Combination therapy with bispecific anti-EGFR / c-Met antibody and anti-PD-1 antibody

Abstract

This invention relates to a combination therapy for modulating the tumor microenvironment and enhancing immune cell infiltration into the tumor microenvironment by using a bispecific anti-EGFR / c-Met antibody in combination with a PD-(L)1 axis inhibitor. This invention also relates to a combination therapy for inhibiting both the EGFR signaling pathway and the MET signaling pathway in tumor cells, and to the targeting of EGFR and MET-expressing tumor cells for destruction by immune effector cells such as natural killer cells and macrophages via antibody-dependent cell-mediated cytotoxicity (ADCC) and trogocytosis mechanisms, respectively.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 451,781, filed on 13 March 2023, and U.S. Provisional Patent Application No. 63 / 459,857, filed on 17 April 2023, the disclosures of which are incorporated herein by reference in their entirety.

[0002] (Reference to electronically submitted sequence listings) The sequence listing for this application, filed as "JBI6783WOPCT1_SL.xml", was created on March 12, 2024, and is 39 kilobytes (KB) in size, and is filed electronically through the U.S. Patent and Trademark Center. This filed sequence listing is part of this Specified and is incorporated herein by reference in its entirety.

[0003] (Field of invention) The present invention relates to a combination therapy and method for treating solid tumors by remodeling the tumor microenvironment and inhibiting EGFR and MET signaling, comprising a bispecific anti-EGFR / c-Met antibody and inhibition of the PD-(L)1 axis. [Background technology]

[0004] Immune checkpoint inhibitors, such as PD-(L)1 axis inhibitors, are effective first-line therapies for solid tumors. However, low response rates and acquired resistance over time have created a need for further treatment options. There is an unmet need for immunotherapies that function to remodel the tumor microenvironment and thus can increase therapeutic responses to various types of cancer, including head and neck cancer and lung cancer, which currently exhibit suboptimal responses to therapy.

[0005] This invention addresses this unmet requirement. [Overview of the project]

[0006] In one embodiment, the present invention relates to a method for treating a solid tumor or mitigating the progression of cancer in a subject who needs to be treated for a solid tumor or whose cancer progression needs to be mitigated, the method comprising administering to the subject (a) a PD-(L)1 axis inhibitor and (b) a bispecific anti-EGFR / c-Met antibody.

[0007] In one embodiment, the present invention relates to a method for enhancing immune cell infiltration into a solid tumor in a subject that requires enhancement of immune cell infiltration into a solid tumor, the method comprising administering to the subject (a) a PD-(L)1 axis inhibitor and (b) a bispecific anti-EGFR / c-Met antibody.

[0008] In one embodiment, the present invention relates to a method for reducing glycolysis or lactate production in the tumor microenvironment in subjects where it is necessary to reduce glycolysis or lactate production in the tumor microenvironment, the method comprising administering to the subject (a) a PD-(L)1 axis inhibitor and (b) a bispecific anti-EGFR / c-Met antibody.

[0009] In one embodiment, the bispecific anti-EGFR / c-Met antibody includes a first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determining region 1 (HCDRl) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determining region 1 (LCDRl) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; and a second domain that binds to c-Met, the second domain comprising the HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12. In one embodiment, the first domain that binds to EGFR comprises the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, and the second domain that binds to c-Met comprises the VH of SEQ ID NO: 15 and the VL of SEQ ID NO: 16. In one embodiment, the bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype. In one embodiment, the bispecific anti-EGFR / c-Met antibody comprises a first heavy chain (HC1) of SEQ ID NO: 17, a first light chain (LC1) of SEQ ID NO: 18, a second heavy chain (HC2) of SEQ ID NO: 19, and a second light chain (LC2) of SEQ ID NO: 20.

[0010] In one embodiment, the PD-(L)1 axis inhibitor is an inhibitory antibody. In one embodiment, the PD-(L)1 axis inhibitor is an anti-PD-1 antibody. In one embodiment, the inhibitory antibody is cetrelimab, pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimuzumab, emvafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, or SHR-1210. In one embodiment, the pembrolizumab antibody includes the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, HCDR3 of SEQ ID NO: 23, light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 26. In one embodiment, the pembrolizumab antibody includes the heavy chain (HC) of SEQ ID NO: 29 and the light chain (LC) of SEQ ID NO: 30. In one embodiment, the cetrelimab antibody includes the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32, HCDR3 of SEQ ID NO: 33, light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 34, LCDR2 of SEQ ID NO: 35, and LCDR3 of SEQ ID NO: 36. In one embodiment, the cetrelimab antibody includes the heavy chain (HC) of SEQ ID NO: 39 and the light chain (LC) of SEQ ID NO: 40.

[0011] In one embodiment, the cancer is an EGFR, c-Met, or EGFR and c-Met expressing cancer associated with wild-type EGFR, EGFR-activating mutations, EGFR gene amplification, elevated levels of circulating HGF, wild-type c-Met, c-Met-activating mutations, c-Met gene amplification, or mutant KRAS, or any combination thereof. In one embodiment, EGFR activating mutations include substitutions of L718Q, G719A, G719X (where X is any amino acid), L861X (where X is any amino acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, L858P, or T790M, deletions of E746-A750, deletions of R748-P753, insertion of Ala(A) between M766 and A767, insertions of Ser, Val, and Ala(SVA) between S768 and V769, and P77 This includes insertions of Asn and Ser(NS) between 2 and H773, insertions of one or more amino acids between D761 and E762, A763 and Y764, Y764 and Y765, M766 and A767, A767 and V768, S768 and V769, V769 and D770, D770 and N771, N771 and P772, P772 and H773, H773 and V774, V774 and C775, one or more deletions or insertions in EGFR exon 20, or any combination thereof. In one embodiment, the mutant KRAS includes substitutions of G12V, G12C, or G12A.

[0012] In one embodiment, the method increases immune cell infiltration into solid tumors. In one embodiment, the immune cells are T cells, B cells, or natural killer cells. In one embodiment, the T cells include CD8+ T cells and CD4+ T cells. In one embodiment, the T cells are CD8+ T cells.

[0013] In one embodiment, this method increases the population of central memory cytotoxic T cells within the tumor microenvironment.

[0014] In one embodiment, the subject is suspected of having or has EGFR, c-Met, or EGFR and c-Met expressing cancer. In one embodiment, the subject is resistant to or has acquired resistance to treatment with a previous anti-cancer therapy.

[0015] In one embodiment, the previous anti-cancer therapy is a kinase inhibitor. In one embodiment, the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL. In one embodiment, the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, lazertinib, poziotinib, cliotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib.

[0016] In one embodiment, the cancer is epithelial cancer, 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, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC). In one embodiment, the cancer is resistant to treatment with a PD-(L)1 axis inhibitor. In one embodiment, the cancer is head and neck squamous cell carcinoma (HNSCC). In one embodiment, the cancer is lung squamous cell carcinoma (LUSC). In one embodiment, the cancer is non-small cell lung cancer (NSCLC).

[0017] In one embodiment, the method further comprises administering to the subject one or more anti-cancer therapies.

[0018] In one embodiment, the one or more anti-cancer therapies are kinase inhibitors. In one embodiment, the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL. In one embodiment, the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, lazertinib, poziotinib, cliotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib.

[0019] In one embodiment, the previous anti-cancer therapy is chemotherapy. In one embodiment, the previous anti-cancer therapy is targeted anti-cancer therapy.

[0020] In one embodiment, the one or more anti-cancer therapies include chemotherapy. In one embodiment, the one or more anti-cancer therapies include targeted cancer therapy.

[0021] In one embodiment, the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor are administered essentially simultaneously at two different injection sites. In one embodiment, the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor are combined prior to administration and administered simultaneously. In one embodiment, the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor are administered continuously over a period of one or more days as part of a multi-stage treatment regimen.

[0022] In one embodiment, the present invention relates to a method for treating a solid tumor, mitigating cancer progression, enhancing immune cell infiltration into a solid tumor, or reducing lactate production in the tumor microenvironment in a subject that requires treatment of a solid tumor, mitigation of cancer progression, enhancement of immune cell infiltration into a solid tumor, or reduction of lactate production in the tumor microenvironment, wherein the method comprises administering a bispecific anti-EGFR / c-Met antibody to the subject, the subject having received prior administration of a PD-(L)1 axis inhibitor.

[0023] In one embodiment, the bispecific anti-EGFR / c-Met antibody includes a first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determining region 1 (HCDRl) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determining region 1 (LCDRl) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; and a second domain that binds to c-Met, the second domain comprising the HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12. In one embodiment, the first domain that binds to EGFR comprises the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, and the second domain that binds to c-Met comprises the VH of SEQ ID NO: 15 and the VL of SEQ ID NO: 16. In one embodiment, the bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype. In one embodiment, the bispecific anti-EGFR / c-Met antibody comprises a first heavy chain (HC1) of SEQ ID NO: 17, a first light chain (LC1) of SEQ ID NO: 18, a second heavy chain (HC2) of SEQ ID NO: 19, and a second light chain (LC2) of SEQ ID NO: 20.

[0024] In one embodiment, the previously administered PD-(L)1 axis inhibitor is an inhibitory antibody. In one embodiment, the pre-administered inhibitory antibody is pembrolizumab (KEYTRUDA®), cetrelimab, nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelelizumab, dostralimab, genolimusumab, emvafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, or SHR-1210.

[0025] In one embodiment, the method enhances immune cell infiltration in a tumor or tumor microenvironment. In one embodiment, the immune cells are T cells, B cells, or natural killer cells. In one embodiment, the immune cells include CD8+ T cells and / or CD4+ T cells. In one embodiment, the immune cells are CD8+ T cells. In one embodiment, the method enhances the population of central memory cytotoxic T cells in the tumor microenvironment.

[0026] In one embodiment, the present invention relates to a method for reducing lactate production in the tumor microenvironment in subjects where it is necessary to reduce lactate production in the tumor microenvironment, the method comprising administering a combination of inhibitors to the subject, wherein the combination of inhibitors is (a) a combination comprising an EGFR inhibitor, a c-Met inhibitor, and a PD-(L)1 axis inhibitor, or (b) a combination comprising an EGFR inhibitor and a c-Met inhibitor, and the subject has received prior administration of a PD-(L)1 axis inhibitor.

[0027] In one embodiment, the combination of an EGFR inhibitor and a c-Met inhibitor includes a bispecific anti-EGFR / c-Met antibody. In one embodiment, the bispecific anti-EGFR / c-Met antibody includes a first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determining region 1 (HCDRl) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determining region 1 (LCDRl) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; and a second domain that binds to c-Met, the second domain comprising the HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12. In one embodiment, the first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, and the second domain that binds to c-Met includes the VH of SEQ ID NO: 15 and the VL of SEQ ID NO: 16. In one embodiment, the bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype. In one embodiment, the bispecific anti-EGFR / c-Met antibody includes the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO: 20.

[0028] In one embodiment, the PD-(L)1 axis inhibitor is an inhibitory antibody. In one embodiment, the PD-(L)1 axis inhibitor is an anti-PD-1 antibody. In one embodiment, the PD-(L)1 axis inhibitory antibody is cetrelimab, pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimusumab, emvafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, or SHR-1210.

[0029] In one embodiment, a population of central memory cytotoxic T cells increases within the tumor microenvironment.

[0030] In one embodiment, the cancer is 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, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC). In one embodiment, the cancer is resistant to treatment with PD-(L)1 axis inhibitors. In one embodiment, the cancer is head and neck squamous cell carcinoma (HNSCC). In one embodiment, the cancer is lung squamous cell carcinoma (LUSC). In one embodiment, the cancer is non-small cell lung cancer (NSCLC).

[0031] In one embodiment, the present invention relates to a kit comprising in two or more containers a first pharmaceutical composition comprising a bispecific anti-EGFR / c-Met antibody and a second pharmaceutical composition comprising a PD-(L)1 axis inhibitor. In one embodiment, the bispecific anti-EGFR / c-Met antibody comprises (a) a first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determining region 1 (HCDRl) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determining region 1 (LCDRl) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; and (b) a second domain that binds to c-Met, the second domain comprising the HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12. In one embodiment, the first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, and the second domain that binds to c-Met includes the VH of SEQ ID NO: 15 and the VL of SEQ ID NO: 16. In one embodiment, the bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype. In one embodiment, the bispecific anti-EGFR / c-Met antibody includes the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO: 20.

[0032] In one embodiment, the PD-(L)1 axis inhibitor is an inhibitory antibody. In one embodiment, the PD-(L)1 axis inhibitor is an anti-PD-1 antibody. In one embodiment, the inhibitory antibody is cetrelimab, pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimuzumab, emvafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, or SHR-1210. In one embodiment, the pembrolizumab antibody includes the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, HCDR3 of SEQ ID NO: 23, light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 26. In one embodiment, the pembrolizumab antibody includes the heavy chain (HC) of SEQ ID NO: 29 and the light chain (LC) of SEQ ID NO: 30. In one embodiment, the cetrelimab antibody includes the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32, HCDR3 of SEQ ID NO: 33, light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 34, LCDR2 of SEQ ID NO: 35, and LCDR3 of SEQ ID NO: 36. In one embodiment, the cetrelimab antibody includes the heavy chain (HC) of SEQ ID NO: 39 and the light chain (LC) of SEQ ID NO: 40.

[0033] In one embodiment, the kit comprises a first pharmaceutical composition comprising a bispecific anti-EGFR / c-Met antibody further comprising a first pharmaceutically acceptable excipient, and a second pharmaceutical composition comprising a PD-(L)1 axis inhibitor further comprising a second pharmaceutically acceptable excipient. [Brief explanation of the drawing]

[0034] [Figure 1]This figure shows the results of immunohistochemistry using anti-EGFR antibodies and anti-MET antibodies against humanized patient-derived xenograft (PDX) tumors of head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma (LUSC). [Figure 2A] This figure shows the clinical profiles of the selected PDX models and experimental designs. [Figure 2B] This graph shows that the LUSC model has primary resistance to pembrolizumab, exhibiting consistent tumor growth despite treatment with pembrolizumab. [Figure 3A] This figure shows the anticancer effect of combination therapy with amivantamab and pembrolizumab in the HNSCC PDX model. [Figure 3B] This graph shows tumor progression in the YHIM-3003 model over 19 days, demonstrating significant tumor regression with combination therapy of amivantasamab (30 mpk) and pembrolizumab (10 mpk) compared to monotherapy with amivantasamab or pembrolizumab (p<0.001). [Figure 4] This figure shows the anticancer effect of combination therapy with amivantamab and pembrolizumab in the LUSC PDX model. [Figure 5] This graph shows data demonstrating persistent tumor regression after administration of amivantamab and in combination with pembrolizumab in the LUSC PDX model. [Figure 6] This image shows an increase in invasive cytotoxic T cells in the tumor after combined treatment. [Figure 7] This graph shows the quantitative analysis of invasive cytotoxic T cells in tumor lesions after combined treatment. TME: Tumor microenvironment, TN: Tumor lesion, ST: Stromal tissue. [Figure 8A] This figure shows data demonstrating that the combination therapy enhanced the central memory subset of cytotoxic T cells in the tumor microenvironment of HNSCC. It is a heatmap of memory T cell subsets (central memory T cells, effector memory T cells, and effector T cells) and activation markers in HNSCC PDX tumor samples. [Figure 8B]This figure shows data demonstrating that the combination therapy enhanced the central memory subset of cytotoxic T cells in the tumor microenvironment of HNSCC. It is a heatmap of memory T cell subsets (central memory T cells, effector memory T cells, and effector T cells) and activation markers in tumor samples from YHIM-2010. [Figure 8C] This figure shows data demonstrating that combination therapy enhanced the central memory subset of cytotoxic T cells in the tumor microenvironment of HNSCC. It also shows that the combination of amivantamab and pembrolizumab had a positive effect in each humanized PDX model, and that both models shared factors that enhanced the CD8+ T central memory subset with combination therapy. [Figure 9] This graph shows data demonstrating that tumor-responsive (CEA-stained) CD8 T cells were abundant in the combination treatment group in HNSCC PDX tumors, and were present in a significantly higher proportion compared to the control group (8.28 + 2.67 and 3.02 + 0.75, respectively, p<0.05). [Figure 10] This figure shows single-cell (scRNA) analysis of an immune population using clustering with Azimuth. [Figure 11-1] This figure shows the scRNA analysis of immune-related transcripts using clustering by Azimuth. [Figure 11-2] This figure shows the scRNA analysis of immune-related transcripts using clustering by Azimuth. [Figure 12] This figure shows data demonstrating that pembrolizumab treatment induced tumor subclusters expressing high levels of EGFR and MET. [Figure 13]This figure shows data demonstrating EGFR / METhigh and EGFR / METlow tumor subclusters. Left panel: Defined tumor subclusters with elevated dual expression of EGFR and MET (EMHIGH), and analyzed for differentially expressed genes (DEG). Right panel: EGFR and MET in EMHIGH and EMLOW tumors, showing increased expression of both markers in the EMHIGH tumor cluster. [Figure 14-1] This graph shows data demonstrating the analysis of differentially expressed genes in populations expressing EGFRHIGH and EGFRLOW, and indicates genes related to immunomodulation, tumor metastasis, drug resistance, and cancer stem cell characteristics. [Figure 14-2] This graph shows data demonstrating the analysis of differentially expressed genes in populations expressing EGFRHIGH and EGFRLOW, and indicates genes related to immunomodulation, tumor metastasis, drug resistance, and cancer stem cell characteristics. [Figure 15] This figure shows data demonstrating the analysis of hypoxia regulators and downstream mediators in populations expressing EGFRhigh and EGFRlow. [Figure 16] This figure shows data demonstrating that EGFR expression was inversely correlated with the infiltration of CD8+ T cells and NK cells in HNSCC and LUSC TCGA. [Figure 17A] This figure shows data demonstrating that EGFR expression was inversely correlated with CD8+ T cell and NK cell infiltration in HNSCC and LUSC TCGA. This figure shows the correlation between EGFR expression and biomarkers based on The Cancer Genome Atlas (TCGA) database. LDHA and SLC16A3 expression were positively correlated with EGFR expression in both HNSCC and LUSC. [Figure 17B]This figure shows data demonstrating that EGFR expression was inversely correlated with CD8+ T cell and NK cell infiltration in HNSCC and LUSC TCGA. The figure (above) shows that LDHA and SLC16A3 expression was significantly increased in the EGFRHIGH / METHIGH tumor subcluster (EMHIGH) of HNSCC PDX. In addition, LDHA and SLC16A3 expression was increased in the pembrolizumab-treated group. [Figure 17C] This figure shows data demonstrating that EGFR expression was inversely correlated with the infiltration of CD8+ T cells and NK cells in HNSCC and LUSC TCGA. This figure also shows that glycolysis regulators (HK2, GPI, ALDOA, PGK1, PGAM1, ENO1, ENO2) were relatively increased in the EMHIGH tumor subcluster (top) and the pembrolizumab-treated group (bottom). [Figure 17D] This figure shows data demonstrating that EGFR expression was inversely correlated with CD8+ T cell and NK cell infiltration in HNSCC and LUSC TCGA. This figure also shows that hypoxia regulators (HIF1A, HDAC1, KDM1A, KDM2A) and downstream signaling markers (CA9, VEGFA, TWIST1) were increased in the EMHIGH tumor subcluster (top) and the pembrolizumab-treated group (bottom). [Figure 17E] This figure shows data demonstrating that EGFR expression was inversely correlated with the infiltration of CD8+ T cells and NK cells in HNSCC and LUSC TCGA. The figure on the left shows that H1703, a LUSC human cancer cell, was treated with IFN-γ for 24 hours, mimicking the physiological response to pembrolizumab in TME. Total protein and surface expression of EGFR and MET showed a strong correlation (right). [Figure 17F]This figure shows data demonstrating that EGFR expression was inversely correlated with the infiltration of CD8+ T cells and NK cells in HNSCC and LUSC TCGA. It also shows the protein expression of EGFR / p-EGFR, MET / p-MET, MCT4 (SLC16A3), and LDHA in H1703 cells 72 hours after amivantamab at 10 mg / mL. The cells were treated with IFN-γ at 100 ng / mL for 24 hours. [Figure 18] This figure shows data demonstrating changes in EGFR and MET expression levels in tumors across different treatment groups. [Figure 19] This figure shows data demonstrating changes in EGFR and MET expression levels in tumors across different treatment groups. [Figure 20A] This figure shows that upregulation of EGFR and MET in HNSCC PDX (YHIM-3003) tumors induced increased expression of immune checkpoint regulators in the EGFRHIGH / METHIGH subcluster (EMHIGH). It is a volcano plot of the top 50 genes in EGFRHIGH / METHIGH compared to the EGFRLOW / METLOW tumor subcluster, analyzed by log2 factor change (FC) relative to p-value. Red dots indicate transcripts with significantly increased factor changes, including MET, PD-L1, and MET regulatory genes. [Figure 20B] This figure shows that upregulation of EGFR and MET in HNSCC PDX (YHIM-3003) tumors induced increased expression of immune checkpoint regulators in the EGFRHIGH / METHIGH subcluster (EMHIGH). This figure also shows that the expression of STAT-4 / PD-L1 (MET, STAT4, CD274), MET-regulated immune checkpoints (BACE2, STK40, PRSS23, DPYD, CAV1, S100A4, PYGL), and MET-related immune checkpoints (HAVCR2, CD276) was generally increased in the EGFRHIGH / METHIGH tumor subcluster compared to the EGFRLOW / METLOW subcluster. [Figure 20C]This figure shows that upregulation of EGFR and MET in HNSCC PDX (YHIM-3003) tumors induced increased expression of immune checkpoint regulators in the EGFRHIGH / METHIGH subcluster (EMHIGH). This figure also shows the expression of MET-related markers in different treatment groups. [Figure 21A] This graph shows exemplary data demonstrating the effect on body weight in YHIM-3003 PDX mice. Treatment began on day 0. Group body weight is expressed as MEAN ± SEM (n=10 mice per group). Data were graphed for all 10 mice studied. Body weight was recorded every other day. SEM, mean, standard error. [Figure 21B] This graph shows exemplary data demonstrating the effect of YHIM-3003 on tumor growth. Treatment began on day 0. Tumor volume is expressed as MEAN ± SEM (n=10 mice per group). The groups were graphed, and all 10 mice were left in the study. Tumor volume was measured every other day. SEM, mean, standard error. [Figure 22A] This figure outlines the synergistic effects of amivantamab and pembrolizumab in combination in an EGFR-high MET-high cancer model. [Figure 22B] This figure outlines the synergistic effects of amivantamab and pembrolizumab in combination in an EGFR-high MET-high cancer model. [Figure 23A] This figure shows the correlation between elevated EGFR / MET expression and poor immune response in patients who did not respond to anti-PD-1, based on Gene Expression Omibus data. [Figure 23B] This figure shows the correlation between elevated EGFR / MET expression and poor immune response in patients who did not respond to anti-PD-1, based on Gene Expression Omibus data. Detailed description of the invention

[0035] The present invention is at least in part based on the finding that targeting of EGFR / c-Met-expressing cells with a bispecific anti-EGFR / c-Met antibody combined with inhibition of the PD-(L)1 axis has a remodeling effect on the tumor immune microenvironment, including a reduction in glycolysis and lactate production within the tumor microenvironment, an increase in the level of CD8+ T cells in the tumor lesion, and an increase in the population of central memory cytotoxic T cells within the tumor microenvironment. The present invention is also based on the finding that the use of the bispecific anti-EGFR / c-Met antibody of the present invention (a) inhibits both the EGFR signaling pathway and the MET signaling pathway in tumor cells, and (b) promotes the targeting of EGFR and MET-expressing tumor cells for destruction by immune effector cells such as natural killer cells and macrophages via antibody-dependent cell-mediated cytotoxicity (ADCC) and trogocytosis mechanisms, respectively.

[0036] Identifying this mechanism provides a basis for selecting patients for therapies that may benefit from combination therapy involving anti-EGFR / c-Met bispecific antibodies and PD-(L)1 axis inhibitors.

[0037] definition All publications cited herein, including but not limited to patents and patent applications, are incorporated herein by reference as if they were fully described.

[0038] The terms used herein should be understood to be used solely to describe specific embodiments and not to limit them. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains.

[0039] Any methods and materials similar to or equivalent to those described herein may be used to carry out the tests of the present invention, and exemplary materials and methods are described herein. The following terms are used in describing and claiming the present invention.

[0040] Where a list is presented, please understand that, unless otherwise stated, each individual element of that list and all combinations of that list are distinct embodiments. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0041] As used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple objects unless otherwise explicitly indicated in the context. For example, the reference "a cell" includes a combination of two or more cells, etc.

[0042] The connecting phrase "and / or" between multiple enumerated elements is understood to encompass both the individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to be included in the meaning and therefore satisfies the requirements of the term "and / or" when used herein. The simultaneous applicability of two or more of the options is also understood to be included in the meaning and therefore satisfies the requirements of the term "and / or."

[0043] The transitional phrases “comprising,” “consisting essentially of,” and “consisting of” are intended to imply the commonly accepted meanings in patent terminology, namely, (i) “comprising” is synonymous with “containing,” “containing,” or “characterizing,” and is comprehensive or non-restrictive, not excluding additional unlisted elements or process steps; (ii) “consisting of” excludes any elements, processes, or components not specified in the claims; and (iii) “consisting essentially of” limits the scope of the claims to specified materials or processes and those that “do not substantially affect the basic and novel features” of the claimed invention. Embodiments described with the phrase “comprising” (or its synonyms) also provide embodiments described independently with “consisting” and “consisting essentially of” as embodiments.

[0044] Terms such as "co-administration," "administered together with," "administered in combination with," and "in combination with" encompass the administration of the selected therapeutic agent or drug to a single patient and are intended to include treatment regimens in which the therapeutic agent or drug is administered by the same or different routes of administration or at the same or different times.

[0045] "Isolated" refers to a homogeneous population of molecules (e.g., synthetic polynucleotides, polypeptides, vectors, or viruses) that has been substantially separated and / or purified from other components of the system in which the molecule is produced, such as recombinant cells, in addition to proteins subjected to at least one purification or isolation step. "Isolated" refers to molecules that are substantially free of other cellular material and / or chemicals, and includes molecules isolated to a higher purity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0046] "To treat," "to treat," or "to treat" a disease or disability such as cancer means achieving one or more of the following: reducing the severity and / or duration of the disability; suppressing the worsening of symptoms characteristic of the disability being treated; limiting or preventing the recurrence of the disability in a person who previously had the disability; or limiting or preventing the recurrence of symptoms in a person who previously had the disability.

[0047] "Preventing," "preventing," "prevention," or "prophylaxis" of a disease or disability means preventing the disability from occurring in the subject.

[0048] "To diagnose" or "diagnose" refers to determining whether a subject has a given disease or condition, or is likely to develop it in the future, or is likely to respond to treatment for a previously diagnosed disease or condition; in other words, it refers to a method of stratifying a patient population based on their likelihood of responding to treatment. Diagnosis is typically made by a physician based on general guidelines for the disease being diagnosed, or other criteria indicating that the subject is likely to respond to a particular treatment.

[0049] "Responsive," "responsive," or "likely responsive" means any kind of improvement or positive response, whether detectable or undetectable, such as reduction or recovery of one or more symptoms, a decrease in the severity of the disease, a stabilized (i.e., non-worsening) disease state, prevention of disease spread, delay or slowing of disease progression, recovery or mitigation of a disease state, and remission (whether partial or total).

[0050] "Newly diagnosed" refers to individuals who have been diagnosed with EGFR or c-Met-expressing cancer but have not yet received treatment for multiple myeloma.

[0051] The "therapeutic dose" refers to the effective amount required to obtain the desired therapeutic outcome in the necessary dosage and duration. The therapeutic dose may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the drug or combination of drugs to induce the desired response in the individual. An example of an effective drug or combination of drugs is, for example, the patient's improved health.

[0052] "Subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, such as non-human primates, mammals and non-mammals including sheep, dogs, cats, horses, cattle, chickens, amphibians, and reptiles. The terms "subject" and "patient" are used interchangeably herein.

[0053] "Approximately" means that a particular value is within the acceptable margin of error as determined by those skilled in the art, which in part depends on how that value is measured or determined, i.e., on the limitations of the measurement system. In the context of a particular assay, result, or embodiment, unless otherwise expressly stated in the example or elsewhere in this specification, "approximately" means within one standard deviation or up to 5%, whichever is greater, according to the practice of the art.

[0054] Cancer refers to the abnormal growth of cells that tend to proliferate uncontrollably and, in some cases, metastasize (spread) to other parts of the patient's body.

[0055] "EGFR or c-Met expressing cancer" refers to cancer that has detectable expression of EGFR or c-Met, or has mutations or amplifications of EGFR or c-Met. EGFR or c-Met expression, amplification, and mutation status can be detected using known methods such as sequencing, fluorescence in situ hybridization, immunohistochemical analysis, flow cytometry, or Western blotting.

[0056] "Epidermal growth factor receptor" or "EGFR" refers to human EGFR (also known as HER1 or ErbB1 (Ullrich et al., Nature 309:418-425, 1984)) having the amino acid sequence shown in GenBank accession number NP_005219, as well as its naturally occurring variants.

[0057] As used herein, “hepatocyte growth factor receptor,” “c-Met,” or “MET” refers to human c-Met and its native variants having the amino acid sequence shown in GenBank accession number NP_001120972.

[0058] A "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.

[0059] A “biosimilar” (of an approved standard / biological product, i.e., a listed medicine) refers to a biological product that is very similar to the standard product, with only minor differences in clinically inactive components, but based on data obtained from (a) to (c) below, there is no clinically significant difference between the biosimilar and the standard product in terms of safety, purity, and potency: (a) analytical studies demonstrating that the biological product is very similar to the standard product, with only minor differences in clinically inactive components; (b) animal studies (including toxicity assessments); and / or (c) clinical trials (multiple) sufficient to demonstrate safety, purity, and potency under one or more appropriate conditions of use, such as the conditions under which the standard product is approved and the conditions under which the standard product is intended to be used, as well as the conditions for granting approval to the biosimilar (including immunogenicity and pharmacokinetic or pharmacodynamic assessments). A biosimilar may be an interchangeable product that can be used as a substitute for the standard product in a pharmacy without intervention from a prescribing healthcare professional. To comply with further standards of "interoperability," biosimilars are required to produce the same clinical outcomes as the standard formulation in any given patient, and if the biosimilar is administered to an individual multiple times, the risk of reduced safety or efficacy from alternating or switching between the biosimilar and the standard formulation must not exceed the risk of using the standard formulation without such alternation or switching. Furthermore, to the extent that the mechanism of action of the standard formulation is known, the biosimilar must utilize the same mechanism of action (multiple) under the proposed conditions of use. In addition, the conditions or conditions of use (multiple) specified, recommended, or presented in the proposed labeling for the biosimilar are already approved for the standard formulation. The route of administration, dosage form, and / or potency of the biosimilar must also be the same as that of the standard formulation, and the biosimilar must be manufactured, processed, packaged, or stored in facilities that meet standards designed to ensure that the biosimilar maintains safety, purity, and potency. Biosimilars may contain one or more post-translational modifications that differ from the standard formulation, such as a different glycosylation profile, which are not expected to alter the performance of the biosimilar.

[0060] "Specific binding," or "specific binding," or "specific binding," refers to an antibody binding to an antigen or an epitope within an antigen with a higher affinity than it does to other antigens. Typically, an antibody binds to an antigen or an epitope within an antigen with a KD of approximately 5 × 10⁻⁸ M or less, for example, approximately 1 × 10⁻⁹ M or less, approximately 1 × 10⁻¹⁰ M or less, approximately 1 × 10⁻¹¹ M or less, or approximately 1 × 10⁻¹² M or less, which is typically at least 100 times smaller than the KD for binding to a non-specific antigen (e.g., BSA, casein). The dissociation constant can be measured using known protocols. However, antibodies that bind to an antigen or an epitope within an antigen may cross-react to other related antigens, such as the same antigen (homolog) from other species, such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus monkey, cyno) or Pan troglodytes (chimpanzee, chimp). Monospecific antibodies bind to one antigen or one epitope, while bispecific antibodies bind to two different antigens or two different epitopes.

[0061] The term "antibody" has a broad meaning and includes monoclonal antibodies, including mouse, human, humanized, and chimeric monoclonal antibodies; antigen-binding fragments; polyspecific antibodies such as bispecific, triplicate, and quadruplicate antibodies; dimers, tetramers, or multimers; single-chain antibodies; domain antibodies; and immunoglobulin molecules, including any other modified forms of immunoglobulin molecules containing antigen-binding sites of the required specificity. A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds, and a multimer thereof (e.g., IgM). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (composed of domains CH1, hinge, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions are interspersed with framework regions (FRs) and can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs). Each VH and VL consists of three CDRs and four FR segments arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0062] The "complementarity-determining region (CDR)" is the region of an antibody that binds to an antigen. CDRs can be defined using various descriptions, such as Kabat (Wu et al. (1970) J Exp Med 132:211-50) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196:901-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thornton (1996) J Bmol Biol 263:800-15). Various descriptions and their correspondences to variable region numbering are documented (see, for example, Lefranc et al. (2003) Dev Comp Immunol 27:55-77; Honegger and Pluckthun, (2001) J Mol Biol 309:657-70; International ImMunoGeneTics (IMGT) database; web resource, see http: / / www_imgt_org). CDRs can be described using available programs such as abYsis by UCL Business PLC. As used herein, the terms “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2”, and “LCDR3” include CDRs defined by any of the Kabat, Chothia, IMGT, or AbM methods described above, unless otherwise expressly stated herein.

[0063] Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of their heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be assigned to one of two distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0064] An "antigen-binding fragment" refers to a portion of an immunoglobulin molecule that binds to an antigen. Antigen-binding fragments may be synthetic polypeptides, enzyme-available polypeptides, or genetically modified polypeptides, and include VH, VL, VH and VL, Fab, F(ab')2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelid VH domains, FR3-CDR3-FR4 moieties, and other smallest recognition units consisting of amino acid residues that reproduce the CDR of an antibody, such as HCDR1, HCDR2, and / or HCDR3, as well as LCDR1, LCDR2, and / or LCDR3. The VH and VL domains can be linked to each other via synthetic linkers to form various types of single-chain antibody designs. When the VH and VL domains are expressed in separate single-chain antibody constructs, the VH / VL domains can form intramolecular or intermolecular pairs to form monovalent antigen-binding sites, such as single-chain Fv (scFv) or diabodies, which are described, for example, in International Publications 1998 / 44001, 1988 / 01649, 1994 / 13804, and 1992 / 01047.

[0065] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules (i.e., individual antibodies within the population are identical except for possible known modifications such as removal of C-terminal lysine from the antibody heavy chain, or post-translational modifications such as amino acid isomerization or amidogenesis, methionine oxidation, or asparagine or glutamine amidogenesis). Monoclonal antibodies typically bind to one antigenic epitope. Bispecific monoclonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific, such as bispecific, and may be monovalent, bivalent, or polyvalent.

[0066] A "humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Because humanized antibodies can contain substitutions in their framework, the framework may not be an exact copy of the expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0067] A “human antibody” refers to an antibody optimized to produce a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody contains a constant region or a portion of a constant region, that constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is obtained from a system using human germline immunoglobulin or a rearranged immunoglobulin gene, the human antibody will contain heavy-chain and light-chain variable regions that are “derived” from a human sequence. Exemplary such systems include human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals, such as mice or rats, that possess human immunoglobulin loci. A “human antibody” typically contains amino acid differences when compared to immunoglobulin expressed in humans, due to differences in the system used to obtain the human antibody and the human immunoglobulin locus, intentional introduction of somatic mutations or substitutions into the framework or CDR, or both. Typically, a “human antibody” is at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a “human antibody” may contain, for example, a consensus framework sequence obtained from human framework sequence analysis described in Knappik et al., (2000) J Mol Biol 296:57-86, or synthetic HCDR3 incorporated into a human immunoglobulin gene library presented on phages, for example, Shi et al., (2010) J Mol Biol 397:385-96 and International Publication No. 2009 / 085462. Antibodies in which at least one CDR originates from a non-human species are not included in the definition of "human antibodies."

[0068] "Recombinant" refers to DNA, antibodies, and other proteins prepared, expressed, produced, or isolated by recombinant means when segments from different sources are combined to produce recombinant DNA, antibodies, or proteins.

[0069] "Bispecificity" refers to an antibody that specifically binds to two different antigens, or to two different epitopes within the same antigen. Bispecific antibodies may cross-react to other related antigens, such as human or monkey antigens, or to the same antigen (homolog) from other species, such as Macaca cynomolgus (e.g., cynomolgus, cyno) or Pan troglodytes, or they may bind to epitopes shared between two or more different antigens.

[0070] "Multispecificity" refers to an antibody that specifically binds to two or more different antigens within the same antigen, or to two or more different epitopes. Multispecific antibodies may cross-react to other related antigens, such as the same antigen (homolog) from other species, such as humans or monkeys, for example, Macaca cynomolgus (e.g., cynomolgus, cyno) or Pan troglodytes, or they may bind to epitopes shared between two or more different antigens.

[0071] "Monocytes" refer to CD14+CD34- mononuclear leukocytes, a type of white blood cell involved in the first line of defense, and are recognized as being capable of differentiating into dendritic cells or macrophage precursors. Monocytes normally migrate within the bloodstream. In response to external stimuli, monocytes secrete cytokines, which are many immunomodulatory substances, and migrate to infected or tumor sites in tissues, where they differentiate into macrophages. Specifically, monocytes express elevated levels of the CD14 surface antigen marker and may express at least one biomarker selected from CD64, CD93, CD180, CD328, CD329, or peanut agglutinin protein (PNA).

[0072] "Enhancing" or "inducing" means enhancing one or more functions or activities of macrophages by more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or by a statistically significant amount, compared to a control (e.g., enhancement in the presence or absence of a drug that enhances macrophage activity).

[0073] An "agonist" is a molecule that, when bound to a cellular protein, induces at least one reaction or activity that would otherwise be induced by the protein's native ligand. A molecule is an agonist if at least one reaction or activity is induced at least 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more than the at least one reaction or activity induced in the absence of the agonist (e.g., a negative control), or if the induction is statistically significant compared to induction in the absence of the agonist.

[0074] An "antagonist" or "inhibitor" is a molecule that, when bound to a cellular protein, inhibits at least one reaction or activity induced by that protein's native ligand. A molecule is an antagonist if at least one reaction or activity is inhibited by at least about 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more than at least one reaction or activity inhibited in the absence of the antagonist (e.g., a negative control), or if the inhibition is statistically significant compared to the inhibition in the absence of the antagonist.

[0075] A "PD-(L)1 axis inhibitor" refers to a molecule that inhibits PD-1 downstream signaling. A PD-(L)1 axis inhibitor may be a molecule that binds to PD-1, PD-L1, or PD-L2.

[0076] "PD-(L)1 axis inhibitor resistance" or "anti-PD-(L)1 axis inhibitor resistance" refers to cancer that did not respond to treatment with PD-(L)1 axis inhibitors.

[0077] "Biological specimen" refers to collections of fluids, cells, or tissues present within a subject, in addition to similar fluids, cells, or tissues isolated from the subject. Exemplary specimens include biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions from secretory tissues and organs, vaginal secretions, ascites, fluids from the pleural cavity, pericardial cavity, peritoneal cavity, abdominal cavity, and other body cavities, fluids recovered by bronchial lavage, synovial fluid, the subject or biological origin such as cell and organ culture media including conditioned media for cells or organs, liquid solutions in contact with lavage solutions, tissue biopsies, tumor tissue biopsies, tumor tissue specimens, fine-needle aspirations, surgically excised tissues, organ cultures, or cell cultures.

[0078] As used in this application, "low fucose" or "low fucose content" refers to an antibody having a fucose content of approximately 1% to 15%.

[0079] As used herein, "normal fucose" or "normal fucose content" refers to an antibody having a fucose content of approximately 50%, typically 80%, or 85% or more.

[0080] Bispecific anti-EGFR / c-Met antibody The bispecific anti-EGFR / c-Met antibodies used in the methods of this disclosure can be generated by Fab arm exchange (or half-body exchange) between two monospecific bivalent antibodies, for example, by introducing substitutions at the heavy chain CH3 interface in each half to facilitate the formation of heterodimers of two antibody halves with different specificities, either in vitro or using co-expression in a cell-free environment. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and dissociation-association of the CH3 domain. The heavy chain disulfide bond in the hinge region of the parent monospecific antibody is reduced. The resulting free cysteine ​​from one of the parent monospecific antibodies forms an intra-heavy chain disulfide bond with a cysteine ​​residue of the second parent monospecific antibody molecule, while simultaneously, the CH3 domain of the parent antibody is released and reformed by dissociation-association. The CH3 domains of the Fab group can be manipulated to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody having two Fab groups or halves, each binding to a different epitope, namely, an epitope at EGFR and an epitope at c-Met. For example, the bispecific antibody of the present invention can be produced using the technique described in International Publication No. 2011 / 131746. For IgG1 antibodies, the mutation F405L in one heavy chain and K409R in the other heavy chain can be used. For IgG2 antibodies, wild-type IgG2 and IgG2 antibodies having the F405L and R409K substitutions may be used. For IgG4 antibodies, wild-type IgG4 and IgG4 antibodies having the F405L and R409K substitutions may be used. To produce a bispecific antibody, a first monospecific bivalent antibody and a second monospecific bivalent antibody are manipulated to have the aforementioned mutations in the Fc region, and the antibodies are incubated together under sufficiently reducing conditions that allow cysteine ​​to undergo disulfide bond isomerization in the hinge region, thereby producing a bispecific antibody by Fab arm exchange. The incubation conditions can, ideally, be returned to non-reducing conditions.Exemplary reducing agents that can be used include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol. For example, incubation can be performed at a temperature of at least 20°C in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol at a pH of 5 to 8, for example, pH 7.0 or pH 7.4, for at least 90 minutes.

[0081] In some embodiments, the bispecific anti-EGFR / c-Met antibody is an IgG1 isotype. There may be some variation within the constant domain of IgG1 (e.g., well-known allotypes), with variations at positions 214, 356, 358, 422, 431, 435, or 436 (residue numbering according to EU numbering) (see, for example, IMGT Web resources; IMGT Repertoire (IG and TR); Proteins and alleles; allotypes). The bispecific anti-EGFR / c-Met antibody may also be any IgG1 allotype known in the art, such as G1m17, G1m3, G1m1, G1m2, G1m27, or G1m28.

[0082] The bispecific anti-EGFR / c-Met antibodies used in the methods of this disclosure can also be generated using designs such as knob-in-hole (Genentech), CrossMAb (Roche), electrostatically-matched (Chugai, Amgen, NovoNordisk, Oncomed), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), and Biclonic (Merus).

[0083] In the "knob-in-hole" technique (see, for example, International Publication No. 2006 / 028936), the select amino acid forming the interface of the human IgG CH3 domain can be mutated at a position that affects CH3 domain interaction to promote heterodimer formation. An amino acid with a small side chain (the hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (the knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of preferential interaction between the heavy chain containing the "hole" and the heavy chain containing the "knob". Exemplary CH3 substitution pairs that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V (represented as the modification position in the first CH3 domain of the first heavy chain / the modification position in the second CH3 domain of the second heavy chain).

[0084] In addition to utilizing a "knob-in-hole" technique for the Fab arm exchange promoter, CrossMAb technology utilizes CH1 / CL domain exchange on one of the half-arms to ensure correct light chain pairing of the resulting bispecific antibody (see, for example, U.S. Patents 8,242,247).

[0085] The full-length bispecific antibodies of the present invention may be generated by using other cross-referencing techniques to exchange variable, constant, or both domains between or within the heavy chain of the bispecific antibody, in one or both groups. Examples of such exchanges include VH-CH1 and VL-CL, VH and VL, CH3 and CL, and CH3 and CH1 as described in International Publications 2009 / 080254, 2009 / 080251, 2009 / 018386, and 2009 / 080252.

[0086] Other methods, such as promoting heavy chain heterodimerization by using electrostatic interactions through the substitution of a positively charged residue on one CH3 surface with a negatively charged residue on a second CH3 surface, may be used as described in U.S. Patent Application Publication 2010 / 0015133, U.S. Patent Application Publication 2009 / 0182127, U.S. Patent Application Publication 2010 / 028637, or U.S. Patent Application Publication 2011 / 0123532. In other methods, heterodimerization is performed by the following substitutions, as described in U.S. Patent Application Publication 2012 / 0149876 or U.S. Patent Application Publication 2013 / 0195849: L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / This can be enhanced by T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W (represented as the modification position in the first CH3 domain of the first heavy chain / the modification position in the second CH3 domain of the second heavy chain).

[0087] The bispecific antibodies of the present invention may be generated using SEEDbody technology. SEEDbody has a selective IgG residue in its constant domain that is substituted with an IgA residue to promote heterodimerization, as described in U.S. Patent Application Publication No. 2007 / 0287170.

[0088] Mutations are typically performed at the DNA level on molecules such as the constant domain of an antibody, using standard methods.

[0089] In some embodiments, the bispecific anti-EGFR / c-Met antibody is used. It includes a first domain that binds to EGFR, which contains the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6; and a second domain that binds to c-Met, which contains the HCDR1 of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDR1 of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12.

[0090] In some embodiments, the first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, and the second domain that binds to c-Met includes the VH of SEQ ID NO: 15 and the VL of SEQ ID NO: 16.

[0091] In some embodiments, the bispecific anti-EGFR / c-Met antibody is an IgG1 isotype. There are several variations within the constant domain of IgG1 (e.g., well-known allotypes), with variations at positions 214, 356, 358, 422, 431, 435, or 436 (residue numbering according to EU numbering) (see, for example, IMGT Web resources; IMGT Repertoire (IG and TR); Proteins and alleles; allotypes). The bispecific anti-EGFR / c-Met antibody may be any IgG1 allotype such as G1m17, G1m3, G1m1, G1m2, G1m27, or G1m28.

[0092] In some embodiments, the bispecific anti-EGFR / c-Met antibody comprises a first heavy chain (HC1) of SEQ ID NO: 17, a first light chain (LC1) of SEQ ID NO: 18, a second heavy chain (HC2) of SEQ ID NO: 19, and a second light chain (LC2) of SEQ ID NO: 20.

[0093] Other publicly available bispecific anti-EGFR / c-Met antibodies may be used in the methods of this disclosure, provided they exhibit similar characteristics to amivantamab, as described in U.S. Patent No. 9,593,164. Bispecific anti-EGFR / c-Met antibodies that can be used in the methods of this disclosure may also be produced by combining a publicly available EGFR-binding VH / VL domain with a c-Met-binding VH / VL domain and testing the resulting bispecific antibody for its characteristics as described in U.S. Patent No. 9,593,164. In some embodiments, the bispecific anti-EGFR / c-Met antibody is a biosimilar.

[0094] In some embodiments, bispecific anti-EGFR / c-Met antibodies have a low fucose content of approximately 1% to 10%. Bispecific anti-EGFR / c-Met antibodies with a low fucose content may be more effective in treating patients with the FcγRIIIa-158F / F or FcγRIIIa-158F / V genotype. Patients can be analyzed for FcγRIIIa polymorphism using routine methods.

[0095] Antibodies with low fucose content can be produced using various methods that have been reported to successfully express relatively highly defucosylated antibodies containing branched complex-type Fc oligosaccharides, such as: control of culture osmotic pressure (Konno et al., Cytotechnology 64(:249-65, 2012), application of variant CHO strain Lec13 as host cell line (Shields et al., J Biol Chem 277:26733-26740, 2002), application of variant CHO strain EB66 as host cell line (Olivier et al., MAbs;2(4), 2010; electronically published before print; PMID:20562582), and application of rat hybridoma cell line YB2 / 0 as host cell line (Shinkawa et al., J Biol Chem This can be achieved by introducing specific small interfering RNA to the α1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-908, 2004), or by co-expression of β-1,4-N-acetylglucosaminyltransferase III and kifunensin, a potent α-mannosidase II or α-mannosidase I inhibitor (Ferrara et al., J Biol Chem 281:5032-5036, 2006, Ferrara et al., Biotechnol Bioeng 93:851-861, 2006, Xhou et al., Biotechnol Bioeng 99:652-65, 2008).

[0096] PD-(L)1 axis inhibitors In one embodiment, the present invention provides a general concept for inhibiting the PD-(L)1 axis. In various embodiments, the composition comprises an inhibitor of one or more genes or proteins in the PD-(L)1 axis. In various embodiments, the present invention comprises compositions and methods for reducing the level or activity of one or more genes or proteins in the PD-(L)1 axis.

[0097] Those skilled in the art will understand, based on the disclosures provided herein, that a decrease in the level or activity of one or more genes or proteins in the PD-(L)1 axis includes a decrease in the expression of biomarkers, including transcription, translation, or both. Those skilled in the art will also understand, once given the teachings of the present invention, that a decrease in the level or activity of one or more genes or proteins in the PD-(L)1 axis includes a decrease in the amount of polypeptide, a decrease in the amount of mRNA, a decrease in transcription, a decrease in translation, or a combination thereof, and also includes a decrease in any activity of one or more genes or proteins in the PD-(L)1 axis.

[0098] Exemplary inhibitors of the PD-(L)1 axis include, but are not limited to, small interfering RNAs (siRNAs), microRNAs, antisense nucleic acids, ribozymes, expression vectors encoding transdominant-negative variants, antibodies, antibody fragments, fusion proteins, aptamers, peptides, and small molecules.

[0099] Those skilled in the art will understand, based on the disclosures provided herein, that one method for reducing the levels of mRNA and / or protein of one or more PD-(L)1 axis proteins in a cell is by reducing or inhibiting the expression of nucleic acids encoding PD-(L)1 axis proteins. Thus, the protein levels of PD-(L)1 axis proteins in cells can be reduced using molecules or compounds that inhibit or reduce gene expression, such as siRNA, antisense molecules, or ribozymes. However, the present invention should not be limited to these examples.

[0100] In one embodiment, RNAi is used to reduce the level or activity of PD-(L)1 axis protein. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types leads to the degradation of complementary mRNA. In cells, long dsRNA is cleaved into short 21-25 nucleotide small interfering RNA, i.e., siRNA, by ribonucleases known as Dicer. The siRNA then assembles with protein components into an RNA-induced silencing complex (RISC), which unravels in the process. The activated RISC then binds to the complementary transcript through base pairing interactions between the siRNA antisense strand and mRNA. The bound mRNA is cleaved, and sequence-specific degradation of the mRNA results in gene silencing. Chemical modification of siRNA can be used to facilitate intravenous systemic delivery. siRNA optimization involves considering the overall G / C content, terminal C / T content, Tm, and nucleotide content of the 3' overhang. Accordingly, the present invention also includes a method for reducing the level of one or more PD-(L)1 axis proteins using RNAi technology.

[0101] In some embodiments, the present invention includes an isolated nucleic acid encoding an inhibitor, such as a protein, antibody, siRNA, or antisense molecule, operably linked to a nucleic acid containing a promoter / regulatory sequence, such that the nucleic acid preferably has the ability to control the expression of the inhibitor encoded by that nucleic acid. Accordingly, the present invention encompasses expression vectors and methods for introducing exogenous DNA into cells, involving the co-expression of exogenous DNA in cells.

[0102] To evaluate inhibitor expression, the expression vector to be introduced into cells may also contain either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via a viral vector. In other embodiments, the selectable marker may be supported on a separate DNA fragment and used in a simultaneous transfection procedure. Both the selectable marker gene and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers are known in the art and include, for example, antibody resistance genes such as neomycin.

[0103] If the inhibitor of the present invention is a small molecule, the small molecule antagonist can be obtained using standard methods known to those skilled in the art. Such methods include chemical organic synthesis or biological means. Biological means include purification from biological sources, recombinant synthesis and in vitro translation systems using methods well known in the art.

[0104] Combinatorial libraries of molecularly diverse compounds that are potentially useful in treating various diseases and conditions are well known in the art, as are methods for constructing such libraries. These methods may utilize a variety of techniques well known to those skilled in the art, including solid-phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear arrangements, deconvolution strategies, tagging techniques, and the generation of unbiased molecular landscapes for read discovery versus biased structures for read development.

[0105] In common methods for small library synthesis, an activated core molecule is condensed with numerous building blocks to produce a combinatorial library of covalently bonded core-building block ensembles. The shape and stiffness of the core determine the orientation of the building blocks in shape space. Libraries can be biased by altering the core, bonds, or building blocks to target characterized biological structures ("focused libraries"), or they can be synthesized with less structural bias using flexible cores.

[0106] In another aspect of the present invention, one or more proteins in the PD-(L)1 axis can be inhibited by inactivating and / or blocking the protein(s). Thus, inhibition of the effect of one or more proteins in the PD-(L)1 axis can be achieved by using transdominant-negative mutants.

[0107] In one embodiment, antibodies specific to one or more proteins in the PD-(L)1 axis may be used. As will be understood by those skilled in the art, any antibody capable of recognizing and binding to the antigen of interest is useful in the present invention. Methods for producing and using antibodies are well known in the art. For example, polyclonal antibodies useful in the present invention are produced by immunizing rabbits according to standard immunological techniques well known in the art. Such techniques involve immunizing animals with a chimeric protein comprising a portion of another protein, such as a maltose-binding protein or a glutathione (GSH)-tagged polypeptide moiety, and / or a portion that makes the antigenic protein of interest immunogenic (e.g., keyhole limpet hemocyanin, the antigen of interest conjugated with KLH), and a portion containing the respective antigenic protein amino acid residues. The chimeric protein is produced by cloning a suitable nucleic acid encoding a marker protein into a plasmid vector suitable for this purpose, such as, but not limited to, pMAL-2 or pCMX.

[0108] Examples of anti-PD-(L)1 axis antibodies include nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimusumab or cetrelimab, or antibodies that bind to PD-L1. PD-L1 antibodies include, but are not limited to, emvafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210. In some embodiments, the anti-PD-(L)1 axis antibody is an anti-PD-1 antibody, such as an antibody that specifically binds to PD-1.

[0109] It is generally known in the art that each anti-PD-(L)1 axis inhibitor antibody has its own unique efficacy and safety profile.

[0110] In some embodiments, the anti-PD-(L)1 axis antibody is, The antibody includes the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, HCDR3 of SEQ ID NO: 23, the light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 26. In some embodiments, the anti-PD-(L)1 axis antibody includes the heavy chain variable region (VH) of SEQ ID NO: 27 and the light chain variable region (VL) of SEQ ID NO: 28. In some embodiments, the anti-PD-(L)1 axis antibody includes the heavy chain (HC) of SEQ ID NO: 29 and the light chain (LC) of SEQ ID NO: 30.

[0111] In some embodiments, the anti-PD-(L)1 axis antibody is, The antibody includes the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32, HCDR3 of SEQ ID NO: 33, the light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 34, LCDR2 of SEQ ID NO: 35, and LCDR3 of SEQ ID NO: 36. In some embodiments, the anti-PD-(L)1 axis antibody includes the heavy chain variable region (VH) of SEQ ID NO: 37 and the light chain variable region (VL) of SEQ ID NO: 38. In some embodiments, the anti-PD-(L)1 axis antibody includes the heavy chain (HC) of SEQ ID NO: 39 and the light chain (LC) of SEQ ID NO: 40.

[0112] However, the present invention should not be construed as being limited solely to methods and compositions comprising these antibodies. Rather, the present invention should be construed as comprising other antibodies, antibody fragments, or antibody mimics against the PD-(L)1 protein or a portion thereof.

[0113] Those skilled in the art will understand, based on the disclosures provided herein, that antibodies can specifically bind to any portion of a PD-(L)1-axis protein(s), and that full-length proteins can be used to produce antibodies specific to that protein. However, the present invention is not limited to the use of full-length proteins as immunogens. Rather, the present invention includes using the immunogenic portion of a protein to produce antibodies that specifically bind to a particular antigen. That is, the present invention includes immunizing an animal with the immunogenic portion or antigenic determinant of an antigen.

[0114] Once the sequence of the specific antigen of interest, as well as a detailed analysis to locate various conserved and non-conserved domains of the protein, is provided, those skilled in the art will understand, based on the disclosures provided herein, how to obtain antibodies specific to various parts of the antigen using methods known in the art or methods to be developed.

[0115] Those skilled in the art will understand, based on the disclosures provided herein, that the present invention involves the use of a single antibody that recognizes a single antigen epitope, but is not limited to the use of a single antibody. Instead, the present invention encompasses the use of at least one antibody, which may be for the same or different antigen protein epitopes.

[0116] The production of polyclonal antibodies is achieved by inoculating a desired animal with an antigen and then isolating antibodies that specifically bind to the antigen from that animal using standard antibody production methods.

[0117] Monoclonal antibodies against full-length proteins or peptide fragments may be prepared using any well-known monoclonal antibody preparation procedure. Large quantities of the desired peptide may also be synthesized using chemical synthesis techniques. Alternatively, the DNA encoding the desired peptide may be cloned and expressed from a suitable promoter sequence in cells suitable for large-scale peptide production. Monoclonal antibodies against peptides are produced from peptide-immunized mice using the standard methods described herein.

[0118] The present invention also includes the use of a humanized antibody that specifically reacts with an epitope of an antigen of interest. The humanized antibody of the present invention has a human framework and has one or more complementarity-determining regions (CDRs) that specifically react with the antigen of interest, typically derived from a mouse antibody. When the antibody used in the present invention is humanized, the antibody may be produced by expressing a recombinant DNA segment encoding heavy and light chain complementarity-determining regions (CDRs) from a donor immunoglobulin that can bind to a desired antigen, such as an epitope on the antigen of interest, conjugated to a DNA segment encoding the receptor human framework region. Generally speaking, the DNA segment will typically include an expression regulatory DNA sequence operably linked to a humanized immunoglobulin coding sequence, which may include a natively related promoter region or a heterologous promoter region. The expression regulatory sequence may be a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells, or the expression regulatory sequence may be a prokaryotic promoter system in a vector capable of transforming or transfecting prokaryotic host cells. Once the vector is incorporated into a suitable host, the host is maintained under conditions favorable for high-level expression of the introduced nucleotide sequence.

[0119] The present invention also includes antibody mimics for functional equivalents of antibodies described herein. Functional equivalents have binding properties comparable to those of antibodies and include, for example, hybridized antibodies and single-chain antibodies, as well as fragments thereof.

[0120] A functional equivalent comprises a polypeptide having an amino acid sequence substantially identical to the amino acid sequence of the variable or hypervariable region of an antibody. A “substantially identical” amino acid sequence is defined herein as a sequence having at least 70%, 80%, 90%, 95%, or 99% (or any integer between 70 and 99) identity to another amino acid sequence, as determined by a sequence similarity search algorithm. A chimeric antibody or other hybrid antibody has a constant region substantially or exclusively derived from the constant region of a human antibody, and a variable region substantially or exclusively derived from the sequence of the variable region of a monoclonal antibody derived from each stable hybridoma.

[0121] A single-chain antibody (scFv) or Fv fragment is a polypeptide consisting of a variable region of the heavy chain of an antibody linked to a variable region of the light chain, with or without an interlinking linker. Therefore, Fv contains an antibody binding site. In one embodiment, scFv can be fused to a half-life extension portion, for example, Fc, using a method known to those skilled in the art.

[0122] The functional equivalent of the antibody of the present invention further comprises an antibody fragment having the same or substantially the same binding properties as the entire antibody. Such a fragment may contain one or both of the Fab fragment or the F(ab')2 fragment. The antibody fragment contains all six complement-determining regions of the entire antibody, but a fragment containing fewer regions than all of such regions (e.g., three, four, or five complement-determining regions) is also functional. The functional equivalent is a member of the IgG immunoglobulin class and its subclasses, but may be any one of the following immunoglobulin classes: IgM, IgA, IgD, or IgE, and their subclasses, or a combination thereof. The heavy chains of various subclasses, such as the IgG subclasses, are responsible for different effector functions, and therefore, by selecting the desired heavy chain constant region, a hybrid antibody with the desired effector function can be produced. Exemplary constant regions are gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), and gamma 4 (IgG4). The light chain steady region can be kappa-type or lambda-type.

[0123] The immunoglobulins of the present invention may be monovalent, divalent, or polyvalent. Monovalent immunoglobulins are dimers (HL) formed from a hybrid heavy chain associated with a hybrid light chain via a disulfide crosslink. Divalent immunoglobulins are tetramers (H2L2) formed from two dimers associated via at least one disulfide crosslink.

[0124] Additional medications In some embodiments, the combination therapy of the present invention comprises the above-mentioned bispecific anti-EGFR / c-Met antibody and PD-(L)1 axis inhibitor in combination with one or more additional therapeutic agents.

[0125] In some embodiments, the combination therapy of the present invention comprises one or more additional anticancer agents. In some embodiments, the additional anticancer agent is GM-CSF, a CD47 antagonist, an anti-CD47 antibody, an HDAC inhibitor, or a CD11b agonist.

[0126] Exemplary CD47 antagonists are CD47 ligand-Fc fusions, such as SIRPα-Fc fusions like TTI621 and anti-CD47 antibodies.

[0127] Exemplary anti-CD47 antibodies are Hu5F9-G4, TI-061, TTI-622, AO-176, IBI-188, ALX-148, SRF-231, CC-90002, and other anti-CD47 antibodies disclosed in International Publication No. 2016 / 081423.

[0128] Exemplary HDAC inhibitors include vorinostat, romidepsin, tidamide, panobinostat, bellinostat, prasinostat, avexinostat, entinostat, bafidemstat, GSK-2879552, licorinostat, idademstat, domatinostat, resminostat, AZD-9468, nanatinostat, CG-200745, mosetinostat, INCB-59872, IMG-7289, tinostamstine, RDN-929, YM-753, HG-146, NBM-BMX, TAK-418, secridemsitat, CKD-504, CKD-506, CC-90011, KA-2507, and citrinostat.

[0129] Commercially available antibodies can be purchased through authorized distributors or pharmacies. The amino acid sequences of small molecules can be found in USAN and / or INN deposits by companies in the CAS registry.

[0130] Treatment method It is well known in this technology field that drug development is an unpredictable area. This lack of predictability is demonstrated, for example, by the requirements of health organizations (such as the Food and Drug Administration) for establishing safe and effective drug regimens for each individual drug candidate in clinical trials. Over the past decade (2011–2020), only 7.9% of all drug candidates in development achieved FDA approval from Phase I clinical trials. See Clinical Development Success Rates and Contributing Factors 2011–2020. Success rates are even lower in oncology, with only 5.3% of oncology drug candidates succeeding.

[0131] In the field of oncology, even for drugs with established dosages for specific indications, the Food and Drug Administration (FDA) recommends further clinical trials to identify optimal dosages for new indications; otherwise, patients may be exposed to unreasonable and significant risks, among other potential drawbacks. See, for example, Optimizing the Dosage of Human Prescription Drugs and Biological Products for the Treatment of Oncologic Diseases; Draft Guidance for Industry; January 2023.

[0132] This disclosure provides a method for treating a subject having a solid tumor, comprising administering a therapeutically effective dose of an isolated bispecific epidermal growth factor receptor (EGFR) / hepatocyte growth factor receptor (c-Met) antibody to the subject in combination with an agent for inhibiting the PD-(L)1 axis in the subject.

[0133] This disclosure provides a method for remodeling the tumor microenvironment of a subject having a solid tumor, comprising administering a therapeutically effective dose of isolated bispecific epidermal growth factor receptor (EGFR) / hepatocyte growth factor receptor (c-Met) antibody to the subject in combination with an agent for inhibiting the PD-(L)1 axis in the subject. In some embodiments, the method comprises inhibiting glycolysis in the tumor microenvironment of a subject having a solid tumor, comprising administering a therapeutically effective dose of isolated bispecific epidermal growth factor receptor (EGFR) / hepatocyte growth factor receptor (c-Met) antibody to the subject in combination with an agent for inhibiting the PD-(L)1 axis in the subject. In some embodiments, the method comprises reducing lactate production in the tumor microenvironment of a subject having a solid tumor, comprising administering a therapeutically effective dose of isolated bispecific epidermal growth factor receptor (EGFR) / hepatocyte growth factor receptor (c-Met) antibody to the subject in combination with an agent for inhibiting the PD-(L)1 axis in the subject.

[0134] In some embodiments, the method comprises inhibiting the EGFR and MET signaling pathways in tumor cells of a subject having a solid tumor, and administering a therapeutically effective dose of isolated bispecific epidermal growth factor receptor (EGFR) / hepatocyte growth factor receptor (c-Met) antibody to the subject in combination with an agent for inhibiting the PD-(L)1 axis in the subject. In some embodiments, the method comprises targeting EGFR and MET-expressing tumor cells for destruction by immune effector cells such as natural killer cells and macrophages via antibody-dependent cell-mediated cytotoxicity (ADCC) and trogocytosis mechanisms, and administering a therapeutically effective dose of isolated bispecific EGFR / c-Met antibody to the subject in combination with an agent for inhibiting the PD-(L)1 axis in the subject.

[0135] This disclosure provides a method for increasing the level of immune cell infiltration into a tumor or tumor microenvironment of a subject having a solid tumor, comprising administering a therapeutically effective dose of an isolated bispecific epidermal growth factor receptor (EGFR) / hepatocyte growth factor receptor (c-Met) antibody to the subject in combination with an agent for inhibiting the PD-(L)1 axis in the subject.

[0136] The “level” of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) can be qualitative (e.g., present or absent) or quantitative (e.g., absolute cell count, relative count, percentage of total cell count (%), or %) of positive cells in the field of view. In some embodiments, the level of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) is above the mean level of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) observed in biological samples derived from healthy subjects. In some embodiments, the level of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) is approximately the 55th percentile of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) observed in biological samples derived from subjects having a similar type and stage of cancer as the subject. In some embodiments, the level of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) is approximately the 60th percentile of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) observed in biological samples derived from the subject having a cancer type and stage equivalent to that of the subject. In some embodiments, the level of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) is approximately the 65th percentile of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) observed in biological samples derived from the subject having a cancer type and stage equivalent to that of the subject. In some embodiments, the level of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) is approximately the 70th percentile of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) observed in biological samples derived from the subject having a cancer type and stage equivalent to that of the subject. In some embodiments, the level of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) is approximately the 75th percentile of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) observed in biological samples derived from the subject having a cancer type and stage equivalent to that of the subject.In some embodiments, the level of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) is approximately the 80th percentile of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) observed in biological samples derived from the subject having a cancer type and stage equivalent to that of the subject. In some embodiments, the level of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) is approximately the 85th percentile of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) observed in biological samples derived from the subject having a cancer type and stage equivalent to that of the subject. In some embodiments, the level of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) is approximately the 90th percentile of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) observed in biological samples derived from the subject having a cancer type and stage equivalent to that of the subject. In some embodiments, the level of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) is approximately the 95th percentile of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) observed in a biological sample derived from the subject having a cancer type and stage equivalent to that of the subject. In some embodiments, the level of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) is approximately the 100th percentile of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) observed in a biological sample derived from the subject having a cancer type and stage equivalent to that of the subject. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a tumor tissue biopsy.

[0137] The levels of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) in the target tumor microenvironment may also be compared to the levels of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) in a biological sample derived from a healthy subject. The increased levels of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) may be, for example, about 1.5 times, about 2 times, about 2.5 times, about 3 times, about 3.5 times, about 4 times, about 4 times, about 5 times, about 5.5 times, about 6 times, about 6.5 times, about 7 times, about 7.5 times, about 8 times, about 8.5 times, about 9 times, or about 10 times higher when compared to the levels of immune cells (e.g., CD8+ T cells, central memory cytotoxic T cells) in a biological sample derived from a healthy subject. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a tissue sample.

[0138] The level of immune cells may be identified, for example, from tumor tissue biopsies obtained from subjects, by immunohistochemistry, evaluating the percentage of areas positively stained using, for example, 4-1BB, CD45RA, PD-1, CD25, CD28, CD3, CD56, FoxP3, CD45, CD4, CCR7, CD8a, and 7-AAD as T cell markers, and comparing this to non-tumor tissue. The level of immune cells may also be identified, for example, from tumor tissue biopsies obtained from subjects with EGFR or c-Met expressing tumors, using immunogene signatures.

[0139] Monocytes may be identified from blood samples derived from subjects with EGFR or c-Met-expressing tumors using, for example, fluorescent cell sorting with monocyte markers such as CD40, CD45RA, CD80, CD86, HLA-DR, CD11c, CD14, CD68, CD45, CD11b, CD19, CD123, CD15, and 7-AAD.

[0140] In some embodiments, the patient's response to the combination therapy of the present invention may be predicted by using the levels of FcγRI or FcγRIIIa to provide more immune cell interactions. For example, in some embodiments, the levels of FcγRI or FcγRIIIa are higher than the average levels of FcγRI or FcγRIIIa observed in biological samples from healthy subjects or in control groups.

[0141] FcγRI or FcγRIIIa levels may be measured using immunohistochemical testing in tumor tissue samples, such as fresh-frozen tumor tissue sections or paraffin-embedded tumor tissue sections. FcγRI or FcγRIIIa levels may also be expressed as the percentage (%) of FcγRI or FcγRIIIa cells in the microscopic field. FcγRI or FcγRIIIa levels may also be measured at the gene expression level using RNA isolated from tumor tissue samples, either as part of an immunogene signature panel or as individual genes.

[0142] In some embodiments, the cancer is 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, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC).

[0143] In some embodiments, the cancer is resistant to treatment with PD-(L)1 axis inhibitors. In some embodiments, the cancer is resistant to treatment with PD-L1 inhibitors. In some embodiments, the cancer is resistant to treatment with anti-PD-L1 antibodies. In some embodiments, the cancer is resistant to treatment with PD-L1 axis inhibitors.

[0144] In some embodiments, the cancer is EGFR or c-Met expressing cancer. In some embodiments, the EGFR or c-Met expressing cancer is epithelial cell carcinoma. In some embodiments, the EGFR or c-Met expressing cancer is breast cancer. In some embodiments, the EGFR or c-Met expressing cancer is ovarian cancer. In some embodiments, the EGFR or c-Met expressing cancer is lung cancer. In some embodiments, the EGFR or c-Met expressing cancer is non-small cell lung cancer (NSCLC). In some embodiments, the EGFR or c-Met expressing cancer is lung adenocarcinoma. In some embodiments, the EGFR or c-Met expressing cancer is small cell lung cancer. In some embodiments, the EGFR or c-Met expressing cancer is colorectal cancer. In some embodiments, the EGFR or c-Met expressing cancer is anal cancer. In some embodiments, the EGFR or c-Met expressing cancer is prostate cancer. In some embodiments, the EGFR or c-Met expressing cancer is kidney cancer. In some embodiments, EGFR or c-Met-expressing cancer is bladder cancer. In some embodiments, EGFR or c-Met-expressing cancer is head and neck cancer. In some embodiments, EGFR or c-Met-expressing cancer is pharyngeal cancer. In some embodiments, EGFR or c-Met-expressing cancer is nasal cancer. In some embodiments, EGFR or c-Met-expressing cancer is pancreatic cancer. In some embodiments, EGFR or c-Met-expressing cancer is skin cancer. In some embodiments, EGFR or c-Met-expressing cancer is oral cancer. In some embodiments, EGFR or c-Met-expressing cancer is tongue cancer. In some embodiments, EGFR or c-Met-expressing cancer is esophageal cancer. In some embodiments, EGFR or c-Met-expressing cancer is vaginal cancer. In some embodiments, EGFR or c-Met-expressing cancer is cervical cancer. In some embodiments, EGFR or c-Met-expressing cancer is spleen cancer. In some embodiments, EGFR or c-Met-expressing cancer is testicular cancer. In some embodiments, EGFR or c-Met-expressing cancer is gastric cancer.In some embodiments, EGFR or c-Met-expressing cancer is thymic cancer. In some embodiments, EGFR or c-Met-expressing cancer is colon cancer. In some embodiments, EGFR or c-Met-expressing cancer is thyroid cancer. In some embodiments, EGFR or c-Met-expressing cancer is liver cancer. In some embodiments, EGFR or c-Met-expressing cancer is hepatocellular carcinoma (HCC). In some embodiments, EGFR or c-Met-expressing cancer is sporadic or hereditary papillary renal cell carcinoma (PRCC).

[0145] In some embodiments, NSCLC includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. In some embodiments, NSCLC cells have an epithelial phenotype. In some embodiments, NSCLC has acquired resistance to treatment with one or more EGFR inhibitors.

[0146] In some embodiments, EGFR or c-Met-expressing cancers are associated with wild-type EGFR, EGFR-activating mutations, EGFR gene amplification, elevated levels of circulating HGF, wild-type c-Met, c-Met-activating mutations, c-Met gene amplification, or mutant KRAS.

[0147] Exemplary EGFR-activating mutations that may be associated with cancer include point mutations, deletion mutations, insertion mutations, inversions, or gene amplifications that increase at least one biological activity of EGFR, such as increased tyrosine kinase activity, formation of receptor homodimers and heterodimers, or enhanced ligand binding. Mutations may be located in any part of the EGFR gene or a regulatory region associated with the EGFR gene, and may include mutations in exons 18, 19, 20, or 21, or mutations in the kinase domain. Other examples of EGFR-activating mutations are known in the art (see, for example, U.S. Patent Application Publication 2005 / 0272083). Information on 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, for example, Hynes and Lane, Nature Reviews Cancer 5:341-354, 2005).

[0148] In some embodiments, EGFR activating mutations include substitutions of L718Q, G719A, G719X (where X is any amino acid), L861X (where X is any amino acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, L858P, or T790M, deletions of E746-A750, deletions of R748-P753, insertion of Ala(A) between M766 and A767, and insertions of Ser, Val, and Ala between S768 and V769. This includes the insertion of (SVA), the insertion of Asn and Ser(NS) between P772 and H773, the insertion of one or more amino acids between D761 and E762, A763 and Y764, Y764 and Y765, M766 and A767, A767 and V768, S768 and V769, V769 and D770, D770 and N771, N771 and P772, P772 and H773, H773 and V774, and V774 and C775, as well as one or more deletions or one or more insertions in EGFR exon 20.

[0149] Exemplary c-Met activating mutations include point mutations, deletion mutations, insertion mutations, inversions, or gene amplifications that increase at least one biological activity of the c-Met protein, such as increased tyrosine kinase activity, formation of receptor homodimers and heterodimers, and enhanced ligand binding. Mutations can be located in any part of the c-Met gene or in gene-related regulatory regions, such as mutations in the kinase domain of c-Met. Exemplary c-Met activating mutations are mutations at residue positions N375, V13, V923, R175, V136, L229, S323, R988, S1058 / T1010, and E168. Methods for detecting mutations or gene amplifications of EGFR and c-Met are well known.

[0150] In some embodiments, the variant KRAS has substitutions of G12V, G12C, or G12A.

[0151] In NSCLC, specific mutations in the EGFR gene are associated with a high response rate (70-80%) to EGFR tyrosine kinase inhibitors (EGFR TKIs). Five amino acid deletions within exon 19 or the point mutation L858R in EGFR are associated with EGFR TKI sensitivity (Nakata and Gotoh, Expert Opin Ther Targets 16:771-781, 2012). These mutations result in ligand-independent activation of EGFR kinase activity. Activated EGFR mutations occur in 10-30% of NSCLC patients and are significantly more common in East Asians, women, non-smokers, and patients with histological findings of adenocarcinoma (Janne and Johnson Clin Cancer Res 12(14 Suppl):4416s-4420s, 2006). EGFR gene amplification is also strongly correlated with the response to EGFR TKI treatment (Cappuzzo et al., J Natl Cancer Inst 97:643-55, 2005). EGFR exon 20 insertion is associated with EGFR TKI resistance.

[0152] The majority of NSCLC patients with EGFR mutations initially respond to EGFR TKI therapy, but virtually all acquire resistance that prevents a sustained response. 50–60% of patients acquire resistance due to a second-site point mutation (T790M) in the kinase domain of EGFR. Nearly 60% of all tumors that become resistant to EGFR tyrosine kinase inhibitors show increased c-Met expression, c-Met amplification, or increased HGF, its only known ligand (Turke et al., Cancer Cell, 17:77-88, 2010).

[0153] In some embodiments, the subject is homozygous for phenylalanine at position 158 of CD16, or heterozygous for both valine and phenylalanine at position 158 of CD16.

[0154] Subjects homozygous for phenylalanine at position 158 of CD16 have the FcγRIIIa-158F / F genotype. Subjects heterozygous for both valine and phenylalanine at position 158 of CD16 have the FcγRIIIa-158F / V genotype. CD16 is also known as Fc gamma receptor IIIa (FcγRIIIa) or low affinity immunoglobulin gamma Fc region receptor III-A isoform. Valine / phenylalanine (V / F) polymorphism at position 158 of the FcγRIIIa protein residue has been shown to affect FcγRIIIa affinity for human IgG. Receptors with FcγRIIIa-158F / F or FcγRIIIa-158F / V polymorphism show reduced Fc binding and therefore reduced ADCC compared to FcγRIIIa-158V / V. The absence or low amount of fucose in human N-linked oligosaccharides improves the antibody's ability to induce ADCC, due to improved binding of the antibody to human FcγRIIIa(CD16) (Shields et al., J Biol Chem 277:26733-40, 2002).

[0155] In some embodiments, subjects have a newly diagnosed EGFR or c-Met-expressing cancer. In some embodiments, subjects with a newly diagnosed EGFR or c-Met-expressing cancer have one or more EGFR exon 20 mutations. Exon 20 mutations (insertions of one or more amino acids) are generally resistant to EGFR tyrosine kinase inhibitors (TKIs) (see, for example, International Publication 2018 / 094225).

[0156] In some embodiments, the subject is resistant to or has acquired resistance to previous anti-cancer therapies.

[0157] In some embodiments, the prior anticancer therapy is chemotherapy, targeted anticancer therapy, or kinase inhibitors.

[0158] In some embodiments, the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL.

[0159] In some embodiments, the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib.

[0160] In some embodiments, the subjects are resistant to or have acquired resistance to EGFR inhibitors. Examples of EGFR inhibitors that cancer may develop resistance to include the anti-EGFR antibodies cetuximab (ERBITUX®), pantanumumab (VECTIBIX®), matuzumab, nimotuzumab; the small molecule EGFR inhibitors erlotinib (TARCEVA®), gefitinib (IRESSA®), EKB-569 (peritinib, irreversible EGFR TKI); and the pan-ErbB and other receptor tyrosine kinase inhibitors lapatinib (EGFR and HER2 inhibitor), peritinib (EGFR and HER2 inhibitor), vandetanib (ZD6474, ZACTIMA®, EGFR, VEGFR2, and RET TKI), PF00299804 (dacomitinib, irreversible pan-ErbB TKI), and CI-1033 (irreversible pan-erbB These include afatinib (BIBW2992, irreversible pan-ErbB TKI), AV-412 (dual EGFR and ErbB2 inhibitor), EXEL-7647 (EGFR, ErbB2, GEVGR, and EphB4 inhibitor), CO-1686 (irreversible mutant-selective EGFR TKI), AZD9291 (irreversible mutant-selective EGFR TKI), and HKI-272 (neratinib, irreversible EGFR / ErbB2 inhibitor).

[0161] Various qualitative and / or quantitative methods can be used to determine whether a subject is resistant to, exhibiting resistance to, or prone to developing resistance to anticancer therapy. Symptoms that may be associated with resistance to anticancer therapy include a decline or steady state of the patient's health, an increase in tumor size, a cessation or slowing of tumor growth reduction, and / or the spread of cancer cells within the body from one location to other organs, tissues, or cells. The re-establishment or worsening of various cancer-related symptoms such as loss of appetite, cognitive impairment, depression, dyspnea, fatigue, hormonal disruption, neutropenia, pain, peripheral neuropathy, and sexual dysfunction may also be indicators that a subject is exhibiting or prone to developing resistance to anticancer therapy. Cancer-related symptoms may vary depending on the type of cancer. For example, symptoms associated with cervical cancer may include abnormal bleeding, abnormally heavy vaginal discharge, pelvic pain unrelated to the normal menstrual cycle, bladder pain or pain during urination, and bleeding during regular menstrual periods, after sexual intercourse, vaginal douching, or pelvic examination. Symptoms associated with lung cancer include persistent cough, hemoptysis, shortness of breath, wheezing chest pain, loss of appetite, unintentional weight loss, and fatigue. Symptoms of liver cancer include loss of appetite and weight, abdominal pain, especially in the upper right abdomen which may extend to the back and shoulders, nausea and vomiting, general weakness and fatigue, hepatomegaly, abdominal distension (ascites), and yellowing of the skin and whites of the eyes (jaundice). Those skilled in oncology can easily identify the symptoms associated with specific types of cancer.

[0162] In some embodiments, the subjects are further subjected to one or more additional anti-cancer therapies.

[0163] In some embodiments, the additional anticancer therapy is chemotherapy, targeted anticancer therapy, or kinase inhibitors.

[0164] In some embodiments, the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL. In some embodiments, the kinase inhibitor is an inhibitor of EGFR. In some embodiments, the kinase inhibitor is an inhibitor of c-Met. In some embodiments, the kinase inhibitor is an inhibitor of HER2. In some embodiments, the kinase inhibitor is an inhibitor of HER3. In some embodiments, the kinase inhibitor is an inhibitor of HER4. In some embodiments, the kinase inhibitor is an inhibitor of VEGFR. In some embodiments, the kinase inhibitor is an inhibitor of or AXL.

[0165] In some embodiments, the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib.

[0166] In some embodiments, the kinase inhibitor is erlotinib. In some embodiments, the kinase inhibitor is gefitinib. In some embodiments, the kinase inhibitor is lapatinib. In some embodiments, the kinase inhibitor is vandetanib. In some embodiments, the kinase inhibitor is afatinib. In some embodiments, the kinase inhibitor is osimertinib. In some embodiments, the kinase inhibitor is razertinib. In some embodiments, the kinase inhibitor is poziotinib. In some embodiments, the kinase inhibitor is criotinib. In some embodiments, the kinase inhibitor is cabozantinib. In some embodiments, the kinase inhibitor is capmatinib. In some embodiments, the kinase inhibitor is axitinib. In some embodiments, the kinase inhibitor is lenvatinib. In some embodiments, the kinase inhibitor is nintedanib. In some embodiments, the kinase inhibitor is regorafenib. In some embodiments, the kinase inhibitor is pazopanib. In some embodiments, the kinase inhibitor is sorafenib. In some embodiments, the kinase inhibitor is sunitinib.

[0167] In the method disclosed herein, anticancer therapies that may be administered in combination with the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor include any one or more chemotherapeutic agents or other anticancer agents known to those skilled in the art. Chemotherapy agents are chemical compounds useful for treating cancer and include growth inhibitors or other cytotoxic agents, such as alkylating agents, antimetabolites, antimicrotubule inhibitors, topoisomerase inhibitors, receptor tyrosine kinase inhibitors, and angiogenesis inhibitors. Examples of chemotherapeutic agents include alkylating agents, e.g., thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates, e.g., busulfan, improsulfan, and piposulfan; aziridines, e.g., benzodopa, carbocon, metsuredopa, and uredopa; ethyleneimines and methylamines, including altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. Lamin (methylamelamine); Nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobembitin, fenestrine, prednimustine, trophosphamide, uracil mustard; Nitrosoureas, e.g., carmustine, chlorozotosine, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as aclasinomycin, actinomycin, autramycin, azaserin, bleomycin, kactinomycin, calicheamicin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, Marcellomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-FU; folic acid analogues Phosphates, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine, etc.; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepicio Stan, testolactone; anti-adrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folic acid; acegraton; aldofamide glycoside; aminolevulinic acid; amsacrin; bestrabusil; bisanthren; edatraxate; defofamine; demecolsin; diaziquan; elfornithine; elliptinium acetate; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamin; mitogluazone; mitoxantrone; mopidamol; nitracrin; pentostatin; fenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; schizophyllan;Spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; members of the taxoid or taxane family, e.g., paclitaxel (TAXOL®, docetaxel (TAXOTERE®)) and its analogs; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristi Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; Xeloda; Ibandronate; CPT-11; Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO); Retinoic acid; Esperamicin; Capecitabine; Sorafenib (NEXAVAR®), Sunitinib (SUTENT®), Pazopanib (VOTR IENT(trademark), Toceranib (PALLADIA(trademark)), Vandetanib (ZACTIMA(trademark)), Cejiranib (RECENTIN(registered trademark)), Regorafenib (BAY73-4506), Axitinib (AG013736), Restaurtinib (CEP-701), Erlotinib (TARCEVA(registered trademark)), Gefitinib (IRESSA(trademark)), Afatinib (BIBW Examples include receptor tyrosine kinase and / or angiogenesis inhibitors, such as lapatinib (TYKERB®), neratinib (HKI-272), and any pharmaceutically acceptable salts, acids, or derivatives thereof. Antihormone agents that act to control or inhibit the hormonal effects on tumors, such as anti-estrogen agents including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifen, LY 117018, onapristone, and toremifene (FARESTON®);Furthermore, antiandrogen agents, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as any pharmaceutically acceptable salts, acids, or derivatives of any of the above, are also included in this definition. Other conventional cytotoxic compounds disclosed in Wiemann et al., 1985, Medical Oncology (Calabresi et al., eds.), Chapter 10, McMillan Publishing, are also applicable to the methods of the present invention.

[0168] Administration Bispecific anti-EGFR / c-Met antibodies and PD-(L)1 axis inhibitors may be administered to the subject together as a mixture, simultaneously as monotherapy, or sequentially as monotherapy in any order.

[0169] In some embodiments, a bispecific anti-EGFR / c-Met antibody is administered prior to the administration of a PD-(L)1 axis inhibitor.

[0170] In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered after administration of a PD-(L)1 axis inhibitor. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered to subjects who have received prior administration of a PD-(L)1 axis inhibitor. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered to subjects who have received prior administration of an anti-PD-(L)1 axis antibody. In some embodiments, the prior administration of the anti-PD-(L)1 axis antibody is provided as a prior cancer therapy or in combination with a prior cancer therapy.

[0171] In some embodiments, a bispecific anti-EGFR / c-Met antibody is administered simultaneously with the administration of a PD-(L)1 axis inhibitor.

[0172] In some embodiments, the combination of a bispecific anti-EGFR / c-Met antibody and a PD-(L)1 axis inhibitor is administered before the administration of one or more additional anticancer agents.

[0173] In some embodiments, the combination of a bispecific anti-EGFR / c-Met antibody and a PD-(L)1 axis inhibitor is administered after the administration of one or more additional anticancer agents.

[0174] In some embodiments, the combination of a bispecific anti-EGFR / c-Met antibody and a PD-(L)1 axis inhibitor is administered concurrently with the administration of one or more additional anticancer agents.

[0175] The time interval between the administration of a bispecific anti-EGFR / c-Met antibody and the administration of a PD-(L)1 axis inhibitor or one or more additional anticancer agents may be several minutes, for example, about 1, 2, 5, 10, 30, or 60 minutes; several hours, for example, about 2, 4, 6, 10, 12, 24, or 36 hours; or for example, about 2, 4, 7, 14, 21, 28, 35, 42, 49, 56 days, or longer.

[0176] In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered following the administration of a PD-(L)1 axis inhibitor for approximately 1, 2, 5, 10, 30, or 60 minutes or several hours, for example, approximately 2, 4, 6, 10, 12, 24, or 36 hours, or for example, approximately 2, 4, 7, 14, 21, 28, 35, 42, 49, or 56 days, or longer. Therefore, in some embodiments, the bispecific anti-EGFR / c-Met antibody is administered to subjects who have previously been administered a PD-(L)1 axis inhibitor.

[0177] In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered approximately 1, 2, 5, 10, 30, or 60 minutes or several hours before administration of the PD-(L)1 axis inhibitor, for example, approximately 2, 4, 6, 10, 12, 24, or 36 hours before, or for example, approximately 2, 4, 7, 14, 21, 28, 35, 42, 49, 56 days before, or more. Therefore, in some embodiments, the PD-(L)1 axis inhibitor is administered to subjects who have previously been administered the bispecific anti-EGFR / c-Met antibody.

[0178] One or more of the following can be administered in a pharmaceutically acceptable carrier: a bispecific anti-EGFR / c-Met antibody, a PD-(L)1-axis inhibitor, or an additional anticancer agent. “Carrier” refers to a diluent, adjuvant, excipient, or vehicle in which the antibody of the present invention is administered together. Such a vehicle may be water and a liquid oil, including oils derived from petroleum, animal, plant, or synthetic sources, such as peanut oil, soybean oil, mineral oil, or sesame oil. For example, a bispecific anti-EGFR / c-Met antibody may be formulated using 0.4% saline and 0.3% glycine. These solutions are sterilized and generally free of particulate matter. They can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). For oral solid dosage forms such as powders, capsules, and tablets, suitable carriers and excipients include starches, sugars, diluents, granulators, lubricants, binders, and disintegrants. Oral solid dosage forms may be coated with substances such as sugars, or enterically coated to modify the primary absorption site. For parenteral administration, the carrier may contain sterile water, and other excipients may be added for increased solubility or preservation. Suspensions or solutions for injection may also be prepared using an aqueous carrier with appropriate additives. Suitable vehicles and formulations (including other human proteins, e.g., human serum albumin) are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691-1092, and in particular pp. 958-989.

[0179] The composition may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and colorants. The concentrations of bispecific anti-EGFR / c-Met antibody and PD-(L)1-axis inhibitor in the pharmaceutical formulation may vary from less than about 0.5% by weight to at least about 1% by weight, and up to 15%, 20%, 30%, 40%, or 50% by weight, and may be selected mainly based on the required dose, fluid volume, viscosity, etc., according to the specific administration mode selected. Pharmaceutical compositions, including solid forms, may contain about 0.1 mg to about 2000 mg of the active ingredient, for example, about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 300 mg, about 500 mg, about 600 mg, or about 1000 mg.

[0180] In some embodiments, amivantamab is administered intravenously. In some embodiments, amivantamab is administered subcutaneously.

[0181] In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in doses ranging from approximately 350 mg to approximately 3000 mg. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in doses ranging from approximately 350 mg to approximately 4650 mg.

[0182] In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in doses of approximately 350 mg, 700 mg, 1050 mg, 1400 mg, 1600 mg, 1750 mg, 2100 mg, 2240 mg, 2400 mg, 3360 mg, 3200 mg, 4320 mg, 3520 mg, or 4640 mg. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in a dose of approximately 350 mg. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in a dose of approximately 700 mg. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in a dose of approximately 1050 mg. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in a dose of approximately 1400 mg. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in a dose of approximately 1600 mg. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in a dose of approximately 1750 mg. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in a dose of approximately 2400 mg. In some embodiments, the antibody is administered in a dose of approximately 3,360 mg. In some embodiments, the antibody is administered in a dose of approximately 3,200 mg. In some embodiments, the antibody is administered in a dose of approximately 4,320 mg. In some embodiments, the antibody is administered in a dose of approximately 3,520 mg. In some embodiments, the antibody is administered in a dose of approximately 4,640 mg. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in a dose of approximately 2240 mg or approximately 3000 mg.

[0183] In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in doses of approximately 700 mg for patients weighing less than 80 kg and 1050 mg for patients weighing 80 kg or more. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in 28-day cycles. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered once a week for the first four weeks (cycle 1). In some embodiments, the first dose is divided over the first two days. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered every two weeks (Q2W), starting from cycle 2. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in doses greater than 700 mg for patients weighing less than 80 kg, starting from cycle 2. In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered in doses greater than 1050 mg for patients weighing 80 kg or more, starting from cycle 2.

[0184] In some embodiments, cetrelimab is administered intravenously. In some embodiments, cetrelimab is administered in a dose of approximately 240 mg. In some embodiments, cetrelimab is administered once every two weeks (Q2W). In some embodiments, cetrelimab is administered once every two weeks (Q2W) in a dose of approximately 240 mg. In some embodiments, cetrelimab is administered in a dose of approximately 480 mg. In some embodiments, cetrelimab is administered once every four weeks (Q4W). In some embodiments, cetrelimab is administered once every four weeks (Q4W) in a dose of approximately 480 mg. In some embodiments, the first dose of cetrelimab is administered on day 2 of cycle 1. In some embodiments, cetrelimab is administered in a 28-day cycle.

[0185] Bispecific anti-EGFR / c-Met antibody sequence An exemplary anti-EGFR / c-Met antibody that can be used in the method of this disclosure is amivantamab. Amivantamab is characterized by the following amino acid sequence: EGFR binding group >Sequence ID 1 (HCDR1, EGFR binding group) TYGMH >Sequence ID 2 (HCDR2, EGFR binding group) VIWDDGSYKYYGDSVKG >Sequence ID 3 (HCDR3, EGFR binding group) DGITMVRGVMKDYFDY >Sequence ID 4 (LCDR1, EGFR binding group) RASQDISSALV >Sequence ID 5 (LCDR2, EGFR binding group) DASSLES >Sequence ID 6 (LCDR3, EGFR binding group) QQFNSYPLT >Sequence ID 7 (HCDR1, c-Met binding group) SYGIS >Sequence ID 8 (HCDR2, c-Met binding group) WISAYNGYTNYAQKLQG >Sequence ID 9 (HCDR3, c-Met binding group) DLRGTNYFDY >Sequence ID 10 (LCDR1, c-Met binding group) RASQGISNWLA >Sequence ID 11 (LCDR2, c-Met binding group) AASSLLS >Sequence ID 12 (LCDR3, c-Met binding group) QQANSFPIT >SEQ ID NO: 13 (VH, EGFR binding group) QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGTLVTVSS >Sequence ID 14 (VL, EGFR binding group) AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK >Sequence ID 15 (VH, c-Met binding group) QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISWVRQAPGHGLEWMGWISAYNGYTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDLRGTNYFDYWGQGTLVTVSS >Sequence ID 16 (VL, c-Met binding group) DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWFQHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPITFGQGTRLEIK >Sequence ID 17 HC1 QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 18 LC1 AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQKPGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 19 HC2 QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISWVRQAPGHGLEWMGWISAYNGYTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDLRGTNYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Sequence ID 20 LC2 DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWFQHKPGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPITFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0186] PD-(L)1 axis inhibitory antibody sequence An exemplary anti-PD-(L)1 axis antibody that can be used in the method of this disclosure is pembrolizumab. Pembrolizumab is characterized by the following amino acid sequence: >Sequence ID 21 (HCDR1) NYYMY >Sequence ID 22 (HCDR2) GINPSNGGTNFNEKFKN >Sequence ID 23 (HCDR3) RDYRFDMGFDY >Sequence ID 24 (LCDR1) RASKGVSTSGYSYLH >Sequence ID 25 (LCDR2) LASYLES >Sequence ID 26 (LCDR3) QHSRDLPLT > Sequence ID 27 (VH) QVQLVQSVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS >Sequence ID 28(VL) EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGGTKVEIK >Sequence ID 29 (HC) QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK >Sequence ID 30 (LC) EIVLTQSPATLLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0187] An exemplary anti-PD-(L)1 axis antibody that can be used in the method of this disclosure is cetrelimab. Cetrelimab is characterized by the following amino acid sequence: Sequence ID 31 (HCDR1) SYAIS Sequence ID 32 (HCDR2) GIIPIFDTANYAQKFQG Sequence ID 33 (HCDR3) PGLAAAYDTGSLDY Sequence ID 34 (LCDR1) RASQSVRSYLA Sequence ID 35 (LCDR2) DASNRAT Sequence ID 36 (LCDR3) QQRNYWPLT Sequence ID 37(VH) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSLDYWGQGTLVTVSS Sequence ID 38(VL) EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNYWPLTFGQGTKVEIK Sequence ID 39(HC) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARPGLAAAYDTGSLDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Sequence ID 40 (LC) EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNYWPLTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0188] Embodiment 1. In a subject requiring treatment of a solid tumor or mitigation of cancer progression, a method for treating a solid tumor or mitigating cancer progression, wherein the method is applied to the subject. (a) PD-(L)1 axis inhibitors, (b) A method comprising administering a bispecific anti-EGFR / c-Met antibody. 2. Bispecific anti-EGFR / c-Met antibodies are, (a) A first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, (b) The method according to Embodiment 1, comprising a second domain that binds to c-Met, the second domain comprising HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12. 3. (a) The first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, (b) The method according to Embodiment 2, wherein the second domain that binds to c-Met includes VH of SEQ ID NO: 15 and VL of SEQ ID NO: 16. 4. The method according to Embodiment 2, wherein the bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype. 5. The method according to Embodiment 4, wherein the bispecific anti-EGFR / c-Met antibody comprises the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO: 20. 5a. The method according to any one of Embodiments 1 to 5, wherein the bispecific anti-EGFR / c-Met antibody is amivantamab. 6. The method according to Embodiment 1, wherein the PD-(L)1 axis inhibitor is an inhibitory antibody. 6a. The method according to Embodiment 1, wherein the PD-(L)1 axis inhibitor is an anti-PD-1 antibody. 7. The method according to Embodiment 6, wherein the inhibitory antibody is selected from the group consisting of cetrelimab, pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimusumab, emvafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210. 8. The method according to Embodiment 7, wherein the pembrolizumab antibody comprises the heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, HCDR3 of SEQ ID NO: 23, light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 26. 8a. The method according to Embodiment 7, wherein the pembrolizumab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 27 and the light chain variable region (VL) of SEQ ID NO: 28. 9. The method according to Embodiment 7, wherein the pembrolizumab antibody comprises the heavy chain (HC) of SEQ ID NO: 29 and the light chain (LC) of SEQ ID NO: 30. 10. The method according to Embodiment 7, wherein the cetrelimab antibody comprises the heavy chain complementation determination region 1 (HCDR1) of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32, HCDR3 of SEQ ID NO: 33, light chain complementation determination region 1 (LCDR1) of SEQ ID NO: 34, LCDR2 of SEQ ID NO: 35, and LCDR3 of SEQ ID NO: 36. 10a. The method according to Embodiment 7, wherein the cetrelimab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 37 and the light chain variable region (VL) of SEQ ID NO: 38. 11. The method according to Embodiment 7, wherein the cetrelimab antibody comprises the heavy chain (HC) of SEQ ID NO: 39 and the light chain (LC) of SEQ ID NO: 40. 12. The method according to Embodiment 1, wherein the cancer is an EGFR, c-Met, or EGFR and c-Met expressing cancer, associated with wild-type EGFR, EGFR-activating mutations, EGFR gene amplification, elevated levels of circulating HGF, wild-type c-Met, c-Met-activating mutations, c-Met gene amplification, or mutant KRAS, or any combination thereof. 13. EGFR activating mutations include substitutions of L718Q, G719A, G719X (where X is any amino acid), L861X (where X is any amino acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, L858P, or T790M, deletions between E746 and A750, deletions between R748 and P753, insertion of Ala(A) between M766 and A767, insertion of Ser, Val, and Ala(SVA) between S768 and V769, and between P772 and H773. The method according to Embodiment 12, comprising insertion of Asn and Ser(NS) into, insertion of one or more amino acids between D761 and E762, A763 and Y764, Y764 and Y765, M766 and A767, A767 and V768, S768 and V769, V769 and D770, D770 and N771, N771 and P772, P772 and H773, H773 and V774, V774 and C775, one or more deletions in EGFR exon 20, or one or more insertions in EGFR exon 20, or any combination thereof. 14. The method according to Embodiment 13, wherein the mutant KRAS includes substitution of G12V, G12C, or G12A. 15. The method according to any one of Embodiments 1 to 14, wherein the method increases immune cell infiltration into a solid tumor, and further, the immune cells are selected from the group consisting of T cells, B cells, and natural killer cells. 16. The method according to Embodiment 15, wherein the T cells include CD8+ T cells and CD4+ T cells. 17. The method according to Embodiment 16, wherein the T cells include CD8+ T cells. 18. The method according to any one of Embodiments 1 to 17, wherein a population of central memory cytotoxic T cells increases within the tumor microenvironment. 19. The method according to any one of Embodiments 1 to 18, relating to a patient suspected of having or having EGFR, c-Met, or EGFR and c-Met expressing cancer. 20. The method according to Embodiment 1, which applies to patients who are resistant to or have acquired resistance to previous anticancer therapy. 21. The method according to Embodiment 20, wherein the previous anticancer therapy was a kinase inhibitor. 22. The method according to Embodiment 21, wherein the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL. 23. The method according to Embodiment 22, wherein the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib. 24. The method according to any one of Embodiments 1 to 23, wherein the cancer is 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, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC). 25. The method according to any one of Embodiments 1 to 24, wherein the cancer is resistant to treatment with a PD-(L)1 axis inhibitor. 26. The method according to any one of Embodiments 1 to 25, wherein the cancer is selected from the group consisting of head and neck squamous cell carcinoma (HNSCC), lung squamous cell carcinoma (LUSC), or non-small cell lung cancer (NSCLC). 27. The method according to any one of Embodiments 1 to 26, further comprising administering one or more anticancer therapies to the subject. 28. The method according to Embodiment 27, wherein one or more anticancer therapies include kinase inhibitors. 29. The method according to Embodiment 28, wherein the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL. 30. The method according to Embodiment 29, wherein the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib. 31. The method according to Embodiment 20, wherein the previous anti-cancer therapy was chemotherapy. 32. The method according to Embodiment 20, wherein the previous anticancer therapy is targeted anticancer therapy. 33. The method according to Embodiment 27, wherein one or more anticancer therapies include chemotherapy. 34. The method according to Embodiment 27, wherein one or more anticancer therapies include targeted anticancer therapy. 35. The method according to any one of Embodiments 1 to 34, wherein a bispecific anti-EGFR / c-Met antibody and a PD-(L)1 axis inhibitor are administered essentially simultaneously to two different injection sites. 36. The method according to any one of Embodiments 1 to 34, wherein a bispecific anti-EGFR / c-Met antibody and a PD-(L)1 axis inhibitor are combined before administration and administered simultaneously. 37. The method according to any one of Embodiments 1 to 34, wherein a bispecific anti-EGFR / c-Met antibody and a PD-(L)1 axis inhibitor are administered sequentially over a period of one day or more as part of a multi-step treatment regimen. 38. A method for enhancing immune cell infiltration into solid tumors in subjects where it is necessary to enhance immune cell infiltration into solid tumors, wherein the method is applied to the subject, (a) PD-(L)1 axis inhibitors, (b) A method comprising administering a bispecific anti-EGFR / c-Met antibody. 39. Bispecific anti-EGFR / c-Met antibodies are, (a) A first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, (b) The method according to Embodiment 38, comprising a second domain that binds to c-Met, the second domain comprising HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12. 40. (a) The first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, (b) The method according to Embodiment 39, wherein the second domain that binds to c-Met includes VH of SEQ ID NO: 15 and VL of SEQ ID NO: 16. 41. The method according to Embodiment 39, wherein the bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype. 42. The method according to Embodiment 41, wherein the bispecific anti-EGFR / c-Met antibody comprises the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO: 20. 43. The method according to Embodiment 38, wherein the PD-(L)1 axis inhibitor is an inhibitory antibody. 43a. The method according to Embodiment 38, wherein the PD-(L)1 axis inhibitor is an anti-PD-1 antibody. 44. The method according to Embodiment 43, wherein the inhibitory antibody is selected from the group consisting of pembrolizumab (KEYTRUDA®), cetrelimab, nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelelizumab, dostralimab, genolimuzumab, emvafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210. 45. The method according to Embodiment 44, wherein the pembrolizumab antibody comprises the heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, HCDR3 of SEQ ID NO: 23, light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 26. 45a. The method according to Embodiment 44, wherein the pembrolizumab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 27 and the light chain variable region (VL) of SEQ ID NO: 28. 46. ​​The method according to Embodiment 44, wherein the pembrolizumab antibody comprises the heavy chain (HC) of SEQ ID NO: 29 and the light chain (LC) of SEQ ID NO: 30. 47. The method according to Embodiment 44, wherein the cetrelimab antibody comprises the heavy chain complementation region 1 (HCDR1) of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32, HCDR3 of SEQ ID NO: 33, light chain complementation region 1 (LCDR1) of SEQ ID NO: 34, LCDR2 of SEQ ID NO: 35, and LCDR3 of SEQ ID NO: 36. 47a. The method according to Embodiment 44, wherein the cetrelimab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 37 and the light chain variable region (VL) of SEQ ID NO: 38. 48. The method according to Embodiment 44, wherein the cetrelimab antibody comprises the heavy chain (HC) of SEQ ID NO: 39 and the light chain (LC) of SEQ ID NO: 40. 49. The method according to Embodiment 38, wherein the cancer is an EGFR, c-Met, or EGFR and c-Met expressing cancer, associated with wild-type EGFR, EGFR-activating mutations, EGFR gene amplification, elevated levels of circulating HGF, wild-type c-Met, c-Met-activating mutations, c-Met gene amplification, or mutant KRAS, or any combination thereof. 50. EGFR activating mutations include substitutions of L718Q, G719A, G719X (where X is any amino acid), L861X (where X is any amino acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, L858P, or T790M, deletions between E746 and A750, deletions between R748 and P753, insertion of Ala(A) between M766 and A767, insertion of Ser, Val, and Ala(SVA) between S768 and V769, and between P772 and H773. The method according to Embodiment 49, comprising insertion of Asn and Ser(NS) into, insertion of one or more amino acids between D761 and E762, A763 and Y764, Y764 and Y765, M766 and A767, A767 and V768, S768 and V769, V769 and D770, D770 and N771, N771 and P772, P772 and H773, H773 and V774, V774 and C775, one or more deletions in EGFR exon 20, or one or more insertions in EGFR exon 20, or any combination thereof. 51. The method according to Embodiment 50, wherein the mutant KRAS includes substitution of G12V, G12C, or G12A. 52. The method according to any one of embodiments 38 to 51, wherein the immune cells are selected from the group consisting of T cells, B cells, and natural killer cells. 53. The method according to Embodiment 52, wherein the T cells include CD8+ T cells and CD4+ T cells. 54. The method according to Embodiment 53, wherein the T cells include CD8+ T cells. 55. The method according to any one of embodiments 38 to 54, wherein a population of central memory cytotoxic T cells increases within the tumor microenvironment. 56. The method according to any one of embodiments 38 to 55, relating to a patient suspected of having or having EGFR, c-Met, or EGFR and c-Met expressing cancer. 57. The method according to Embodiment 38, wherein the subject is resistant to or has acquired resistance to previous anticancer therapy. 58. The method according to Embodiment 57, wherein the previous anticancer therapy is a kinase inhibitor. 59. The method according to Embodiment 58, wherein the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL. 60. The method according to Embodiment 59, wherein the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib. 61. The method according to any one of Embodiments 38 to 60, wherein the cancer is 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, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC). 62. The method according to any one of embodiments 38 to 61, wherein the cancer is resistant to treatment with a PD-(L)1 axis inhibitor. 63. The method according to any one of embodiments 38 to 62, wherein the cancer is selected from the group consisting of head and neck squamous cell carcinoma (HNSCC), lung squamous cell carcinoma (LUSC), or non-small cell lung cancer (NSCLC). 64. The method according to any one of embodiments 38 to 63, further comprising administering one or more anticancer therapies to the subject. 65. The method according to Embodiment 64, wherein one or more anticancer therapies include kinase inhibitors. 66. The method according to Embodiment 65, wherein the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL. 67. The method according to Embodiment 66, wherein the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib. 68. The method according to Embodiment 57, wherein the previous anti-cancer therapy was chemotherapy. 69. The method according to Embodiment 57, wherein the previous anticancer therapy is targeted anticancer therapy. 70. The method according to Embodiment 64, wherein one or more anticancer therapies include chemotherapy. 71. The method according to Embodiment 64, wherein one or more anticancer therapies include targeted anticancer therapy. 72. The method according to any one of embodiments 38 to 71, wherein a bispecific anti-EGFR / c-Met antibody and a PD-(L)1 axis inhibitor are administered essentially simultaneously to two different injection sites. 73. The method according to any one of embodiments 38 to 71, wherein a bispecific anti-EGFR / c-Met antibody and a PD-(L)1 axis inhibitor are combined before administration and administered simultaneously. 74. The method according to any one of embodiments 38 to 71, wherein a bispecific anti-EGFR / c-Met antibody and a PD-(L)1 axis inhibitor are administered sequentially over a period of one day or more as part of a multi-step treatment regimen. 75. A method for reducing glycolysis or lactate production in the tumor microenvironment in subjects where it is necessary to reduce glycolysis or lactate production in the tumor microenvironment, wherein the subject (a) PD-(L)1 axis inhibitors, (b) A method comprising administering a bispecific anti-EGFR / c-Met antibody. 76. Bispecific anti-EGFR / c-Met antibodies are, (a) A first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, (b) The method according to Embodiment 75, comprising a second domain that binds to c-Met, the second domain comprising HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12. 77. (a) The first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, (b) The method according to Embodiment 76, wherein the second domain that binds to c-Met includes VH of SEQ ID NO: 15 and VL of SEQ ID NO: 16. 78. The method according to embodiment 76, wherein the bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype. 79. The method according to Embodiment 78, wherein the bispecific anti-EGFR / c-Met antibody comprises the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO: 20. 80. The method according to Embodiment 75, wherein the PD-(L)1 axis inhibitor is an inhibitory antibody. 80a. The method according to Embodiment 75, wherein the PD-(L)1 axis inhibitor is an anti-PD-1 antibody. 81. The method according to Embodiment 80, wherein the inhibitory antibody is selected from the group consisting of pembrolizumab (KEYTRUDA®), cetrelimab, nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelelizumab, dostralimab, genolimusumab, emvafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210. 82. The method according to Embodiment 81, wherein the pembrolizumab antibody comprises the heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, HCDR3 of SEQ ID NO: 23, light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 26. 82a. The method according to Embodiment 81, wherein the pembrolizumab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 27 and the light chain variable region (VL) of SEQ ID NO: 28. 83. The method according to Embodiment 81, wherein the pembrolizumab antibody comprises the heavy chain (HC) of SEQ ID NO: 29 and the light chain (LC) of SEQ ID NO: 30. 84. The method according to Embodiment 81, wherein the cetrelimab antibody comprises the heavy chain complementation determination region 1 (HCDR1) of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32, HCDR3 of SEQ ID NO: 33, light chain complementation determination region 1 (LCDR1) of SEQ ID NO: 34, LCDR2 of SEQ ID NO: 35, and LCDR3 of SEQ ID NO: 36. 84a. The method according to Embodiment 81, wherein the cetrelimab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 37 and the light chain variable region (VL) of SEQ ID NO: 38. 85. The method according to Embodiment 81, wherein the cetrelimab antibody comprises the heavy chain (HC) of SEQ ID NO: 39 and the light chain (LC) of SEQ ID NO: 40. 86. The method according to Embodiment 75, wherein the cancer is an EGFR, c-Met, or EGFR and c-Met expressing cancer associated with wild-type EGFR, EGFR-activating mutations, EGFR gene amplification, elevated levels of circulating HGF, wild-type c-Met, c-Met-activating mutations, c-Met gene amplification, or mutant KRAS, or any combination thereof. 87. EGFR activating mutations include substitutions of L718Q, G719A, G719X (where X is any amino acid), L861X (where X is any amino acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, L858P, or T790M, deletions between E746 and A750, deletions between R748 and P753, insertion of Ala(A) between M766 and A767, insertion of Ser, Val, and Ala(SVA) between S768 and V769, and between P772 and H773. The method according to Embodiment 86, comprising insertion of Asn and Ser(NS) into, insertion of one or more amino acids between D761 and E762, A763 and Y764, Y764 and Y765, M766 and A767, A767 and V768, S768 and V769, V769 and D770, D770 and N771, N771 and P772, P772 and H773, H773 and V774, V774 and C775, one or more deletions in EGFR exon 20, or one or more insertions in EGFR exon 20, or any combination thereof. 88. The method according to Embodiment 87, wherein the mutant KRAS includes substitution of G12V, G12C, or G12A. 89. The method according to any one of embodiments 75 to 88, wherein the method increases immune cell infiltration into a solid tumor, and further, the immune cells are selected from the group consisting of T cells, B cells, and natural killer cells. 90. The method according to Embodiment 89, wherein the T cells include CD8+ T cells and CD4+ T cells. 91. The method according to Embodiment 90, wherein the T cells include CD8+ T cells. 92. The method according to any one of embodiments 75 to 91, wherein a population of central memory cytotoxic T cells increases within the tumor microenvironment. 93. The subject is anyone suspected of having or having EGFR, c-Met, or EGFR and c-Met expressing cancer, and the method according to any one of Embodiments 75 to 92. 94. The subject is resistant to or has acquired resistance to treatment with a previous anti-cancer therapy, and the method according to Embodiment 75. 95. The previous anti-cancer therapy is a kinase inhibitor, and the method according to Embodiment 94. 96. The kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL, and the method according to Embodiment 95. 97. The kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, lazertinib, poziotinib, crizotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib, and the method according to Embodiment 96. 98. The cancer is epithelial cell cancer, 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 cancer (HCC), or sporadic or hereditary papillary renal cell cancer (PRCC), and the method according to any one of Embodiments 75 to 97. 99. The cancer is resistant to treatment with a PD-(L)1 axis inhibitor, and the method according to any one of Embodiments 75 to 98. 100. The cancer is selected from the group consisting of head and neck squamous cell carcinoma (HNSCC), lung squamous cell carcinoma (LUSC), or non-small cell lung cancer (NSCLC), and the method according to any one of Embodiments 75 to 99. 101. The method according to any one of Embodiments 75 to 100, further comprising administering to the subject one or more anti-cancer therapies. 102. The one or more anti-cancer therapies include a kinase inhibitor, and the method according to Embodiment 101. 103. The method according to Embodiment 102, wherein the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL. 104. The method according to Embodiment 103, wherein the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib. 105. The previous anti-cancer therapy is chemotherapy, as described in Embodiment 94. 106. The method according to Embodiment 94, wherein the previous anticancer therapy is a targeted anticancer therapy. 107. The method according to Embodiment 101, wherein one or more anticancer therapies include chemotherapy. 108. The method according to Embodiment 101, wherein one or more anticancer therapies include targeted anticancer therapy. 109. The method according to any one of embodiments 75 to 108, wherein a bispecific anti-EGFR / c-Met antibody and a PD-(L)1 axis inhibitor are administered essentially simultaneously to two different injection sites. 110. The method according to any one of Embodiments 75 to 108, wherein a bispecific anti-EGFR / c-Met antibody and a PD-(L)1 axis inhibitor are combined before administration and administered simultaneously. 111. The method according to any one of Embodiments 75 to 108, wherein a bispecific anti-EGFR / c-Met antibody and a PD-(L)1 axis inhibitor are administered sequentially over a period of one day or more as part of a multi-step treatment regimen. 112. A method for treating a solid tumor, mitigating cancer progression, enhancing immune cell infiltration into a solid tumor, or reducing lactate production in the tumor microenvironment in subjects requiring treatment of a solid tumor, mitigating cancer progression, enhancing immune cell infiltration into a solid tumor, or reducing lactate production in the tumor microenvironment, the method comprising administering a bispecific anti-EGFR / c-Met antibody to a subject, wherein the subject has received prior administration of a PD-(L)1 axis inhibitor. 113. Bispecific anti-EGFR / c-Met antibodies are, (a) A first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, (b) The method according to Embodiment 112, comprising a second domain that binds to c-Met, the second domain comprising HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12. 114. (a) The first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, (b) The method according to Embodiment 113, wherein the second domain that binds to c-Met includes VH of SEQ ID NO: 15 and VL of SEQ ID NO: 16. 115. The method according to Embodiment 113, wherein the bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype. 116. The method according to Embodiment 113, wherein the bispecific anti-EGFR / c-Met antibody comprises the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO: 20. 117. The method according to Embodiment 112, wherein the pre-administered PD-(L)1 axis inhibitor is an inhibitory antibody. 117a. The method according to Embodiment 112, wherein the PD-(L)1 axis inhibitor is an anti-PD-1 antibody. 118. The method according to Embodiment 117, wherein the pre-administered inhibitory antibody is selected from the group consisting of pembrolizumab (KEYTRUDA®), cetrelimab, nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimusumab, emvafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210. 119. The method according to any one of embodiments 112 to 118, wherein the immune cells are selected from the group consisting of T cells, B cells, and natural killer cells. 120. The method according to Embodiment 119, wherein the immune cells include CD8+ T cells and CD4+ T cells. 121. The method according to Embodiment 120, wherein the immune cells include CD8+ T cells. 122. The method according to any one of Embodiments 112-120, wherein a population of central memory cytotoxic T cells increases within the tumor microenvironment. 123. A method for reducing lactate production in the tumor microenvironment in subjects requiring reduction of lactate production in the tumor microenvironment, the method comprising administering a combination of inhibitors to the subject, the combination of inhibitors being: (a) A combination including an EGFR inhibitor, a c-Met inhibitor, and a PD-(L)1 axis inhibitor, (b) A method selected from the group consisting of a combination including an EGFR inhibitor and a c-Met inhibitor, wherein the subject has received prior administration of a PD-(L)1 axis inhibitor. 124. The combination of an EGFR inhibitor and a c-Met inhibitor is the method according to Embodiment 123, comprising a bispecific anti-EGFR / c-Met antibody. 125. Bispecific anti-EGFR / c-Met antibodies are, (a) A first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, (b) The method according to Embodiment 124, comprising a second domain that binds to c-Met, the second domain comprising HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12. 126. (a) The first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, (b) The method according to Embodiment 125, wherein the second domain that binds to c-Met includes VH of SEQ ID NO: 15 and VL of SEQ ID NO: 16. 127. The method according to Embodiment 124, wherein the bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype. 128. The method according to Embodiment 124, wherein the bispecific anti-EGFR / c-Met antibody comprises the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO: 20. 129. The method according to Embodiment 124, wherein the PD-(L)1 axis inhibitor is an inhibitory antibody. 129a. The method according to Embodiment 124, wherein the PD-(L)1 axis inhibitor is an anti-PD-1 antibody. 130. The method according to Embodiment 129, wherein the inhibitory antibody is selected from the group consisting of pembrolizumab (KEYTRUDA®), cetrelimab, nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelelizumab, dostralimab, genolimusumab, emvafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210. 131. The method according to any one of embodiments 123-130, wherein a population of central memory cytotoxic T cells increases within the tumor microenvironment. 132. The method according to any one of Embodiments 123 to 131, wherein the cancer is 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, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC). 133. The method according to any one of embodiments 123 to 132, wherein the cancer is resistant to treatment with a PD-(L)1 axis inhibitor. 134. The method according to any one of Embodiments 123 to 133, wherein the cancer is selected from the group consisting of head and neck squamous cell carcinoma (HNSCC), lung squamous cell carcinoma (LUSC), or non-small cell lung cancer (NSCLC). 135. A kit comprising a first pharmaceutical composition containing a bispecific anti-EGFR / c-Met antibody and a second pharmaceutical composition containing a PD-(L)1 axis inhibitor, in two or more containers. 136. Bispecific anti-EGFR / c-Met antibodies are, (a) A first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDR1) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, (b) The kit according to Embodiment 135, comprising a second domain that binds to c-Met, the second domain comprising HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12. 137. (a) The first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, (b) The kit according to Embodiment 135, wherein the second domain that binds to c-Met includes VH of SEQ ID NO: 15 and VL of SEQ ID NO: 16. 138. The kit according to Embodiment 135, wherein the bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype. 139. The kit according to Embodiment 135, wherein the bispecific anti-EGFR / c-Met antibody comprises the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO: 20. 140. The PD-(L)1 axis inhibitor is an inhibitory antibody, as described in Embodiment 135 of the kit. 140a. The method according to Embodiment 135, wherein the PD-(L)1 axis inhibitor is an anti-PD-1 antibody. 141. The inhibitory antibody is selected from the group consisting of pembrolizumab (KEYTRUDA®), cetrelimab, nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimuzumab, emvafolimab, atezolizumab (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210, as described in Embodiment 140. 142. The kit according to Embodiment 141, wherein the pembrolizumab antibody comprises the heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, HCDR3 of SEQ ID NO: 23, light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO: 26. 142a. The kit according to Embodiment 141, wherein the pembrolizumab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 27 and the light chain variable region (VL) of SEQ ID NO: 28. 143. The pembrolizumab antibody is a kit according to Embodiment 141, comprising the heavy chain (HC) of SEQ ID NO: 29 and the light chain (LC) of SEQ ID NO: 30. 144. The kit according to Embodiment 141, wherein the cetrelimab antibody comprises the heavy chain complementation region 1 (HCDR1) of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32, HCDR3 of SEQ ID NO: 33, light chain complementation region 1 (LCDR1) of SEQ ID NO: 34, LCDR2 of SEQ ID NO: 35, and LCDR3 of SEQ ID NO: 36. 144a. The method according to Embodiment 141, wherein the cetrelimab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 37 and the light chain variable region (VL) of SEQ ID NO: 38. 145. The kit according to Embodiment 141, comprising the cetrelimab antibody heavy chain (HC) of SEQ ID NO: 39 and the light chain (LC) of SEQ ID NO: 40. The first pharmaceutical composition containing the bispecific anti-EGFR / c-Met antibody further comprises a first pharmaceutically acceptable excipient, and the second pharmaceutical composition containing a PD-(L)1 axis inhibitor further comprises a second pharmaceutically acceptable excipient, the kit according to any one of embodiments 135 to 145

[0189] Next, the present invention will be described with reference to the following specific non-limiting examples.

[0190] Example 1. Combination activity of amivantamab and pembrolizumab in head and neck squamous cell carcinoma and lung squamous cell carcinoma expressing wild-type EGFR and MET Amivantamab is a bispecific human immunoglobulin (Ig) G1 Duobody® that binds to the epidermal growth factor receptor (EGFR) and the mesenchymal-epithelial transition (cMet) receptor.

[0191] To further explore its antitumor activity in wild-type EGFR head and neck cancer, the efficacy of amivantamab as a single agent and in combination with pembrolizumab was investigated in a head and neck cancer patient-derived xenograft (PDX) model YHIM-3003.

[0192] Amivantamab or pembrolizumab alone only moderately delays tumor growth in this model, but the combination of amivantamab (10 mg / kg, BIW) and pembrolizumab (10 mg / kg, Q5D) significantly inhibited the growth of YHIM-3003 tumors at the end of the study, while all regimens including the combination were well tolerated throughout the treatment process.

[0193] This study demonstrated the advantage of the combination of amivantamab and pembrolizumab by effectively remodeling the tumor immune microenvironment and provided strong preclinical evidence for clinically combining amivantamab and PD-1 blockade therapy.

[0194] The materials and methods used in the experiment are described below.

[0195] Test drug and control Amivantamab: Amivantamab was diluted in anti-septic grade HBSS solution for intraperitoneal injection at a dose of 10 mg / kg per mouse.

[0196] Pembrolizumab (anti-PD-1): Anti-PD-1 was diluted in an anti-septic grade HBSS solution for intraperitoneal injection at a dose of 10 mg / kg per mouse.

[0197] The test substance formulation was prepared weekly and divided into aliquots for each dispensing. The prepared aliquots were stored at 4°C until needed.

[0198] Selection of patient-derived xenografts. PDX library tumors were screened for EGFR and MET expression by immunohistochemistry (Figure 1). Immunohistochemical staining for EGFR and MET was quantified using intensity scores and compared between models before selecting tumors with dual expression of EGFR and MET from among LUSC and HNSCC. According to the intensity scores, the HNSCC model (YHIM-3003) showed co-expression of EGFR (mean score of 157.8 + 21.3) and MET (mean score of 21.4 + 12.6). The LUSC model (YHIM-2010) showed expression of both EGFR (mean score of 199.4 + 48.82) and MET (mean score of 161.4 + 34.1). The LUSC model was previously considered insensitive to pembrolizumab treatment (Figures 2A and 2B). In addition, YHIM-3003 and YHIM-2010 showed the most consistent and stable tumor development compared to the other models.

[0199] Animal models PDX mouse models were created using 6-8 week old female SCID (NOG) mice obtained from Orient Bio. After removing necrotic and supporting tissue from core biopsy specimens, small tumor tissue samples (3mm x 3mm x 3mm) from each patient were subcutaneously transplanted into 1-2 mice. Once the tumors exceeded 1.5cm in diameter, they were surgically removed. The tumors were cut into small pieces (3mm x 3mm x 3mm) and re-transplanted into hCD34 humanized mice (Jackson Laboratories, Sacramento, CA, USA).

[0200] All animals were monitored and examined in detail for their general health. Humanized mice were also screened for infection. The animals were housed in groups of five in individually ventilated cages in an isolation area for one week. Healthy animals were selected and transported to the laboratory.

[0201] The procedures, including the management and use of animals in the study, were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC number, 2021-0112) prior to implementation. During the study, the management and use of animals followed the principles outlined in the Guide for the Care and Use of Laboratory Animals, 8th Edition, 2010 (National Research Council).

[0202] If an animal was found to be in a critical condition with severe clinical signs, or to have lost more than 15% of its body weight in one day, or more than 20% of its body weight from its pre-test level, treatment of the animal was terminated and the animal was humanely euthanized.

[0203] [Table 1]

[0204] [Table 2]

[0205] [Table 3]

[0206] [Table 4]

[0207] Sample preparation Blood, tumor, and spleen samples were collected on day 5 of the in vivo experiment. Tumor samples were partially excised. A portion of the tumor tissue was fixed in 10% formalin and made into FFPE blocks to generate multiple slides for IHC. The remaining portion was divided and used for single-cell RNA sequencing and flow cytometry. Spleen samples were processed for FFPE blocks and flow cytometry. For flow cytometry analysis, the tumor was shredded into small pieces using a sterile surgical blade in PBS, enzymatically dissociated into single cells using collagenase (Worthington Biochemical, New Jersey, US) at 37°C for 1 hour, and filtered through a 70 μm cell filter. Spleen samples were dissociated by gently tapping with a syringe stopper in PBS, and then filtered through a 70 μm cell strainer. The dissociated samples were frozen in FBS + 10% DMSO and stored at -80°C until required for analysis.

[0208] In vivo drug treatment and preparation Drug treatment was initiated when the tumor size reached 200 mm3 in both models. For LUSC PDX (YHIM-2010), amivantamab was administered in combination with pembrolizumab (10 mpk, ip, Q5D) at doses of 10 mpk and 30 mpk (ip, BIW). During the HNSCC PDX (YHIM-3003) experiment, 10 mpk of amivantamab was administered in combination with pembrolizumab. Treatment groups consisted of vehicle, amivantamab, pembrolizumab, and combinations. Each group consisted of 15 mice; 5 mice were ethically sacrificed on day 5, and the remaining 10 mice continued treatment until the end of the survival assay. Amivantamab and pembrolizumab were prepared for intraperitoneal injection by dilution with anti-septic grade Hanks' Balanced Sat Solution (HBSS, Life Technologies, NY, USA). The drug solutions were prepared on the day of administration and vigorously mixed before injection.

[0209] multiplex immunohistochemistry Intracellular hemoglobin (IHC) was performed using an automated staining system (BOND Rx, Leica Biosystems). Briefly, 4 mm paraffin-embedded tumor sections were deparaffinized and rehydrated. The slides were then subjected to heat-induced epitope recovery at 100°C for 20 minutes using citrate buffer. Antibodies were used at a 1:100 dilution, and hematoxylin solution was used for counterstaining. The stained slides were visualized using Vectra Polaris and Phenochart programs. The mean H score was calculated for all IHC slide images, and the statistical significance of the p-value (<0.05) was analyzed. The T lymphocyte panel targets were PD-1, GZMB, FOXP3, CD4, CD8, and pan-CK, with detection fluorescence at 620 nm, 570 nm, 520 nm, 690 nm, 480 nm, and 780 nm, respectively (Table 5). The bone marrow panel targets consisted of CD16, PD-L1, CD163, CD11c, CD68, and pan-CK at 620nm, 570nm, 520nm, 690nm, 480nm, and 780nm, respectively.

[0210] [Table 5]

[0211] Complete slides were visualized using the Vectra Automated Quantitative Pathology Imaging System (Vectra 3.0.5, PerkinElmer) and analyzed using inForm Advanced Image Analysis Software (2.6.0) and Phenochart (1.1.0, PerkinElmer). Regions of interest (ROIs) were selected in the multi-stained scan images to distinguish specific areas of tumor tissue via a user-trained classification algorithm. Within the segmented regions of the tumor, the characteristics of individual cells were identified using a nuclear-based cell classification algorithm. The classified cell types were quantified and analyzed by converting the number of cells per area and the number of cells in total cells into quantitative values ​​represented by an "intensity score".

[0212] Flow cytometry analysis of immune cells Tumor cells and splenocytes were collected on day 5 in vivo and analyzed by flow cytometry. For immunoassays, dissociated single cells were washed with FACS buffer (PBS containing 1% BSA, 0.01% sodium azide, and 0.5 mM EDTA) and blocked with FcR blocking reagent (Miltenyi Biotec) at room temperature for 20 minutes. Fixation and permeabilization for intracellular staining were prepared using True-Nuclear® transcription factor buffer at room temperature for 30 minutes. Multicolor flow cytometry analysis was performed using BD LSRfortessa® X-20 (BD Bioscience, New Jersey, USA). FlowJo (FlowJo, LLC) software was used for data acquisition and analysis. Screening of tumor-reactive T cells was performed using CEA and MAGEA antibodies.

[0213] cell line LUSC cell lines, EBC-1 (HTB-60) and H1703 (CRL-5889), were purchased from the American Type Culture Collection (ATCC; Inc. Manassas, VA, USA). The cells were cultured and maintained in a humidified incubator containing 5% CO2 in HyClone® RPMI-1640 (Cytiva, Massachusetts, US) supplemented with 10% fecal bovine serum (Cytiva) and 1% antibiotic / antifungal solution (Cytiva).

[0214] Western blot reagents The following primary antibodies were used for protein detection. Anti-actin (#A3854, 1:1000) was purchased from Sigma Aldrich. Anti-EGFR (#2232, 1:1000), anti-p-EGFR (#2234, 1:1000), anti-LDHA (#3582, 1:1000), anti-MET (#8198, 1:1000), and anti-p-MET (#3077, 1:1000) were purchased from Cell Signaling Technology. Anti-SLC16A3 / MCT4 (ab74109, 1:1000) was purchased from Abcam. For protein detection, we used secondary HRP conjugate anti-rabbit IgG (#7074, 1:1000) or anti-mouse IgG (#7076, 1:1000) purchased from Cell Signaling Technology, and the ECL System (#1707062, Bio-Rad).

[0215] Single-cell RNA sequencing and processing Mean weight loss and percentage change were calculated for each group. If there was toxicity to the treatment, it was evaluated based on weight loss. All statistical calculations were performed using Prism 9.0 (Graph Pad Software Inc, USA). Statistical comparisons of tumor volume and survival curves between treatment groups were calculated using two-way ANOVA followed by Tukey's multiple comparison test (p-values ​​less than 0.05 were considered statistically significant).

[0216] Tumor samples were dissociated using the gentleMACS Human Tumor Dissociation Kit (Miltenyi Biotec) and then processed according to the manufacturer's guidelines (10X Genomics, California, US) before single-cell library preparation. The volume of each sample was calculated for target capture of 10,000 cells. The processed samples were prepared for gene expression analysis using Chromium Single Cell 5' Reagent Kits (10X Genomics). The single-cell libraries were then outsourced for single-cell RNA sequencing at Macrogen (Korea). Sequencing was performed to achieve a read depth of over 50,000. The FASTQ files were then processed for mapping based on mm10 and hg19 to distinguish between mouse and human genomes. Read counting and merging of samples were performed via Cell Ranger (7.1.0). Scanpy was used for further quality control, including filtering (minimum 200 genes in at least three cells with less than 20% mitochondrial reads as a cutoff), cell normalization (Harmony), batch correction, and clustering. Gene expression analysis in HNSCC and LUSC models was visualized using heatmaps, violin plots, and dot plots with Seurat (version 4.9.9).

[0217] Filtering and normalization of scRNA sequencing data Four quality measures—mitochondrial gene density (less than 20%), minimum gene count (less than 200), and minimum cell count (less than 3)—were applied to the raw gene-cell-barcode matrix of each cell. After basic filtering, patient data with fewer than 30 cells per patient were excluded from the analysis. The data were scaled and normalized. Batch correction algorithms were used, employing harmony and relative expression levels for the remaining cells. Gene subsets were used for subsequent analyses.

[0218] Cell type identification and analysis Primary cell type annotation using the Azimuth lung database (https: / / azimuth_hubmapconsortium_org / ) was validated using cell type signatures obtained by CITE-seq. Each detailed cluster was partitioned using the Seurat FindClusters algorithm. Cell type marker expression across cluster subsets was analyzed via tSNE and UMAP. The top 50 genes in heterologous EGFR and MET high tumor clusters were analyzed for the top 50 differential gene expression (DEG) markers. Among the analyzed genes, key immunomodulatory factors were marked in volcano plots.

[0219] Tumor-associated tetramer assay Tetrameric peptides were prepared, and the proportion of tumor-specific T lymphocytes was compared between treatment groups. MAGEA2 (HLA-A * 24:02 EYLQLVFGI) and CEA (HLA-A * The preparation of Flex-TTM tetramers (24:02 TYACFVSNL) was outsourced by Biolegend (California, US). The preparation of Flex-TTM tetramers is available online, for example, on the Biolegend website.

[0220] calculation The tumor volume was calculated using the following formula: Tumor Volume (TV, mm3) = (L × W² / 2); where "L" is the length of the tumor (the longest dimension of the tumor) and "W" is the width of the tumor measured with calipers (the longest dimension of the tumor perpendicular to L).

[0221] The percentage ΔTGI was defined as the difference in tumor volume between the treatment group and the control group, and was calculated as ([(TVc-TVc0)-(TVt-TVt0)] / (TVc-TVc0))×100. In the formula, "TVc" is the mean tumor volume of the given control group on a specified day, "TVc0" is the mean initial tumor volume (day 0) of the given control group, "TVt" is the mean tumor volume of the treatment group on a specified day, and "TVt0" is the mean initial tumor volume (day 0) of the treatment group.

[0222] In the case of tumor regression, an additional metric, tumor regression (TR), is calculated as (1 - mean (TVt / TV0)) × 100, where TVt and TV0 are defined in the same way as above.

[0223] Data Analysis In each study, statistical comparisons were performed by referencing the vehicle group up to the final day of the study, including the day when all 10 mice remained in each group. A difference between groups was considered statistically significant when p ≤ 0.05. Statistical significance of tumor volume and body weight was calculated using linear mixed-effects analysis in R software version 4.0.3 (using Janssen's in-house developed Shiny application, In Vivo Longitudinal Data Analysis Version 4.8), with treatment and time as fixed effects and time as a continuous variable (Pinheiro J, Bates D. Mixed-effects models in S and S-Plus. Heidelberg, Germany: Springer; 2000). If individual longitudinal response trajectories were not linear, logarithmic transformation (cardin 10) was performed. Using the information derived from this model, pairwise comparisons of treatments over time were performed against the control group or between all treatment groups.

[0224] The experimental results are explained here.

[0225] Antitumor efficacy of amivantamab in combination with pembrolizumab in humanized mouse models of head and neck squamous epithelium and lung squamous epithelium. EGFR and MET-expressing tumors obtained from patients with head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma (LUSC) were transplanted into Hu-CD34-NSG to establish a humanized patient-derived xenograft (PDX) model. The tumor model characteristics are shown in Table 6.

[0226] [Table 6]

[0227] Tumor-bearing PDX cells were treated with a vehicle, pembrolizumab (10mpk, Q5D, n=10), amivantamab (10mpk, BIW, n=10), or a combination of pembrolizumab and amivantamab (n=10) (Figure 2A). Immunomodulatory responses within the tumor microenvironment (TME) were analyzed using multiplexed immunohistochemistry, flow cytometry, and single-cell RNA sequencing.

[0228] The combination therapy of amivantamab and pembrolizumab demonstrated a significant reduction in tumor volume (p<0.001) compared to vehicle or monotherapy in both HNSCC and LUSC models (Figures 3-5). The synergistic benefit of the combination therapy of amivantamab and pembrolizumab compared to monotherapy was observed in in vivo experiments of tumor-bearing PDX models of HNSCC (YHIM-3003) and LUSC (YHIM-2010). In HNSCC PDX, the combination therapy of amivantamab and pembrolizumab demonstrated a significant reduction in tumor volume compared to vehicle or single-group therapy (p<0.001, Figures 3A-3B). The highest tumor growth inhibition (TGI) rate was observed mainly in the combination therapy group (Figure 3B). TGI exceeding 40% was observed in 70% of the combination therapy groups, but only 20% of the amivantamab group experienced TGI exceeding 40% (Figure 3B). TGI did not exceed 40% in the pembrolizumab group.

[0229] Furthermore, significantly longer survival was observed in the combination therapy group compared to the vehicle therapy group (p<0.0001). In addition, longer survival was observed in the combination therapy group compared to the monotherapy group. At the end of day 19, 40% of the combination group were alive, in contrast to pembrolizumab and amivantamab monotherapy, in which all mice died on days 17 and 19, respectively (Figure 3A).

[0230] In the LUSC PDX, amivantamab administered at the same dose of 30 mpk in combination with pembrolizumab significantly reduced tumor volume compared to the pembrolizumab group (p<0.001, Figure 5). Interestingly, no rebound in tumor growth was observed in either the amivantamab group or the combination group after discontinuation of drug administration on day 47 (Figure 5). Amivantamab appeared to enhance the immune activity of lymphocytes, including a subset of T cells with memory phenotypes, and prevented recurrence of tumor development after treatment discontinuation. However, amivantamab alone at 30 mpk effectively reduced tumor volume (Figure 5). This suggests that the dose of amivantamab needs to be reduced to evaluate the synergistic effect of combination therapy with pembrolizumab. Amivantamab administered in combination with pembrolizumab at a modified dose of 10 mpk significantly reduced tumor volume compared to amivantamab and pembrolizumab monotherapy (p<0.05 and p<0.001, respectively, Figure 4). The combination therapy also resulted in improved survival over 61 days. All mice in the vehicle and pembrolizumab groups died on days 47 and 53, respectively, while 70% and 40% of mice in the combination and amivantamab groups survived to day 61 (Figure 4).

[0231] Tumor-infiltrating CD8+ T cells were significantly higher in tumors treated with the concomitant therapy. Multispectral imaging of tumors showed a significant increase in granzyme B-producing CD8+ T cells within the tumor in the combination therapy group (p<0.01) (Figures 6-7). Landscape of the tumor microenvironment (TME) within HNSCC PDX tumors revealed that immune cell infiltration in the tumor and stromal areas was enhanced by combination treatment with amivantamab and pembrolizumab (Figure 6). The proportion of granzyme B-expressing (GZMB) CD8 T cells in the tumor foci of the TME was significantly increased in the combination therapy group compared to the control group (18.81±10.36 and 4.09±1.55, respectively, p<0.05, Figure 7). Although there was no statistically significant difference between the groups in the stroma, an increasing trend was observed in the combination therapy group. In general, the GZMB+CD8 T cell population increased (in terms of total TME area) with combination treatment of amivantamab and pembrolizumab in YHIM-3003. In contrast, no significant results or trends were observed in the proportion of Foxp3-regulating CD4 T cells between treatment groups. These results suggest that amivantamab and pembrolizumab synergistically enhanced the infiltration of active cytotoxic CD8 T cells into the TME and suppressed tumor cell proliferation.

[0232] Antitumor phenotypic changes in T cell populations induced by combination therapy Further analysis of the T cell subset suggested that central memory type CD8+ T cells increased with concomitant treatment. This group also showed significantly higher levels of CEA tetramer-positive CD8+ T cells in the tumor (p<0.01), suggesting that cytotoxic T cells recognizing tumor-specific antigens enhanced the anti-tumor immune response (Figures 8-9).

[0233] To compare changes in lymphocyte populations between treatment groups, changes in immune cell phenotypes were analyzed using flow cytometry. The combination treatment groups significantly increased the proportion of central memory CD8 T cells in HNSCC PDX (13.38±8.80 and 2.11±2.34, p<0.05, Figure 8A) and LUSC PDX (24.00±8.26 and 10.55±4.47, p<0.05, Figure 8B) compared to pembrolizumab monotherapy. T cell analysis identified a common trend between the HNSCC PDX and LUSC PDX models in the central memory subset of cytotoxic CD8 T cells (Figure 8C). No recurrence of tumorigenesis was observed after treatment with high-dose amivantamab in combination with pembrolizumab (Figure 5). This observation initially suggested that combination therapy may stimulate a T cell memory subset during the immune response to tumorigenesis. Immunoprofiling of humanized PDX tumors showed that the combination of amivantamab and pembrolizumab enhanced the induction of T cell memory subpopulations (i.e., central memory CD8 T cells, Figures 3A-3C).

[0234] Splenocytes from YHIM-3003 mice were treated and stained with a tetrameric antibody to detect T lymphocytes capable of recognizing the tumor-specific antigen CEA (carcinoembryonic antigen). The combination therapy group had a significantly higher proportion of CEA-expressing tumor-reactive CD8 T cells compared to the vehicle group (8.28±2.67 and 3.02±0.75, respectively, p<0.05, Figure 9). The combination therapy group also had the highest proportion compared to amivantamab (6.17±1.98) and pembrolizumab (6.20±3.05, Figure 9).

[0235] Pembrolizumab monotherapy induces an EGFR / MET high subcluster that upregulates genes involved in lactate production and immunosuppression. Single-cell RNA sequencing analysis of HNSCC tumors shows that EGFR high MET high The clusters were shown to be concentrated in TME after pembrolizumab treatment. EGFR high METhigh The subcluster had elevated glycolytic genes and lactate pathway-related genes compared with the cluster, showing an increase low in MET low The lactate transporter, MCT4 (SLC16A3) gene, and LDHA gene were dramatically increased in the EGFR high / MET high cluster (Figs. 10 - 19). The elevated lactate pathway may lead to immune evasion in tumors and potentially weaken the activity of pembrolizumab. Interestingly, combination treatment with amivantamab was able to reduce the EGFR high / MET high subcluster and effectively control the tumor by creating a favorable immune TME.

[0236] Specifically, in HNSCC PDX tumors, the subcluster with high EGFR expression simultaneously increased the MET level (Fig. 12). Interestingly, single-agent pembrolizumab treatment induced a higher density of EGFR- and MET-dual-expressing subclusters compared with other treatment groups (Fig. 12). Tumor subclusters with high-level expression of both EGFR and MET (EMHIGH) were defined, and the top 50 genes were analyzed to identify the expression-variable genes (DEGs) in the EGFR high / MET high and EGFR low / MET low subclusters (Figs. 13 and 14). Among the top genes, ANXA1, ARF1, HLA-E, LDHA, SLC16A3, S100A11, and TPT1 were important immune regulatory factors showing significant fold changes, and LDHA and SLC16A3 showed 128- and 25-log fold changes, respectively (p < 0.05, log2 fold change ≥ 1, Fig. 14), indicating that these markers were significantly elevated in the EGFR low / MET low subcluster compared with the EGFR high / MET high subcluster. These markers also showed significant elevation in the EGFR of LUSC PDX tumors HIGHThese were key factors in the subcluster (data not shown). This study identified lactate dehydrogenase A (LDHA) and SLC16A3 as core genes with regulatory functions in biological processes of glucose metabolism that can interfere with immune surveillance and response within the TME. Elevated EGFR in pembrolizumab-treated HNSCC PDX was also confirmed by multiplex IHC (Figure 18). EGFR intensity in tumors of the pembrolizumab group was found to have an intensity score of 699±31.6, which was significantly higher than the scores in the vehicle, amivantamab, and combination groups (180±33.1, 88±9.5, and 182±29.4, p<0.01, Figure 19). This suggests that the majority of tumors actively upregulated EGFR and MET expression as one of the multifaceted responses to pembrolizumab. Upregulation of EGFR and MET in tumor cells may have contributed to resistance to pembrolizumab monotherapy (Figure 3A).

[0237] Amivantamab downregulates genes induced by the EGFR / MET high subcluster and promotes antitumor immunity. Based on the publicly available database of The Cancer Genome Atlas (TCGA), EGFR expression in both HNSCC and LUSC was significantly correlated with LDHA and SLC16A3 expression (p<0.05, Figure 17A), suggesting that regulation of EGFR and glucose metabolism simultaneously contributes to TME favorable to tumor progression. In this study, LDHA and SLC16A3 expression was significantly higher in the EGFRHIGH / METHIGH subcluster of HNSCC PDX tumors compared to the EGFRLOW / METLOW subcluster (p<0.05, Figure 17B). In addition, LDHA and SLC16A3 expression was significantly higher in the pembrolizumab group (Figure 17B). Transcriptional upregulation of other glycolysis markers was also predominantly increased in the EGFRHIGH / METHIGH subcluster and the pembrolizumab group (HK2, GPI, ALDOA, PGK1, PGAM1, ENO1, and ENO2, Figure 17C). Furthermore, key regulators of hypoxia showed the same trend across the EGFRHIGH / METHIGH subcluster and treatment groups (HIF1A, HDAC1, KDM1A, KDM2A, CA9, VEGFA, and TWIST1, Figure 17D), suggesting that tumors in pembrolizumab-treated HNSCC PDX mice may have an acidic environment due to their preference for anaerobic glucose metabolism in the TME.

[0238] Correlations between the translational and surface expression of EGFR and MET were also confirmed in HNSCC and LUSC cell lines. T cells secrete IFN-γ, which functions as an autocrine / parasecretory molecule in response to pembrolizumab at tumor sites. Therefore, cell lines were treated with IFN-γ to replicate the immunostimulation in TME of a PDX model induced by pembrolizumab treatment. At the protein level, upregulation of EGFR and MET in selected HNSCC and LUSC cell lines after IFN-γ showed a moderate correlation, while the expression of pEGFR and pMET showed a strong positive correlation (r=0.9824, p<0.0001, Figure 17E). In addition, IFN-γ-induced surface expression of EGFR and MET showed a positive correlation (r=0.9507, p<0.005, Figure 17E). In H1703, IFN-γ-mediated upregulation of EGFR and MET, as well as pEGFR and pMET, was inhibited by amivantamab. Amivantamab also reduced the expression of SLC16A3 (monocarboxylic acid transporter 4, MCT4) in H1703 in the presence of IFN-γ (Figure 17F).

[0239] Amibantamab also works with EGFR HIGH MET HIGH In tumor subclusters, immune checkpoint-related markers, including PD-L1, were reduced. DEG analysis of HNSCC PDX tumors also identifies MET regulatory genes and other immune checkpoint markers, as well as EGFR HIGH / MET HIGH The study showed an increase in tumor subclusters. Significantly increased MET-related genes were BACE2, CD274, CD276, DPYD, PRSS23, PYGL, STK40, and S100A4 (Figure 20A). In addition to the elevation of MET regulatory genes, the MET-STAT4-PD-L1 axis showed an increase in EGFR HIGH / MET HIGH Expression was significantly increased in the tumor group (Figure 20B). Expression of these markers appeared to be decreased in the combination therapy group, suggesting that the combination of amivantamab and pembrolizumab interfered with downstream signaling of MET regulatory genes (Figure 20C).

[0240] Efficacy and effect of amivantamab alone or in combination with pembrolizumab on body weight in the YHIM-3003 PDX model. In the PDX model YHIM-3003, the efficacy of amivantamab (10 mg / kg, ip, BIW), pembrolizumab (10 mg / kg, ip, Q5D), or combination therapy was evaluated for 19 days. No weight loss (Figure 21A) or adverse clinical signs were observed during the treatment period, indicating that amivantamab and pembrolizumab treatments were well tolerated in the indicated administration regimens.

[0241] Amivantamab or pembrolizumab monotherapy had only a slight effect on tumor growth rate, with percentage ΔTGI of 15.64% and 10.26%, respectively, at day 19 in the PDX model YHIM-3003 (Figure 21B, Table 7). In contrast, the combination of amivantamab and pembrolizumab induced stronger inhibition of tumor growth, with a ΔTGI of 57.37% (p<0.0001) compared to the vehicle control.

[0242] [Table 7] NA: Not applicable; TGI: Tumor growth inhibition; TR: Tumor regression. The percentage ΔTGI was calculated and compared on day 28. Significance was calculated over time up to day 28.

[0243] The objective of this study was to evaluate the antitumor activity of amivantamab delivered as monotherapy or in combination with the anti-PD-1 antibody pembrolizumab in a humanized patient-derived xenograft model. Amivantamab or pembrolizumab alone only moderately delayed tumor growth in this model, while the combination of amivantamab (10 mg / kg, BIW) and pembrolizumab (10 mg / kg, Q5D) significantly inhibited YHIM-3003 tumor growth at the end of the study. All regimens, including the combination, were well tolerated throughout the entire course of treatment.

[0244] Consideration This study demonstrated the benefits of combining amivantamab and pembrolizumab by effectively remodeling the tumor immune microenvironment, providing strong preclinical rationale for the clinical combination of amivantamab and PD-1 blockade therapy. Indeed, there is an ongoing study of combination therapy with amivantamab and cetrelimab in patients with metastatic NSCLC (PolyDamas, NCT05908734), and clinical results are awaited.

[0245] The synergistic effect of amivantamab and pembrolizumab was observed in treatment of humanized PDX mouse models with pembrolizumab-insensitive HNSCC (YHIM-3003) and LUSC (YHIM-2010) tumors. Through a comprehensive analysis of the immunogenic mechanisms underlying the synergistic effect, we noted that the combination treatment enhanced the stimulation of the central memory subset of cytotoxic CD8+ T cells in both PDX models and induced tumor-responsive T cells in HNSCC PDX. The central memory subset of CD8+ T cells exhibited superior in vivo and in vitro antitumor immunity compared to effector memory T cells. In addition to modulating T cells, the combination treatment increased the proportion of granzyme B-expressing cytotoxic CD8+ T cells in the TME, suggesting that the combination treatment promoted the infiltration of physiologically active CD8+ T cells into the tumor lesion. These data suggest that combination therapy may ultimately enhance T cell-mediated tumor killing in tumors that were previously unresponsive to immunotherapy.

[0246] The inventors subsequently analyzed the single-cell transcriptome landscape of tumors and revealed that tumor cells exhibit relatively high EGFR and MET expression in response to pembrolizumab treatment alone. EGFR signaling in cancer is associated with overall metabolism favorable to hyperglycolytic tumors. The inventors have identified EGFR HIGHThe study demonstrated that tumor subclusters were clearly elevated in the pembrolizumab-treated group compared to the combination therapy group in both HNSCC PDX and LUSC PDX, and that lactate-producing biomarkers, including LDHA and SLC16A3, were significantly higher.

[0247] The findings of this study suggest that pembrolizumab may promote a tumor immune microenvironment with "Warburg phenotype" metabolic characteristics in tumors. LDHA is essential for the conversion of pyruvate to lactate, while SLC16A3 promotes lactate exchange between cells and the extracellular matrix (ECM). EGFR HIGH Tumor cells likely produced lactate and actively excreted it into the extracellular matrix (ECM) of the tumor mesenter. While lactate provides metabolic fuel for cancer cells as well as immune cells that kill tumors, such as T cells and NK cells, lactate accumulation due to enhanced glycolysis significantly impairs the ability of CD8+ T cells and NK cells to infiltrate tumor sites. Several studies have also highlighted that lactate suppresses the cytotoxic activity of CD8+ T cells by inhibiting IFN-γ production, which is crucial for promoting tumor killing by CD8+ T cells and NK cells. This suggests that tumors with high EGFR expression may be unresponsive to immunotherapy by disrupting the essential mechanisms of cytotoxic T cells and NK cells in the TME, and subsequently losing function. Indeed, pembrolizumab monotherapy in HNSCC expressing high levels of EGFR and MET has an objective response rate of 16.9% and a median progression-free survival of 2.3 months.

[0248] In addition to creating a metabolic environment favorable for tumor persistence, we found that pembrolizumab treatment also increases the expression of MET-related immune checkpoint markers and PD-L1. Pembrolizumab treatment alone appeared to induce upregulation of immune checkpoints in tumor cells, contributing to greater resistance to T cell death and evasion of the immune response through enhanced signaling via the MET-STAT4-PD-L1 axis. We hypothesize that the HNSCC PDX model became insensitive to anti-PD-1 immunotherapy alone by developing a multifaceted bypass mechanism that upregulates the MET signaling pathway in addition to amplifying EGFR. These data suggest that amivantamab reduces the immunosuppressive effect of pembrolizumab on immune cells in TME. Significant reductions in glycolysis biomarkers and MET-regulated immune checkpoint biomarkers in the combination therapy group restored infiltration and activation of invasive cytotoxic CD8 T cells and NK cells, which was also evident in in vivo models and comprehensive immunoprofiling analyses. These phenomena were less pronounced in the lung squamous cell carcinoma model, as co-expression of EGFR and MET in the tumors was not very abundant. However, gene expression analyses showed similar trends for EGFR and MET.

[0249] Upon binding to major histocompatibility complex (MHI) molecules, T cells secrete IFN-γ to enhance the antitumor activity of surrounding T lymphocytes. IFN-γ signaling also promotes MET activation, inducing immune checkpoints through enhancement of the MET-STAT4-PD-L1 axis in tumor cells, providing tumor cells with immune evasion mechanisms. This also applies to the MET-STAT4-PD-L1 axis and MET-related immune checkpoints, particularly in pembrolizumab-treated EGFR. HIGH Our own research has also shown that these levels were elevated in tumors. These changes may promote tumor growth by enabling immune tolerance and may affect the response to immune checkpoint inhibitors.

[0250] In conclusion, this study found that amivantamab is effective in reducing EGFR in pembrolizumab-insensitive humanized HNSCC PDX models. HIGH MET HIGH This study revealed that amivantamab reduces the immunosuppressive effects induced by pembrolizumab in tumors. The combination of amivantamab and pembrolizumab significantly reduced tumor volume in HNSCC and LUSC tumor-bearing PDX models compared to amivantamab or pembrolizumab alone. This study demonstrated that the combination therapy greatly enhanced antitumor phenotypic changes in invasive CD8 T cells and central memory T cells in TMEs. Single-cell RNA transcriptome analysis showed that pembrolizumab alone induced tumor invasion mechanisms by upregulating EGFR and MET signaling. However, when pembrolizumab was administered in combination with amivantamab, the same biomarkers were reduced. This study highlights the rationale for combination therapy of amivantamab and PD-1 blocking immunotherapy, which may be applicable in clinical treatment regimens for solid advanced tumors.

[0251] Overall, the data suggest that amivantamab in combination with PD-(L)1 axis inhibitors such as pembrolizumab may provide synergistic antitumor effects in EGFR and MET wild-type head and neck cancers where the benefits of anti-PD-1 therapy are limited. Our study demonstrated the benefits of combining amivantamab with PD-(L)1 axis inhibitors such as pembrolizumab by effectively remodeling the TME, and provided a rationale for the clinical combination of amivantamab and PD-(L)1 axis inhibitors.

[0252] Example 2. Amivantamab, an EGFR-MET bispecific drug, in combination with the anti-PD-1 drug cetrelimab in patients with advanced non-small cell lung cancer: Phase 1 / 2 PolyDamas study Background: Patients with advanced non-small cell lung cancer (NSCLC) with EGFR mutations are treated with third-generation TKIs. However, EGFR activation and TKI treatment can contribute to the upregulation of PD-1 / PD-L1 and promote treatment resistance. Amivantamab (ami) is effective against EGFR-mutated advanced NSCLC, either as monotherapy or in combination. Cetrelimab (cet) is an anti-PD-1 monoclonal antibody with clinical activity in previously treated NSCLC (Felip Cancer Chemother Pharmacol 2022;J.89(4):499-514). Simultaneous targeting of the innate and adaptive immune systems by ami and cet may improve the antitumor activity compared to either drug alone. The PolyDamas trial (NCT05908734) aims to identify the recommended phase 2 combination dose (RP2CD) and evaluate the antitumor effect of ami + cet in patients with advanced NSCLC.

[0253] Methods: This open-label, multicenter interventional study will have a combination dose selection phase followed by an expansion phase. Primary endpoints include safety (Phase 1 dose selection) and investigator-reported objective response rate using RECIST 1.1 criteria (Phase 2 dose expansion). For dose selection, 20 patients with advanced NSCLC, with or without known driver mutations, who have progressed during or after standard chemotherapy will be enrolled. Dosage will be in 28-day cycles, starting with a low dose of ami (700 mg IV; 1050 mg for patients weighing ≥80 kg), administered once weekly for the first four weeks (Cycle 1) (initial dose as a divided infusion over the first two days), and then every two weeks thereafter (Cycle 2 and beyond). cet is the first dose given on day 2 of Cycle 1, administered IV at 240 mg Q2W. Dose escalation / decreasing will be based on observation of dose-limiting toxicity. RP2CD is selected through Bayesian optimal spacing design using a 3+3 design run-in.

[0254] The Phase 2 expanded cohorts will enroll 30 patients each after RP2CD identification. Cohort A will enroll patients with advanced NSCLC having EGFR exon 19 deletion or L858R mutation and who have experienced disease progression with third-generation TKIs and platinum-based chemotherapy. Cohort B will enroll patients with untreated wild-type advanced NSCLC (no known driver mutations) and a PD-L1 tumor score of ≥50%. Secondary endpoints for Phase 2 include duration of response, disease control rate, progression-free survival, and overall survival.

[0255] This study is inherently exploratory and hypothetical. There are no formal statistical hypotheses for either phase. In both phases, patients will continue the study treatment until disease progression, unacceptable toxicity, or other criteria for discontinuation of treatment are met. The study is currently enrolling patients, with a target enrollment of 80 patients in total.

Claims

1. A method for treating a solid tumor or mitigating the progression of cancer in a subject requiring treatment of a solid tumor or mitigating the progression of cancer, wherein the method is applied to the subject. (a) PD-(L)1 axis inhibitors, (b) A method comprising administering a bispecific anti-EGFR / c-Met antibody.

2. The aforementioned bispecific anti-EGFR / c-Met antibody is (a) A first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determination region 1 (HCDRl) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDRl) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, (b) The method according to claim 1, comprising a second domain that binds to c-Met, the second domain comprising HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO:

12.

3. (a) The first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, (b) The method according to claim 2, wherein the second domain bound to c-Met includes VH of SEQ ID NO: 15 and VL of SEQ ID NO:

16.

4. The method according to claim 2, wherein the bispecific anti-EGFR / c-Met antibody is an IgG1 isotype.

5. The method according to claim 4, wherein the bispecific anti-EGFR / c-Met antibody comprises the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO:

20.

6. The method according to claim 1, wherein the PD-(L)1 axis inhibitor is an inhibitory antibody.

7. The aforementioned inhibitory antibodies include cetrelimab, pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimusumab, emvafolimab, and atezolizumab (T The method according to claim 6, wherein a drug selected from the group consisting of ECENTRIQ®, durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210.

8. The method according to claim 7, wherein the cetrelimab antibody comprises the heavy chain complementation determination region 1 (HCDRl) of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32, HCDR3 of SEQ ID NO: 33, light chain complementation determination region 1 (LCDRl) of SEQ ID NO: 34, LCDR2 of SEQ ID NO: 35, and LCDR3 of SEQ ID NO:

36.

9. The method according to claim 7, wherein the cetrelimab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 37 and the light chain variable region (VL) of SEQ ID NO: 38, or comprises the heavy chain (HC) of SEQ ID NO: 39 and the light chain (LC) of SEQ ID NO:

40.

10. The method according to claim 7, wherein the pembrolizumab antibody comprises the heavy chain complementarity determining region 1 (HCDRl) of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, HCDR3 of SEQ ID NO: 23, light chain complementarity determining region 1 (LCDRl) of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO:

26.

11. The method according to claim 7, wherein the pembrolizumab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 27 and the light chain variable region (VL) of SEQ ID NO: 28, or comprises the heavy chain (HC) of SEQ ID NO: 29 and the light chain (LC) of SEQ ID NO:

30.

12. The method according to claim 1, wherein the cancer is an EGFR, c-Met, or EGFR and c-Met expressing cancer associated with wild-type EGFR, EGFR activating mutation, EGFR gene amplification, elevated levels of circulating HGF, wild-type c-Met, c-Met activating mutation, c-Met gene amplification, or mutant KRAS, or any combination thereof.

13. The aforementioned EGFR activating mutations include substitutions of L718Q, G719A, G719X (where X is any amino acid), L861X (where X is any amino acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, L858P, or T790M, deletions of E746-A750, deletions of R748-P753, insertion of Ala(A) between M766 and A767, insertion of Ser, Val, and Ala(SVA) between S768 and V769, and between P772 and H773. The method according to claim 12, comprising insertion of Asn and Ser(NS) into, insertion of one or more amino acids between D761 and E762, A763 and Y764, Y764 and Y765, M766 and A767, A767 and V768, S768 and V769, V769 and D770, D770 and N771, N771 and P772, P772 and H773, H773 and V774, V774 and C775, one or more deletions in EGFR exon 20, or one or more insertions in EGFR exon 20, or any combination thereof.

14. The method according to claim 13, wherein the mutant KRAS includes substitution of G12V, G12C, or G12A.

15. The method according to any one of claims 1 to 14, wherein the method increases immune cell infiltration into a solid tumor, and the immune cells are further selected from the group consisting of T cells, B cells, and natural killer cells.

16. The method according to claim 15, wherein the T cells include CD8+ T cells and CD4+ T cells.

17. The method according to claim 16, wherein the T cells include CD8+ T cells.

18. The method according to any one of claims 1 to 17, wherein a population of central memory cytotoxic T cells increases within the tumor microenvironment.

19. The method according to any one of claims 1 to 18, wherein the subject is suspected of having or having EGFR, c-Met, or EGFR and c-Met expressing cancer.

20. The method according to claim 1, wherein the subject is resistant to or has acquired resistance to treatment with previous anticancer therapies.

21. The method according to claim 20, wherein the aforementioned prior anticancer therapy is a kinase inhibitor.

22. The method according to claim 21, wherein the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL.

23. The method according to claim 22, wherein the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib.

24. The method according to any one of claims 1 to 23, wherein the cancer is 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, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC).

25. The method according to any one of claims 1 to 24, wherein the cancer is resistant to treatment with a PD-(L)1 axis inhibitor.

26. The method according to any one of claims 1 to 25, wherein the cancer is selected from the group consisting of head and neck squamous cell carcinoma (HNSCC), lung squamous cell carcinoma (LUSC), or non-small cell lung cancer (NSCLC).

27. The method according to any one of claims 1 to 26, further comprising administering one or more anticancer therapies to the subject.

28. The method according to claim 27, wherein the one or more anticancer therapies include kinase inhibitors.

29. The method according to claim 28, wherein the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL.

30. The method according to claim 29, wherein the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib.

31. The method according to claim 20, wherein the aforementioned prior anti-cancer therapy is chemotherapy.

32. The method according to claim 20, wherein the aforementioned prior anticancer therapy is targeted anticancer therapy.

33. The method according to claim 27, wherein the one or more anticancer therapies include chemotherapy.

34. The method according to claim 27, wherein the one or more anticancer therapies include targeted anticancer therapy.

35. The method according to any one of claims 1 to 34, wherein the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor are administered essentially simultaneously to two different injection sites.

36. The method according to any one of claims 1 to 34, wherein the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor are combined before administration and administered simultaneously.

37. The method according to any one of claims 1 to 34, wherein the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor are administered continuously over a period of one day or more as part of a multi-step treatment regimen.

38. A method for enhancing immune cell infiltration into a solid tumor in a subject requiring enhancement of immune cell infiltration into the solid tumor, wherein the method is applied to the subject, (a) PD-(L)1 axis inhibitors, (b) A method comprising administering a bispecific anti-EGFR / c-Met antibody.

39. The aforementioned bispecific anti-EGFR / c-Met antibody is (a) A first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determination region 1 (HCDRl) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDRl) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, (b) The method according to claim 38, comprising a second domain that binds to c-Met, the second domain comprising HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO:

12.

40. (a) The first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, (b) The method according to claim 39, wherein the second domain bound to c-Met includes VH of SEQ ID NO: 15 and VL of SEQ ID NO:

16.

41. The method according to claim 39, wherein the bispecific anti-EGFR / c-Met antibody is an IgG1 isotype.

42. The method according to claim 41, wherein the bispecific anti-EGFR / c-Met antibody comprises the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO:

20.

43. The method according to claim 38, wherein the PD-(L)1 axis inhibitor is an inhibitory antibody.

44. The aforementioned inhibitory antibodies include cetrelimab, pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimusumab, emvafolimab, and atezolizumab (TE The method according to claim 43, wherein a drug selected from the group consisting of CENTRIQ®, durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210.

45. The method according to claim 44, wherein the cetrelimab antibody comprises the heavy chain complementation determination region 1 (HCDRl) of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32, HCDR3 of SEQ ID NO: 33, light chain complementation determination region 1 (LCDRl) of SEQ ID NO: 34, LCDR2 of SEQ ID NO: 35, and LCDR3 of SEQ ID NO:

36.

46. The method according to claim 44, wherein the cetrelimab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 37 and the light chain variable region (VL) of SEQ ID NO: 38, or comprises the heavy chain (HC) of SEQ ID NO: 39 and the light chain (LC) of SEQ ID NO:

40.

47. The method according to claim 44, wherein the pembrolizumab antibody comprises the heavy chain complementarity determining region 1 (HCDRl) of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, HCDR3 of SEQ ID NO: 23, light chain complementarity determining region 1 (LCDRl) of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO:

26.

48. The method according to claim 44, wherein the pembrolizumab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 27 and the light chain variable region (VL) of SEQ ID NO: 28, or comprises the heavy chain (HC) of SEQ ID NO: 29 and the light chain (LC) of SEQ ID NO:

30.

49. The method according to claim 38, wherein the cancer is an EGFR, c-Met, or EGFR and c-Met expressing cancer associated with wild-type EGFR, EGFR activating mutation, EGFR gene amplification, elevated levels of circulating HGF, wild-type c-Met, c-Met activating mutation, c-Met gene amplification, or mutant KRAS, or any combination thereof.

50. The aforementioned EGFR activating mutations include substitutions of L718Q, G719A, G719X (where X is any amino acid), L861X (where X is any amino acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, L858P, or T790M, deletions of E746-A750, deletions of R748-P753, insertion of Ala(A) between M766 and A767, insertion of Ser, Val, and Ala(SVA) between S768 and V769, and between P772 and H773. The method according to claim 49, comprising insertion of Asn and Ser(NS) into, insertion of one or more amino acids between D761 and E762, A763 and Y764, Y764 and Y765, M766 and A767, A767 and V768, S768 and V769, V769 and D770, D770 and N771, N771 and P772, P772 and H773, H773 and V774, V774 and C775, one or more deletions in EGFR exon 20, or one or more insertions in EGFR exon 20, or any combination thereof.

51. The method according to claim 50, wherein the mutant KRAS includes substitution of G12V, G12C, or G12A.

52. The method according to any one of claims 38 to 51, wherein the immune cells are selected from the group consisting of T cells, B cells, and natural killer cells.

53. The method according to claim 52, wherein the T cells include CD8+ T cells and CD4+ T cells.

54. The method according to claim 53, wherein the T cells include CD8+ T cells.

55. The method according to any one of claims 38 to 54, wherein a population of central memory cytotoxic T cells increases within the tumor microenvironment.

56. The method according to any one of claims 38 to 55, wherein the subject is suspected of having or having EGFR, c-Met, or EGFR and c-Met expressing cancer.

57. The method according to claim 38, wherein the subject is resistant to or has acquired resistance to treatment with previous anticancer therapies.

58. The method according to claim 57, wherein the aforementioned prior anticancer therapy is a kinase inhibitor.

59. The method according to claim 58, wherein the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL.

60. The method according to claim 59, wherein the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib.

61. The method according to any one of claims 38 to 60, wherein the cancer is 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, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC).

62. The method according to any one of claims 38 to 61, wherein the cancer is resistant to treatment with a PD-(L)1 axis inhibitor.

63. The method according to any one of claims 38 to 62, wherein the cancer is selected from the group consisting of head and neck squamous cell carcinoma (HNSCC), lung squamous cell carcinoma (LUSC), or non-small cell lung cancer (NSCLC).

64. The method according to any one of claims 38 to 63, further comprising administering one or more anticancer therapies to the subject.

65. The method according to claim 64, wherein the one or more anticancer therapies include kinase inhibitors.

66. The method according to claim 65, wherein the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL.

67. The method according to claim 66, wherein the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib.

68. The method according to claim 57, wherein the aforementioned prior anti-cancer therapy is chemotherapy.

69. The method according to claim 57, wherein the aforementioned prior anticancer therapy is targeted anticancer therapy.

70. The method according to claim 64, wherein the one or more anticancer therapies include chemotherapy.

71. The method according to claim 64, wherein the one or more anticancer therapies include targeted anticancer therapy.

72. The method according to any one of claims 38 to 71, wherein the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor are administered essentially simultaneously to two different injection sites.

73. The method according to any one of claims 38 to 71, wherein the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor are combined before administration and administered simultaneously.

74. The method according to any one of claims 38 to 71, wherein the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor are administered continuously over a period of one day or more as part of a multi-step treatment regimen.

75. A method for reducing glycolysis or lactate production in the tumor microenvironment of a subject that requires reduction of glycolysis or lactate production in the tumor microenvironment, wherein the method applies to the subject: (a) PD-(L)1 axis inhibitors, (b) A method comprising administering a bispecific anti-EGFR / c-Met antibody.

76. The aforementioned bispecific anti-EGFR / c-Met antibody is (a) A first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determination region 1 (HCDRl) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDRl) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, (b) The method according to claim 75, comprising a second domain that binds to c-Met, the second domain comprising HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO:

12.

77. (a) The first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, (b) The method according to claim 76, wherein the second domain bound to c-Met includes VH of SEQ ID NO: 15 and VL of SEQ ID NO:

16.

78. The method according to claim 76, wherein the bispecific anti-EGFR / c-Met antibody is the IgG1 isotype.

79. The method according to claim 78, wherein the bispecific anti-EGFR / c-Met antibody comprises the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO:

20.

80. The method according to claim 75, wherein the PD-(L)1 axis inhibitor is an inhibitory antibody.

81. The aforementioned inhibitory antibodies include cetrelimab, pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimusumab, emvafolimab, and atezolizumab (TE The method according to claim 80, wherein a drug selected from the group consisting of CENTRIQ®, durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210.

82. The method according to claim 81, wherein the cetrelimab antibody comprises the heavy chain complementation determination region 1 (HCDRl) of SEQ ID NO: 31, HCDR2 of SEQ ID NO: 32, HCDR3 of SEQ ID NO: 33, light chain complementation determination region 1 (LCDRl) of SEQ ID NO: 34, LCDR2 of SEQ ID NO: 35, and LCDR3 of SEQ ID NO:

36.

83. The method according to claim 81, wherein the cetrelimab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 37 and the light chain variable region (VL) of SEQ ID NO: 38, or comprises the heavy chain (HC) of SEQ ID NO: 39 and the light chain (LC) of SEQ ID NO:

40.

84. The method according to claim 81, wherein the pembrolizumab antibody comprises the heavy chain complementarity determining region 1 (HCDRl) of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, HCDR3 of SEQ ID NO: 23, light chain complementarity determining region 1 (LCDRl) of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO:

26.

85. The method according to claim 81, wherein the pembrolizumab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 27 and the light chain variable region (VL) of SEQ ID NO: 28, or comprises the heavy chain (HC) of SEQ ID NO: 29 and the light chain (LC) of SEQ ID NO:

30.

86. The method according to claim 75, wherein the cancer is an EGFR, c-Met, or EGFR and c-Met expressing cancer associated with wild-type EGFR, EGFR activating mutation, EGFR gene amplification, elevated levels of circulating HGF, wild-type c-Met, c-Met activating mutation, c-Met gene amplification, or mutant KRAS, or any combination thereof.

87. The aforementioned EGFR activating mutations include substitutions of L718Q, G719A, G719X (where X is any amino acid), L861X (where X is any amino acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, L858P, or T790M, deletions of E746-A750, deletions of R748-P753, insertion of Ala(A) between M766 and A767, insertion of Ser, Val, and Ala(SVA) between S768 and V769, and between P772 and H773. The method according to claim 86, comprising insertion of Asn and Ser(NS) into, insertion of one or more amino acids between D761 and E762, A763 and Y764, Y764 and Y765, M766 and A767, A767 and V768, S768 and V769, V769 and D770, D770 and N771, N771 and P772, P772 and H773, H773 and V774, V774 and C775, one or more deletions in EGFR exon 20, or one or more insertions in EGFR exon 20, or any combination thereof.

88. The method according to claim 87, wherein the mutant KRAS includes substitution of G12V, G12C, or G12A.

89. The method according to any one of claims 75 to 88, wherein the method increases immune cell infiltration into a solid tumor, and the immune cells are further selected from the group consisting of T cells, B cells, and natural killer cells.

90. The method according to claim 89, wherein the T cells include CD8+ T cells and CD4+ T cells.

91. The method according to claim 90, wherein the T cells include CD8+ T cells.

92. The method according to any one of claims 75 to 91, wherein a population of central memory cytotoxic T cells increases within the tumor microenvironment.

93. The method according to any one of claims 75 to 92, wherein the subject is suspected of having or having EGFR, c-Met, or EGFR and c-Met expressing cancer.

94. The method according to claim 75, wherein the subject is resistant to or has acquired resistance to treatment with previous anticancer therapies.

95. The method according to claim 94, wherein the aforementioned prior anticancer therapy is a kinase inhibitor.

96. The method according to claim 95, wherein the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL.

97. The method according to claim 96, wherein the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib.

98. The method according to any one of claims 75 to 97, wherein the cancer is 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, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC).

99. The method according to any one of claims 75 to 98, wherein the cancer is resistant to treatment with a PD-(L)1 axis inhibitor.

100. The method according to any one of claims 75 to 99, wherein the cancer is selected from the group consisting of head and neck squamous cell carcinoma (HNSCC), lung squamous cell carcinoma (LUSC), or non-small cell lung cancer (NSCLC).

101. The method according to any one of claims 75 to 100, further comprising administering one or more anticancer therapies to the subject.

102. The method according to claim 101, wherein the one or more anticancer therapies include a kinase inhibitor.

103. The method according to claim 102, wherein the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL.

104. The method according to claim 103, wherein the kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, razertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, or sunitinib.

105. The method according to claim 94, wherein the aforementioned prior anti-cancer therapy is chemotherapy.

106. The method according to claim 94, wherein the aforementioned prior anticancer therapy is targeted anticancer therapy.

107. The method according to claim 101, wherein the one or more anti-cancer therapies include chemotherapy.

108. The method according to claim 101, wherein the one or more anticancer therapies include targeted anticancer therapy.

109. The method according to any one of claims 75 to 108, wherein the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor are administered essentially simultaneously to two different injection sites.

110. The method according to any one of claims 75 to 108, wherein the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor are combined before administration and administered simultaneously.

111. The method according to any one of claims 75 to 108, wherein the bispecific anti-EGFR / c-Met antibody and the PD-(L)1 axis inhibitor are administered continuously over a period of one day or more as part of a multi-step treatment regimen.

112. A method for treating a solid tumor, mitigating cancer progression, enhancing immune cell infiltration into a solid tumor, or reducing lactate production in the tumor microenvironment in a subject that requires treatment of a solid tumor, mitigation of cancer progression, enhancement of immune cell infiltration into a solid tumor, or reduction of lactate production in the tumor microenvironment, wherein the method comprises administering a bispecific anti-EGFR / c-Met antibody to the subject, the subject having received prior administration of a PD-(L)1 axis inhibitor.

113. The aforementioned bispecific anti-EGFR / c-Met antibody is (a) A first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determination region 1 (HCDRl) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDRl) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, (b) The method according to claim 112, comprising a second domain that binds to c-Met, the second domain comprising HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO:

12.

114. (a) The first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, (b) The method according to claim 113, wherein the second domain bound to c-Met includes VH of SEQ ID NO: 15 and VL of SEQ ID NO:

16.

115. The method according to claim 113, wherein the bispecific anti-EGFR / c-Met antibody is an IgG1 isotype.

116. The method according to claim 113, wherein the bispecific anti-EGFR / c-Met antibody comprises the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO:

20.

117. The method according to claim 112, wherein the pre-administered PD-(L)1 axis inhibitor is an inhibitory antibody.

118. The aforementioned pre-administered inhibitory antibodies are cetrelimab, pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimusumab, emvafolimab, and atezolizumab. The method according to claim 117, wherein a drug selected from the group consisting of (TECENTRIQ®), durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210.

119. The method according to any one of claims 112 to 118, wherein the immune cells are selected from the group consisting of T cells, B cells, and natural killer cells.

120. The aforementioned immune cells are CD8 ＋ T cells and CD4 ＋ The method according to claim 119, comprising T cells.

121. The aforementioned immune cells are CD8 ＋ The method according to claim 120, comprising T cells.

122. The method according to any one of claims 112 to 120, wherein a population of central memory cytotoxic T cells increases within the tumor microenvironment.

123. A method for reducing lactate production in the tumor microenvironment in a subject requiring reduction of lactate production in the tumor microenvironment, the method comprising administering a combination of inhibitors to the subject, wherein the combination of inhibitors is (a) A combination including an EGFR inhibitor, a c-Met inhibitor and a PD-(L) monoaxial inhibitor, (b) A method selected from the group consisting of a combination of an EGFR inhibitor and a c-Met inhibitor, wherein the subject has received prior administration of a PD-(L)1 axis inhibitor.

124. The method according to claim 123, wherein the combination of an EGFR inhibitor and a c-Met inhibitor comprises a bispecific anti-EGFR / c-Met antibody.

125. The aforementioned bispecific anti-EGFR / c-Met antibody is (a) A first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determination region 1 (HCDRl) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDRl) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, (b) The method according to claim 124, comprising a second domain that binds to c-Met, the second domain comprising HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO:

12.

126. (a) The first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, (b) The method according to claim 125, wherein the second domain bound to c-Met includes VH of SEQ ID NO: 15 and VL of SEQ ID NO:

16.

127. The method according to claim 124, wherein the bispecific anti-EGFR / c-Met antibody is an IgG1 isotype.

128. The method according to claim 124, wherein the bispecific anti-EGFR / c-Met antibody comprises the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO:

20.

129. The method according to claim 124, wherein the PD-(L)1 axis inhibitor is an inhibitory antibody.

130. The aforementioned inhibitory antibodies include cetrelimab, pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimusumab, emvafolimab, and atezolizumab (TE The method according to claim 129, wherein a drug selected from the group consisting of CENTRIQ®, durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210.

131. The method according to any one of claims 123 to 130, wherein a population of central memory cytotoxic T cells increases within the tumor microenvironment.

132. The method according to any one of claims 123 to 131, wherein the cancer is 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, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC).

133. The method according to any one of claims 123 to 132, wherein the cancer is resistant to treatment with a PD-(L)1 axis inhibitor.

134. The method according to any one of claims 123 to 133, wherein the cancer is selected from the group consisting of head and neck squamous cell carcinoma (HNSCC), lung squamous cell carcinoma (LUSC), or non-small cell lung cancer (NSCLC).

135. A kit comprising a first pharmaceutical composition containing a bispecific anti-EGFR / c-Met antibody and a second pharmaceutical composition containing a PD-(L)1 axis inhibitor, in two or more containers.

136. The aforementioned bispecific anti-EGFR / c-Met antibody is (a) A first domain that binds to EGFR, the first domain comprising the heavy chain complementarity determination region 1 (HCDRl) of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, HCDR3 of SEQ ID NO: 3, light chain complementarity determination region 1 (LCDRl) of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6, (b) A second domain that binds to c-Met, the second domain comprising HCDRl of SEQ ID NO: 7, HCDR2 of SEQ ID NO: 8, HCDR3 of SEQ ID NO: 9, LCDRl of SEQ ID NO: 10, LCDR2 of SEQ ID NO: 11, and LCDR3 of SEQ ID NO: 12, the kit according to claim 135.

137. (a) The first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, (b) The kit according to claim 135, wherein the second domain that binds to c-Met includes VH of SEQ ID NO: 15 and VL of SEQ ID NO:

16.

138. The kit according to claim 135, wherein the bispecific anti-EGFR / c-Met antibody is an IgG1 isotype.

139. The kit according to claim 135, wherein the bispecific anti-EGFR / c-Met antibody comprises the first heavy chain (HC1) of SEQ ID NO: 17, the first light chain (LC1) of SEQ ID NO: 18, the second heavy chain (HC2) of SEQ ID NO: 19, and the second light chain (LC2) of SEQ ID NO:

20.

140. The kit according to claim 135, wherein the PD-(L)1 axis inhibitor is an inhibitory antibody.

141. The aforementioned inhibitory antibodies include cetrelimab, pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), cintilimab, semiprimab (LIBTAYO®), tripolibamab, tislerizumab, spartalizumab, chamelerizumab, dostralimab, genolimusumab, emvafolimab, and atezolizumab (TEC). The kit according to claim 140, selected from the group consisting of ENTRIQ®, durvalumab (IMFINZI®), avelumab (BAVENCIIO®), REGN2810, pizilizumab, MEDI0680, PDR001, PF-06801591, BGB-A317, TSR-042, and SHR-1210.

142. The kit according to claim 141, wherein the cetrelimab antibody comprises the heavy chain complementation region 1 (HCDRl) of SEQ ID NO: 31, the HCDR2 of SEQ ID NO: 32, the HCDR3 of SEQ ID NO: 33, the light chain complementation region 1 (LCDRl) of SEQ ID NO: 34, the LCDR2 of SEQ ID NO: 35, and the LCDR3 of SEQ ID NO:

36.

143. The kit according to claim 141, wherein the cetrelimab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 37 and the light chain variable region (VL) of SEQ ID NO: 38, or comprises the heavy chain (HC) of SEQ ID NO: 39 and the light chain (LC) of SEQ ID NO:

40.

144. The kit according to claim 141, wherein the pembrolizumab antibody comprises the heavy chain complementarity determining region 1 (HCDRl) of SEQ ID NO: 21, HCDR2 of SEQ ID NO: 22, HCDR3 of SEQ ID NO: 23, light chain complementarity determining region 1 (LCDRl) of SEQ ID NO: 24, LCDR2 of SEQ ID NO: 25, and LCDR3 of SEQ ID NO:

26.

145. The kit according to claim 141, wherein the pembrolizumab antibody comprises the heavy chain variable region (VH) of SEQ ID NO: 27 and the light chain variable region (VL) of SEQ ID NO: 28, or comprises the heavy chain (HC) of SEQ ID NO: 29 and the light chain (LC) of SEQ ID NO:

30.

146. The kit according to any one of claims 135 to 145, wherein the first pharmaceutical composition comprising the bispecific anti-EGFR / c-Met antibody further comprises a first pharmaceutically acceptable excipient, and the second pharmaceutical composition comprising the PD-(L)1 axis inhibitor further comprises a second pharmaceutically acceptable excipient.

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