EGFR / c-met bispecific antibody and use thereof

Abstract

Disclosed is a bispecific antibody against EGFR and MET. By modifying the amino acid sequences of the Fc segments of the binding arms, a KIH structure is formed between the EGFR binding arm and the cMet binding arm of the bispecific antibody so as to be assembled into a heterodimer, which enhances the ADCC activity of the bispecific antibody, or improves the inhibition strength for ligand-mediated antigen phosphorylation. The prepared bispecific antibody is simple to prepare, has a stable structure and has a better tumor suppression effect, and thus has good market prospects.

Description

EGFR / c-Met bispecific antibodies and their applications Technical Field

[0001] The present invention relates to bispecific antibodies that bind to human epidermal growth factor receptor (EGFR) and human proto-oncogene receptor tyrosine kinase (c-Met), pharmaceutical compositions containing the same, and their use in preparing drugs for treating diseases or tumors involving cells with abnormal EGFR and c-Met, and drugs for inhibiting the growth or metastasis of tumors or cancer cells expressing EGFR and / or c-Met in a subject. Background Art

[0002] In 2020, lung cancer caused 1.8 million deaths worldwide, accounting for 18% of the 9.96 million total global cancer deaths. According to 2020 statistics, lung cancer accounted for 17.9% of new cancer cases in China, ranking first among all new cancer cases. Lung cancer also caused 710,000 deaths in China in 2020, representing 23.8% of all cancer deaths. NSCLC accounts for 80%-85% of all lung cancer cases. The most common driver mutation in NSCLC is EGFR mutation, particularly in Asian non-smokers, where the prevalence of EGFR mutations can reach as high as 40%-60%. A range of targeted therapies have been developed for different mutation types in this target. Gefitinib and erlotinib, as well as the EGFR monoclonal antibody cetuximab combined with afatinib, have demonstrated promising results in the treatment of NSCLC patients harboring EGFR mutations. However, nearly all patients experience varying degrees of tumor progression and inevitable acquired resistance after 6-12 months of EGFR-TKI treatment, ultimately leading to tumor recurrence or progression. The development of acquired resistance to EGFR-TKI limits the application of EGFR-targeted drugs, and the development of EGFR / c-Met dual-target inhibitors is expected to improve cancer efficacy and overcome resistance.

[0003] There are multiple mechanisms of acquired resistance to EGFR-TKI. Clinically, it has been found that among the many mechanisms of acquired resistance to EGFR-TKI, abnormal activation of the stem cell growth factor (HGF) / c-Met signaling pathway plays an important role (Nie Lin, International Journal of Oncology, 2016, 43(09): 703-705.). In addition, multiple literature reports show that c-Met is closely related to EGFR resistance. Among all secondary mutations, c-Met occupies a very important position, and about 25% of patients will develop c-Met secondary mutations. Studies have found that among NSCLC patients with EGFR mutations and acquired resistance to EGFR-TKI, about 20% of patients have c-Met amplification, and this is often accompanied by high expression of c-Met protein. According to research reports, in c-Met amplified resistant lung cancer cells, c-Met inhibitors can significantly increase the sensitivity of EGFR-TKI resistant lung cancer cells, but c-Met inhibitors alone have no significant effect. For these patients, the combination of c-Met inhibitors and EGFR inhibitors is of great significance for maintaining the efficacy of EGFR in targeted therapy of NSCLC.

[0004] EGFR and c-Met, two driver genes, complement each other in terms of targeted drug resistance. In other words, one reason for the failure of EGFR-targeted drugs is c-Met pathway activation, while the failure of c-Met-targeted drugs is also partially due to EGFR pathway activation. Therefore, dual-target specific inhibitory products that can simultaneously inhibit EGFR and c-Met have become a very important research and development direction in market demand.

[0005] Co-administration of anti-c-Met and anti-EGFR mAbs requires the injection of two separate products or a single injection of a co-formulation of the two different antibodies. Two injections allow for flexibility in dosage and timing, but present inconveniences for patients in terms of compliance and pain. Co-formulations can also provide some flexibility in dosage, but finding formulation conditions that ensure chemical and physical stability of both antibodies is often challenging or impossible due to their distinct molecular characteristics. Bispecific antibodies that simultaneously inhibit both EGFR and c-Met are emerging as an important development direction for addressing acquired resistance to EGFR-TKIs.

[0006] Currently, only one EGFR / c-Met bispecific antibody has been approved for marketing worldwide: amivantamab (Chinese name: eivantamab), developed by Janssen Pharmaceuticals, a subsidiary of Johnson & Johnson Pharmaceuticals. This offers new hope for patients. It is generally accepted in the art that when following the teachings of the art, including WO199509917, and attempting to generate viable bispecific antibodies, technicians often encounter problems related to the chemical and physical stability of the resulting bispecific antibodies. Amino acid changes are required in the resulting bispecific antibodies to fully overcome these problems. The amino acid sequence and structure of the antibody are important technical issues affecting the chemical and physical stability of bispecific antibodies. The art cannot demonstrate the need for amino acid changes, nor can it predict the actual amino acid sequence changes required to overcome the resulting problems. Furthermore, the required changes are often unconventional, unpredictable, or not derived from common knowledge. Similarly, bispecific antibodies generated from known antibodies are often found to be less satisfactory in at least one important functional pharmacokinetic or pharmacodynamic property when compared to their parent antibodies. There remains an unmet market demand for EGFR / c-Met bispecific antibody products in the cancer treatment field.

[0007] To address the issues of EGFR-TKI resistance and high-dose toxicity, thereby achieving enhanced efficacy and continuous improvement in safety, the field of cancer treatment still expects antibody engineering to obtain more novel anti-EGFR / c-Met bispecific antibodies as candidate functional molecules.

[0008] Summary of the Invention

[0009] To overcome the problems encountered with immunotherapy using anti-EGFR, anti-c-Met, or HGF monoclonal antibodies, the present invention provides novel bispecific antibodies that can simultaneously specifically bind to EGFR and block the binding of epidermal growth factor (EGF) to EGFR, and specifically bind to c-Met and block the binding of hepatocyte growth factor (HGF) to c-Met. The present invention also provides methods for preparing these bispecific antibodies, pharmaceutical compositions containing these bispecific antibodies, and the use of these bispecific antibodies in the preparation of pharmaceuticals.

[0010] In one aspect, the present invention provides an isolated bispecific antibody comprising an antigen-binding arm A that can specifically bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and block the binding of epidermal growth factor (EGF) to EGFR, and an antigen-binding arm B that can specifically bind to the extracellular portion of human proto-oncogene receptor tyrosine kinase (c-Met) and block the binding of hepatocyte growth factor (HGF) to c-Met; wherein the antigen-binding arm A comprises light chain A (LCA) and heavy chain A (HCA), and the antigen-binding arm B comprises light chain B (LCB) and heavy chain B (HCB).

[0011] Wherein, the LCA comprises a light chain variable region A (VLA), and the LCB comprises a light chain variable region B (VLB); the HCA comprises a heavy chain variable region A (VHA) and a heavy chain constant region A (HCACHA), and the HCB comprises a heavy chain variable region B (VHB) and a heavy chain constant region B (HCBCHB); wherein, the VLA and the VHA pair to form a binding site that specifically binds to EGFR, and the VLB and the VHB pair to form a binding site that specifically binds to c-Met.

[0012] In one embodiment, the HCA comprises at least one amino acid substitution in the HCACHA, and the HCB comprises at least one amino acid substitution in the HCBCHB, and the amino acid substitution in the HCACHA and the amino acid substitution in the HCBCHB occur at different amino acid residue positions when the residues are numbered according to the EU index.

[0013] In other embodiments, the present invention provides an EGFR / c-Met bispecific antibody, wherein, in an assay for EGF-activated EGFR autophosphorylation using the non-small cell lung cancer HCC827 cell line, the c-Met phosphorylation levels in the test group receiving the EGFR / c-Met bispecific antibody of the present invention and the control group not stimulated with HGF are similar; the EGFR phosphorylation levels in the test group receiving the EGFR / c-Met bispecific antibody of the present invention and the control group not stimulated with EGF are similar; the EGFR / c-Met bispecific antibody of the present invention inhibits EGF-activated EGFR autophosphorylation and HGF-activated c-Met autophosphorylation in the non-small cell lung cancer HCC827 cell line by at least 60%, at least 80%, or even at least 100%, thereby inhibiting downstream signaling activated by both EGFR and c-Met phosphorylation. The absorbance at 665 nm represents the total number of EGFR phosphorylation, and the absorbance at 615 nm represents the total number of EGFR proteins. The TR-FRET ratio is equal to the absorbance at 665 divided by the absorbance at 615 multiplied by 1000. The TR-FRET ratio is used to represent the level of EGFR autophosphorylation.

[0014] In other embodiments, the present invention provides an EGFR / c-Met bispecific antibody, wherein the antibody inhibits phosphorylation of EGFR at Y168 in the non-small cell lung cancer HCC827 cell line with a TR-FRET ratio of 33, which is close to that of the IgG1 group without HGF stimulation, and inhibits phosphorylation of c-Met at Y1234 / Y1235 in the non-small cell lung cancer HCC827 cell line with a TR-FRET ratio of 27, which is close to that of the IgG1 group without HGF stimulation.

[0015] In other embodiments, the present invention provides a c-MetEGFR / c-Met bispecific antibody that binds to EGFR as shown in SEQ ID NO:58 and to c-Met as shown in SEQ ID NO:60.

[0016] In other embodiments, the present invention provides an EGFR / c-Met bispecific antibody that has a stronger ability to inhibit the activity of MKN45, HCC827, or NCI-H1975-HGF cells than a combination of an anti-EGFR monoclonal antibody and an anti-c-MetG monoclonal antibody.

[0017] In other embodiments, the present invention provides an EGFR / c-Met bispecific antibody that inhibits the growth of EGFR and c-Met double-positive tumor cells. After adding the EGFR / c-Met bispecific antibody, the viability of human pancreatic cancer cells KP4, non-small cell lung cancer Kras mutant cells A549, human gastric cancer c-Met gene amplification cells MKN45, and non-small cell lung cancer EGFR mutant cells HCC827 is reduced by 40%-50%.

[0018] In other embodiments, the present invention provides EGFR / c-Met bispecific antibodies that can significantly inhibit the proliferation of human tumor cells KP4, A549, MKN45, and HCC827. When the bispecific antibody is administered at a dose of 250-500 μg / mL, the cell viability is reduced by as much as 10%, 20%, 30%, or 40% after 72 hours, which is comparable to the addition of and the marketed drug Avanta, and even has a stronger inhibitory ability.

[0019] In some embodiments, in the antigen-binding arm A, the LCA comprises the amino acid sequence shown in SEQ ID NO: 1.

[0020] In some embodiments, in the antigen-binding arm A, the HCA comprises the amino acid sequence shown in SEQ ID NO: 2.

[0021] In some embodiments, in the antigen-binding arm B, the LCB comprises the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, in the antigen-binding arm B, the LCB comprises the amino acid sequence shown in SEQ ID NO: 23.

[0022] In some embodiments, in the antigen-binding arm B, the HCB comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, in the antigen-binding arm B, the HCB comprises the amino acid sequence shown in SEQ ID NO: 24.

[0023] In some embodiments, the antigen binding arm A is a fusion peptide having an LCA-linker-HCA structure, and the antigen binding arm B is a fusion peptide having an LCB-linker-HCB structure, wherein "-" represents a peptide bond.

[0024] In some embodiments, the linker is a short peptide structure represented by (GGGS)n. In some embodiments, n is any integer between 4 and 10.

[0025] In some embodiments, n is any integer between 4 and 8.

[0026] In some embodiments, n is any integer between 4 and 6.

[0027] In a second aspect, the present invention provides a bispecific antibody comprising an antigen-binding arm A that specifically targets EGFR and an antigen-binding arm B that specifically targets c-Met, wherein the binding arm A comprises, from N-terminus to C-terminus, ScFab(A), a hinge region connecting the C-terminus of the ScFab(A) and the N-terminus of the Fc(A), and Fc(A), to form a ScFab(A)-hinge region-Fc(A) structure; the binding arm B comprises, from N-terminus to C-terminus, ScFab(B), a hinge region connecting the C-terminus of the ScFab(B) and the N-terminus of the Fc(B), and Fc(B), to form a ScFab(B)-hinge region-Fc(B) structure; wherein the Fc(A) comprises a combination of amino acid mutations that form a knob structure, the Fc(B) comprises a combination of amino acid mutations that form a hole structure, the knob structure of the Fc(A) and the hole structure of the Fc(B) interact to form a KIH structure, and the binding arm A and the binding arm B form a heterodimer through the KIH structure.

[0028] In one embodiment, the ScFab (A) comprises a light chain variable region A (VLA), a light chain constant region A (CLA), a linker and a first heavy chain constant region A (CH1A) from N-terminus to C-terminus, the Fc (A) comprises a second heavy chain constant region A and a third heavy chain constant region A (CH2ACH3A), the ScFab (B) comprises a light chain variable region B (VLB), a light chain constant region B (CLB), a linker and a first heavy chain constant region B (CH1B) from N-terminus to C-terminus, and the Fc (B) comprises a second heavy chain constant region B and a third heavy chain constant region B (CH2BCH3B).

[0029] In one embodiment, the Fc(A) comprises a combination of amino acid mutations that form a knob structure as shown in SEQ ID NO:11.

[0030] In one embodiment, the Fc(B) comprises a combination of amino acid mutations that form a hole structure as shown in SEQ ID NO: 22.

[0031] In one embodiment, the Fc(B) comprises the amino acid sequence shown in SEQ ID NO: 33 that forms a hole structure.

[0032] In one embodiment, the antigen binding arm A and the binding arm B comprise a linker selected from the amino acid sequence shown in SEQ ID NO: 9, 20 or 31.

[0033] In some embodiments, the antigen binding arm A comprises the amino acid sequence shown in SEQ ID NO:4.

[0034] In some embodiments, the antigen binding arm B comprises the amino acid sequence shown in SEQ ID NO:15.

[0035] In some embodiments, the antigen binding arm B comprises the amino acid sequence shown in SEQ ID NO:26.

[0036] In some embodiments, the CHA comprises the amino acid mutation combination S354C and T366W that form a knob structure, and the CHB comprises the amino acid mutation combination Y349C, T366S, L368A and Y407V that form a hole structure.

[0037] In a preferred embodiment, CHA and CHB comprise the constant region of an IgG1 antibody, preferably the constant region of a human IgG1 antibody.

[0038] In other embodiments, the present invention provides bispecific EGFR / c-Met antibodies comprising certain heavy chain CDRs and light chain CDRs, VHA, VLA, VHB, VLB, HCA, LCA, HCB, and LCB sequences.

[0039] In some embodiments, the CH2 regions of the bispecific antibody Fc(A) and Fc(B) can be engineered to alter the ADCC and / or CDC activity of the antibody, or to maintain the ADCC and / or CDC activity of the antibody.

[0040] In a preferred embodiment, CH2 of the bispecific antibody Fc(A) and Fc(B) comprises amino acid mutations that result in enhanced ADCC and / or CDC activity.

[0041] In a preferred embodiment, the amino acid modification that results in enhanced ADCC and / or CDC activity comprises amino acid modification of a defucosylated amino acid that reduces the ratio of core fucose glycoforms.

[0042] In some embodiments, the EGFR / c-Met described in any of the aforementioned aspects, wherein the binding site formed by the pairing of LCA and HCA that bind to human EGFR can also bind to mouse EGFR and cynomolgus EGFR.

[0043] In a fourth aspect, the present invention further provides an isolated nucleic acid molecule encoding the anti-EGFR / c-Met bispecific antibody or fragment thereof according to any of the aforementioned aspects, a recombinant vector comprising the isolated nucleic acid molecule, and a host cell comprising the recombinant vector.

[0044] In a fifth aspect, the present invention also provides a method for preparing the bispecific antibody described in any of the preceding aspects, comprising the steps of expressing an engineered mammalian host cell carrying a vector comprising a nucleic acid encoding the binding arm of the anti-EGFR / c-Met bispecific antibody of the present invention and purifying the bispecific antibody expressed by the host cell.

[0045] In a sixth aspect, the present invention further provides a composition comprising one or more of the bispecific antibodies, drug conjugates, nucleic acid molecules, recombinant vectors, and recombinant cell drugs selected from any of the aforementioned aspects.

[0046] In a seventh aspect, the present invention further provides the bispecific antibody, nucleic acid molecule, recombinant vector, host cell and pharmaceutical composition of any of the aforementioned aspects, which are used to treat diseases or tumors involving abnormal EGFR and c-Met cells.

[0047] In an eighth aspect, the present invention further provides the bispecific antibody, nucleic acid molecule, recombinant vector, host cell and pharmaceutical composition described in any of the foregoing items, for use in treating diseases or tumors involving abnormal EGFR and c-Met cells.

[0048] In a ninth aspect, the present invention further provides the use of any of the aforementioned bispecific antibodies, nucleic acid molecules, recombinant vectors, host cells, and pharmaceutical compositions in the preparation of drugs for treating diseases or tumors involving abnormal EGFR and c-Met cells.

[0049] In some embodiments, the disease is a disease involving cells expressing abnormal EGFR and c-Met; and the tumor is an EGFR-positive tumor, a c-Met-positive tumor, or an EGFR and c-Met double-positive tumor.

[0050] In some embodiments, the tumor is resistant to treatment with an EGFR tyrosine kinase inhibitor.

[0051] In a tenth aspect, a method is provided for evaluating the autophosphorylation levels of EGFR and c-Met in tumor cells by measuring the TR-FRET ratio (665 nm absorbance / 615 nm absorbance) with and without the addition of the bispecific antibody of the present invention, and determining the inhibitory ability of the antibody of the present invention on the ligand HGF-mediated c-Met autophosphorylation. Beneficial effects:

[0052] The bispecific antibody of the present invention has any of the following biological functions: (1) the binding arm A specifically binds to domain III of human EGFR, thereby blocking the binding of EGF to EGFR; the binding arm B specifically binds to human c-Met, thereby blocking the binding of HGF to c-Met; wherein the KD of the c-Met binding arm may be at least 10 times smaller than the Kd of the EGFR binding arm.

[0053] (2) Inhibits EGF-mediated EGFR autophosphorylation and HGF-mediated c-Met autophosphorylation in non-small cell lung cancer HCC827;

[0054] The addition of the bispecific antibody of the present invention significantly reduced the TR-FRET ratio (665 nm absorbance / 615 nm absorbance) measured relative to the TR-FRET measured without the bispecific antibody, approaching the phosphorylation level mediated by HGF without ligand. The bispecific antibody also reduced EGFR autophosphorylation (P-EGFR(Y1068)) in HCC827 cells by at least 90%, demonstrating superior inhibitory efficacy compared to amivantamab.

[0055] The TR-FRET ratio (665 nm absorbance / 615 nm absorbance) measured with the bispecific antibody of the present invention was significantly reduced compared to that measured without the bispecific antibody, approaching the phosphorylation level mediated by HGF without ligand. The bispecific antibody also reduced c-Met autophosphorylation (Pc-Met (Y1234 / Y1235)) in HCC827 cells by at least 90%. The inhibitory strength was superior to that of amivantamab.

[0056] (3) It can inhibit the growth and proliferation of human tumor cells and has a synergistic effect. Its inhibitory effect on the proliferation of EGFR and c-Met double-positive tumor cells is stronger than the combination of two targeted monoclonal antibodies;

[0057] (4) has reduced fucose content, mediating antibody-dependent cellular cytotoxicity (ADCC);

[0058] (6) mediate antibody-dependent phagocytosis (ADCP);

[0059] (7) Mediate the internalization of EGFR and c-Met target proteins;

[0060] (8) Specific binding to cynomolgus monkey c-Met and EGFR proteins;

[0061] (9) It has significant therapeutic effect on tumors, with a TGI higher than 60%.

[0062] Based on the above functional activities, the present invention provides a new targeted therapy option for the field of cancer treatment, especially for cancers and tumors related to abnormal overexpression of c-Met and EGFR, and provides a new treatment for the drug resistance problem of traditional EGFR targeted therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1. Inhibition of c-Met autophosphorylation by the bispecific antibodies of the present invention. Absorbance at 665 nm represents the signal intensity of anti-c-Met-P-Y1234 / Y1235, while absorbance at 615 nm represents the signal intensity of anti-c-Met (as a reference). The ratio of 665 / 615 represents the relative level of c-Met autophosphorylation. Two replicate wells were set up for each dosing concentration.

[0064] Figure 2 shows analysis of the binding activity of the bispecific antibodies of the present invention to the c-Met target protein. A, Dose-dependent binding curve of the anti-EGFR / c-Met antibody to human c-Met protein detected by ELISA. B, Dose-dependent binding curve of the anti-EGFR / c-Met antibody to CHO-K1 cells overexpressing human c-Met detected by FACS. The abscissa represents the antibody concentration (logarithmic value), and the ordinate represents the antibody binding signal intensity.

[0065] Figure 3 shows biochemical activity analysis of the bispecific antibodies of the present invention blocking the binding of the ligand HGF to c-Met. A, Dose-dependent binding curve of the anti-EGFR / c-Met antibody blocking HGF binding to human c-Met protein as measured by ELISA. B, Dose-dependent binding curve of the anti-EGFR / c-Met antibody blocking HGF binding to CHO-K1 cells overexpressing human c-Met as measured by FACS. The abscissa represents antibody concentration (logarithmic value), and the ordinate represents the binding signal intensity of the ligand HGF.

[0066] Figure 4 shows analysis of the binding activity of the bispecific antibodies of the present invention against the EGFR target protein. A, Dose-dependent binding curve of the anti-EGFR / c-Met antibody to human EGFR protein detected by ELISA. B, Dose-dependent binding curve of the anti-EGFR / c-Met antibody to CHO-K1 cells overexpressing human EGFR detected by FACS. The abscissa represents the antibody concentration (logarithmic value), and the ordinate represents the antibody binding signal intensity.

[0067] Figure 5 shows biochemical activity analysis of the bispecific antibodies of the present invention blocking the binding of the ligand EGF to EGFR. A, ELISA analysis of the dose-dependent binding curve of the anti-EGFR / c-Met antibody blocking EGF binding to human EGFR protein. B, FACS analysis of the dose-dependent binding curve of the anti-EGFR / c-Met antibody blocking EGF binding to CHO-K1 cells overexpressing human EGFR. The abscissa represents the antibody concentration (logarithmic value), and the ordinate represents the binding signal intensity of the ligand EGF.

[0068] FIG6 shows the antibody concentration gradient binding curves of the present invention to EGFR and c-Met double positive cells MKN45.

[0069] Figure 7 shows the binding curves of the bispecific antibody of the present invention against the ligands HGF and EGF and MKN45 cells. A, FACS detection of the antibody's antagonistic binding to EGF and MKN45 cells; B, FACS detection of the antibody's antagonistic binding to HGF and MKN45 cells.

[0070] Figure 8 shows inhibition of target protein signaling pathways by the bispecific antibodies of the present invention. A. The antibodies of the present invention inhibit HGF-activated c-Met phosphorylation. The absorbance at a wavelength of 665 nm represents the signal intensity of anti-c-Met-P-Y1234 / Y1235, the absorbance at a wavelength of 615 nm represents the signal intensity of anti-c-Met (as a reference), and 665 / 615 represents the relative level of c-Met autophosphorylation. Two replicate wells were set up in parallel for each dosing concentration. B. The antibodies of the present invention inhibit EGF-activated EGFR phosphorylation. The absorbance at a wavelength of 665 nm represents the signal intensity of anti-EGFR-P-Y1068, the absorbance at a wavelength of 615 nm represents the signal intensity of anti-EGFR (as a reference), and 665 / 615 represents the relative level of c-Met autophosphorylation. Two replicate wells were set up in parallel for each dosing concentration.

[0071] Figure 9 Evaluation of antibody-dependent ADCC activity of the bispecific antibodies of the present invention. A, In vitro ADCC activity of NK cells against H1975 cells (antibody-dependent); B, In vitro ADCC activity of NK cells against HCC827 cells (antibody-dependent). The vertical axis represents the percentage of cytotoxicity (%), calculated as (TRF value of the experimental group - TRF value of the spontaneous pore) / (TRF value of the maximum release pore - TRF value of the spontaneous pore) * 100.

[0072] Figure 10: Evaluation of antibody-dependent ADCP activity. A, ADCP evaluation on H1975 cells; B, ADCP evaluation on HCC827 cells. The vertical axis represents the level of phagocytosis, calculated by the number of cells positive for both PCB450 and PE as determined by flow cytometry. The percentage of phagocytosis is the sum of the number of cells positive for both PCB450 and PE divided by the number of cells positive for PCB450.

[0073] FIG11 : Antibodies of the present invention inhibit the proliferation of tumor cell lines in vitro.

[0074] A inhibits the proliferation of tumor cell line KP4,

[0075] B inhibits the proliferation of tumor cell line A549,

[0076] C inhibits the proliferation of tumor cell line MKN45,

[0077] D inhibits the proliferation of tumor cell line HCC827-HGF.

[0078] Figure 12 ELISA binding analysis of the bispecific antibodies of the present invention to cynomolgus monkey c-Met and EGFR proteins. (A, ELISA binding analysis of the antibodies of the present invention to cynomolgus monkey c-Met protein. B, ELISA binding analysis of the antibodies of the present invention to cynomolgus monkey c-Met protein).

[0079] FIG13 The bispecific antibody of the present invention has a stronger inhibitory effect on MKN45 cell proliferation in vitro than the combination of single-target antibodies.

[0080] FIG14 shows that the bispecific antibody of the present invention has a stronger inhibitory effect on HCC827-HGF cell proliferation in vitro than the combination of single-target antibodies.

[0081] FIG15 : The bispecific antibody of the present invention has a stronger inhibitory effect on H1975-HGF cell proliferation in vitro than the combination of single-target antibodies.

[0082] Figure 16 Detection of cellular internalization levels caused by the bispecific antibodies of the present invention. A. FACS detection of internalization of the antibodies of the present invention after treatment with gradient concentrations of antibodies. B and C. The Live-Cell Analysis System was used to detect the internalization level of fluorescent-labeled antibodies at the same concentration (5 μg / ml) over a period of 48 consecutive hours (1 hour interval each time).

[0083] FIG17 Inhibition of HCC827 xenograft tumor growth by bispecifics of the present invention.

[0084] FIG18 Inhibition of HCC827-HGF (overexpressing HGF) xenograft tumor growth by the bispecifics of the present invention. DETAILED DESCRIPTION

[0085] The present invention is based at least in part on the unexpected discovery that the bispecific antibodies of the present invention that specifically bind to EGFR and c-Met can provide beneficial effects of synergistic effects in specifically binding to EGFR and inhibiting binding of the tumor cell ligand EGF and antigen EGFR, specifically binding to c-Met and inhibiting binding of the ligand HGF to the antigen c-Met, inhibiting phosphorylation pathways downstream of EGFR and c-Met on the surface of tumor cells, and inhibiting tumor cell growth and proliferation and tumor killing activity, compared to small molecule EGFR and / or c-Met inhibitors, or EGFR monoclonal antibodies and c-Met monoclonal antibodies, or a combination of EGFR monoclonal antibodies and c-Met monoclonal antibodies; that the bispecific antibodies of the present invention can completely inhibit ligand-mediated phosphorylation of EGFR and c-Met antigens; and that when compared to a monospecific agent that binds to EGFR and a monospecific agent that binds to c-Met, or a mixture thereof, the bispecific antibodies that specifically bind to EGFR and c-Met can provide significantly improved synergistic effects of tumor cell inhibition and more effectively inhibit tumor growth.

[0086] The present invention provides bispecific antibodies capable of specifically binding to EGFR and c-Met, polynucleotides encoding the bispecific antibodies of the present invention or their complementary nucleic acids, vectors, host cells, pharmaceutical compositions comprising the bispecific antibodies of the present invention or their encoding nucleic acids or vectors, as well as methods for preparing and using the same.

[0087] In the context of this disclosure, the following definitions are provided:

[0088] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature and techniques used in connection with the cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The aforementioned techniques and procedures are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature and laboratory procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0089] As used in this disclosure, unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0090] the term

[0091] As used herein, the term "substitution" or "substitutional" or "mutation" or "mutant" refers to the alteration, deletion or insertion of one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to produce a variant of the sequence.

[0092] As used herein, "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, such as substitutions, insertions, or deletions.

[0093] As used herein, "synthetic" or "non-natural" or "artificial" refers to a polynucleotide or polypeptide molecule that does not occur in nature.

[0094] The phrase "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated bispecific antibody that specifically binds EGFR and c-Met is substantially free of antibodies that specifically bind antigens other than human EGFR and c-Met). However, an isolated antibody that specifically binds EGFR and c-Met may have cross-reactivity with other antigens, such as orthologs of human EGFR and / or c-Met, such as cynomolgus macaque (Macaca fascicularis) (rhesus monkey) EGFR and / or c-Met. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0095] As used herein, the term "specifically binds" or "specific binding" refers to the ability of a bispecific agent that specifically binds to EGFR and c-Met, or an EGFR / c-Met bispecific antibody of the invention, to bind to a predetermined antigen with a dissociation constant (KD) (M) of about 1E-6 or less, e.g., about 1E-7 or less, about 1E-8 or less, about 1E-9 or less, about 1E-10 or less, about 1E-11 or less, about 1E-12 or less, or about 1E-13 or less. Typically, a bispecific agent that specifically binds to EGFR and c-Met, or an EGFR / c-Met bispecific antibody of the invention, binds to a predetermined antigen (i.e., EGFR or c-Met) with a KD that is at least ten-fold less than its KD for a nonspecific antigen (e.g., BSA or casein) as measured by surface plasmon resonance using, for example, a Proteon instrument (BioRad). Thus, a specific agent comprising a specific binding affinity for EGFR and c-Met, or an EGFR / c-Met bispecific antibody of the present invention, specifically binds each of EGFR and c-Met with a binding affinity (KD) of at least about 1E-6 or less, e.g., about 1E-7 or less, about 1E-8 or less, about 1E-9 or less, about 1E-10 or less, about 1E-11 or less, about 1E-12 or less, or about 1E-13 or less. However, a bispecific agent comprising a specific binding affinity for EGFR and c-Met, or an EGFR / c-Met bispecific antibody of the present invention, that specifically binds to a predetermined antigen may have cross-reactivity with other related antigens, e.g., the same predetermined antigen from other species (homologs).

[0096] The term "stability" refers to the ability of a molecule to remain in a folded state under physiological conditions so that it retains at least one of its normal functional activities, eg, binding to a predetermined antigen such as EGFR or c-Met.

[0097] As used herein, the terms "epidermal growth factor receptor," "EGFR," "ErbBl," and "EGF receptor" are used interchangeably herein to refer to the EGFR protein (see, e.g., EGFR-ECD-HIS (Gene ID: 1956, AA25-645). As used herein, "EGFR extracellular domain" or "EGFR-ECD" refers to the extracellular EGFR domain that is anchored on the cell membrane or in circulation, including fragments thereof, the amino acid sequence of which is shown in SEQ ID NO: 58, and the encoding nucleic acid thereof is shown in SEQ ID NO: 59. EGFR is one of four receptor tyrosine kinases. kinase, RTK) (called Her- or cErbB-1, -2, -3 and -4) family. In one embodiment, the extracellular domain of EGFR can include four domains: "domain I" (from about 1-158 amino acid residues), "domain II" (amino acid residues 159-336), "domain III" (amino acid residues 337-470), and "domain IV" (amino acid residues 471-645), wherein the boundaries can have about 1-3 amino acid variations; two of the four subdomains are involved in ligand binding, and one of them is involved in homodimerization and heterodimerization (Ferguson (2008)). EGFR integrates extracellular signals from multiple ligands to produce multiple intracellular responses (Yarden at al. 2001; and Jorrisen et al. 2003). et al. 2003). EGFR is involved in several human epithelial malignancies, particularly breast cancer, bladder cancer, non-small cell lung cancer, lung cancer, colon cancer, ovarian cancer, head and neck cancer, and brain cancer; activating mutations in the gene, as well as overexpression of the receptor and its ligands, form an autocrine activation loop (for details, see Robertson et al. 2000). Therefore, this RTK has been widely used as a target for cancer therapy. Small molecule inhibitors targeting RTKs and monoclonal antibodies (mAbs) directed against the extracellular ligand-binding domain have shown several clinical successes.

[0098] As used herein, "ligand of EGFR" encompasses all (e.g., physiological) ligands of EGFR, including EGF, TGF-α, heparin-binding EGF (HB-EGF), amphiregulin (AR), and epiregulin (EPI) (reviewed by Olayioye MA et al.; EMBO J (2000) Vol 19: pp 3159-3167). An exemplary EGF has an amino acid sequence as shown in SEQ ID NO: 56).

[0099] c-Met, also known as tyrosine protein kinase c-Met or hepatocyte growth factor receptor (c-Met), is a protein encoded by the c-Met gene in humans. This protein has tyrosine kinase activity. As used herein, "hepatocyte growth factor receptor" or "c-Met" refers to human c-Met and its natural variants having the amino acid sequence set forth in SEQ ID NO:60 or GenBank Accession No. NP001120972. "Hepatocyte growth factor" (HGF), as used herein, refers to the well-known human HGF having the amino acid sequence set forth in SEQ ID NO:61, which is cleaved to form a dimer of α and β chains linked by disulfide bonds. The primary single-chain precursor protein is post-translationally cleaved to produce α and β subunits, which are linked by disulfide bonds to form the mature receptor. The terms "c-Met polypeptide," "c-Met receptor," "c-Met," "HGF receptor," or "c-Met" are used interchangeably herein and, unless otherwise indicated, are intended to refer to a human receptor tyrosine kinase, as well as functionally active mutant forms thereof, that binds to human hepatocyte growth factor. The structure of c-Met comprises an extracellular domain (ECD) comprising SEMA (Sema domain), PSI (Plexin, semaphorin, and integrin domain), and IPT (four immunoglobulin, Plexin, and transcription factor domains); and an intracellular domain comprising a transmembrane region (TM), a juxtamembrane domain (JM), and a kinase domain (KD). Specific examples of c-Met include, for example, a human polypeptide encoded by the nucleotide sequence provided in GenBank Accession No. NM_000245, or a human protein encoded by the polypeptide sequence provided in GenBank Accession No. NP_000236.

[0100] As used herein, the c-Met sequence used for functional detection of the bispecific antibody that specifically binds to EGFR and c-Met of the present invention is shown in SEQ ID NO: 60, and the encoding nucleic acid thereof is shown in SEQ ID NO: 61.

[0101] As used herein, "hepatocyte growth factor" (HGF) refers to the well-known human HGF having the amino acid sequence set forth in SEQ ID NO: 57, which is cleaved to form a dimer of α and β chains linked by disulfide bonds. "Blocking binding" or "inhibiting binding," as used interchangeably herein, refers to the ability of a bispecific agent that specifically binds to EGFR and c-Met, or an EGFR / c-Met bispecific antibody of the present invention, to block or inhibit the binding of an EGFR ligand (such as EGF) to EGFR and / or the binding of HGF to c-Met, and encompasses both partial and complete blocking / inhibition. Blocking / inhibiting the binding of an EGFR ligand (such as EGF) to EGFR and / or the binding of an HGF ligand to c-Met by a bispecific agent that specifically binds to EGFR and c-Met, or a bispecific EGFR / c-Met antibody of the present invention, partially or completely reduces the normal level of EGFR signaling and / or c-Met signaling, compared to when the binding of the EGFR ligand to EGFR and / or HGF to c-Met is not blocked or inhibited. A bispecific agent that specifically binds to EGFR and c-Met, or an EGFR / c-Met bispecific antibody of the invention, "blocks binding" of an EGFR ligand (such as EGF binding to EGFR and / or HGF binding to c-Met) when the inhibition is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Inhibition of binding can be measured using well-known methods, such as ELISA or FACS, to measure the level of inhibition of EGF binding to intracellular EGFR or to cell surface EGFR by a bispecific antibody of the invention, or to measure the level of inhibition of HGF binding to intracellular c-Met or to cell surface c-Met by a bispecific antibody of the invention.

[0102] The term "bispecific agent that specifically binds to EGFR and c-Met" refers to a molecule comprising a first domain that specifically binds to EGFR and a second domain that specifically binds to c-Met. An exemplary agent that specifically binds to EGFR and c-Met is a bispecific antibody.

[0103] As used herein, the term "EGFR / c-Met bispecific antibody" refers to a bispecific antibody comprising a binding arm that binds to EGFR and a binding arm that binds to c-Met, wherein binding arm A and binding arm B each comprise a light chain and a heavy chain, respectively. Each light chain comprises a variable region VL and a light chain constant region CL, and each heavy chain comprises VH and a first heavy chain constant region (CH1), a second heavy chain constant region (CH2), and a third heavy chain constant region (CH3). The two binding arms of different specificities are covalently linked together to form a heterodimer via a KIH structure formed between the heavy chain CH3s of the two binding arms. The domains that specifically bind to EGFR and c-Met are typically referred to as a VHA / VLA pair and a VHB / VLB pair, respectively, and the bispecific anti-EGFR / c-Met antibody is monovalent with respect to binding to EGFR and c-Met.

[0104] The antibodies of the present invention disclosed herein preferably comprise a binding arm A comprising a light chain A targeting EGFR and a heavy chain A connected by a linker, and a binding arm B comprising a light chain B targeting c-Met and a heavy chain B connected by a linker. Binding arms A and B form a KIH structure through heavy chain interactions, further forming a heterodimeric bispecific antibody. Preferably, the antibodies of the present invention are of human IgG type, preferably human IgG1 subclass. Such antibodies of the present invention disclosed herein can exhibit good ADCC properties and a long half-life after administration to humans, and CH3 engineering technologies are available that can provide modified heavy chains. As used herein, the term "EGFR / c-Met bispecific molecule" refers to a molecule comprising a binding arm A that binds to EGFR and a different binding arm B that binds to c-Met, the two domains being covalently linked together directly or via a linker. Exemplary EGFR / c-Met-binding bispecific molecules comprise a binding arm A that specifically binds to EGFR and a binding arm B that specifically binds to c-Met.

[0105] As used herein, the term "bispecific anti-EGFR / c-Met antibody" or "bispecific EGFR / c-Met antibody" refers to a bispecific antibody having a first domain that specifically binds to EGFR and a second domain that specifically binds to c-Met. The domains that specifically bind to EGFR and c-Met are typically a VH / VL pair, and the bispecific anti-EGFR / c-Met antibody is monovalent with respect to binding to EGFR and c-Met.

[0106] In one aspect, the bispecific EGFR / c-Met antibody of the present invention can bind to the domain III junction of human EGFR and inhibit and block the binding of the E ligand EGF to EGFR, and can bind to the variable domain of c-Met to block the binding of the ligand HGR to c-Met; the KD of the c-Met antibody may be at least 10 times smaller than the KD of the EGFR antibody.

[0107] The bispecific EGFR / c-Met antibodies of the present invention can block / inhibit the binding of an EGFR ligand (such as EGF) to EGFR and / or the binding of a c-Met ligand (such as HGF) to c-Met, resulting in partial or complete reduction of EGFR signaling and / or c-Met signaling relative to normal levels, compared to when EGF ligand binding to EGFR and / or HGF ligand binding to c-Met are not blocked or inhibited. The EGFR / c-Met bispecific antibodies of the invention that specifically bind to EGFR and c-Met block the binding of an EGFR ligand, such as EGF, to EGFR and / or the binding of a c-Met ligand, such as HGF, to c-Met when the inhibition is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0108] The term "EGFR signaling" refers to signal transduction induced by the binding of an EGFR ligand to EGFR, resulting in autophosphorylation of at least one tyrosine residue in EGFR. An exemplary EGFR ligand is EGF. Upon EGFR binding, at least one tyrosine residue including position 1068 is autophosphorylated.

[0109] As used herein, "neutralizes EGFR signaling" or "blocks EGFR signaling" refers to the ability of the EGFR / c-Met bispecific antibodies of the present invention that specifically bind to EGFR and c-Met to inhibit EGFR signaling induced by an EGFR ligand, such as EGF, by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0110] The term "c-Met signaling" refers to signal transduction induced by HGF binding to c-Met, resulting in autophosphorylation of at least one tyrosine residue in c-Met. Typically, upon HGF binding, at least one tyrosine residue at positions 1230, 1234, 1235, or 1349 is autophosphorylated.

[0111] As used herein, "neutralizes c-Met signaling" or "blocks c-Met signaling" refers to the ability of a bispecific EGFR / c-Met antibody to inhibit HGF-induced c-Met signaling by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0112] Abnormally activated c-Met can induce tumor growth, the formation of new blood vessels that supply nutrients to tumors (angiogenesis), and the spread of cancer to other organs (metastasis). c-Met is deregulated in many types of human malignancies, including kidney cancer, liver cancer, stomach cancer, breast cancer, and brain cancer.

[0113] As used interchangeably herein, "overexpression" and "overexpressed" refer to cancer cells or malignant cells having measurably higher levels of EGFR and / or c-Met on their surface compared to normal cells of the same tissue type. Such overexpression can be caused by gene amplification or by increased transcription or translation. EGFR and / or c-Met is considered to be overexpressed when the level of EGFR and / or c-Met on the surface of a cell is at least 1.5-fold higher than that of a normal cell.

[0114] As used herein, with respect to cell surface expression of c-Met or EGFR on a tumor or cell line, the terms "low," "medium," and "high" are intended to mean less than about 300,000, greater than about 300,000, and greater than about 1 million receptors per cell, respectively.

[0115] As used herein, the term "linker molecule" or "linker" or "linker" preferably refers to a peptide linker. Peptide linkers, as used in certain embodiments of the present invention, are used to link antibodies, antigen-binding sites, and / or antibody fragments comprising different antigen-binding sites (e.g., scFab, VH domain, and / or VL domain) to form the bispecific antibodies of the present invention. Preferably, the peptide linker is a glycine-rich peptide having at least 5 amino acids, preferably at least 10 amino acids, and more preferably 10-50 amino acids. In some embodiments of the invention, the glycine-rich peptide linker is (GxS)n, wherein G=glycine, S=serine, (x=3 and n=3, 4, 5 or 6) or (x=4 and n=2, 3, 4 or 5) (i.e., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9) or GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 20), respectively).

[0116] In some embodiments, additional glycine or threonine, such as GSTG, TG, GG, or GGGT, can also be added to either end of the (GxS)n formatted glycine-rich peptide linker.

[0117] Bispecific antibodies preferably contain a modified constant domain (Fc portion). Antibodies of the invention, such as those disclosed herein, can have good ADCC properties and a good half-life after administration to humans. Furthermore, CH3 modification technology exists that can provide modified heavy chains that preferentially form heterodimers over homodimers upon co-expression in clonal cells. The ADCC activity of EGFR / c-Met bispecific antibodies can also be improved using techniques known to those skilled in the art.

[0118] Unless expressly stated otherwise, throughout this specification the numbering of amino acid residues in the constant region of antibodies is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition.

[0119] "Humanized antibodies" refer to antibodies in which the antigen-binding site is derived from a non-human species and the variable region framework is derived from a human immunoglobulin sequence. Humanized antibodies may include substitutions in the framework region, so that the framework may not be an exact copy of an expressed human immunoglobulin or germline gene sequence.

[0120] "Human-adapted" antibodies or "human framework-adapted (HFA)" antibodies are humanized antibodies derived from acceptor human frameworks selected based on maximum CDR to FR similarity, length compatibility, and sequence similarity to the CDR1 and CDR2 loops and a portion of the light chain CDR3 loop.

[0121] "Human antibodies" refer to antibodies having heavy and light chain variable regions in which both the framework and antigen-binding sites are derived from human sequences. If the antibody comprises a constant region, the constant region is also derived from human sequences. Among other things, the variable regions of the antibodies can be obtained from systems that use human germline immunoglobulins or rearranged immunoglobulin genes, such systems including human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice carrying human immunoglobulin gene libraries as described herein. "Human antibodies" may comprise a consensus framework sequence derived from human framework sequence analysis, and do not include antibodies whose antigen-binding sites are derived from non-human species.

[0122] Isolated humanized antibodies can be synthetic. Although derived from human immunoglobulin sequences, human antibodies can be generated using systems such as phage display incorporating synthetic CDRs and / or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties, thereby producing antibodies that do not naturally exist within the human antibody germline repertoire in vivo.

[0123] As used herein, the term "engineered antibody" or "recombinant antibody" includes all antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below), antibodies isolated from host cells transformed to express the antibody, antibodies isolated from recombinant combinatorial antibody libraries, as well as antibodies prepared, expressed, created, or isolated by any other means involving splicing together human immunoglobulin gene sequences with other DNA sequences, or antibodies generated in vitro using Fab arm exchange.

[0124] As used herein, the term "substantially identical" means that the two antibody variable region amino acid sequences being compared are identical or have "insubstantial differences". Insubstantial differences are substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in the antibody variable region sequence that do not adversely affect the antibody properties. Amino acid sequences substantially identical to the variable region sequences disclosed herein are within the scope of the present invention. In some embodiments, the sequence identity may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Percent identity can be determined, for example, by pairwise alignment using the default settings of the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carlsbad, CA). The protein sequences of the present invention can be used as query sequences for performing searches of public or patent databases, for example to identify related sequences. Exemplary programs for performing such searches are the XBLAST or BLASTP programs (http: / / www.ncbi.nlm.nih.gov) or the GenomeQuest™ (GenomeQuest, Westborough, MA) suite using default settings.

[0125] Exemplary antibodies of the present invention bind to EGFR and c-Met (preferably human EGFR and human c-Met). The EGFR / c-Met bispecific antibodies of the present invention disclosed herein bind to EGFR and, under otherwise identical conditions, bind to the cognate receptors ErbB-2 and ErbB-4 from the same species with an EC50 at least 100-fold lower. Binding is preferably assessed using binding of cellularly expressed antigens. For example, antibodies that bind to EGFR and c-Met may also bind to other proteins if such other proteins contain the same epitope. Thus, the term "binding" does not exclude binding of an antibody to another protein or proteins containing the same epitope. Such binding is generally referred to as cross-reactivity. The EGFR / c-Met bispecific antibodies of the present invention disclosed herein preferably bind to human EGFR and cynomolgus EGFR. EGFR / c-Met bispecific antibodies generally do not bind to other proteins other than EGFR and / or c-Met on the cell membrane in humans (preferably adults). The antibodies according to the invention disclosed herein are generally capable of binding to EGFR with a binding affinity (ie, equilibrium dissociation constant, Kd) of at least 1E-6 M, as outlined in more detail below.

[0126] As used herein, the term "in combination" means that two or more therapeutic agents may be co-administered to a subject in a mixture, simultaneously as a single agent, or sequentially in any order as a single agent.

[0127] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.

[0128] Unless expressly stated otherwise, throughout this specification, the numbering of amino acid residues in antibody constant regions is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991).

[0129] Material composition

[0130] In one aspect, the present invention provides a bispecific antibody that specifically binds to EGFR and c-Met, comprising a binding arm A that can specifically bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a binding arm B that can specifically bind to the extracellular portion of human proto-oncogene receptor tyrosine kinase (c-Met), wherein the binding arm A can also bind to mouse EGFR and c-Met, cynomolgus monkey EGFR and c-Met, or both.

[0131] Binding arm A binds to EGFR with an affinity equivalent to the KD (M) of 2.0E-8 for nimotuzumab. Binding arm A preferably binds to EGFR (EGFR-ECD-HIS (Gene ID: 1956, AA25-645, SEQ ID NO: 58) with a KD of 10E-7 to 10E-9, preferably a KD of 10E-8 to 10E-9. Binding arm B preferably binds to c-Met with a KD (M) of 10E-8 or less, preferably 10E-9 to 10E-10. The affinity of binding arm B for c-Met is preferably higher than the affinity of binding arm A for EGFR, i.e., in this preferred embodiment, the KD of the antibody against c-Met is less than the Kd of the antibody against EGFR.

[0132] In a preferred embodiment, the KD of binding arm B for c-Met is at least 5-fold less, and preferably at least 10-fold less, than the KD of binding arm A for EGFR. In this embodiment, this affinity level allows the bispecific antibodies of the invention disclosed herein to preferentially bind to EGFR on cells and block the binding of the ligand HGF to c-Met.

[0133] Function of EGFR-specific binding arm A

[0134] In some embodiments, the binding arm A that specifically binds to EGFR comprises a variable domain having an amino acid sequence derived from JH001 or a variant thereof as described herein, preferably binding to EGFR domain III (see Chinese Patent Application No. 2020105770075). The binding arm A derived from JH001 can inhibit the binding of the ligand EGF to EGFR. The bispecific antibodies of the present invention described herein that have cross-reactivity with human, mouse, and cynomolgus monkey EGFR allow for use in xenograft studies with human cancer models.

[0135] The binding arm A of the bispecific antibody of the present invention binds to EGFR with high affinity and inhibits EGFR signal transduction, and can provide beneficial effects compared with small molecule EGFR inhibitors in terms of specificity and inhibition of the increased autophosphorylation level of EGFR mediated by the ligand EGF and the signal transduction cascade reaction triggered by the occurrence and development of tumors, as well as synergistic effects in tumor cell growth inhibition and killing compared with conventional monospecific antibodies and their combination therapeutic agents.

[0136] In one exemplary embodiment, the variable domain of the binding arm A of the isolated bispecific antibody can bind to EGFR, preferably derived from the variable domain of a bivalent monospecific antibody that targets EGFR and inhibits EGF-induced cell death in HCC827 non-small cell lung cancer cells at a level comparable to that of JH001 monoclonal antibody or a combination thereof, though inhibition is generally less than 100%. Preferably, the bispecific antibodies of the invention are measured for inhibition of EGF-induced cell death in tumor cells, such as H292, H1975, HCC827, and D773_V774 insHBa / F3. Without being bound by theory, it is believed that binding of the binding arm A of the bispecific antibodies of the invention to EGFR blocks the binding of EGF to EGFR.

[0137] In one embodiment, the level of extracellular EGFR binding of the EGFR / c-Met bispecific antibody is determined by measuring an ELISA binding EC50 value. The binding arm A of the bispecific antibody of the present invention can bind to EGFR with an ELISA binding EC50 of less than about 150 ng / mL, less than about 120 ng / mL, less than about 100 ng / mL, less than about 80 ng / mL, less than about 50 ng / mL, less than about 30 ng / mL, or less than 10 ng / mL. The method for determining the ELISA binding EC50 can be found in the Examples.

[0138] In one embodiment, the level of cell surface EGFR binding by the EGFR / c-Met bispecific antibody is determined by measuring a FACS binding EC50 value. The binding arm A of the bispecific antibody of the present invention can bind to EGFR with a FACS binding EC50 of less than about 1500 ng / mL, less than about 1200 ng / mL, less than about 1000 ng / mL, less than about 800 ng / mL, less than about 500 ng / mL, less than about 300 ng / mL, or less than 150 ng / mL. The FACS binding EC50 can be determined by referring to the methods described in the Examples.

[0139] In one embodiment, the ELISA blocking IC50 value method is used to characterize the level of extracellular blocking of the ligand EGF binding to EGFR by the EGFR / c-Met bispecific antibody. The binding arm A of the bispecific antibody of the present invention can block EGF binding to EGFR with an ELISA blocking IC50 value of less than about 4000 ng / mL, less than about 3500 ng / mL, less than about 3000 ng / mL, less than about 2000 ng / mL, less than about 1500 ng / mL, less than about 1000 ng / mL, or less than 500 ng / mL. Exemplary EGFR / c-Met bispecific antibody binding arm A can block EGF binding to EGFR with an ELISA blocking IC50 value between about 400 ng / mL and 2200 ng / mL, such as an EGFR-binding arm A having the amino acid sequence of SEQ ID NO:4. The ELISA blocking IC50 determination method can be found in the Examples.

[0140] In one embodiment, the level of cell surface blocking of the EGFR / c-Met bispecific antibody against the binding of the ligand EGF to EGFR is determined using a FACS blocking IC50 method. Binding Arm A of the bispecific antibody of the present invention can be measured to inhibit EGF binding to EGFR at a FACS blocking IC50 value of less than about 8000 ng / mL, less than about 7000 ng / mL, less than about 6000 ng / mL, less than about 5000 ng / mL, less than about 4000 ng / mL, less than about 3500 ng / mL, less than 3000 ng / mL, less than 2500 ng / mL, or less than 2000 ng / mL. The FACS blocking IC50 method for determining the IC50 value can be found in the Examples.

[0141] One embodiment involves measuring EGFR autophosphorylation levels. Using the EGFR Phosphorylation (Y1068) Assay Kit, the absorbance at 665 nm represents the total phosphorylated C-Met, and the absorbance at 615 nm represents the total C-Met protein. The TR-FRET ratio is equal to the absorbance at 665 nm divided by the absorbance at 615 nm, multiplied by 1000. The TR-FRET ratio is used to represent the level of C-Met autophosphorylation. The TR-FRET ratio is calculated from non-small cell lung cancer cells (HCC827) and used to assess the extent to which the antibodies of the present invention inhibit EGFR-involved signaling pathways.

[0142] Binding Arm A of the bispecific antibody of the present invention can inhibit the phosphorylation activity of EGF on EGFR by 80%-100% compared to the TR-FRET ratio of EGF binding to EGFR using the same assay conditions in the absence of the bispecific antibody of the present invention.

[0143] The binding arm A of the bispecific antibody of the present invention can inhibit the EGFR signaling level by at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% compared to the signaling level when the same assay conditions are used in the absence of co-incubation comprising the EGFR / c-Met bispecific antibody of the present invention.

[0144] The combination of ligand EGF and EGFR can stimulate the activation of the internal cytoplasmic tyrosine kinase domain of receptor EGFR dimerization, autophosphorylation, receptor, and the initiation of the signal transduction and transactivation pathway of the regulation and control of multiple participation DNA synthesis (gene activation) and cell cycle progression or division. The inhibition of EGFR signal conduction can cause one or more EGFR downstream signal conduction pathways to be suppressed, so neutralization of EGFR can have various effects, including the inhibition of cell proliferation and differentiation, angiogenesis, cell movement and metastasis. EGFR signal conduction can be measured using various well-known methods, such as measuring the autophosphorylation of receptor at any one of tyrosine Y1068, Y1148 and Y1173.

[0145] In an exemplary embodiment, the level of extracellular binding of the bispecific antibody of the present invention to human EGFR is detected by biolayer interferometry (BLI). The binding arm A of the bispecific antibody of the present invention binds to human EGFR with a dissociation constant (KD) (M) of less than about 8E-8, such as less than about 6E-8, less than about 5E-8, less than about 3E-8, ​​less than about 2E-8, or less than about 1E-8.

[0146] In some embodiments, the binding arm A of the bispecific antibodies of the present invention binds to human EGFR with a KD (M) between about 2E-8 and about 4E-9. The affinity of the binding arm A for EGFR can be determined experimentally using any suitable method (e.g., as described in the Examples herein). The affinity of a particular binding arm A-antigen interaction measured may vary if measured under different conditions (e.g., osmolarity, pH). Therefore, measurements of affinity and other antigen binding parameters (e.g., KD, Kon, Koff) are preferably performed using a standardized solution of the antigen and a standardized buffer, such as the buffer described herein.

[0147] The bispecific antibodies of the invention as disclosed herein can inhibit EGF-induced growth of EGF-responsive cells without inducing the significant common toxicities associated with high-affinity bivalent EGFR antibodies, which makes the antibodies ideally suited for combination with TKIs, which have their own toxicity profiles, the induced growth being preferably measured using an assay as described in the Examples. Some growth also occurs in the case of inhibitory antibodies.

[0148] Function of c-Met-specific binding arm B

[0149] The anti-EGFR / c-Met bispecific antibody of the present invention has a binding arm B that specifically binds to the hepatocyte growth factor receptor (c-Met) and blocks the binding of hepatocyte growth factor (HGF) to the binding arm B of c-Met.

[0150] In one embodiment, the bispecific antibodies of the present invention, whose binding arm B specifically binds to c-Met with high affinity and inhibits c-Met signaling, can provide beneficial effects compared to small molecule c-Met inhibitors, including improved specificity and inhibition of HGF-mediated c-Met autophosphorylation and the signaling cascade that triggers tumorigenesis and progression. Furthermore, they can exhibit synergistic effects in tumor cell growth inhibition and killing compared to conventional monospecific antibodies and combination therapeutics. Binding arm B is monovalent, thereby preventing the undesirable receptor clustering and activation that can occur with other bivalent molecules.

[0151] The variable domain that can bind to c-Met in binding arm B is preferably derived from the variable domain of a bivalent monospecific antibody that targets c-Met and can inhibit EGF-induced death of non-small cell lung cancer cells HCC827, with an inhibition level equivalent to or close to that of Amivantama monoclonal antibody or its combination, and the inhibition is usually not 100%.

[0152] In one embodiment, the level of HGF-mediated c-Met autophosphorylation in the presence and absence of an antibody of the present invention is measured using a c-Met phosphorylation (Y1234 / Y1235) assay kit. The level of HGF-mediated c-Met autophosphorylation in the presence and absence of an antibody of the present invention is measured by calculating the ratio of absorbance at 665 nm / absorbance at 615 nm (TR-TRET ratio).

[0153] In an exemplary embodiment, the EGFR / c-Met bispecific antibody of the present invention inhibits HGF-mediated c-Met autophosphorylation, resulting in a reduction in the TR-TRET ratio of at least 40%, and preferably at least 60% (85-100%). Without being bound by theory, the significant reduction in the TR-TRET ratio can be interpreted as the binding of the antibody to c-Met blocking the binding of HGF to c-Met, thereby inhibiting HGF-mediated c-Met phosphorylation.

[0154] In one embodiment, when used in combination with a TKI, the bispecific antibodies of the invention as disclosed herein can inhibit the growth of EGFR-TKI-resistant tumor cells induced by HGF and EGF / HGF. In one exemplary embodiment, the bispecific antibodies of the invention as disclosed herein can inhibit the growth of EGFR-TKI-resistant tumor cell lines KP4, A549, MKN45, and HCC827-HGF induced by HGF and EGF / HGF. The TKI is preferably gefitinib.

[0155] In some embodiments, the c-Met-specific binding arm B of the bispecific antibody of the present invention preferably comprises an amino acid sequence derived from the variable domain of onartuzumab or ABT-700, or a variant thereof, as described herein, and preferably blocks the binding of the ligand HGR to c-Met. When, in the presence of a saturating amount of the binding arm B, under half-maximal binding conditions, the bispecific antibody of the present invention inhibits the binding of the ligand HGR to c-Met, resulting in a decrease in the TR-TRET ratio by at least 85-100%, the variable domain of the binding arm B is considered to block the binding of the ligand HGF to c-Met. The c-Met-specific binding arm B may preferably bind to the sema domain of c-Met.

[0156] The c-Met-specific binding arm B of the present invention can compete with HGF for binding to c-Met.

[0157] In one embodiment, the binding arm B of the bispecific antibody of the present invention can specifically bind to c-Met. Exemplarily, in an ELISA binding IC50 assay of extracellular c-Met binding, the binding arm B of the bispecific antibody of the present invention can bind to c-Met with an ELISA binding EC50 value of less than about 30 ng / mL, or less than about 25 ng / mL, or less than about 20 ng / mL, or less than about 150 ng / mL, or less than about 10 ng / mL, less than about 8 ng / mL, or less than about 5 ng / mL. Exemplarily, the binding arm B of the bispecific antibody of the present invention can bind to c-Met with a FACS binding EC50 value of less than about 600 ng / mL, or less than about 500 ng / mL, or less than about 400 ng / mL, or less than about 300 ng / mL, or less than about 200 ng / mL, or less than about 150 ng / mL, or less than about 130 ng / mL, or less than 100 ng / mL.

[0158] In one embodiment, the binding arm B of the bispecific antibody of the present invention has an inhibitory effect on the binding of HGF to c-Met. Exemplary, in an extracellular ELISA blocking IC50 assay for inhibition of HGF binding to c-Met, the binding arm B of the bispecific antibody of the present invention may inhibit the binding of HGF to c-Met with an ELISA blocking IC50 value of less than about 600 ng / mL, or less than about 500 ng / mL, or less than about 400 ng / mL, or less than about 300 ng / mL, or less than about 200 ng / mL, less than about 150 ng / mL, or less than about 100 ng / mL. Exemplarily, the binding arm B of the bispecific antibody of the present invention can block the binding of HGF to c-Met with a FACS blocking IC50 value of less than about 17,000 ng / mL, or less than about 15,000 ng / mL, or less than about 12,000 ng / mL, or less than about 10,000 ng / mL, or less than about 8,000 ng / mL, less than about 6,000 ng / ml, or less than about 4,000 ng / mL, or less than about 2,000 ng / mL, or less than about 1,500 ng / mL, or less than about 1,000 ng / mL.

[0159] The binding arm B of the present invention can inhibit the binding level of HGF to c-Met by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, compared to the binding of HGF to c-Met when the same assay conditions are used in the absence of the binding arm B of the bispecific antibody of the present invention.

[0160] The binding arm B of the bispecific antibody of the present invention can inhibit the level of c-Met signaling by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, compared to the signaling level when the same assay conditions are used in the absence of the binding arm B of the bispecific antibody of the present invention.

[0161] HGF binding to c-Met stimulates receptor dimerization, autophosphorylation, activation of the receptor's internal cytoplasmic tyrosine kinase domain, and multiple signal transduction and transactivation pathways involved in the regulation of DNA synthesis (gene activation) and cell cycle progression or division. Inhibition of c-Met signaling can lead to inhibition of one or more c-Met downstream signaling pathways, and thus neutralization of c-Met can have various effects, including inhibition of cell proliferation and differentiation, angiogenesis, cell motility, and metastasis.

[0162] c-Met signaling can be measured using various well-known methods. One embodiment, for example, is by measuring autophosphorylation of the receptor at at least one tyrosine residue, Y1234, Y1235, and / or phosphorylation of a natural or synthetic substrate. One embodiment can detect phosphorylation using an ELISA assay using an antibody specific for phosphotyrosine, such as the c-Met Phosphorylation (Y1068) Detection Kit assay described herein.

[0163] In one embodiment, a c-Met phosphorylation (Y1234 / Y1235) assay kit is used to measure the level of HGF-mediated c-Met autophosphorylation in the presence and absence of an antibody of the present invention. The ratio of absorbance at 665 nm / absorbance at 615 nm (TR-TRET ratio) is calculated to determine the level of HGF-mediated c-Met autophosphorylation in the presence and absence of an antibody of the present invention. In one exemplary embodiment, inhibition of HGF-mediated c-Met autophosphorylation by the EGFR / c-Met bispecific antibody of the present invention results in a reduction in the TR-TRET ratio of at least 40%, preferably at least 60%, more preferably at least 85%, or 100%. Without being bound by theory, the significant reduction in the TR-TRET ratio can be interpreted as the binding of the antibody to c-Met blocking HGF binding to c-Met, thereby inhibiting HGF-mediated c-Met phosphorylation. In one embodiment, the binding arm B of the present invention binds to a molecule with a dissociation constant (KD) of equal to or less than about 1.0E-8, 6.0E-9, 3.0E-9, 1.0E-9, 8.0E-10, 5.0E-10, and 4.0E-10, as determined by surface plasmon resonance, as practiced by one skilled in the art. D ) binds to human c-Met. The affinity of binding arm B for c-Met can be determined experimentally using any suitable method (e.g., as described in the Examples herein, using the c-Met phosphorylation (Y1234 / Y1235) assay kit from Bioauxilium (THUNDER TM Phospho-c-Met (Y1234 / Y1235) TR-FRET Cell Signaling Assay Kit) detects the level of c-Met autophosphorylation. The measured affinity of a specific binding arm B-antigen interaction may vary if measured under different conditions (e.g., osmolarity, pH). Therefore, affinity and other antigen binding parameters (e.g., K D , K on , K off ) are preferably measured using standardized solutions of protein scaffold and antigen, and a standardized buffer, such as the buffer described herein.

[0164] Function of bispecific antibodies that specifically bind to EGFR and c-Met

[0165] In one embodiment, the present invention provides an EGFR / c-Met bispecific antibody that inhibits EGF-mediated EGFR autophosphorylation and HGF-mediated c-Met autophosphorylation. In one embodiment, the inhibitory effect of the EGFR / c-Met bispecific antibody of the present invention on EGF-stimulated EGFR phosphorylation in non-small cell lung cancer HCC827 cells was determined using an EGFR phosphorylation (Y1068) assay kit. Exemplarily, using IgG1 as a control, the reduction in EGFR phosphorylation levels caused by 50 μg / mL of the EGFR / c-Met bispecific antibody of the present invention and 10 ng / mL of EGF-mFC-stimulated EGFR was determined. The absorbance at 665 nm represents the total number of EGFR phosphorylated proteins, and the absorbance at 615 nm represents the total number of EGFR proteins. The TR-FRET ratio is equal to the absorbance at 665 divided by the absorbance at 615 multiplied by 1000. The TR-FRET ratio is used to indicate the level of EGFR autophosphorylation. Binding Arm A of the EGFR / c-Met bispecific antibody inhibited EGF-induced phosphorylation of EGFR at residue Y1068 with a TR-FRET ratio (665 nm absorbance*1000 / 615 nm absorbance) of approximately 25-30, a result close to the EGFR phosphorylation level in the absence of EGF. In one embodiment, the inhibitory effect of the EGFR / c-Met bispecific antibody of the present invention on HGF-his-stimulated c-Met phosphorylation in HCC827 cells was determined using a c-Met phosphorylation (Y1234 / Y1235) assay kit. For example, the reduction in c-Met phosphorylation levels stimulated by 100 ng / mL HGF-his-stimulated c-Met using 50 μg / mL of the EGFR / c-Met bispecific antibody of the present invention was measured. The absorbance at 665 nm represents the total phosphorylated c-Met, and the absorbance at 615 nm represents the total c-Met protein. The TR-FRET ratio is equal to the absorbance at 665 nm divided by the absorbance at 615 nm multiplied by 1000. The TR-FRET ratio is used to represent the level of c-Met autophosphorylation. Binding Arm B of the bispecific EGFR / c-Met antibody inhibited HGF-induced c-Met phosphorylation at c-Met residues Y1234 / Y1235 with a TR-FRET ratio (absorbance at 665 nm*1000 / absorbance at 615 nm) of approximately 35-40, which is close to the level of c-Met phosphorylation in the absence of HGF induction. In an exemplary embodiment, the EGFR / c-Met bispecific antibody of the present invention inhibits EGF-mediated EGFR autophosphorylation and HGF-mediated c-Met autophosphorylation at a level comparable to that of Avanta.

[0166] In one embodiment, the present invention provides an EGFR / c-Met bispecific antibody that specifically binds to EGFR / c-Met double-positive tumor cells. In one embodiment of specific binding to double-positive tumor cells, the binding arm A of the EGFR / c-Met bispecific antibody of the present invention binds to EGFR double-positive cells with an ELISA binding EC50 value of 300 ng / mL-1500 ng / mL, preferably 300-800 ng / mL, and more preferably 300-400 ng / mL. In one exemplary embodiment, the double-positive tumor cells are MKN45 cells.

[0167] In one embodiment, the present invention provides that the binding arm A of the EGFR / c-Met bispecific antibody can antagonize the binding of EGF to EGFR / c-Met double-positive tumor cells, while the binding arm B can antagonize the binding of HGF to EGFR / c-Met double-positive tumor cells.

[0168] In one embodiment of blocking specific binding to double-positive tumor cells, the binding arm A of the EGFR / c-Met bispecific antibody of the present invention can inhibit EGF binding to EGFR on the surface of double-positive cells with a FACS blocking IC50 value of about 800-4000 ng / mL, preferably 1000-3000 ng / mL, and more preferably 1500-2000 ng / mL; the binding arm B can inhibit HGF binding to c-Met on the surface of double-positive cells with a FACS blocking IC50 value of about 2000 to about -10000 ng / mL, preferably 2000-6000 ng / mL, and more preferably 2000-3500 ng / mL. In one exemplary embodiment, the double-positive tumor cells are MKN45 cells.

[0169] In another embodiment, the EGFR / c-Met bispecific antibody of the present invention is a defucosylated anti-HGF / EGFR antibody, which has a significant ability to mediate NK cell cytotoxicity (ADCC) on target cells.

[0170] In another embodiment, the EGFR / c-Met bispecific antibody of the present invention mediates antibody-dependent phagocytosis (ADCP) in tumor cells.

[0171] In one embodiment, the tumor cells mediated by ADCP by the EGFR / c-Met bispecific antibody of the present invention are preferably HCC827 cells.

[0172] In one embodiment, the cell having phagocytic activity is THP-1 (a monocytic cell line derived from human peripheral blood).

[0173] In another embodiment, the bispecific antibody of the invention has high affinity for both EGFR and c-Met antigens.

[0174] In an exemplary embodiment, when the affinity of the bispecific antibody of the present invention for EGFR is detected based on biolayer interferometry (BLI), the binding arm A of the bispecific antibody of the present invention binds to human EGFR with a KD (M) of approximately less than 5.5E-9 to approximately less than 3.5E-9, preferably a KD (M) of 3.6E-9 to 4.5E-9, and most preferably a KD (M) of 3.9E-9 to 4.2E-9; and the binding arm B binds to human c-Met with a KD (M) of approximately less than 1.5E-9 to less than 4.3E-10, preferably 3.0E-9 to less than 4.30E-10.

[0175] In an assay using non-small cell lung cancer HCC827 cells and detecting the TR-FRET ratio (665 nm absorbance*1000 / 615 nm absorbance) from HCC827 cells using an EGFR phosphorylation (Y1068) assay kit with or without incubation with binding arm A, the binding arm A of the EGFR / c-Met bispecific antibody of the present invention can block EGF binding to EGFR with a FACS blocking IC50 value of about 300 to about -2000 ng / mL, preferably 1000-1100 ng / mL, and inhibit EGF-mediated EGFR autophosphorylation with a TR-FRET ratio of about 25-28. The binding arm A in the EGFR / c-Met bispecific antibody of the present invention may inhibit the binding of EGF to EGFR by at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% compared to the binding of EGF to EGFR using the same assay conditions in the absence of binding arm A.

[0176] In an assay using non-small cell lung cancer HCC827 cells and incubating with or without the binding arm B using a c-Met phosphorylation (Y1234 / Y1235) assay kit to detect the TR-FRET ratio (665 nm absorbance*1000 / 615 nm absorbance) from the HCC827 cells, the binding arm B in the EGFR / c-Met bispecific antibody of the present invention can inhibit HGF binding to c-Met with a FACS blocking IC50 value of about 2000-7000 ng / mL, preferably 2000-3000 ng / mL, and inhibit HGF-mediated c-Met autophosphorylation with a TR-FRET ratio of about 30-35. The binding arm B in the EGFR / c-Met bispecific antibody can block the binding of HGF to c-Met by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% compared to the binding of HGF to c-Met in the absence of binding arm B using the same assay conditions.

[0177] The EGFR / c-Met bispecific antibodies of the present invention may inhibit EGFR and / or c-Met signaling by at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% compared to the level of signaling using the same assay conditions in the absence of the EGFR / c-Met bispecific antibodies of the present invention.

[0178] EGFR and c-Met signaling of the binding arms can be measured using the methods described above.

[0179] The EGFR / c-Met bispecific antibodies of the present invention can inhibit tumor cell proliferation in vitro. The EGFR / c-Met bispecific antibodies of the present invention, comprising a binding arm A that specifically binds EGFR and a binding arm B that specifically binds c-Met, provide significantly enhanced synergistic inhibition of EGFR and c-Met signaling and tumor cell proliferation, compared to the inhibition observed with a mixture of an EGFR monoclonal antibody and a c-Met monoclonal antibody. In one embodiment, synergistic inhibition can be assessed, for example, by measuring and comparing the decrease in tumor cell viability caused by a mixture comprising an EGFR / c-Met bispecific antibody and two monospecific antibodies (one that binds EGFR and the other that binds c-Met).

[0180] In one embodiment, wherein When the bispecific molecules of the present invention inhibit tumor cell proliferation, the cell viability of the tumor cells is comparable to that measured when inhibited by amivantanab, and even lower than that measured when inhibited by ervanta. This indicates that the EGFR / c-Met bispecific antibody of the present invention can synergistically inhibit HCC827 cell proliferation compared to a mixture of an EGFR antibody and a c-Met antibody.

[0181] In one embodiment, the bispecific antibodies of the invention as disclosed herein can inhibit the growth of tumor cells in vitro. In one exemplary embodiment, the bispecific antibodies of the invention as disclosed herein can inhibit the proliferation of tumor cell lines. In some embodiments, the tumor cell lines include KP4, A549, MKN45, and HCC827-HGF.

[0182] In one embodiment, the bispecific antibodies of the present invention disclosed herein can mediate the internalization of EGFR and c-Met target proteins in tumor cells with a higher internalization efficiency than monoclonal antibodies against EGFR or c-Met. For example, the tumor cells shown are MKN45 cells.

[0183] In one embodiment, the bispecific antibodies of the present invention as disclosed herein can also specifically bind to cynomolgus monkey C-MET and EGFR proteins.

[0184] In one embodiment, the bispecific antibodies of the invention as disclosed herein have therapeutic effects on inhibiting tumor growth.

[0185] Exemplarily, the tumor is non-small cell lung cancer HCC827 (EGFR exon 19 deletion mutation).

[0186] Exemplarily, the tumor is HCC827-HGF.

[0187] Assembly of parent antibodies

[0188] Table 1 shows an exemplary parent antibody of the bispecific antibody of the present invention that specifically binds to the binding arm A of the bispecific antibody, an exemplary parent antibody that specifically binds to the binding arm B of the bispecific antibody, and an exemplary bispecific antibody produced by assembly.

[0189] Table 1 Parent anti-EGFR antibody binding arm A and exemplary parent anti-c-Met antibody binding arm B specifically binding to c-Met and exemplary bispecific antibodies generated by assembly

[0190] In one embodiment, the assembled fragments of the EGFR-specific binding arm A are derived from a marketed EGFR-specific monoclonal antibody.

[0191] In one embodiment, the EGFR-specific monoclonal antibody drugs on the market and under development include but are not limited to Nimotuzumab, Cetuximab, Necitumumab, Panitumumab, Demupitamab, Modotuximab, Pimurutamab, and QL1203.

[0192] In one embodiment, the assembled fragment of the c-Met-specific binding arm B is derived from a marketed c-Met-specific monoclonal antibody.

[0193] In one embodiment, the marketed and investigational c-Met-specific monoclonal antibodies include, but are not limited to, Onartuzumab, ABT-700, Emibetuzumab, Telisotuzumab vedotin, HLX-55 monoclonal antibody, SAIT-301, and RC-108. In one embodiment, an exemplary anti-EGFR parent antibody that can be used to engineer the binding arm A of the bispecific molecule is JH001 (derived from the present applicant's patent application No. 2020105770075).

[0194] In one embodiment, an exemplary anti-c-Met parent antibody that can be used to engineer the binding arm B of the bispecific molecule is, for example, Onartuzumab. In one embodiment, an exemplary anti-c-Met parent antibody that can be used to engineer the bispecific molecule is ABT-700.

[0195] In one embodiment, the binding arm A of the bispecific antibody specifically binding to EGFR and c-Met of the present invention can be derived from an existing monospecific EGFR antibody, such as JH001 (derived from the applicant's patent application No. 2020105770075), and the binding arm B can be derived from an existing monospecific c-Met antibody, such as Onartuzumab. In an exemplary embodiment, the bispecific antibody specifically binding to EGFR and c-Met of the present invention is BT470.

[0196] In one embodiment, the bispecific antibody of the present invention that specifically binds to EGFR and c-Met can have binding arm A derived from an existing monospecific EGFR antibody, such as JH001 (derived from the present applicant's patent application No. 2020105770075), and binding arm B can be derived from an existing monospecific c-Met antibody, such as ABT-700. In one exemplary embodiment, the bispecific antibody of the present invention that specifically binds to EGFR and c-Met is BT468.

[0197] The amino acid sequence of the binding arm A of the bispecific antibody embodiment of the present invention and the amino acid sequence of the parent antibody are shown in Example 2.2.

[0198] Structure of the binding arm

[0199] In one embodiment, the bispecific antibody of the present invention that specifically binds to EGFR and c-Met comprises a binding arm A that specifically binds to EGFR and a binding arm B that specifically binds to c-Met, wherein the binding arm A and the antigen-binding site of an anti-EGFR antibody, or a VHA / VLA pair, or an LCA / HCA pair, or a single fusion peptide chain consisting of an antibody's ScFab(A) and Fc(A) pair, are formed through interaction of their respective heavy chains to form a KIH structure. The binding arm B can be considered to be the antigen-binding site of an anti-c-Met antibody, or a VHB / VLB pair, or an LCB / HCB pair, or a single fusion peptide chain consisting of an ScFab(B) and Fc(B) pair. The binding arms A and B of the EGFR / c-Met bispecific antibody of the present invention can be obtained by further engineering existing monospecific anti-EGFR and anti-c-Met antibodies. For example, the EGFR / c-Met bispecific antibody of the present invention can use the EGFR monoclonal antibody (JH001) of the applicant's patent application 2020105770075 as a source to provide the antigen-binding domain and functional fragment to design the binding arm A, and use the marketed c-Met monoclonal antibody such as Onartuzumab or ABT-700 as the basis to provide the antigen-binding domain and functional fragment to design the binding arm B, and further introduce the knob mutation into the Fc (A) of the binding arm A and the hole mutation into the Fc (B) of the binding arm B, so that the binding arm A and the binding arm B interact through Fc (A) and Fc (B) to form a KIH structure, thereby producing the EGFR / c-Met bispecific antibody with a heterodimeric structure of the present invention.

[0200] Light chain-linker-heavy chain

[0201] On the one hand, the present invention provides a bispecific antibody, which comprises a binding arm A that specifically binds to EGFR and a binding arm B that specifically binds to c-Met, wherein the binding arm A and the binding arm B are single-chain binding peptides comprising all functional fragments of the full-length antibody, the binding arm A is a fusion peptide formed by a light chain A (LCA) and a heavy chain A (HCA) connected by a linker, wherein the light chain A (LCA), the linker and the heavy chain A (HCA) are sequentially connected from the N-terminus to the C-terminus to form a fusion peptide with the structural formula LCA-linker-HCA, and the binding arm B is a fusion peptide formed by a light chain B (LCB) and a heavy chain B (HCB) connected by a linker, wherein the light chain B (LCB), the linker and the heavy chain B (HCB) are sequentially connected from the N-terminus to the C-terminus to form a fusion peptide with the structural formula LCB-linker-HCB, wherein "-" is a peptide bond.

[0202] In one embodiment, the linker has an amino acid sequence as shown in (GGGGS)n, (such as any one of SEQ ID NOs: 9, 20, and 31); exemplarily, the linker has an amino acid sequence as shown in SEQ ID NO: 31; exemplarily, the linker has an amino acid sequence as shown in SEQ ID NO: 20; exemplarily, the linker has an amino acid sequence as shown in SEQ ID NO: 9.

[0203] In one embodiment, the LCA and HCA of the binding arm A and the LCB and HCB of the binding arm B preferably comprise amino acid sequences having amino acid sequences as shown in SEQ ID NOs: 1 and 2 derived from JH001 and sequences having SEQ ID NOs: 23 and 24, respectively, derived from the ABT-700 sequence; in one embodiment, the linker has an amino acid sequence as shown in SEQ ID NO: 20; and the bispecific antibody in at least one exemplary embodiment is named BT468.

[0204] In one embodiment, the LCA and HCA of the binding arm A and the LCB and HCB of the binding arm B respectively comprise the amino acid sequences shown in SEQ ID NOs: 1 and 2 derived from JH001 and the sequences shown in SEQ ID NOs: 12 and 13 derived from Onartuzumab. In one embodiment, the linker has the amino acid sequence shown in SEQ ID NO: 20; the bispecific antibody in at least one exemplary embodiment is named BT470.

[0205] In some embodiments, binding arm A and binding arm B respectively use EGFR monoclonal antibody and c-Met monoclonal antibody commonly used in the market as the source of assembly fragments to prepare bispecific antibodies BT59, BT460, BT468 and BT469.

[0206] In one aspect, the present invention provides a bispecific antibody comprising a binding arm A and a binding arm B, wherein the binding arm A and the binding arm B respectively have a heavy chain variable region A / light chain variable region A (VHA / VLA) combination that specifically binds to the extracellular portion of EGFR and a VHB / VLB combination that specifically binds to the extracellular portion of c-Met, thereby binding to both EGFR antigen and c-Met antigen.

[0207] In a preferred embodiment, the binding arm A and the binding arm B of the bispecific antibody of the present invention are single-chain binding peptides comprising all functional fragments of the full-length antibody, wherein the binding arm A is a fusion peptide of the structural formula VLA-CLA-linker-VHA-CHA and the binding arm B is a fusion peptide of the structural formula VLB-CLB-linker-VHBA-CHB, wherein "-" represents a peptide bond.

[0208] In an exemplary embodiment, in the binding arm A of the bispecific antibody of the present invention, the VLA and the VHA comprise VLACDR1, VLACDR3 and VLACDR3, VHACDR1, VHACDR2 and VHACDR3 as shown in Table 2, respectively.

[0209] In one embodiment, the constituent fragments of the binding arm A are derived from JH001.

[0210] Table 2 Exemplary CDR sequences of binding arm A

[0211] In one embodiment, preferably, the binding arm A comprises a VLA chain having a different framework than the CDR region of the VLA shown in SEQ ID NO:5 and a VHA chain having a different framework than the CDR region of the VHA shown in SEQ ID NO:7. The different frameworks can be the framework of another human VH or a framework of a different mammal.

[0212] In the bispecific antibody described herein, the binding arm A that specifically binds to the extracellular portion of EGFR comprises a heavy chain variable region A / light chain variable region A (VHA / VLA) combination.

[0213] In one embodiment, the binding arm A of the present invention that specifically binds to EGFR and inhibits EGFR autophosphorylation comprises a VLA and a VHA selected from the group consisting of those listed in Table 3.

[0214] Table 3 Exemplary variable region amino acid sequences of binding arm A of the EGFR / c-Met bispecific antibodies of the present invention

[0215] In an exemplary embodiment, the binding arm B that specifically binds to c-Met comprises VLBCDR1, VLBCDR3 and VLBCDR3, VHBCDR1, VHBCDR2 and VHBCDR3 selected from the group consisting of the amino acid sequences shown in Table 4.

[0216] Table 4 Exemplary CDR sequences included in binding arm B

[0217] In the bispecific antibody described herein, the binding arm B that specifically binds to c-Met comprises a heavy chain variable region B / light chain variable region B (VHB / VLB) combination.

[0218] In one embodiment, the binding arm B of the present invention that specifically binds to c-Met and inhibits autophosphorylation of c-Met comprises a VLA and a VHA selected from the group consisting of those listed in Table 5.

[0219] Table 5 Sequences of VLB and VHB parent antibodies binding arm B

[0220] In one embodiment, the light chain B (LCB) and heavy chain B (HCB) may each have the CDR regions of VLB as set forth in SEQ ID NO: 27 but a different framework, and the CDR regions of VHB as set forth in SEQ ID NO: 29 but a different framework, respectively. In one embodiment, the light chain B (LCB) and heavy chain B (HCB) may each have the CDR regions of VLB as set forth in SEQ ID NO: 16 but a different framework, and the CDR regions of VHB as set forth in SEQ ID NO: 18 but a different framework, respectively. The different frameworks may be those of another human VHB or those of a different mammal.

[0221] In one embodiment, VLA and VHA of the binding arm A are derived from the parent antibody JH001, as shown in SEQ ID NOs: 5 and 7, respectively, and VLB and VHB of the binding arm B are derived from onartuzumab, as shown in SEQ ID NOs: 16 and 18, respectively. At least one exemplary bispecific antibody is BT470.

[0222] In one embodiment, VLA and VHA of the binding arm A are derived from the parent antibody JH001, as shown in SEQ ID NOs: 5 and 7, respectively, and VLB and VHB of the binding arm B are derived from ABT-700, as shown in SEQ ID NOs: 27 and 29, respectively. At least one exemplary bispecific antibody is BT468.

[0223] In a preferred embodiment, the linker comprises the linker connecting light chain A and heavy chain A shown in SEQ ID NO: 20.

[0224] The binding properties of the binding arm may reside primarily in the exact sequence of the CDR3 region, which is generally supported by the sequence of the CDR1 and CDR2 regions in the variable domain combined with the appropriate structure of the variable domain as a whole. The amino acid sequences of the CDR regions as described herein are determined using the Kabat definition.

[0225] In at least one embodiment, the LCA and HCA of the EGFR binding site of binding arm A comprise the JH001 light chain variable region A (VLA-JH001) as set forth in SEQ ID NO: 5 and the JH001 heavy chain variable region A (VHA-JH001) as set forth in SEQ ID NO: 7, respectively. In at least one embodiment, the VLA-JH001 preferably comprises the VLA CDR1, VLA CDR2, and VLA CDR3 sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively, and the VHA-JH001 preferably comprises the VH CDR1, VHA CDR2, and VHA CDR3 sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively. In at least one exemplary embodiment, the human or humanized bispecific antibodies of the present invention as disclosed herein are BT470 and BT468.

[0226] In at least one embodiment, the c-Met binding site of the binding arm B comprises a light chain B (LCB) and a heavy chain B (HCB) derived from Onartuzumab as shown in SEQ ID NO: 12 and 13, respectively, and a linker linking the light chain B and heavy chain B as shown in SEQ ID NO: 20.

[0227] In at least one embodiment, the LCB and HCB of the c-Met binding site of arm B comprise a light chain variable region B derived from Onartuzumab (VLB-Onar) with SEQ ID NO: 16 and a heavy chain variable region B derived from Onartuzumab (VHB-Onar) with SEQ ID NO: 18, respectively. An exemplary human or humanized bispecific antibody of the invention as disclosed herein is BT470.

[0228] In one embodiment, the light chain variable region (VLB-Onar) containing the c-Met binding site preferably comprises the sequences of the VLB CDR1, VLB CDR2, and VLB CDR3 regions derived from the VLB chain of onartuzumab, as shown in SEQ ID NOs: 40, 41, and 42, respectively. The heavy chain variable region (VHB) containing the c-Met binding site preferably comprises the sequences of the VHB CDR1, VHB CDR2, and VHB CDR3 regions derived from onartuzumab, as shown in SEQ ID NOs: 43, 44, and 45, respectively. CDR grafting can also be used to generate a VH chain having the CDR regions of the VLB chains set forth in SEQ ID NOs: 40, 41, and 42 or the CDR regions of the VHB chains set forth in SEQ ID NOs: 43, 44, and 45, but with a different framework. The different framework can be that of another human VH or a framework from a different mammal. Exemplary, the human or humanized bispecific antibody of the present invention as disclosed herein is BT470, which comprises an antigen-binding arm A that binds to the first antigen-binding site of EGFR and an antigen-binding arm B that binds to the second antigen-binding site of c-Met. Exemplary, the human or humanized bispecific antibody of the present invention as disclosed herein is BT470. In at least one embodiment, the binding arm A comprises LCA and HCA derived from JH001 (as shown in SEQ ID NOs: 1 and 2, respectively) that specifically bind to the extracellular domain III of EGFR, and a linker connecting the LCA and HCA as shown in SEQ ID NO: 20, and the binding arm B comprises light chain B and heavy chain B derived from ABT-700 (as shown in SEQ ID NOs: 23 and 24, and a linker connecting the LCB and HCB as shown in SEQ ID NO: 20).

[0229] In at least one embodiment, the EGFR binding site comprises the JH001 light chain variable region A (VLA-JH001) as set forth in SEQ ID NO:5 and the JH001 heavy chain variable region A (VHA-JH001) as set forth in SEQ ID NO:7. The light chain variable region A (VLA-JH001) preferably comprises the VLA CDR1, VLA CDR2, and VL CDR3 sequences set forth in SEQ ID NOs:34, 35, and 36, respectively, and the heavy chain variable region A (VHA-JH001) preferably comprises the VH CDR1, VHA CDR2, and VHA CDR3 sequences set forth in SEQ ID NOs:37, 38, and 39, respectively. Furthermore, the antigen-binding arm B comprising the c-Met binding site comprises a light chain B and a heavy chain B, each of which specifically binds to c-Met, as set forth in SEQ ID NOs:12 and 13, respectively, and a linker connecting the light chain B and heavy chain B, as set forth in SEQ ID NO:20.

[0230] In at least one embodiment, the c-Met binding site comprises the ABT-700 light chain variable region B (VLB-ABT-700) and heavy chain variable region B (VHB-ABT-700) as shown in SEQ ID NOs: 27 and 29. In at least one embodiment, the light chain variable region B (VLB-ABT-700) comprising the c-Met binding site preferably comprises the sequences of the VLB CDR1, VLB CDR2, and VLB CDR3 regions of the VLB chain of ABT-700 as shown in SEQ ID NOs: 46, 47, and 48, respectively, and the heavy chain variable region (VHB-ABT-700) comprising the c-Met binding site preferably comprises the sequences of the VHB CDR1, VHB CDR2, and VHB CDR3 regions of the VHB chain of ABT-700 as shown in SEQ ID NOs: 49, 50, and 51, respectively. CDR grafting can also be used to generate a VH chain having the CDR regions of the VHA chains shown in SEQ ID NOs: 46, 47, and 48, or the CDR regions of the VHB chains shown in SEQ ID NOs: 49, 50, and 51, but with a different framework. The different framework can be that of another human VH, or a framework from a different mammal. An exemplary human or humanized bispecific antibody of the invention as disclosed herein is BT468.

[0231] constant region

[0232] The bispecific antibodies of the present invention disclosed herein are preferably for use in humans. A preferred embodiment of the bispecific antibodies of the present invention is a human or humanized antibody. As disclosed herein, the bispecific antibodies of the present invention are preferably heterodimeric bispecific antibodies formed by an interaction between a binding arm comprising a fusion peptide of a light chain-linker-heavy chain structure targeting EGFR and a binding arm comprising a fusion peptide of a light chain-linker-heavy chain structure targeting c-Met via the CH3 of the heavy chain. The light and heavy chains of the binding arms A and B of the bispecific antibodies of the present invention disclosed herein further comprise a light chain constant region and a heavy chain constant region, and the constant regions are preferably human constant regions.

[0233] In some embodiments, the binding arm of the EGBFR / c-Met bispecific antibody of the present invention is an IgG antibody.

[0234] In some embodiments, the binding arm of the EGBFR / c-Met bispecific antibody of the present invention can be an IgG1, IgG2, IgG3, or IgG4 antibody.

[0235] In some embodiments, the binding arms of the EGBFR / c-Met bispecific antibodies of the present invention are preferably IgG1 antibodies, and more preferably human IgG1. The light and heavy chains of the binding arms A and B of the bispecific antibodies according to the present invention as disclosed herein preferably comprise constant regions of human antibodies.

[0236] Full-length IgG1 antibodies are preferred because they generally have a good half-life, and for reasons of immunogenicity, it is desirable to maintain complete proximity to the autologous (human) molecule. Some changes in the IgG1 constant region occur naturally and / or are allowed without changing the immunological properties of the resulting antibody. Such features are described, for example, in the context of CH2 and CH3 herein. The constant region may comprise one or more, preferably no more than 10, preferably no more than 5 amino acid differences from the constant region of a naturally occurring human antibody. Preferably, the constant portion is derived entirely from naturally occurring human antibodies. Such antibodies of the present invention as disclosed herein can have good ADCC properties by heavy chain modification through constant region amino acid mutations, and have a good half-life after administration to the human body. And there are CH3 transformation technologies that can provide modified heavy chains, which, after co-expression in cloned cells, preferentially form heterodimers compared to homodimers. The ADCC activity of antibodies can also be improved by techniques known to those skilled in the art.

[0237] ScFab-Fc structure

[0238] In one aspect, the present invention provides a bispecific antibody comprising a binding arm A that can bind to the extracellular portion of human epidermal growth factor receptor (EGFR) and a binding arm B that can bind to the extracellular portion of human proto-oncogene receptor tyrosine kinase (c-Met); wherein the binding arm A comprises a ScFab (A) segment and an Fc (A) segment comprising a CH2A segment domain and a CH3A segment domain from the N-terminus to the C-terminus, and the ScFab (A) is connected to the N-terminus of the CH2A segment domain of the Fc (A) through a hinge region, forming a ScFab (A)-hinge region-Fc (A) structure; the binding arm B comprises a ScFab (B) segment and an Fc (B) segment comprising a CH2B segment domain and a CH3B segment domain from the N-terminus to the C-terminus, and the ScFab hinge region (B) is connected to the N-terminus of the CH2B segment domain of the Fc (B) through the hinge region structure, forming a ScFab (B)-hinge region-Fc (B) structure.

[0239] In one embodiment, the ScFab (A) comprises, from N-terminus to C-terminus, a light chain variable region A (VLA), a light chain constant region A (CLA), a linker, a heavy chain variable region A (VHA), and a first heavy chain constant region A (CH1A), forming a VLA-CLA-linker-VHA-CH1A structure, and the Fc (A) consists of the second and third constant regions (CH2ACH3A) of HCA, and comprises a combination of amino acid mutations that form a knob structure; the ScFab (B) comprises, from N-terminus to C-terminus, a light chain variable region B (VLB), a light chain constant region B (CLB), a linker, a heavy chain variable region B (VHB), and a first heavy chain constant region B (CH1B), forming a VLB-CLB-linker-VHB-CH1B structure, and the Fc (B) consists of the second and third constant regions (CH2BCH3B) of HCB, and comprises a combination of amino acid mutations that form a knob structure; wherein the CH3A and the CH3B interact to form a KIH structure that prevents heavy chain mispairing.

[0240] In some embodiments, the light chain constant region CLA comprised by the ScFab (A) and the light chain constant region CLB comprised by the ScFab (B) are κ-type light chains, preferably human κ-type light chains.

[0241] In some embodiments, CH1A comprised by ScFab (A) and CH1B comprised by ScFab (B) are of IgG subtype.

[0242] In some embodiments, CH1A comprised by ScFab (A) and CH1B comprised by ScFab (B) are CH1 of human IgG subtype.

[0243] In some embodiments, the CH1A and Fc(A) comprised by ScFab(A), and the CH1B and Fc(B) comprised by ScFab(B) are human IgG1 subtype 1.

[0244] In some embodiments, CH1A contained in ScFab (A) and CH1B contained in ScFab (B) are the amino acid sequences of CH1 of human IgG1 subtype shown in SEQ ID NO: 63.

[0245] Table 6 describes the SEQ ID NOs of the amino acid sequences of the variable and constant regions of the parent antibodies from which the exemplary EGFR-specific binding arm A and the exemplary c-Met-specific binding arm B disclosed herein are derived.

[0246] Table 6 SEQ ID NOs of amino acid sequences of variable and constant regions of parent antibodies from which exemplary EGFR binding arm A and exemplary c-Met binding arm B are derived:

[0247] In one embodiment, the VLA, CLA, linker, VHA, and CH1A in the ScFab (A) preferably comprise amino acid sequences derived from JH001 as shown in SEQ ID NOs: 5, 6, 9, 7, and 63, respectively; the Fc (A) comprising a knob mutation comprises a variant of the Fc segment amino acid sequence derived from the parent antibody JH001 as shown in SEQ ID NO: 11; in the ScFab (B), the VLB, CLB, linker, VHB, and CH1B preferably comprise amino acid sequences derived from Onartuzumab as shown in SEQ ID NOs: 16, 17, 20, 18, and 63, respectively; and the Fc (B) comprising a combination of amino acid mutations forming a hole structure comprises an Fc segment amino acid sequence variant derived from the parent antibody Onartuzumab as shown in SEQ ID NO: 22. In an exemplary embodiment, the ScFab (A), the hinge region A and the Fc (A) comprise the amino acid sequences shown in SEQ ID NOs: 3, 10 and 11, respectively, derived from JH001, and the ScFab (B), the hinge region B and the Fc (B) comprise the amino acid sequences shown in SEQ ID NOs: 14, 21 and 22, respectively, derived from Onartuzumab, e.g., BT470.

[0248] In one embodiment, in the ScFab (A), the VLA, CLA, linker, VHA, and CH1A preferably comprise the amino acid sequences of SEQ ID NOs: 5, 6, 9, 7, and 63 derived from JH001, respectively; the Fc (A) comprising a knob mutation comprises a variant of the Fc segment amino acid sequence derived from the parent antibody JH001 as shown in SEQ ID NO: 11; in the ScFab (B), the VLB, CLB, linker, VHB, and CH1B preferably comprise the amino acid sequences of SEQ ID NOs: 27, 28, 31, 29, and 63 derived from ABT-700, respectively; and the Fc (B) comprising a combination of amino acid mutations forming a hole structure comprises a variant of the Fc segment amino acid sequence derived from the parent antibody ABT-700 as shown in SEQ ID NO: 33. In an exemplary embodiment, the ScFab (A), the hinge region A and the Fc (A) comprise the amino acid sequences shown in SEQ ID NOs: 3, 10 and 11, respectively, derived from JH001, and the ScFab (B), the hinge region B and the Fc (B) comprise the amino acid sequences shown in SEQ ID NOs: 25, 32 and 33, respectively, derived from ABT-700, e.g., BT468.

[0249] In a preferred embodiment, the Fc(A) of binding arm A comprises a CH2ACH3A segment derived from the constant region CHA of heavy chain A as shown in SEQ ID NO: 2, and the Fc(B) of binding arm B comprises a CH2BCH3B segment derived from the constant region CHB of heavy chain B as shown in SEQ ID NO: 13. At least one exemplary embodiment is BT470.

[0250] In a preferred embodiment, the Fc(A) of binding arm A comprises a CH2ACH3A segment derived from the constant region CHA of heavy chain A as shown in SEQ ID NO: 2, and the Fc(B) of binding arm B comprises a CH2BCH3B segment derived from the constant region CHB of heavy chain B as shown in SEQ ID NO: 24. At least one exemplary embodiment is BT468.

[0251] In some embodiments, the binding arm A has a structure as shown in the formula VLA-CLA-linker-VHA-CH1A-Fc(A); wherein VLA is a light chain variable region that specifically binds to EGFR, CLA is a constant region of a light chain that specifically binds to EGFR, VHA is a heavy chain variable region that specifically binds to EGFR, CH1 is the first constant region of a heavy chain that specifically binds to EGFR, and Fc(A) comprises the CH2ACH3A region of heavy chain A that specifically binds to EGFR. In some embodiments, the binding arm B has a structure as shown in the formula VLB-CLB-linker-VHB-CH1B-Fc(B); wherein VLB is a light chain variable region that specifically binds to c-Met, CLB is a constant region of a light chain that specifically binds to c-Met, VHB is a heavy chain variable region that specifically binds to c-Met, CH1B is the first constant region of a heavy chain that specifically binds to c-Met, and Fc(B) comprises the CH2BCH3B region of heavy chain B that specifically binds to c-Met.

[0252] In one embodiment, the antigen-binding arm A comprises light chain complementary determining regions LACDR1, LACDR2 and LACDR3 and heavy chain complementary determining regions HACDR1, HACDR2 and HACDR3, the antigen-binding arm B comprises light chain complementary determining regions LBCDR1, LBCDR2 and LBCDR3, and the VHB comprises heavy chain complementary determining regions HBCDR1, HBCDR2 and HBCDR3; wherein the antigen-binding arm A and the antigen-binding arm interact to form a KIH structure, and the EGFR / C-Met bispecific antibody is a heterodimer formed by the antigen-binding arm A and the antigen-binding arm B through the KIH structure;

[0253] Wherein, the LACDR1, LACDR2 and LACDR3, HACDR1, HACDR2 and HACDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 27, 28, 29, 30, 31 and 32, and the LBCDR1, LBCDR2 and LBCDR3, HBCDR1, HBCDR2 and HBCDR3 respectively comprise the amino acid sequences shown in SEQ ID NOs: 33, 34, 35, 36, 37 and 38, or respectively comprise the amino acid sequences shown in SEQ ID NOs: 33, 34, 35, 36, 37 and 38.

[0254] The linker of binding arm A and binding arm B is a chemical structure with a connecting function, preferably a connecting peptide, more preferably a peptide structure of a (GSSS) n structure. In some embodiments, in the bispecific antibodies of the present invention, the linker in the binding arm is preferably a short peptide. In some embodiments, the amino acid sequence of the connecting peptide is selected from but not limited to the following sequences: (GGGGS) 4 (SEQ ID NO: 9), GGGGSGGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 20), GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31). In some embodiments, in the bispecific antibodies of the present invention, the amino acid sequence of the linker in the binding arm is preferably GGGGSGGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 20). In some embodiments, in the bispecific antibodies of the present invention, the amino acid sequence of the linker in the binding arm is more preferably (GGGGS) 4 (SEQ ID NO: 9). In some embodiments, in the bispecific antibody of the present invention, the amino acid sequence of the linker in the binding arm is more preferably (GGGGS)6 (SEQ ID NO: 20).

[0255] In the preparation of the EGFR / c-Met bispecific antibodies of the present invention, the probability of light and heavy chain mispairing is generally reduced and prevented by modifying the constant region of the heavy chain so that it is more favorable for heterodimerization (i.e., dimerization of the heavy chain in combination with another heavy chain / light chain) than homodimerization. In a preferred embodiment, the bispecific antibodies of the present invention as disclosed herein comprise two different immunoglobulin heavy chains having compatible heterodimerization domains. In order to increase the percentage of the desired bispecific antibody product, the present invention uses CH3 modification, or the compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain, as defined below. Various methods in which heterodimerization of the heavy chain can be achieved are described in the art. One preferred method is to generate a "knob into hole (KIH)" bispecific antibody (see US13 / 866,747, now authorized as US9,248,181) and the like. In one embodiment, binding arm A of a heavy chain A / light chain A combination comprising variable domains that bind to EGFR comprises a knob variant structure of heavy chain A, and binding arm B of a heavy chain B / light chain B combination comprising variable domains that bind to c-Met comprises a hole variant structure of heavy chain B. The knob variant of heavy chain A that binds to EGFR and the hole variant of heavy chain B that binds to c-Met do not form homodimers.

[0256] The preferred knob mutation is the amino acid substitution S354C, T366W in the CH3A domain of the binding arm A (knob "variant" heavy chain) and the preferred hole mutation is the amino acid substitution Y349C, T366S, L368A, Y407V (hole variant" heavy chain) in the binding arm B, or vice versa. Due to the repulsion between the charged residues in the CH3-CH3 interface between the same heavy chains, the homodimerization of the CH3 variant heavy chain is not conducive. According to the spatial complementarity and hydrophobic interaction, the formation of heterodimers is guided, the interchain disulfide bonds are increased to reduce homodimer impurities, and the knob variant heavy chain and the hole variant heavy chain preferentially pair to form heterodimers, effectively preventing heavy chain mispairing. Furthermore, each antigen-binding arm is connected by 4-10 consecutive groups, preferably 6 consecutive groups of GGGGS sequences (GGGGS). n The structural linker connects the paired light and heavy chains, solving the light chain mispairing problem of bispecific antibodies. Through the above measures, the EGFR and c-Met bispecific antibodies of this method can effectively prevent the mispairing of the light and heavy chains with different targets to produce non-target bispecific antibodies.

[0257] In one embodiment, the bispecific antibody of the present invention is an antibody with reduced effector function. In one embodiment, the antibody with reduced effector function is preferably an IgG antibody comprising a modified CH2 and / or lower hinge region, for example to reduce Fc-receptor interactions or reduce C1q binding. In some embodiments, the antibody of the present invention is an IgG1 antibody with a mutated CH2 and / or lower hinge domain, such that the interaction of the bispecific IgG antibody with the Fc-γ receptor is reduced. The antibody comprising a mutated CH2 region is preferably an IgG1 antibody. Such a mutated IgG1 CH2 and / or lower hinge domain preferably comprises an amino acid substitution at position 235 and / or position 236 (EU numbering), preferably L235A and / or G236A substitution.

[0258] The Fc region mediates the effector functions of antibodies, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellphagocytosis (ADCP). The use of Fc fusion proteins in therapeutic antibodies can achieve the desired reduction or enhancement of effector functions.

[0259] For example, in some embodiments, when an immune response is to be activated, enhanced, or stimulated, reduced effector function may be desired. Antibodies with reduced effector function can be used to target cell surface molecules of immune cells, etc.

[0260] As in some embodiments, the antibodies of the present invention disclosed herein preferably have enhanced effector function. The bispecific antibodies of the present invention as disclosed herein preferably have antibody-dependent cell-mediated cytotoxicity (ADCC). In one aspect, the bispecific antibodies of the present invention as disclosed herein show improved ADCC activity. Methods for achieving ADCC enhancement in the field of antibody engineering include, for example, glycoengineering (Kyowa Hakko / Biowa, GlycArt (Roche) and Eureka Therapeutics) and mutagenesis, all of which attempt to improve the binding of Fc to low-affinity activated FcγRIIIa, and / or reduce the binding to low-affinity inhibitory FcγRIIb. Preferably, a technique for enhancing the ADCC of antibodies used in the present invention is non-fucosylated amino acid mutation.

[0261] The present invention also provides non-fucosylated EGFR / c-Met bispecific antibodies. The bispecific antibodies disclosed herein preferably contain reduced amounts of fucosylated N-linked carbohydrate structures in the Fc region when compared to the same antibodies produced in normal CHO cells. The bispecific antibodies of the invention disclosed herein are preferably non-fucosylated to enhance ADCC activity.

[0262] Exemplary EGFR / c-Met bispecific antibodies

[0263] In an exemplary embodiment, in the EGFR / c-Met bispecific antibody of the present invention, the binding arm A that specifically binds to the EGFR extracellular domain III comprises a ScFab (A) as shown in SEQ ID NO: 3 and an Fc (A) as shown in SEQ ID NO: 11 derived from JH001, and a hinge region connecting the ScFab (A) and the Fc (A) as shown in SEQ ID NO: 10, wherein the ScFab (A) comprises VLA, CLA, linker, VHA, and CH1A as shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 7, and SEQ ID NO: 63, respectively; the Fc (A) comprises the CH2ACH3A segment of the HCA as shown in SEQ ID NO: 2; the binding arm B that specifically binds to c-Met comprises a ScFab (B) as shown in SEQ ID NO: 25 and an Fc (B) as shown in SEQ ID NO: 33 derived from ABT-700, and a hinge region as shown in SEQ ID NO: 10. NO:20 shows a linker connecting ScFab (B) and Fc (B), wherein ScFab (B) comprises VLB, CLB, VHB and CH1B as shown in SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:20, SEQ ID NO:29 and SEQ ID NO:63, respectively; the Fc (B) comprises the CH2BCH3B segment in HCB as shown in SEQ ID NO:24, and at least one embodiment is the bispecific antibody BT468 of the present invention disclosed herein.

[0264] In an exemplary embodiment, in the EGFR / c-Met bispecific antibody of the present invention, the binding arm A that specifically binds to the EGFR extracellular domain III comprises a ScFab (A) as shown in SEQ ID NO: 3 and an Fc (A) as shown in SEQ ID NO: 11 derived from JH001, and a hinge region connecting the ScFab (A) and the Fc (A) as shown in SEQ ID NO: 10, wherein the ScFab (A) comprises VLA, CLA, linker, VHA and CH1A as shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 7 and SEQ ID NO: 63, respectively; the Fc (A) comprises the CH2ACH3A segment in the HCA as shown in SEQ ID NO: 2; the binding arm B that specifically binds to c-Met comprises a ScFab (B) as shown in SEQ ID NO: 14 and an Fc (B) as shown in SEQ ID NO: 22 derived from Onartuzumab, and a hinge region as shown in SEQ ID NO: 10 connected to the ScFab (A) and the Fc (A). NO:20 shows a linker connecting ScFab (B) and Fc (B), wherein ScFab (B) comprises VLB, CLB, VHB and CH1B as shown in SEQ ID NOs:16, 17, 20, 18 and 63, respectively; the Fc (B) comprises the CH2BCH3B segment in HCB as shown in SEQ ID NO:13, and at least one embodiment is the bispecific antibody BT470 of the present invention disclosed herein.

[0265] The properties of EGFR / c-Met bispecific antibodies having conservative substitutions and / or additional substitutions in their Fc region can be tested using the methods described herein.

[0266] In certain embodiments described herein, the immune effector properties of the EGFR / c-Met bispecific antibody can be enhanced or silenced by Fc engineering using techniques known to those skilled in the art. For example, Fc effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc., can be provided and / or controlled by modifying residues in the Fc that contribute to these activities.

[0267] The ability of monoclonal antibodies to induce ADCC can be enhanced by engineering their oligosaccharide components. Human IgG1 or IgG3 is N-glycosylated at Asn297 with most glycans in the biantennary G0, G0F, G1, G1F, G2, or G2F forms, for example, well known in the art. Antibodies produced by non-engineered CHO cells typically have a glycan fucose content of about at least 85%. Removal of core fucose from the biantennary complex-type oligosaccharide attached to the Fc region enhances the ADCC of the antibody via improved FcγRIIIa binding without altering antigen binding or CDC activity. The binding arms of such antibodies can be achieved using various methods that result in the successful expression of relatively highly defucosylated antibodies with biantennary complex-type Fc oligosaccharides, such as control of culture osmolarity, use of a variant CHO line Lec13 as a host cell line, use of a variant CHO line EB66 as a host cell line, use of a rat hybridoma cell line YB2 / 0 as a host cell line, introduction of small interfering RNA specific for the α1,6-fucosyltransferase (FUT8) gene, or co-expression of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or the potent α-mannosidase I inhibitor kifunine. An exemplary embodiment of the EGFR / c-Met bispecific antibody of the present invention is to use engineered CHO-K1 cells (exemplarily designated FUT8-17 cells) modified with the α1,6-fucosyltransferase (FUT8) gene using gene editing technology to produce the EGFR / c-Met bispecific antibody of the present invention with enhanced ADCC.

[0268] In some embodiments described herein, ADCC elicited by EGFR / c-Met bispecific antibodies can also be enhanced by certain substitutions in the antibody Fc.

[0269] In the present application, "low fucose" or "low fucose content" refers to an antibody with a fucose content between about 1% and 15%.

[0270] In the present application, "normal fucose" or "normal fucose content" refers to an antibody having a fucose content of about more than 50%, usually about more than 80% or more than 85%.

[0271] Biological function

[0272] The c-Met / EGFR bispecific antibodies of the aforementioned aspects of the present invention have the following biological functions:

[0273] (1) The binding arm A specifically binds to domain III of human EGFR, thereby blocking the binding of EGF to EGFR; the binding arm B specifically binds to human c-Met, thereby blocking the binding of HGF to c-Met; wherein, the KD of the c-Met binding arm binding to c-Met may be at least 10 times smaller than the Kd of the EGFR binding arm binding to EGFR.

[0274] (2) The humanized bispecific antibody inhibits ligand EGF-mediated EGFR autophosphorylation and ligand HGF-mediated c-Met autophosphorylation in non-small cell lung cancer HCC827;

[0275] (3) It can inhibit the growth and proliferation of human tumor cells and has a synergistic effect. Its inhibitory effect on the proliferation of EGFR and c-Met double-positive tumor cells is stronger than the combination of two targeted monoclonal antibodies.

[0276] (4) has reduced fucose content, mediating antibody-dependent cellular cytotoxicity (ADCC);

[0277] (6) mediate antibody-dependent phagocytosis (ADCP);

[0278] (7) Mediate the internalization of EGFR and c-Met target proteins;

[0279] (8) Specific binding to cynomolgus monkey c-Met and EGFR proteins;

[0280] (9) It has significant therapeutic effect on tumors, with a TGI higher than 60%.

[0281] In a preferred embodiment, the EGFR-specific variable domain of the bispecific antibody of the present invention inhibits the phosphorylation of Y1068 of EGFR.

[0282] In a preferred embodiment, the c-Met-specific variable domain of the bispecific antibody of the present invention inhibits the phosphorylation of Y1234 / Y1235 of c-Met.

[0283] In the determination of EGFR autophosphorylation levels, the TR-FRET ratio (665 nm absorbance / 615 nm absorbance) measured with the addition of the c-Met / EGFR bispecific antibody of the present invention was significantly reduced, almost equivalent to the TR-FRET ratio measured without the addition of the bispecific antibody of the present invention. The phosphorylation level of EGFR in the assay sample containing the c-Met / EGFR bispecific antibody of the present invention was close to the phosphorylation level of EGFR mediated by the absence of ligand EGF. The c-Met / EGFR bispecific antibody of the present invention caused a reduction of EGFR autophosphorylation (P-EGFR (Y1068)) in HCC827 cells by no less than 90%, and the inhibitory strength was superior to that of Evantor.

[0284] In an assay of c-Met autophosphorylation levels, the TR-FRET ratio (665 nm absorbance / 615 nm absorbance) measured with the addition of the bispecific antibody of the present invention was significantly reduced relative to the TR-FRET measured without the addition of the bispecific antibody of the present invention. The phosphorylation level of c-Met in the assay sample containing the c-Met / EGFR bispecific antibody of the present invention approached the phosphorylation level mediated by HGF in the absence of ligand. The c-Met / EGFR bispecific antibody of the present invention caused a reduction of c-Met autophosphorylation (Pc-Met(Y1234 / Y1235)) in HCC827 cells by at least 90%, and exhibited superior inhibitory strength to that of Evantor.

[0285] In some embodiments, the activity of the c-Met / EGFR bispecific antibodies of the present invention in inhibiting EGFR autophosphorylation (P-EGFR (Y1068)) is ranked from high to low preferably as BT470>Evantor>BT461.

[0286] In some embodiments, the activity of the c-Met / EGFR bispecific antibody of the present invention in inhibiting c-Met autophosphorylation (Pc-Met (Y1234 / Y1235)) is preferably BT470>BT461>BT468>Evantor from high to low.

[0287] In one embodiment, the bispecific antibodies of the invention inhibit tumor cell growth and proliferation.

[0288] In one embodiment, the bispecific antibodies of the invention inhibit tumor cell growth and proliferation, and cell viability is reduced to about 60% of the viability of cells in the absence of antibody inhibition.

[0289] In some exemplary embodiments, the c-Met / EGFR bispecific antibodies BT461, BT468, and BT470 of the present invention significantly inhibit the proliferation of human tumor cells KP4, A549, MKN45, and HCC827 at multiple gradient concentrations, demonstrating comparable or even stronger inhibitory activity than the marketed drug Avanta. In some embodiments, the tumor cells whose proliferation is inhibited by the c-Met / EGFR bispecific antibodies of the present invention include, but are not limited to, H292, H1975, HCC827, or D773_V774insHBa / F3 cells.

[0290] In some embodiments, the bispecific antibodies of the present invention specifically bind to human c-Met protein with high affinity.

[0291] In some preferred embodiments, the bispecific antibodies of the present invention bind to human EGFR-HIS with an equilibrium dissociation constant (KD) (kd / ka) (AHC2 sensor) of no greater than 3E-8, ​​or no greater than 2E-8, or no greater than 9.20E-9, or no greater than 5.0E-9, or no greater than 3.6E-9. An exemplary antibody is BT470. In some preferred embodiments, the bispecific antibodies of the present invention specifically bind to human c-Met protein with a KD (M) (AHC2 sensor) of no greater than 5E-8, or no greater than 2E-8, no greater than 5E-9, or no greater than 2E-9, or no greater than 1E-9, or no greater than 8E-10, or no greater than 5E-10. An exemplary embodiment is BT470.

[0292] In some embodiments, the bispecific antibody of the present invention has specific binding activity that specifically targets EGFR and c-Met antigens, and blocking activity that inhibits the binding of the ligand EGF to EGFR and blocks the binding of the ligand HGF to c-Met.

[0293] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention has an extracellular c-Met binding EC50 of no higher than 40 ng / mL by ELISA, preferably no higher than 30 ng / mL, more preferably no higher than 25 ng / mL, even more preferably no higher than 20 ng / mL, even more preferably no higher than 15 ng / mL, and most preferably no higher than 8 ng / mL. Exemplary embodiments include BT470 and BT468.

[0294] In some embodiments, the c-Met / EGFR bispecific antibody of the present invention binds to cell surface c-Met with a FACS binding EC50 of no higher than 1000 ng / mL, preferably no higher than 800 ng / mL, more preferably no higher than 600 ng / mL, even more preferably no higher than 500 ng / mL, even more preferably no higher than 400 ng / mL, even more preferably no higher than 300 ng / mL, even more preferably no higher than 200 ng / mL, and most preferably no higher than 150 ng / mL. Exemplary embodiments include BT470 and BT468.

[0295] In some embodiments, the bispecific antibodies of the present invention block the specific binding of the ligand HGF to the human c-Met protein.

[0296] In some preferred embodiments, the bispecific antibodies of the present invention have an ELISA blocking IC50 of 600 ng / mL or less, preferably 500 ng / mL or less, more preferably 400 ng / mL or less, even more preferably 300 ng / mL or less, and most preferably 200 ng / mL or less, for antagonizing the binding of the ligand HGF to the human c-Met protein. Examples of these include BT470 and BT468.

[0297] In some embodiments, the c-Met / EGFR bispecific antibody of the present invention antagonizes the binding of the ligand HGF to c-Met by FACS blocking IC50 of no higher than 17,000 ng / mL, preferably no higher than 15,000 ng / mL, more preferably no higher than 10,000 ng / mL, more preferably no higher than 8,000 ng / mL, more preferably no higher than 3,000 ng / mL, more preferably no higher than 2,000 ng / mL, and most preferably no higher than 1,500 ng / mL. Exemplary antibodies include BT470 and BT468.

[0298] In some exemplary embodiments, the c-Met / EGFR bispecific antibodies of the present invention inhibit c-Met autophosphorylation at levels ranging from high to low, preferably BT470 and BT468, and most preferably BT470.

[0299] In some embodiments, the c-Met / EGFR bispecific antibody of the present invention has an extracellular ELISA binding EC50 of EGFR protein of no higher than 100 ng / mL, preferably no higher than 90 ng / mL, more preferably no higher than 80 ng / mL, even more preferably no higher than 70 ng / mL, even more preferably no higher than 50 ng / mL, and most preferably no higher than 35 ng / mL. Some exemplary embodiments include BT470 and BT468.

[0300] In some embodiments, the c-Met / EGFR bispecific antibody of the present invention binds to cell surface EGFR membrane protein with a FACS binding EC50 of no higher than 2000 ng / mL, preferably no higher than 1500 ng / mL, more preferably no higher than 1000 ng / mL, more preferably no higher than 800 ng / mL, more preferably no higher than 600 ng / mL, even more preferably no higher than 400 ng / mL, and even more preferably no higher than 300 ng / mL. An exemplary embodiment is BT470 and BT468.

[0301] In some embodiments, the c-Met / EGFR bispecific antibody of the present invention preferably has the binding activity to EGFR as BT468 and BT470, more preferably BT468, and most preferably BT468.

[0302] In some embodiments, the c-Met / EGFR bispecific antibody of the present invention blocks EGF ligand binding to EGFR with an ELISA blocking IC50 of no greater than 2500 ng / mL, preferably no greater than 2000 ng / mL, more preferably no greater than 1500 ng / mL, even more preferably no greater than 1000 ng / mL, and even more preferably no greater than 800 ng / mL. Further preferably, no greater than 600 ng / mL, and most preferably no greater than 500 ng / mL. Exemplary embodiments include BT4700 and BT468.

[0303] In some embodiments, the c-Met / EGFR bispecific antibody of the present invention blocks EGF ligand binding to cell membrane surface EGFR protein with a FACS blocking IC50 of no higher than 8000 ng / mL, preferably no higher than 7000 ng / mL, more preferably no higher than 6000 ng / mL, more preferably no higher than 5000 ng / mL, even more preferably no higher than 4000 ng / mL, even more preferably no higher than 3000 ng / mL, even more preferably no higher than 2000 ng / mL, and most preferably no higher than 1500 ng / mL. Exemplary embodiments such as BT4680 and BT470

[0304] In some preferred embodiments, the EGFR / c-Met bispecific antibody can specifically bind to double-positive cells.

[0305] In some preferred embodiments, the EGFR / c-Met can specifically bind to the Shuangyang cell line MKN45.

[0306] In some preferred embodiments, the EGFR / c-Met bispecific antibody binds to Shuangyang MKN45 cells with a FACS binding EC50 of no higher than 1200 ng / mL, preferably no higher than 1000 ng / mL, or preferably no higher than 900 ng / mL, or preferably no higher than 800 ng / mL, or preferably no higher than 600, or no higher than 400 ng / mL, or no higher than 360 ng / mL. In an exemplary embodiment, the EGFR / c-Met antibody, such as BT470, binds to Shuangyang MKN45 cells with a FACS binding EC50 of no higher than 360 ng / mL.

[0307] In some embodiments, the EGFR / c-Met bispecific antibodies of the present invention that are active in binding to EGFR and c-Met double positive cells are preferably BT468 and BT470, more preferably BT468 and most preferably BT468.

[0308] In some embodiments, the EGFR-binding arm and the c-Met-binding arm of the EGFR / c-Met bispecific antibody of the present invention bind to EGFR / c-Met double-positive tumor cells at a level comparable to that of ervantuzumab.

[0309] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention has an antagonistic ability to antagonize the binding of the ligand HGF to c-Met and antagonize the binding of EGF to EGFR at a level comparable to that of ervantuzumab.

[0310] In some preferred embodiments, the EGFR / c-Met bispecific antibody of the present invention blocks the specific binding of the ligands HGF and EGF to the double positive cell tumor cells. In one embodiment, the tumor cells are MKN cells.

[0311] In some preferred embodiments, the EGFR / c-Met bispecific antibody of the present invention blocks the specific binding of the ligand EGF to Shuangyang MKN45 cells by FACS blocking IC50 of no more than 3500 ng / mL. In some preferred embodiments, the EGFR / c-Met bispecific antibody blocks the specific binding of the ligand EGF to Shuangyang MKN45 cells by ELISA blocking IC50 of no more than 3000 ng / mL. In some preferred embodiments, the EGFR / c-Met bispecific antibody of the present invention blocks the specific binding of the ligand HGF to Shuangyang MKN45 cells by ELISA blocking IC50 of no more than 2500 ng / mL. In some preferred embodiments, the EGFR / c-Met bispecific antibody of the present invention blocks the specific binding of the ligand EGF to Shuangyang MKN45 cells by ELISA blocking IC50 of no more than 2000 ng / mL. In some preferred embodiments, the EGFR / c-Met bispecific antibody of the present invention blocks the specific binding of the ligand EGF to Shuangyang MKN45 cells by ELISA blocking IC50 of no more than 1500 ng / mL. In one exemplary preferred embodiment, the EGFR / c-Met antibody of the present invention blocks the specific binding of the ligand EGF to the Shuangyang MKN45 cell line by an ELISA blocking IEC50 of no greater than 1000 ng / mL. In one exemplary embodiment, the EGFR / c-Met bispecific antibody, such as BT470, blocks the specific binding of the ligand EGF to the Shuangyang MKN45 cell line by a FACS blocking IC50 of no greater than 900 ng / mL.

[0312] In some preferred embodiments, the EGFR / c-Met bispecific antibody blocks the specific binding of the ligand HGF to the Shuangyang MKN45 cells by an ELISA blocking IC50 of no more than 7000 ng / mL. In some preferred embodiments, the EGFR / c-Met bispecific antibody blocks the specific binding of the ligand HGF to the Shuangyang MKN45 cells by an ELISA blocking IC50 of no more than 6000 ng / mL. In some preferred embodiments, the EGFR / c-Met bispecific antibody blocks the specific binding of the ligand HGF to the Shuangyang MKN45 cells by an ELISA blocking IC50 of no more than 5000 ng / mL. In some preferred embodiments, the EGFR / c-Met bispecific antibody blocks the specific binding of the ligand HGF to the Shuangyang MKN45 cells by an ELISA blocking IC50 of no more than 4000 ng / mL. In some preferred embodiments, the EGFR / c-Met bispecific antibody blocks the specific binding of the ligand HGF to the Shuangyang MKN45 cells by an ELISA blocking IC50 of no more than 3000 ng / mL. In some preferred embodiments, the EGFR / c-Met bispecific antibody blocks the specific binding of the ligand HGF to the Shuangyang MKN45 cells by ELISA with an IC50 of no greater than 2500 ng / mL. In some preferred embodiments, the EGFR / c-Met bispecific antibody blocks the specific binding of the ligand HGF to the Shuangyang MKN45 cells by ELISA with an IC50 of no greater than 2000 ng / mL. In one exemplary embodiment, the EGFR / c-Met bispecific antibody blocks the specific binding of the ligand HGF to the Shuangyang MKN45 cells by FACS with an IC50 of no greater than 2500 ng / mL, such as BT470.

[0313] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention completely inhibits EGF-activated EGFR autophosphorylation and inhibits HGF-activated c-Met autophosphorylation.

[0314] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention inhibits HGF-activated c-Met phosphorylation, and the TR-FRET ratio measured using a c-Met phosphorylation (Y1234 / Y1235) assay kit is 36-38%, which is close to the negative control measurement value without HGF activation.

[0315] In some embodiments, the inhibitory activity of the EGFR / c-Met bispecific antibody of the present invention in inhibiting EGF-activated EGFR phosphorylation is tested using an EGFR phosphorylation (Y1068) assay kit, and the TR-FRET ratio is 26-29%, which is close to the negative control measurement value without HGF activation.

[0316] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention is an antibody in which some amino acids in the Fc region undergo de-fucose mutation.

[0317] In some embodiments, the c-Met / EGFR bispecific antibody comprising a defucosylating amino acid mutation of the present invention has a significant NK-mediated cytotoxic effect (ADCC effect) on target cells.

[0318] In some embodiments, the EGFR / c-Met bispecific antibodies with defucosylated amino acid mutations of the present invention mediate antibody-dependent cellular cytotoxicity (ADCC) at a level comparable to that of Avanta. In some embodiments, the EGFR / c-Met bispecific antibodies with defucosylated amino acid mutations of the present invention mediate antibody-dependent cellular cytotoxicity (ADCC) against H1975 tumor cells by natural killer (NK) cells. In some embodiments, the EGFR / c-Met bispecific antibodies with defucosylated amino acid mutations of the present invention mediate antibody-dependent cellular cytotoxicity (ADCC) against HCC827 cells by natural killer (NK) cells.

[0319] In some embodiments, the c-Met / EGFR bispecific antibodies of the present invention comprising a fucose amino acid mutation can mediate phagocytosis (ADCP) of tumor cells by phagocytes. In some embodiments, the phagocytes are human peripheral blood mononuclear cell lines. In some embodiments, the ADCP effect is mediated on H1975 cells. In some embodiments, the ADCP effect is mediated on HCC827 cells. Preferably, the c-Met / EGFR bispecific antibodies of the present invention comprising a fucose amino acid mutation can mediate antibody-dependent phagocytosis (ADCP) of HCC827 cells by the human peripheral blood mononuclear cell line THP-1. In some embodiments, the ADCP effect mediated by the EGFR / c-Met bispecific antibodies of the present invention is comparable to that of the drug Avanta.

[0320] In some embodiments, the EGFR / c-Met bispecific antibodies of the present invention are capable of inhibiting the proliferation of tumor cell lines.

[0321] In some embodiments, the EGFR / c-Met bispecific antibodies of the present invention can inhibit the proliferation of tumor cells, including but not limited to pancreatic cancer cell lines, non-small cell lung cancer cells, and gastric cancer cells, with comparable or even stronger inhibitory activity than the marketed drug Avanta. In one embodiment, the gastric cancer cells are MKN45 cells. In one embodiment, the non-small cell lung cancer cells include EGFR mutant cells HCC827-HGF (transfected to overexpress human HGF) and NCI-H1975-HGF (transfected to overexpress human HGF).

[0322] In some embodiments, the EGFR / c-Met bispecific antibodies of the present invention inhibit tumor cell growth in vitro at a concentration of 400-600 μg / mL. In some embodiments, the EGFR / c-Met bispecific antibodies of the present invention reduce the viability of MKN tumor cells to 50%-60% of the pre-inhibition level at a concentration of 400-600 μg / mL, compared to a 62-65% reduction in viability of MKN cells resulting from combined inhibition with the monoclonal antibody derived from the binding arm. In some embodiments, the EGFR / c-Met bispecific antibodies of the present invention reduce the viability of HCC827-HGF cells to 50%-60% of the pre-inhibition level at a concentration of 400-600 μg / mL, compared to a 60-70% reduction in viability of HCC827-HGF cells resulting from combined inhibition with the monoclonal antibody derived from the binding arm. In some embodiments, when the EGFR / c-Met bispecific antibody of the present invention is at a concentration of 400-600 μg / mL, the viability of NCI-H1975-HGF cell growth is reduced to 80% of the level before inhibition, compared to the combined inhibition of the monoclonal antibody derived from the binding arm, which causes the viability of NCI-H1975-HGF cell growth to decrease to 85% of the level before inhibition.

[0323] In some embodiments, the bispecific antibodies of the present invention exhibit a stronger inhibitory effect on tumor cell proliferation than EGFR monoclonal antibodies, c-Met monoclonal antibodies, or a combination of EGFR and c-Met monoclonal antibodies. In one exemplary embodiment, the bispecific antibodies of the present invention inhibit tumor cell growth, resulting in a decrease in tumor cell viability, which is lower than the viability of tumor cells inhibited by a control monoclonal antibody combination of JH001 and Onar. The bispecific antibodies of the present invention exhibit synergistic efficacy in inhibiting tumors in vitro.

[0324] In some embodiments, the bispecific antibodies of the present invention have a stronger inhibitory effect on the proliferation of gastric cancer and non-small cell lung cancer compared to EGFR monoclonal antibody, c-Met monoclonal antibody, or the combination of EGFR monoclonal antibody and c-Met monoclonal antibody, and have a synergistic effect in the in vitro tumor inhibition efficacy.

[0325] In some embodiments, the bispecific antibodies of the present invention can promote internalization of the target proteins EGFR and c-Met with greater efficiency than monoclonal antibodies against EGFR or c-Met; they also exhibit a synergistic effect relative to dual-target internalization mediated by monoclonal antibodies against EGFR, c-Met, or the combination of monoclonal antibodies against EGFR and c-Met. In some embodiments, preferred bispecific antibodies of the present invention with high levels of target protein internalization are BT470 and BT468.

[0326] In some embodiments, the bispecific antibodies of the present invention have therapeutic effects of inhibiting tumor growth in vivo.

[0327] In some embodiments, the bispecific antibodies of the present invention have therapeutic efficacy in inhibiting the growth of non-small cell lung cancer tumors.

[0328] In some embodiments, the bispecific antibodies of the invention inhibit non-small cell lung cancer tumor growth by TGI of 60%-90%.

[0329] In some embodiments, the bispecific antibodies of the invention inhibit the TGI of non-small cell lung cancer tumor growth by 70%-90%.

[0330] In some embodiments, the bispecific antibodies of the invention inhibit non-small cell lung cancer tumor growth by 80%-90% TGI.

[0331] In some embodiments, the bispecific antibodies of the invention inhibit non-small cell lung cancer tumor growth by a TGI of 85%-90%.

[0332] In some embodiments, the EGFR / c-Met bispecific antibody of the present invention has cross-binding activity to cynomolgus monkey EGFR and c-Met.

[0333] Nucleic acid, vector, host cell and method for preparing EGFR / c-Met bispecific antibody

[0334] In one aspect, the present invention provides an isolated nucleic acid comprising nucleic acids encoding the binding arm A and the binding arm B of the EGFR / c-Met bispecific antibody according to the aforementioned aspects.

[0335] In one embodiment, the present invention provides a nucleic acid encoding 1# encoding the EGFR / c-Met bispecific antibody described in the aforementioned aspects, wherein the nucleic acid 1# comprises a nucleic acid 2# encoding a binding arm A that specifically binds to EGFR and a nucleic acid 3# encoding a binding arm B that specifically binds to c-Met, wherein the nucleic acid 2# is a nucleic acid comprising a light chain A-linker-heavy chain A structure encoding the binding arm A or a variant thereof having 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, and the nucleic acid 3# is a nucleic acid comprising a light chain A-linker-heavy chain A structure encoding the binding arm A, and a variant thereof having 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof, and the nucleic acid 3# is a nucleic acid comprising a light chain B encoding the binding arm B, respectively. The nucleic acid of B-linker-heavy chain B structure or its variant having 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof; the nucleotide sequences 2# and 3# respectively comprise CH3 domain mutations (L234A, L235A, S354C, T366W) forming a Knob structure of heavy chain A (HCA) binding arm A, and CH3 mutations (L234A, L235A, Y349C, T366S, L368A, Y407V) forming a Hole structure of heavy chain B (HCB) binding arm B.

[0336] In a preferred embodiment, the nucleotide sequence encoding the binding arm of the EGFR / c-Met bispecific antibody is codon-optimized for the species and / or cell type of the antibody-producing cell. For example, for engineered Chinese hamster cell (CHO) production, the nucleic acid sequence of the molecule is codon-optimized for use in Chinese hamster cells.

[0337] In a preferred embodiment, nucleic acid encoding 2# is a nucleic acid encoding binding arm A that specifically binds to EGFR, and its nucleotide sequence is as shown in SEQ ID NO: 52. Nucleic acid encoding 3# is a nucleic acid encoding binding arm B that specifically binds to c-Met, and its encoding nucleotide sequence is as shown in SEQ ID NO: 53. At least one exemplary embodiment is BT470.

[0338] In a preferred embodiment, the encoding nucleic acid 2# is a nucleic acid encoding the binding arm A that specifically binds to EGFR, and its nucleotide sequence is shown in SEQ ID NO:52. The encoding nucleic acid 3# is a nucleic acid encoding the binding arm B that specifically binds to c-Met, and its nucleotide sequence is shown in SEQ ID NO:54. At least one exemplary embodiment is BT468.

[0339] Among them, the 2# nucleic acid contains a codon-optimized nucleotide sequence encoding an amino acid combination of S354C and T366W that forms a knob structure, and the 3# nucleic acid contains a codon-optimized nucleotide sequence encoding an amino acid combination of Y349C, T366S, L368A, and Y407V that forms a hole structure.

[0340] In certain embodiments, the encoding nucleic acid comprises an encoding nucleic acid for producing a linker whose expression is beneficial for producing a bispecific antibody or a binding arm thereof (shown in SEQ ID NO: 62).

[0341] In some preferred embodiments, the nucleic acid molecule undergoes mutations that facilitate the expression of a codon-optimized amino acid sequence.

[0342] In a preferred embodiment, codon optimization is performed for the species and / or cell type of the antibody-producing cells.

[0343] In one aspect, the present invention provides methods for producing the EGFR / c-Met bispecific antibodies described in the aforementioned aspects. Bispecific antibodies can be produced using a variety of methods. In one embodiment, the production method involves expressing nucleic acids encoding two different light chain-linker-heavy chain fusion peptides in cells. The two fusion peptide expression products interact within the cells through the heavy chain Fc regions to form a KIH structure, thereby generating the EGFR / c-Met bispecific antibodies described herein. The antibodies produced in this manner will typically comprise dimers of binding arms of light chain and heavy chain pairs with two different binding specificities for EGFR and c-Met, respectively. The bispecific antibodies can then be purified from the culture product. The ratio of bispecific antibodies to other antibodies produced by the cells can be increased in a variety of ways.

[0344] In one embodiment, the method for preparing the aforementioned EGFR / c-Met bispecific antibody comprises the steps of: synthesizing nucleic acids 2# and 3# encoding binding arm A and binding arm B, respectively, constructing recombinant vectors 2 and 3 encoding nucleic acids expressing the EGFR binding arm A and c-Met binding arm B, respectively; generating recombinant vectors 1 carrying recombinant nucleic acids 1# of binding arm A and binding arm B by enzyme ligation; transfecting the recombinant vectors 1 into host cells, culturing the host cells to produce a fermentation product of the bispecific antibody of the present invention, and purifying the protein product produced by the cells to obtain the EGFR / c-Met bispecific antibody of the present invention. The binding arm A of the bispecific antibody may have the properties described above for the binding arm that binds EGFR, and the binding arm B of the bispecific molecule may have the properties described above for the binding arm that binds c-Met.

[0345] One embodiment provides a method for producing a bispecific antibody of the present invention from a single cell, wherein the bispecific antibody comprises two CH3 domains capable of forming an interface, the method comprising:

[0346] 1) Designing nucleic acid 2# encoding binding arm A and binding arm B as shown in SEQ ID NO: 52 and nucleic acid 3# encoding binding arm B as shown in SEQ ID NO: 53 or 54, respectively, and constructing recombinant expression vectors 1 and 2 of nucleic acid 2# and 3#, respectively;

[0347] 2) Enzymatically cleaving the linked nucleic acids 2# and 3#' to obtain a recombinant expression vector 3 containing the nucleic acid 1# encoding the binding arm A and the binding arm B;

[0348] 3) transforming a mammalian host cell with the recombinant expression vector 3 to obtain a recombinant mammalian expression cell 3 expressing nucleic acids 2# and 3#;

[0349] 4) fermenting and culturing the mammalian recombinant expression cells obtained in step 3);

[0350] 5) extracting and purifying the expression product of the mammalian recombinant expression cell 3 to obtain the EGFR / c-Met bispecific antibody of the present invention.

[0351] A nucleic acid encoding the EGFR / c-Met bispecific antibody described in the aforementioned aspects is provided in the cell; wherein nucleic acid molecules 2# and 3# provide nucleotide sequences encoding amino acid sequences for preferential pairing between the heavy chain containing the CH3A domain and the heavy chain containing the CH3B domain; the method further comprises culturing the host cell, allowing expression of the two nucleic acid molecules, and harvesting the bispecific antibody from the culture. Nucleic acids 2# and 3# can be provided separately or as part of the same nucleic acid molecule, vector, or gene delivery vector and can be integrated into the same site in the host cell genome, or nucleic acid molecules 2# and 3# can be provided separately to the cell.

[0352] The bispecific antibodies produced by these methods are also part of the present invention. The CH3 heterodimerization domain is preferably a human IgG1 heterodimerization domain. The heavy chain constant region comprising the CH3 heterodimerization domain is preferably an IgG1 constant region.

[0353] The bispecific antibodies of the present invention disclosed herein are heterodimers formed by two binding arms A and B, each of which is a fusion peptide single chain formed by a light chain, a linker, and a heavy chain, respectively, and formed by a KIH structure formed between mutated heavy chains. Preferably, the heterodimer is formed by a KIH structure formed between mutated heavy chain lower hinge regions.

[0354] In some embodiments, the bispecific antibodies of the present invention as disclosed herein are bispecific antibodies in which two binding arms A and B, each formed by a fusion peptide single chain connected by a hinge region, are formed by a KIH structure formed by interchain interaction between the heavy chain CH3 portion, preferably, a heterodimer formed by a KIH structure formed between CH2CH3 variants of the mutated heavy chain lower hinge region. The heavy chain is modified with CH3 to ensure efficient heterodimerization and bispecific antibody formation.

[0355] In one embodiment, the present invention provides a method for producing an antibody, comprising culturing a cell of the present invention as disclosed herein and harvesting the antibody from the culture. Preferably, the cells are cultured in a serum-free medium. Preferably, the cells are adapted for growth in suspension. Also provided are antibodies obtainable by the method for producing an antibody according to the present invention as disclosed herein. Preferably, the antibody is purified from the culture medium. Preferably, the antibody is affinity purified.

[0356] Nucleic acids 2# and 3# encoding the bispecific antibody binding arm A and binding arm B of the present invention can be prepared and assembled into complete single-stranded or double-stranded molecules by chemical synthesis (such as solid-phase polynucleotide synthesis on an automated polynucleotide synthesizer). Alternatively, the polynucleotides of the present invention can also be prepared by other techniques, such as conventional cloning followed by PCR. The techniques for preparing or obtaining polynucleotides with given known sequences are well known in the art. The polynucleotides of the present invention may contain at least one non-coding sequence, such as a promoter or enhancer sequence, an intron, a polyadenylation signal, a cis sequence that promotes RepA binding, and the like. The polynucleotide sequence may also contain additional sequences encoding additional amino acids, which encode, for example, a tag or label sequence, such as a histidine tag or HA tag to facilitate protein purification or detection; a signal sequence; a fusion protein partner such as RepA, Fc; or a phage coat protein such as pIX or pill. The nucleic acid molecules used in the present invention as disclosed herein are typically, but not absolutely, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Alternative nucleic acids can be used by those skilled in the art.

[0357] In one aspect, the present invention provides vectors comprising the aforementioned polynucleotides. Such vectors can be plasmid vectors, viral vectors, baculovirus expression vectors, transposon-based vectors, or any other vector suitable for introducing the polynucleotides of the present invention into a given organism or genetic background by any means. For example, the polynucleotides encoding the heavy and light chains of the bispecific antibodies of the present invention can be inserted into an expression vector. In one embodiment, the binding arm A comprising the EGFR-specific light chain, linker, and heavy chain can be cloned in a first expression vector, and the binding arm B comprising the c-Met-specific light chain, linker, and heavy chain can be cloned in a second expression vector. The DNA fragments encoding the binding arm A and the binding arm B can be operably linked as two protein expression units to control sequences in an expression vector that ensure the expression of the binding arm A and the binding arm B of the bispecific antibody of the present invention. Such control sequences include a signal sequence, a promoter (e.g., a naturally associated or heterologous promoter), an enhancer element, and a transcription termination sequence, and can be selected to be compatible with the host cell selected for antibody expression. Once the vector has been incorporated into an appropriate host, the host may be maintained under conditions suitable for high-level expression of the protein encoded by the incorporated synthetic polynucleotide.

[0358] In one embodiment, the present invention uses PEE6.4 and PEE12.4 expression vector systems to express antibody variants.

[0359] In one aspect, the present invention provides engineered host cells comprising an antibody according to the invention as disclosed herein and / or an engineered nucleic acid encoding the invention as disclosed herein. Bispecific antibodies can be produced by (transiently) transfecting a host cell with a plasmid encoding the antibody.

[0360] The nucleic acid examples disclosed herein are contained in cells. When the nucleic acid is expressed in the cells, the cells can produce antibodies according to the present invention as disclosed herein. Preferably, the cells (typically in vitro, isolated or recombinant cells) produce the antibodies.

[0361] It should be understood that the host cell is intended to refer not only to the specific subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may differ from the parent cell, but are still included within the scope of the term "host cell" as used herein. Such host cells can be eukaryotic cells, prokaryotic cells, or plant cells.

[0362] Exemplary eukaryotic cells can be mammals, insects, birds or other animal sources, more preferably mammalian cells, more preferably primate cells, most preferably human cells. Mammalian eukaryotic cells include immortalized cell lines, such as hybridoma or myeloma cell lines such as SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581). Other available cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHOK1SV (Lonza Biologics, Walkersville, MD), CHO-K1 (ATCC CRL-61). Cells of the present invention as disclosed herein are, for example, CHO cells, 293F cells, NS0 cells or PER-C6™ cells, or another cell type known for their suitability for antibody production for clinical purposes.

[0363] In a particularly preferred embodiment, the cells are CHO cells.

[0364] The present invention also provides cell cultures comprising the cells of the present invention as disclosed herein. Cell lines for large-scale production of antibodies have been developed, for example for clinical applications. A preferred embodiment includes the use of cell lines developed for large-scale production of antibodies for the production of antibodies of the present invention as disclosed herein, the cell lines preferably comprising cells for producing antibodies.

[0365] In one embodiment, the cell for producing the antibody of the present invention comprises a nucleic acid molecule encoding VLA as shown in SEQ ID NO: 5, VHA as shown in SEQ ID NO: 7, and / or encoding light chain A (LCA) as shown in SEQ ID NO: 1, heavy chain A (HCA) as shown in SEQ ID NO: 2; and further comprises a nucleic acid molecule encoding VLB as shown in SEQ ID NO: 16 or 27, VHB as shown in SEQ ID NO: 18 or 29, and / or encoding light chain LCB as shown in SEQ ID NO: 12 or 23, heavy chain B (HCB) as shown in SEQ ID NO: 13 or 24.

[0366] In a preferred embodiment, the cell for producing the antibody of the present invention comprises a nucleotide sequence encoding the binding arm A that specifically binds to EGFR as shown in SEQ ID NO:4, as shown in SEQ ID NO:52, and a nucleotide sequence encoding the binding arm B that specifically binds to c-Met as shown in SEQ ID NO:15, as shown in SEQ ID NO:53, and at least one exemplary embodiment is BT470. In a preferred embodiment, the cell for producing the antibody of the present invention comprises a nucleotide sequence encoding the binding arm A that specifically binds to EGFR as shown in SEQ ID NO:4, as shown in SEQ ID NO:52, and a nucleotide sequence encoding the binding arm B that specifically binds to c-Met as shown in SEQ ID NO:26, as shown in SEQ ID NO:54, and at least one exemplary embodiment is BT468.

[0367] In a particularly preferred embodiment, the host cell is a CHO cell, such as an expiCHO S cell. Preferably, the antibody variants are expressed using the PEE6.4 and PEE12.4 expression vector systems.

[0368] In an exemplary embodiment, the EGFR / c-Met bispecific antibody of the present invention can be produced by the following steps:

[0369] 1. Designing nucleic acid 2# encoding the binding arm A of the anti-EGFR antibody heavy chain containing mutations (S354C, T366W), forming a knob structure, and nucleic acid 3# encoding the binding arm B of the anti-c-Met antibody heavy chain containing mutations Y349C, T366S, L368A, and Y407V;

[0370] 2. The nucleotide sequences 2# and 3# of the nucleic acids encoding the two peptide chains of binding arm A and binding arm B were respectively constructed into the PEE6.4 and PEE12.4 expression vectors by SalI / NotI enzyme digestion and ligation; thus, the single-arm plasmids PEE6.4 containing binding arm B and PEE12.4 containing binding arm A were obtained respectively;

[0371] 3. The two plasmids were ligated with SalI / NotI enzyme (NEB) to obtain the PEE12.4 recombinant plasmid that co-expressed anti-c-Met and anti-EGFR;

[0372] 4. Transfect the recombinant PEE12.4 expression plasmid obtained in step 3, comprising two segments co-expressing the anti-c-Met binding arm B and the anti-EGFR binding arm A, into mammalian expression cells, such as CHO-K1 cells, to express the EGFR / c-Met bispecific antibody of the present invention;

[0373] 5. Purification of expressed protein Preferably, the antibody is affinity purified.

[0374] In some embodiments, the c-Met / EGFR bispecific antibody of the present invention produced by the preparation method comprises a fucose-free amino acid modification.

[0375] Removal of core fucose from the biantennary complex oligosaccharide attached to the Fc region enhances the antibody's ADCC via improved FcγRIIIa binding without altering antigen binding or CDC activity. Successful expression of relatively highly defucosylated antibodies with biantennary complex Fc oligosaccharides can be achieved using various methods, such as control of culture osmolarity, the variant CHO line Lec13, the rat hybridoma cell line YB2 / 0 as a host cell line, and the introduction of small interfering RNA specific for the α-1,6-fucosyltransferase (FUT8) gene. In one exemplary embodiment, small interfering RNA specific for the α-1,6-fucosyltransferase (FUT8) gene was introduced using gene editing technology to achieve relatively high expression levels of defucosylated antibodies.

[0376] As used in this application, "low fucose" or "low fucose content" refers to an antibody with a fucose content between about 1% and 15%.

[0377] As used herein, "normal fucose" or "normal fucose content" refers to an antibody having a fucose content of more than about 50%, typically more than about 80% or more than 85%.

[0378] Methods for evaluating anti-tumor levels

[0379] In one embodiment, the evaluation method comprises scoring the tumor proliferation inhibitory ability of the bispecific antibody based on the level of inhibition of c-Met autophosphorylation.

[0380] In one embodiment, the screening method is to use a (Y1234 / Y1235) assay kit to determine the level of c-Met / EGFR bispecific antibody inhibition of c-Met phosphorylation and autophosphorylation, wherein the c-Met phosphorylation inhibition ability is positively correlated with the anti-tumor ability of the bispecific vector.

[0381] In one embodiment, the screening method is to use an EGFR phosphorylation (Y1068) assay kit to determine the level of EGFR phosphorylation inhibition by the c-Met / EGFR bispecific antibody to be tested.

[0382] In one embodiment, the tumor includes non-small cell carcinoma, gastric cancer, and pancreatic cancer.

[0383] In one embodiment, the screening method is to determine the level of inhibition of the aforementioned bispecific antibody on the growth or viability of human tumor cells. In an exemplary embodiment, the tumor comprises non-small cell carcinoma.

[0384] In one embodiment, the inhibition level of EGFR autophosphorylation, the inhibition level of c-Met autophosphorylation, and the anti-tumor level of the bispecific antibody are positively correlated.

[0385] Pharmaceutical composition

[0386] In one aspect, the present invention provides a pharmaceutical composition comprising the aforementioned EGFR / c-Met bispecific antibody of the present invention and a pharmaceutically acceptable carrier.

[0387] The EGFR / c-Met bispecific antibodies of the present invention can be prepared as pharmaceutical compositions comprising an effective amount of a domain, molecule, or antibody as an active ingredient in a pharmaceutically acceptable carrier. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active compound 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.

[0388] In one aspect, the present invention provides an EGFR / c-Met bispecific antibody for use in treating EGFR and / or c-Met positive tumors.

[0389] In one aspect, the present invention provides an EGFR / c-Met bispecific antibody or a pharmaceutical composition comprising the same for use in treating EGFR and / or c-Met-positive tumors. The EGFR / c-Met bispecific antibody or a pharmaceutical composition comprising the same for therapeutic use can be administered by any suitable route of delivery of the agent to the host, such as parenteral administration, for example, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal, intravaginal, rectal); formulations in tablets, capsules, solutions, powders, gels, granules; and formulations contained in syringes, implantable devices, osmotic pumps, cartridges, mini-pumps; or other methods well known in the art as understood by a skilled artisan. Site-specific administration can be achieved, for example, by 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.

[0390] In one embodiment, the EGFR / c-Met bispecific antibody of the present invention can be administered parenterally by intravenous (IV) infusion or bolus injection, intramuscular injection, subcutaneous injection, or intraperitoneal injection.

[0391] Patients may be given an intravenous infusion for as little as 15 minutes, but more typically for 30 minutes, 60 minutes, 90 minutes, or even 2 or 3 hours. The bispecific EGFR / c-Met antibodies of the invention may also be injected directly into the site of the disease (e.g., the tumor itself). The dose administered to a patient suffering from cancer should be sufficient to alleviate or at least partially inhibit the disease being treated (a "therapeutically effective amount"), and may sometimes be 0.1 to 10 mg / kg body weight, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg, but may be even higher, such as 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg. A fixed unit dose, such as 50, 100, 200, 500, or 1000 mg, may also be administered, or the dose may be based on the surface area of ​​the patient, such as 400, 300, 250, 200, or 100 mg / m 2 Typically, 1 to 8 doses (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) can be administered to treat cancer, but 10, 12, 20, or more doses can be given. Administration of the EGFR / c-Met bispecific antibody of the present invention can be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months, or longer. Repeated courses of treatment are possible, as are long-term administrations. Repeated administrations can be with the same dose or with different doses.

[0392] In one embodiment, the pharmaceutical composition further comprises a second therapeutic agent. In one embodiment, the second therapeutic agent is a chemotherapeutic agent. In one embodiment, the second therapeutic agent is a targeted anticancer therapy.

[0393] The EGFR / c-Met bispecific antibody of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use. This technology has been proven to be effective for conventional protein preparation and can employ well-known lyophilization and reconstitution techniques.

[0394] The EGFR / c-Met bispecific antibody of the present invention can be administered simultaneously, sequentially or separately in combination with a second therapeutic agent.

[0395] The EGFR / c-Met bispecific antibodies of the present invention can be administered together with any one or more of chemotherapeutic agents or other anti-cancer therapeutic agents known to those skilled in the art. Chemotherapeutic agents are compounds useful for treating cancer and include growth inhibitors or other cytotoxic agents, and include alkylating agents, antimetabolites, antimicrotubule inhibitors, topoisomerase inhibitors, receptor tyrosine kinase inhibitors, angiogenesis inhibitors, and the like. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, poprosulfan and piposulfan; aziridines such as benzodopa, carboquinone, c-Meturedopa and uredopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, naphthyl mustard, cholephosphamide, estramustine, ifosfamide, mechlorethamine, chlorambucil hydrochloride, melphalan, nebiquinone, phenylephrine, pinene; chloramphenicol, trofosamide, uracil nitrogen mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclarubicin, dactinomycin, anthramycin, azaserine, bleomycin, actinomycin C, calicheamicin, carabicin, carminomycin, carmomycin, chromomycin, actinomycin D, daunorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, mexicomycin, mitomycin, mycophenolic acid, nogamycin, olivomycin, peplomycin, potfir oxazolidinone, ... antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; colcemid; diacrazone; elfornithine; elliptonium acetate; etoglucose; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguanidine; mitoxantrone; mopidarol; nitropropane; pentostatin; phenac-Met; pirarubicin;Podophyllic acid; 2-ethylhydrazide; procarbazine; razoxane; cisoflavone; spirogermanamine; tricholonone; triazoline; 2,2′,2″-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; a member of the taxane family, such as paclitaxel ( Docetaxel and its analogs; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novocycline; teniposide; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethyl Ornithine (DMFO); retinoic acid; esperamicins; capecitabine; inhibitors of receptor tyrosine kinases and / or angiogenesis, including sorafenib, sunitinib, pazopanib (VOTRIENT™), tocitanib (PALLADIA™), vandetanib (ZACTIMA™), cediranib (BAY73-4506), axitinib (AG013736), lestaurinib (CEP-701), erlotinib, gefitinib (IRESSA™), BIBW2992 (TOVOK™), lapatinib, neratinib (HKI-272), etc., and pharmaceutically acceptable salts, acids or derivatives of any of the above substances. And pharmaceutically acceptable salts, acids or derivatives of any of the above substances. Other conventional cytotoxic compounds (such as Wiemann et al., 1985, published in Medical Compounds such as those disclosed in Oncology (Calabresi et al., eds.), Chapter 10, McMillan Publishing) can also be used in the methods of the present invention.

[0396] In one embodiment, exemplary chemotherapeutic agents that can be used in combination with the EGFR / c-Met bispecific antibodies of the present invention include tyrosine kinase inhibitors and targeted anti-cancer therapies such as Gefitinib and Tarceva (erlotinib) and other antagonists of HER2, HER3, HER4 or VEGF.

[0397] When an appropriate treatment regimen for a second therapeutic agent has been established, that regimen can be used in combination with the EGFR / c-Met bispecific antibody of the present invention.

[0398] The EGFR / c-Met bispecific antibodies of the present invention, optionally in combination with a second therapeutic agent, can be administered in conjunction with any form of radiation therapy and / or surgery, including external beam radiation, intensity modulated radiation therapy (IMRT), and any form of radiosurgery, including Gamma Knife, CyberKnife, linear accelerators, and interstitial radiation (e.g., implanted radioactive seeds, GliaSite balloons). Combination with radiation therapy may be particularly useful for head and neck cancer and brain tumors.

[0399] The present invention particularly describes antibodies that can simultaneously target both EGFR and c-Met receptors and can potently inhibit the proliferation of cancer cell lines in vitro and inhibit tumor growth in vivo. The bispecific antibodies of the invention as disclosed herein can combine a low toxicity profile with high efficacy. The antibodies of the invention as disclosed herein can be used for the targeted treatment of various types of EGFR and / or c-Met-positive tumors. When compared to monoclonal antibodies that bind to the same antigen with both arms, either alone or in combination, the antibodies of the invention as disclosed herein can have an improved therapeutic window. When compared to the nimotuzumab antibody, the EGFR / c-Met bispecific antibodies of the invention as disclosed herein can exhibit superior growth inhibition in vitro, in vivo, or in combination.

[0400] Uses of the bispecific antibodies of the present invention

[0401] The EGFR / c-Met bispecific antibodies of the present invention can be used to diagnose, monitor, regulate, treat, alleviate, help prevent, or alleviate the symptoms of human diseases or specific pathologies that result in abnormal overexpression of EGFR and / or c-Met in cells, tissues, organs, tumors, or generally in a host. The methods of the present invention can be used to treat animal patients of any classification, preferably mammals, and more preferably humans.

[0402] One aspect of the present invention is to provide use of the bispecific antibody of the present invention for preparing a medicament for inhibiting the growth or proliferation of cells that abnormally overexpress EGFR and / or c-Met, wherein the method for administering the medicament comprises contacting patient cells with the isolated EGFR / c-Met bispecific antibody of the present invention.

[0403] In one embodiment, the method for administering the drug comprises administering to a subject an effective amount of a drug comprising the isolated EGFR / c-Met bispecific antibody of the present invention, such that the growth or metastasis of tumors or cancer cells expressing EGFR and / or c-Met is inhibited.

[0404] In one embodiment, the method of administering the medicament comprises administering a therapeutically effective amount of the isolated EGFR / c-Met bispecific antibody of the present invention to a patient in need thereof for a period of time sufficient to treat the cancer.

[0405] In one embodiment, the cancer is resistant to or has acquired resistance to treatment with one or more EGFR inhibitors.

[0406] In one embodiment, the subject may be resistant to, or have acquired resistance to, erlotinib, gefitinib, afatinib, CO-1686, AZD9192, or EKB-569 (pelitinib, an irreversible EGFR-TKI), pan-ErbB; and other receptor tyrosine kinase inhibitors, or the anti-EGFR antibody nimotuzumab.

[0407] In one embodiment, the method for determining whether the subject is resistant to EGFR inhibitor treatment, forms resistance to EGFR inhibitor treatment, or is susceptible to forming resistance to EGFR inhibitor treatment includes, for example, a decline or stabilization of the patient's health, an increase in tumor size, a stagnation or slowing of the decline in tumor growth, and / or the spread of cancer cells from one location to other organs, tissues, or cells in the body. The re-establishment or worsening of various symptoms associated with cancer may also indicate that the subject has formed or is susceptible to forming resistance to EGFR inhibitors, such as anorexia, cognitive dysfunction, depression, dyspnea, fatigue, hormonal imbalance, neutropenia, pain, peripheral neuropathy, and sexual dysfunction. Symptoms associated with cancer may vary depending on the type of cancer. A person skilled in the art of oncology can easily identify symptoms associated with a particular type of cancer.

[0408] In one embodiment, the other means for determining whether a subject has developed resistance to an EGFR inhibitor include examining EGFR phosphorylation, ERK1 / 2 phosphorylation, and / or AKT phosphorylation in cancer cells, wherein increased phosphorylation may indicate that the subject has developed or is susceptible to developing resistance to an EGFR inhibitor. Identification of subjects who have developed resistance to an EGFR inhibitor may involve detecting elevated c-Met expression levels or increased c-Met activity, such as caused by elevated levels of circulating HGF, activating mutations in the c-Met gene, or c-Met gene amplification.

[0409] In one embodiment, the cancer is associated with EGFR mutation, residue deletion, EGFR amplification, or c-Met amplification or mutation.

[0410] In one aspect, the present invention provides EGFR / c-Met bispecific antibodies that can be used to treat any disease or disorder characterized by aberrant activation or production of EGFR, c-Met, EGF, soluble EGFR, soluble c-Met or other EGFR ligands, or HGF, or a disorder associated with EGFR or c-Met expression, which may or may not involve malignancy or cancer, wherein aberrant activation and / or production of EGFR, c-Met, EGF or other EGFR ligands, or HGF occurs in cells or tissues of a subject suffering from or susceptible to the disease or disorder.

[0411] An EGFR / c-Met bispecific antibody of the present invention comprises a binding arm A and a binding arm B, and can be used to treat tumors, including cancer and benign tumors.

[0412] In some embodiments, exemplary cancers suitable for treatment by the EGFR / c-Met bispecific antibodies of the present invention include cancers that overexpress EGFR and / or c-Met and are associated with increased EGFR activity and / or expression levels (such as, EGFR activating mutations, EGFR gene amplification, or ligand-mediated EGFR activation) and increased c-Met activity and / or expression levels (such as, c-Met activating mutations, c-Met gene amplification, or HGF-mediated cc-Met activation, or mutant KRAS).

[0413] In some embodiments, exemplary EGFR activating mutations that may be associated with cancer include point mutations, deletion mutations, insertion mutations, inversions, or gene amplifications, which result in an increase in at least one biological activity associated with EGFR, such as increased tyrosine kinase activity, the formation of receptor homodimers and heterodimers, enhanced ligand binding, etc. The mutation may be located in any part of the EGFR gene or in a regulatory region associated with the EGFR gene, and include mutations in exons 18, 19, 20, or 21, or mutations in the kinase domain. Exemplary activating EGFR mutations are G719A, L861X (X is any amino acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, L858P or T790M substitutions, the deletion of E746-A750, the deletion of R748-P753, the insertion of Ala between M766 and A767, the insertion of SVA (Ser, Val, Ala) between S768 and V769, and the insertion of NS (Asn, Ser) between P772 and H773. Other examples of EGFR activating mutations are known in the art (see, e.g., U.S. Patent Publication US2005 / 0272083). Information about EGFR and other ErbB receptors, including receptor homodimers and heterodimers, receptor ligands, autophosphorylation sites, and signaling molecules involved in ErbB-mediated signaling, is known in the art (see, e.g., Hynes and Lane, Nature Reviews Cancer 5:341-354, 2005).

[0414] Methods for detecting EGFR and c-Met mutations or gene amplifications are well known in the art.

[0415] In another embodiment of the present invention, the patient has an NSCLC tumor or tumor metastasis with an activating EGFR mutation or EGFR gene amplification, and the method of administering the EGFR / c-Met bispecific antibody of the present invention to the patient comprises administering a therapeutically effective amount of the EGFR / c-Met bispecific antibody of the present invention to the patient.

[0416] In one aspect, the present invention further provides a method of treating a patient suffering from cancer, the method comprising administering to a patient in need thereof a therapeutically effective amount of the EGFR / c-Met bispecific antibody of the present invention for a period of time sufficient to treat the cancer, wherein the cancer is associated with EGFR mutation, EGFR amplification, or c-Met amplification.

[0417] In some embodiments, the cancer is associated with wild-type EGFR and wild-type c-Met.

[0418] In some embodiments, the cancer is associated with wild-type EGFR and c-Met amplification.

[0419] In some embodiments, the patient has NSCLC associated with EGFR amplification and wild-type c-Met.

[0420] In some embodiments, the patient has NSCLC associated with EGFR amplification and c-Met amplification.

[0421] In some embodiments, patients are treated with the EGFR / c-Met bispecific antibodies of the present invention. When the tumor is associated with EGFR amplification, wild-type c-Met, and / or c-Met amplification, the EGFR / c-Met bispecific antibodies of the present invention show therapeutic efficacy in in vivo tumor animal models.

[0422] In one embodiment, the EGFR / c-Met bispecific antibodies of the present invention can be used to prepare a medicament for treating non-small cell lung cancer (NSCLC), including squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. In some embodiments, the NSCLC cells have an epithelial phenotype. In some embodiments, the NSCLC has acquired resistance to treatment with one or more EGFR inhibitors.

[0423] In NSCLC, specific mutations in the EGFR gene are associated with high response rates (70-80%) to EGFR tyrosine kinase inhibitors (EGFR-TKIs). Although most NSCLC patients with EGFR mutations initially respond to EGFR TKI therapy, nearly all develop resistance that prevents a sustained response. Nearly 60% of all tumors resistant to EGFR tyrosine kinase inhibitors have increased c-Met expression, amplified c-Met genes, or increased expression of its only known ligand, HGF (Turke et al., Cancer Cell, 17:77-88, 2010).

[0424] The present invention also provides the use of an antibody of the present invention for the preparation of a medicament for treating a subject having a tumor or at risk of having such a tumor, wherein the method of administering the medicament comprises administering to a subject in need thereof a therapeutically effective amount of an antibody or pharmaceutical composition as disclosed herein. The tumor is preferably an EGFR single-positive tumor, a c-Met single-positive tumor, or an EGFR / c-Met double-positive tumor. Prior to initiating the treatment, the method preferably further comprises determining whether the subject has such an EGFR, c-Met, or EGFR / c-Met positive tumor.

[0425] The invention as disclosed herein is applicable to a wide range of cancers. Exemplary cancers suitable for treatment by the bispecific molecules of the invention, such as the EGFR / c-Met bispecific antibodies of the invention, include epithelial cell carcinoma, breast cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, small cell lung cancer, colorectal 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, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymic cancer, colon cancer, thyroid cancer, liver cancer (hepatocellular carcinoma (HCC)), or sporadic or hereditary papillary renal cell carcinoma (PRCC).

[0426] In an exemplary embodiment, the cancer is non-small cell lung cancer.

[0427] In an exemplary embodiment, the cancer is gastric cancer.

[0428] In an exemplary embodiment, the cancer is pancreatic cancer.

[0429] The present invention also provides the EGFR / c-Met bispecific antibody or pharmaceutical composition of the present invention as disclosed herein for use in treating a subject having or at risk of having an EGFR, c-Met or EGFR / c-Met positive tumor.

[0430] In a preferred embodiment, the present invention can be used to treat a subject having a tumor, preferably an EGFR / c-Met-positive cancer, preferably a tumor / cancer having an EGFR RTK-resistant phenotype, an EGFR monoclonal antibody-resistant phenotype, or a combination thereof. The subject is preferably a human subject. In one embodiment, the subject is preferably a subject suitable for antibody therapy using an EGFR-specific antibody, such as nimotuzumab.

[0431] EGFR-positive tumors are typically tumors that harbor EGFR-activating mutations. EGFR-activating mutations are EGFR mutations that result in activation of the EGF / EGFR signaling pathway. EGFR-activating mutations can be important in the cancerous state of a tumor. One way such tumors can become insensitive to EGFR-targeted therapies is through activation of the HGF / c-Met signaling pathway. The tumor can be an HGF-dependent tumor. Activation of the c-Met / HGF signaling pathway is one way EGFR-positive tumors can escape treatment with EGFR-targeted therapies. The c-Met / HGF pathway can be activated in a variety of ways. Various methods of activation have been described in the art, some of which are detailed herein. The antibodies of the invention as disclosed herein are particularly suitable for treating tumors in which activation of the c-Met / HGF signaling pathway is associated with the presence or excess of HGF. Such c-Met-positive tumors are referred to as HGF-dependent tumors or HGF-dependent tumors. The antibodies of the invention as disclosed herein can also be used to at least partially inhibit this potential escape mechanism of EGFR-positive tumors. Additionally, such tumors may escape EGFR-targeted therapy through the selective outgrowth of tumor cells in which the c-Met / HGF signaling pathway is activated. Such cells may be present at the onset of EGFR-targeted therapy. Such cells may have a selective growth advantage over HGF / c-Met signaling-negative tumor cells. The tumor may be one in which the HGF / c-Met signaling pathway is activated. The tumor may be associated with elevated levels of hepatocyte growth factor (HGF) or overexpression of the HGF receptor c-Met. The tumor may be one in which growth is driven by EGF and / or HGF. If a signaling pathway in tumor cells is activated in response to the presence of a growth factor, and removal of the growth factor results in inhibition of tumor cell growth, this indicates that the tumor is driven by a certain growth factor. This reduction can be measured by decreased cell division and / or induced cell killing, such as apoptosis. If, in the presence of HGF, the tumor grows or grows faster under conditions that would otherwise allow tumor growth, then the tumor is an HGF-associated tumor.

[0432] When an affinity is mentioned herein as being higher than another affinity, the Kd is lower than the other Kd. For the avoidance of doubt, a Kd of 10E-9 is lower than a Kd of 10e-8 M. An antibody with a Kd (M) of 10E-9 M has a higher affinity for its target than one with a Kd of 10E-8.

[0433] The citation of a patent document or other matter herein is not to be taken as an admission that the document or matter is known or that the information it contains was part of the common general knowledge as at the priority date of any claim.

[0434] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments; however, it should be understood that the scope of the invention as disclosed herein may include embodiments having a combination of all or some of the described features.

[0435] Also provided herein are articles of manufacture or kits comprising bispecific antibodies and immune cells. The articles of manufacture or kits may also include a package insert containing instructions for using the immune cells to treat cancer associated with aberrant EGFR or c-Met activation in an individual, delay cancer progression in an individual, or enhance immune function in an individual suffering from cancer. Any of the antigen-specific immune cells described herein may be included in the articles of manufacture or kits. Suitable containers include, for example, bottles, vials, bags, and syringes. Containers can be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or polyolefin, Hastelloy). In some embodiments, the container holds the formulation, and a label on or associated with the container may indicate instructions for use. The articles of manufacture or kits may also include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and a package insert with instructions for use. In some embodiments, the articles of manufacture further include one or more additional pharmaceutical agents (e.g., chemotherapeutic agents and anti-tumor agents). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0436] Specific embodiments

[0437] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0438] Experimental methods in the following embodiments where specific conditions are not specified were performed according to conventional conditions or the conditions recommended by the raw material or product manufacturers. Alternatively, experimental methods described in biotechnology textbooks such as Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory, Current Methods in Molecular Biology, and Cell Biology were used. Reagents where the source is not specified were purchased from commercial sources.

[0439] The reagents and raw materials used in the present invention are commercially available.

[0440] Example 1 Construction, expression and purification of human EGFR recombinant protein

[0441] 1.1 Construction of expression vector for human EGFR recombinant protein

[0442] In this example, an expression vector of EGFR-ECD-HIS (Gene ID: 1956, AA25-645, SEQ ID NO: 58, encoding nucleic acid SEQ ID NO: 59) was constructed, wherein the C-terminus of EGFR-ECD was connected to a his tag and cloned into the mammalian expression vector PHR (Ningbo Testo Biotechnology Co., Ltd., TSPLA10685) through restriction enzyme sites.

[0443] 1.2 Expression and purification of EGFR-ECD-HIS

[0444] Expansion: Adjust the cell density of expi293 cells (ThermoFisher) to 6-8×10 5 cell / ml, 1% culture, 37℃ 8% CO2 shaker culture for 24h, microscopic examination again, survival rate>95%, cell concentration at 1.2×10 6 cell / ml.

[0445] Transfection and EGFR-ECD-HIS protein expression: pHR-EGFR-ECD-HIS plasmid DNA encoding the EGFR-ECD extracellular domain protein was transiently transfected into expi293F cells (ThermoFisher, Catalog No. A30221) using the ExpiFectamine293 transfection kit (Gibco, Catalog No. A14524) as described by the manufacturer. The transfected Expi293 cells were cultured in expression medium for 7 days, and the cell supernatant was collected.

[0446] EGFR-ECD-HIS protein purification: The resulting cell supernatant was centrifuged (6000 × g, 10 mins) and filtered (using a 0.45 μm filter) for purification. Purification conditions were as follows: prepacked column: HIS-Excel prepacked column (GE, 17-3712-05), equilibrated with 10 column volumes of PBS buffer; instrument: AKTA; flow rate: 1 mL / min; contaminant protein eluent: PBS-10 imidazole; target protein eluent: PBS-500 imidazole; elution order: elution with contaminant protein eluent, followed by elution with target protein eluent. The resulting eluate was buffer exchanged with PBS through a 10 kDa ultrafiltration tube and then concentrated. The resulting protein was measured for concentration using a NanoDropOne micro-spectrophotometer and for purity using an HPLC-SEC system. The assay procedures were performed according to the NanoDropOne micro-spectrophotometer's instructions.

[0447] Example 2 Combination, Construction and Expression of Anti-c-Met / EGFR Bispecific Antibodies

[0448] 2.1 Obtaining the sequences of anti-EGFR binding arm A and anti-c-Met binding arm B:

[0449] (1) Sources of anti-c-Met binding arm B and its heavy and light chain sequences: The sources of anti-c-Met antibodies and their heavy and light chain sequences include Onartuzumab from Genentech and ABT-700 from Pierre Fabre. The amino acid sequences of the heavy and light chain segments of Onartuzumab are from patent application CN102942631A, and the amino acid sequences of the heavy and light chain segments of ABT-700 are from patent application CN103183739B. The variable region sequences constituting the antigen binding site of binding arm B are shown in Table 3. The assembled fragments, sequence numbers, and combinations of binding arm B are shown in Table 6. The sequences contained in binding arm B are shown in Table 8.

[0450] (2) Sources of the heavy and light chain sequences of the anti-EGFR binding arm A: The amino acid sequence selected was the humanized anti-EGFR monoclonal antibody JH001 (24H7) sequence developed by the applicant's team and Biotech Pharmaceuticals itself, of which the amino acid sequences of each segment of the heavy and light chains of JH001 were from patent application CN202010577007.5.

[0451] The variable region sequences that make up the antigen binding site of binding arm A are shown in Table 5 , the assembled fragments, sequence numbers, and combinations of binding arm A are shown in Table 6 , and the sequences contained in binding arm B are shown in Table 7 .

[0452] Table 7 Amino acid sequence of binding arm A

[0453] Table 8 Amino acid sequence of binding arm B

[0454] 2.2 Binding arm combinations of anti-c-Met / EGFR bispecific antibodies

[0455] The binding arm combinations of the anti-c-Met / EGFR bispecific antibody are shown in Table 1.

[0456] Table 1

[0457] A total of six anti-c-Met / EGFR antibodies were generated: BT459, BT460, BT461, BT468, BT469, and BT470. The assembled fragment sequences of the EGFR-binding arms and c-Met-binding arms of BT468 and BT470 are shown in Tables 7 and 8.

[0458] 2.3 Construction and expression of anti-c-Met / EGFR dual antibody

[0459] Bispecific antibody plasmid design: The construction of the anti-c-Met / EGFR antibody is based on Roche's KIH (knob into hole) technology. The CH3 domain of the anti-EGFR antibody heavy chain was mutated by amino acids (S354C, T366W) using genetic engineering technology to form a knob structure, and the CH3 of the anti-c-Met antibody heavy chain was mutated (Y349C, T366S, L368A, Y407V) to form a hole structure. The formation of heterodimers is guided by spatial complementarity and hydrophobic interactions. This group of mutations can increase interchain disulfide bonds and reduce homodimer impurities, which can effectively prevent heavy chain mispairing. The paired light and heavy chains are connected by a continuous (GGGGS)6 structure linker, solving the light chain mispairing problem of bispecific antibodies.

[0460] Construction of bispecific antibody expression plasmids: The nucleotide sequences encoding the two binding arm peptide chains of the anti-EGFR / c-Met bispecific antibody (1# and 2#, see Table 9) were constructed into PEE6.4 and PEE12.4 expression vectors, respectively, to obtain plasmid 1 expressing the anti-EGFR binding arm A and plasmid 2 expressing the anti-c-Met binding arm B, respectively. The two plasmids were then ligated with SalI / NotI enzymes (NEB), and the two nucleotide sequences were constructed into the same expression vector to obtain a recombinant PEE12.4 expression vector that co-expresses anti-c-Met and anti-EGFR.

[0461] Expression and purification of the bispecific antibody: pHR plasmid DNA encoding the bispecific antibody protein was transiently transfected into expiCHO S cells (Thermo Fisher) using the ExpiFectamine CHO Transfection Kit (Gibco) as described by the manufacturer. The transfected ExpiCHO-S cells were cultured in ExpiCHO-S expression medium for 10 days, and the cell supernatant was collected. The resulting cell supernatant (CHO cells, Chinese hamster ovary cells, ATCC, CRL-2092) was centrifuged (6000 × g, 10 min) and filtered (using a 0.45 μm filter) for purification. Purification conditions were as follows: Instrument: AKTA; Flow rate: 1 mL / min; Prepacked column: Hitrap MabSelect SuReProtein A (GE, #11-0034-93-GEC), equilibrated with 10 volumes of buffer 1 (PBS, Solebol, P1010, pH 7.4); Eluent A: buffer 1 (PBS, pH 7.4); Eluent B: buffer 3 (100 mM glycine, 150 mM NaCl, pH 3.0); Elution order: Elution with eluent A (approximately 30 mL) until the A280 value stabilized, followed by elution with eluent B. The eluate corresponding to the peak with an A280 value above 30 was collected, and 15 μL / mL of neutralization buffer (1 M Tris-HCl, pH 9.0) was added to the collection tube in advance to maintain the pH of the resulting antibody solution at 7.0-8.0. The antibody solution was buffer exchanged with PBS and concentrated using a 10KD ultrafiltration tube. The protein concentration was determined using Nanodrop and the purity was determined using HPLC-SEC.

[0462] BT459, BT460, BT468, BT469, BT470 and BT461 were expressed and purified using the above-mentioned eukaryotic cell expression vector expression and purification method, and BT468, BT470 and BT461 were further subjected to subsequent binding, blocking level determination, EGFR, c-Megt autophosphorylation inhibition level, ADCC, ADCP, inhibition of tumor cell growth and in vivo tumor treatment functional determination.

[0463] Table 9 Encoding nucleic acid of binding arm

[0464] Example 3 Preliminary Screening of Anti-c-Met / EGFR Bispecific Antibodies

[0465] 3.1 Inhibition of c-Met autophosphorylation

[0466] The c-Met phosphorylation (Y1234 / Y1235) assay kit from Bioauxilium (Cat. No. KIT-c-MetP-500, THUNDER TM Phospho-c-Met (Y1234 / Y1235) TR-FRET Cell Signaling Assay Kit) was used to detect the level of c-Met autophosphorylation. Non-small cell lung cancer HCC827 (Punosai #CL-0094) cells grown to 80% confluency were washed with PBS, and trypsin / EDTA (0.25%, Gibco, catalog number 25200-072) was added. After centrifugation, the cells were resuspended in complete culture medium RPMI1640 (10% FBS). The cells resuspended in culture medium (RPMI1640 / 10% FBS) were seeded in 96-well plates (costar catalog number 3599) (2×10 4 Cells were incubated at 37°C for 6 hours, washed with PBS, and placed in starvation medium (RPMI1640 / 1% FBS) (Gibco, catalog number 72400047) for 6 hours. The 96-well plates containing cells were divided into negative control groups and experimental groups. IgG1 (Sino Biologics HG1K) at a concentration of 5 μg / mL and the anti-c-Met / EGFR antibodies (BT459, BT460, BT461, BT468, BT469, and BT470) obtained in Example 2 were added to the wells of the negative control group and the wells of the experimental group, respectively. The cells were pre-incubated at 37°C for 6 hours. After 1 hour, 100 ng / mL HGF-his was added for stimulation for 15 minutes. The lysis buffer provided in the detection kit was then added (50 μL / well) and shaken (400 rpm) for 30 minutes. 18 μL of the mixture was transferred from each well to a white opaque plate (Costar, 3917). Detection buffer (6 μL) was then added. The reaction was incubated at room temperature in the dark for 1 hour. Centrifugation was performed at 1000 rpm for 2 minutes, and detection was performed using a TR-FRET microplate reader (PerkinElmer, Envision-2105). The absorbance at 665 nm represents the total number of phosphorylated c-Met proteins, while the absorbance at 615 nm represents the total number of c-Met proteins. The TR-FRET ratio is calculated by dividing the absorbance at 665 by the absorbance at 615, multiplied by 1000. The TR-FRET ratio is used to characterize the level of c-Met autophosphorylation. The specific test results are shown in Figure 1. BT459, BT460, BT461, BT468, BT469, and BT470 are all able to inhibit the level of c-Met autophosphorylation, and the order of their inhibitory abilities is: BT470>BT460>BT461=BT469>BT468=BT459.

[0467] 3.2 Inhibition of human tumor cell growth or viability

[0468] The digested cells H292 (EGFR wild type, ATCC#CRL-1848), H1975 (EGFR L858R / T790M mutation, ATCC#CRL-5908), HCC827 (EGFR19 exon deletion, Purnosai #CL-0094), D773_V774insHBa / F3 (EGFR20 exon insertion, Nanjing Kebai CBP73224) cells were seeded in 96-well plates (2000 cells / well) and cultured overnight in complete medium (RPMI1640+10% FBS); after removing the medium from the 96-well plates, 100 μL of IgG1, BT459, BT460, BT461, BT468, BT469, and BT470 diluted to a concentration of 250 μg / mL with complete cell culture medium were added, and after culturing in an incubator at a concentration of 5% CO2 and a temperature of 37°C for 72 h, an equal volume of 2% IgG1, BT459, BT460, BT461, BT468, BT469, and BT470 were added to each well. Reagent (Vazyme, Novozymes, Cat. No. DD1102) was added and shaken for 5 min until the cell clusters were completely lysed. After incubation for 25 min, 100 μL of the mixture was transferred to an opaque plate with a white bottom and detected using a TR-FRET microplate reader (PerkinElmer, model, cat. No.). The results are shown in Table 10.

[0469] Table 10 Proliferation inhibition ability score, the higher the score, the higher the proliferation inhibition ability, positive value means inhibition of proliferation, negative value means no inhibition of proliferation

[0470] The results are shown in Table 10. From the inhibition score, BT459 cannot inhibit the proliferation of H1975 cells, BT460 cannot inhibit the proliferation of H1975 and D773_V774insHBa / F3 cells, BT469 has a low inhibition on the proliferation of D773_V774insHBa / F3 cells (ranking), while BT461, BT468, and BT470 have a high level of proliferation inhibition on these four human tumor cells.

[0471] Example 4 Binding of the Antibody of the Present Invention to Human c-Met Target Protein

[0472] 4.1 ELISA-binding activity analysis

[0473] c-Met-HIS (Bipsys, c-Met-H5227) was diluted to 2 μg / mL using coating solution (8 ml of 0.2 mol / L NaCO3 and 17 ml of 0.2 mol / L NaHCO3, then 75 ml of distilled water and adjusted to pH 9.6). 50 μL / well of the coating solution was added to the ELISA plate and incubated at 37°C for 2 hours. Discard the liquid in the wells and wash five times using a plate washer with 200 μL of washing solution (PBST, PBS diluted with 0.05% Tween-20). Add 200 μL of blocking solution (PBST + 5% skim milk powder) to each well and incubate overnight at 4°C. Discard the liquid in the wells and wash five times using a plate washer (Tecan, Hydrospeed) with 200 μL of washing solution. Add 50 μL of 20 μg / mL anti-c-Met / EGFR dual antibody (BT461, BT468, BT470) in a 5-fold dilution series (7 levels), and set up a blank control. Incubate at 37°C for 60 min. Discard the remaining liquid in the wells and wash five times with 200 μL of washing buffer using a plate washer. Add 50 μL of Goat anti-human IgG-Fc secondary antibody (Jackson, 1:10,000 dilution) to each well and incubate for 60 min at 37°C. Discard the remaining liquid in the wells and wash five times with 200 μL of washing buffer using a plate washer. Add 50 μL of color development solution per well and incubate at 37°C for 15 min. Terminate the reaction with 50 μL of 2 mol / L H2SO4, and read the OD450 value on a microplate reader. ELISA binding activity analysis results are shown in Table 11 and Figure 2A.

[0474] 4.2 FACS-binding activity analysis

[0475] To further test the specificity of these antibodies binding to c-Met membrane protein on the cell surface, we stably overexpressed human c-Met membrane protein in CHO-K1 cells (hamster ovary cell substrain, ATCC#CCL-61) by plasmid electroporation and drug screening. The CHO-K1-c-Met cells overexpressing human c-Met were digested and counted, resuspended in a 96-well plate (3E5 cells were added to each well), and washed with PBS (twice); a blank control group and an experimental group were set up on the 96-well plate, and antibodies BT461, BT468, and BT470 (at a concentration of 20 μg / mL) were added to the wells of the experimental group and diluted into 7 gradients with a 5-fold concentration using a gradient dilution method; the 96-well plate was placed at 4°C for 30 minutes, centrifuged (1000 rpm, 2 minutes), and the wells were removed. The cells were washed twice with PBS (200 μL / well). 50 μL of PE-anti-human-IgG-Fc-Secondary Antibody (Jackson, 1:10,000 dilution) was added to each well and incubated at 4°C for 30 minutes. The cells were centrifuged at 1000 rpm for 2 minutes, and the liquid was removed. The cells were washed twice with PBS (200 μL / well). 200 μL of PBS was added to each well, and the cells were resuspended and analyzed by flow cytometry. The results of FACS binding activity analysis are shown in Table 11 and Figure 2B.

[0476] Table 11 Analysis of c-Met binding activity of the bispecific antibodies of the present invention

[0477] As can be seen from Table 11 and Figure 2, antibodies BT461, BT468, and BT470 can specifically bind to human c-Met protein, and the order of binding levels (the lower the EC50, the higher the binding) is BT470>BT461>BT468.

[0478] Example 5 Blocking activity on the binding of HGF to c-Met

[0479] 5.1 ELISA-blocking activity analysis

[0480] c-Met-hfc (Bepsis, product number c-Met-H5256) was coated with a coating solution (8 ml of 0.2 mol / L Na2CO3, 0.2 mol / L NaHCO3 17ml mixed, then add 75ml distilled water, adjust the pH to 9.6) diluted to 2μg / mL, and coated into the ELISA plate wells at a volume of 50μL / well, incubated at 37°C for 2hrs, removed the liquid and washed with washing solution (PBST, PBS diluent with 0.05% Tween-20) (200μL / time, 5 times); added 200μL blocking solution to each well, and incubated at 4°C overnight, removed the liquid and washed with washing solution (200μL / time, 5 times); set up a blank control group and an experimental group on the ELISA plate, added 25μL of HGF-HIS (Bipusys, product number HGF-H52H3) at a concentration of 200ng / mL and antibody BT4 at a concentration of 100μg / mL to the wells of the experimental group. 61, BT468, and BT470 were diluted into seven 5-fold gradients using a gradient dilution method and incubated at 37°C for 120 min. After removal of the liquid, the wells were washed with wash buffer (200 μL / well, five times). Goat anti-human his-secondary antibody (Jackson, Catalog No. 109-005-098, 1:10,000 dilution) was added to each well. After removal of the liquid, the wells were washed with wash buffer (200 μL / well, five times). The color development solution (50 μL / well) was added to each well. After incubation at 37°C for 15 min, the reaction was terminated by adding H2SO4 aqueous solution (50 μL / well, 2 mol / L), and the OD450 value was read on a microplate reader. The results of the ELISA blocking activity analysis are shown in Table 12 and Figure 3A.

[0481] 5.2 FACS-blocking activity analysis

[0482] CHO-K1-c-Met cells overexpressing c-Met were digested and counted, resuspended in a 96-well plate (3E5 cells were added to each well), and washed twice with PBS. A blank control group and an experimental group were set up on the 96-well plate. HGF-HIS (5 μg / mL, 25 μL) and bispecific antibodies BT461, BT468, and BT470 (100 μg / mL) were added to the wells of the experimental group and diluted into 7 gradients at 5-fold concentrations. The 96-well plate was incubated at 4°C for 30 mins and centrifuged (1000 rpm). The cells were washed twice with PBS (200 μL / well) after removing the liquid from the wells. 6xHis-Monoclonal-Antibody FITC (25 μL, Invitrogen, diluted 1:100) was added to each well and incubated at 4°C for 30 minutes. The cells were centrifuged at 1000 rpm for 2 minutes, removed the liquid, and washed twice with PBS (200 μL / well). 200 μL of PBS was added to each well, and the cells were resuspended and counted using a flow cytometer. FACS analysis results are shown in Table 12 and Figure 3B.

[0483] Table 12. Analysis of blocking activity on HGF binding to c-Met

[0484] As shown in Table 12 and Figure 3A, antibodies BT461, BT468, and BT470 all block the binding of the HGF ligand to the c-Met receptor, with the blocking ability ranking as BT470 > BT461 > BT468 (lower IC50, higher blocking ability). Furthermore, as shown in Table 12 and Figure 3B, at the same concentration, the blocking ability of antibody BT470 is stronger than that of BT468.

[0485] Example 6 Binding to human EGFR protein

[0486] 6.1 ELISA-Binding Activity Analysis

[0487] The EGFR-his prepared in Example 1 was diluted to 2 μg / mL using a coating solution (8 ml of 0.2 mol / L Na2CO3 and 17 ml of 0.2 mol / L NaHCO3, then 75 ml of distilled water and adjusted to pH 9.6). 50 μL / well of the coating solution was added to the ELISA plate and incubated at 37°C for 2 hours. Discard the liquid in the wells and wash five times using a plate washer with 200 μL of washing solution. Add 200 μL of blocking solution to each well and incubate at 4°C overnight. Discard the liquid in the wells and wash five times using a plate washer with 200 μL of washing solution. Add 50 μL of 20 μg / mL anti-c-Met / EGFR dual antibody (BT461, BT468, BT470) to each well in a 5-fold dilution series (7 levels). Set up two replicate wells for each gradient, along with a blank control. Incubate at 37°C for 60 min. Discard the remaining liquid from the wells and wash five times with 200 μL of wash buffer using a plate washer. Add 50 μL of Goat anti-human IgG-Fc secondary antibody (Jackson, Cat. No. 109-005-098, 1:10,000 dilution) to each well and incubate at 37°C for 60 min. Discard the remaining liquid from the wells and wash five times with 200 μL of wash buffer using a plate washer. Add 50 μL of color development solution to each well and incubate at 37°C for 15 min. The reaction was terminated by adding 50 μL / well of 2 mol / L H2SO4, and the OD450 value was read on a microplate reader. The results of the ELISA binding activity analysis are shown in Table 13 and Figure 4A.

[0488] 6.2 FACS-binding activity analysis

[0489] The specificity of these antibodies binding to the EGFR membrane protein on the cell surface was tested.

[0490] Following the same steps as in Example 1, human EGFR membrane protein was stably overexpressed in CHO-K1 cells by plasmid electroporation and drug addition screening. The CHO-K1-EGFR cells overexpressing human EGFR were digested and counted, resuspended in a 96-well plate (3E5 cells were added to each well), and washed twice with PBS. A blank control group and an experimental group were set up on the 96-well plate. Anti-c-Met / EGFR antibodies BT461, BT468, and BT470 (at a concentration of 20 μg / mL) were added to the wells of the experimental group and diluted into 7 gradients using a 5-fold concentration gradient method. The 96-well plate was incubated at 4°C for 30 minutes and centrifuged (1000 rpm for 2 minutes). After removing the liquid from the wells, the cells were washed twice with PBS (200 μL / well). 50 μL of PE-anti-human-IgG-Fc-Secondary Antibody (Jackson, 1:10,000 dilution) was added to each well and incubated at 4°C for 30 minutes. The cells were centrifuged at 1000 rpm for 2 minutes, and the liquid was removed. The cells were washed twice with PBS (200 μL / well). 200 μL of PBS was added to each well, and the cells were resuspended and analyzed by flow cytometry. The results of FACS binding activity analysis are shown in Table 13 and Figure 4B.

[0491] Table 13 Analysis of EGFR binding activity of the bispecific antibodies of the present invention

[0492] As can be seen from Table 13 and Figure 4, antibodies BT468 and BT470 can specifically bind to human EGFR protein, and the order of binding levels (the lower the EC50, the higher the binding) is BT468>BT461>BT470.

[0493] Example 7 Blocking Activity of EGF Binding to EGFR

[0494] 7.1 ELISA-Blocking Activity Analysis

[0495] EGF-mFC (Bepsis, product number EGF-H525b) was diluted to 5 μg / mL with coating solution (8 ml of 0.2 mol / L Na2CO3 and 17 ml of 0.2 mol / L NaHCO3 were mixed, and then 75 ml of distilled water was added to adjust the pH to 9.6). The mixture was coated onto the ELISA plate at a volume of 50 μL / well and incubated at 37°C for 2 hours. After removing the liquid, the plate was washed with washing solution (PBST, 0.05% Tween-20 was added to the PBS diluent) (200 μL / time, 5 times); 200 μL of blocking solution was added to each well, and the plate was incubated at 4°C overnight. After removing the liquid, the plate was washed with washing solution (200 μL / time, 5 times); a blank control group and an experimental group were set up on the ELISA plate, and EGF was added to the wells of the experimental group. R-his (25 μL, 4 μg / mL) and anti-c-Met / EGFR antibody were diluted into 7 gradients using a 5-fold concentration gradient method and incubated at 37°C for 120 min. After removing the liquid, the wells were washed with washing solution (200 μL / time, 5 times); Goat-anti-HIS-Fc-Secondary-Antibody (Jackson, 1:10000 dilution) was added to each well, and after removing the liquid, the wells were washed with washing solution (200 μL / time, 5 times); the color development solution (50 μL / well) was added to each well, and after incubation at 37°C for 15 min, H2SO4 aqueous solution (50 μL / well, 2 mol / L) was added to terminate the reaction, and the OD450 value was read on a microplate reader. The results of the ELISA-blocking activity analysis are shown in Table 14 and Figure 5A.

[0496] 7.2 FACS-blocking activity analysis

[0497] CHO-K1-EGFR cells overexpressing EGFR were digested and counted, resuspended in a 96-well plate (3E5 cells were added to each well), and washed twice with PBS. A blank control group and an experimental group were set up on the 96-well plate. EGF-mFC (2 μg / mL, 25 μL) and anti-c-Met / EGFR antibody (500 μg / mL) were added to the wells of the experimental group and diluted into 7 gradients according to 5-fold concentration. The 96-well plate was placed at 4°C for 30 minutes and centrifuged (1000 rpm, 2 minutes). The plate was then washed twice with PBS (200 μL / well). PE-anti-Mouse-IgG-Fc-Secondary Antibody (50 μL, Jackson, 1:200 dilution) was added to each well and incubated at 4°C for 30 minutes. The cells were centrifuged at 1000 rpm for 2 minutes, and the liquid was removed. The cells were washed twice with PBS (200 μL / well). PBS was added to each well (200 μL / well), and the cells were resuspended and counted using a flow cytometer. The results of the FACS binding activity analysis are shown in Table 14 and Figure 5B.

[0498] Table 14 Analysis of the blocking activity of the anti-c-Met / EGFR bispecific antibodies of the present invention on EGF binding to EGFR

[0499] As can be seen from Table 14 and Figure 5, antibodies BT461, BT468, and BT470 can all block the binding of the ligand EGF to the receptor EGFR, and the blocking levels (the lower the IC50, the higher the blocking ability) are ranked as BT468>BT461>BT470.

[0500] Example 8 Specific binding to EGFR / c-Met double positive tumor cells MKN45 cells

[0501] MKN45 cells (Shan En Biotech, catalog number SNL-173) that highly express EGFR and c-Met membrane proteins were digested and counted, resuspended in a 96-well plate (3E5 cells were added to each well), and washed twice with PBS. A blank control group and an experimental group were set up on the 96-well plate. Anti-c-Met / EGFR antibodies BT461, BT468, BT470, and a control (Avantuo, Johnson, batch number MAS0001) (at a concentration of 20 μg / mL) were added to the wells of the experimental group and diluted into 7 gradients with a 5-fold concentration using a gradient dilution method. The 96-well plate was incubated at 4°C for 30 minutes. s, centrifuged (1000 rpm, 2 mins), removed the liquid from the wells, and washed twice with PBS (200 μL / well). PE-anti-human-IgG-Fc-Secondary Antibody (Jackson, 1:10,000 dilution, 50 μL) was added to each well and incubated at 4°C for 30 min. Centrifuged (1000 rpm, 2 mins), removed the liquid, and washed twice with PBS (200 μL / well). PBS was added to each well (200 μL / well), the cells were resuspended, and read using a flow cytometer. The results of FACS binding activity analysis are shown in Table 15 and Figure 6.

[0502] Table 15 EC50 of the binding of the bispecific antibodies of the present invention to the double-positive cells

[0503] As can be seen from Table 15 and Figure 6, the binding of antibodies BT461, BT468, BT470, and Avanto to MKN45 is dependent on the antibody concentration gradient, and the order of binding levels (the lower the EC50, the higher the binding) is Avanto > BT470 > BT468 > BT461.

[0504] Example 9 Antagonizing the Binding of Ligands HGF and EGF to MKN45 Cells

[0505] Antagonizing the binding of EGF to MKN45 cells: Referring to the experimental procedure of Example 7.2, FACS detection was performed to detect the antagonism of the binding of the ligand EGF to MKN45 cells by the antibodies BT461, BT468, BT470 of the present invention and the control antibody Avanta. The results are shown in Table 16 and Figure 7A below.

[0506] Antagonizing the binding of HGF to MKN45 cells: Referring to the experimental procedure of Example 5.2, FACS detection was performed to detect the antagonism of the binding of the ligand HGF to MKN45 cells by the antibodies BT461, BT468, BT470 of the present invention and the control antibody Avanta. The results are shown in Table 16 and Figure 7B below.

[0507] Table 16 IC50 of the antibodies of the present invention blocking the binding of ligands EGF and HGF to double-positive cells

[0508] Example 10 The antibodies of the present invention inhibit EGFR autophosphorylation and c-Met autophosphorylation

[0509] Phosphorylation of EGFR and c-Met activates the same downstream signaling pathways, thereby promoting tumor cell proliferation, survival, invasion, and metastasis. To investigate whether an anti-c-Met / EGFR bispecific antibody could inhibit the c-Met and EGFR signaling pathways, we measured the levels of EGFR and c-Met autophosphorylation.

[0510] 10.1 Inhibition of EGF-activated EGFR phosphorylation

[0511] [Corrected 03.01.2024 according to rule 26] The EGFR phosphorylation (Y1068) assay kit from Bioauxilium (Cat. No. KIT-EGFRP-500, THUNDER TMEGFR autophosphorylation levels were detected using the Phospho-EGFR (Y1068) TR-FRET Cell Signaling Assay Kit. Non-small cell lung cancer HCC827 cells grown to 80% confluency were washed with PBS, incubated with trypsin / EDTA (0.25%, Gibco, Cat. No. 25200-072), centrifuged, and resuspended in RPMI1640 complete culture medium (10% FBS). The cells resuspended in culture medium (RPMI1640 / 10% FBS) were seeded into 96-well plates (manufacturer costar product number 3599) (1×104 cells / well), incubated at 37°C for 6 hours, washed with PBS, and placed in starvation medium (RPMI1640 / 1% FBS) (brand Gibco, product number 72400047) for 6 hours; the 96-well plates containing cells were divided into negative control groups and experimental groups, and IgG1 (Sino-Bio HG1K) at a concentration of 50 μg / mL and the anti-c-Met / EGFR antibody (BT4) obtained in Example 2 were added to the wells of the negative control group and the wells of the experimental group, respectively. 61, BT468, BT470); preincubate at 37°C for 1 hour, then stimulate with 10 ng / mL EGF-mFC for 15 minutes. Add the lysis buffer from the detection kit (50 μL / well) and shake (400 rpm) for 30 minutes. Transfer 18 μL of the mixture from each well to a white opaque plate (Costar, 3917), then add Detection Buffer (6 μL). Incubate at room temperature in the dark for 1 hour, centrifuge (1000 rpm) for 2 minutes, and detect using a TR-FRET microplate reader (PerkinElmer, Envision-2105). The absorbance at 665 nm represents the total number of EGFR phosphorylated proteins, and the absorbance at 615 nm represents the total number of EGFR proteins. The TR-FRET ratio is equal to the absorbance at 665 divided by the absorbance at 615, multiplied by 1000. The TR-FRET ratio is used to indicate the level of EGFR autophosphorylation. The specific test results are shown in Figure 8B. The EGFR-specific ligand EGF mediates the activation of strong EGFR autophosphorylation in HCC827 cells. Both JH001 and Taixinsheng antibodies can significantly inhibit the level of EGFR phosphorylation activated by EGF, while antibodies BT461 and BT470 almost completely retain the inhibitory function of their anti-EGFR antibody arms on EGF-activated EGFR phosphorylation. The order of inhibition of EGF-mediated pEGFR is: BT470>Evantuo BT461, and 468 has no inhibitory ability.

[0512] 10.2 Inhibition of HGF-activated c-Met phosphorylation

[0513] [Corrected 03.01.2024 according to rule 26] The c-Met phosphorylation (Y1234 / Y1235) assay kit from Bioauxilium (Cat. No. KIT-c-MetP-500, THUNDER TM Phospho-c-Met (Y1234 / Y1235) TR-FRET Cell Signaling Assay Kit) was used to detect the level of c-Met autophosphorylation. Non-small cell lung cancer HCC827 (Punosai #CL-0094) cells grown to 80% confluency were washed with PBS, and trypsin / EDTA (0.25%, Gibco, catalog number 25200-072) was added. After centrifugation, the cells were resuspended in complete culture medium RPMI1640 (10% FBS). The cells resuspended in culture medium (RPMI1640 / 10% FBS) were seeded in 96-well plates (costar catalog number 3599) (2×10 4 Cells were incubated at 37°C for 6 hours, washed with PBS, and incubated in starvation medium (RPMI1640 / 1% FBS) (Gibco, catalog number 72400047) for 6 hours. The 96-well plates containing cells were divided into negative control and experimental groups. IgG1 (Sino-Bio HG1K) and anti-EGFR / c-Met bispecific antibodies (BT461, BT468, and BT470) at a concentration of 50 μg / mL were added to the wells of the negative control and experimental groups, respectively. The cells were pre-incubated at 37°C for 1 hour, and then the cells were added. Cells were stimulated with 100 ng / mL HGF-his for 15 minutes, then the lysis buffer from the detection kit was added (50 μL / well) and shaken (400 rpm) for 30 minutes. 18 μL of the mixture was transferred from each well to a white opaque plate (Costar, 3917), followed by the addition of 6 μL of detection buffer. The cells were incubated at room temperature in the dark for 1 hour, centrifuged at 1000 rpm for 2 minutes, and assayed using a TR-FRET microplate reader (PerkinElmer, Envision-2105). The absorbance at 665 nm represents the total number of phosphorylated c-Met, while the absorbance at 615 nm represents the total number of c-Met proteins. The TR-FRET ratio is calculated by dividing the absorbance at 665 by the absorbance at 615, multiplied by 1000. This ratio is used to characterize the level of c-Met autophosphorylation. The specific test results are shown in Figure 8A. The c-Met-specific ligand HGF mediates and activates strong c-Met autophosphorylation in HCC827 cells. The antibodies BT468 and BT470 of the present invention can both significantly inhibit the level of c-Met autophosphorylation. The order of the ability to inhibit HGF-mediated c-Met is: BT470>BT461>BT468>Evantor.

[0514] Example 11 Evaluation of the efficacy of ADCC mediated by the antibody of the present invention

[0515] Using the CHO-K1 (FUT8 knockout, knocking out the FUT8 gene completely eliminates fucose from the antibody Fc terminus, thereby enhancing the ADCC activity of the antibody drug. Our company independently knocked out the FUT8 gene in CHO-K1 cells and named the host cells FUT8-17 cells) cell line, defucosylated anti-HGF / EGFR antibodies (BT468, BT470) were expressed according to the steps of Example 1 to detect whether the defucosylated antibodies BT468 and BT470 mediated antibody-dependent cellular cytotoxicity (ADCC).

[0516] 1. NK cell preparation: Thaw NK cells, resuspend in complete medium (1640 + 10% FBS), add hIL2 at a final concentration of 10 ng / mL and culture overnight (hIL2 stock concentration is 400 μg / mL, i.e. 1:40,000);

[0517] 2. Target Cell Labeling: Target cells were labeled using the PERKINELMER kit: Lysis buffer, 4005-0010; Eu-solution, C135-100; DELFIA BATDA reagent, C136-100. HCC827 cells were harvested from the logarithmic growth phase, washed once with culture medium, and resuspended to a cell density of 1E6 cells / mL. 1 mL of the suspension was then added with 1.7 μL of BATDA and incubated at 37°C for 30 min. The cells were then washed twice with PBS + 20 mM HEPES at 1100 rpm for 4 min each time.

[0518] 3. Target Cell Plating: Resuspend the labeled cells in 1640 complete medium (supplemented with 1mM sulfinpyrazone) and adjust the cell density to 1E5 cells / mL. Plate the cell suspension as shown in the table below, adding 100μL of cells per well to a V-bottom clear 96-well plate. For sulfinpyrazone preparation: Dissolve 404.48mg of sulfinpyrazone powder in 5mL of acetone to obtain 200mM sulfinpyrazone. Filter through a 0.22μM filter. The mixture will be a suspension, not completely dissolved.

[0519] 4. Antibody loading: Dilute the antibody to the corresponding concentration, from low to high concentration, and add 50 μL per well to the corresponding well plate; mix well and place at 37°C, 5% CO2, and incubate for 30 minutes;

[0520] 5. NK cell plating: Harvest NK cells and adjust the cell density to 2.5E5 cells / mL using 1640 complete medium (supplemented with 1 mM sulfinpyrazone). 100 μL was added to each well of G4-G23 (effector-target ratio 2.5:1). 250 μL was added to G1 well, 150 μL to G2 and G3 wells, and 50 μL of 1640 complete medium (supplemented with 1 mM sulfinpyrazone) was added to G4 well. 25 μL of lysis buffer (HBSS + 0.5% Triton X-100) was added to the maximum release well (G3). Incubate at 37°C, 5% CO2 for 4 h.

[0521] 6. TRF Assay: After the incubation period, remove the 96-well plate and equilibrate to room temperature. Centrifuge at 1500 rpm for 5 minutes. Aspirate 20 μL of supernatant into a PE-bottomed white plate. Add 200 μL of europium. Shake the plate in the dark for 15 minutes before measuring TRF. The assay program is: Delay (μm) 600, Window time 100, Number of flashes 200.

[0522] 7. Calculation of cell killing toxicity percentage (%): (TRF value of experimental group - TRF value of spontaneous pore) / (TRF value of maximum release pore - TRF value of spontaneous pore) * 100.

[0523] The results are shown in FIG9 . The antibodies BT461, BT468, and BT470 (without fucose) of the present invention significantly mediate NK cell cytotoxicity (ADCC) on target cells, and the ADCC effect mediated by them is slightly lower than that of Avanta.

[0524] Example 12 Antibodies of the present invention mediate antibody-dependent phagocytosis (ADCP)

[0525] The ADCP effects of the antibodies BT461, BT468, and BT470 of the present invention on H1975 and HCC827 cells were evaluated using THP-1 cells (a human peripheral blood mononuclear cell line). The experimental steps are as follows:

[0526] 1. Target cell labeling: Target cells (H1975 cells and HCC827 cells) were collected and washed once with 10 mL of PBS. The cells were centrifuged at 1000 rpm for 5 min and resuspended in PBS. The cell density was adjusted to 1E6 cells / mL. 0.4 μL of CTY (CellTrace TM Yellow, thermo, Catalog No. C34573, PE channel), incubate at 37°C for 10 min, then add 2 volumes of complete culture medium to terminate the staining reaction; 2E4 unlabeled H1975 cells are taken per well for compensation.

[0527] 2. Target cell plating: Adjust the density of the labeled target cells to 1E6 cells / mL using 1640 complete medium. Take the cell suspension and plate it as shown in the table below. Except for G1, G2, and G3, add 50 μl of cells to each well of a round-bottom transparent 96-well plate.

[0528] 3. Antibody loading: Dilute the antibody to the corresponding concentration, from low to high concentration, add 50 μL per well to the corresponding well plate, mix well, and place at 37°C, 5% CO2, and pre-incubate for 30 minutes.

[0529] 4. Effector cell incubation: Collect THP-1 cells, add 10 mL PBS to wash the cells, centrifuge at 1000 rpm for 5 min, resuspend in PBS, adjust the cell density to 1E6 cells / mL, add 0.2 μL CTV (CellTrace TM Violet, thermo, product number C34571, PCB450 channel), incubated in a 37°C incubator for 10 min, and then added 2 times the volume of complete culture medium to terminate the staining reaction; 2E4 unlabeled THP-1 cells were taken per well for compensation.

[0530] 5. Plating Effector Cells: After centrifugation, adjust the density to 2E5 cells / mL using 1640 complete medium. Plate the cell suspension as shown in the table below, adding 50 μL of cells to each well of a round-bottom, transparent 96-well plate. Incubate at 37°C, 5% CO2 for 2.5 hours. Add 50 μL of cells to each well of a round-bottom, transparent 96-well plate.

[0531] 6. Test on the instrument: After the time is up, add 150μL 1% BSA-PBS to each well to wash the cells and centrifuge at 1000rpm for 3 minutes. Wash once. Add 150μL 1% BSA-PBS to each well to resuspend the cells and test on the instrument as soon as possible. Before testing on the instrument, the experimental wells need to be resuspended by pipetting. Shake the plate once every 2 wells and collect 100μL sample. CellTrace TM Violet selects PCB450 channel, CellTrace TM Yellow selects the PE channel.

[0532] 7. Calculation of phagocytosis level: Detect the number of PCB450 and PE double-positive cells by flow cytometry. The phagocytosis percentage is the sum of the number of double-positive cells divided by the number of PCB450-positive cells.

[0533] As shown in Figure 10, the antibodies BT461, BT468, and BT470 of the present invention can mediate the ADCP effect of THP-1 on target cells H1975 and HCC827. The ADCP effect mediated by the BT470 antibody is stronger than that of the BT461 and BT468 antibodies, and is comparable to that of the drug Evanta.

[0534] Example 13 Inhibition of tumor cell line proliferation in vitro

[0535] In this example, several human tumor cell lines were selected, including human pancreatic cancer cell line KP4 (Nanjing Kebai Biotechnology, product number CBP60541), non-small cell lung cancer Kras mutant cell line A549 (ATCC#CCL-185), human gastric cancer c-Met gene amplified cell line MKN45 (Shang En Biotechnology, product number SNL-173), non-small cell lung cancer EGFR mutant cell line HCC827, and the like. The effects of the antibodies of the present invention on the proliferation of these cell lines were detected using a kit (Novozymes, DD1102).

[0536] Tumor cells growing in the logarithmic phase were obtained, digested and inoculated into 96-well plates (2000 cells / well), and cultured overnight in complete culture medium. After removing the culture medium from the 96-well plates, 100 μL of anti-c-Met / EGFR antibodies BT461, BT468, BT470, and control (Evantor, Johnson, batch number MAS0001) diluted with complete cell culture medium were added (initial concentration was 500 μg / mL) and diluted to 5 gradients with a 2-fold concentration using a gradient dilution method. After culturing in an incubator at a concentration of 5% CO2 and a temperature of 37°C for 72 h, an equal volume of 50 μg / mL was added to each well. Reagent (Vazyme, Novozyme, catalog number DD1102), shake and mix for 5 minutes until the cell cluster is completely lysed. After incubation for 25 minutes, 100 μL of the mixture is transferred to a white opaque plate and detected using a TR-FRET enzyme reader (PerkinElmer, model, catalog number). The results are shown in Figure 11. The antibodies BT468 and BT470 of the present invention can significantly inhibit the proliferation of human tumor cells KP4, A549, MKN45, and HCC827 at multiple gradient concentrations, and are comparable to the marketed drug Avantuo, or even have stronger inhibitory ability. Among them, the inhibitory ability of BT470 in KP4 cells is greater than that of BT468 and Avantuo; in HCC827 cells, the inhibitory ability of BT470 is greater than that of BT468; in A549 cells and MKN45 cells, the proliferation inhibition ability of the antibodies of the present invention is not much different.

[0537] Example 14 Specific binding to cynomolgus monkey c-Met and EGFR proteins

[0538] Cross-species binding was detected using ELISA. Recombinant proteins (mouse EGFR-his, mouse c-Met-his, cynomolgus macaque EGFR-his, cynomolgus macaque c-Met-his, rat EGFR-his, and rat c-Met-hFC) were diluted to 2 μg / mL in coating buffer and added to each well of the ELISA plate at a volume of 50 μL / well. The plate was incubated at 37°C for 2 hours. The liquid was discarded and the wells were washed five times with 200 μL of washing buffer per well. 200 μL of blocking buffer was added to each well and incubated overnight at 4°C. The liquid was discarded and the wells were washed five times with 200 μL of washing buffer per well. Add 50 μL of anti-c-Met / EGFR antibodies BT461, BT468, and BT470, or a control (Evantor, Johnson & Johnson, batch number MAS0001) at a 20 μg / mL concentration, in a 5-fold dilution, across seven antibody steps. A blank control was also included. Incubate at 37°C for 60 min. Discard the remaining liquid from the wells, and wash five times with 200 μL of wash buffer using a plate washer. Add 50 μL of Goat anti-human IgG-Fc-Secondary Antibody (1:10,000 dilution) to each well. Incubate at 37°C for 60 min. Discard the remaining liquid from the wells, and wash five times with 200 μL of wash buffer using a plate washer. Add 50 μL of color development solution to each well, and incubate at 37°C for 15 min. Add 50 μL / well 2 mol / L H2SO4 to terminate the reaction, and read the OD450 value on a microplate reader.

[0539] FIG12 ELISA binding of the antibody BT470 of the present invention to cynomolgus monkey c-Met and EGFR proteins

[0540] Table 117 Species cross-test results

[0541] The results are shown in FIG12 and Table 17. The antibody BT470 of the present invention has cross-binding activity to cynomolgus monkey EGFR and c-Met.

[0542] Example 15 Detection of the affinity between the antibody of the present invention and the antigen based on biolayer interferometry (BLI)

[0543] 15.1 Binding Kinetics of the Bispecific Antibodies of the Present Invention to Recombinant Human EGFR-His

[0544] EGFR-His (ACRO, Cat. No. EGR-H5222) was serially diluted from 100 nM in PBST and serially diluted two-fold to seven concentration points using a zero-concentration reference well. The c-Met / EGFR bispecific antibody BT470 and control antibodies Avanto, Taixinsheng, and JH001 were diluted to 5 μg / mL. A molecular interaction instrument (Fortebio, Sartorius, Model: Octet R8) was operated at 30°C and a shake speed of 1000 rpm. The antibody was captured using a coated AHC2 probe (Sartorius, Cat. No. 18-5142) with a capture time of 180 s. The sample was bound to the serially diluted sample with a binding time of 120 s and a dissociation time of 300 s. The sample was then regenerated three times with regeneration buffer (10 mM glycine, pH 1.7) for 30 s each. Detection was performed using ForteBio's Octet System. After obtaining the sensorgram data, the binding constant (ka) and dissociation constant (kd) were analyzed using Octet BLI Analysis software, and the ideal binding and dissociation curves were fitted to calculate the equilibrium dissociation constant KD (kd / ka) between the antibody and antigen. The results are shown in Table 18.

[0545] Table 18 EGFR-HIS affinity (AHC2 sensor)

[0546] 15.2 Binding Kinetics of the Bispecific Antibodies of the Present Invention to Recombinant Human c-Met-His

[0547] The binding kinetics of the bispecific antibody to recombinant human c-Met-His were measured using amino-coupling on the Octet Molecular Interaction Analyzer. The experimental protocol was as follows: 1. Pre-wet the AR2G sensor (Sartorius, 18-5092). 2. Baseline: Add 200 μL of buffer (1× PBS, 0.02% Tween-20, 0.1% BSA) to a black 96-well plate and run the first baseline step for 60 seconds. 3. Activation: Activate with 40 mM EDC (Sartorius, 18-1033) and 10 mM NHS (Sartorius, 18-1067) at a 1:1 ratio for 300 seconds. 4. Sample Loading: Dilute the protein to the desired concentration with NaNc and immobilize the c-Met-His loading sample onto the sensor. 5. Baseline: Add 200uL of buffer (1×PBS, 0.02% Tween-20, 0.1% BSA) to a black 96-well plate and run the second baseline step for 120 seconds. 6. Quenching: Quench with 1.0M Ethanolamine-HCL (Sartorius, 18-1072) for 300 seconds. 7. Binding: Use the protein-coated sensor to bind to the gradient-diluted antibody sample for 600 seconds. 8. Dissociation: Run the dissociation step for 800 seconds. After obtaining the sensorgram data, use Octet BLI Analysis software to analyze the binding constant (ka) and dissociation constant (kd). Use kinetic fitting to fit the ideal binding and dissociation curves. The results are shown in Table 19 below:

[0548] Table 19c-Met-HIS affinity (amino coupling)

[0549] Example 16 Synergistic Effect in Inhibiting Cell Proliferation: Bispecific Antibodies Are More Effective Than Combinations

[0550] Similar to the experimental method of "Example 13 In vitro inhibition of tumor cell line proliferation", this example selected multiple human tumor cell lines, including human gastric cancer c-Met gene amplified cells MKN45 (Shan En Biotechnology, Product No. SNL-173), non-small cell lung cancer EGFR mutation cells HCC827-HGF (transfected to overexpress human HGF) and NCI-H1975-HGF (transfected to overexpress human HGF), and used The effect of the antibodies of the present invention on the proliferation of these cell lines was detected using a kit (Novozymes, DD1102). Briefly, these cells were exposed to a combination of therapeutic antibodies: BT470, JH001 & ONAR-B12 (ONAR monovalent antibody and inactive antibody B12) diluted in complete cell culture medium, with an initial concentration of 500 μg / mL (when used in combination, equivalent monovalent antibody amount was converted). The cells were then diluted to 5 gradients of 2-fold concentration using a gradient dilution method. After 72 hours of incubation in an incubator at 37°C and a 5% CO2 atmosphere, an equal volume of 50 μg / mL was added to each well. Reagent (Vazyme, Novozymes, Cat. No. DD1102) was added and shaken for 5 min until the cell clusters were completely lysed. After incubation for 25 min, 100 μL of the mixture was transferred to an opaque plate with a white bottom and detected using a TR-FRET microplate reader (PerkinElmer, model, cat. no.).

[0551] The results are shown in Figures 13, 14 and 15. In the three cell lines of MKN45, HCC827-HGF and NCI-H1975-HGF, the ability of BT470 dual antibody to inhibit tumor cell activity is stronger than that of its related monoclonal antibody combination (JH001 & ONAR-B12), which indicates that BT470 has a synergistic effect in inhibiting tumors in vitro.

[0552] Example 17 Bis-antibody mediated target protein internalization

[0553] 17.1 Using pHAb Probe to Draw Internalization Curves of Antibody Concentration Gradients

[0554] 100 μg of Goat-anti-human-IgG-Fc-Secondary-Antibody was dissolved in buffer using an ultrafiltration tube. 500 μL of 10 mM sodium bicarbonate (pH 8.5) was added and the mixture was centrifuged at 15,000 rpm at 4°C for 10 minutes. Repeat twice. 25 μL of a 1:1 DMSO-water mixture was added to 250 μg of pHAb Reactive Dyes (Promega, stored at -80°C) to obtain a 10 mg / mL dye stock solution. The pHAb-reactive dye was conjugated to the antibody at an antibody-to-dye ratio of 100 μg:1.2 μL and allowed to bind on a shaker at room temperature for 60 minutes. Unreacted dye was removed using a desalting column. The column was centrifuged at 900 g for 2 minutes to equilibrate. The dye-antibody conjugated mixture was then dripped onto the center of the slant side of the desalting column and centrifuged at 900 g for 2 minutes, facing outward. The sample was collected and assayed for antibody concentration and dye-to-antibody ratio.

[0555] MKN45 cells were counted and 3E5 cells were added to each well of a U-bottom 96-well plate. The plates were centrifuged at 1000 rpm for 2 minutes and washed twice with PBS. Antibodies BT461, BT468, BT470, Amivantamab, JH001, nimotuzumab, BT264 (anti-c-Met arm sequence of Evanta), and IgG1 were serially diluted to a starting concentration of 20 μg / mL. Five-fold dilutions were added to each well for seven steps, with 50 μL of antibody added. A blank control was also included. The plates were incubated at 4°C for 30 minutes. After centrifugation at 1000 rpm for 2 minutes, the liquid was discarded, and the plates were washed twice with 200 μL of PBS, all on ice. Fifty μL of dye-conjugated secondary antibody (diluted 1:200) was added to each well. One plate was incubated at 37°C and the other plate was incubated at 4°C. After 4 hours, the plates were washed twice with PBS and resuspended in 200 μL of PBS. Flow cytometry detection (PE fluorescence excitation and reception)

[0556] [Corrected 03.01.2024 according to Rule 26] The results are shown in Figure 16A. Antibodies BT461, BT468, BT470, and Amivantamab promoted target protein internalization with higher internalization efficiency than the monoclonal antibodies against EGFR or c-Met: JH001, BT264, and nimotuzumab. The internalization efficiency mediated by the bispecific antibodies was ranked as Amivantamab > BT470 > BT461 > BT468.

[0557] 17.2 Utilization Live-Cell Analysis System draws the internalization curve of the antibody over time

[0558] Count the viable MKN45 cells and resuspend them in 1640 medium containing 10% FBS to adjust the density to 5×104 / ml. Use a pipette to add 100 μl of cells (5000 cells / well) to each well of a flat-bottom 96-well culture plate and place in a CO2 incubator overnight to allow the cells to adhere. The next day, add the antibody to be tested and the pH probe ( Human Fabfluor-pH Antibody Labeling Dye for Antibody Internalization (Sartorius, Cat. No. 4722) was prepared at a concentration of 20 μg / ml using 10% FBS culture medium. 30 μl of antibody and 30 μl of pH probe were mixed in a 1:1 ratio and incubated in a 37°C incubator in the dark for 30 min. 50 μl of the incubated primary antibody-secondary antibody complex was added to the wells inoculated with cells to avoid air bubbles. Place the cell culture plate in the wells. In the Live-Cell Analysis System, take pictures every hour. Data were analyzed using integrated software, and graphs were drawn with the time point as the X-axis and the fluorescence intensity as the Y-axis.

[0559] The results, as shown in Figures 16B and 16C, show that antibodies BT461, BT470, and Amivantamab promote target protein internalization, with the internalization efficiency of the bispecific antibodies ranked as Amivantamab = BT470 > BT461. The results also show that the internalization efficiency of the bispecific antibody BT470 is greater than that of the monoclonal antibodies against EGFR or c-Met, JH001 and nimotuzumab, indicating a synergistic effect of the bispecific antibody BT470 in dual-target internalization.

[0560] Example 18 Tumor Efficacy Study Using Bis-Antibody Molecules

[0561] The in vivo anti-tumor activity of the anti-bispecific antibodies was evaluated in a mouse xenograft model (NSG, Vital River, 6-week-old, female) of human non-small cell lung cancer HCC827 (EGFR exon 19 deletion mutation) or HCC827-HGF.

[0562] Human HCC827 cells were propagated as monolayer cultures in vitro using RPM1640 + 10% FBS as complete culture medium. Cells in the logarithmic growth phase were digested and resuspended in serum-free culture medium / Matrigel (1:1) to obtain a suspension. Cells were plated at 5×10 6 The mice were inoculated subcutaneously (sc) on the right side of the back to form tumors. Ten days after implantation, the mice were divided into three groups according to the tumor volume. The average tumor volume in each group was 80-100 mm 3 Around 30 days, the drug administration was started. 0.1 mg / kg of HGF-his mixed IgG1 or antibody molecules were administered intraperitoneally (IP) twice a week for three weeks.

[0563] The efficacy of the treatment was also evaluated using HCC827-HGF cells, which are HCC827 cells transfected to express human HGF (hepatocyte growth factor). These cells were cultured at 5×10 6 The mice were inoculated subcutaneously (sc) on the right side of the back to form tumors. Ten days after implantation, the mice were divided into three groups according to the tumor volume. The average tumor volume in each group was 80-100 mm 3 IgG1 or antibody molecules were administered intraperitoneally (IP) twice a week for three weeks.

[0564] The experimental results are shown in Figures 17 and 18. In the HCC827 tumor model (exogenously administered human HGF-His) and the HCC827-HGF tumor model, BT470 has the therapeutic effect of inhibiting tumor growth. The tumor inhibition effect %TGI after 21 days of administration is shown in Table 20 below:

[0565] Table 20 Tumor inhibition efficacy %TGI after 21 days of administration

Claims

1. An isolated bispecific antibody comprising an antigen binding arm A that can specifically bind to human epidermal growth factor receptor (EGFR) and prevent epidermal growth factor (EGF) from binding to EGF and an antigen binding arm B that can specifically bind to the extracellular portion of human proto-oncogene receptor tyrosine kinase (C-Met) and prevent hepatocyte growth factor (HGF) from binding to C-Met, wherein: The antigen binding arm A comprises light chain A (LCA) and heavy chain A (HCA), and the antigen binding arm B comprises light chain B (LCB) and heavy chain B (HCB), and is characterized in that the (LCA), (HCA), (LCB) and (HCB) are composed of amino acid sequences SEQ ID NO: 1, 2, 12 and 13, respectively.

2. The bispecific antibody according to claim 1, wherein the bispecific antibody is capable of completely inhibiting the autophosphorylation of EGFR mediated by the ligand EGF and the autophosphorylation of C-Met mediated by the ligand HGF in the HCC827 cell line, or the bispecific antibody inhibits the phosphorylation of the Y1068 site of EGFR and simultaneously inhibits the phosphorylation of C-Met at the Y1234 / Y1235 sites.

3. The bispecific antibody according to claim 1, wherein The bispecific antibody can inhibit the growth and proliferation of human tumor cells and has a synergistic effect. The inhibitory effect of the bispecific antibody on the proliferation of EGFR and C-Met double-positive tumor cells is stronger than the combination of two targeted monoclonal antibodies; Or further, the bispecific antibody can mediate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP).

4. The isolated bispecific antibody according to any one of claims 1 to 3, wherein The antigen binding arm A is a fusion peptide having an LCA-linker-HCA structure, and the binding arm B is a fusion peptide having an LCB-linker-HCB structure, wherein the linker is a short peptide having a (GGGS)n structure, wherein n is any integer between 4 and 10.

5. An isolated EGFR / C-Met bispecific antibody, comprising an antigen-binding arm A specific for epidermal growth factor receptor (EGFR) and an antigen-binding arm B specific for human proto-oncogene receptor tyrosine kinase (C-Met), wherein the antigen-binding arm A is a ScFab (A)-hinge region-Fc (A) structure formed by connecting a ScFab (A) as shown in SEQ ID NO: 3 and an Fc (A) as shown in SEQ ID NO: 11 and comprising CH2ACH3A, and the antigen-binding arm B is a ScFab (B)-hinge region-Fc (B) structure formed by connecting a ScFab (B) as shown in SEQ ID NO: 14 or 25 and an Fc (B) as shown in SEQ ID NO: 22 or 33 and comprising CH2BCH3B, wherein: The Fc(A) comprises a combination of mutant amino acids that form a Knob structure, and the Fc(B) comprises a combination of mutant amino acids that form a hole structure. The knob structure of the Fc(A) and the hole structure of the Fc(B) interact to form a KIH structure, and the antigen binding arm A and the antigen binding arm B form a heterodimer through the KIH structure.

6. The isolated EGFR / C-Met bispecific antibody according to any one of claims 1 to 5, wherein The antigen binding arm A comprises the amino acid sequence shown in SEQ ID NO: 4, and the antigen binding arm B comprises the amino acid sequence shown in SEQ ID NO: 15 or 26, respectively.

7. The bispecific antibody of any one of claims 1 to 6, wherein the bispecific antibody can bind to human EGFR and cynomolgus monkey EGFR, and the bispecific antibody can bind to human c-Met and cynomolgus monkey HGFR.

8. An isolated synthetic nucleic acid molecule comprising the nucleic acid encoding the antigen binding arm A and antigen binding arm B, LCA, HCA, LCB and HCB according to any one of claims 1 to 7, wherein the nucleic acid molecule encoding the antigen binding arm A comprises the nucleotide sequence shown in SEQ ID NO: 52, and the nucleic acid molecule encoding the antigen binding arm B comprises the nucleotide sequence shown in SEQ ID NO: 53 or 54.

9. A vector comprising the polynucleotide according to claim 7.

10. An isolated host cell comprising the vector of claim 9.

11. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

12. Use of the bispecific antibody according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8, and the pharmaceutical composition according to claim 11 in the preparation of a drug for treating a disease or tumor involving abnormal EGFR and MET cells and a drug for inhibiting the growth or metastasis of tumors or cancer cells expressing EGFR and / or c-Met in a subject, wherein: The treatment includes: The antibodies inhibit the growth of HGF-induced HGF growth-responsive cells, and the antibodies inhibit the growth of EGF-induced EGF growth-responsive cells; Wherein, the tumor is an EGFR-positive tumor, a C-Met-positive tumor, or an EGFR- and C-Met-double-positive tumor; The disease or tumor of EGFR and MET abnormal cells is associated with EGFR activating mutations, EGFR gene amplification, elevated levels of circulating HGF, c-Met activating mutations, c-Met gene amplification, or mutant KRAS; wherein the EGFR activating mutation is selected from G719A, G719X, L861X, L858R, E746K, L747S, E749Q, A750P, A755V, V765M, L858P or T790M substitution, deletion of E746-A750, deletion of R748-P753, insertion of alanine between M766 and A767, insertion of serine, valine and alanine between S768 and V769, and insertion of asparagine and serine between P772 and H773, wherein X is any amino acid; Wherein, the mutant KRAS has a G12V or G12C substitution; or optionally, the mutant KRAS has a G12V substitution.

13. The use according to claim 12, wherein the cancer is selected from epithelial cell cancer, breast cancer, ovarian cancer, lung adenocarcinoma, small cell lung cancer, colorectal cancer, anal cancer, prostate cancer, bladder cancer, pharyngeal cancer, nasal cancer, pancreatic cancer, skin cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer, testicular cancer, gastric cancer, thymus cancer, thyroid cancer, hepatocellular carcinoma, or sporadic or hereditary papillary renal cell carcinoma, or optionally, the cancer is non-small cell lung cancer, pancreatic cancer, gastric cancer, colon cancer, liver cancer; Or optionally, wherein the drug comprises a second therapeutic agent, the second treatment comprises a chemotherapeutic agent and a targeted anticancer therapy; optionally, the chemotherapeutic agent or the targeted anticancer therapy is a tyrosine kinase inhibitor of EGFR, c-Met, HER2, HER3, HER4 or VEGFR; or optionally, where The tyrosine kinase inhibitor is erlotinib, gefitinib or afatinib.

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