constant region, with or without key framework residues; or (c) transplanting the entire non-human variable domain, but “masking” the non-human portion with a human-like portion by replacing surface residues.

[0497] In some embodiments, the coding nucleic acid sequences of the heavy chain variable region (VHA) and light chain variable region (VLA) of the humanized anti-Trop2 antibody of the present invention were obtained by CDR transplantation and reverse mutation.

[0498] In one exemplary embodiment, the method for designing the encoding nucleic acids of humanized antibodies VHA and VLA includes: firstly, inputting the light and heavy chain sequences of the mouse antibody into an antibody structure simulation and optimization analysis platform, searching for homologous proteins in the variable region, comparing the results of several databases, selecting the heavy chain template and light chain template with the highest homology in the germLine framework, and transplanting the CDR sequence of the mouse antibody light and heavy chain variable regions into the germLine framework sequence. After the CDR transplantation is completed, several key sites are selected from the humanized sequences generated from each template for reversion mutation, finally obtaining the encoding nucleic acids of multiple humanized heavy chain variable region sequences and multiple humanized light chain variable region sequences; constructing expression vectors from the selected HA' and LA encoding nucleic acids, combining and transfecting them to obtain the final humanized anti-Trop2 antibody of the present invention.

[0499] For the preparation of Trop2 monoclonal antibodies or their Trop2 binding fragments, once the coding nucleic acids encoding DNA fragments encoding VHA and / or VLA are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques. For example, isolated DNA regions encoding VHA can be converted into full-length heavy chain genes by operably ligating the VHA-coding region to another DNA molecule (CH1, CH2, and CH3) encoding the human IgG heavy chain constant region. The sequences of human heavy chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be the human IgG1, IgG2, IgG3, or IgG4 constant region, preferably the IgG1 or IgG4 heavy chain constant region, and most preferably the human IgG4 heavy chain constant region. For Fab fragment heavy chain genes, VHA-coding DNA can be efficiently ligated to another DNA molecule encoding only the heavy chain CH1 constant region. In this document, "operably ligated" means that the ligation of the two DNA fragments should keep the amino acid sequences encoded by the two DNA fragments within the reading frame.

[0500] In some preferred embodiments, the sequences of the heavy chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification.

[0501] The light chain constant region can be the human κ or human λ constant region.

[0502] In some preferred embodiments, the light chain constant region may be the human κ constant region. The sequence of the human light chain constant region gene is known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification.

[0503] In some preferred embodiments, the full-length heavy chain encoding nucleic acid of the chimeric anti-Trop2 antibody of the present invention is generated by placing the encoding nucleic acid of the VHA of the murine antibody at the N-terminus of the constant region of the human IgG4 heavy chain; the full-length light chain encoding nucleic acid is generated by placing the encoding nucleic acid of the VLA of the murine antibody at the N-terminus of the constant region of the human light chain kappa.

[0504] In some exemplary preferred embodiments, the full-length heavy chain encoding nucleic acid of the humanized anti-Trop2 antibody of the present invention is a full-length heavy chain A'(HA') that encodes the VHA of the humanized anti-Trop2 antibody or its variant VHA, which is designed by humanizing the VHA encoding nucleic acid of the mouse anti-Trop2 antibody heavy chain variable region, and is placed at the N-terminus of the encoding nucleic acid of the humanized anti-Trop2 antibody VLA, which is designed by humanizing the VLA encoding nucleic acid of the mouse anti-Trop2 antibody light chain variable region, and is placed at the N-terminus of the encoding nucleic acid of the humanized anti-Trop2 antibody kappa constant region.

[0505] In some exemplary embodiments, the humanized anti-Trop2 antibody variable region design method of the present invention includes: using a variable region CDR transplantation method combined with key amino acid back mutation technology to humanize the variable region sequence of Trop2 mouse monoclonal antibody 44E7; the humanization design includes: using bioinformatics analysis, through sequence alignment, finding the antibody germline sequence most similar to the light and heavy chains of mouse monoclonal antibody 44E7 in a known database, transplanting the CDR sequences of VHA and VLA of mouse monoclonal antibody 44E7 into the human antibody frame region (FR); then using BLAST analysis to determine the homologous structure with the highest structural similarity to the variable region sequence of mouse monoclonal antibody 44E7; then establishing a three-dimensional structural model after antibody light and heavy chain variable region sequence alignment; finally, finding key amino acids through the three-dimensional structure and performing back mutations to obtain the coding nucleic acid of the humanized VHA sequence and the coding nucleic acid of the humanized VLA sequence.

[0506] In some embodiments, the chimeric antibody comprises VHA and VLA as shown in SEQ ID NO:7 and SEQ ID NO:15, respectively.

[0507] In some embodiments, the humanized antibody comprises VHA and VLA as shown in any one of SEQ ID NO:8-14 and any one of SEQ ID NO:16-21, respectively.

[0508] In some embodiments, the chimeric antibody comprises HA and LA as shown in SEQ ID NO:22 and SEQ ID NO:30, respectively.

[0509] In some embodiments, the humanized antibody comprises HA and LA as shown in any one of SEQ ID NO:23-29 and any one of SEQ ID NO:31-36, respectively.

[0510] (2) Construction of full-length heavy chain and full-length light chain expression vectors:

[0511] Plasmid vectors containing nucleic acids encoding the human IgG4 heavy chain constant region and the light chain kappa constant region were digested with enzymes, respectively. Using genetic engineering methods, the nucleic acids encoding VHA and VLA of chimeric antibodies or humanized anti-Trop2 antibodies were ligated to the N-terminus of the nucleic acids encoding the human IgG4 heavy chain constant region and the light chain kappa constant region, respectively. This yielded expression vectors containing fusion gene fragments encoding the full-length heavy chain of chimeric anti-Trop2 antibodies or humanized anti-Trop2 antibodies and recombinant expression vectors containing fusion gene fragments encoding the humanized light chain.

[0512] (3) Transiently transfect host cells to obtain engineered recombinant host cells expressing the humanized anti-Trop2 antibody of the present invention;

[0513] (4) When the recombinant host cells obtained from (3) reach a cell viability of ≤70%, collect the culture supernatant;

[0514] (5) The target antibody was purified from the culture supernatant using a Protein A affinity chromatography column.

[0515] Preparation method of anti-Trop2 / HER2 bispecific antibody

[0516] In one aspect, the present invention provides a method for preparing an anti-Trop2 / HER2 bispecific antibody.

[0517] In some embodiments, the present invention provides an anti-Trop2 / HER2 bispecific antibody prepared using genetic engineering methods.

[0518] In some embodiments, the method for preparing the anti-Trop2 / HER2 bispecific antibody includes the following steps:

[0519] First, the coding nucleic acids for the first antigen-binding arm specific to Trop2 and the second antigen-binding arm specific to HER2 were designed and synthesized separately. Then, the gene fragments fused from the coding nucleic acids for the first antigen-binding arm specific to Trop2 and the second antigen-binding arm specific to HER2 were constructed into expression vectors using genetic engineering methods, resulting in single-arm expression vectors specifically binding to Trop2 and HER2. The single-arm expression vectors were then transiently transfected into host cells, and the resulting recombinant cells were cultured and purified to obtain the Trop2 / bispecific antibody of this invention.

[0520] In the preparation of the anti-Trop2 / HER2 bispecific antibody of the present invention, the fusion nucleic acid encoding the light chain A (LA) and the linker, and the nucleic acid encoding the heavy chain A (HA) can be inserted into the same or different expression vectors respectively. In a preferred embodiment, the nucleic acid encoding the variable region is used to generate a full-length antibody gene of any antibody isotype. By operably linking the nucleic acid encoding the VLA segment to the nucleic acid encoding the CLA segment in the vector, and operably linking the nucleic acid encoding the VHA segment to the nucleic acid encoding the CHA end in the vector, a binding arm structure is generated that encodes the C-terminus of anti-Trop2 VL linked to the N-terminus of the human κ chain constant region. The C-terminus of the κ chain constant region is followed by a (G4S)6 linker. The C-terminus of the linker is followed by the N-terminus of the Trop2 antibody VH. The C-terminus of anti-Trop2 VH is linked to the binding arm structure of the N-terminus of the human IgG1 constant region. The nucleic acid encoding the fusion peptide A containing the polynucleotide sequence shown in SEQ ID NO:80, which is sequentially linked with VLA, CLA, linker, VHA, and CHA, is linked into a nucleic acid fusion product and inserted into an expression vector to generate an expression vector containing a nucleic acid encoding the first antigen binding arm specific to Trop2, as shown in SEQ ID NO:80.

[0521] In some embodiments, the nucleotide sequences encoding nucleic acids for VLA and VHA are shown as SEQ ID NO:73 and SEQ ID NO:66, respectively, as shown in Table 15 of Example 15.2, and the nucleotide sequence encoding nucleic acid for binding arm A is shown as SEQ ID NO:82.

[0522] In some embodiments, the nucleotide sequences encoding nucleic acids for VLB and VHB are shown as SEQ ID NO:84 and SEQ ID NO:85, respectively, as shown in Table 15 of Example 15.2, and the nucleotide sequence encoding nucleic acid for binding arm A is shown as SEQ ID NO:83.

[0523] In the preparation of the anti-Trop2 / HER2 bispecific antibody of the present invention, the nucleic acid encoding light chain B (LB) and the nucleic acid encoding the linker, and the nucleic acid encoding heavy chain B (HB) can be inserted into the same or different expression vectors respectively. In a preferred embodiment, the nucleic acid encoding the variable region is used to generate a full-length antibody gene of any antibody isotype. By operably linking the nucleic acid encoding the VHB segment to the pre-CHB segment encoding nucleic acid in the vector and operably linking the nucleic acid encoding the VLB segment to the pre-CLB segment encoding nucleic acid in the vector, a binding arm structure is generated where the C-terminus of the anti-HER2 VL is linked to the N-terminus of the human κ chain constant region, the C-terminus of the κ chain constant region is followed by a (G4S)4 linker, the C-terminus of the linker is followed by the N-terminus of the HER2 antibody VH, and the C-terminus of the anti-Trop2 VH is linked to the N-terminus of the human IgG1 constant region. The nucleic acid encoding the structure of the fusion peptide B containing the polynucleotide sequence shown in SEQ ID NO:83, which is sequentially linked with VLB, CLAB, linker, VHB, and CHB, is linked into a nucleic acid fusion product and inserted into the expression vector to generate an expression vector containing the nucleic acid encoding the second antigen binding arm specific to HER2, as shown in SEQ ID NO:83.

[0524] In some implementations, for example, the linker encodes the amino acid sequence (Gly4-Ser). 4-10 .

[0525] In some implementations, for example, the linker encodes the amino acid sequence (Gly4-Ser). 4-6 .

[0526] Alternatively or additionally, the recombinant expression vector can encode a signal peptide that promotes the secretion of antibody chains from host cells. The genes encoding LA and HA of the anti-Trop2 antibody can be cloned into a vector containing the signal peptide, so that the signal peptide is linked within the N-terminal frame of the anti-Trop2 antibody chain gene. Similarly, the genes encoding LB and HB of the anti-HER2 antibody can be cloned into a vector containing the signal peptide, so that the signal peptide is linked within the N-terminal frame of the anti-HER2 antibody chain gene.

[0527] Signal peptides can be immunoglobulin signal peptides or heterologous signal peptides (i.e., signal peptides derived from non-immunoglobulins).

[0528] In some preferred embodiments, the polynucleotides encoding HA and LA, which encode monoclonal antibodies binding to the Trop2 protein as described in this invention, can be cloned into an expression vector, with each nucleotide sequence ligated downstream of a suitable promoter. For example, each polynucleotide sequence encoding HA and LA can be operatively ligated to a different promoter, or the nucleotide sequences encoding HA and LA can be operatively ligated to a single promoter such that HA and LA can both be expressed by the same promoter.

[0529] In some preferred embodiments, the polynucleotides encoding HB and LB, which encode monoclonal antibodies against the anti-HER2 protein as described in this invention, can be cloned into an expression vector, with each nucleotide sequence ligated downstream of a suitable promoter. For example, each polynucleotide sequence encoding HB and LB can be operatively ligated to a different promoter, or the nucleotide sequences encoding HB and LB can be operatively ligated to a single promoter such that HB and LB can both be expressed by the same promoter.

[0530] In some embodiments, the pHR expression vector is pHr-hκ, such as... Figure 19 As shown.

[0531] In some embodiments, the pHR expression vector is pHr-hIgG4, such as... Figure 20 As shown.

[0532] In some embodiments, the pHR expression vector is pHr-hIgG1, such as... Figure 21 As shown.

[0533] In some embodiments, the nucleic acid encoding the Trop2-specific first antigen-binding arm of the anti-Trop2 / HER2 bispecific antibody protein as described in this invention and the nucleic acid encoding the HER2-specific second antigen-binding arm can be constructed onto two vectors, or ligated into a single expression vector, which can be introduced into the same host cell.

[0534] In a preferred embodiment, when the encoding nucleic acids of the first antigen-binding arm specific to Trop2 and the second antigen-binding arm specific to HER2 are ligated into the same expression vector, the resulting recombinant expression vector is expressed in the host cell. Each antigen-binding arm can be isolated from the host cell expressing it, and the isolated first antigen-binding arm specific to Trop2 and the second antigen-binding arm specific to HER2 can interact in the host cell to form a KIH structure. The KIH structure is further assembled into the heterodimeric anti-Trop2 / HER2 bispecific antibody of the present invention, and purified to obtain the anti-Trop2 / HER2 bispecific antibody of the present invention.

[0535] In one embodiment, the recombinant vector used to express the anti-Trop2 / HER2 bispecific antibody of the present invention is a pHr expression vector, and the host cell is Expi CHO-S cells.

[0536] The choice of the aforementioned expression vector / promoter depends on the type of host cell used to produce the antibody, which is well known in the art and will not be described in detail here.

[0537] Methods for introducing nucleic acids or vectors into mammalian cells are known in the art. The vectors can be transferred into cells by physical, chemical or biological methods. For example, for eukaryotic hosts, DNA transfection methods including but not limited to the following can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0538] In an exemplary embodiment of the present invention, a nucleic acid encoding a heavy or light chain containing an anti-Trop2 antibody or its antigen-binding fragment, or an expression vector pHr containing VHA-encoded nucleic acid and VLA-encoded nucleic acid, is transiently transfected into host cells via a CHO-s transient transfection expression system.

[0539] The antibody product of the anti-Trop2 antibody or its antigen-binding fragment encoding the heavy chain A (HA) or light chain A (LA) of the recombinant host cell, or the host cell containing a nucleic acid fusion product encoding the Trop2 binding arm and the HER2 binding arm, can be expressed intracellularly, on the cell membrane, or secreted extracellularly. In some preferred embodiments, the aforementioned expressed anti-Trop2 humanized antibody or anti-Trop2 / HER2 bispecific antibody of the present invention is secreted extracellularly into the host cell.

[0540] In some exemplary embodiments, the obtained recombinant host cells can be cultured using conventional methods to express the antibodies encoded by the nucleic acid molecules of the present invention. Depending on the host cells used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period. If desired, the target recombinant protein can be separated and purified from the culture medium by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof.

[0541] In one embodiment, the preparation of the chimeric or humanized anti-Trop2 antibody involves constructing a gene fragment encoding a fused nucleic acid of heavy chain A and light chain A into a pHr expression vector with an N-terminal signal peptide, resulting in an expression plasmid for the anti-Trop2 antibody. The CHO-s transient transduction expression system is then used to generate engineered cells expressing the anti-Trop2 antibody.

[0542] In one embodiment, a single-arm expression plasmid A with a Trop2 binding arm is obtained by constructing a gene fragment fused at 5' to 3' with three coding nucleic acids encoding LA, linker and HA respectively into a pHr expression vector with a signal peptide at the N-terminus.

[0543] In one embodiment, a single-arm expression plasmid B with a HER2 binding arm is obtained by constructing a gene fragment fused at 5' to 3' with three coding nucleic acids encoding LB, linker and HB respectively into a pHr expression vector with a signal peptide at the N-terminus.

[0544] The CHO-s transient transfection expression system was then used to generate anti-Trop2 antibodies. The recombinant pHr expression vectors A and B, encoding the first and second antigen-binding arms, were then transiently transfected into Expi CHO-S cells using transfection reagents to obtain engineered Expi CHO-S cells that produce the anti-TROP2 / HER2 bispecific antibody of this invention.

[0545] The aforementioned Expi CHO-S cells expressing anti-Trop2 antibody were cultured continuously until the cell viability was ≤70%, and the culture supernatant was collected. The target chimeric or humanized anti-Trop2 antibody was purified from the culture supernatant using a Protein A affinity chromatography column.

[0546] In one embodiment, the preparation of the anti-Trop2 / HER2 bispecific antibody involves constructing a gene fragment encoding the first antigen-binding arm specific to Trop2 and the second antigen-binding arm specific to HER2 into a pHr expression vector with an N-terminal signal peptide, resulting in single-arm anti-Trop2 and anti-HER2 antibody plasmids. This pHr expression vector is then transfected into Expi CHO-S cells. The obtained Expi CHO-S cells expressing the anti-Trop2 antibody are continuously cultured until cell viability is ≤70%, and the culture supernatant is collected. The Trop2 / HER2 bispecific antibody of the present invention is purified from the culture supernatant using a Protein A affinity chromatography column.

[0547] Functional activity of anti-Trop2 antibodies or their antigen-binding fragments

[0548] The anti-Trop2 antibodies of the present invention can be evaluated using a variety of their physical properties and detected by ELISA, FACS, antigen cross-binding assay, and biofilm thin-layer interferometry (BLI).

[0549] In some embodiments, the anti-Trop2 antibody of the present invention, its antigen-binding fragment, or a mutant thereof can specifically bind to human Trop2.

[0550] In some embodiments, the anti-Trop2 antibody is a chimeric antibody, and the chimeric antibody CHI44E7 is found to bind to human Trop2 protein (e.g., human Trop2 with the amino acid sequence shown in SEQ ID NO:63) in vitro at an EC50 binding rate of about 0.79 (nM), or about 0.68 (nM), or about 0.54 (nM), or preferably about 0.42 (nM), or preferably about 0.35 (nM), or preferably about 0.28 (nM), or most preferably about 0.24 (nM), as determined by ELISA.

[0551] In some embodiments, the anti-Trop2 antibody is a chimeric antibody, and the chimeric antibody CHI44E7 is determined by FACS to bind to EC50 and human Trop2 (e.g., human Trop2 with the amino acid sequence shown in SEQ ID NO: 63) on the cell membrane at a concentration of about no more than 4.89 (nM), or about no more than 4.68 (nM), or about no more than 4.59 (nM), or about no more than 4.36 (nM), or about no more than 4.28 (nM), or about no more than 4.14 (nM), or about no more than 4.02 (nM), or about no more than 3.96 (nM), or most preferably about no more than 3.82 (nM).

[0552] In some embodiments, the chimeric anti-Trop2 antibody of the present invention, its antigen-binding fragment or its mutant or derivative thereof, can have cross-binding activity against Trop2 in humans and cynomolgus monkeys.

[0553] In some embodiments, the anti-Trop2 antibody is a chimeric antibody. An exemplary chimeric antibody, CHI44E7, binds to the cynomolgus monkey Trop2 protein via ELISA with EC50 at a concentration of approximately no higher than 0.348 (nM), or approximately no higher than 0.315 (nM), or approximately no higher than 0.296 (nM), or approximately no higher than 0.278 (nM), or approximately no higher than 0.246 (nM), or approximately no higher than 0.228 (nM), or most preferably approximately no higher than 0.202 (nM).

[0554] In some embodiments, the anti-Trop2 antibody is a chimeric antibody, and the exemplary chimeric antibody CHI44E7 cannot bind to the same family protein EpCAM.

[0555] In some embodiments, the anti-Trop2 antibody is a chimeric antibody, and the exemplary chimeric antibody CHI44E7 has an affinity for human Trop2 protein that is close to but slightly lower than that for the reference antibody huRS7.

[0556] In some exemplary embodiments, the KD(M) of the chimeric antibody CHI44E7 against human Trop2 protein was determined using biofilm thin-layer interferometry (BLI) to be no higher than approximately 3.76E-09, or no higher than approximately 2.98E-09, or no higher than approximately 2.66E-09, or no higher than approximately 2.48E-09, or no higher than approximately 2.36E-09, preferably no higher than 2.25E-09.

[0557] In some embodiments, the anti-Trop2 antibody is a humanized antibody.

[0558] In some exemplary embodiments, the humanized anti-Trop2 antibody includes hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9.

[0559] In some embodiments, the humanized anti-Trop2 antibodies hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9 are measured by ELISA at levels not exceeding or greater than 0.45 (nM), or not exceeding or greater than 0.38 (nM), or not exceeding or greater than 0.33 (nM), or not exceeding or greater than 0.32 (nM), or not exceeding or greater than 0.25 (nM), or not exceeding or greater than 0.20 (nM), or not exceeding or greater than 0.15 (nM) in vitro by EC50 binding, which is close to the in vitro Trop2 binding level of the parental chimeric antibody CHI44E7.

[0560] In some embodiments, the humanized anti-Trop2 antibodies, such as hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9, have a high affinity for cynomolgus monkey Trop2 protein comparable to that of the chimeric antibody ch44E7. ELISIA measurements show that the ELISIA binding EC50 of the humanized anti-Trop2 antibody to cynomolgus monkey Trop2 protein is approximately no higher than 0.85 (nM), or approximately no higher than 0.76 (nM), or approximately no higher than 0.65 (nM), or approximately no higher than 0.54 (nM), or approximately no higher than 0.43 (nM), or preferably approximately no higher than 0.32 (nM), which is close to the cross-binding level of the parental chimeric antibody to cynomolgus monkey Trop2.

[0561] In some embodiments, the humanized anti-Trop2 antibodies, such as hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9, do not bind to mouse Trop2.

[0562] In some embodiments, the humanized anti-Trop2 antibody is capable of binding to Trop2 expressed on the surface of tumor cells in vitro.

[0563] In some embodiments, the humanized anti-Trop2 antibodies of the present invention, such as hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9, are capable of binding to Trop2 expressed on the surface of CHO-K1 cells in vitro.

[0564] In some embodiments, FACS assay was used to detect the binding of the humanized anti-Trop2 antibodies of the present invention, such as hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9, to Trop2 (i.e., CHO-hTrop2) expressed on the surface of CHO-K1 cells. Combined with EC50 of approximately 12.5 (nM), or approximately 10.9 (nM), or approximately 10.216 (nM), or approximately 9.53 (nM), or approximately 8.65 (nM), or approximately 8.01 (nM), or approximately 7.52 (nM), or approximately 6.81 (nM), or approximately 5.92 (nM), or approximately 5.38 (nM).

[0565] In some embodiments, the humanized antibody has a high affinity for human Trop2 protein comparable to that of the chimeric antibody ch44E7.

[0566] In some embodiments, the humanized anti-Trop2 antibodies, such as hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9, have a higher affinity for human Trop2 than the maternal chimeric antibody ch44E7.

[0567] In some embodiments, the affinity of the humanized anti-Trop2 antibodies, such as hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9, for binding to human Trop2 protein is determined using the BLI method at a KD(M) not higher than 1.72E-08, or not higher than 1.56E-08, or not higher than 1.32E-08, or not higher than 1.22E-08, or not higher than 1.02E-08, or not higher than 9.50E-09, or not higher than 6.25E-09, or not higher than 3.15E-09.

[0568] In some exemplary embodiments, the affinity results of the humanized anti-Trop2 antibody are shown in Table 12 of Example 9.

[0569] In some embodiments, the humanized anti-Trop2 antibody of the present invention, its antigen-binding fragment, or a mutant thereof does not cross-bind with other proteins of the same protein family.

[0570] In some embodiments, the humanized anti-Trop2 antibody of the present invention, its antigen-binding fragment, or a mutant thereof does not cross-bind with EpCAM of the same protein family.

[0571] In some embodiments, the humanized anti-Trop2 antibody of the present invention, its antigen-binding fragment or a mutant thereof, has activity that promotes tumor cell endocytosis.

[0572] In some exemplary embodiments, flow cytometry is used to detect the average fluorescence signal intensity of cell PE, and endpoint mapping is used to detect the endocytic activity of Trop2 antibodies such as hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9.

[0573] In some exemplary embodiments, BxPC-3 cells were incubated with a mixture of the present invention's humanized anti-Trop2 antibody and labeled goat anti-human Fc secondary antibody. The mean fluorescence signal intensity of the cell PE was detected by flow cytometer and the endocytic activity of the Trop2 antibody was plotted.

[0574] In some exemplary embodiments, the average fluorescence signal intensity of PE measured in BxPC-3 cell samples incorporating the humanized anti-Trop2 antibodies of the present invention, such as hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9, was 187,500-272,500.

[0575] In some exemplary embodiments, the average fluorescence signal intensity of PE measured in BxPC-3 cell samples incorporating the humanized anti-Trop2 antibodies of the present invention, such as hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9, was higher than that measured in BxPC-3 cell samples incorporating the maternal chimeric antibody ch44E7.

[0576] In some embodiments, the humanized anti-Trop2 antibody of the present invention, its antigen-binding fragment or mutant thereof, has a stronger tumor cell endocytosis-promoting activity than the maternal chimeric antibody.

[0577] Functional characteristics of anti-Trop2 monoclonal antibodies

[0578] The anti-Trop2 antibody of the present invention can bind to human Trop2 protein with high affinity, can bind to human Trop2 protein expressed in the in vitro environment and on the cell surface, cross-binds Trop2 protein of cynomolgus monkey in vitro, and has no cross-binding activity against EpCAM of the same protein family.

[0579] The anti-Trop2 antibodies of the present invention can be characterized by their various physical properties in order to detect and / or distinguish their different categories.

[0580] The term "antibody that specifically binds to human Trop2" as used in this article refers to antibodies that bind to human Trop2 protein (and Trop2 protein that may be derived from one or more non-human species) but do not substantially bind to non-human Trop2 protein.

[0581] In some embodiments, the anti-Trop2 antibody is a chimeric antibody.

[0582] In some embodiments, the exemplary chimeric anti-Trop2 antibody ch44E7 of the present invention binds to human Trop2 protein with “high affinity,” that is, with a KD(M) of 3.68E-9 or less, more preferably 3.46E-9 or less, more preferably 3.16E-9 or less, more preferably 2.88E-9 or less, more preferably 2.66E-9 or less, more preferably 2.38E-9 or less, and more preferably 2.25E-9 or less, which is more than the affinity for human Trop2 protein.

[0583] In some embodiments, the anti-Trop2 antibody is a humanized antibody.

[0584] In some embodiments, the humanized anti-Trop2 antibodies hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9 of the present invention bind to human Trop2 protein with "high affinity," i.e., with a KD(M) of 2.52E-08 or less, more preferably 2.32E-08 or less, and even more preferably... The affinity to human Trop2 protein is selected as 1.98E-08 or less, more preferably 1.66E-08 or less, more preferably 1.28E-08 or less, more preferably 1.18E-08 or less, more preferably 1.05E-08 or less, more preferably 9.60E-09 or less, more preferably 6.32E-09 or less, and most preferably below 3.12E-09, which has an affinity for human Trop2 protein comparable to or higher than that of the parental chimeric antibody ch44E7.

[0585] "Basically not binding to non-human Trop2" means that the KD(M) value of binding to non-human Trop2 is 1.0E-06 or greater, more preferably 1.0E-05 or greater, more preferably 1.0E-04 or greater, more preferably 1.0E-03 or greater, and even more preferably 1.0E-02 or greater.

[0586] As used herein, “KD” is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods well established in the art. The preferred detection method for determining the antibody KD is by using surface plasmon resonance, preferably using a biosensor system such as the Biacore™ system. The terms “Kassoc” or “Ka”, as used herein, refer to the binding rate of a specific antibody-antigen interaction, while the terms “Kdis” or “Kd”, as used herein, refer to the dissociation rate of a specific antibody-antigen interaction.

[0587] In vitro binding of hTrop2

[0588] The binding specificity of the anti-Trop2 antibody of the present invention can be determined by in vitro assays, such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry.

[0589] In some embodiments, the anti-Trop2 antibody is a chimeric antibody.

[0590] In some embodiments, the chimeric anti-Trop2 antibody or its antigen-binding fragment can bind to Trop2 in both humans and cynomolgus monkeys at an ELISA concentration of about 0.90 nM or lower via EC50 specific binding.

[0591] In some embodiments, the anti-Trop2 antibody is a chimeric antibody, wherein the exemplary chimeric antibody ch44E7 can bind to the human Trop2 protein at an ELISA concentration of about no more than 0.86 (nM), or preferably about no more than 0.78 (nM), or preferably about no more than 0.65 (nM), or preferably about no more than 0.4 (nM), or preferably about no more than 0.36 (nM), or most preferably about no more than 0.24 (nM), with slightly weaker binding activity than the reference antibody huRS7.

[0592] In some embodiments, the anti-Trop2 antibody is a chimeric antibody, wherein the exemplary chimeric antibody ch44E7, as measured by ELISA, cross-binds with Trop2 in cynomolgus monkeys at a binding EC50 of no more than 0.22 (nM).

[0593] In some embodiments, the anti-Trop2 antibody is a humanized antibody.

[0594] In some embodiments, the humanized anti-Trop2 antibodies hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9 of the present invention bind specifically to recombinant human Trop2 protein in vitro via ELISA with EC50 at concentrations not higher than, or about 0.382 (nM), 0.370 (nM), or not higher than 0.339 (nM), or not higher than 0.318 (nM), or not higher than 0.260 (nM), or not higher than 0.220 (nM), or not higher than 0.198 (nM), or not higher than 0.152 (nM).

[0595] In some embodiments, the humanized anti-Trop2 antibodies hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9 of the present invention specifically bind to the Trop2 protein of cynomolgus monkeys in vitro via ELISA with EC50 at concentrations not exceeding 0.99 (nM), 0.85 (nM), 0.67 (nM), 0.55 (nM), 0.48 (nM), 0.40 (nM), 0.35 (nM), or 0.33 (nM).

[0596] The humanized anti-Trop2 antibodies hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8 and hu44E7-9 of the present invention do not cross-bind with mouse Trop2.

[0597] Combining Trop2 expressed on the cell surface

[0598] In some embodiments, the anti-Trop2 antibody is a chimeric antibody, wherein the exemplary chimeric antibody ch44E7 is found to bind to human Trop2 expressed on the CHO-K1 cell membrane at a binding EC50 of no more than about 8.6 (nM), or preferably no more than about 7.80 (nM), or preferably no more than about 6.50 (nM), or preferably no more than about 5.08 (nM), or preferably no more than about 4.05 (nM), or most preferably no more than 3.80 (nM), as measured by FACS.

[0599] In some embodiments, the anti-Trop2 antibody is a humanized antibody.

[0600] In some embodiments, FACS measurements of the humanized anti-Trop2 antibodies hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9 of the present invention showed values ​​not exceeding 14.60 (nM), or not exceeding 13.50 (nM), or not exceeding 12.35 (nM), or not exceeding 10 (nM). EC50 binds specifically to the human Trop2 protein on the surface of CHO-K1 cells in vitro at concentrations of 0.80 (nM), or no higher than 10.20 (nM), or no higher than 9.50 (nM), or no higher than 8.05 (nM), or no higher than 7.95 (nM), or no higher than 7.35 (nM), or no higher than 6.25 (nM), or no higher than 5.96 (nM), or no higher than 5.26 (nM).

[0601] It does not cross-bind with EpCAM, a protein in the same family.

[0602] In some embodiments, the exemplary chimeric anti-Trop2 antibody ch44E7 and the reference antibody huRS7 of the present invention cannot bind to the recombinant human EpCAM protein.

[0603] In some embodiments, the exemplary humanized anti-Trop2 antibodies hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9, as well as the reference antibody huRS7, cannot bind to the recombinant human EpCAM protein.

[0604] In some embodiments, the exemplary humanized anti-Trop2 antibody of the present invention further promotes the endocytic activity of tumor cells.

[0605] In some embodiments, the exemplary humanized anti-Trop2 antibodies hu44E7-1, hu44E7-2, hu44E7-3, hu44E7-4, hu44E7-5, hu44E7-6, hu44E7-7, hu44E7-8, and hu44E7-9 of the present invention have endocytic activity.

[0606] In some embodiments, the average fluorescence signal intensity of the PE cells generated by the endocytosis of the humanized anti-Trop2 antibody of the present invention, as determined by the FACS endpoint assay, is 170,000-2,750,000.

[0607] Functional activity of anti-Trop2 / HER2 bispecific antibody

[0608] The anti-Trop2 / HER2 bispecific antibody of the present invention has one or more of the following activities:

[0609] (1) It can bind to human and monkey Trop2 protein;

[0610] (2) It binds with high affinity to human Trop2 and human HER2 in vitro, respectively;

[0611] (3) Simultaneously binds to Trop2 on the surface of Trop2-positive cells and / or HER2 expressed on the surface of HER2-positive cells;

[0612] (4) It has the activity of promoting tumor cell endocytosis, and its endocytosis activity is higher than that of the parent monoclonal antibody hu44E7-3 and Herceptin. The two binding arms have a synergistic effect in terms of endocytosis activity.

[0613] (5) It can inhibit the growth and proliferation of Trop2-positive and / or HER2-positive tumors in humans and animals. (6) It has pM-level Trop2-dependent killing activity against Trop2-positive tumor cells.

[0614] In some embodiments, the anti-Trop2 / HER2 bispecific antibody of the present invention can bind to human Trop2-His and human HER2 in vitro, respectively.

[0615] In some exemplary embodiments, the EC50 of the anti-Trop2 / HER2 bispecific antibody binding to recombinant human Trop2 in vitro, as measured by ELISA, is approximately no higher than 2.08 (nM), or approximately no higher than 1.96 (nM), or approximately no higher than 1.85 (nM), or approximately no higher than 1.76 (nM), or approximately no higher than 1.64 (nM), or approximately no higher than 1.52 (nM), or approximately no higher than 1.45 (nM), which is weaker than the parental humanized anti-Trop2 antibody hu44E7-3, differing by nearly 20 times.

[0616] In some exemplary embodiments, the EC50 of the anti-Trop2 / HER2 bispecific antibody binding to recombinant human Her2 protein in vitro, as measured by ELISA, is approximately no higher than 0.145 (nM), or approximately no higher than 0.136 (nM), or approximately no higher than 0.124 (nM), or approximately no higher than 0.112 (nM), or approximately no higher than 0.104 (nM), or approximately no higher than 0.092 (nM), or approximately no higher than 0.086 (nM), which is quite close to the EC50 of Herceptin binding to HER2.

[0617] In some exemplary embodiments, the anti-Trop2 / HER2 bispecific antibody has comparable binding activity to the human HER2 protein and to the parent antibody of the HER2 binding arm.

[0618] In some embodiments, the anti-Trop2 / HER2 bispecific antibody of the present invention binds to cells expressing Trop2 and cells expressing human HER2.

[0619] In some embodiments, the anti-human Trop2 / HER2 bispecific antibody can bind to tumor cells expressing human Trop2 and HER2 on their cell surface.

[0620] In some embodiments, the anti-human Trop2 / HER2 bispecific antibody can bind to cell lines that highly express Trop2 and low express Her2.

[0621] In some embodiments, the Trop2-overexpressing and Her2-underexpressing cell line is MDA-MB-468 cells.

[0622] In some embodiments, the EC50 of the anti-human Trop2 / HER2 bispecific antibody bound to MDA-MB-468 cells, as determined by FACS, is approximately no higher than 145 nM.

[0623] In some embodiments, the binding activity of the anti-human Trop2 / HER2 bispecific antibody to Trop2-high and Her2-low expression cell lines, as measured by FACS, is weaker than that of the anti-Trop2 parental antibody hu44E7-3, but stronger than that of the anti-HER2 parental antibody Herceptin.

[0624] In some embodiments, the anti-human Trop2 / HER2 bispecific antibody can bind to cell lines that express low levels of Trop2 and high levels of Her2. In some embodiments, the Trop2-low and Her2-high cell line is HCC1569.

[0625] In some embodiments, the EC50 of the anti-human Trop2 / HER2 bispecific antibody bound to HCC1569 cells, as determined by FACS, is approximately no higher than 7.720 nM.

[0626] In some embodiments, FACS analysis showed that the binding activity of the anti-human Trop2 / HER2 bispecific antibody to Trop2-low and Her2-high expression cell lines was significantly stronger than that of the anti-Trop2 parental antibody hu44E7-3 and slightly weaker than that of the anti-HER2 parental antibody Herceptin, but the binding amount was higher than that of the anti-HER2 parental antibody Herceptin.

[0627] In some embodiments, the anti-human Trop2 / HER2 bispecific antibody can bind to cell lines that highly express Trop2 and Her2.

[0628] In some embodiments, the Trop2-overexpressing and Her2-overexpressing cell line is the HCC1954 cell line.

[0629] In some embodiments, the EC50 of the anti-human Trop2 / HER2 bispecific antibody bound to the HCC1954 cell line, as determined by FACS, is approximately no higher than 9.70 nM.

[0630] In some embodiments, the EC50 of the binding of the anti-human Trop2 / HER2 bispecific antibody to Trop2-high and Her2-high cell lines, as determined by FACS, is slightly higher than that of the parental antibodies hu44E7-3 and Herceptin, but the binding amount is also higher.

[0631] In some embodiments, the anti-Trop2 / HER2 bispecific antibody has high affinity for both Trop2 and HER2 in vitro.

[0632] In some embodiments, the equilibrium dissociation constants (KD) of the Trop2×Her2bsAb antibody binding to human Trop2 protein and human Her2 protein are determined using biofilm thin-layer interferometry (BLI).

[0633] In some embodiments, the KD(M) of the Trop2×Her2 bsAb antibody of the present invention binding to human HER2 protein, as measured by the BLI method, is approximately no higher than 2.46E-09, or approximately no higher than 2.38E-09, or approximately no higher than 2.25E-09, or approximately no higher than 2.18E-09, or approximately no higher than 2.08E-09, with the most preferred value being no higher than 2.01E-09, which is comparable to the parent antibody Herceptin.

[0634] In some embodiments, the KD(M) of the Trop2×Her2 bsAb antibody of the present invention binding to human Trop2 protein, as measured by the BLI method, is approximately no higher than 2.42E-08, or approximately no higher than 2.36E-09, or approximately no higher than 2.25E-08, or approximately no higher than 1.18E-08, or approximately no higher than 1.08E-08, with the most preferred value being no higher than 1.05E-08, which is comparable to the parent antibody hu44E7-3.

[0635] In some embodiments, the anti-Trop2 / HER2HER2 bispecific antibody has endocytic activity against tumor cells.

[0636] In some embodiments, the endocytic activity of the antibody of the present invention is evaluated by measuring the average PE fluorescence value of the Trop2×Her2 bsAb bispecific antibody in NCI-N87 cells using the FACS endpoint method.

[0637] In some exemplary embodiments, an endpoint method is used to detect the endocytic activity of the Trop2×Her2 bsAb bispecific antibody and the parent monoclonal antibody of the present invention. Specific implementation methods of the endpoint method are described in the examples.

[0638] In some embodiments, the anti-Trop2 / HER2 bispecific antibody has endocytic activity in NCI-N87 cells.

[0639] In some implementations, the average PE fluorescence value of the Trop2×Her2 bsAb bispecific antibody of the present invention, measured by the endpoint method using FACS, was approximately 250,000 to 280,000 during endocytosis detection in NCI-N87 cells, which is higher than that of the parental monoclonal antibodies Herceptin and hu44E7-3.

[0640] In some embodiments, the anti-Trop2 / HER2 bispecific antibody exhibits endocytic activity in HCC1954 cells.

[0641] In some implementations, the average PE fluorescence value of the Trop2×Her2 bsAb bispecific antibody of the present invention, measured by the endpoint method using FACS, was approximately 400,000 to 450,000 during endocytosis detection in HCC1954 cells, which is higher than that of the parental monoclonal antibodies Herceptin and hu44E7-3.

[0642] In some embodiments, the Trop2-binding arm and HE HER2-binding arm of the anti-Trop2 / HER2 bispecific antibody have a synergistic effect in terms of endocytic activity.

[0643] Functional activity of Trop2 / HER2 bispecific antibody-drug conjugate

[0644] In one exemplary embodiment, the Trop2 / HER2 bispecific ADC of the present invention is a Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE.

[0645] In some embodiments, the Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE of the present invention has the following biological activities:

[0646] (1) Specifically binds to Trop2 and HER2 in vitro;

[0647] (2) It can specifically bind to Trop2 and HER2-positive cells;

[0648] (3) It has killing activity against Trop-positive and HER2-positive cells in vitro;

[0649] (4) It has an inhibitory effect on tumor cell growth in animal models of subcutaneous transplantation of tumor cell lines.

[0650] In vitro killing activity against tumor cells

[0651] In some embodiments, the Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE of the present invention, which specifically binds to Trop2 and HER2, has a strong killing effect on Trop2 and HER2 double-positive tumor cells.

[0652] In some embodiments, the Trop2 and HER2 double-positive tumor cells are tumor cells with high Her2 expression and moderate Trop2 expression.

[0653] In some embodiments, the Her2-overexpressing, Trop2-medium-expressing tumor cells are human gastric cancer cells.

[0654] In some embodiments, the tumor cells with high Her2 expression and moderate Trop2 expression are NCI-N87 cells.

[0655] In some embodiments, the Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE inhibits NCI-N87 cells with an IC50 of approximately no higher than 1.056 (nM), or preferably no higher than 0.918 (nM), or preferably no higher than 0.812 (nM), or preferably no higher than 0.805 (nM), or preferably no higher than 0.793 (nM), or most preferably no higher than 0.762 (nM), which is superior to parental monoclonal antibody ADCs such as Herceptin-ADC and hu44E7-3-ADC.

[0656] In some embodiments, in the in vitro killing activity assay, the survival rate of NCI-N87 cells after adding the Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE was 20-30%, which was lower than the survival rate of NCI-N87 cells after adding parental monoclonal antibodies to ADCs such as Herceptin-ADC and hu44E7-3-ADC, respectively. Furthermore, the rate of decrease in NCI-N87 cell survival rate caused by Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE was higher than the rate of decrease in NCI-N87 cell survival rate caused by parental antibody hu44E7-3-ADC.

[0657] In some embodiments, the Trop2 and HER2 double-positive tumor cells are tumor cells with very low Her2 expression levels and high Trop2 expression.

[0658] In some embodiments, the tumor cells with very low Her2 expression levels and high Trop2 expression are human breast cancer cells.

[0659] In some embodiments, the tumor cells with very low Her2 expression levels and high Trop2 expression are MDA-MB-468 cells.

[0660] In some embodiments, the Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE inhibits MDA-MB-468 cells with an IC50 of approximately no higher than 23.89 (nM), or preferably no higher than 23.56 (nM), or preferably no higher than 22.98 (nM), or preferably no higher than 22.76 (nM), or preferably no higher than 22.52 (nM), or most preferably no higher than 22 (nM), which is significantly superior to parental monoclonal antibody ADCs such as Herceptin-ADC.

[0661] In some embodiments, in the in vitro killing activity assay, the survival rate of MDA-MB-468 cells after adding the Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE was 10-15%, which was comparable to the survival rate of MDA-MB-468 cells after adding hu44E7-3-ADC, but lower than the survival rate of MDA-MB-468 cells after adding the parental monoclonal antibody Herceptin-ADC; and the rate of decrease in MDA-MB-468 cell survival rate caused by Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE was higher than the rate of decrease in MDA-MB-468 cell survival rate caused by the survival rate of MDA-MB-468 cells after adding the parental antibody hu44E7-3-ADC.

[0662] In some embodiments, the Trop2 and HER2 double-positive tumor cells are tumor cells with very low Trop2 expression levels and high HER2 expression levels.

[0663] In some embodiments, the tumor cells with very low Trop2 expression levels and high HER2 expression are human breast cancer cells.

[0664] In some embodiments, the tumor cells with very low Trop2 expression levels and high HER2 expression are HCC1569 breast cancer cells.

[0665] In some embodiments, the Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE inhibits HCC1569 cells with an IC50 of approximately no higher than 6.36 (nM), or preferably no higher than 6.28 (nM), or preferably no higher than 6.12 (nM), or preferably no higher than 5.98 (nM), or preferably no higher than 5.86 (nM), or most preferably no higher than 5.75 (nM), which is significantly better than hu44E7-3-ADC and comparable to Herceptin-ADC.

[0666] In some embodiments, in the in vitro killing activity assay, the survival rate of HCC1569 cells after adding the Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE was 25%-35%, which was comparable to the survival rate of MDA-MB-468 cells after adding the parental monoclonal antibody Herceptin-ADC, but lower than the survival rate of MDA-MB-468 cells after adding the parental monoclonal antibody hu44E7-3-AD; and the rate of decrease in MDA-MB-468 cell survival rate caused by Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE was higher than the rate of decrease in MDA-MB-468 cell survival rate caused by the survival rate of MDA-MB-468 cells after adding the parental antibody hu44E7-3-ADC.

[0667] In some implementations, in NCI-N87 cells with high Her2 expression and moderate Trop2 expression, Trop2×Her2bsADC showed comparable and stronger cytotoxic activity to Herceptin-MMAE than hu44E7-3-MMAE.

[0668] In some implementations, in triple-negative breast cancer cells with very low Her2 expression levels, the killing effects, from highest to lowest, were hu44E7-3-MMAE, Trop2×Her2 bsADC, and Herceptin-MMAE.

[0669] In some implementations, in HCC1569 breast cancer cells with low Trop2 expression, the killing effects, from highest to lowest, were Herceptin-MMAE, Trop2×Her2 bsADC, and hu44E7-3-MMAE.

[0670] In some implementations, the Trop2×Her2 bsADC of the present invention has broader applicability to a variety of tumor cells compared to anti-Trop2 monospecific and HER2 monospecific maternal monoclonal antibody ADCs.

[0671] In some embodiments, the anti-Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE of the present invention can inhibit tumor growth and proliferation in animal models of Trop2-positive and HER2-positive tumors.

[0672] In some embodiments, the anti-Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE of the present invention can inhibit tumor growth and proliferation in a mouse model of breast cancer.

[0673] In some embodiments, the anti-Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE of the present invention can inhibit tumor growth and proliferation in a mouse tumor animal model of NCI-N87 human gastric cancer cells.

[0674] Tumor growth inhibition value (TGI) is used to evaluate the inhibitory effect of a test drug on tumor growth in vivo (i.e., in animal experiments). The TGI value is calculated using formula (I).

[0675] TGI = (1 - tumor weight in the treatment group / tumor weight in the control group) * 100%, (I).

[0676] In some embodiments, administration of the present invention's anti-Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE at a dose level of 0.1-10 mg / mL in animals inhibited the growth and proliferation of NCI-N87 human gastric cancer cells in a mouse tumor animal model of NCI-N87 human gastric cancer cells.

[0677] In some embodiments, administration of the present invention's anti-Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE at a dose level of 0.5-5 mg / mL to animals inhibited the growth and proliferation of NCI-N87 human gastric cancer cells in a mouse tumor animal model.

[0678] In some embodiments, administration of the present invention's anti-Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE at a dose level of 1-3 mg / mL in animals inhibited the growth and proliferation of NCI-N87 human gastric cancer cells in a mouse tumor animal model of NCI-N87 human gastric cancer cells.

[0679] In some embodiments, administration of the present invention's anti-Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE at a dose of 2 mg / mL in vivo inhibited the growth and proliferation of NCI-N87 human gastric cancer cells in a mouse tumor animal model of NCI-N87 human gastric cancer cells.

[0680] In some embodiments, the TGI of the anti-Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE of the present invention after 6 doses in the NCI-N87 model is 141%, which is close to the TGI of parental monoclonal antibody ADCs such as hu44E7-3-MMAE and Trop2×Her2 bsADC.

[0681] In some embodiments, the in vivo antitumor efficacy of the anti-Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE of the present invention in the NCI-N87 model is close.

[0682] Security of Trop2×Her2bsADC

[0683] In some embodiments, the anti-Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE of the present invention has no effect on the weight of animals and is comparable to Herceptin-MMAE and hu44E7-3-MMAE.

[0684] In some embodiments, no significant hematologic toxicity was observed with the anti-Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE of the present invention.

[0685] In some embodiments, the blood routine parameters such as neutrophil percentage, lymphocyte percentage and red blood cell count of the NCI-N87 model treated with the anti-Trop2 / HER2 dual antibody-MC-VC-PAB-MMAE of the present invention are comparable to those of the model treated with Herceptin-MMAE and hu44E7-3-MMAE.

[0686] In some embodiments, the anti-Trop2 / HER2 bispecific antibody-MC-VC-PAB-MMAE of the present invention causes varying degrees of elevation of gamma-glutamyl transferase, alanine aminotransferase, creatine kinase, and aspartate aminotransferase in a human gastric cancer subcutaneous xenograft animal model. Among these, the effect of Trop2×Her2 bsADC is less than that of Herceptin-MMAE and hu44E7-3-MMAE.

[0687] Pharmaceutical Composition

[0688] In one aspect, the present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable effective dose of an anti-Trop2 antibody or an antigen-binding fragment thereof, or a mutant or derivative thereof, of the foregoing aspects of the present invention as an active ingredient, or an anti-Trop2 / HER2 bispecific antibody of the present invention, or an anti-Trop2 / HER2 bispecific antibody-drug conjugate of the present invention, and a pharmaceutically acceptable carrier.

[0689] "Pharmaceuticalally acceptable effective dose" refers to the dose listed in a pharmacopoeia or other recognized pharmacopoeia for use in animals, especially for humans.

[0690] "Pharmaceutical acceptable carriers" are generally non-toxic solid, semi-solid, or liquid fillers, diluents, encapsulating materials, or any type of excipients that are well-known to those skilled in the art of pharmacy.

[0691] In some embodiments, the pharmaceutical composition formulation of the present invention may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0692] Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high concentrations of drugs.

[0693] In some embodiments, the composition is formulated according to conventional procedures to be a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic buffer solution. These components are usually provided individually or mixed together in unit dosage forms, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active agent. When administered by infusion, the composition can be prepared with an infusion bottle containing sterile pharmaceutical-grade water or physiological saline. When administered by injection, a single ampoule of sterile water for injection or physiological saline can be provided, allowing for mixing prior to administration.

[0694] The dosage of the pharmaceutical composition formulation of the present invention can be specifically determined by the clinician based on the reduction of the severity of disease symptoms, the increase of the frequency and duration of the asymptomatic period, or the ability to prevent damage or incapacitation caused by the disease.

[0695] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or bolus). Depending on the route of administration, the active ingredient may be encapsulated in a material to protect it from acids and other natural conditions that may inactivate it; alternatively, the antibody of the present invention may be administered via a non-parenteral route, such as topical, epidermal, or mucosal administration, or local application.

[0696] In some embodiments, the pharmaceutical compositions of the present invention may also be further manufactured as sustained-release agents, including implants, transdermal patches, and microcapsule delivery systems.

[0697] In some embodiments, the pharmaceutical composition may be administered via medical devices, such as (1) needle-free subcutaneous injection devices; (3) transdermal drug delivery devices; and (5) permeation devices.

[0698] Applications in the preparation of antibody-drug conjugates

[0699] In some embodiments, the anti-Trop2 antibody described in the foregoing aspects of the present invention, its antigen-binding fragment or variant, and / or the anti-Trop2 / HER2 bispecific antibody described in the foregoing aspects, its antigen-binding fragment, can be used to prepare antibody-drug conjugates.

[0700] In some embodiments, the antibody-drug conjugate may comprise an anti-Trop2 antibody or an anti-Trop2 / HER2 bispecific antibody as described above, and a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG conjugated to the anti-Trop2 antibody or the anti-Trop2 / HER2 bispecific antibody.

[0701] In some embodiments, those skilled in the art can crosslink the anti-Trop2 antibody or anti-Trop2 / HER2 bispecific antibody of the present invention with a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalating agents, DNA crosslinking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents, preferably SN-38.

[0702] In ADCs, antibodies and therapeutic agents are preferably cross-linked via a cleavable linker, such as a peptide linker, disulfide linker, or hydrazone linker. More preferably, the linker is a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu.

[0703] In some embodiments, the anti-Trop2 / HER2 bispecific antibody may bind to or fuse with a therapeutic agent, wherein the therapeutic agent may include a detectable marker, such as a radioactive marker, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent (which may be a drug or a toxin), an ultrasound enhancer, or a non-radioactive marker.

[0704] In some embodiments, the therapeutic agent is a cytotoxic agent (which may be a drug or a toxin).

[0705] The technique of conjugating different therapeutic agents to antibodies is well known to those skilled in the art.

[0706] Using the aforementioned anti-Trop2 antibody or anti / Trop2 / HER2 bispecific antibody, those skilled in the art can detectably label it by conjugating it with a chemiluminescent compound, and then determine the presence of the chemiluminescently labeled antigen-binding peptide by detecting the luminescence that occurs during the chemical reaction.

[0707] Uses in the preparation of drugs for the prevention and treatment of cancer.

[0708] On the one hand, the present invention provides the use of the aforementioned anti-Trop2 antibody, variant or derivative thereof, anti-Trop2 / HER2 bispecific antibody or its antigen-binding fragment, antibody-drug conjugate or composition in the preparation of medicaments for the prevention and treatment of certain tumors or cancers.

[0709] In some embodiments, the present invention provides the anti-Trop2 antibody described in the foregoing aspects and / or the anti-Trop2 / HER2 bispecific antibody described in the foregoing aspects for the preparation of a medicament for the prevention or treatment of diseases related to Trop2 overexpression or Trop2 and HER2 overexpression or dysfunction, or diseases targeting Trop2 or Trop2 and HER2.

[0710] In some embodiments, the present invention provides the anti-Trop2 antibody described in the foregoing aspects and / or the anti-Trop2 / HER2 bispecific antibody described in the foregoing aspects, which are used to prepare a drug for killing cells that overexpress Trop2 or simultaneously overexpress Trop2 and HER2.

[0711] The coding sequences of anti-Trop2 monoclonal antibodies and anti-Trop2 / HER2 bispecific antibodies, nucleic acid constructs, anti-Trop2 / HER2 bispecific antibody ADCs and cells, and compositions comprising the products described herein can all be used to prepare drugs for the prevention or treatment of the various conditions and diseases described herein, wherein the conditions and diseases are diseases or conditions related to abnormal expression of Trop2 or Trop2 and HER2, and are diseases directly or indirectly caused by abnormal expression of Trop2 or abnormal dual targets of Trop2 and HER2, generally referring to diseases directly caused by Trop2 overexpression or abnormal Trop2 and HER2, such as cancer or tumors.

[0712] In some embodiments, the tumor with Trop2 overexpression or dysfunction, or with abnormal overexpression of Trop2 and HER2, is preferably a solid tumor.

[0713] In some embodiments, the solid tumors include, but are not limited to, breast cancer, colorectal cancer, gastric adenocarcinoma, esophageal cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, ovarian epithelial cancer, prostate cancer, pancreatic ductal adenocarcinoma, head and neck cancer, squamous cell carcinoma, renal cell carcinoma, bladder tumor, cervical cancer, endometrial cancer, follicular thyroid cancer, and glioblastoma multiforme.

[0714] In some implementations, the Trop2 overexpressing cancers include any one or more of the following cancers: thyroid cancer, papillary carcinoma, breast cancer, squamous cell carcinoma of the lung, endometrial cancer, prostate cancer, colon cancer, lung adenocarcinoma, ovarian epithelial cancer (59%), gastric cancer, pancreatic cancer, and non-small cell lung cancer.

[0715] In some implementations, the Trop2 overexpressing cancers include any one or more of these cancers, such as breast cancer, gastric cancer, pancreatic cancer, and non-small cell lung cancer.

[0716] In some embodiments, the Trop2 and HER2 overexpressing cancers are thyroid cancer, urothelial carcinoma, breast cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, lung cancer, endometrial cancer, skin cancer, stomach cancer, esophageal cancer, pancreatic cancer, prostate cancer, liver cancer, lymphoma, or glioma.

[0717] In some embodiments, the Trop2 and HER2 overexpressing cancers are cervical cancer, prostate cancer, thyroid cancer, urothelial carcinoma, head and neck cancer, endometrial cancer, ovarian cancer, lung cancer, breast cancer, carcinoid cancer, skin cancer, liver cancer, or testicular cancer.

[0718] In some implementations, the Trop2 and HER2 overexpressing cancers are pancreatic cancer, lung cancer, gastric cancer, prostate cancer, and breast cancer.

[0719] Treatment methods and usage

[0720] In some embodiments, the anti-Trop2 antibody, its antigen-binding fragment or mutant thereof, or the anti-Trop2 / HER2 bispecific antibody or its antigen-binding fragment thereof of the present invention can be used as a treatment for diseases with Trop2 overexpression or dysfunction.

[0721] In some implementations, the disease characterized by Trop2 overexpression or dysfunction is cancer, in which Trop2 is overexpressed in tumor cells.

[0722] In some implementations, the treatment method requires administering an effective amount of the invented antibody to the patient.

[0723] In some embodiments, the present invention provides a method for treating diseases associated with Trop2 overexpression or dysfunction, or Trop2-targeted cancers, in patients in need.

[0724] In some embodiments, the present invention provides a method for treating diseases associated with Trop2 and HER2 overexpression or dysfunction, or cancers targeting both Trop2 and HER2, in patients in need.

[0725] In some implementations, the Trop2 overexpressing cancers include any one or more of the following cancers: thyroid cancer, papillary carcinoma, breast cancer, squamous cell carcinoma of the lung, endometrial cancer, prostate cancer, colon cancer, lung adenocarcinoma, ovarian epithelial cancer (59%), gastric cancer, pancreatic cancer, and non-small cell lung cancer.

[0726] In some implementations, the Trop2 overexpressing cancers include any one or more of these cancers, such as breast cancer, gastric cancer, pancreatic cancer, and non-small cell lung cancer.

[0727] In some embodiments, the Trop2 and HER2 overexpressing cancers are thyroid cancer, urothelial carcinoma, breast cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, lung cancer, endometrial cancer, skin cancer, stomach cancer, esophageal cancer, pancreatic cancer, prostate cancer, liver cancer, lymphoma, or glioma.

[0728] In some embodiments, the Trop2 and HER2 overexpressing cancers are cervical cancer, prostate cancer, thyroid cancer, urothelial carcinoma, head and neck cancer, endometrial cancer, ovarian cancer, lung cancer, breast cancer, carcinoid cancer, skin cancer, liver cancer, or testicular cancer.

[0729] In some implementations, the Trop2 and HER2 overexpressing cancers are pancreatic cancer, lung cancer, stomach cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, skin cancer, or brain cancer.

[0730] In some embodiments, the treatment methods further involve the administration of the anti-Trop2 antibody, anti-Trop2 / HER2 bispecific antibody, or anti-Trop2 / HER2 bispecific ADC of the present invention to a patient (e.g., a human or tumor treatment animal model) to treat one or more of the cancers or tumor diseases or conditions described herein with abnormal expression of Trop2 or Trop2 and HER2.

[0731] In one implementation, the treatment involves administering an effective amount of an anti-Trop2 antibody, an anti-Trop2 / HER2 bispecific antibody, or an anti-Trop2 / HER2 bispecific ADC to the patient.

[0732] In some implementations, at least one of the patient's cancer cells (e.g., stromal cells) expresses, overexpresses, or is induced to express the Trop2 tumor antigen.

[0733] In some implementations, at least one of the patient's cancer cells (e.g., stromal cells) express, overexpress, or are induced to express Trop2 and HER2 tumor antigens.

[0734] Methods of administration of formulations comprising the anti-Trop2 antibody, antigen-binding fragment, mutant, or anti-Trop2 / HER2 bispecific antibody or its anti-Trop2 / HER2 ADC or anti-Trop2 / HER2 bispecific antibody or its anti-Trop2 / HER2 ADC include, but are not limited to, parenteral routes such as intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous. The formulation can be administered via any convenient route, such as by infusion or rapid concentration, absorbed through the epithelial or mucosal skin layer (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding peptides of this disclosure can be administered parenterally. As used herein, the term "parenteral" refers to administration methods including, but not limited to, intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0735] The anti-Trop2 antibody, encoding nucleic acid, or antibody-drug conjugate of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as associated cytokines or cell populations. In this regard, the pharmaceutical composition can be prepared by mixing an active pharmaceutical agent of desired purity with an optional pharmaceutically acceptable carrier in the form of a lyophilized formulation or an aqueous solution. The pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and may include at least one of buffers (e.g., neutral buffered saline, sulfate buffered saline), antioxidants, preservatives, isotonic agents, stabilizers, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), and surfactants. Furthermore, for the pharmaceutical composition to be used for in vivo administration, it must be sterile. The pharmaceutical composition can be sterilized by filtration through a sterile filter membrane.

[0736] In the treatment of tumors that overexpress or are induced to express Trop2, or tumors that simultaneously and abnormally overexpress Trop2 and HER2, the specific dosage and dosing regimen for any particular patient will depend on a variety of factors. The dosage and treatment regimen of the Trop2 antibody, anti-Trop2 / HER2 bispecific antibody or its anti-Trop2 / HER2 ADC, or composition of the present invention administered during treatment can be determined by those skilled in the art and clinicians based on factors including the specific antibody used, its variant or derivative, the patient's age, weight, general health condition, sex and diet, as well as the timing of administration, excretion rate, drug combination and the severity of the specific disease being treated, combined with the individual patient to be treated, route of administration, type of formulation, characteristics of the compound used, severity of the disease and expected effect, and in conjunction with pharmacological and pharmacokinetic principles well known in the art.

[0737] "Therapeutic effective dose" or "therapeutic effective amount" refers to the amount of a therapeutic agent applied that will alleviate one or more symptoms of the disease being treated to a certain extent.

[0738] The amount of antibody active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the target population and the specific route of administration, and is generally the amount of the composition that produces a therapeutic effect. Typically, in one hundred percent, this amount will range from about 0.01% to about 99% of the active ingredient combined with a pharmaceutically acceptable carrier. The composition can be administered in any convenient manner.

[0739] Skilled practitioners and clinicians in the art can adjust the dosing regimen to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus injection can be administered, several separate doses can be administered over time, or the dose can be proportionally reduced or increased depending on the urgency of the treatment situation. Parenteral compositions can be formulated into dosing units to facilitate administration and uniformity of dosage.

[0740] The effective therapeutic dose can be initially estimated using the IC50 determined from cell culture experiments. The dose can then be established in animal models to achieve a range of circulating plasma concentrations, including the IC50 value determined in cell culture experiments. This information can be used to more accurately determine the useful dose in humans. The specific composition, route of administration, and dosage can be selected by the physician based on the patient's condition.

[0741] Dosing regimens can be adjusted to achieve the desired optimal response (e.g., therapeutic or preventative response). For example, a single bolus injection can be administered, several separate doses can be administered over time (multiple, repeated, or maintenance), and the dose can be proportionally reduced or increased depending on the treatment outcome. Formulating non-gastrointestinal administration formulations into unit dosage units is particularly advantageous for ease of administration and maintaining dosage consistency. The unit dosage unit form herein refers to a physically discrete unit suitable as a single dose for treating mammalian subjects; each unit dose contains a predetermined amount of active compound calculated to achieve the desired therapeutic effect, along with the required drug carrier. The specification of the unit dosage unit form of this disclosure will be primarily determined by the unique characteristics of the antibody and the specific therapeutic or preventative effect to be achieved.

[0742] Therefore, those skilled in the art will understand that, based on the disclosure provided herein, dosages and dosing regimens are adapted according to methods well-known in the therapeutic field. That is, the maximum tolerable dose can be readily determined, and the effective dose can be determined if it provides a detectable therapeutic benefit to the subject, as can the required timing of administration of each agent to provide a detectable therapeutic benefit to the subject. Therefore, while certain dosages and dosing regimens are illustrated herein, these examples do not in any way limit the dosages and dosing regimens that may be provided to subjects when implementing this disclosure.

[0743] In various embodiments, the antibody or antigen-binding fragment thereof disclosed herein may be administered at approximately 1-200 mg / subject, 1-150 mg / subject, or 1-100 mg / subject. The total monthly dose may be administered as a single dose or in divided doses, and may exceed the typical range given herein at the physician's discretion.

[0744] In some embodiments, the construct is administered to a subject at a dose of at least about 5 mg / kg. In some embodiments, the construct is administered to a human at a dose equivalent to at least 5 mg / kg in mice.

[0745] In various embodiments, the total dose administered will result in plasma antibody concentrations within the following ranges: for example, about 1 to 1000 μg / ml, about 1 to 750 μg / ml, about 1 to 500 μg / ml, about 1 to 250 μg / ml, about 10 to 1000 μg / ml, about 10 to 750 μg / ml, about 10 to 500 μg / ml, about 10 to 250 μg / ml, about 20 to 1000 μg / ml, about 20 to 750 μg / ml, about 20 to 500 μg / ml, about 20 to 250 μg / ml, about 30 to 1000 μg / ml, about 30 to 750 μg / ml, about 30 to 500 μg / ml, about 30 to 250 μg / ml.

[0746] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated, and may include single or multiple doses. It should be further understood that for any given subject, the specific dosage regimen should be adjusted over time based on individual needs and the professional judgment of the person responsible for or supervising the administration, and the dosage ranges described herein are exemplary and not intended to limit the scope or application of the claimed compositions. Furthermore, the dosing regimen of the compositions disclosed herein can be based on a variety of factors, including disease type, subject age, weight, sex, subject's medical condition, disease severity, route of administration, and the specific antibody used. Therefore, dosing regimens can vary considerably but can be routinely determined using standard methods. For example, the dosage can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory test results. Therefore, this disclosure includes a gradual escalation of the subject's dosage as determined by a skilled technician. Determining appropriate dosages and regimens is well known in the relevant art, and those skilled in the art will be able to understand and apply these methods once the teachings disclosed herein are received.

[0747] The toxicity and therapeutic index of the pharmaceutical compositions of this application can be determined in cell culture or laboratory animals using standard pharmaceutical procedures, for example, to determine the LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective to 50% of the population). The ratio of the toxic dose to the therapeutically effective dose is called the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compositions with a larger therapeutic index are preferred.

[0748] For repeated dosing over several days or longer, depending on the condition, treatment continues until the desired symptom suppression is achieved or until a sufficient therapeutic level is reached, such as pain relief. Exemplary dosing regimens include an initial dose of about 2 mg / kg, followed by a maintenance dose of about 1 mg / kg of bispecific anti-HER2 / Trop2 antibody weekly, or a maintenance dose of about 1 mg / kg every other week. However, other dosing regimens may be useful, depending on the pharmacokinetic decay pattern the practitioner wishes to achieve. For example, in some embodiments, dosing from one to four times per week is considered. Progression of this therapy is easily monitored using standard techniques and detection methods. Dosing regimens (including the HER2 / Trop2 antagonist used) can vary over time. In various embodiments, the appropriate dose of the HER2 / Trop2 antagonist antibody will depend on the bispecific anti-HER2 / Trop2 antibody (or a combination thereof) used, the type and severity of the headache to be treated (e.g., migraine), whether the drug is used for preventative or therapeutic purposes, prior treatment, the patient's clinical history and response to the drug, and the attending physician's judgment. Typically, clinicians will administer bispecific anti-HER2 / Trop2 antibodies until the desired outcome is achieved. The dosage and / or frequency may vary throughout the treatment process.

[0749] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be relieved. It should be further understood that, for any given subject, the specific dosage regimen should be adjusted over time based on individual needs and the professional judgment of the person administering or supervising the administration of the pharmaceutical composition, and the dosage ranges described herein are exemplary and not intended to limit the scope or practice of the claimed compositions.

[0750] In various embodiments, the single or multiple administration of the pharmaceutical composition depends on the dose and frequency required and tolerated by the subject. In any case, the composition should provide a sufficient amount of at least one antibody disclosed herein or an antigen-binding fragment thereof to effectively treat the subject. This dose may be administered once, or it may be given periodically until a therapeutic outcome is achieved or until treatment needs to be discontinued due to side effects.

[0751] The frequency of administration of the antibody or its antigen-binding fragment pharmaceutical composition depends on the nature of the treatment and the specific disease being treated. Subjects may be treated periodically, such as weekly or monthly, until the desired therapeutic outcome is achieved. Exemplary dosing frequencies include, but are not limited to: once weekly without interruption; once weekly, every other week; once every two weeks; once every three weeks; once weekly for two consecutive weeks, then monthly; once weekly for three consecutive weeks, then monthly; once monthly; once every two months; once every three months; once every four months; once every five months; or once every six months, or once a year. Combination therapy

[0752] In another aspect, the present invention provides a combination therapy in which the pharmaceutical composition of the present invention is co-administered with one or more additional therapies that effectively inhibit tumor growth in a subject, said additional therapies including one or more of the following: immunotherapy, chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, vaccination regimens and stem cell transplantation, wherein the combination therapy provides increased tumor cell killing, i.e., when co-administered, there is a synergistic effect between the isolated antibody or antigen-binding fragment and the other therapy.

[0753] Immunotherapy options include treatment with agonist, antagonist, or blocker antibodies against co-stimulatory or co-inhibitory molecules (immune checkpoints) such as PD-1, PD-L1, OX-40, CD137, GITR, LAG3, TIM-3, and VISTA.

[0754] When the bispecific antibodies disclosed herein are administered in combination with another conventional antitumor agent, either concurrently or sequentially, such antibodies or antigen-binding fragments can enhance the therapeutic effect of the antitumor agent or overcome cellular resistance to such antitumor agents. This allows for a reduction in the dosage of the antitumor agent, thereby reducing undesirable side effects, or restoring the effectiveness of the antitumor agent in resistant T cells.

[0755] Many antitumor compounds can cause adverse side effects. It has been shown that when two or more different treatments are combined, these treatments can work synergistically and allow for a reduction in the dosage of each treatment, thereby reducing the harmful side effects produced by each compound at higher doses. In other cases, malignancies that are unresponsive to other treatments may respond to a combination of two or more different therapies.

[0756] In some implementations, the drugs that can be used in combination with antitumor therapy are chemical plasmid tumor compounds.

[0757] These chemotherapeutic antitumor compounds can be classified according to their mechanisms of action into the following categories: antimetabolites / anticancer agents, purine analogs, folic acid antagonists and related inhibitors, antiproliferative / antimitotic agents, microtubule disruptors, DNA damage agents, antiproliferative / antimitotic alkylating agents, antiproliferative / antimitotic antimetabolites, immunosuppressants, antiangiogenic compounds, corticosteroids, growth factor signal transduction kinase inhibitors, and mitochondrial dysfunction inducers.

[0758] In some embodiments, the present invention provides a method for inhibiting tumor growth in a subject, the method comprising administering to the subject a pharmaceutical composition of the present disclosure and one or more additional antibodies, including anti-LAG-3 antibodies, anti-PD-L1 antibodies, and anti-PD-1 antibodies. In some embodiments, the subject is a human. Trop2 pathway blockade may also be further combined with standard cancer treatment.

[0759] In some implementations, the subject is a human being.

[0760] In some embodiments, the pharmaceutical composition may contain at least one additive selected from: a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, a growth inhibitor, and an active pharmaceutical agent required for the specific indication to be treated. The specific amount of the additive may be adjusted as needed. Administration may include injection. The compositions described herein may be administered to patients subcutaneously, intradermally, intratumorally, intravenously, or intraperitoneally.

[0761] In one embodiment, the composition of the present invention is administered to a patient via intradermal or subcutaneous injection.

[0762] In other embodiments, the compositions of the present invention are preferably administered via intravenous injection. The T-cell compositions may be injected directly into the tumor site.

[0763] The dosage of the composition can range from about 0.0001 to 100 mg / kg. The "therapeuticly effective dose" of the anti-Trop2 antibody or its antigen-binding moiety, bispecific molecule, CAR-T cell, oncolytic virus, immunoconjugate, nucleic acid molecule, expression vector, or host cell administered in cancer treatment is preferably determined based on the need to reduce the severity of disease symptoms, increase the frequency and duration of asymptomatic periods, or prevent damage or disability caused by disease suffering. For example, for the treatment of tumor-bearing subjects, the "therapeuticly effective dose" preferably eliminates symptoms by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% relative to untreated subjects.

[0764] The therapeutic composition comprising the anti-Trop2 antibody or anti-Trop2 / HER2 bispecific antibody of the present invention can be administered via a medical device.

[0765] In some embodiments, the monoclonal antibodies of the present invention can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibodies of the present disclosure or their antigen-binding portions cross the blood-brain barrier, they can be formulated in liposomes, which may additionally contain a targeting portion to enhance selective transport to specific cells or organs.

[0766] The combination of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as a separate composition of each agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0767] Furthermore, if more than one dose of combination therapy is administered consecutively, the order of administration can be reversed or the same order can be maintained at each administration point. Sequential administration can be combined with simultaneous administration, or any combination thereof.

[0768] In this article, "anti-tumor effect" refers to a biological effect that can be represented by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various cancer-related physiological symptoms.

[0769] The terms "patient," "subject," and "individual" are used interchangeably in this article to refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans.

[0770] Although the invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the spirit and scope of the invention as defined by the appended claims, as detailed in the specific examples provided below, by combining Herceptin antibodies or Herceptin scFv with anti-Trop2 antibodies or anti-Trop2 scFv to generate highly effective bispecific antibodies. Many different bispecific antibody configurations have been generated and tested. The unique bispecific antibodies provided exhibit superior biological activity compared to any of the monospecific anti-HER2 and monospecific anti-Trop2 antibodies tested. In many assays, the bispecific antibodies also showed synergistic activity superior to monospecific anti-HER2 antibodies. Bispecific anti-HER2 / Trop2 antibodies conjugated with cytotoxic agents such as MMAE have shown excellent in vitro and in vivo antitumor activity. These data suggest that the bispecific antibodies may have therapeutic uses, particularly as ADCs or as ADCC-enhancing antibodies, for the treatment of cancers expressing HER2 and / or Trop2, including patients currently unsuitable for trastuzumab or pertuzumab treatment. Furthermore, the bispecific antibody of the present invention can be used to treat cancer patients who have failed existing anti-HER2 and / or anti-Trop2 therapies.

[0771] Although the invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations may be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

[0772] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0773] All figures and references, Genbank sequences, patents and published patent applications cited in this application are expressly incorporated herein by reference.

[0774] Experimental methods not specifically described in the following examples are generally performed under standard conditions, such as those described in Molecular Cloning: A Laboratory Manual (4th Edition) published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer.

[0775] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0776] Example

[0777] Example 1: Construction of a human Trop2 overexpressing cell line

[0778] Human Trop2-overexpressing cell lines were constructed using liposome transfection. Lipofectamine was used... TM The human Trop2-expressing pCMV3 plasmid (Sinochem, HG10428-NY) was transfected into CHO-K1 cells (hamster ovary cell subline, ATCC#CCL-61) using a 3000 transfection reagent (Invitrogen, L3000008). Specifically, the transfection was performed at a rate of 4 × 10⁻⁶ cells per day prior to transfection. 5 Cell / well quantity: CHO-K1 cells were seeded into 6-well plates and cultured overnight to allow cell adhesion. The next day, the transfection was performed according to the reagent manufacturer's instructions, using 2.5 μg of DNA. After transfection, the cells were placed in a CO2 incubator. After 48 hours, the medium was replaced with one containing Hygromycin B (Invitrogen, 10687010). After 10 days of antibiotic-resistant selection, a mixed cell pool expressing human Trop2 was obtained. The mixed cell pool was then seeded using the limiting dilution method to obtain monoclonal cell lines. The isolated monoclonal cell lines were identified using flow cytometry (FACS). Specifically, the monoclonal cells to be identified were digested and washed twice with FACS buffer (PBS + 1% BSA), and 50 μL of anti-HA-tagged mouse antibody (Sinochem, 100028-MM10) was added. The cells were incubated at 4°C for 30 min. After centrifugation at 500g for 3 min, the supernatant was discarded. Add 200 μL of FACS buffer to each well and wash twice. Add 50 μL of PE-labeled goat anti-mouse Fc secondary antibody (Jackson, 115-115-164) to each well and incubate at 4°C for 30 min. Centrifuge at 500g for 3 min and discard the supernatant. Add 200 μL of FACS buffer to each well and wash twice. Resuspend the cells in 50 μL of FACS buffer to each well and identify the Trop2 expression level in monoclonal cells using a flow cytometer (Beckman Coulter Biotechnology Co., Ltd., A00-1-1102).

[0779] Figure 1 A showed that the proportion of Trop2-positive cells in the obtained Trop2-expressing mixed cell pool reached 78.6%. Figure 1 B shows that human Trop2-overexpressing cell clone 1E11 was successfully obtained and named CHO-hTrop2.

[0780] The amino acid sequence of hTrop2 used in the human Trop2 overexpression cell line of this invention is shown in SEQ ID NO:86, and the nucleic acid it encodes is shown in SEQ ID NO::87.

[0781] The amino acid sequence of hTrop2 used in the human Trop2 overexpression cell line of this invention is shown in SEQ ID NO:86, and the nucleic acid it encodes is shown in SEQ ID NO::87.

[0782] MPLLLLLPLL WAGALAYPYD VPDYAGGGGS HTAAQDNCTC PTNKMTVCSP DGPGGRCQCRALGSGMAVDC

[0783] STLTSKCLLL KARMSAPKNA RTLVRPSEHA LVDNDGLYDP DCDPEGRFKA RQCNQTSVCWCVNSVGVRRT

[0784] DKGDLSLRCD ELVRTHHILIDLRHRPTAGA FNHSDLDAEL RRLFRERYRL HPKFVAAVHYEQPTIQIELR

[0785] QNTSQKAAGD VDIGDAAYYF ERDIKGESLF QGRGGLDLRV RGEPLQVERT LIYYLDEIPPKFSMKRLTAGLIAVIVVVVV ALVAGMAVLV ITNRRKSGKY KKVEIKELGE LRKEPSL (SEQ ID NO: 86).

[0786] atgccactgc tgctcttgct gcctctgctt tgggctggag ctctggctta tccttacgacgtgcctgact

[0787] acgccggtgg aggcggtagc cacacggccg cgcaggacaa ctgcacgtgt cccaccaacaagatgaccgt

[0788] gtgcagcccc gacggccccg gcggccgctg ccagtgccgc gcgctgggct cgggcatggcggtcgactgc

[0789] tccacgctga cctccaagtg tctgctgctc aaggcgcgca tgagcgcccc caagaacgcccgcacgctgg

[0790] tgcggccgag tgagcacgcg ctcgtggaca acgatggcct ctacgacccc gactgcgaccccgagggccg

[0791] cttcaaggcg cgccagtgca accagacgtc ggtgtgctgg tgcgtgaact cggtgggcgtgcgccgcacg

[0792] gacaagggcg acctgagcct acgctgcgat gagctggtgc gcacccacca catcctcattgacctgcgcc

[0793] accgccccac cgccggcgcc ttcaaccact cagacctgga cgccgagctg aggcggctcttccgcgagcg

[0794] ctatcggctg caccccaagt tcgtggcggc cgtgcactac gagcagccca ccatccagatcgagctgcgg

[0795] cagaacacgt ctcagaaggc cgccggtgac gtggatatcg gcgatgccgc ctactacttcgagagggaca

[0796] tcaagggcga gtctctattc cagggccgcg gcggcctgga cttgcgcgtg cgcggagaacccctgcaggt

[0797] ggagcgcacg ctcatctatt acctggacga gattcccccg aagttctcca tgaagcgcctcaccgccggc

[0798] ctcatcgccg tcatcgtggt ggtcgtggtg gccctcgtcg ccggcatggc cgtcctggtgatcaccaacc

[0799] ggagaaagtc ggggaagtac aagaaggtgg agatcaagga actggggggag ttgagaaaggaaccgagctt gtaa (SEQ ID NO: 87).

[0800] Example 2: Generation and Identification of Anti-Trop2 Monoclonal Antibody

[0801] This invention utilizes hybridoma technology to screen for novel anti-Trop2 antibodies. Specifically, recombinant human Trop2 protein (Bepsys, TR2-H5223) with a 6×His tag fused to its C-terminus was emulsified with a rapid adjuvant (Bio-Long, KX0210041) and then immunized with BalB / C mice (purchased from Spiefol Biotechnology Co., Ltd.). After serum titer testing, mice that met the fusion criteria were euthanized, spleens were collected, and single-cell suspensions were prepared. Hybridomas were generated by electrofusion with SP20 myeloma cells (Nanjing Kebai, CBP60881). The fused cells were seeded into 96-well cell culture plates and cultured for 10 days. After 10 days, the supernatant was collected for ELISA and FACS binding activity screening to obtain the parent clone 44E7 capable of binding human Trop2. Single clones with Trop2 binding activity were isolated from the mixed cell pool of the parent clone using limiting dilution and sequenced. The heavy chain variable region and light chain variable region sequences of the mouse clone 44E7 were obtained by sequencing, and the amino acid sequences of its CDR are shown in Table 1.

[0802] Table 1 shows the VHA CDR and VLA CDR of the Trop2 antibody of this invention as defined by the Kabat numbering system.

[0803] Serial Number CDR Serial Number sequence (a) VHACDR1 SEQ ID NO:1 DYNMH (b) VHACDR2 SEQ ID NO:2 YIYPYNGGSAYNQKFKN (c) VHACDR3 SEQ ID NO:3 EDYGYDPGYYFDY (d) VLACDR1 SEQ ID NO:4 RSSQSLVHSNGNSYLH (e) VLACDR2 SEQ ID NO:5 KVSNRFS (f) VLACDR3 SEQ ID NO:6 SQSTHVWT

[0804] Example 3 Construction, Expression and Purification of Recombinant Trop2 Chimeric Antibody

[0805] First, the heavy chain and light chain variable regions of the 44E7 murine antibody were placed at the N-terminus of the human IgG4 heavy chain constant region (Uniprot ID: P01861) and light chain kappa constant region (Uniprot ID: P01834), respectively. The resulting fusion gene fragment was then constructed into a pHr expression vector with an N-terminal signal peptide. The obtained light and heavy chain expression vectors were simultaneously transfected into Expi CHO-S cells (Gibco, A29133) using a transfection reagent (Gibco, A29129). Specifically, the cell density was adjusted the day before transfection to achieve a density of 6 × 10⁶ cells on the day of transfection. 6 / mL. Take 4% OptiPRO TMSFM complexing medium (purchased from Gibco) was used as the transfection buffer to dilute plasmid DNA to 0.8 μg / mL. This was then mixed with ExpiFectamine. TM After mixing the CHO reagent (purchased from Gibco), gently pour the resulting cationic transfection reagent / DNA mixture into the Expi CHO-S cell suspension, mix gently, and incubate overnight at 37°C with 5% CO2. After 18-22 hours of overnight incubation, add 24% of the transfected culture volume of ExpiCHO reagent to the culture flask. TM Excipients (purchased from Gibco) and 0.6% ExpiCHO by volume of the transfected culture. TM The cell culture supernatant was collected after gently mixing with an enhancer (purchased from Gibco) and continuous culturing until day 10 or when cell viability was ≤70%. The target antibody was purified from the culture supernatant using a Protein A affinity chromatography column. Specifically, before purification, the collected cell supernatant was centrifuged at 10,000 rpm for 10 min and filtered through a 0.22 μm filter. The Protein A column was equilibrated with 5 column volumes of equilibration buffer (10 mM PB, pH 7.0). The filtered supernatant was added to the purification column and equilibrated with 10 column volumes of equilibration buffer. 5 mL of elution buffer (100 mM Gly-NaCl, pH 3.8) was added, and the eluent was collected. The antibody concentration was determined using the A280 method, and the antibody purity was determined by SEC-HPLC. The recombinant chimeric antibody ch44E7 with a purity greater than 95% was then concentrated by ultrafiltration.

[0806] The amino acid sequences and encoding nucleic acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of ch44E7, as well as the amino acid sequences of the heavy chain and light chain, are as follows:

[0807] The amino acid sequence of VH in ch44E7:

[0808] EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMHWVKQSHGKSLEWIGYIYPYNGGSAYNQKFKNKATLTIDISSSTA YMELRSLSSEDSAVYYCAREDYGYDPGYYFDYWGQGTTLTVSS (SEQ ID NO: 7).

[0809] The amino acid sequence of VL in ch44E7:

[0810] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNSYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLK ISRVEAEDLGIYFCSQSTHVWTFGGGTKLEIK(SEQ ID NO:15)。

[0811] EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMHWVKQSHGKSLEWIGYIYPYNGGSAYNQKFKNKATLTIDISSSTA

[0812] YMELRSLSSEDSAVYYCAREDYGYDPGYYFDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

[0813] VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFL

[0814] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL

[0815] NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:22)。

[0816] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNSYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLK

[0817] ISRVEAEDLGIYFCSQSTHVWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD

[0818] NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0819] (SEQ ID NO:30)

[0820] Nucleic acid encoding the VH of ch44E7:

[0821] GAGGTCCAGCTTCAGCAGTCAGGACCTGAGCTGGTGAAACCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTTCTGGAT

[0822] ACACATTCACTGACTACAACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTTATCC

[0823] TTACAATGGTGGTTCTGCTTACAACCAGAAGTTCAAGAACAAGGCCACATTGACTATAGACATTTCCTCCAGCACAGCC

[0824] TACATGGAACTCCGCAGCCTGTCATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGAGGACTATGGTTACGACCCGGGCTACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO:63)

[0825] Nucleic acid encoding the VL of ch44E7:

[0826] GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTC

[0827] AGAGCCTTGTACACAGTAATGGAAACTCCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGAT

[0828] CTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGATTCAGTGGCAGTGGGTCAGGGACAGATTTCACACTCAAG

[0829] ATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTTCTGCTCTCAAAGTACTCATGTGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 71).

[0830] Example 4: Trop2 chimeric antibody ELISA binding activity analysis

[0831] This embodiment utilizes the ELISA method to detect the binding activity of the chimeric antibody ch44E7 to Trop2 protein. Specifically, recombinant human Trop2-His protein (Bipsys, TR2-H5223) was diluted to 1 μg / mL with coating buffer (a mixture of 8 mL of 0.2 mol / L Na2CO3 and 17 mL of 0.2 mol / L NaHCO3, then 75 mL of distilled water was added to adjust the pH to 9.6). 50 μL of this solution was added to each well of a Corning ELISA plate (3590) and incubated at 37°C for 2 hours. After discarding the liquid in the wells, 200 μL of washing buffer (PBS + 0.5‰ Tween-20) was added to each well, and the plate was washed three times. Then, 200 μL of blocking buffer (PBS + 0.5‰ Tween-20 + 5% skim milk powder) was added to each well, and the plate was incubated at 37°C for 2 hours. After discarding the liquid in the wells, 200 μL of washing buffer was added to each well, and the plate was washed three times. Add 50 μL of serially diluted ch44E7 antibody and reference antibody huRS7 (sequence derived from patent CN100360567C) to the wells of an ELISA plate. The initial antibody concentration was 80 nM, and each well was diluted 5-fold for a total of 8 concentration points. Incubate at 37°C for 60 min. After discarding the liquid in the wells, add 200 μL of washing buffer to each well and wash 3 times. Add 50 μL of HRP-labeled goat anti-human Fc secondary antibody (Jackson, 109-035-170) to each well and incubate at 37°C for 60 min. After discarding the liquid in the wells, add 200 μL of washing buffer to each well and wash 3 times. Add 50 μL of chromogenic buffer (Sigma, T4444) to each well and incubate at room temperature for 2 min. Then, add 50 μL of 2 mol / L H2SO4 to terminate the reaction. Read the OD using a Thermo Scientific Microplate Reader (MμLtiskan FC, 8D-030A). 450 Numerical value. Based on OD 450 Plot the numerical values ​​against antibody concentrations.

[0832] Figure 2 The results showed that ch44E7 could bind to recombinantly expressed human Trop2 protein, but its binding activity was slightly weaker than that of the reference antibody huRS7. The EC50 value of the ch44E7 binding curve was about twice that of the latter.

[0833] Example 5: FACS Binding Activity Analysis of Trop2 Chimeric Antibody

[0834] This example tested the binding activity of the ch44E7 antibody to the Trop2 protein on the cell surface. Specifically, the CHO-hTrop2 cell line obtained in Example 1 was digested into a single-cell suspension using trypsin. The suspension was washed twice with FACS buffer (PBS + 1% BSA), and then the density was adjusted to 2 × 10⁻⁶ cells / cell. 6 Add 100 μL / well to each well of a U-bottom 96-well plate (BIOFIL, 002096). Centrifuge at 500g for 3 min, discard the supernatant, and add 50 μL of serially diluted ch44E7 chimeric antibody and reference antibody huRS7 to each well. The initial antibody concentration is 400 nM, and each well is diluted 5-fold for a total of 8 concentration points. Incubate at 4°C for 30 min, centrifuge at 500g for 3 min, and discard the supernatant. Add 200 μL of FACS buffer to each well and wash twice. Add 50 μL of PE-labeled goat anti-human Fc secondary antibody (Jackson, 109-115-098) to each well and incubate at 4°C for 30 min. Centrifuge at 500g for 3 min, discard the supernatant, and add 200 μL of FACS buffer to each well and wash twice. Cells were resuspended in 50 μL of FACS buffer in each well, and the fluorescence intensity of the cells was detected using a flow cytometer (Beckman Coulter Biotechnology Co., Ltd., A00-1-1102). A graph was plotted with antibody concentration on the x-axis and PE fluorescence area on the y-axis.

[0835] Figure 3 The results showed that both ch44E7 and the reference antibody huRS7 could bind to Trop2 on the cell membrane, and the binding activity of the ch44E7 chimeric antibody was slightly lower than that of the reference antibody huRS7.

[0836] Example 6: Species Cross-Binding Activity Analysis of Trop2 Chimeric Antibodies

[0837] This embodiment utilizes the ELISA method to detect the cross-binding activity of ch44E7 with Trop2 proteins from different species. The specific procedure is as follows: Mouse Trop2-His (TR2-M52H6) and monkey Trop2-His (TR2-R52H3) were diluted to 1 μg / mL with coating buffer and added to each well at 50 μL, incubating at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well, washing three times. 200 μL of blocking buffer was added to each well, and incubation was carried out at 37°C for 2 hours. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well, washing three times. 50 μL of serially diluted ch44E7 antibody and reference antibody huRS7 were added to each well of the ELISA plate, with an initial antibody concentration of 80 nM, and each antibody was diluted 5-fold sequentially, resulting in 8 concentration points. The plates were incubated at 37°C for 60 min. After discarding the liquid in each well, add 200 μL of washing buffer to each well and wash three times. Add 50 μL of HRP-labeled goat anti-human Fc secondary antibody to each well and incubate at 37°C for 60 min. After discarding the liquid in each well, add 200 μL of washing buffer to each well and wash three times. Add 50 μL of chromogenic buffer to each well and incubate at room temperature for 2 min. Then add 50 μL of 2 mol / L H₂SO₄ to stop the reaction and read the OD450 value on a microplate reader. Plot the antibody concentration on the x-axis, OD... 450 Plot the values ​​on the ordinate.

[0838] like Figure 4 As shown, similar to the reference antibody huRS7, the ch44E7 antibody can bind to recombinant monkey Trop2 protein. However, the binding activity of ch44E7 to monkey Trop2 protein is slightly weaker than that of huRS7, with its EC50 value being approximately twice that of the latter. Binding experiments with mouse Trop2 protein showed that neither ch44E7 nor huRS7 exhibited cross-binding activity with mouse Trop2 protein.

[0839] Example 7: Analysis of cross-binding activity between Trop2 chimeric antibody and EpCAM

[0840] This embodiment examines whether the ch44E7 antibody cross-binds with EpCAM, a member of the Trop2 family. Specifically, recombinant human EpCAM-His (EPM-H5223) was diluted to 1 μg / mL with coating buffer and added to 50 μL / well of an ELISA plate, incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. 200 μL of blocking buffer was added to each well, and the plate was incubated at 37°C for 2 hours. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing three times. 50 μL of serially diluted ch44E7 antibody and reference antibody huRS7 were added to the wells of the ELISA plate, with an initial antibody concentration of 80 nM, and each well was diluted 5-fold for a total of 8 concentration points. The plates were incubated at 37°C for 60 min. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing three times. Add 50 μL of HRP-labeled goat anti-human Fc secondary antibody to each well and incubate at 37°C for 60 min. Discard the liquid in the wells and add 200 μL of washing buffer to each well, washing three times. Add 50 μL of chromogenic solution to each well and incubate at room temperature for 2 min. Then, add 50 μL of 2 mol / L H2SO4 to stop the reaction and read the OD on a microplate reader. 450 Numerical values. OD is plotted on the x-axis as antibody concentration. 450 Plot the values ​​on the ordinate.

[0841] Figure 5 The results showed that neither the ch44E7 antibody nor the reference antibody huRS7 could bind to the recombinant human EpCAM protein.

[0842] Example 8: Trop2 chimeric antibody affinity assay

[0843] In this embodiment, the equilibrium dissociation constant (KD) of the Trop2 chimeric antibody binding to human Trop2 protein was determined using biofilm thin-layer interferometry (BLI). The specific procedures were as follows: One hour before the experiment, the AHC2 sensor (Sartorius, 18-5142) was immersed in PBST solution (10 mM PBS + 0.1% BSA + 0.2‰ Tween-20, pH 7.4). The recombinant human Trop2-His protein was serially diluted with PBST, starting at 100 nM and then sequentially diluted 2-fold, resulting in 7 concentration points. A zero-concentration control well was also included. The chimeric antibody ch44E7 and the reference antibody huRS7 were diluted to 5 μg / mL. The Fortebio molecular interaction analyzer (Sartorius, Octet R8) was set to the following operating conditions: temperature 30°C, Shake speed 1000 rpm. Antibodies were captured using a pre-coated AHC2 sensor for 180 s; binding to serially diluted antibody samples was performed for 120 s; dissociation was performed for 300 s; and regeneration was carried out for 30 s with regeneration buffer (10 mM glycine, pH 1.7). After obtaining the sensor data, the binding constant (ka) and dissociation constant (kd) were analyzed using Octet BLI Analysis software. An ideal binding-dissociation curve was fitted, and the equilibrium dissociation constant KD (kd / ka) between the antibody and antigen was calculated. Table 2 shows the affinity c of ch44E7.

[0844] Table 2. Equilibrium dissociation constants for Trop2 antibody detection by BLI method

[0845] Antibody Ka(1 / Ms) Kd(1 / s) KD(M) huRS7 3.10E+05 4.58E-04 1.48E-09 ch44E7 2.19E+05 4.91E-04 2.24E-09

[0846] Example 9: Trop2 mouse anti-humanization modification

[0847] This embodiment employs a method combining complementary determining region (CDR) transplantation with key amino acid back mutation technology to humanize the variable region sequence of Trop2 murine monoclonal antibody 44E7. Using bioinformatics analysis and sequence alignment, the most similar antibody germline sequence to the light and heavy chains of murine monoclonal antibody 44E7 was found in a known database. The CDR sequence of the light and heavy chains of murine monoclonal antibody 44E7 was then transplanted into the human antibody frame region (FR). BLAST analysis was used to identify the homologous structure with the highest sequence similarity to the variable region of the murine monoclonal antibody 44E7. A three-dimensional structural model was constructed after sequence alignment of the antibody's light and heavy chain variable regions. Key amino acids were then identified through the 3D structure and reversed through mutation, resulting in 7 humanized heavy chain variable region sequences and 6 humanized light chain variable region sequences (see Tables 3 and 4, respectively). The amino acid sequences of the heavy and light chains are shown in Tables 6 and 7, respectively. Examples of light and heavy chain variable region combinations for antibodies are shown in Table 5, and light and heavy chain combinations are shown in Table 8. The coding nucleic acid sequences for the heavy and light chain variable regions are shown in Tables 9 and 10, respectively. Light and heavy chain expression vectors were constructed based on Tables 9 and 10, and antibody expression was performed according to the light and heavy chain combinations in Tables 5, 8, and 11.

[0848] Table 3. Heavy chain variable region A (VHA) of exemplary anti-Trop2 antibodies of the present invention.

[0849]

[0850] Table 4. Light chain variable region A (VLA) of exemplary anti-Trop2 antibodies of the present invention.

[0851]

[0852]

[0853] Table 5. Combinations of heavy chain variable region A (VHA) and light chain variable region A (VLA) of exemplary antibodies of the present invention.

[0854]

[0855]

[0856] Table 6. Heavy chain A' (HA') of the exemplary anti-Trop2 antibody of the present invention.

[0857]

[0858]

[0859]

[0860] Table 7 Light chain A (LA) of the exemplary anti-Trop2 antibody of the present invention

[0861]

[0862]

[0863] Table 8. Exemplary heavy and light chain combinations of chimeric anti-Trop2 antibodies and humanized anti-Trop2 antibodies.

[0864]

[0865]

[0866] Table 9. Nucleic acids encoding the heavy chain variable region of chimeric antibodies, humanized antibodies, and positive controls.

[0867]

[0868]

[0869] Table 10. Nucleic acids encoding the heavy chain variable region of chimeric antibodies, humanized antibodies, and positive controls.

[0870]

[0871]

[0872] Table 11 Combination of the light and heavy chain coding nucleic acid sequences of humanized anti-Trop2 antibodies (SEQ ID NO:)

[0873]

[0874]

[0875] The affinity of the 44E7 chimeric antibody and the humanized antibody was determined using biofilm thin-layer interferometry (BLI), as detailed in Example 8. The experimental results are shown in Table 12. Compared to the parent chimeric antibody, the affinity of the humanized antibody was reduced to varying degrees.

[0876] Table 12. Equilibrium dissociation constants for Trop2 antibody detection by BLI method

[0877] Antibody Ka(1 / Ms) Kd(1 / s) KD(M) ch44E7 1.86E+05 5.87E-04 3.16E-09 hu44E7-1 2.05E+05 3.45E-03 1.69E-08 hu44E7-2 2.15E+05 2.22E-03 1.04E-08 hu44E7-3 2.07E+05 3.28E-03 1.58E-08 hu44E7-4 1.89E+05 1.79E-03 9.52E-09 hu44E7-5 2.50E+05 3.08E-03 1.23E-08 hu44E7-6 2.25E+05 2.87E-03 1.28E-08 hu44E7-7 1.99E+05 1.25E-03 6.29E-09 hu44E7-8 2.88E+05 5.47E-03 1.90E-08 hu44E7-9 2.53E+05 3.07E-03 1.21E-08

[0878] Example 10: ELISA Binding Activity Analysis of Humanized Trop2 Antibody

[0879] This embodiment utilizes the ELISA method to detect the binding activity of humanized 44E7 antibody to Trop2 protein. Specifically, the recombinant human Trop2-His protein was diluted to 1 μg / mL with coating buffer and added to each well at 50 μL, incubating at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. 200 μL of blocking buffer was added to each well, and the plate was incubated at 37°C for 2 hours. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing three times. 50 μL of serially diluted ch44E7 antibody and humanized antibody hu44E7 with different light and heavy chain combinations were added to each well of the ELISA plate. The initial antibody concentration was 100 nM, and the plate was diluted 5-fold sequentially, resulting in 8 concentration points. The plate was incubated at 37°C for 60 min. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. Add 50 μL of HRP-labeled goat anti-human Fc secondary antibody to each well and incubate at 37°C for 60 min. Discard the liquid in the wells and add 200 μL of washing buffer to each well, washing three times. Add 50 μL of chromogenic solution to each well and incubate at room temperature for 2 min. Then add 50 μL of 2 mol / L H2SO4 to stop the reaction and read the OD on a microplate reader. 450 Numerical values. OD is plotted on the x-axis as antibody concentration. 450 Plot the values ​​on the ordinate.

[0880] according to Figure 6 The results showed that, compared with the maternal chimeric antibody, the binding activity of the humanized antibody to the recombinant human Trop2 protein was weakened to varying degrees.

[0881] Example 11: FACS Binding Activity Analysis of Humanized Trop2 Antibody

[0882] This embodiment detects the binding activity of humanized antibodies to the Trop2 protein on the cell surface. Specifically, CHO-hTrop2 cells digested into a single-cell state were washed twice with FACS buffer, and then the density was adjusted to 2 × 10⁻⁶ cells / cells. 6Add 100 μL of the antibody to each well of a U-bottom 96-well plate at a concentration of 800 nM. Centrifuge at 500 g for 3 min and discard the supernatant. Add 50 μL of serially diluted ch44E7 chimeric antibody and humanized hu44E7 antibody with different light and heavy chain combinations to each well. The initial antibody concentration is 800 nM, and the antibody is diluted 5-fold sequentially, resulting in 8 concentration spots. Incubate at 4 °C for 30 min. Centrifuge at 500 g for 3 min and discard the supernatant. Add 200 μL of FACS buffer to each well and wash twice. Add 50 μL of PE-labeled goat anti-human Fc secondary antibody to each well and incubate at 4 °C for 30 min. Centrifuge at 500 g for 3 min and discard the supernatant. Add 200 μL of FACS buffer to each well and wash twice. Resuspend the cells in 50 μL of FACS buffer to each well and detect the fluorescence intensity of the cells using flow cytometry. Plot the antibody concentration on the x-axis and the PE fluorescence area on the y-axis.

[0883] according to Figure 7 The results showed that, except for hu3F8 H5L1 and hu3F8 H5L2, the binding activity of the other humanized antibodies to human Trop2 at the cellular level was close to that of the maternal chimeric antibodies.

[0884] Example 12: Species Cross-Binding Activity Analysis of Humanized Trop2 Antibody

[0885] This embodiment detects the cross-binding activity of humanized antibodies with Trop2 proteins from different species. Specifically, recombinant mouse Trop2-His protein and monkey Trop2-His protein were diluted to 1 μg / mL with coating buffer and added to 50 μL / well of an ELISA plate, incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing 3 times. 200 μL of blocking buffer was added to each well, and incubated at 37°C for 2 hours. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing 3 times. 50 μL of serially diluted 44E7 chimeric antibody and humanized antibodies with different light and heavy chain combinations were added to the wells of the ELISA plate. The initial antibody concentration was 400 nM, and the antibodies were diluted 5-fold sequentially, for a total of 10 concentration points. The plates were incubated at 37°C for 60 min. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing 3 times. Add 50 μL of HRP-labeled goat anti-human Fc secondary antibody to each well and incubate at 37°C for 60 min. Discard the liquid in the wells, add 200 μL of washing buffer to each well, and wash 3 times. Add 50 μL of chromogenic solution to each well and incubate at room temperature for 2 min. Then add 50 μL of 2 mol / L H2SO4 to stop the reaction and read the OD450 value on a microplate reader. Plot the antibody concentration on the x-axis, OD450... 450 Plot the values ​​on the ordinate.

[0886] The results are as follows Figure 8As shown, the humanized antibody retained its binding activity against monkey Trop2, but still had no binding activity against mouse Trop2.

[0887] Example 13: Cross-binding activity analysis of humanized Trop2 antibody with EpCAM

[0888] Dilute human EpCAM-His to 1 μg / mL with coating buffer and add 50 μL / well to each well of an ELISA plate. Incubate at 37°C for 2 hours. Discard the liquid in the wells and add 200 μL of washing buffer to each well, washing 3 times. Add 200 μL of blocking buffer to each well and incubate at 37°C for 2 hours. Discard the liquid in the wells and add 200 μL of washing buffer to each well, washing 3 times. Add 50 μL of serially diluted 44E7 chimeric antibody and humanized antibodies with different light and heavy chain combinations to each well of the ELISA plate. The initial antibody concentration is 400 nM, and the antibody is diluted 5-fold sequentially, for a total of 8 concentration points. Incubate at 37°C for 60 min. Discard the liquid in the wells and add 200 μL of washing buffer to each well, washing 3 times. Add 50 μL of HRP-labeled goat anti-human Fc secondary antibody to each well and incubate at 37°C for 60 min. Discard the liquid in the wells and add 200 μL of washing buffer to each well, washing 3 times. Add 50 μL of chromogenic solution to each well and incubate at room temperature for 2 min. Then, add 50 μL of 2 mol / L H2SO4 to terminate the reaction. Read the OD values ​​on a microplate reader. 450 Numerical values. OD is plotted on the x-axis as antibody concentration. 450 Plot the values ​​on the ordinate.

[0889] The results are as follows Figure 9 As shown, the humanized antibody did not bind to human EpCAM.

[0890] Example 14: Detection of endocytic activity of humanized Trop2 antibody

[0891] This embodiment uses an endpoint method to detect the endocytic activity of the Trop2 antibody. Specifically, the density of digested BxPC-3 cells (Wuhan Shangen Biotechnology, SNL-099) was adjusted to 1×10⁻⁶ cells one day before the experiment. 5The antibody was seeded at a rate of 50 μL / well into 96-well cell culture plates and cultured overnight at 37°C with 5% CO2 to allow cell adhesion. The next day, the antibody was diluted to 24 μg / mL, and the goat anti-human Fc secondary antibody (SSA015) labeled with pHAb Amine and Thiol Reactive Dyes (Promega, G9841) was diluted to 72 μg / mL. 25 μL of the antibody and 25 μL of the labeled goat anti-human Fc secondary antibody were mixed and incubated at room temperature in the dark for 30 min before being added to the corresponding cell wells. After culturing at 37°C with 5% CO2 for 24 hours, the cells were digested with trypsin and washed twice with FACS buffer. The mean fluorescence intensity of the PE signal in the cells was detected by flow cytometry and plotted.

[0892] Figure 10 The results showed that the endocytic activity of humanized antibodies with different combinations of light and heavy chains varied. Among the humanized antibodies, hu44E7-3 showed the highest endocytic activity.

[0893] Example 15 Construction of Trop2×Her2 bispecific antibody vector and protein expression and purification

[0894] 15.1 Construction of Trop2×Her2 bispecific antibody expression vector

[0895] In this embodiment, an anti-Trop2×Her2 bispecific antibody was constructed and named Trop2×Her2 bsAb. The specific structure of the bispecific antibody is as follows: The bispecific antibody consists of two peptide chains, where peptide chain 1# is the amino acid sequence for binding Trop2. Specifically, the C-terminus of the anti-Trop2 VL is linked to the N-terminus of the human κ chain constant region, followed by a (G4S)6 linker. The C-terminus of the linker is then linked to the N-terminus of the Trop2 antibody VH, and the C-terminus of the anti-Trop2 VH is linked to the N-terminus of the human IgG1 constant region; peptide chain 2# is the amino acid sequence for binding Her2. Specifically, the C-terminus of the Herceptin VL (sequence derived from the patent) is linked to the N-terminus derived from the human κ chain constant region, followed by a (G4S)6 linker. The C-terminus of the linker is then linked to the N-terminus of the Herceptin VH, and the C-terminus of the Herceptin VH is linked to the N-terminus of the human IgG1 constant region.

[0896] A series of mutations were performed on the Fc of this bispecific antibody. Specifically, the knock-in-hole mutation, invented by Genentech in the 1990s, was used to prevent heavy chain mismatches. In one antibody heavy chain (knob chain), serine S at position 354 was mutated to cysteine ​​C, and serine T at position 366 was mutated to tryptophan W (S354C and T366W). In the other heavy chain (Hole chain), tyrosine Y at position 349 was mutated to cysteine ​​C, serine T at position 366 was mutated to serine S, leucine L at position 368 was mutated to alanine A, and amino acid 407 was mutated from tyrosine Y to valine V (Y349C, T366S, L368A, and Y407V). The constructed anti-Trop2 and anti-Her2 gene fragments were then constructed into the pHr expression vector, specifically, the Trop2 antibody sequence was placed in the knock-in chain, and the Herceptin sequence was placed in the hole chain. Finally, single-arm antibody plasmids against Trop2 and Her2 were obtained. Simultaneously, the variable regions of Herceptin and hu44E7-3 were constructed into the human IgG1 constant region backbone for expression and purification, respectively, and the resulting antibodies served as parental monoclonal antibody controls.

[0897] The amino acid sequences of the fragments contained in the anti-Trop2 binding arm and the anti-Her2 binding arm are shown in Tables 13 and 14, and the encoding nucleic acid sequences are shown in Table 15.

[0898] Table 13. Fragment sequence of the first antigen-binding arm A of Trop2×Her2 bsAb with Trop2 specificity and its SEQ ID NO:.

[0899]

[0900]

[0901] Table 14. Fragment sequences of the HER2-specific second binding arm B of Trop2×Her2 bsAb and their SEQ ID NO:

[0902]

[0903]

[0904] Table 15. Nucleic acid sequences encoding Trop2 binding arm A and HER2 binding arm B.

[0905]

[0906]

[0907]

[0908] 15.2 Expression and purification of Trop2×Her2 bispecific antibody

[0909] Bispecific antibodies were generated using the CHO-S transient transfection expression system. Specifically, Expi CHO-S cells were passaged according to the required transfection volume, ensuring a cell density of 6 × 10⁶ cells / mL on the day of transfection. 6 / mL. Take 4% OptiPRO TM SFM complexing medium was used as the transfection buffer. 0.8 μg of plasmid DNA was added to each milliliter of transfection buffer, mixed well, and then ExpiFectamine was added. TM Mix CHO reagent thoroughly, then gently pour the cationic transfection reagent / DNA mixture into the Expi CHO-S cell suspension. Gently mix and incubate overnight at 37°C with 5% CO2. After 18-22 hours of overnight incubation, add 24% of the transfected culture volume of ExpiCHO reagent to the culture flask. TM ExpiCHO was added as excipient and 0.6% of the volume of the transfected culture. TM Add the enhancer, mix gently, and culture continuously until day 10 or when cell viability is ≤70%, then collect the culture supernatant. Purify the target antibody from the culture supernatant using a Protein A affinity chromatography column. Specifically, before purification, centrifuge the collected cell supernatant at 10000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter. Equilibrate the Protein A column with 5 column volumes of equilibration buffer. Add the filtered supernatant to the purification column and equilibrate with 10 column volumes of equilibration buffer. Add 5 mL of elution buffer and collect the eluent. The antibody concentration is detected using the A280 method, and the antibody purity is determined by SEC-HPLC. The obtained bispecific antibody is then concentrated by ultrafiltration to finally obtain Trop2×Her2 bsAb.

[0910] Example 16: Trop2×Her2 Bispecific Antibody ELISA Binding Activity Analysis

[0911] Recombinant human Trop2-His (TR2-H5223) and human Her2-His (HE2-H5225) proteins were diluted to 2 μg / mL with coating buffer and added to each well at 50 μL. The plates were incubated at 37°C for 2 hours. The liquid in the wells was discarded, and 200 μL of washing buffer was added to each well for washing three times. 200 μL of blocking buffer was added to each well, and the plates were incubated at 37°C for 2 hours. The liquid in the wells was discarded again, and 200 μL of washing buffer was added to each well for washing three times. Herceptin, hu44E7-3, and Trop2×Her2 bsAb were serially diluted 5-fold, starting at 500 nM, for a total of 11 concentration points. Serially diluted Herceptin and Trop2×Her2 bsAb were incubated with the coated Her2-His protein, and serially diluted hu44E7-3 and Trop2×Her2 bsAb were incubated with the coated Trop2-His protein. The incubation conditions were 37°C for 60 min. After discarding the liquid in the wells, 200 μL of washing buffer was added to each well, and the mixture was washed three times. 50 μL of HRP-labeled goat anti-human Fc secondary antibody was added to each well, and the mixture was incubated at 37°C for 60 min. After discarding the liquid in the wells, 200 μL of washing buffer was added to each well, and the mixture was washed three times. 50 μL of chromogenic buffer was added to each well, and the mixture was incubated at room temperature for 2 min. Then, 50 μL of 2 mol / L H2SO4 was added to stop the reaction, and the OD values ​​were read on a microplate reader. 450 Numerical values. OD is plotted on the x-axis as antibody concentration. 450 Plot the values ​​on the ordinate.

[0912] The results are as follows Figure 11 As shown, Trop2×Her2 bsAb and Herceptin exhibit comparable binding activities to recombinant human Her2 protein, with very similar EC50 values ​​in their two binding curves. However, compared to hu44E7-3, Trop2×Her2 bsAb shows a significant difference in binding activity to recombinant human Trop2 protein. The binding activity of Trop2×Her2 bsAb is significantly weaker than that of hu44E7-3, with the former's EC50 being approximately 20 times that of the latter.

[0913] Example 17: FACS Binding Activity Analysis of Trop2×Her2 Bispecific Antibody

[0914] This embodiment tested the binding activity of the antibody with tumor cell lines expressing different levels of Trop2 and Her2. Specifically, MDA-MB-468 cells (Wuhan Shangen Biotechnology, SNL-061), HCC1569 cells (Nanjing Kebai Biotechnology, CBP60372), and HCC1954 cells (Shenzhen Huatuo Biotechnology, HTX2453) were digested into single-cell suspensions using trypsin. After digestion was terminated with complete culture medium, the cells were washed twice with FACS buffer, and then the cell density was adjusted to 2 × 10⁶ cells / cells. 6 Add 100 μL of antibody per well to each of the following 96-well U-bottom plates. Centrifuge at 500g for 3 min, discard the supernatant, and add 50 μL of serially diluted Herceptin, hu44E7-3, and Trop2×Her2 bsAb to each well. The initial antibody concentration is 400 nM, with 5-fold dilutions for a total of 8 concentration points. Incubate at 4℃ for 30 min. Centrifuge at 500g for 3 min, discard the supernatant. Add 200 μL of FACS buffer to each well and wash twice. Add 50 μL of PE-labeled goat anti-human Fc secondary antibody to each well and incubate at 4℃ for 30 min. Centrifuge at 500g for 3 min, discard the supernatant. Add 200 μL of FACS buffer to each well and wash twice. Resuspend the cells in 50 μL of FACS buffer to each well and detect the fluorescence intensity of the cells using flow cytometry. Plot the antibody concentration on the x-axis and the PE fluorescence area on the y-axis.

[0915] The results are as follows Figure 12 As shown, for the MDA-MB-468 cell line with high Trop2 expression and low Her2 expression, the binding activity of Trop2×Her2bsAb was weaker than that of hu44E7-3, but stronger than that of Herceptin. For the HCC1569 cell line with low Trop2 expression and high Her2 expression, the binding activity of Trop2×Her2 bsAb was significantly stronger than that of hu44E7-3. Although the EC50 value of Trop2×Her2 bsAb was slightly higher than that of Herceptin, its maximum binding amount was also higher. For the HCC1954 cell line with high Trop2 expression and high Her2 expression, although the EC50 of Trop2×Her2 bsAb was slightly higher than that of the two parent monoclonal antibodies, its maximum binding amount was the highest among the three detection antibodies.

[0916] Example 18 Trop2×Her2 Bispecific Antibody Affinity Detection

[0917] The equilibrium dissociation constants (KD) of the Trop2×Her2 bsAb antibody binding to human Trop2 and human Her2 proteins were determined using biofilm thin-layer interferometry (BLI). The specific procedures were as follows: Before the experiment, the AHC2 sensor (Sartorius, 18-5142) was immersed in PBST solution (10 mM PBS + 0.1% BSA + 0.2‰ Tween-20, pH 7.4). Human Trop2-His (Bepsys, TR2-H5223) was serially diluted with PBST, starting at 100 nM and then sequentially diluted 2-fold, for a total of 7 concentration points. A zero-concentration control well was also included. The antibody to be tested was diluted to 5 μg / mL. The Fortebio molecular interaction analyzer (Sartorius, Octet R8) was set to the following operating conditions: temperature 30℃, shake speed 1000 rpm. Antibodies were captured using a pre-coated AHC2 probe for 180 s; binding to serially diluted antibody samples was performed for 120 s; dissociation was performed for 300 s; and regeneration was repeated three times with regeneration buffer (10 mM glycine, pH 1.7) for 30 s each time. Detection was performed using ForteBio's Octet System. After obtaining the sensor data, the binding constant (ka) and dissociation constant (kd) were analyzed using Octet BLI Analysis software. An ideal binding-dissociation curve was fitted, and the equilibrium dissociation constant KD (kd / ka) between the antibody and antigen was calculated. Affinity test results are shown in Tables 16 and 17.

[0918] Table 16 Results of antibody-recombinant human Trop2-His protein affinity assay

[0919] Antibody Ka(1 / Ms) Kd(1 / s) KD(M) hu44E7-3 1.38E+05 1.72E-03 1.25E-08 Trop2×Her2bsAb 1.97E+05 2.01E-03 1.02E-08

[0920] Table 17 Results of antibody-recombinant human Her2-His protein affinity assay

[0921] Antibody Ka(1 / Ms) Kd(1 / s) KD(M) Herceptin 4.31E+05 6.52E-04 1.51E-09 Trop2×Her2bsAb 5.20E+05 1.03E-03 1.99E-09

[0922] Example 19: Detection of Trop2×Her2 Bispecific Antibody Internalization Activity

[0923] This embodiment uses an endpoint method to compare the endocytic activity of Trop2×Her2 bsAb bispecific antibody and the parent monoclonal antibody. Specifically, the digested NCI-N87 cells (Wuhan Shangen Biotechnology, SNL-106) and HCC1954 cells (Shenzhen Huatuo Biotechnology, HTX2453) were adjusted to a density of 1×10⁻⁶ cells one day before the experiment. 5The antibody was seeded at a rate of 50 μL / well into 96-well cell culture plates and cultured overnight at 37°C with 5% CO2 to allow cell adhesion. The next day, the antibody was diluted to 24 μg / mL, and the goat anti-human Fc secondary antibody (SSA015) labeled with pHAb Amine and Thiol Reactive Dyes (Promega, G9841) was diluted to 72 μg / mL. 25 μL of the antibody and 25 μL of the labeled goat anti-human Fc secondary antibody were mixed and incubated at room temperature in the dark for 30 min before being added to the corresponding cell wells. After culturing at 37°C with 5% CO2 for 24 hours, the cells were digested with trypsin and washed twice with FACS buffer. The mean fluorescence intensity of the PE signal in the cells was detected by flow cytometry and plotted.

[0924] Figure 13 The results showed that in NCI-N87 cells, Trop2×Her2 bsAb had higher endocytic activity than the parental monoclonal antibody hu44E7-3 and Herceptin, indicating that the two antibodies had a synergistic effect in endocytic activity.

[0925] Example 20 Preparation of Antibody-Drug Conjugates

[0926] Antibody-drug conjugates were prepared by linking MC-VC-PAB-MMAE (Med-ZY-B003, Medicilon Biopharmaceuticals) to Herceptin, hu44E7-3, and Trop2×Her2 bsAb antibody molecules using a thiol conjugation method. The chemical structure of MC-VC-PAB-MMAE is shown below. Figure 13 The specific operation is as follows: First, the disulfide bonds between antibody chains are reduced using TCEP. Specifically, 2 mL of 2.5 mg / mL antibody solution is added to a 15 mL centrifuge tube; 50 mM TCEP aqueous solution is added while stirring continuously at room temperature. The TCEP solution can be drawn up with a pipette tip and slowly added while stirring continuously for 1 min to obtain a homogeneous solution. The TCEP to antibody ratio is 6.5 equiv, and the mixture is incubated at 37°C for 2 h. After the reaction, the MMAE-linker is coupled to the antibody at a ratio of 15 equiv. Specifically, the MMAE-linker is diluted to 5 mM with room temperature DMSO solution. The antibody reaction solution is removed, and 5% (v / v) DMSO solution is added, stirring continuously for 1 min; the drug-linker solution is divided into 5 portions and steadily added to the reaction solution while stirring for 1 min to obtain a homogeneous solution; the mixture is then placed in a 25°C water bath and reacted for 3 h. After the reaction, the mixture is centrifuged using a desalting centrifuge column (Thermo Scientific). TM(89893) Remove free MMAE-linkers. Concentrate the filtered ADC solution. Filter the concentrated ADC solution through a 0.22 μm sterile filter in a sterile laminar flow hood for sterilization. Determine the concentration of the ADC solution using UV chromatography. Detect the purity of the ADC solution using SEC-HPLC and the drug-to-antibody ratio (DAR) of the conjugated ADC using HIC-HPLC. Table 18 summarizes the average DAR value of the ADC molecules obtained by HIC assay and the purity results obtained by SEC assay.

[0927] Table 18. Detection results of DAR value and purity of ADC molecules

[0928] DAR(HIC) Purity (SEC) Herceptin-MMAE 5.61 81.56％ hu44E7-3-MMAE 4.46 95.50％ Trop2×Her2bsADC 5.54 94.29％

[0929] The chemical structure of MC-VC-PAB-MMAE is as follows: Figure 14 As shown.

[0930] Example 21: In vitro tumor-killing activity assay of Trop2×Her2 bispecific antibody-drug conjugate.

[0931] This embodiment tested the in vitro killing activity of the prepared antibody-drug conjugate against different tumor cell lines. The specific procedures were as follows: One day before the experiment, NCI-N87 cells (human gastric cancer cells), MDA-MB-468 cells (human breast cancer cells), and HCC1569 cells (human breast cancer cell line) were digested into single-cell suspensions using trypsin, and then the cell density was adjusted to 2*10⁻⁶ cells / cells using a medium containing 20% ​​FBS. 4 Cells were seeded at a rate of 2000 cells / well into 96-well cell culture plates and cultured overnight to allow cell adhesion. On the day of the experiment, the antibody-drug conjugate to be tested was taken and diluted with basal medium to prepare a 2× stock solution. The initial concentration of the stock solution in each well was 200 nM. Then, it was serially diluted 3-fold with basal medium to a total of 9 concentration points. A medium control was also set up. The serially diluted antibody-drug conjugate was mixed with the culture medium in the cell culture wells at a 1:1 ratio and then incubated in a 37°C CO2 incubator for 5 days. After 5 days, cell proliferation was detected using the Cell Counting-Lite 3D kit (Novizan, DD1102-02). The results are shown below. Figure 15 .

[0932] Figure 15The results showed that Trop2×Her2 bsADC exhibited significant cytotoxic activity against all three tumor cell lines. Specifically, in NCI-N87 cells with high Her2 expression and moderate Trop2 expression, Trop2×Her2 bsADC showed comparable cytotoxic activity to Herceptin-MMAE and was stronger than hu44E7-3-MMAE. In triple-negative breast cancer cells with very low Her2 expression, the cytotoxic effect, from highest to lowest, was hu44E7-3-MMAE, Trop2×Her2 bsADC, and Herceptin-MMAE. In HCC1569 breast cancer cells with low Trop2 expression, the cytotoxic effect, from highest to lowest, was Herceptin-MMAE, Trop2×Her2bsADC, and hu44E7-3-MMAE. In conclusion, compared to the parental monoclonal antibody ADC control, Trop2×Her2 bsADC has broader applicability against various tumor cell lines.

[0933] Example 22: In vivo efficacy of Trop2×Her2 bispecific antibody-drug conjugate

[0934] This embodiment uses CB17.SCID mice to establish an animal model of NCI-N87 human gastric cancer cell subcutaneous xenografts and observes the inhibitory effect of Trop2×Her2 bsADC on NCI-N87 cell subcutaneous xenografts. Specifically, after the CB17.SCID mice (Speford (Beijing) Biotechnology Co., Ltd.) passed quarantine, NCI-N87 cells in the logarithmic growth phase were collected and the concentration was adjusted to 10. 8 The cell mixture was prepared at a 1:1 ratio with an equal volume of matrix gel and injected subcutaneously into the right anterior axilla of each mouse. The cells were inoculated at a concentration of approximately 100–150 mm². 3 Animals were randomly divided into five groups of six each, based on tumor volume: PBS solvent group, Trop2×Her2 bsAb group, Herceptin-MMAE group, hu44E7-3-MMAE group, and Trop2×Her2bsADC group. After grouping, drug administration began (D1). The drug was prepared at 0.4 mg / mL with PBS and administered intravenously at a dose of 2 mg / kg twice weekly for 18 consecutive days. Tumor volume was measured twice weekly, and animal weight, complete blood count, and blood biochemical parameters were recorded.

[0935] according to Figure 16As shown, in the NCI-N87 model, Herceptin-MMAE, hu44E7-3-MMAE, and Trop2×Her2bsADC showed similar efficacy, with TGIs of 148%, 142%, and 141% after 6 doses, respectively. Trop2×Her2bsAb had no inhibitory effect on the subcutaneous xenograft model, with a TGI of only 6%. Regarding safety, the three antibody-drug conjugates had no effect on the animals' body weight. Figure 17 ), and no obvious blood toxicity was observed. Figure 18 Antibody-drug conjugates (ADCs) can cause varying degrees of elevation in gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), creatine kinase (creatine kinase), and aspartate aminotransferase (AST). The effect of Trop2×Her2 bsADC is less than that of Herceptin-MMAE and hu44E7-3-MMAE, indicating that Trop2×Her2 bsADC has a better safety profile. Figure 19 ).

Claims

1. An antibody against trophoblast cell surface antigen 2 (Trop2), its antigen-binding fragment, or a mutant thereof, comprising a heavy chain variable region (VHA) and a light chain variable region (VLA), wherein, The VHA includes VHA CDR1 shown in SEQ ID NO:1, VHA CDR2 shown in SEQ ID NO:2, and VHA CDR3 shown in SEQ ID NO:3, and the light VLA includes VLA CDR1 shown in SEQ ID NO:4, VLA CDR2 shown in SEQ ID NO:5, and VLA CDR3 shown in SEQ ID NO:

6.

2. The anti-Trop2 antibody of claim 1, its antigen-binding fragment, or a mutant thereof, wherein, The VHA comprises an amino acid sequence as shown in any one of SEQ ID NO: 7-14, or an amino acid sequence having a similarity of not less than 85% to an amino acid sequence shown in any one of SEQ ID NO: 7-14; and / or the VLA comprises an amino acid sequence as shown in any one of SEQ ID NO: 15-21, or an amino acid sequence having a similarity of not less than 85% to an amino acid sequence shown in any one of SEQ ID NO: 15-21.

3. An anti-Trop2 antibody, its antigen-binding fragment, or a mutant thereof, comprising a heavy chain (HA') and a light chain (LA), wherein the HA' comprises an amino acid sequence as shown in any one of SEQ ID NO: 22-29, or an amino acid sequence having at least 85% similarity to an amino acid sequence shown in any one of SEQ ID NO: 22-29; and / or the LA comprises an amino acid sequence as shown in any one of SEQ ID NO: 30-36, or an amino acid sequence having at least 85% similarity to an amino acid sequence shown in any one of SEQ ID NO: 30-36.

4. A bispecific antibody comprising an antigen-binding arm A that specifically binds to Trop2 and an antigen-binding arm B that specifically binds to another tumor surface antigen, wherein said antigen-binding arm A comprises VHA and VLA from the antibody, its antigen-binding fragment, or a mutant thereof, as described in any one of claims 1-3.

5. A bispecific antibody comprising an antigen-binding arm A that specifically binds to Trop2 and an antigen-binding arm B that specifically binds to HER2; wherein, The antigen-binding arm A comprises a heavy chain variable region A (VHA) and a light chain variable region A (VLA), wherein the VHA comprises VHA CDR1 shown in SEQ ID NO:1, VHA CDR2 shown in SEQ ID NO:2, and VHA CDR3 shown in SEQ ID NO:3, and the VLA comprises VLA CDR1 shown in SEQ ID NO:4, VLA CDR2 shown in SEQ ID NO:5, and VLA CDR3 shown in SEQ ID NO:6; The second antigen-binding arm comprises a heavy chain variable region B (VHB) and a light chain variable region B (VLB), wherein the VHB comprises VHB CDR1 shown in SEQ ID NO:39, VHB CDR2 shown in SEQ ID NO:40, and VHB CDR3 shown in SEQ ID NO:41, and the VLB comprises VLB CDR1 shown in SEQ ID NO:42, VLB CDR2 shown in SEQ ID NO:43, and VLB CDR3 shown in SEQ ID NO:

44.

6. The bispecific antibody according to claim 5, wherein, The VLA and VHA respectively contain the amino acid sequences shown in SEQ ID NO:17 and SEQ ID NO:10, and the VLB and VHB respectively contain the amino acid sequences shown in SEQ ID NO:50 and 52.

7. A bispecific antibody comprising an antigen-binding arm A that specifically binds to Trop2 and an antigen-binding arm B that specifically binds to HER2; wherein, The antigen-binding arm A comprises heavy chain A (HA) and light chain A (VLA), and the antigen-binding arm B comprises heavy chain B (HB) and light chain A (LA), wherein the LA and the LB comprise amino acid sequences as shown in SEQ ID NO:32 and 56, the HA comprises Fc(A), and the Fc(A) comprises a knob mutation; the HB comprises Fc(B), and the Fc(B) comprises a hole mutation, as shown. The Fc(A) and the Fc(B) interact to form a KIH structure, and the binding arm A and the binding arm B assemble into a heterodimer through the KIH structure.

8. The bispecific antibody according to claim 7, wherein, The HAs each contain an amino acid sequence as shown in SEQ ID NO:48; the HBs each contain an amino acid sequence as shown in SEQ ID NO:

57.

9. The bispecific antibody according to claim 8, wherein the LA and the HA are linked by a linker, and the LB and the HB are linked by a linker, wherein, The linker is a linker peptide having the structure shown in (GGGGS)n; wherein n is any integer selected from 1 to 10; or preferably, n = 6, and the linker peptide has an amino acid sequence as shown in SEQ ID NO:

58.

10. An antibody-drug conjugate comprising a bispecific antibody according to any one of claims 4-9 and a cytotoxic therapeutic agent, wherein the bispecific antibody is linked to the cytotoxic drug portion via a linker structure.

11. The antibody-drug conjugate of claim 10, wherein, The connector structure is MC-VC-PAB.

12. The antibody-drug conjugate of claim 11, wherein, The cytotoxic drug is partially selected from DNA alkylating agents, DNA topoisomerase inhibitors, microtubule disruptors, RNApol II inhibitors, and microtubule destabilizers, wherein the microtubule destabilizer is selected from auristatin and maytansine.

13. The antibody-drug conjugate of claim 12, wherein, The cytotoxic drug is partly MMAE.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) An active ingredient selected from the group consisting of: an anti-TROP2 antibody according to any one of claims 1-3, an antigen-binding fragment thereof or a mutant thereof, an anti-Trop2 ADC, or a bispecific antibody binding to Trop2 and HER2 according to any one of claims 4-9, or an antibody-drug conjugate according to any one of claims 10-13, and (ii) Pharmaceutically acceptable carriers.

15. Any one or more of the following applications of the anti-Trop2 antibody according to any one of claims 1-3, or the bispecific antibody binding TROP2 and HER2 according to any one of claims 4-9, the antibody-drug conjugate according to claims 10-13, or the pharmaceutical composition according to claim 14: (1) Use in the preparation of drugs for the prevention or treatment of diseases related to Trop2 overexpression or dysfunction, or tumors targeting Trop2, diseases related to HER2 overexpression or dysfunction, or diseases with both Trop2 and HER2 positive. (2) Application in the preparation of drugs for killing cells that are overexpressing Trop2, or overexpressing HER2, or co-overexpressing Trop2 and HER2; (4) Application in the preparation of drugs for treating Trop2-positive or HER2-positive or Trop2 / HER2-positive tumors; (5) Application in the preparation of Trop2 / HER2 bispecifically binding antibody-drug conjugates; (6) Application in the preparation of drugs suitable for treating HER2-resistant tumors.

16. The application as described in claim 13, wherein, The diseases associated with Trop2 overexpression or dysfunction are solid tumors, wherein the solid tumors include or are selected from colorectal cancer, rectal cancer, gastric cancer, invasive endometrial adenocarcinoma, ovarian cancer, squamous bladder cancer, choriocarcinoma, bronchial cancer, breast cancer, cervical cancer, pancreatic cancer, seminal vesicle cancer, or non-small cell lung cancer; or the solid tumors are preferably Trop2-positive epithelial carcinomas, wherein the Trop2-positive epithelial carcinomas are breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer; The diseases associated with abnormal HER2 expression or function are selected from cancers including gastroesophageal junction cancer, breast cancer, bladder cancer, ampullary cancer, small bowel cancer, cervical cancer, endometrial cancer, non-small cell lung cancer, such as ovarian cancer, gastric cancer, lung adenocarcinoma, and invasive uterine cancer, such as serous endometrial cancer. The tumors that are double-positive for trop2 and HER2 include or are selected from breast cancer, gastric cancer and non-small cell lung cancer.

17. A method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof of any one of claims 1-3 and 4-8, or an antibody-drug conjugate of any one of claims 10 or 11.

18. A method for killing tumor cells, the method comprising: The tumor cells are contacted with an effective amount of the composition, said composition comprising an antibody or antigen-binding fragment thereof of any one of claims 1-3 and 4-8, or an antibody-drug conjugate of any one of claims 10 or 11.

19. The method according to claim 17 or 18, wherein the tumor is a solid tumor, the solid tumor being selected from solid tumors overexpressing Trop2, solid tumors overexpressing HER2; or solid tumors overexpressing both Trop2 and HER2; wherein, The solid tumors overexpressing Trop2 are selected from one of the following: colon cancer, rectal cancer, gastric cancer, invasive endometrial adenocarcinoma, ovarian cancer, squamous bladder cancer, choriocarcinoma, bronchial cancer, breast cancer, cervical cancer, pancreatic cancer, seminal vesicle cancer, and non-small cell lung cancer; the solid tumors are preferably Trop2-positive epithelial carcinomas, wherein the Trop2-positive epithelial carcinomas are breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer; The diseases associated with abnormal HER2 expression or function are selected from cancers including gastroesophageal junction cancer, breast cancer, bladder cancer, ampullary cancer, small bowel cancer, cervical cancer, endometrial cancer, non-small cell lung cancer, such as ovarian cancer, gastric cancer, lung adenocarcinoma, and invasive uterine cancer, such as serous endometrial cancer. The tumors that are double-positive for trop2 and HER2 include or are selected from breast cancer, gastric cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, lung cancer, renal cancer, and non-small cell lung cancer.

20. The method according to any one of claims 17-19, wherein the solid tumor is a solid tumor overexpressing Trop2 and HER2, and the solid tumor overexpressing Trop2 and HER2 is selected from breast cancer, gastric cancer, colorectal cancer, kidney cancer, cervical cancer, ovarian cancer, lung cancer, renal cell carcinoma, and non-small cell lung cancer.

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