Use of bispecific antibody in preparation of drug for treating cancers

Abstract

Provided is a use of a bispecific antibody in preparation of a drug for treating cancers, and in particular a use in preparation of a drug for treating advanced solid tumors.

Description

Use of a bispecific antibody in the preparation of a drug for treating cancer

[0001] This application claims priority to Chinese Patent Application No. 2023112834484, filed September 28, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention belongs to the field of medicine and relates to the use of a bispecific antibody in the preparation of a drug for treating cancer. The bispecific antibody is a drug that simultaneously inhibits the human epidermal growth factor receptor and tyrosine kinase Met dual targets. Based on its unique structure and novel mechanism of action, the bispecific antibody has the potential to achieve clinical benefits that are difficult to achieve with single drugs or combination drugs. Background Art

[0003] EGFR, a member of the human epidermal growth factor receptor (HER) family, is widely distributed on the surface of epithelial, mesenchymal, and neuronal tissues and cells. Under physiological conditions, binding to its ligand (such as epidermal growth factor (EGF)) promotes cell differentiation, proliferation, migration, and survival. Under pathological conditions, EGFR acts as an oncogene, promoting tumor development and progression through gene mutation and upregulation of expression. Common EGFR mutations occur in exons 18-21, primarily exon 19 deletion (Exon 19del), exon 21 L858R point mutation (L858R), and exon 20 insertion mutation (Exon 20ins). EGFR mutations or overexpression are highly prevalent in NSCLC (50% mutations), head and neck cancer (80% overexpression), colon cancer (25% mutations), and breast cancer (11% overexpression / mutation). Several drugs targeting EGFR have demonstrated clinical efficacy in these indications.

[0004] The MET gene is a proto-oncogene that encodes the mesenchymal epidermal transition factor (c-MET), also known as the hepatocyte growth factor receptor (HGFR). c-MET / HGFR also belongs to the receptor tyrosine kinase (RTK) family and is a transmembrane tyrosine kinase receptor. After binding to its ligand hepatocyte growth factor (HGF), it can induce MET dimerization and phosphorylation of its substrates, thereby activating downstream signaling pathways and participating in the regulation of cell proliferation, differentiation, migration, angiogenesis and other processes.

[0005] The c-MET pathway is closely linked to EGFR resistance. MET gene expression bypasses the inhibited EGFR phosphorylation kinase pathway, providing a bypass pathway by activating ERBB3 and the PI3K / AKT pathway downstream of MET. MET activates and promotes downstream signal transduction through this bypass pathway, preventing EGFR TKI-induced cell apoptosis while promoting tumor cell proliferation, ultimately leading to EGFR TKI resistance. Therefore, simultaneous inhibition of the EGFR and c-MET pathways is essential to overcome EGFR TKI resistance caused by MET pathway activation. This provides a theoretical basis for the combined use of c-MET pathway inhibition and EGFR pathway inhibition in the treatment of advanced solid tumors.

[0006] Due to its unique mutation structure, EGFR Exon 20ins is insensitive to existing first- to third-generation EGFR TKIs, and chemotherapy and immunotherapy are also ineffective. The 2022 CSCO guidelines still recommend platinum-based chemotherapy as the standard first-line treatment. Currently, there is no standard treatment for patients with advanced solid tumors that develop EGFR resistance due to c-MET pathway activation. Therefore, finding effective treatments is a pressing clinical need.

[0007] Summary of the Invention

[0008] The present invention provides use of a bispecific antibody that specifically binds to c-Met and EGFR in preparing a drug for preventing or treating cancer.

[0009] The present invention also provides a use of a pharmaceutical composition of a bispecific antibody that specifically binds to c-Met and EGFR in the preparation of a drug for preventing or treating cancer.

[0010] The present invention also provides a bispecific antibody that specifically binds to c-Met and EGFR, or a pharmaceutical composition of the bispecific antibody, and its use in combination with an EGFR inhibitor in the preparation of a drug for preventing or treating cancer.

[0011] In some embodiments, the EGFR inhibitor is selected from erlotinib, gefitinib, befotertinib, osimertinib, almonertinib, furmonertinib, oritinib, rezetinib, avitinib, olmutinib, rociletinib; preferably almonertinib and osimertinib.

[0012] In some embodiments, the aforementioned bispecific antibody or bispecific antibody pharmaceutical composition and the EGFR inhibitor are contained in different preparations as active ingredients, and are administered simultaneously, concurrently, sequentially, continuously, alternately, or separately.

[0013] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment directed against a first target, c-Met, and a second antibody or antigen-binding fragment directed against a second target, EGFR; wherein the first antibody or antigen-binding fragment comprises a HCDR1 as set forth in SEQ ID NO:01, a HCDR2 as set forth in SEQ ID NO:02, a HCDR3 as set forth in SEQ ID NO:03; and, a LCDR1 as set forth in SEQ ID NO:04, a LCDR2 as set forth in SEQ ID NO:05, and a LCDR3 as set forth in SEQ ID NO:06; wherein the second antibody or antigen-binding fragment comprises a HCDR1 as set forth in SEQ ID NO:09, a HCDR2 as set forth in SEQ ID NO:10, a HCDR3 as set forth in SEQ ID NO:11; and, a LCDR1 as set forth in SEQ ID NO:12, a LCDR2 as set forth in SEQ ID NO:13, and a LCDR3 as set forth in SEQ ID NO:14.

[0014] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment directed against a first target, c-Met, and a second antibody or antigen-binding fragment directed against a second target, EGFR, wherein the first antibody or antigen-binding fragment comprises a heavy chain variable region as set forth in SEQ ID NO:07 and a light chain variable region as set forth in SEQ ID NO:08; and the second antibody or antigen-binding fragment comprises a heavy chain variable region as set forth in SEQ ID NO:15 and a light chain variable region as set forth in SEQ ID NO:16.

[0015] In some embodiments, the c-Met binding domain of the bispecific antibody is a Fab region formed by a first heavy chain and a first light chain, the EGFR binding domain is a Fab region formed by a second heavy chain and a second light chain, and the first heavy chain and the second heavy chain bind to each other to form a heterodimeric Fc region.

[0016] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment directed against a first target, c-Met, and a second antibody or antigen-binding fragment directed against a second target, EGFR, wherein the first antibody or antigen-binding fragment comprises a heavy chain as set forth in SEQ ID NO: 21 and a light chain as set forth in SEQ ID NO: 22; and the second antibody or antigen-binding fragment comprises a heavy chain as set forth in SEQ ID NO: 23 and a light chain as set forth in SEQ ID NO: 24.

[0017] In some embodiments, the cancer is a cancer expressing c-Met and / or EGFR.

[0018] In some embodiments, the aforementioned cancer is a c-Met-expressing cancer.

[0019] In some embodiments, the cancer is an EGFR-expressing cancer.

[0020] In some embodiments, the aforementioned cancer is a cancer expressing c-Met and EGFR.

[0021] In some embodiments, the cancer expressing c-Met or / and EGFR is mediated by wild-type EGFR, EGFR activating mutations, EGFR gene amplification, elevated levels of circulating HGF, wild-type c-Met, c-Met activating mutations, c-Met gene amplification, MET gene amplification, or mutant KRAS, preferably mediated by EGFR activating mutations, MET gene amplification.

[0022] The exemplary EGFR activating mutation that can be associated with cancer includes point mutation, deletion mutation, insertion mutation, inversion or gene amplification that causes at least one biological activity of EGFR to increase (such as tyrosine kinase activity raising, receptor homodimer and heterodimer formation, ligand binding enhancement etc.).Sudden change can be located in any part of the regulatory region of EGFR gene or associated with EGFR gene, and includes the mutation in exon 18,19,20 or 21 or the mutation in kinase domain.Other examples of EGFR activating mutation are known in the art (see, for example, U.S. Patent Publication US2005 / 0272083).Information about EGFR and other ErbB receptors (including receptor homodimer and heterodimer, receptor ligand, autophosphorylation site and the signal transduction molecule that participates in the signal transduction of ErbB mediation) is known in the art (see, for example, Hynes and Lane, Nature Reviews Cancer 5:341-354,2005).

[0023] In some embodiments, the cancer is a cancer expressing EGFR or / and c-Met mediated by an EGFR activating mutation. In some embodiments, the EGFR activating mutation comprises at least one mutation selected from the group consisting of: G719X (X is any amino acid), L861X (X is any amino acid).

[0024] In some embodiments, the EGFR activating mutation comprises at least one mutation selected from the group consisting of: L718Q, L718V, G719A, G719S, G719C, G719D, L858R, L858P, L861Q, G724S, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, S768I, or T790M substitution; deletion of E746-A750; deletion of R748-P753; insertion of Ala (A) between M766 and A767; insertion of Ser, Val, and Ala (SVA) between S768 and V769; Asn and Ser (NS) between P772 and H773 insertion of one or more amino acids between D761 and E762, between A763 and Y764, between Y764 and Y765, between M766 and A767, between A767 and V768, between S768 and V769, between V769 and D770, between D770 and N771, between N771 and P772, between P772 and H773, between H773 and V774, between V774 and C775; deletion of one or more amino acids in EGFR exon 19; or insertion of one or more amino acids in EGFR exon 19; one or more deletions in EGFR exon 20; or one or more insertions in EGFR exon 20; or any combination thereof.

[0025] In some embodiments, the EGFR activating mutation comprises at least one mutation selected from the group consisting of: L858R mutation, T790M mutation, L861Q mutation, one or more insertion mutations in EGFR exon 20, and one or more amino acid deletion mutations in EGFR exon 19. In some embodiments, the above-mentioned cancer is selected from solid tumors, preferably advanced solid tumors.

[0026] In some embodiments, the cancer is selected from epithelial cell cancer, breast cancer, ovarian cancer, lung cancer, rectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, endometrial cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer or melanoma, preferably lung cancer or liver cancer.

[0027] In some embodiments, the cancer is selected from non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer, small cell lung cancer, sarcomatoid carcinoma of the lung, and hepatocellular carcinoma (HCC).

[0028] In a preferred embodiment, the cancer is selected from non-squamous non-small cell lung cancer. In a preferred embodiment, the cancer is selected from locally advanced or metastatic non-squamous non-small cell lung cancer.

[0029] In a preferred embodiment, the above-mentioned cancer is selected from locally advanced or metastatic non-small cell lung cancer, carrying an EGFR activating mutation including at least one mutation selected from the group consisting of: L858R mutation, T790M mutation, L861Q mutation, C797S mutation, one or more insertion mutations in EGFR exon 20, and one or more amino acid deletion mutations in EGFR exon 19.

[0030] In a preferred embodiment, the above-mentioned cancer is selected from locally advanced or metastatic non-squamous non-small cell lung cancer, carrying an EGFR activating mutation including at least one mutation selected from the group consisting of: L858R mutation, one or more insertion mutations in EGFR exon 20, and one or more amino acid deletion mutations in EGFR exon 19.

[0031] In a preferred embodiment, the cancer is selected from locally advanced or metastatic non-squamous non-small cell lung cancer comprising a deletion mutation of one or more amino acids in EGFR exon 19 or an L858R mutation.

[0032] In a preferred embodiment, the cancer is selected from locally advanced or metastatic non-small cell lung cancer comprising one or more insertion mutations in exon 20 of EGFR.

[0033] In some embodiments, the dosage of the EGFR inhibitor is 10-500 mg, preferably 10-200 mg, more preferably 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg or 200 mg.

[0034] In some embodiments, the EGFR inhibitor is administered twice a day, once a day, once every two days, or once every three days, preferably once a day or once every two days.

[0035] In a preferred embodiment, the dosage of the EGFR inhibitor is 110 mg, and the administration frequency is once a day.

[0036] The bispecific anti-EGFR / c-Met antibody can be administered in a pharmaceutically acceptable excipient, diluent, or carrier.

[0037] "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the antibody of the invention is administered. Such vehicles can be liquids, such as water and oils, including those derived from petroleum, animals, plants, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine can be used to formulate bispecific anti-EGFR / c-Met antibodies. These solutions are sterile and generally free of particulate matter. They can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). For parenteral administration, the carrier may include sterile water, and other excipients may be added to increase solubility or preservative properties. Injectable suspensions or solutions can also be prepared using aqueous carriers together with suitable additives. Suitable vehicles and formulations include other human proteins (e.g., human serum albumin).

[0038] In some embodiments, the bispecific antibody composition unit concentration is about 1 mg / mL to about 100 mg / mL, preferably about 5 mg / mL to about 50 mg / mL, and more preferably about 10 mg / mL to about 30 mg / mL.

[0039] In some embodiments, the bispecific antibody pharmaceutical composition unit concentration is about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL.

[0040] In some embodiments, the specifications of the bispecific antibody pharmaceutical composition are 10 mL:10 mg to 10 mL:1000 mg per vial, preferably 10 mL:100 mg to 10 mL:500 mg, more preferably 10 mL:100 mg, 10 mL:200 mg, 10 mL:300 mg, 10 mL:400 mg, 10 mL:500 mg.

[0041] The administration method can be any suitable route for delivering the above-mentioned bispecific anti-EGFRxc-Met antibodies to the host, such as parenteral administration, for example, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, vaginal, rectal), using formulations in the form of tablets, capsules, solutions, powders, gels, granules; and contained in syringes, implant devices, osmotic pumps, cartridges, mini-pumps; or other methods well known in the art as understood by the skilled person. Site-specific administration can be achieved, for example, by intratumoral, parenteral, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracavitary, intracerebellar, intracerebroventricular, intracolonic, intracervical, intragastric, intrahepatic, intracardiac, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, oral, sublingual, intranasal, or transdermal delivery.

[0042] In some embodiments, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered at a dose of between about 100 mg to about 5000 mg.

[0043] In some embodiments, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered at a dose of between about 400 mg to about 2000 mg.

[0044] In some embodiments, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered at about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg, about 650 mg, about 660 mg, about 670 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg g, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg, about 850 mg, about 860 mg, about 870 mg, about 880 mg, about 890 mg, about 900 mg, about 910 mg, about 920 mg, about 930 mg, about 940 mg, about 950 mg, about 960 mg, about 970 mg, about 980 mg, about 990 mg, about 1000 mg, about 1010 mg, about 1020 mg, about 1030 mg, about 1040 mg, about 105 0mg, about 1060mg, about 1070mg, about 1080mg, about 1090mg, about 1100mg, about 1110mg, about 1120mg, about 1130mg, about 1140mg, about 1150mg, about 1160mg, about 1170mg, about 1180mg, about 1190mg, about 1200mg, about 1210mg, about 1220mg, about 1230mg, about 1240mg, about 1250mg, about 1260mg, about 1270mg, about 1280mg, about 1290mg, about 1300mg, about 1310mg, about 1320mg, about 1330mg, about 1340mg, about 1350mg, about 1360 mg, about 1370 mg, about 1380 mg, about 1390 mg, about 1400 mg, about 1410 mg, about 1420 mg, about 1430 mg, about 1440 mg, about 1450 mg, about 1460 mg, about 1470 mg, about 1480 mg, about 1490 mg, about 1500 mg, about 1510 mg, about 1520 mg, about 1530 mg, about 1540 mg, about 1550 mg, about 1560 mg, about 1570 mg, about 1580 mg, about 1590 mg, about 1600 mg, about 1610 mg, 1620 mg, about 1630 mg, about 1640 mg, about 1650 mg, about 1660 mg, about 1670 mg,or about 2000 mg.

[0045] In a preferred embodiment, the above-mentioned bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered at a dose of 400 mg, 800 mg, 1000 mg, 1200 mg, 1400 mg, 1600 mg, 1800 mg, or 2000 mg.

[0046] In a preferred embodiment, the above-mentioned bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered at a dose of 400 mg, 800 mg, 1200 mg, 1600 mg, or 2000 mg.

[0047] In some embodiments, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered once daily. In some embodiments, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered once every two days. In some embodiments, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered once every three days. In some embodiments, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered twice weekly. In some embodiments, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered once weekly. In some embodiments, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered once every two weeks. In some embodiments, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered once every three weeks. In some embodiments, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered once monthly.

[0048] In a preferred embodiment, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered once a week for the first treatment cycle and then once every two weeks, with every four weeks being one treatment cycle.

[0049] In a preferred embodiment, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered once a week for the first treatment cycle and then once every three weeks, with each three weeks constituting one treatment cycle.

[0050] In preferred embodiments, the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is administered at a dose of 400 mg, 800 mg, 1200 mg, 1600 mg, or 2000 mg; the dosing frequency is once a week for the first treatment cycle, once every two weeks starting from the second treatment cycle, with each four-week treatment cycle constituting one treatment cycle. The present invention provides a method for treating or preventing cancer, comprising administering the aforementioned bispecific antibody or pharmaceutical composition to a subject in need thereof.

[0051] The present invention provides a method for treating or preventing cancer, comprising administering the aforementioned bispecific antibody or bispecific antibody pharmaceutical composition and the aforementioned EGFR inhibitor in combination to a subject in need thereof, wherein the combined administration can be simultaneous, concurrent, sequential, continuous, alternating or separate administration.

[0052] In some embodiments, the above-mentioned cancer is a solid tumor, preferably epithelial cell cancer, breast cancer, ovarian cancer, lung cancer, rectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, endometrial cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, colon cancer, thyroid cancer, liver cancer or melanoma, more preferably non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer, small cell lung cancer, lung sarcoma-like cancer, hepatocellular carcinoma.

[0053] In a preferred embodiment, the cancer is locally advanced or metastatic non-small cell lung cancer, preferably locally advanced or metastatic non-small cell lung cancer with EGFR mutation.

[0054] In some embodiments, the above-mentioned EGFR mutation includes at least one mutation selected from the group consisting of: L858R mutation, T790M mutation, L861Q mutation, C797S mutation, one or more insertion mutations in EGFR exon 20, and one or more amino acid deletion mutations in EGFR exon 19.

[0055] In a preferred embodiment, the cancer is non-small cell lung cancer with a deletion mutation of one or more amino acids in EGFR exon 19 or an L858R mutation.

[0056] In vitro pharmacodynamic test results showed that the bispecific anti-EGFRxc-Met antibody of the present invention or its combination can bind to cynomolgus monkey EGFR and c-MET, and enhanced ADCC activity can be detected. In vivo pharmacodynamic test results showed that the bispecific anti-EGFRxc-Met of the present invention has a tumor inhibition effect superior to Amivantamab, a similar drug already on the market. The tumor inhibition effect of the bispecific anti-EGFRxc-Met of the present invention combined with osimertinib is significantly stronger than that of the two single drugs, demonstrating a good combined synergistic effect; no new toxicity was observed in the animals in the combined drug group. All animals in the EGFR triple mutation and EGFR-20 exon insertion mutation tumor models were well tolerated and effectively inhibited tumor growth. It has biological activity and therapeutic potential for the treatment of advanced malignant tumors.

[0057] In some embodiments, the bispecific antibodies comprise a first antibody or antigen-binding fragment directed against a first target, c-Met, and a second antibody or antigen-binding fragment directed against a second target, EGFR, wherein the first antibody or antigen-binding fragment comprises a heavy chain variable region as set forth in SEQ ID NO: 07 or a light chain variable region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 07, and a light chain variable region as set forth in SEQ ID NO: 08 or a light chain variable region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 08; and the second antibody or antigen-binding fragment comprises a light chain variable region as set forth in SEQ ID NO: 15 or a light chain variable region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15. NO:15 has a heavy chain variable region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, and a light chain variable region as set forth in SEQ ID NO:16 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:16.

[0058] In some embodiments, the bispecific antibody comprises a first antibody or antigen-binding fragment directed against a first target, c-Met, and a second antibody or antigen-binding fragment directed against a second target, EGFR, the first antibody or antigen-binding fragment comprising a heavy chain as set forth in, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 21, and a light chain as set forth in, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 22; and the second antibody or antigen-binding fragment comprising a heavy chain as set forth in, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 23, and a light chain as set forth in, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 24. A light chain as set forth in SEQ ID NO:24 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24.

[0059] In some embodiments, the EGFR activating mutation is T790M / L858R, D770-N771 ins_SVD, Del19 / T790M / C797S, or exon 19 deletion.

[0060] In some embodiments, the bispecific antibody pharmaceutical composition unit concentration is 1 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL.

[0061] In some embodiments, the bispecific antibody pharmaceutical composition further comprises a buffer and sucrose.

[0062] In some embodiments, the buffer is citric acid-sodium citrate, and the concentration is 5-150 mM, such as 10 mM; the concentration of sucrose is 20-60 mg / mL, such as 40 mg / mL.

[0063] In some embodiments, the first dose of the bispecific anti-EGFR / c-Met antibody or pharmaceutical composition thereof is more than 400 mg and is administered on the first and second days of the first cycle, with 400 mg administered on the first day and the remaining dose on the second day. The second and subsequent doses do not need to be administered in divided doses.

[0064] The present invention provides a bispecific antibody pharmaceutical composition, which is the above-mentioned bispecific antibody pharmaceutical composition.

[0065] In a preferred embodiment, the bispecific antibody pharmaceutical composition further comprises an EGFR inhibitor, wherein the EGFR inhibitor is selected from one or more of almonertinib, erlotinib, gefitinib, befotertinib, osimertinib, furmonertinib, oritinib, rezetinib, avitinib, olmutinib and rociletinib; preferably almonertinib or osimertinib.

[0066] The present invention provides a bispecific antibody pharmaceutical composition for treating or preventing cancer, wherein the bispecific antibody pharmaceutical composition is the above-mentioned bispecific antibody pharmaceutical composition.

[0067] In a preferred embodiment, the bispecific antibody pharmaceutical composition further comprises an EGFR inhibitor, wherein the EGFR inhibitor is selected from one or more of almonertinib, erlotinib, gefitinib, befotertinib, osimertinib, furmonertinib, oritinib, rezetinib, avitinib, olmutinib and rociletinib; preferably almonertinib or osimertinib. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 shows the quality identification results of the bispecific antibody EGFRxc-Met-1. Part A of Figure 1 shows the purity of the bispecific antibody obtained by affinity purification, and Part B of Figure 1 shows the proportion of correctly paired products of the purified bispecific antibody.

[0069] Figure 2 shows the effect of the combination of bispecific antibody EGFRxc-Met-1 and ametinib on the growth of subcutaneous xenograft tumors in PC-9 nude mice. DETAILED DESCRIPTION

[0070] Detailed Description of the Invention

[0071] 1. Terminology

[0072] In order to make the present application easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this application belongs.

[0073] When a list is provided, it is understood that each individual element of the list and each combination of the list is a separate embodiment unless otherwise indicated. For example, a list of embodiments presented as "A, B, or C" will be understood to include embodiments "A," "B," "C," "A or B," "A or C," "B or C," or "A, B, or C."

[0074] "About" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular assay, result, or embodiment, unless expressly stated otherwise in the examples or elsewhere in the specification, "about" means within one standard deviation or up to 5%, whichever is greater, as is customary in the art.

[0075] The three-letter and one-letter codes for amino acids used herein are as described in J. Biol. Chem, 243, p3558 (1968).

[0076] The term "antibody" as used herein refers to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and order of the constant region of the heavy chains of immunoglobulins differ, resulting in different antigenicity. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same class, Ig can be further divided into different subclasses based on the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on differences in the constant region. Each of the five Ig classes can have either kappa or lambda chains.

[0077] In the present application, the antibody light chain variable region described herein may further comprise a light chain constant region, wherein the light chain constant region comprises a human or murine κ, λ chain or a variant thereof.

[0078] In the present application, the antibody heavy chain variable region described herein may further comprise a heavy chain constant region, wherein the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0079] The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region (V region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region (C region). The variable region comprises three hypervariable regions (HVRs) and four framework regions (FRs), whose sequences are relatively conserved. These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from amino to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues in the VL and VH regions of the antibodies or antigen-binding fragments described herein conform to the known Kabat numbering convention and Kabat or ABM definition convention (http: / / bioinf.org.uk / abs / ).

[0080] The term "EGFR" as used herein refers to human epidermal growth factor receptor 1, also known as ErbB-1 or HER1. EGFR is a receptor tyrosine kinase that comprises an extracellular ligand-binding domain, a membrane-spanning domain, and an intracellular kinase domain. Upon binding to its ligands (e.g., epidermal growth factor (EGF) and transforming growth factor α (TGFα)), EGFR forms homodimers or heterodimers with other ErbB receptors, and its kinase function is activated, leading to autophosphorylation of several tyrosine residues in the intracellular domain. Anti-EGFR antibodies are antibodies that specifically bind to EGFR. In certain embodiments, anti-EGFR antibodies can interfere with or inhibit the activation of EGFR, for example, by preventing ligand binding and / or receptor dimerization. Upon activation, EGFR can transmit signals to downstream effectors, including PI3-K, RAS-RAF-MAPK P44 / P42, and protein kinase C signaling pathways, ultimately transmitting the signals to the cell nucleus to regulate cell proliferation.

[0081] As used herein, the term "EGFR exon 20 insertion mutation" or "EGFR exon 20 mutation," or "EGFR Exon20ins," or "Exon20ins" refers to a mutation comprising at least one insertion or duplication of amino acids 762 to 823 of human EGFR. EGFR exon 20 insertion mutations can be detected using known methods, such as sequencing, including next-generation sequencing (NGS), fluorescence in situ hybridization, immunohistochemistry, flow cytometry, or Western blotting.

[0082] "EGFR and / or c-Met-expressing cancer" refers to a cancer that has detectable EGFR and / or c-Met expression or has EGFR and / or c-Met mutation or amplification. EGFR or c-Met expression, amplification, and mutation status can be detected using known methods, such as sequencing, including next-generation sequencing, fluorescence in situ hybridization, immunohistochemistry, flow cytometry, or Western blotting.

[0083] The term "recombinant human antibody" includes human antibodies prepared, expressed, created or isolated by recombinant means, and the techniques and methods involved are well known in the art, such as:

[0084] 1. Antibodies isolated from transgenic or transchromosomal animals (e.g., mice) expressing human immunoglobulin genes or hybridomas prepared therefrom;

[0085] 2. Antibodies isolated from host cells transformed to express the antibody, such as transfectomas;

[0086] 3. Antibodies isolated from a recombinant combinatorial human antibody library; and

[0087] 4. Antibodies prepared, expressed, created, or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.

[0088] Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by the germline genes, but also include subsequent rearrangements and mutations that occur, such as during antibody maturation.

[0089] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into the human antibody variable region framework. Humanized antibodies can overcome the disadvantage of chimeric antibodies, which often induce strong immune responses due to the presence of large amounts of mouse protein components. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the human antibody variable region to maintain activity.

[0090] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogs, which generally include at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of a parental antibody. Antibody fragments retain at least some of the binding specificity of the parental antibody. Typically, when activity is expressed on a molar basis, an antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody for the target. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23: 1126-11.

[0091] A "Fab fragment" consists of a light chain and the CH1 and variable region of a heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0092] The "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domain.

[0093] A "Fab' fragment" contains one light chain and a portion of one heavy chain including the VH domain and the CH1 domain as well as the region between the CH1 and CH2 domains, such that an interchain disulfide bond can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0094] A "F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0095] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0096] The term "multispecific antibody" is used in its broadest sense to encompass antibodies with multiple epitope specificities. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multiple epitope specificities; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable domains, each binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments that are covalently or non-covalently linked together, etc.

[0097] The term "single-chain antibody" refers to a single-chain recombinant protein composed of the heavy chain variable region VH and the light chain variable region VL of an antibody connected by a connecting peptide. It is the smallest antibody fragment with a complete antigen binding site.

[0098] The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment can target the same or different antigens.

[0099] The term "single-arm antibody" as described herein refers to an antigen-binding fragment comprising a Fab segment and an Fc segment, typically a Fab segment comprising a heavy chain (e.g., VH and CH1) and a light chain (e.g., VL and CL), and an Fc segment comprising a constant region (e.g., CH2 and CH3). The Fab segment and the Fc segment can be connected by a linker or not. The single-arm antibody of the present invention can be prepared or synthesized by a variety of methods. For example, the sequence encoding the Fab heavy chain and the sequence encoding the Fc are constructed into the same vector, and the sequence encoding the Fab light chain is constructed into another vector, and the two vectors are separately transformed into host cells to obtain a single-arm antibody.

[0100] The term "antigen binding site" in this application refers to the three-dimensional site recognized by the antibody or antigen-binding fragment of this application.

[0101] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope generally comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitope is bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0102] The terms "specific binding" and "selective binding" used in this application refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when human EGFR or c-Met is used as an analyte and an antibody is used as a ligand, the antibody binds to the epitope at a specific binding site of less than 10 μg / L when measured by surface plasmon resonance (SPR) technology in an instrument. -7 M or even smaller equilibrium dissociation constant (K D ) binds to a predetermined antigen, and its affinity for binding to the predetermined antigen is at least twice that of its affinity for binding to a nonspecific antigen (such as BSA, etc.) other than the predetermined antigen or a closely related antigen. The term "antibody that recognizes an antigen" is used interchangeably with the term "specifically binding antibody" herein.

[0103] The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking. Inhibition / blocking of a ligand preferably reduces or alters the normal level or type of activity that would occur upon ligand binding in the absence of inhibition or blocking. Inhibition and blocking are also intended to encompass any measurable decrease in ligand binding affinity when in contact with the antibody, compared to the ligand not in contact with the antibody.

[0104] The term "inhibit growth" (eg, with respect to cells) is intended to include any measurable decrease in cell growth.

[0105] The terms "induce an immune response" and "enhance an immune response" are used interchangeably and refer to the stimulation of an immune response (ie, passive or adaptive) to a specific antigen. The term "induce" with respect to inducing CDC or ADCC refers to the stimulation of specific direct cell killing mechanisms.

[0106] "ADCC," as used herein, stands for antibody-dependent cell-mediated cytotoxicity, and refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the antibody's Fc region. Modification of the Fc region of IgG can enhance, reduce, or eliminate the antibody's ADCC effector function. Such modifications involve mutations in the heavy chain constant region of the antibody.

[0107] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found in, for example, Chapters 5-8 and 15 of the Cold Spring Harbor Laboratory Manual of Antibody Laboratory Techniques. For example, mice can be immunized with human BCMA or fragments thereof, and the resulting antibodies can be renatured, purified, and subjected to amino acid sequencing using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described herein are engineered to have one or more human FR regions added to the non-human CDR regions using genetic engineering methods. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website at http: / / imgt.cines.fr or from the Journal of Immunoglobulins, 2001, ISBN 012441351.

[0108] The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified using conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred prior art, mammalian expression systems can lead to glycosylation of antibodies, especially in F C The highly conserved N-terminus of the region is expressed. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product must be immediately frozen, such as at -70°C, or lyophilized.

[0109] The term "pharmaceutical composition" is a product comprising one or more active ingredients (e.g., antibodies, small molecule drugs) in optionally specified amounts, as well as any product produced directly or indirectly by combining one or more active ingredients in optionally specified amounts. The different active ingredients in the pharmaceutical composition can be administered independently in separate formulations, including administration simultaneously or at different time points for combined synergistic effect. In the present disclosure, "pharmaceutical composition" and "preparation" are not mutually exclusive.

[0110] The term "combination" or "combination" is a mode of administration that refers to the administration of at least one dose of a bispecific antibody or a bispecific antibody pharmaceutical composition and at least one dose of an additional therapeutic agent within a certain time limit, wherein both substances show a pharmacological effect. The time limit can be within a dosing cycle, preferably within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours, more preferably within 12 hours. The bispecific antibody or bispecific antibody pharmaceutical composition and the additional therapeutic agent can be administered simultaneously or sequentially. This period includes such treatments, wherein the bispecific antibody or bispecific antibody pharmaceutical composition and the additional therapeutic agent are administered by the same route of administration or different routes of administration. The combined administration mode described in the present application is selected from simultaneous administration, independent formulation and co-administration, or independent formulation and sequential administration.

[0111] The terms "administer," "give," and "treat," when applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administer," "give," and "treat" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treat," when applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0112] The term "treatment" means administering an internal or external therapeutic agent, such as any of the antibodies of the present application, to a patient who has one or more symptoms of a disease, and it is known that the therapeutic agent has a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptom. Although the embodiments of the present application (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating the symptoms of the target disease in every patient, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0113] The term "preventing" a disease or disorder means preventing the disorder from occurring in a subject.

[0114] The term "diagnosis" refers to a method of determining whether a subject has a given disease or condition, is likely to develop a given disease or condition in the future, or is likely to respond to treatment for a previously diagnosed disease or condition (i.e., to stratify a patient population according to likelihood of response to treatment). Diagnosis is typically performed by a physician based on general guidelines for the disease to be diagnosed or other criteria indicating that a subject is likely to respond to a particular treatment.

[0115] The term "consisting essentially of" or variations thereof as used throughout the specification and claims means that all recited elements or groups of elements are included, and optionally other elements of similar or different properties from the recited elements, which do not significantly alter the basic or novel properties of a specified dosage regimen, method, or composition.

[0116] As used herein, the term "naturally occurring" as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide sequence or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is naturally occurring.

[0117] The term "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or signs of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0118] The term "exogenous" refers to substances that are produced outside the body of an organism, cell, or human, depending on the context.

[0119] The term "endogenous" refers to a substance that is produced within a cell, organism, or human body in context.

[0120] The term "homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0121] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It should also be understood that, due to deliberate or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny that possess the same function or biological activity as that screened for in the originally transformed cell are included. Where a different designation is intended, this is clear from the context.

[0122] "Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the specified sequence can but need not be present.

[0123] DETAILED DESCRIPTION

[0124] The following examples further illustrate the present application, but these examples are not intended to limit the scope of this application. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[0125] Example 1 Construction and preparation of bispecific antibodies

[0126] Monoclonal antibody screening

[0127] The sequence of the anti-c-MET antibody-1 used in this application was obtained by immunizing mice with the mesenchymal epidermal transition factor (c-MET) antigen (purchased from AcroBiosystems), extracting total RNA, performing reverse transcription, and screening a yeast display library constructed by PCR amplification. The sequence of Antibody-1 was also obtained by sequencing, where the CDR sequences of the antibody were determined using the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0128] Specifically, a fully human anti-c-MET antibody library was constructed from five immunized mice, with a diversity of 3×10 7 Human c-MET protein was labeled using a biotin labeling kit (purchased from Thermo) according to the product instructions. Yeast cells that specifically bind to c-MET were enriched using MACS, and after multiple rounds of flow cytometry sorting, yeast cells that specifically bind to the biotin-labeled c-MET protein with high affinity were obtained. Using the final sorted yeast cells as templates, the antibody heavy and light chain sequences were retrieved and constructed into expression vectors for antibody production.

[0129] Table 1. cMET Antibody-1 Sequence Information

[0130] After antibody preparation, ForteBio affinity assays were performed according to a previously reported method (Estep, P et al., High-throughput method for antibody-antigen affinity and epitope binding determination. MAbs, 2013. 5(2): p. 270-8). The results showed that anti-c-MET antibody-1 had good binding activity to human c-MET protein and monkey c-MET protein.

[0131] Table 2. Affinity of candidate molecules for human c-Met

[0132] Table 3. Affinity of candidate molecules for monkey c-Met

[0133] Construction of anti-cMet and EGFR dual antibodies

[0134] According to the method described in the patent (patent application number: 201611016435.0), nucleotide sequences encoding the variable regions of anti-EGFR and anti-cMet antibodies were synthesized and linked to constant regions capable of spontaneously forming heterodimers. The anti-cMet antibody was c-MET antibody-1 obtained by the above screening, and the heavy and light chain variable regions of the anti-EGFR antibody were disclosed in patent number: WO02 / 100348. The first amino acid Q in the heavy chain variable region was mutated to E, and the first amino acid A in the light chain variable region was mutated to D. Their sequences are shown in Table 4.

[0135] Table 4. EGFR antibody sequence information

[0136] Using CH1 / CL-preferring mutations (patent number: WO2021 / 067404A2) and Knob-in-Hole technology, the EGFRxcMET-1 molecule was constructed. The heavy chain variable regions of the anti-cMet antibody and anti-EGFR antibody were constructed into the CH1 mutant heavy chain constant regions CH SET1 (SEQ ID NO: 17) and CH SET2 (SEQ ID NO: 18), respectively. The light chain variable regions of the anti-cMet antibody and anti-EGFR antibody were constructed into the CL mutant light chain constant regions CL SET1 (SEQ ID NO: 19) and CL SET2 (SEQ ID NO: 20), respectively. A 1+1 asymmetric anti-EGFRxcMet bispecific antibody cell line was constructed.

[0137] Table 5. EGFRxcMet bispecific antibody sequence information

[0138] The vectors pCHO2.0-GS-Puro-H1-L1, containing nucleotide sequences encoding the heavy chain (SEQ ID NO: 21) and light chain (SEQ NO: 22) of an anti-c-Met antibody, and pCHO2.0-GS-Puro-H2-L2, containing nucleotide sequences encoding the heavy chain (SEQ ID NO: 23) and light chain (SEQ ID NO: 24) of an anti-EGFR antibody, were co-transfected into the CHOS-ADP Fut8 KO (defucosylation knockout) host cell line by electroporation. Puromycin and MSX pressure were used to select the cells to obtain high-yielding minipools. A single round of limiting dilution and single cloning analysis identified high-yielding, stable clones. The cloned cell line, 6F5, was confirmed as a recombinant engineered cell line by comparing its growth, protein expression, and physicochemical properties.

[0139] Dynamis AGT Medium was used as the basal medium, and the inoculation density was (1.0±0.2)×10 6 cells / mL, and 5.0±0.5% (w / w) of the initial culture weight of 7a was added as feed on the 3rd, 5th, 7th, 9th, and 11th days, respectively, and 0.5±0.05% (w / w) of the initial culture weight of 7b was added as feed. The dissolved oxygen was set at 40%, the initial culture temperature was 36.5°C, and the temperature was lowered to 33.0°C on the 4th day. Every day, 300g / kg of glucose concentrate was added to the glucose concentration in the cell fluid to reach 6.0g / L based on the glucose concentration test results, except on the day of harvest. The culture was terminated on the 14th day or when the cell viability was less than 80%. During the culture process, cell density and viability were detected using Vicell (Beckman Company). Starting from the 7th day, the antibody production was detected every day using Cedex (Roche Company). The results are shown in Table 4, and the expression level of the bispecific antibody reached 2.432g / L.

[0140] The supernatant was collected and the target protein was purified using protein A magnetic beads (purchased from GenScript) sorting. The magnetic beads were resuspended in an appropriate volume of binding buffer (PBS + 0.1% Tween 20, pH 7.4) (1-4 times the volume of the magnetic beads) and added to the sample to be purified. Incubate at room temperature for 1 hour with gentle shaking during the period. The sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed 3 times with binding buffer. Elution buffer (0.1M sodium citrate, pH 3.2) was added according to 3-5 times the volume of the magnetic beads and shaken at room temperature for 5-10 minutes. The beads were returned to the magnetic stand, the elution buffer was collected, and the mixture was transferred to a collection tube to which neutralization buffer (1M Tris, pH 8.54) had been added and mixed to obtain the bispecific antibody sample EGFRxcMET-1.

[0141] Protein purity was determined by HPLC. The HPLC method was as follows: mobile phase: 150 mM Na₂HPO₄·12H₂O, pH 7.0. Chromatographic conditions: detection wavelength 280 nm, column temperature 25°C, flow rate 0.5 mL / min, detection time: 30 min, TSK gel G3000SWXL column. SEC results, shown in Figure 1, Part A, indicate a purity of 98.18% for the bispecific antibody obtained by one-step affinity purification.

[0142] The heavy and light chain pairing of the protein was determined by high-performance liquid chromatography-mass spectrometry using a Vanquish UHPLC system (Thermo), a Q Exactive mass spectrometer (Thermo), and a Waters ACQUITY UPLC BEH C4 column, 2.1 mm × 10 mm. A 50 μg sample was diluted to 25 μL with ultrapure water. After centrifugation, 20 μL of the sample was transferred to a 10 vial, and 5 μL was injected. The intact molecular weight was analyzed by LC-MS. Chromatographic conditions were: column temperature: 80°C; UV detection wavelength: 280 nm; flow rate: 0.3 mL / min; mobile phase A: aqueous solution (containing 0.1% formic acid); mobile phase B: acetonitrile solution (containing 0.1% formic acid). Mass spectrometry parameters were: ESI source: ion transfer tube temperature 320°C, voltage 3.8 kV, gas flow rate 36 L / min; mode: positive ion Full MS; resolution: 17500; scan range: 600-4000 m / z. As shown in Figure 1, Part B, the purified bispecific antibody showed >98% correct pairing.

[0143] Example 2 Preparation of Anti-EGFRxc-Met Bispecific Antibody Injection

[0144] Using citric acid-sodium citrate (abbreviated as: CA) buffer, bispecific antibody liquid formulations with concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg / mL were prepared, and sucrose was optionally added.

[0145] Table 6. EGFRxcMet bispecific antibody injection ingredients

[0146] Prepare 1 kg of 10 mM citric acid-sodium citrate buffer by weighing 0.282 g of citric acid monohydrate (C6H8O7·H2O) and 2.547 g of sodium citrate dihydrate (C6H5Na3O7·2H2O). Add water to 1 kg and filter the buffer. Replace EGFRxcMET-1 with the prepared 10 mM citric acid-sodium citrate buffer via dialysis. Add sucrose mother liquor to prepare the target formulations, with protein concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 mg / mL. Filter using a 0.22 μm PVDF filter in a biosafety cabinet. Dispense into vials, stopper, and cap to prepare bispecific antibody injections.

[0147] Example 3 Binding activity of anti-EGFRxc-Met bispecific antibody to EGFR and c-Met positive tumor cells

[0148] A375 cells are human malignant melanoma cells purchased from (Addexbio) with low expression of EGFR and c-Met receptors. H292 cells are human lung adenocarcinoma cells purchased from (procell) with higher expression of EGFR and c-Met receptors. HCC827 cells are human non-small cell lung cancer cells purchased from (Chinese Academy of Sciences Cell Collection), with high expression of EGFR and c-Met receptors. The expanded cultured cells were adjusted to an appropriate cell density and added to a 96-well flow cytometer. After centrifugation, the gradient diluted sample to be tested was added and incubated at 4°C for 1 hour. Wash twice with PBS, add the corresponding fluorescent secondary antibody diluted to the appropriate concentration, incubate at 4°C for 30 minutes, and wash twice with PBS. Resuspend the cells in PBS, detect on a CytoFlex flow cytometer, and calculate the corresponding MFI.

[0149] For tumor cells with different EGFR and MET expression peaks, the EGFRxc-Met bispecific antibody candidate EGFRxcMET-1 showed strong cell binding activity, with a binding strength of EC 50 The specific values ​​are shown in Table 7 below.

[0150] Table 7. Antibody binding activity to tumor cells

[0151] Example 4 Anti-EGFRxc-Met bispecific antibody induces ADCC effect

[0152] A375 cells are human malignant melanoma cells purchased from Addexbio, with low expression of EGFR and c-Met receptors. H1975 cells are human lung adenocarcinoma cells purchased from Addexbio, with high expression of EGFR and c-Met receptors. HCC827 cells are human non-small cell lung cancer cells purchased from the Chinese Academy of Sciences Cell Bank, with high expression of EGFR and c-Met receptors. The expanded cultured A375 / H1975 / HCC827 cells were resuspended in 1640 medium at a cell density of 1.2×10 6 cells / mL; CD16a-NFAT-Luc Jurkat reporter gene cells (self-made, CD16a and NFAT-Luc gene sequences were constructed in the pCDNA3.1 vector, then transfected into Jurkat cells, and the CD16a-NFAT-Luc Jurkat reporter gene cells were obtained by antibiotic resistance pressure screening) were resuspended in 1640 medium and the cell density was adjusted to 6×10 6 cells / mL cells / mL; the antibody was diluted to 300 nM with 1640 medium, then diluted 4-fold in a series, 25 μL of the above-mentioned diluted antibody was added to each well of a 96-well cell culture plate, and 25 μL of the above-mentioned target cells were added to each well, mixed, and incubated at room temperature for 30 min. Then, 25 μL of the above-mentioned effector cells were added to each well, mixed, and incubated at 37°C, 5% CO2 for 6 h. Then, 75 μL of Bio-turbo reagent (purchased from Ruian) was added to each well, mixed, and the chemiluminescent signal was collected using a microplate reader.

[0153] The EGFRxc-Met bispecific antibody EGFRxMET-1 induced ADCC effect and showed dependence on the amount of antigen expression (HCC827>H1975>A375).

[0154] Example 5 Tumor Inhibitory Effects of Different Doses of EGFRxc-Met Bispecific Antibodies in H292 Tumor-Bearing CB-17 SCID Mouse Model

[0155] This study established a tumor-bearing model by subcutaneously inoculating CB-17 SCID mice with human lung cancer H292 cells (highly expressing EGFR / c-MET). This model can be used to evaluate the anti-tumor and disease-related safety profiles of the test article's mechanism of action. Four groups of six mice were intraperitoneally injected with PBS and three different doses of EGFRxcMET-1 (2, 8, and 32 mg / kg) every four to six days for a total of three doses. The safety and growth inhibitory effects of different doses of the bispecific antibody were evaluated in a subcutaneously inoculated CB-17 SCID mouse model of human lung cancer H292 cells.

[0156] The tumors of mice in the negative control PBS group grew rapidly, and the average tumor volume reached 900 mm at the end of the experiment (D30). 3 The above results indicate that the tumor model of this experiment was successfully established.

[0157] At the end of the experiment, the tumor volumes of the PBS group and the 2, 8, and 32 mg / kg EGFRxcMET-1 groups were 930.82 mm 3 、587.42mm 3 (P<0.05), 65.03mm 3 (P<0.0001), 45.35 mm 3 Compared with the negative control PBS group, each dose group of EGFRxcMET-1 inhibited tumor growth in a dose-dependent manner, with TGIs of 45%, 113%, and 115%, respectively. Tumor weights were 0.53g, 0.30g (P<0.01), 0.03g (P<0.0001), and 0.01g (P<0.0001), respectively. The results are shown in Table 8. No abnormalities were observed in the mice in each group. Compared with the control group, no significant decrease in body weight was observed in any of the treated groups. Gross dissection of the major organs of the mice in each group at the end of the study revealed no significant abnormalities, indicating that the drugs in each group did not cause significant toxicity in the mice at the doses used in this study.

[0158] Table 8. Effects of each treatment group on the volume, weight and TGI of H292 subcutaneous transplanted tumors at the end of the experiment (D30) Note: Compared with the PBS group, “NS” P>0.05; “*” P<0.05; “**” P<0.01; “****” P<0.0001

[0159] Example 6 Study on the Growth Inhibitory Effect of Osimertinib Combined with Subcutaneous Inoculation of H1975-HGF Tumor Model in NOD / SCID Mice

[0160] In this study, a tumor model was established by subcutaneously inoculating NOD / SCID mice with H1975-HGF (highly expressing EGFR (T790M / L858R) and c-MET) human lung cancer cells. This model can be used to evaluate the antitumor effects related to the mechanism of action of the test article and the safety profile in the disease state. Six groups of six mice were divided into each group and received intraperitoneal injections of PBS, 3 mg / kg rivrevant, 3 mg / kg EGFRxcMET-1, 3 mg / kg osimertinib, 3 mg / kg rivrevant combined with 3 mg / kg osimertinib, and 3 mg / kg EGFRxcMET-1 combined with 3 mg / kg osimertinib. Osimertinib was administered once daily for a total of six doses, while EGFRxcMET-1 / rivrevant was administered every other day for a total of two doses. To investigate the growth inhibitory effect and safety of EGFRxcMET-1 combined with osimertinib on the H1975-HGF tumor model inoculated subcutaneously in NOD / SCID mice.

[0161] Taking into account the endpoints of tumor volume, TGI, and tumor weight, one-sixth of the tumors in the EGFRxcMET-1 combined with osimertinib group regressed, while none of the other groups showed tumor regression. The tumor inhibition effect of EGFRxcMET-1 combined with osimertinib was significantly stronger than either drug alone, demonstrating a strong synergistic effect. Furthermore, the tumor inhibition effect of EGFRxcMET-1 combined with osimertinib was superior to that of ervantumab combined with osimertinib.

[0162] Table 9. Effects of each treatment group on H1975-HGF tumor volume, weight and TGI Note: Compared with the PBS group, “NS” P>0.05; “*” P<0.05; “**” P<0.01; “***” P<0.001; “****” P<0.0001; N / A, no statistical analysis was performed.

[0163] Example 7 In vivo pharmacodynamic study of Ba / F3 EGFR D770-N771 ins_SVD nude mouse subcutaneous transplant tumor model

[0164] In a subcutaneous xenograft tumor model of Ba / F3 EGFR D770-N771 ins_SVD nude mice harboring an EGFR exon 20 insertion mutation, 10 mg / kg of ervantumab and 3 and 10 mg / kg of EGFRxcMET-1 were administered intraperitoneally twice weekly for two weeks (a total of four doses). The corresponding tumor inhibition rates (TGI) were 53.35%, 49.23%, and 58.41%, respectively. At the same dose (10 mg / kg), EGFRxcMET-1 demonstrated comparable tumor inhibition efficacy to ervantumab and was well tolerated by all animals.

[0165] Table 10. Evaluation of anti-tumor efficacy on Ba / F3 EGFR-D770-N771ins_SVD xenograft tumor model

[0166] Example 8 In vivo pharmacodynamic study of Ba / F3 EGFR-Del19 / T790M / C797S nude mouse subcutaneous transplant tumor model

[0167] In a subcutaneous xenograft tumor model of nude mice harboring triple EGFR mutations (Ba / F3 EGFR-Del19 / T790M / C797S), 10 mg / kg of ervantumab and 3, 10, and 30 mg / kg of EGFRxcMET-1 were administered intraperitoneally twice weekly for two weeks (a total of four doses). The corresponding tumor inhibition rates (TGI) were 97.17%, 75.81%, 96.57%, and 121.57%, respectively. EGFRxcMET-1 demonstrated a significant tumor inhibition effect with a favorable dose-response relationship. At the same dose (10 mg / kg), the tumor inhibition effect was comparable to that of ervantumab and was well tolerated by all animals.

[0168] Table 11. Evaluation of anti-tumor efficacy in Ba / F3 EGFR-Del19 / T790M / C797S xenograft tumor model

[0169] Example 9 In vivo pharmacodynamic study of ametinib combined with human non-small cell lung cancer PC-9 cell line derived subcutaneous transplanted tumor model in nude mice

[0170] This study evaluated the synergistic antitumor effect of combined therapy with EGFRxc-Met bispecific antibody and ametinib in a nude mouse subcutaneous xenograft tumor model derived from the non-small cell lung cancer PC-9 cell line harboring EGFR exon 19 deletion mutation.

[0171] PC-9 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. After the cells were fully grown, they were divided into flasks and passaged. Cells in the logarithmic growth phase were collected and counted. The cells were resuspended in PBS to a final concentration of 5.0×10 7 cells / mL and then inoculated. 0.1mL PC-9 cell suspension (containing 5×10 6 The cells were subcutaneously inoculated on the right back of 8-10 week old BALB / c nude mice. The tumor growth was observed and the animals were randomly divided into groups according to tumor volume and body weight. The average tumor volume at the time of grouping was 328 mm 3 The day of administration was defined as Day 1. The remaining animals were euthanized at the end of the experiment. This experiment was divided into 5 groups, with 1 animal in each group. The experimental design was as follows:

[0172] Table 12. Experimental design of PC-9 nude mouse subcutaneous xenograft tumor model a. Dosing volume: 10 μL / g based on mouse body weight. If body weight loss exceeds 15%, the dosing regimen should be adjusted accordingly. bpo: oral administration; ip: intraperitoneal administration; c. qd: once daily; biw: twice weekly.

[0173] The mice in the Vehicle solvent control and IgG control groups were euthanized on the 29th day after continuous administration; the EGFRxcMET-1 group was euthanized on the 60th day after continuous administration; the ametinib group and the two-drug combination group were discontinued after 90 days of continuous administration, and euthanized on the 65th day after discontinuation of the drug. The tumor volume data at each time point in each group are expressed as the mean and standard error (SEM). Based on the tumor volume data at different time points in each group, Dunnett's multiple comparisons test in Two-way or One-way ANOVA was used to analyze the differences between the groups. All data were analyzed using GraphPad Prism 9, and p < 0.05 was considered to be significantly different. The tumor inhibition rate of mice in each group on Day 22 after administration is shown in Table 13, and the tumor inhibition rate of mice in each group at the efficacy endpoint is shown in Table 14.

[0174] Table 13. Evaluation of the anti-tumor efficacy of the combination of bispecific antibodies and ametinib in the PC-9 xenograft tumor model (22 days after administration) Note: The data in the table are all from the 22nd day.

[0175] Table 14. Evaluation of the anti-tumor efficacy of the combination of bispecific antibodies and ametinib in the PC-9 xenograft tumor model (efficacy endpoint on day 155) Note: The data in the table are the endpoints of the efficacy of each group on day 155.

[0176] The efficacy evaluation of the ametinib monotherapy group and the EGFRxcMET-1 + ametinib combination group ended on Day 155, and the mouse tumors were removed and photographed. In the ametinib monotherapy group, after 3 weeks of administration, the average tumor volume was 18.79 mm 3 The TGI was 194.28%, and the average tumor volume of the whole group continued to shrink. From the 78th day, the average tumor volume was less than 2mm 3 , 7 mice had complete tumor regression. After drug withdrawal, the tumors that had regressed in the ametinib monotherapy group recurred and continued to grow. The average tumor volume at the end point of drug efficacy reached 903.91mm 3 , and among the 7 mice whose tumors had completely regressed before, 4 had relapsed and 7 mice had tumor burden at the efficacy endpoint (7 / 10). For the EGFRxcMET-1 + ametinib combination group, after 3 weeks of continuous administration, the average tumor volume was 12.60mm 3 The TGI was 196.17%, and the average tumor volume of the whole group continued to shrink. From the 78th day, the average tumor volume was less than 1mm 3 , 9 mice had complete tumor regression. After drug withdrawal, only 3 mice had tumor recurrence, but the size of the tumor was always around 5mm. 3 The average tumor volume of the combined group was 111.77 mm 3 Only one mouse in the whole group was tumor-bearing (1 / 10). The tumor volumes of each group at different time points are shown in Figure 2.

[0177] EGFRxcMET-1 demonstrated significant tumor inhibition in a nude mouse subcutaneous xenograft model derived from the human non-small cell lung cancer PC-9 cell line harboring an EGFR exon 19 deletion mutation. The combination of EGFRxcMET-1 and ametinib significantly enhanced the tumor inhibition compared with EGFRxcMET-1 alone, and significantly delayed tumor recurrence and growth compared with ametinib alone.

[0178] Example 10 Phase I Clinical Study of Safety, Tolerability, Pharmacokinetics, and Efficacy of EGFRxc-Met Bispecific Antibody in Patients with Advanced Solid Tumors

[0179] 1. Research Objectives

[0180] To evaluate the safety and tolerability, PK characteristics, efficacy, immunogenicity, and other safety indicators of EGFRxcMET-1 in subjects with locally advanced or metastatic NSCLC and EGFR mutations. To evaluate the efficacy, safety, PK (pharmacokinetic) characteristics, immunogenicity, and other efficacy indicators of EGFRxcMET-1 in subjects with advanced solid tumors.

[0181] 2. Study Drug

[0182] The test drug was EGFRxcMET-1 injection, the sequence of which is as described in SEQ ID NOs: 21-24 of the present invention. Dosage form: injection, specifications: 10 mL: 200 mg / vial. All study drugs used in this trial were provided by Shanghai Hansen Biopharmaceutical Technology Co., Ltd.

[0183] 3. Dosage regimen

[0184] The dose-escalation trial will administer EGFRxcMET-1 to target subjects via intravenous infusion. The dose escalation will begin with a preset starting dose of 400 mg and increase sequentially to 400 mg, 800 mg, 1200 mg, 1600 mg, and 2000 mg.

[0185] The Q2W dosing regimen: Subjects received an intravenous infusion once weekly starting on Day 1 of Cycle 1 (C1D1), and every two weeks starting in Cycle 2, with each four-week treatment cycle. For the 400mg dose group, the first dose was administered on C1D1. For the 800mg and higher dose groups, the first dose was administered on both C1D1 and C1D2, with 400mg administered on C1D1 and the remaining dose on C1D2. The second and subsequent doses do not require split doses.

[0186] Q3W dosing regimen: Subjects received the drug via intravenous drip once weekly on C1D1, C1D8, C1D15, and C2D1, followed by a three-week treatment cycle. The first dose was administered on C1D1 and C1D2, with 400 mg administered on C1D1 and the remaining dose on C1D2. The second and subsequent doses did not require split dosing.

[0187] 4. Inclusion criteria

[0188] Phase Ia: Subjects with histologically or cytologically confirmed locally advanced or metastatic NSCLC who have failed or are intolerant to standard treatment or are unable to obtain standard treatment and carry EGFR activating mutations.

[0189] Phase Ib: Cohort 1 includes patients with histologically or cytologically confirmed locally advanced or metastatic NSCLC who have failed or are intolerant to platinum-based chemotherapy and harbor EGFR Exon 20ins mutations. Cohort 2 includes patients with other advanced solid tumors who have failed or are intolerant to standard treatment based on histological or cytological confirmation.

[0190] 5. Clinical trial results

[0191] Of the 19 subjects enrolled, 14 underwent dose-escalation studies (1 in the 400 mg Q2W group, 3 in the 800 mg Q2W group, 4 in the 1200 mg Q2W group, and 6 in the 1600 mg Q2W group), and 5 underwent dose-expansion studies (4 in the 800 mg Q2W group and 1 in the 2000 mg Q3W group).

[0192] Dose escalation has been completed across four dose cohorts, with no DLT (dose-limiting toxicity) events observed. The maximum tolerated dose (MTD) of EGFRxcMET-1 has not been reached. Among 16 efficacy-evaluable subjects, one experienced a partial response (PR) and eight experienced stable disease (SD), with five subjects experiencing target lesion reduction, for a disease control rate (DCR) of 56.3%.

[0193] In summary, the current research results show that EGFRxcMET-1 has an anti-tumor effect in subjects with advanced EGFR activating mutation NSCLC who have failed or are intolerant to previous standard treatment, and preliminary anti-tumor activity was observed in all dose groups.

[0194] Example 11 Phase Ib / III Clinical Study of the Safety, Efficacy, Pharmacokinetics, and Immunogenicity of an EGFRxc-Met Bispecific Antibody Combined with Ametinib Mesylate Tablets in Patients with Advanced Non-Squamous Non-Small Cell Lung Cancer

[0195] 1. Research Objectives

[0196] To evaluate the efficacy, safety, tolerability, PK characteristics, and immunogenicity of different dose levels of EGFRxcMET-1 combined with ametinib in subjects with advanced non-squamous NSCLC harboring EGFR mutations who have not received prior systemic anti-tumor therapy.

[0197] 2. Study Drug

[0198] The drug under investigation was EGFRxcMET-1 injection, listed in SEQ ID NOs: 21-24 of the present invention. Dosage form: injection, specifications: 10 mL: 200 mg / vial. All study drugs used in this trial were provided by Shanghai Hansen Biopharmaceutical Technology Co., Ltd.

[0199] The study drug was ametinib mesylate tablets, dosage form: tablets, strength: 55 mg / tablet. All study drugs used in this trial were provided by Shanghai Hansen Biopharmaceutical Technology Co., Ltd.

[0200] 3. Dosage regimen

[0201] Two doses of EGFRxcMET-1 injection (800mg and 1200mg) were administered via intravenous drip once weekly starting on Cycle 1 (C1D1) and every two weeks starting in Cycle 2. Each four-week treatment cycle consisted of one dose. The first dose was administered on Cycles 1 and 2 (400mg on Cycle 1), and the remaining dose was administered on Cycle 2. The second and subsequent doses did not require split dosing.

[0202] Ametinib was administered orally at a dose of 110 mg once daily starting from C1D1, with a treatment cycle of 4 weeks.

[0203] 4. Inclusion criteria

[0204] Patients must have histologically or cytologically confirmed locally advanced or metastatic non-squamous NSCLC and carry an EGFR-sensitizing mutation (L858R mutation and / or exon 19 deletion mutation), either alone or in combination with other EGFR mutations. Patients must not have received prior systemic anti-cancer therapy or palliative lung therapy at the advanced stage.

[0205] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of a bispecific antibody in the preparation of a drug for preventing or treating cancer, characterized in that: The bispecific antibody comprises a first antibody or antigen-binding fragment against a first target, c-Met, and a second antibody or antigen-binding fragment against a second target, EGFR; wherein the first antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:01, HCDR2 as shown in SEQ ID NO:02, HCDR3 as shown in SEQ ID NO:03; and LCDR1 as shown in SEQ ID NO:04, LCDR2 as shown in SEQ ID NO:05, LCDR3 as shown in SEQ ID NO:06; The second antibody or antigen-binding fragment comprises HCDR1 as shown in SEQ ID NO:09, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:11; and LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:

14.

2. The use according to claim 1, characterized in that The c-Met binding domain of the bispecific antibody is a Fab region formed by a first heavy chain and a first light chain, the EGFR binding domain is a Fab region formed by a second heavy chain and a second light chain, and the first heavy chain and the second heavy chain are combined with each other to form a heterodimer Fc region.

3. Use of a bispecific antibody pharmaceutical composition in the preparation of a drug for preventing or treating cancer, characterized in that: The bispecific antibody pharmaceutical composition comprises the bispecific antibody for use according to claim 1 or 2 and one or more pharmaceutically acceptable excipients, diluents or carriers.

4. The use according to any one of claims 1 to 3, characterized in that The bispecific antibody or bispecific antibody pharmaceutical composition is used in combination with an EGFR inhibitor to prepare the drug.

5. The use according to claim 4, characterized in that The EGFR inhibitor is selected from one or more of almonertinib, erlotinib, gefitinib, befotertinib, osimertinib, furmonertinib, oritinib, rezetinib, avitinib, olmutinib and rociletinib; preferably almonertinib or osimertinib.

6. The use according to any one of claims 1 to 5, characterized in that The first antibody or antigen-binding fragment comprises a heavy chain variable region as set forth in SEQ ID NO:07 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:07, and a light chain variable region as set forth in SEQ ID NO:08 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:08; the second antibody or antigen-binding fragment comprises a heavy chain variable region as set forth in SEQ ID NO:15 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:15, and a light chain variable region as set forth in SEQ ID NO:16 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

17. NO:16 has a light chain variable region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

7. The use according to any one of claims 1 to 6, characterized in that The first antibody or antigen-binding fragment comprises a heavy chain as set forth in SEQ ID NO:21, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:21, and a light chain as set forth in SEQ ID NO:22, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:22; the second antibody or antigen-binding fragment comprises a heavy chain as set forth in SEQ ID NO:23, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:24, and a light chain as set forth in SEQ ID NO:25, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:

26. NO:24 has a light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

8. The use according to any one of claims 1 to 7, characterized in that The bispecific antibody, bispecific antibody pharmaceutical composition and EGFR inhibitor are contained in different preparations as active ingredients, respectively, and are administered simultaneously, concurrently, sequentially, continuously, alternately or separately.

9. The use according to any one of claims 1 to 8, characterized in that The cancer is selected from solid tumors, preferably advanced solid tumors.

10. The use according to any one of claims 1 to 9, characterized in that The cancer is a cancer expressing c-Met and / or EGFR.

11. The use according to claim 10, characterized in that The cancer expressing c-Met and / or EGFR is mediated by wild-type EGFR, EGFR activating mutation, EGFR gene amplification, increased levels of circulating HGF, wild-type c-Met, c-Met activating mutation, c-Met gene amplification, MET gene amplification, or mutant KRAS, preferably mediated by EGFR activating mutation, MET gene amplification.

12. The use according to claim 11, characterized in that The EGFR activating mutation comprises at least one mutation selected from the group consisting of: L718Q, L718V, G719A, G719S, G719C, G719D, L858R, L858P, L861Q, G724S, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, S768I or T790M substitution; deletion of E746-A750; deletion of R748-P753; insertion of Ala (A) between M766 and A767; insertion of Ser, Val and Ala (SVA) between S768 and V769; insertion of Asn and Ser (NS) between P772 and H773; Insertion of one or more amino acids between D761 and E762, between A763 and Y764, between Y764 and Y765, between M766 and A767, between A767 and V768, between S768 and V769, between V769 and D770, between D770 and N771, between N771 and P772, between P772 and H773, between H773 and V774, between V774 and C775; deletion of one or more amino acids in EGFR exon 19; or insertion of one or more amino acids in EGFR exon 19; deletion of one or more amino acids in EGFR exon 20; or insertion of one or more amino acids in EGFR exon 20.

13. The use according to any one of claims 1 to 12, characterized in that The cancer is selected from epithelial cell cancer, breast cancer, ovarian cancer, lung cancer, rectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, endometrial cancer, cervical cancer, spleen cancer, testicular cancer, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer or melanoma, preferably lung cancer or liver cancer.

14. The use according to claim 12, characterized in that The cancer is non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer, small cell lung cancer, lung sarcomatoid carcinoma or hepatocellular carcinoma (HCC); Preferably, the EGFR activating mutation is T790M / L858R, D770-N771 ins_SVD, Del19 / T790M / C797S or exon 19 deletion.

15. The use according to claim 14, characterized in that The cancer is non-squamous non-small cell lung cancer.

16. The use according to any one of claims 4 to 15, characterized in that The dosage of the EGFR inhibitor is 10-500 mg, preferably 10-200 mg, more preferably 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg or 200 mg.

17. The use according to any one of claims 4 to 16, characterized in that The EGFR inhibitor is administered twice a day, once a day, once every two days, or once every three days, preferably once a day or once every two days.

18. The use according to any one of claims 3 to 17, characterized in that The bispecific antibody pharmaceutical composition has a unit concentration of about 1 mg / mL to about 100 mg / mL, preferably about 5 mg / mL to about 50 mg / mL, more preferably about 10 mg / mL to about 30 mg / mL; for example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mg / mL; Preferably, the bispecific antibody pharmaceutical composition further comprises a buffer and sucrose; More preferably, the buffer is citric acid-sodium citrate with a concentration of 5-150 mM, such as 10 mM; the concentration of sucrose is 20-60 mg / mL, such as 40 mg / mL.

19. The use according to any one of claims 3 to 18, characterized in that The bispecific antibody pharmaceutical composition has a specification of 10 mL: 10 mg to 10 mL: 1000 mg per vial, preferably 10 mL: 100 mg to 10 mL: 500 mg, more preferably 10 mL: 100 mg, 10 mL: 200 mg, 10 mL: 300 mg, 10 mL: 400 mg or 10 mL: 500 mg.

20. The use according to any one of claims 1 to 19, characterized in that The dosage of the bispecific antibody or bispecific antibody pharmaceutical composition is about 100 mg to about 5000 mg, preferably 400 mg to about 2000 mg, and more preferably 400 mg, 800 mg, 1000 mg, 1200 mg, 1400 mg, 1600 mg, 1800 mg or 2000 mg.

21. The use according to any one of claims 1 to 20, characterized in that The administration frequency of the bispecific antibody or bispecific antibody pharmaceutical composition is once a day, once every two days, once every three days, twice a week, once a week, once every two weeks, once every three weeks, or once a month; preferably once a week, once every two weeks, or once every three weeks.

22. The use according to claim 21, characterized in that The administration frequency of the bispecific antibody or bispecific antibody pharmaceutical composition is: 1) Once a week in the first treatment cycle and once every two weeks from the second treatment cycle onwards, with each four weeks being a treatment cycle; or 2) Once a week in the first treatment cycle and once every three weeks from the second treatment cycle onwards, with every three weeks being one treatment cycle; Preferably, if the first dose is more than 400 mg, it is administered on the first and second days of the first cycle, with 400 mg administered on the first day and the remaining dose on the second day. The second and subsequent doses do not need to be administered in divided doses.

23. A method for treating or preventing cancer, characterized in that: The method comprises administering the bispecific antibody or bispecific antibody pharmaceutical composition for use according to any one of claims 1 to 22 to a subject in need thereof; Preferably, the administration frequency of the bispecific antibody or bispecific antibody pharmaceutical composition is the administration frequency in the use according to any one of claims 1 to 22; and / or the administration dosage is the administration dosage in the use according to claim 20.

24. The method of claim 23, wherein: The method further comprises administering to a subject in need thereof the EGFR inhibitor for use as described in any one of claims 4 to 17 in combination, wherein the combined administration is simultaneous, concurrent, sequential, continuous, alternating or separate administration; Preferably, the administration frequency of the EGFR inhibitor is the administration frequency in the use according to any one of claims 17 to 22; and / or, the administration dosage is the administration dosage in the use according to claim 16.

25. The method according to claim 23 or 24, characterized in that The cancer is a solid tumor; preferably, epithelial cell cancer, breast cancer, ovarian cancer, lung cancer, rectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, endometrial cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer or melanoma; more preferably, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer, small cell lung cancer, lung sarcomatoid cancer or hepatocellular carcinoma.

26. A bispecific antibody pharmaceutical composition, characterized in that: The bispecific antibody pharmaceutical composition is the bispecific antibody pharmaceutical composition for use according to any one of claims 3 to 22; Preferably, the bispecific antibody pharmaceutical composition further comprises an EGFR inhibitor, wherein the EGFR inhibitor is selected from one or more of almonertinib, erlotinib, gefitinib, befotertinib, osimertinib, furmonertinib, oritinib, rezetinib, avitinib, olmutinib and rociletinib; preferably almonertinib or osimertinib.

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