Combinations with epidermal growth factor receptor tyrosine kinase inhibitors for treating cancer

Abstract

The present disclosure relates to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) and anti-EGFR / cMET antibody molecules for use in combination in the treatment of cancer (e.g., non-small cell lung cancer [NSCLC]).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 367,068, filed June 27, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0002] Reference to sequence listings submitted electronically This application incorporates by reference the Sequence Listing, which is submitted herewith in computer readable format (CRF) as a text file entitled "EGFCM-400-WO-PCT," created on Jun. 5, 2023, and having a size of 75,711 bytes.

[0003] The present specification relates to an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) for use in the treatment of cancer (e.g., non-small cell lung cancer [NSCLC]), the EGFR TKI for use in combination with an anti-EGFR / cMET inhibitor molecule. [Background technology]

[0004] Several drugs against the epidermal growth factor receptor (EGFR) are approved or in clinical development. For example, two first-generation (erlotinib and gefitinib), two second-generation (afatinib and dacomitinib), and a third-generation (osimertinib) tyrosine kinase inhibitors (TKIs) are currently available for the management of EGFR mutation-positive non-small cell lung cancer (NSCLC). All of these TKIs are effective in patients with NSCLC whose tumors harbor an in-frame deletion in exon 19 and an L858R point mutation in exon 21 of EGFR. These two mutations account for approximately 90% of all EGFR mutations. In approximately 50% of patients, resistance to first- and second-generation TKIs is mediated by the acquisition of the "gatekeeper" mutation, T790M. Currently, osimertinib is the only registered EGFR TKI that is active against exon 19 deletions and L858R mutations, regardless of the presence of T790M mutations. However, patients treated with osimertinib eventually progress due to the development of acquired resistance, primarily due to other resistance mechanisms. Therefore, there remains a need to develop novel therapies to treat cancer, especially for patients whose disease has progressed after treatment with third-generation EGFR TKIs.

[0005] cMET, the gene product of the proto-oncogene MET, is a receptor tyrosine kinase expressed primarily on the surface of epithelial cells. Abnormal expression and dysregulation of the c-MET pathway have been reported in a variety of human cancers, including non-small cell lung cancer, colorectal cancer, gastrointestinal cancer, head and neck cancer, pancreatic cancer, renal cancer, and hepatocellular carcinoma, among others (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4). There is a large and growing body of literature demonstrating crosstalk and direct interaction between the EGFR and cMET signaling pathways, and this crosstalk functionally translates into resistance to EGFR- and cMET-targeted therapies in clinical settings (Non-Patent Document 5; Non-Patent Document 6; Non-Patent Document 7).

[0006] Antibody-drug conjugates (ADCs) are being investigated as a means to overcome limitations associated with the treatment of cMET- and EGFR-expressing cancers. One EGFR-directed ADC, depatukizumab mafodotin (ABT-414), is in Phase III clinical development by AbbVie for glioblastoma (NPL 8). ABT-414 was previously tested in Phase II trials for several additional solid tumor indications (ClinicalTrials.gov: NCT01741727). ADCs have shown only limited efficacy in these indications at tolerated doses, and troublesome ocular toxicity has been frequently observed in treated patients (NPL 9). A second-generation EGFR ADC, ABBV-221, was in clinical development but was discontinued due to safety concerns (NPL 10; NPL 11). One c-MET-targeting ADC, telisotuzumab vedotin (ABBV-399), is in phase II clinical development in patients with non-small cell lung cancer (NSCLC) whose tumors express high levels of c-MET, both as monotherapy and in combination with the EGFR inhibitor erlotinib (NPL 12; NPL 13). The combination of a c-MET ADC and an EGFR TKI demonstrated clinical activity in phase I trials in this selected patient population, with peripheral neuropathy and skin rash being the most common treatment-related adverse events (NPL 12; NPL 11).

[0007] Bispecific antibodies targeting EGFR and cMET have also been developed and are undergoing clinical trials in the treatment of patients with advanced NSCLC, both as monotherapy and in combination with third-generation EGFR TKIs (ClinicalTrials.gov: NCT02609776).

[0008] The nature of bispecific antibodies allows for fine tuning of the interaction between each target, potentially affecting the overall properties of the molecule and generating ADCs with an acceptable therapeutic window (Non-Patent Document 14). This concept has been tested in vitro for EGFR and c-MET, but researchers have yet to demonstrate in vivo the concept of improved safety or efficacy compared to the EGFR and c-MET ADCs mentioned above (Non-Patent Document 15).

[0009] Therefore, there is a need for new therapeutic strategies targeting EGFR- and cMET-expressing cancers that exhibit both efficacy and an acceptable safety profile. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Organ,SLand MSTsao,An overview of the c-MET signaling pathway.Therapeutic advances in medical oncology,2011.3(1 Suppl):p.S7-S19 [Non-patent document 2] Birchmeier, C., et al., Met, metastasis, motility and more. Nat Rev Mol Cell Biol, 2003.4:p.915-925 [Non-patent document 3] Mo,HNand P.Liu,Targeting MET in cancer therapy.Chronic Dis Transl Med,2017.3(3):p.148-153 [Non-patent document 4] Sierra, JRand M.-S.Tsao, c-MET as a potential therapeutic target and biomarker in cancer. Therapeutic Advances in Medical Oncology, 2011.3(1 suppl):p.S21-S35 [Non-Patent Document 5] McDermott, U., et al., Acquired Resistance of Non-Small Cell Lung Cancer Cells to MET Kinase Inhibition Is Mediated by a Switch to Epidermal Growth Factor Receptor Dependency. Cancer research, 2010. 70(4): p. 1625-1634

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Non-Patent Document 14

[0011] The inventors recognized that developing anti-EGFR / cMET antibody molecules that bind to EGFR with low affinity (e.g., bind to human EGFR with a dissociation constant (Kd) of 10 nM or greater) could be used in combination with EGFR TKIs known to be effective treatments for cancer (e.g., NSCLC).

[0012] As shown herein, it has been demonstrated that anti-EGFR / cMET antibody molecules containing such low-affinity EGFR-binding domains conjugated to drugs (antibody-drug conjugates ("ADCs")) exhibit reduced on-target toxicity in normal tissues, such as skin toxicity, and therefore an improved safety profile compared to conjugates containing an EGFR antigen-binding domain that binds human EGFR with high affinity.

[0013] Furthermore, ADCs containing this low-affinity EGFR-binding domain used in combination with the third-generation TKI osimertinib have been demonstrated to effectively treat a range of EGFR-mutant cancer models, including those that have developed resistance to osimertinib. Thus, it is believed that the combination of the antibody molecules disclosed herein with an EGFR TKI may provide a safe and effective treatment for EGFR-related cancers, for example, in patients who have developed resistance to an EGFR TKI.

[0014]

[0010] In one aspect, provided herein is an EGFR TKI for use in treating cancer in a human patient, wherein the EGFR TKI is administered in combination with an anti-EGFR / cMET antibody molecule, wherein the anti-EGFR / cMET antibody molecule comprises an EGFR-binding domain and a cMET-binding domain, and the EGFR-binding domain is a. The complementarity determining regions (CDRs) of: i. HCDR1 having the amino acid sequence of SEQ ID NO: 1; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2; iii. an HCDR3 having the amino acid sequence of SEQ ID NO: 3, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; a heavy chain variable (VH) region comprising: b. The following CDR: i. LCDR1 having the amino acid sequence of SEQ ID NO: 4; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; The light chain variable (VL) region contains Includes.

[0015] In another aspect, provided herein is an anti-EGFR / cMET antibody molecule for use in treating cancer in a human patient, the anti-EGFR / cMET antibody molecule being administered in combination with an EGFR TKI, the anti-EGFR / cMET antibody molecule comprising an EGFR-binding domain and a cMET-binding domain, wherein the EGFR-binding domain is a. The complementarity determining regions (CDRs) of: i. HCDR1 having the amino acid sequence of SEQ ID NO: 1; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2; iii. an HCDR3 having the amino acid sequence of SEQ ID NO: 3, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; a heavy chain variable (VH) region comprising: b. The following CDR: i. LCDR1 having the amino acid sequence of SEQ ID NO: 4; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; The light chain variable (VL) region contains Includes.

[0016] In another aspect, provided herein is a method of treating cancer in a human patient comprising administering an EGFR / cMET antibody molecule in combination with an EGFR TKI, wherein the anti-EGFR / cMET antibody molecule comprises an EGFR-binding domain and a cMET-binding domain, and the EGFR-binding domain is a. The complementarity determining regions (CDRs) of: i. HCDR1 having the amino acid sequence of SEQ ID NO: 1; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2; iii. an HCDR3 having the amino acid sequence of SEQ ID NO: 3, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; a heavy chain variable (VH) region comprising: b. The following CDR: i. LCDR1 having the amino acid sequence of SEQ ID NO: 4; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; The light chain variable (VL) region contains Includes:

[0017] In another aspect, provided herein is a pharmaceutical combination of an EGFR / cMET antibody molecule and an EGFR TKI, wherein the anti-EGFR / cMET antibody molecule comprises an EGFR-binding domain and a cMET-binding domain, and the EGFR-binding domain is a. The complementarity determining regions (CDRs) of: i. HCDR1 having the amino acid sequence of SEQ ID NO: 1; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2; iii. an HCDR3 having the amino acid sequence of SEQ ID NO: 3, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; a heavy chain variable (VH) region comprising: b. The following CDR: i. LCDR1 having the amino acid sequence of SEQ ID NO: 4; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof in which one, two, or three amino acids in one or more of LCDR1, LCDR2, or LCDR3 are replaced with another amino acid; The light chain variable (VL) region contains Includes:

[0018] In some examples, the administration of the EGFR TKI and the anti-EGFR / cMET antibody molecule is separate, sequential, or simultaneous.

[0019] Examples of EGFR TKIs suitable for use in the claimed combination therapies are described below. In some examples, the EGFR TKI is osimertinib or a pharmaceutically acceptable salt thereof.

[0020] In some examples, the anti-EGFR binding domain comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2, an HCDR3 having the amino acid sequence of SEQ ID NO: 3, an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6. In some examples, the anti-EGFR binding domain comprises a VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, and a VL region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0021] In some instances, the anti-cMET binding domain comprises: a. The complementarity determining regions (CDRs) of: i. HCDR1 having the amino acid sequence of SEQ ID NO: 24; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 25; iii. an HCDR3 having the amino acid sequence of SEQ ID NO: 26, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; a heavy chain variable (VH) region comprising: b. The following CDR: i. LCDR1 having the amino acid sequence of SEQ ID NO: 27; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 28; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 29, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; The light chain variable (VL) region contains Includes.

[0022] In some examples, the anti-cMet binding domain comprises an HCDR1 having the amino acid sequence of SEQ ID NO: 24, an HCDR2 having the amino acid sequence of SEQ ID NO: 25, an HCDR3 having the amino acid sequence of SEQ ID NO: 26, an LCDR1 having the amino acid sequence of SEQ ID NO: 27, an LCDR2 having the amino acid sequence of SEQ ID NO: 28, and an LCDR3 having the amino acid sequence of SEQ ID NO: 29. In some examples, the anti-cMET binding domain comprises a VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 38, and a VL region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0023] In some examples, the antibody molecule is conjugated to a drug, including a cytotoxin, a radioisotope, an immunomodulator, a cytokine, a lymphokine, a chemokine, a growth factor, a tumor necrosis factor, a hormone, a hormone antagonist, an enzyme, an oligonucleotide, DNA, RNA, siRNA, RNAi, microRNA, a photoactive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide, a lipid, a carbohydrate, a chelator, or a combination thereof. In some examples, the drug is a topoisomerase I inhibitor, as further described herein.

[0024] In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some examples, the non-small cell lung cancer is EGFR mutation-positive NSCLC. EGFR mutation-positive NSCLC includes those containing activating mutations such as the L858R mutation and / or one or more deletions in exon 19 of the EGFR gene, as well as mutations associated with EGFR TKI resistance, such as an insertion in exon 20 of the EGFR gene. As shown herein, the combination of EGFR-cMET complex and osimertinib has demonstrated efficacy across a variety of EGFR-mutated cancers, including those classified as osimertinib-resistant.

[0025] Examples and experiments illustrating the principles of the present disclosure will now be described with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0026] [Figure 1A] Diagrammatic representation of the RAA22 / B09-57 DuetMab. Showing the anti-EGFR RAA22 Fab, anti-cMET B09-57 Fab, and the hole and knob heavy chains. The structural drawing is a composite of the individual domain structures. [Figure 1B] Diagrammatic representation of the QD6 / B09-57 DuetMab. Showing the anti-EGFR QD6 Fab, anti-cMET B09-57 Fab, and the hole and knob heavy chains. The structural drawing is a composite of the individual domain structures. [Figure 2] Simultaneous binding studies in an antigen capture format were performed by Octet analysis. Human cMET antigen-loaded sensors were exposed to sequential association and dissociation interactions first with the antibody and then with the human EGFR antigen. Ass = association, Diss = dissociation, NI-NTA = nickel-nitrilotriacetic acid. [Figure 3A]ELISA results demonstrating cross-reactivity of EGFR and c-Met species. The high-affinity monospecific EGFR IgG (QD6) and the monovalent bispecific EGFR / c-Met DuetMAb (QD6 / B09) bound to human, cynomolgus, and mouse EGFR. The low-affinity monospecific EGFR IgG (RAA22) showed weaker binding to human, cynomolgus, and mouse EGFR compared to QD6, and the corresponding monovalent bispecific EGFR / c-Met DuetMAb (RAA22 / B09) showed even weaker binding to human and cynomolgus EGFR compared to the bivalent parent IgG (RAA22), with negligible binding to mouse EGFR. The monospecific c-Met IgG (B09) and all bispecific variants showed comparable binding to human and cynomolgus c-Met, but no detectable binding to mouse c-Met. [Figure 3B] ELISA results demonstrating EGFR and c-Met family specificity. None of the antibodies tested showed appreciable binding to any of the EGFR HER family proteins (HER2, HER3, or HER4) or to any of the c-Met family members (Ron (CD136) or Semaphorin 3a). [Figure 4A] Internalization profile of QD6 / B09 DuetMab and its respective single-arm control antibody. The internalization profile is shown by the time course of the membrane and cytoplasmic signals of each construct. The QD6 / B09 set was acquired using an Opera confocal fluorescence microscope. [Figure 4B] Internalization profiles of the RAA22 / B09 DuetMab and its respective single-arm control antibody. The internalization profiles are shown by the time course of the membrane and cytoplasmic signals of each construct. Sets were acquired using a Zeiss spinning disk confocal fluorescence microscope. The identical profiles of QD6 / B09 and QD6 / IgG indicate an internalization mode driven by the EGFR arm of the QD6 / B09 DuetMab, while the RAA22 / B09 DuetMab requires engagement of both the EGFR and c-MET arms for efficient internalization. [Figure 5A] Internalization profile of the RAA22 / B09-AZD1508 ADC in cells expressing moderate and high levels of the target c-MET and EGFR cell surface receptors. Membrane, cytoplasmic, and total signals for the RAA22 / B09-AF647 ADC in H1975 cells are shown. One representative experiment out of two is shown. H1975 cells show a concomitant decrease in total and membrane intensity, indicating dissociation of the antibody from the cell surface. [Figure 5B] Equivalent to Figure 8A, but for HCC827 cells. HCC827 cells show a stable total signal over the entire time of the experiment. The decrease in membrane signal is due to antibody internalization. [Figure 6A] Internalization of the RAA22 / B09-AZD1508 ADC single-arm control antibody in HCC827 cells. The intensity profile of the RAA22 / IgG single-arm is approximately 10-fold lower than RAA22 / B09 due to weaker binding to EGFR via the single-arm linkage. [Figure 6B] The B09 / IgG single arm dissociates from the cell membrane as shown by the simultaneous decrease in total and membrane signals over time. [Figure 7] Analysis of the relative contribution of individual antibody arms to the cytotoxic activity of bispecific ADCs. NCI H1975 cells were pretreated with excess unarmed parental antibodies to block EGFR or cMET. The EGFR-cMET ADC (RAA22 / B09-AZ1508) was then added to the cells in a 4x serial dilution series with final concentrations ranging from 67 nM to 0.0009 nM. Treated cells were cultured for 72 hours in a humidified incubator at 37°C and 5% CO2. Metabolic activity was determined using a CellTiter-Glo luminescent viability assay (Promega). Data were plotted as percent metabolic activity relative to the untreated control. IC50 values were determined using logistic nonlinear regression analysis between maximum viability (untreated cells) and maximum response (peak of inhibition) using GraphPad Prism software. [Figure 8]Further evaluation of individual antibody arms for bispecific ADC activity. Monospecific, monovalent ADCs were constructed by pairing each binding arm with a non-binding isotype control arm (R347) to generate an EGFR ADC (RAA22 / R347-AZ1508) and an anti-cMET ADC (B09 / R347-AZ1508). The ADCs were added to NCI H1975 cells in a 4x serial dilution series with final concentrations ranging from 67 nM to 0.0009 nM. Percent metabolic activity was measured as described in Figure 7. [Figure 9A] Mouse PDX studies were conducted to determine the efficacy of high-affinity (QD6 / B09-AZ1508) and low-affinity (RAA22 / B09-AZ1508) EGFR-cMET ADCs in patient xenograft (PDX) models of multiple human cancers in immunocompromised mice. Each compound was tested at a single dose level of 3 mg / kg in a single mouse for each PDX model representing a different human tumor. Tumor growth rates relative to untreated control tumors (T / C%) were calculated for tumors that grew larger than the initial volume, and tumor regression rates were calculated for tumors that showed a decrease in size compared to the initial tumor volume. (A) shows a direct comparison of the high-affinity ADC and the low-affinity ADC in each model. [Figure 9B] 1 shows a waterfall plot of high affinity ADCs ranked by potency. [Figure 9C] 1 shows a waterfall plot of low affinity ADCs ranked by potency. [Figure 10]A dose-ranging in vivo efficacy study in PDX models was performed in athymic nude mice unilaterally implanted with tumor fragments harvested from the host animals in the flank. As shown in the figure, the high-affinity EGFR-cMET ADC (QD6 / B09-AZ1508) was tested at dose levels of 1 mg / kg and 2 mg / kg, and the reduced-affinity variant for EGFR (RAA22 / B09-AZ1508) was tested at doses of 1 mg / kg, 2 mg / kg, and 3 mg / kg. Tumor volume measurements were performed twice weekly after the start of treatment and plotted as a line graph of tumor volume over time. Error bars indicate the standard error of the mean (SEM). Inset images show immunohistochemical staining of EGFR and cMET for each model from tumor tissue harvested from early passages of the model. [Figure 11A] In vivo efficacy of EGFR-cMET bispecific ADC in subcutaneous and orthotopic pancreatic PDX models. A) Subcutaneous MEDI-PANC-08 PDX model, ● - untreated, ■ - R347-AZ1508 (3 mg / kg-Q1W x 4), ▲ - QD6 / B09-1508 (1 mg / kg-Q1W x 4), ▼ - QD6 / B09-1508 (2 mg / kg-Q1W x 4), and

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[0023] Figure 1 shows a waterfall plot of multiple EGFR mutant NSCLC patient tumor tissue xenograft model response to treatment with osimertinib at 25 mg / kg. The x-axis shows the best response from baseline over the study period. The y-axis intercept line indicates 30% regression from baseline, which defines the response. [Figure 22B]

[0023] Figure 1 shows a waterfall plot of multiple EGFR-mutated NSCLC patient tumor tissue xenograft model responses to treatment with 2 mg / kg EGFR-cMET™ ADC. The x-axis shows the best response from baseline over the study period. The y-axis intercept line indicates 30% regression from baseline, which defines response. [Figure 22C]

[0023] Figure 1 shows a waterfall plot of multiple EGFR-mutated NSCLC patient tumor tissue xenograft model responses to treatment with a combination of 25 mg / kg osimertinib and 2 mg / kg EGFR-cMET™ ADC. The x-axis shows the best response from baseline over the study period. The y-axis intercept line indicates 30% regression from baseline, which defines response. DETAILED DESCRIPTION OF THE INVENTION

[0027] Aspects and examples of the present disclosure will now be described with reference to the accompanying drawings. Further aspects and disclosures will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0028] target EGFR Human EGFR (also known as proto-oncogene c-ErbB-1, receptor tyrosine protein kinase erbB-1 and EC 2.7.10.1) is a protein identified by UniProt P00533. Alternative splicing of the mRNA encoded by the human EGFR gene (also known as ERBB, ERBB1, and HER1) results in four isoforms: isoform 1 (UniProt: P00533-1, v2 (last sequence update: November 1, 1997)), isoform 2 (UniProt: P00533-2, v1) (which contains substitutions F404L and L405S relative to isoform 1 and lacks the amino acid sequence corresponding to positions 406-1210 in isoform 1), isoform 3 (UniProt: P00533-3, v1) (which contains substitutions at positions 628-705 in isoform 1 and lacks the amino acid sequence corresponding to positions 706-1210 in isoform 1), and isoform 4 (UniProt: P00533-4) (which contains substitution C628S relative to isoform 1 and lacks the amino acid sequence corresponding to positions 629-1210 in isoform 1).

[0029] The structure and function of EGFR are reviewed, for example, in Ferguson, Annu Rev Biophys. (2008) 37:353-373. EGFR is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family). The receptor contains a large extracellular region, a single transmembrane domain, an intracellular juxtamembrane domain, a tyrosine kinase domain, and a C-terminal regulatory region. Binding of EGFR to a ligand induces receptor dimerization and autophosphorylation of several tyrosine residues (Y992, Y1045, Y1068, Y1148, and Y1173) in the C-terminal regulatory region of EGFR.

[0030] Aberrant EGFR expression / activity has been implicated in many diseases, including nervous system disorders and many cancers.

[0031] As used herein, "EGFR" refers to EGFR from any species, including EGFR isoforms, fragments, variants, or homologs from any species.

[0032] cMET Human cMET (c-Met, also known as hepatocyte growth factor receptor (HGFR) or tyrosine protein kinase Met) is a protein identified by UniProt P08581. Alternative splicing of the mRNA encoded by the human MET gene results in three isoforms: isoform 1 (UniProt: P08581-1, v4 (last sequence update: July 7, 2009)), isoform 2 (UniProt: P08581-2) (which contains the amino acid sequence "STWWKEPLNIVSFLFCFAS" inserted at position 755 of isoform 1), and isoform 3 (UniProt: P08581-3), also known as soluble met variant 4, which contains the amino acid sequence "RHVNIALIQR" substituted for positions 755-764 of isoform 1 and lacks the amino acid sequence corresponding to positions 765-1390 of isoform 1).

[0033] The structure of cMET is reviewed, for example, in Gherardi, 2003, which is incorporated herein by reference in its entirety. cMET is a heterodimer composed of a disulfide-linked alpha chain (50 kDa) and a beta chain (145 kDa). cMET contains an N-terminal Sema domain that mediates binding to hepatocyte growth factor (HGF) and an intracellular kinase domain. Ligand binding at the cell surface induces autophosphorylation of cMET on its intracellular domain, which provides docking sites for downstream signaling molecules and activation of several signaling cascades.

[0034] cMET is expressed in normal tissues on the surface of epithelial cells. Overexpression of cMET has been observed in many human tumors and cancers and is often associated with a metastatic phenotype and poor prognosis. Examples of cancers in which high levels of cMET expression have been observed include non-small cell lung cancer (NSCLC), pancreatic cancer, colorectal cancer, head and neck squamous cell carcinoma, breast cancer, and esophagogastric cancer. Coexpression of EGFR and cMET is commonly observed in these cancers.

[0035] antibody molecule The present disclosure provides antibody molecules, which may be provided in isolated form, meaning that they are free of contaminants, such as antibodies capable of binding to other polypeptides and / or serum components.

[0036] The term "antibody molecule" describes an immunoglobulin, whether naturally produced or partially or wholly synthetically produced. An antibody molecule may be a human or humanized antibody molecule. An antibody molecule may be a monoclonal antibody molecule. Examples of antibodies include immunoglobulin isotypes such as immunoglobulin G (IgG), including their isotypic subclasses such as IgG1, IgG2, IgG3, and IgG4, as well as fragments thereof.

[0037] The term "antibody molecule," as used herein, includes antibody fragments, provided that they are shown to bind to the relevant target molecule. Examples of antibody fragments include Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, and single domain antibodies (e.g., VhH). Unless the context requires otherwise, the term "antibody molecule," as used herein, is therefore equivalent to "antibody molecule or fragment thereof."

[0038] Antibody molecules and methods for constructing and using them are well known in the art and are described, for example, in Holliger & Hudson, Nature Biotechnology 23(9):1126-1136 (2005). It is possible to take monoclonal and other antibody molecules and use techniques of recombinant DNA technology to generate other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques can involve transferring the CDRs or variable regions of one antibody molecule into a different antibody molecule (see EP-A-184187, GB-A-2188638A, and EP-A-239400).

[0039] In view of current technology related to monoclonal antibody technology, antibody molecules can be prepared for most antigens. The antigen-binding domain can be a portion of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single-chain Fv fragment (scFv)). Monoclonal antibodies suitable for a selected antigen can be prepared by known techniques, such as those described in "Monoclonal Antibodies; A Manual of Techniques" by H. Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies; Techniques and Applications" by J.G.R. Hurrell (CRC Press, 1982). Chimeric antibodies are described in Neuberger, 1988.

[0040] An antibody molecule according to the present disclosure comprises an antigen-binding domain (also referred to herein as a "binding domain"). An "antigen-binding domain" or "binding domain" describes the portion of a molecule that binds to all or part of a target antigen. If the antigen is large, an antibody may bind only to a specific portion of the antigen, which portion is called an epitope. An antibody antigen-binding site may be provided by one or more antibody variable domains. The antigen-binding site of an antibody optionally comprises a light chain variable (VL) region and a heavy chain variable (VH) region. The VH and VL regions of an antigen-binding domain together constitute the Fv region.

[0041] An antigen-binding domain generally contains six complementarity-determining regions (CDRs): three in the VH region (HCDR1, HCDR2, and HCDR3) and three in the VL region (LCDR1, LCDR2, and LCDR3). Together, the six CDRs define the paratope of the antigen-binding domain (the portion of the antigen-binding domain that binds to the target antigen).

[0042] The VH and VL regions each contain framework regions (FRs) on either side of each CDR, which provide a scaffold for the CDRs. From N- to C-terminus, the VH region contains the following structure: N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-C-terminus, and the VL region contains the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus.

[0043] There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat, 1991, Chothia, 1987, IMGT numbering (described in LeFranc, 2015), and VBASE2 (described in Retter, 2005). The CDRs and FRs of the VH and VL regions of the antibody molecules described herein are defined according to Kabat (Kabat, 1991).

[0044] Antibody molecules comprising at least two antigen-binding domains, each of which is capable of binding to a different target, may be referred to as "bispecific antibody molecules." In contrast, antibody molecules that bind only to a single target (e.g., EGFR or c-MET) are referred to as "monospecific antibody molecules." The present disclosure relates to bispecific antibody molecules comprising an EGFR-binding domain and a cMET-binding domain.

[0045] Anti-EGFR binding domain A binding domain that binds to EGFR (anti-EGFR binding domain) typically comprises the CDRs of an antibody molecule capable of binding to EGFR. In some examples, a binding domain that binds to EGFR further comprises the FRs of an antibody molecule capable of binding to EGFR. That is, in some examples, a binding domain that binds to EGFR comprises the VH region and the VL region of an antibody molecule capable of binding to EGFR.

[0046] In some examples, the binding domain that binds to EGFR comprises a VH region and a VL region that is or is derived from the VH / VL region of an EGFR-binding antibody clone described herein (i.e., anti-EGFR antibody clone RAA22 or QD6). In some examples, the binding domain that binds to EGFR comprises a VH region and a VL region that is or is derived from the VH / VL region of RAA22.

[0047] In some examples, the binding domain that binds to EGFR comprises three HCDRs or three LCDRs, optionally three VH CDRs and three VL CDRs of the anti-EGFR antibody clone RAA22 or QD6, optionally RAA22. The VH and VL domain sequences of antibodies RAA22 and QD6 are described herein, and therefore the three VH domain CDRs and three VL domain CDRs of the antibodies can be determined from the sequences.

[0048] In some examples, the binding domain that binds to EGFR comprises a VH region set forth in (1) or (2) below: (1) a VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 1; HCDR2 having the amino acid sequence of SEQ ID NO: 2, an HCDR3 having the amino acid sequence of SEQ ID NO: 3; or a variant thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; or (2) a VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 1; HCDR2 having the amino acid sequence of SEQ ID NO: 7, an HCDR3 having the amino acid sequence of SEQ ID NO: 3; or A variant thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid.

[0049] In some examples, the binding domain that binds to EGFR comprises the VH region described in (1) above.

[0050] In some examples, the binding domain that binds to EGFR comprises the VH region described in (1) or (2) above, and this VH region further comprises the FR described in (3) below: (3) HFR1 having the amino acid sequence of SEQ ID NO: 8; HFR2 having the amino acid sequence of SEQ ID NO: 9, HFR3 having the amino acid sequence of SEQ ID NO: 10; HFR4 having the amino acid sequence of SEQ ID NO: 11, or A variant thereof in which one, two, or three amino acids in one or more of HFR1, HFR2, HFR3, or HFR4 are replaced with another amino acid.

[0051] In some examples, the binding domain that binds to EGFR comprises a VH region comprising the CDRs described in (1) or (2) above and the FRs described in (3) above.

[0052] In some examples, the binding domain that binds to EGFR comprises a VH region set forth in (4) or (5) below: (4) A VH region comprising the CDR described in (1) and the FR described in (3), (5) A VH region comprising the CDR described in (2) and the FR described in (3).

[0053] In some examples, the binding domain that binds to EGFR comprises the VH region described in (4) above.

[0054] In some examples, the binding domain that binds to EGFR comprises a VH region set forth in (6) or (7) below: (6) A VH region comprising an amino acid sequence having at least 70% sequence identity, for example at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 16. (7) A VH region comprising an amino acid sequence having at least 70% sequence identity, for example at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 18.

[0055] In some examples, the binding domain that binds to EGFR comprises the VH region described in (6) above.

[0056] In some examples, the binding domain that binds to EGFR comprises a VL region described in (8) or (9) below: (8) a VL region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO: 4; LCDR2 having the amino acid sequence of SEQ ID NO: 5; LCDR3 having the amino acid sequence of SEQ ID NO: 6, or A variant thereof in which one, two, or three amino acids in one or more of LCDR1, LCDR2, or LCDR3 are replaced with another amino acid. (9) A VL region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO: 4; LCDR2 having the amino acid sequence of SEQ ID NO: 66; LCDR3 having the amino acid sequence of SEQ ID NO: 67, or A variant thereof in which one, two, or three amino acids in one or more of LCDR1, LCDR2, or LCDR3 are replaced with another amino acid.

[0057] In some examples, the binding domain that binds to EGFR comprises the VL region described in (8) above.

[0058] In some examples, the binding domain that binds to EGFR comprises a VL region described in (8) or (9) above, and this VL region further comprises a FR described in (10) below: (10) LFR1 having the amino acid sequence of SEQ ID NO: 12; LFR2 having the amino acid sequence of SEQ ID NO: 13; LFR3 having the amino acid sequence of SEQ ID NO: 14; LFR4 having the amino acid sequence of SEQ ID NO: 15, or A variant in which one, two, or three amino acids in one or more of LFR1, LFR2, LFR3, or LFR4 are replaced with another amino acid.

[0059] In some examples, the binding domain that binds to EGFR comprises a VL region comprising the CDRs described in (8) or (9) above and the FRs described in (10) above.

[0060] In some examples, the binding domain that binds to EGFR comprises a VL region described in (11) or (12) below: (11) A VL region comprising the CDR described in (8) and the FR described in (10). (12) A VL region comprising the CDR described in (9) and the FR described in (10).

[0061] In some examples, the binding domain that binds to EGFR comprises the VL region described in (11) above.

[0062] In some examples, the binding domain that binds to EGFR comprises a VL region described in (13) or (14) below: (13) A VL region comprising an amino acid sequence having at least 70% sequence identity, for example at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 20. (14) A VL region comprising an amino acid sequence having at least 70% sequence identity, for example at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 22.

[0063] In some examples, the binding domain that binds to EGFR comprises the VL region described in (13) above.

[0064] In some examples, the binding domain that binds to EGFR comprises the VH region described in any one of (1) to (7) above and the VL region described in any one of (8) to (14) above. In some examples, the binding domain comprises the VH region described in any one of (1), (4), and (6) and the VL region described in any one of (8), (11), and (13). In other examples, the binding domain comprises the VH region described in any one of (2), (5), and (7) and the VL region described in any one of (9), (12), and (14).

[0065] Anti-cMET antigen-binding domain A binding domain that binds to cMET (anti-cMET binding domain) typically comprises the CDRs of an antibody molecule capable of binding to cMET. In some examples, a binding domain that binds to cMET further comprises the FRs of an antibody molecule capable of binding to cMET. That is, in some examples, a binding domain that binds to cMET comprises the VH region and the VL region of an antibody molecule capable of binding to cMET.

[0066] In some examples, the binding domain that binds to cMET comprises a VH region and a VL region that is or is derived from a cMET-binding antibody clone described herein (i.e., anti-cMET antibody clone B09-GL).

[0067] In some examples, a binding domain that binds to cMET comprises three HCDRs or three LCDRs, and optionally three VH CDRs and three VL CDRs, of the cMET-binding antibody clone B09-GL. The VH and VL domain sequences of antibody B09-GL are described herein, and thus the three VH domain CDRs and three VL domain CDRs of the antibody can be determined from the sequences.

[0068] In some examples, the binding domain that binds to cMET comprises a VH region set forth in (15) below: (15) A VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 24, HCDR2 having the amino acid sequence of SEQ ID NO: 25, an HCDR3 having the amino acid sequence of SEQ ID NO: 26, or A variant thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid.

[0069] In some examples, the binding domain that binds to cMET comprises the VH region described in (15) above, and this VH region further comprises the FR described in (16) below: (16) HFR1 having the amino acid sequence of SEQ ID NO: 30; HFR2 having the amino acid sequence of SEQ ID NO: 31; HFR3 having the amino acid sequence of SEQ ID NO: 32; HFR4 having the amino acid sequence of SEQ ID NO: 33, or A variant thereof in which one, two, or three amino acids in one or more of HFR1, HFR2, HFR3, or HFR4 are replaced with another amino acid.

[0070] In some examples, the binding domain that binds to cMET comprises a VH set forth in (17) below: (17) A VH region comprising the CDRs according to (15) and the FRs according to (16).

[0071] In some examples, the binding domain that binds to cMET comprises a VH region set forth in (18) below: (18) A VH region comprising an amino acid sequence having at least 70% sequence identity, for example at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 38.

[0072] In some examples, the binding domain that binds to cMET comprises a VL region set forth in (19) below: (19) A VL region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO: 27; LCDR2 having the amino acid sequence of SEQ ID NO: 28; LCDR3 having the amino acid sequence of SEQ ID NO: 29, or A variant thereof in which one, two, or three amino acids in one or more of LCDR1, LCDR2, or LCDR3 are replaced with another amino acid.

[0073] In some examples, the antigen-binding domain that binds to cMET comprises the VL region described in (19) above, and this VL region further comprises the FR described in (20) below: (20) LFR1 having the amino acid sequence of SEQ ID NO: 34; LFR2 having the amino acid sequence of SEQ ID NO: 35; LFR3 having the amino acid sequence of SEQ ID NO: 36; LFR4 having the amino acid sequence of SEQ ID NO: 37, or A variant in which one, two, or three amino acids in one or more of LFR1, LFR2, LFR3, or LFR4 are replaced with another amino acid.

[0074] In some examples, the binding domain that binds to cMET comprises a VL region set forth in (21) below: (21) A VL region comprising the CDRs described in (19) and the FRs described in (20).

[0075] In some examples, the binding domain that binds to cMET comprises a VL region set forth in (22) below: (22) A VL region comprising an amino acid sequence having at least 70% sequence identity, for example at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 40.

[0076] In some examples, the binding domain that binds to cMET comprises a VH region described in any one of (15) to (18) above and a VL region described in any one of (19) to (22) above.

[0077] CDR replacement In examples according to the present disclosure, one or more amino acids (eg, one, two, or three) are substituted with another amino acid.

[0078] Naturally occurring residues can be divided into the following classes based on common side chain properties: 1) Non-polar, aliphatic: glycine (G), methionine (M), alanine (A), valine (V), leucine (L), isoleucine (I), 2) Polar, uncharged: cysteine (C), serine (S), threonine (T), asparagine (N), glutamine (Q), proline (P), (3) Acidic (negative charge): aspartic acid (D), glutamic acid (E), (4) Basic (positively charged): histidine (H), lysine (K), arginine (R), (5) Aromatic: tryptophan (W), tyrosine (Y), phenylalanine (F).

[0079] The amino acid substitution may be a conservative amino acid substitution. A conservative amino acid substitution may involve the exchange of a member of one of these classes with another member of the same class. For example, a conservative amino acid substitution may be the substitution of an acidic amino acid (glutamic acid (E)) for an acidic amino acid (aspartic acid (D)).

[0080] In some embodiments, substitutions may be function-conservative, i.e., in some embodiments, substitutions may not affect (or not substantially affect) one or more functional properties (e.g., binding affinity) of the antigen-binding domain comprising the substitution, compared to the equivalent unsubstituted antigen-binding domain.

[0081] constant region In some examples, the antibody molecules described herein comprise an immunoglobulin heavy chain constant (CH) region. In some examples, the CH is or is derived from a heavy chain constant sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM.

[0082] In some examples, the CH region is the constant of human immunoglobulin G1 (IGHG1; UniProt: P01857-1(v1); SEQ ID NO: 42) or a fragment thereof.

[0083] In some examples, the CH region comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46, 63 or 64. In some examples, the CH region comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of SEQ ID NO: 63 or 64.

[0084] In some examples, the antibody molecule comprises a heavy chain comprising, or consisting of, a VH region described herein and a CH region described herein.

[0085] In some examples, the antibody molecules described herein comprise an immunoglobulin light chain constant (CL) region or a fragment thereof. In some examples, the CL region is or is derived from a kappa CL region set forth in SEQ ID NO: 47 or SEQ ID NO: 48. In some examples, the CL region is or is derived from a lambda CL region set forth in SEQ ID NO: 49 or SEQ ID NO: 65. In some examples, the antibody molecules comprise a first CL region that is or is derived from a kappa CL region set forth in SEQ ID NO: 47 or 48 and a second CL region that is or is derived from a lambda CL region set forth in SEQ ID NO: 49 or 65.

[0086] In some examples, the antibody molecules described herein comprise: a first heavy chain (wherein the first heavy chain comprises a VH region of an anti-EGFR binding domain and a first heavy chain constant (CH) region or a fragment thereof); a first light chain (wherein the first light chain comprises a VL region of an anti-EGFR binding domain and a first light chain constant (CL) region or a fragment thereof); a second heavy chain, wherein the second heavy chain comprises a VH region of an anti-cMET binding domain and a second heavy chain constant (CH) region or a fragment thereof; a second light chain, wherein the second light chain comprises a VL region of an anti-cMET binding domain and a second light chain constant (CL) region or a fragment thereof.

[0087] The first and second CH regions can be the same or different. In other words, the first and second CH regions can form homodimers or heterodimers. For example, an asymmetric bispecific antibody molecule has different first and second CH regions, as described in more detail below. The first and second CL regions can be the same or different. In some examples, the first CL region is or is derived from a kappa CL region set forth in SEQ ID NO: 47 or 48, and the second CL region is or is derived from a lambda CL region set forth in SEQ ID NO: 49 or 65.

[0088] It will be understood that when an antibody molecule comprises a first VH region and a first CH region, these regions together form the first heavy chain of the antibody molecule, i.e., the first VH region and the first CH region are linked to one another. Similarly, a second VH region and a second CH region form the second heavy chain of the antibody molecule, a first VL region and a first CL region form the first light chain of the antibody molecule, and a second VL region and a second CL region form the second light chain of the antibody molecule.

[0089] In some examples, the antibody molecule comprises a heavy chain having an amino acid sequence with at least 70% sequence identity, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of the B-09-GL heavy chain set forth in SEQ ID NO:50, the QD6 heavy chain set forth in SEQ ID NO:53, the RAA22 heavy chain set forth in SEQ ID NO:56, the heavy chain set forth in SEQ ID NO:59 or the heavy chain set forth in SEQ ID NO:60.

[0090] In some examples, the antibody molecule comprises a first and a second heavy chain, (i) the first heavy chain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of the B-09-GL heavy chain set forth in SEQ ID NO: 56; (ii) The second heavy chain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of the RAA22 heavy chain set forth in SEQ ID NO: 50.

[0091] In some examples, the antibody molecule comprises a first and a second heavy chain, (i) the first heavy chain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of the heavy chain set forth in SEQ ID NO: 59; (ii) the second heavy chain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of the heavy chain set forth in SEQ ID NO: 60.

[0092] In some examples, the antibody molecules described herein comprise a light chain that comprises, or consists of, a VL region described herein and a CL region described herein.

[0093] In some examples, the antibody molecules described herein comprise a light chain having an amino acid sequence with at least 70% sequence identity, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of the B-09-GL light chain set forth in SEQ ID NO: 52, the QD6 light chain set forth in SEQ ID NO: 55, the RAA22 light chain set forth in SEQ ID NO: 58, the light chain set forth in SEQ ID NO: 61 or the light chain set forth in SEQ ID NO: 62.

[0094] In some examples, the antibody molecules described herein comprise a first and a second light chain, (i) the first light chain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of the RAA22 light chain set forth in SEQ ID NO: 58; (ii) The second light chain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of the B-09-GL light chain set forth in SEQ ID NO: 52.

[0095] In some examples, the antibody molecules described herein comprise a first and a second light chain, (i) the first light chain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of the light chain set forth in SEQ ID NO: 61; (ii) the second light chain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, to the amino acid sequence of the light chain set forth in SEQ ID NO: 62.

[0096] The CH, CL, heavy chain and / or light chain of the antibody molecules described herein may comprise one or more modifications that, for example, abrogate or reduce Fc effector function, promote the formation of heterodimeric antibody molecules, increase the efficiency of pairing of cognate heavy and light chains, and / or aid in complex formation as described in more detail below. The modified CH, CL, heavy chain and light chain may be referred to as modified CH, modified CL, modified heavy chain and modified light chain, respectively.

[0097] The antibody molecule may contain mutations in the CH region of the heavy chain to reduce or abolish binding of the antibody molecule to one or more Fcγ receptors, such as FcγRI, FcγRIIa, FcγRIIb, or FcγRIII, and / or complement. Such mutations abrogate or reduce Fc effector function. Mutations that reduce or abolish binding of antibody molecules to one or more Fcγ receptors and complement are known, including, for example, the L234F / L235E / P331S "triple mutation" or "TM" described in Organesyan, 2008. Other mutations known to modulate antibody effector function are described, for example, in Wang, 2018.

[0098] Thus, in some examples, the first and / or second heavy chain comprises a phenylalanine (F) at position 234, a glutamic acid (E) at position 235, and a serine (S) at position 331, numbered according to the EU index. For example, one or both of the first and second heavy chains may comprise a CH region having an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 42, and may comprise a phenylalanine (F) at position 234, a glutamic acid (E) at position 235, and a serine (S) at position 331, numbered according to the EU index. As shown in the Examples (e.g., Example 12), the inclusion of a TM in the heavy chain has been demonstrated to improve the pharmacokinetic properties of exemplary antibody molecules and ADCs.

[0099] Examples of CH regions containing the triple mutation are SEQ ID NOs: 63 and 64. Thus, in some examples, one of the first and second heavy chains comprises a CH region having an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 63, and the other heavy chain comprises a CH region having an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 64, wherein one or both of the CH regions comprises a phenylalanine at position 234, a glutamic acid at position 235, and a serine at position 331, numbering according to the EU index.

[0100] Examples of heavy chains comprising a CH region containing a triple mutation are SEQ ID NOs: 59 and 60. Thus, in some examples, one of the first and second heavy chains has an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 59, and the other heavy chain has an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 60, wherein one or both of the heavy chains comprise a phenylalanine at position 234, a glutamic acid at position 235, and a serine at position 331, where numbering is according to the EU index.

[0101] The VL and CL regions and the VH and CH1 regions of an antibody molecule together make up the Fab region. The remainder of the antibody molecule makes up the Fc region.

[0102] Unless otherwise specified, amino acid residue positions of constant domains, including positions of substitutions, deletions and insertions, in amino acid sequences described herein are numbered according to EU numbering (Edelman, 2007).

[0103] Bispecific construction Bispecific antibody molecules can be provided in any suitable configuration. Suitable configurations for the bispecific antibody molecules described herein and methods for producing them are described in Kontermann, MAbs 2012, 4(2):182-197 and Kontermann and Brinkmann 2015, 20(7):838-847, both of which are incorporated herein by reference in their entirety. See in particular Figure 2 in Kontermann MAbs 2012, 4(2):182-19.

[0104] Bispecific antibody molecules can also be generated from existing antibodies by chemical conjugation, e.g., to link two IgG molecules or two Fab′ fragments using homo- or heterobifunctional coupling reagents as described in Graziano and Guptill, Methods Mol Biol. 2004(283):71-85.

[0105] In some embodiments, the bispecific antibody molecule may be an immunoglobulin G-like (IgG-like) bispecific antibody molecule. An IgG-like bispecific antibody molecule may comprise an Fv region, Fab region, or sVD specific for one antigen and an Fv / Fab / sVD and Fc region specific for another antigen. An IgG-like bispecific antibody molecule may be symmetric or asymmetric. In one example, the bispecific antibody molecule is asymmetric.

[0106] Symmetric IgG-like bispecific antibody molecules generally contain antigen-binding domains fused to the N- or C-terminus of the heavy or light chain of an IgG molecule, for example, in the form of scFv fragments or variable single domains. A distinctive feature of these symmetric IgG-like bispecific antibody molecules is that they contain two identical heavy chains. Furthermore, symmetric IgG-like bispecific antibody molecules are typically bivalent for each epitope. As used herein, valency refers to the number of antigen-binding regions of an antibody molecule that can bind to a single epitope. A monoclonal monospecific IgG antibody molecule is bivalent for a single epitope. That is, it contains two antigen-binding domains, each of which can bind to an epitope on a single target molecule. A symmetric IgG-like bispecific antibody molecule is bivalent for each epitope. That is, it typically contains four antigen-binding domains, two of which can bind to a first epitope on the target molecule and two of which can bind to a second epitope on the target molecule.

[0107] Examples of symmetric IgG-like bispecific antibody molecules include DVD-IgG, IgG-scFv, scFv-IgG, scFv4-Ig, IgG-scFab, scFab-IgG, IgG-sVD, sVD-IgG, 2-in-1-IgG, mAb 2 , tandemab common LCs, which can be formed by methods known in the art, such as chemical cross-linking, somatic cell hybridization, or redox methods.

[0108] In contrast, asymmetric IgG-like bispecific antibody molecules are typically monovalent for each target. For example, as described in Klein, 2012, the concept of monovalent bispecific IgGs is believed to have a unique therapeutic niche because they (i) do not undergo receptor homodimerization, (ii) potentially reduce toxicity to non-target tissues by losing the avidity of each antigen, and (iii) have better selectivity when both antigens are selectively restricted or abundantly expressed on target cells. Thus, in some embodiments, the antibody molecule is an asymmetric IgG-like bispecific antibody molecule.

[0109] Asymmetric IgG-like bispecific antibody molecules involve the heterodimerization of two distinct heavy chains and the precise pairing of cognate light and heavy chains. Heavy chain heterodimerization can be addressed by several techniques, including knob-into-hole, CH3 electrostatic steering, and CH3 chains with engineered domains and leucine zipper replacement. Correct light and heavy chain pairing can be ensured using one of these heavy chain heterodimerization techniques, along with the use of a common light chain, domain crossover between CH1 and CL, linking the heavy and light chains with a linker, assembling heavy-light chain dimers in vitro from two separate monoclonal antibodies, interfacial engineering of the entire Fab domain, or disulfide engineering of the CH1 / CL interface.

[0110] Examples of asymmetric IgG-like bispecific antibody molecules include DuetMab, kih IgG, kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pairing and SEED bodies.

[0111] In some examples, antibody molecules contain one or more modifications in one or more of the CH1, CH2, and CH3 domains that promote the formation of heterodimeric antibody molecules. For example, the DuetMab antibody molecule described above contains one or more modifications in one or more of the CH1, CH2, and CH3 domains that promote the formation of heterodimeric antibody molecules. This can include the knob-into-hole (KiH) strategy, which relies on single amino acid substitutions in the CH3 domain to promote heavy chain heterodimerization, as described in Ridgway, 1996. In knob variant heavy chain CH3s, small amino acids are replaced with larger amino acids, while in hole variants, large amino acids are replaced with small amino acids. Additional modifications can also be introduced to stabilize the association between heavy chains.

[0112] CH3 modifications that enhance heterodimerization include, for example, Y407V / T366S / L368A on one heavy chain and T366W on the other heavy chain; S354C / T366W on one heavy chain and Y349C / Y407V / T366S / L368A on the other heavy chain, where constant region numbering is according to the EU index.

[0113] Other examples of CH3 modifications to enhance heterodimerization are described, for example, in Table 1 of Brinkmann and Kontermann, 2017 MABS 9(2), 182-212, which is specifically incorporated herein by reference.

[0114] In some examples, the antibody molecule comprises first and second heavy chains that form a heterodimer, wherein one of the first and second heavy chains comprises a cysteine (C) residue at position 354 and a tryptophan (W) residue at position 366, and the other heavy chain comprises a cysteine (C) residue at position 349, a valine (V) residue at position 407, a serine (S) at position 366, and an alanine (A) at position 368, and the numbering of the constant regions is according to the EU index. For example, one of the first and second heavy chains may have the sequence set forth in SEQ ID NO: 42 and further comprise a cysteine (C) residue at position 354 and a tryptophan (W) residue at position 366, and the other heavy chain has the sequence set forth in SEQ ID NO: 42 and further comprise a cysteine (C) residue at position 349, a valine (V) residue at position 407, a serine (S) at position 366, and an alanine (A) at position 368, and the numbering of the constant regions is according to the EU index.

[0115] In some instances, the antibody molecule (i) a first heavy chain comprising a first modified CH3 region, wherein the first modified CH3 region comprises a cysteine (C) residue at position 354 and a tryptophan (W) residue at position 366; and (ii) a second heavy chain comprising a second modified CH3 region, wherein the second modified CH3 region comprises a cysteine (C) residue at position 349, a valine (V) residue at position 407, a serine (S) residue at position 366, and an alanine (A) residue at position 368. where the numbering of the constant regions is according to the EU index.

[0116] A particular representative configuration of an asymmetric IgG-like bispecific antibody molecule is referred to as a "DuetMab." DuetMab antibody molecules use KIH technology to heterodimerize two different heavy chains, replacing one native disulfide bond at the CH1-CL interface with an engineered disulfide bond to increase the efficiency of pairing between cognate heavy and light chains. Disclosures regarding DuetMab can be found, for example, in U.S. Pat. No. 9,527,927 and Mazor, 2015, which are incorporated herein by reference in their entireties.

[0117] In some instances, the antibody molecule (a) a modified CH region, wherein the modified heavy chain comprises a substitution of a native non-cysteine amino acid with a cysteine amino acid; and (b) a corresponding modified CL region, wherein the modified CL comprises a substitution of a native non-cysteine amino acid with a cysteine amino acid. Including, (i) the first heavy chain comprises a modified CH region and the first light chain comprises a corresponding modified CL region; or (ii) the second heavy chain comprises a modified CH region and the second light chain comprises a corresponding modified CL region;

[0118] In some examples, a substituted cysteine in a modified CH region resulting from the substitution of a native non-cysteine amino acid with a cysteine amino acid and a corresponding substituted cysteine in a modified CL region resulting from the substitution of a native non-cysteine amino acid with a cysteine amino acid can form a disulfide bond.

[0119] In some examples, the modified CH region comprises a substitution of a native non-cysteine amino acid at position 126 with a cysteine amino acid, and the corresponding modified CL region comprises a substitution of a native non-cysteine amino acid at position 121 with a cysteine, where the numbering of the constant regions is according to the EU index.

[0120] In some examples, the modified CH region comprises a substitution of the native non-cysteine amino acid at position 126 with a cysteine amino acid and a substitution of the native cysteine amino acid at position 219 with a non-cysteine amino acid, e.g., with valine, and the corresponding modified CL region comprises a substitution of the native non-cysteine amino acid at position 121 with a cysteine and a substitution of the native cysteine amino acid at position 214 with a non-cysteine amino acid, e.g., with valine, where the numbering of the constant regions is according to the EU index.

[0121] In some examples, the antibody molecule comprises a second CH region and a second corresponding light chain, wherein the second CH region and the second corresponding CL do not contain substitutions of naturally occurring non-cysteine amino acids with cysteine amino acids and do not contain substitutions of naturally occurring cysteines with non-cysteine amino acids.

[0122] Complex The antibody molecule may be conjugated to a drug. In this case, the antibody molecule may be referred to as a "conjugate" or "antibody-drug conjugate." Such conjugates have application in the treatment and / or diagnosis of diseases as described herein. As used herein, the drug may be referred to as a "payload" or "warhead."

[0123] In some examples, the drug comprises a cytotoxin, a radioisotope, an immunomodulatory agent, a cytokine, a lymphokine, a chemokine, a growth factor, a tumor necrosis factor, a hormone, a hormone antagonist, an enzyme, an oligonucleotide, DNA, RNA, siRNA, RNAi, microRNA, a photoactive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide, a lipid, a carbohydrate, a chelator, or a combination thereof.

[0124] A cytotoxin is a compound that can induce the death of a targeted cell. In the context of antibody-drug conjugates, the cytotoxin is typically delivered to the targeted cell by the antibody molecule, where it is released intracellularly and induces cell death. The use of cytotoxins in antibody-drug conjugates is described, for example, in Chalouni and Doll 2018 J Exp Clin Cancer Res. 37(1):20. In some examples, the cytotoxin is a tubulysin, an auristatin, a maytansinoid, a topoisomerase inhibitor, or a pyrrolobenzodiazepine (PBD).

[0125] In certain examples, the cytotoxin is or includes tubulysin. Tubulysins are a class of cytostatic tetrapeptides containing isoleucine and three other complex unnatural amino acids: Mep (RN-mepipecolic acid), Tuv (tubuvaline), and Tut (tubulylosin) or Tup (tubuphenylalanine). Tubulysins are highly potent cytotoxic molecules, potent against multidrug-resistant cell lines (Domling, 2005). These compounds exhibit high cytotoxicity in tests against a panel of cancer cell lines, with IC values in the low picomolar range; therefore, they are of interest as anticancer therapeutics. See, e.g., WO 2012 / 019123. Tubulysin conjugates are disclosed, for example, in U.S. Pat. No. 7,776,814. In some examples, the tubulysin is tubulysin A, which has the following chemical structure: [ka]

[0126] In some examples, the tubulysin is tubulysin 1508, also known as "AZ1508," and is described in more detail in WO 2015 / 157594. Tubulysin 1508 has the following chemical structure: [ka]

[0127] In some instances, the cytotoxin is or includes a topoisomerase inhibitor. As used herein, the term "topoisomerase inhibitor" refers to a cytotoxic agent that inhibits one or more activities of topoisomerase enzymes (topoisomerase I and II), which play a key role in DNA replication and transcription by regulating DNA supercoiling. Therefore, antibody-drug conjugates containing a topoisomerase inhibitor as a cytotoxin are expected to disrupt normal processes involving DNA, thus resulting in cell death. Conjugates containing topoisomerase inhibitors are effective against various tumor-containing cell lines and have demonstrated anticancer activity in clinical trials. See, for example, Ogitani, 2016a; Ogitani, 2016b; Cardillo, 2015, and Bardia, 2017.

[0128] In some examples, the antibody molecule is conjugated to a topoisomerase I inhibitor. Representative examples of topoisomerase I inhibitors include, but are not limited to, camptothecin and its analogs topotecan, irinotecan, belotecan, exatecan, lurotecan, and sinotecan. Representative examples of topoisomerase II inhibitors include, but are not limited to, amsacrine, daunorubicin, doxorubicin, epipodophyllotoxin, ellipticine, epirubicin, etoposide, razoxane, and teniposide.

[0129] An example of the chemical structure of camptothecin is: [ka]

[0130] General examples of suitable topoisomerase I inhibitors are shown in the following compounds: [ka] The compound is designated as A*.

[0131] In some examples, the compound (e.g., A*) is provided with a linker (which may be referred to as a "ligand unit" or "cell binding agent" (CBA)) for linking to an antibody molecule described herein. Suitably, the linker is cleavably linked (e.g., conjugated) to an amino residue, e.g., an amino acid of an antibody molecule described herein.

[0132] The design and selection of linkers for use in conjugates is known in the art and is described, for example, in Beck, 2017. The linkers used herein can be any of the linkers described in Beck, 2017.

[0133] More particularly, examples of suitable topoisomerase I inhibitors are the following compounds having formula "I": [ka] and salts and solvates thereof, wherein R L is a linker for linking to an antibody molecule described herein, said linker optionally being selected from: (ia): [ka] (In the formula, Q is [ka] (In the formula, Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue. and X is [ka] (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, and at least b1 or b2=0 (i.e., only one of b1 and b2 does not have to be 0), and at least c1 or c2=0 (i.e., only one of c1 and c2 does not have to be 0)). and G L is a linker (e.g., a ligand unit or cell binding agent) for linking to an antibody or antigen-binding fragment thereof described herein, or (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; and e is 0 or 1).

[0134] formula [ka] Now, the superscript sign C(=O) and NH indicates the group to which the atom is attached. For example, an NH group is shown attached to a carbonyl (not part of the depicted moiety) and a carbonyl is shown attached to an NH group (not part of the depicted moiety).

[0135] It will be appreciated by those skilled in the art that more than one of the above agents (eg, topoisomerase I inhibitors) may be conjugated to the antibody molecule.

[0136] For example, conjugates (e.g., antibody-drug conjugates) of the present disclosure may be represented by the general formula IV: L-(D L ) p (IV) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody molecule (e.g., a Ligand unit or CBA) described herein, and D L is a drug with a linker (eg, a drug linker unit), and p is an integer from 1 to 20.

[0137] In some instances, D L is a topoisomerase I inhibitor with a linker, represented by formula III: [ka]

[0138] R LL is a linker (e.g., a Ligand unit) that is linked to an antibody molecule described herein, which linker optionally comprises (ia'): [ka] wherein Q and X are as defined above, and G LL is a linker (e.g., a Ligand unit or CBA) that is linked to an antibody molecule described herein, and (ib'): [ka] (In the formula, R L1 and R L2 is as defined above) is selected from.

[0139] Drug loading is represented by p, the number of topoisomerase I inhibitors (e.g., drug units) per antibody molecule (e.g., ligand unit). Drug loading can range from 1 to 20 drug units (D) per ligand unit. In the case of a composition, p represents the average drug loading of the conjugates in the composition, and p ranges from 1 to 20. In some examples, when the drug is a topoisomerase inhibitor, p ranges from 2 to 8, optionally 4 to 8, e.g., 5 to 7 or 5.5 to 6.5. As described in the Examples, ADCs containing the topoisomerase I inhibitor SG3932 were produced with an average DAR of 6 + / - 6%.

[0140] Thus, the present disclosure encompasses conjugates comprising an antibody molecule (e.g., a Ligand unit or CBA) described herein covalently linked to at least one topoisomerase I inhibitor (e.g., a Drug unit such as A* exemplified above), which optionally includes a linker (e.g., a Linker unit), such as R L and / or R LL The ADC is linked to the antibody molecule via a linker such as those described above. In other words, the present disclosure encompasses antibody molecules (e.g., Ligand units or CBAs) described herein, optionally having one or more topoisomerase I inhibitors attached thereto via a linker (e.g., Drug-Linker units). The antibody molecules described in detail above (representing Ligand units or CBAs) are targeting agents that bind to targeting moieties. More specifically, the antibody molecules can specifically bind, for example, to EGFR and cMET on target cells, thereby delivering the Drug units to the target cells. Accordingly, the present invention also provides methods for treating, for example, various cancers and other disorders (e.g., cancers / disorders associated with the presence of cells, such as cancer cells, that express EGFR and cMET) with ADCs. Such methods are described in further detail below.

[0141] Q X In one example, Q is an amino acid residue. The amino acid can be a natural amino acid or an unnatural amino acid. For example, Q can be selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, where Cit is citrulline.

[0142] In one example, Q comprises a dipeptide residue. The amino acids in the dipeptide can be any combination of natural and unnatural amino acids. In some embodiments, the dipeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. In that case, the dipeptide is the recognition site for cathepsin.

[0143] In one example, Q is NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , NH -Val-Cit- C=O , NH -Phe-Cit- C=O , NH -Leu-Cit- C=O , NH -Ile-Cit- C=O , NH -Phe-Arg- C=O , NH -Trp-Cit- C=O , and NH -Gly-Val- C=O and Cit is citrulline.

[0144] In one example, Q is NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , and NH -Val-Cit- C=O is selected from.

[0145] In one example, Q is NH -Phe-Lys- C=O , NH -Val-Cit- C=O or NH -Val-Ala- C=O is selected from.

[0146] Other suitable dipeptide combinations include: NH -Gly-Gly- C=O , NH -Gly-Val- C=O , NH -Pro-Pro- C=O , and NH -Val-Glu- C=O Examples include:

[0147] Other dipeptide combinations, such as those described in Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, which is incorporated herein by reference, may also be used.

[0148] In some examples, Q is a tripeptide residue. The amino acids in the tripeptide can be any combination of natural and unnatural amino acids. In some embodiments, the tripeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. In that case, the tripeptide is the recognition site for cathepsin. Particularly interesting tripeptide linkers are: NH -Glu-Val-Ala- C=O , NH -Glu-Val-Cit- C=O , NH -αGlu-Val-Ala- C=O , NH -αGlu-Val-Cit- C=O is.

[0149] In some examples, Q is a tetrapeptide residue. The amino acids in the tetrapeptide can be any combination of natural and unnatural amino acids. In some embodiments, the tetrapeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the tetrapeptide is the site of action for cathepsin-mediated cleavage. In that case, the tetrapeptide is the recognition site for cathepsin. Tetrapeptide linkers of particular interest are: NH -Gly-Gly-Phe-Gly C=O , and NH -Gly-Phe-Gly-Gly C=O is.

[0150] In some instances, the tetrapeptide is NH -Gly-Gly-Phe-Gly C=O is.

[0151] In the above representation of peptide residues: NH - represents the N-terminus of the residue, C=Orepresents the C-terminus of the residue. The C-terminus is bound to the NH of A*.

[0152] Glu is a glutamic acid residue, i.e., [ka] represents αGlu is a glutamic acid residue when bound via the α chain, i.e. [ka] Represents.

[0153] In one example, where appropriate, the amino acid side chains are chemically protected. The side chain protecting groups can be as described above. The protected amino acid sequence can be cleaved by an enzyme. For example, a dipeptide sequence containing a Boc-side-chain-protected Lys residue can be cleaved by cathepsin.

[0154] Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem catalogue and are discussed above.

[0155] G L G L may be selected from the following:

[0156] [Table 1]

[0157] [Table 2]

[0158] In the formula, Ar is C 5~6 represents an arylene group, such as phenylene; X' represents C 1~4 Represents alkyl. In some instances, G L is G L1-1 and G L1-2In some of these examples, G L is G L1-1 is.

[0159] G LL G LL may be selected from the following:

[0160] [Table 3]

[0161] [Table 4]

[0162] In the formula, Ar is C 5~6 represents an arylene group, such as phenylene; X' represents C 1~4 CBA represents a cell binding agent or ligand unit. In some examples, G LL is G LL1-1 and G LL1-2 In some of these examples, G LL is G LL1-1 is.

[0163] X X is optionally [ka] (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c=0 or 1, d=0 to 5, at least b1 or b2=0, and at least c1 or c2=0) is.

[0164] a can be 0, 1, 2, 3, 4, or 5. In some examples, a is 0 to 3. In some of these examples, a is 0 or 1. In a further example, a is 0.

[0165] b1 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, b1 is 0 to 12. In some of these examples, b1 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0166] b2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, b2 is 0 to 12. In some of these examples, b2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8. Preferably, only one of b1 and b2 may not be 0.

[0167] c1 can be 0 or 1. c2 can be 0 or 1. Preferably, only one of c1 and c2 may not be 0.

[0168] d can be 0, 1, 2, 3, 4, or 5. In some examples, d is 0 to 3. In some of these examples, d is 1 or 2. In a further example, d is 2. In a further example, d is 5.

[0169] In some examples of X, a is 0, b1 is 0, c1 is 1, c2 is 0, d is 2, and b2 can be 0 to 8. In some of these examples, b2 is 0, 2, 3, 4, 5, or 8. In some examples of X, a is 1, b2 is 0, c1 is 0, c2 is 0, d is 0, and b1 can be 0 to 8. In some of these examples, b1 is 0, 2, 3, 4, 5, or 8. In some examples of X, a is 0, b1 is 0, c1 is 0, c2 is 0, d is 1, and b2 can be 0 to 8. In some of these examples, b2 is 0, 2, 3, 4, 5, or 8. In some examples of X, b1 is 0, b2 is 0, c1 is 0, c2 is 0, and one of a and d is 0. The other of a and d is 1 to 5. In some of these examples, the other of a and d is 1. In other of these examples, the other of a and d is 5. In some examples of X, a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 can be 0 to 8. In some of these examples, b2 is 0, 2, 3, 4, 5, or 8.

[0170] In some instances, R L is of formula Ib. In some examples, R LL is of formula Ib'.

[0171] R L1 and R L2 are independently selected from H and methyl, or may be taken together with the carbon atom to which they are attached to form a cyclopropylene or cyclobutylene group.

[0172] In some instances, R L1 and R L2 Both of R and R are H. In some instances, R L1 is H and R L2 is methyl. In some instances, R L1 and R L2 Both of the are methyl.

[0173] In some instances, R L1and R L2 together with the carbon atom to which they are attached form a cyclopropylene group. In some instances, R L1 and R L2 together with the carbon atom to which they are attached form a cyclobutylene group.

[0174] In group Ib, in some instances, e is 0. In other instances, e is 1, and the nitro group can be at any available position on the ring. In some of these instances, it is at the ortho position. In other of these instances, it is at the para position.

[0175] In some examples where the compounds described herein are provided as a single enantiomer or in enantiomerically enriched form, the enantiomerically enriched form has a high enantiomeric ratio of greater than 60:40, 70:30, 80:20, or 90:10. In further examples, the enantiomeric ratio is greater than 95:5, greater than 97:3, or greater than 99:1.

[0176] In some instances, R L is selected from the following:

[0177] [Table 5]

[0178] [Table 6]

[0179] [Table 7]

[0180] In some instances, R LL is the above R L In some examples, the compound of Formula I is a group derived from a group of Formula I P : [ka] and salts and solvates thereof, wherein R LP is a linker for linking to an antibody or antigen-binding fragment thereof described herein, said linker comprising: (ia): [ka] (In the formula, Q P teeth, [ka] (In the formula, Q XP Q P is an amino acid residue, a dipeptide residue, or a tripeptide residue) and X P teeth, [ka] (In the formula, aP=0 to 5, bP=0 to 16, cP=0 or 1, and dP=0 to 5) and G L is a linker for linking to an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein), (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; and e is 0 or 1) is selected from.

[0181] aP can be 0, 1, 2, 3, 4, or 5. In some examples, aP is 0 to 3. In some of these examples, aP is 0 or 1. In a further example, aP is 0.

[0182] bP can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, b is 0 to 12. In some of these examples, bP is 0 to 8, and can be 0, 2, 4, or 8.

[0183] cP can be 0 or 1.

[0184] dP can be 0, 1, 2, 3, 4, or 5. In some examples, dP is 0 to 3. In some of these examples, dP is 1 or 2. In a further example, dP is 2.

[0185] X P In some examples, aP is 0, cP is 1, dP is 2, and bP can be 0 to 8. In some of these examples, bP is 0, 4, or 8.

[0186] Q above for compounds of formula I X Examples of (e.g., where appropriate) Q XP can be applied to.

[0187] G above for compounds of formula I L , R L1 , R L2 Examples of e and e are of formula I P This can be applied to compounds of the following formula:

[0188] In some examples, the conjugate of formula IV is P : L-(D LP ) p (IV P ) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein; and D LP is represented by formula III P : [ka] and a topoisomerase I inhibitor (e.g., a drug linker unit) represented by R LLP is a linker that is attached to the antibody or antigen-binding fragment thereof (e.g., a ligand unit), said linker comprising: (ia'): [ka] (In the formula, Q P and X P is as defined above, and G LL is a linker that is linked to an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein), and (ib'): [ka] (In the formula, R L1 and R L2 is as defined above) is selected from p is an integer of 1 to 20.

[0189] In some examples, the compound of Formula I is P2 : [ka] and salts and solvates thereof, wherein R LP2 is a linker for linking to an antibody or antigen-binding fragment thereof described herein, said linker comprising: (ia) [ka] (In the formula, Q is [ka] (In the formula, Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue. and XP2 teeth, [ka] (In the formula, aP2=0 to 5, b1P2=0 to 16, b2P2=0 to 16, cP2=0 or 1, dP2=0 to 5, and at least b1P2 or b2P2=0 (i.e., only one of b1 and b2 does not have to be 0)) and G L is a linker for linking to an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein), (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; and e is 0 or 1) is selected from.

[0190] aP2 can be 0, 1, 2, 3, 4, or 5. In some examples, aP2 is 0 to 3. In some of these examples, aP2 is 0 or 1. In a further example, aP2 is 0.

[0191] b1P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, b1P2 is 0 to 12. In some of these examples, b1P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0192] b2P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, b2P2 is 0 to 12. In some of these examples, b2P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0193] In some examples, only one of b1P2 and b2P2 may not be zero.

[0194] cP2 can be 0 or 1.

[0195] dP2 can be 0, 1, 2, 3, 4, or 5. In some examples, dP2 is 0-3. In some of these examples, dP2 is 1 or 2. In a further example, dP2 is 2. In a further example, dP2 is 5.

[0196] X P2 In some examples, aP2 is 0, b1P2 is 0, cP2 is 1, dP2 is 2, and b2P2 can be 0 to 8. In some of these examples, b2P2 is 0, 2, 3, 4, 5, or 8. X P2 In some examples, aP2 is 1, b2P2 is 0, cP2 is 0, dP2 is 0, and b1P2 can be 0 to 8. In some of these examples, b1P2 is 0, 2, 3, 4, 5, or 8. P2 In some examples, aP2 is 0, b1P2 is 0, cP2 is 0, dP2 is 1, and b2P2 can be 0 to 8. In some of these examples, b2P2 is 0, 2, 3, 4, 5, or 8. X P2 In some examples, b1P2 is 0, b2P2 is 0, cP2 is 0, and one of aP2 and dP2 is 0. The other of aP2 and d is 1 to 5. In some of these examples, the other of aP2 and d is 1. In other of these examples, the other of aP2 and dP2 is 5.

[0197] Q above for compounds of formula I X Examples of compounds of formula Ia include (e.g., where appropriate): P2 Q X can be applied to.

[0198] G above for compounds of formula I L , R L1 , R L2 Examples of e and e are of formula I P2 The present invention can be applied to the compounds of the present invention.

[0199] In some examples, the conjugate of formula IV is P2 : L-(D LP2 ) p (IV P2 ) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein; and D LP2 is represented by formula III P2 : [ka] and a topoisomerase I inhibitor (e.g., a drug linker unit) represented by R LLP2 is a linker that is attached to the antibody or antigen-binding fragment thereof (e.g., a ligand unit), said linker comprising: (ia'): [ka] (Wherein, Q and X P2 is as defined above, and G LL is a linker attached to the antibody or antigen-binding fragment thereof), and (ib'): [ka] (In the formula, R L1 and R L2 is as defined above) is selected from p is an integer of 1 to 20.

[0200] Particularly suitable topoisomerase I inhibitors include those having the formula: [ka]

[0201] In some examples, the antibody molecules described herein are conjugated to a topoisomerase I inhibitor (e.g., SG3932) having the following formula: [ka]

[0202] For the avoidance of doubt, the number "8" indicates that the structure within the enclosed brackets is repeated 8 times. Thus, an alternative representation of SG3932 is: [ka] is.

[0203] Another way to describe the SG4010 is: [ka] is.

[0204] Another expression for SG4057 is: [ka] is.

[0205] Another expression for SG4052 is: [ka] is.

[0206] Any of the antibodies or antigen-binding fragments thereof described herein can be conjugated to one or more of the above-described topoisomerase I inhibitors.

[0207] Synthesis of Topoisomerase I Inhibitors For comparison, one general synthetic route for the preparation of exemplary topoisomerase I inhibitors is now described.

[0208] R L However, compounds of formula Ia are compounds of formula 2: [ka] (In the formula, R L* is -QH) From the compound of formula 3: [ka] or its activated form. Such a reaction can be carried out under amide coupling conditions. Compounds of formula 2 can be synthesized by coupling compounds of formula 4: [ka] (In the formula, R L*prot -Q-Prot N (In the formula, Prot N is an amine protecting group) Compounds of formula 4 can be synthesized using the Friedlander reaction by deprotection of a compound of formula 5: [ka] The compound of formula 5 can be synthesized by coupling with compound A3. [ka] Compounds of formula 6 can be synthesized from the compound I7 by removing the trifluoroacetamide protecting group. L*prot Compounds of formula I (wherein R L is of formula Ia or Ib) can be obtained from compound I11 to compound R L It can be synthesized by coupling the -OH or its activated form.

[0209] Amine protecting groups: Amine protecting groups are well known to those skilled in the art, and in particular reference is made to the disclosure of suitable protecting groups in Greene's Protecting Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pages 696-871.

[0210] Drug loading (p) is the average number of drugs (e.g., tubulysin or topoisomerase inhibitors) per antibody molecule. In compositions of the present disclosure, drug loading ranges from 1 to 20 drugs (D) per antibody molecule. For example, drug loading can range from 1 to 10 drugs (D) per antibody molecule, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drugs are covalently attached to the antibody molecule. Conjugate compositions include populations of antibody molecules conjugated with drugs in the range of 1 to 10. When the compounds of the present disclosure are conjugated to lysine, drug loading can range from 1 to 80 drugs (D) per antibody molecule, with preferred upper limits of 40, 20, 10, or 8. Conjugate compositions include populations of antibody molecules conjugated with drugs in the range of 1 to 80, 1 to 40, 1 to 20, 1 to 10, or 1 to 8.

[0211] The average number of drugs per antibody molecule in conjugates prepared from a conjugation reaction can be characterized by conventional methods, such as UV, reverse-phase HPLC, HIC, mass spectrometry, ELISA assays, and electrophoresis. The quantitative distribution of conjugates with respect to p can also be determined. ELISA can determine the average value of p in a particular preparation of conjugates (Hamblett, 2004; Sanderson, 2005). However, the distribution of p(drug) values cannot be distinguished due to the antibody-antigen binding and detection limits of ELISA. An ELISA assay for detecting conjugates does not reveal where on the antibody the drug moiety is attached, e.g., to the heavy or light chain fragment or to a specific amino acid residue. In some instances, separation, purification, and characterization of homogeneous conjugates with a specific p value from conjugates with different drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis. These techniques are also applicable to other types of conjugates.

[0212] In some conjugates, p may be limited by the number of binding sites on the antibody molecule: for example, an antibody molecule may have only one or more cysteine thiol groups, or may have only one or more sufficiently reactive thiol groups to which a linker may be attached.

[0213] Typically, fewer drugs than the theoretical maximum are conjugated to an antibody molecule during the conjugation reaction. An antibody molecule may contain, for example, many lysine residues that do not react with the linker (L). Only the most reactive lysine groups can react with amine-reactive linker reagents. Also, only the most reactive cysteine thiol groups can react with thiol-reactive linker reagents. Generally, antibody molecules contain few, if any, free reactive cysteine thiol groups that can be linked to a drug moiety. Most cysteine thiol residues in antibody molecules in conjugates exist as disulfide bridges and must be reduced using a reducing agent such as dithiothreitol (DTT) or TCEP under partial or full reducing conditions. The loading capacity (drug / antibody ratio) of the conjugate can be controlled in several different ways, including: (i) limiting the molar excess of drug linker relative to antibody, (ii) limiting the time or temperature of the conjugation reaction, and (iii) partial or limited reducing conditions for cysteine thiol modification.

[0214] Certain antibody molecules have reducible interchain disulfides, i.e., cysteine bridges. Antibody molecules can be made reactive for conjugation with linker reagents by treating them with a reducing agent such as DTT (dithiothreitol). Thus, theoretically, each cysteine bridge becomes two reactive thiol nucleophiles. This process, also known as "classical conjugation," distinguishes it from methods in which conjugation occurs at engineered cysteines at specific sites in the antibody molecule. Additional nucleophilic groups can be introduced into antibodies by converting amines to thiols through the reaction of lysines with 2-iminothiolane (Traut's reagent).

[0215] ADCs in which drugs are randomly conjugated to natural cysteine residues are prepared by classical conjugation, involving partial reduction of the antibody followed by reaction with the desired linker-drug. For example, the antibody is partially reduced by adding approximately 3 molar equivalents of DTT at pH 8.0 to a concentration of 5 mg / mL, followed by incubation at approximately 37°C for approximately 2 hours. The reduction reaction is then cooled on ice, and excess DTT is removed, for example, by diafiltration. The linker-drug can then be added at a linker-drug / thiol molar ratio of approximately 1:10. The conjugation reaction can be carried out in the presence of approximately 10% v / v DMSO. After conjugation, excess free cysteine (approximately 2x molar ratio to linker-drug) can be added to quench unreacted linker-drug to generate the cysteine-linker-drug adduct. The reaction mixture is then purified (e.g., by hydrophobic interaction chromatography) and subjected to buffer exchange with PBS. Drug loading distribution can be determined using standard methods such as hydrophobic interaction chromatography and reduced reversed-phase chromatography, as described elsewhere.

[0216] Methods for preparing conjugates using direct conjugation with solvent-accessible thiols generated by reduction of antibody interchain disulfide bridges containing N-alkylmaleimides are known. Another method conjugates drugs to the primary amines of lysine using N-hydroxysuccinimide esters. Such methods are reviewed, for example, in Gebleux and Casi, Pharmacol Ther (2016) 167:48-59, which is incorporated herein by reference in its entirety.

[0217] Alternatively or additionally to the classical conjugation methods described above, it is also possible to use site-specific conjugation, in which the drug loading and conjugation site are controlled. This can be achieved, for example, by engineering cysteines at specific residues, replacing residues with unnatural amino acids with bioorthogonal reactivity, or by enzymatic ligation approaches. One method of site-specific conjugation is described in Dimasi, 2017, which is incorporated herein by reference in its entirety, and involves inserting cysteines into antibody molecules at specific positions.

[0218] Cysteine amino acids can be engineered into reactive sites on antibody molecules without forming interchain or intermolecular disulfide bonds (Junutula, 2008; Dornan, 2009; U.S. Patent No. 7,521,541; U.S. Patent No. 7,723,485; WO 2009 / 052249). The engineered cysteine thiol can react with a drug-linker of the present invention bearing a thiol-reactive electrophilic group (e.g., maleimide or α-haloamide) to form a conjugate containing a cysteine-engineered antibody molecule and a drug. Thus, the drug placement can be designed, controlled, and known. The engineered cysteine thiol group typically reacts with a thiol-reactive linker reagent or drug-linker reagent in high yield, allowing for control of drug loading. IgG antibodies can be engineered to introduce cysteine amino acids by substitution at a single site on the heavy or light chain, providing two new cysteines on a symmetric antibody. If desired, drug loadings approaching 2 can be achieved in a nearly uniform complexation product.

[0219] In some examples, the antibody molecule of the conjugate of the present disclosure comprises a CH region, and the drug is chemically conjugated to a cysteine amino acid inserted between positions 239 and 240 of the CH region, where the numbering of the constant region is according to the EU index. Thus, the linkage between the antibody molecule and the drug can be made via this inserted cysteine amino acid and the terminal maleimide group on the linker.

[0220] Examples of CH regions containing a cysteine amino acid inserted between positions 239 and 240 in the CH region are SEQ ID NO: 43 and SEQ ID NO: 45. Examples of heavy chains containing a CH region containing a cysteine amino acid inserted between positions 239 and 240 in the CH region are SEQ ID NO: 50, 53 and 56.

[0221] In other examples, the conjugated antibody molecule does not contain any amino acid residues inserted in the CH region. In particular examples, the conjugated antibody molecule does not contain an inserted cysteine amino acid in the CH region (e.g., between positions 239 and 240 (where constant region numbering is according to the EU index)). As demonstrated in the Examples (e.g., Example 12), an inserted cysteine is not required when classical conjugation is used to conjugate the drug-linker to a native cysteine.

[0222] Examples of CH regions that do not have any amino acid residues inserted therein are SEQ ID NOs: 44, 46, 63, and 64. Examples of heavy chains that include CH regions that do not have any amino acid residues inserted therein are SEQ ID NOs: 51, 54, 57, 59, and 60.

[0223] When two or more nucleophilic or electrophilic groups on an antibody molecule react with a drug-linker intermediate or linker reagent followed by a drug reagent, the resulting product is a mixture of conjugate compounds with a distribution of drugs attached to the antibody (e.g., 1, 2, 3, etc.). Liquid chromatography methods such as polymeric reversed-phase chromatography (PLRP) and hydrophobic interaction chromatography (HIC) can separate compounds in a mixture by drug loading. Although preparations of conjugates with a single drug loading (p) can be isolated, these single drug loading conjugates may still be heterogeneous mixtures because the drugs may be attached via linkers to different sites on the antibody molecule.

[0224] Thus, the conjugate compositions of the present disclosure include a mixture of antibody-drug conjugate compounds in which the antibody bears one or more drug moieties (e.g., tubulysin or topoisomerase inhibitors), which may be attached to the antibody molecule at various amino acid residues.

[0225] In some examples, the average number of Tubulysin drug moieties per antibody molecule ranges from 1 to 8. In some examples, the range is selected from 1 to 6, 1 to 4, 1 to 3, optionally 1 to 2, 1.5 to 2, 1.8 to 2, e.g., 1.9 to 2.

[0226] As already described above, in some examples, the antibody molecules of the ADCs may comprise one or more mutations in the CH region of the heavy chain that reduce or abolish binding of the antibody molecule to one or more Fcγ receptors. Thus, the first and / or second heavy chains of the ADCs described herein comprise a phenylalanine (F) at position 234, a glutamic acid (E) at position 235, and a serine (S) at position 331, numbering according to the EU index.

[0227] Functional properties of antibody molecules and conjugates The antibody molecules and conjugates described herein may be characterized by reference to particular functional properties.

[0228] binding affinity The antibody molecules and conjugates described herein may be characterized by an antigen-binding domain that binds to EGFR with a specific affinity for EGFR and / or an antigen-binding domain that binds to c-Met with a specific affinity for c-Met. The binding affinity of an antibody molecule or conjugate to a cognate antigen, such as human, mouse, or cynomolgus monkey EGFR or c-Met, can be determined by surface plasmon resonance (SPR), for example, using Biacore. Binding affinity can be determined using an antibody molecule, for example, as part of a bispecific antibody molecule comprising a first antigen-binding domain that binds to EGFR and a second antigen-binding domain that binds to c-Met. Alternatively, binding affinity can be determined using an antibody molecule that is monospecific for EGFR or c-Met. In some examples, binding affinity is determined using Biacore, as described in Example 2.1.

[0229] Binding affinity is typically measured by Kd (the equilibrium dissociation constant between antigen-binding domain and its antigen).As is well understood, the lower the Kd value, the higher the binding affinity of antigen-binding domain.For example, an antigen-binding domain that binds to a target with a Kd of 10nM is considered to bind to said target with higher affinity than an antigen-binding domain that binds to the same target with a Kd of 100nM.

[0230] A reference to human EGFR may refer to a polypeptide comprising the extracellular domain of EGFR, for example, having the amino acid sequence set forth in SEQ ID NO: 68. A reference to mouse EGFR may refer to a polypeptide produced from the molecule available from SinoBiological under catalog number 51091-M08H. A reference to cynomolgus EGFR may refer to the amino acid sequence set forth in SEQ ID NO: 69. A reference to human c-Met may refer to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 70. A reference to mouse c-Met may refer to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 90 or a polypeptide produced from the molecule available from SinoBiological under catalog number 50622-M08H. A reference to cynomolgus c-Met may refer to the amino acid sequence set forth in SEQ ID NO: 71.

[0231] EGFR affinity The antibody molecules and conjugates described herein may contain a binding domain that binds to EGFR with low affinity. EGFR is known to be expressed at low levels in normal tissues, such as skin. Antibody molecules and conjugates that bind to EGFR with low affinity are advantageously expected to exhibit low on-target toxicity in normal tissues while still being able to target tumors that express EGFR at high levels, resulting in an improved safety profile. Furthermore, as shown herein, conjugates containing this low-affinity EGFR-binding domain are more effective in treating cancer than conjugates containing EGFR-binding domains with higher affinity.

[0232] The binding domain that binds to EGFR may bind to human EGFR with an affinity having a Kd of 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, or 40 nM or more. Alternatively, the binding domain that binds to EGFR may bind to human EGFR with a Kd of 10 to 100 nM, 20 to 100 nM, 30 to 100 nM, 40 to 100 nM, 10 to 80 nM, 20 to 80 nM, 30 to 80 nM, 40 to 80 nM, 10 to 70 nM, 20 to 70 nM, 30 to 70 nM, 40 to 70 nM, 10 to 60 nM, 20 to 60 nM, 30 to 60 nM, 40 to 60 nM, 10 to 50 nM, 20 to 50 nM, 30 to 50 nM, or 40 to 50 nM.

[0233] The binding domain that binds to EGFR may bind to human EGFR with an affinity lower than that of a binding domain comprising the heavy and light chain sequences of the antibody molecule QD6 set forth in SEQ ID NOs: 53 and 55, respectively.

[0234] For example, an antigen-binding domain that binds to EGFR may bind to human EGFR with an affinity having a Kd that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or 7-fold higher than the Kd for binding to human EGFR of a binding domain comprising the heavy chain and light chain sequences of the antibody molecule QD6 set forth in SEQ ID NOs: 53 and 55, respectively. Alternatively, an antigen-binding domain that binds to EGFR may bind to human EGFR with an affinity having a Kd that is 2- to 10-fold higher, 3- to 10-fold higher, 4- to 10-fold higher, 5- to 10-fold higher, 6- to 10-fold higher, 7- to 10-fold higher, 2- to 9-fold higher, 3- to 9-fold higher, 4- to 9-fold higher, 5- to 9-fold higher, 6- to 9-fold higher, 7- to 9-fold higher, 2- to 8-fold higher, 3- to 8-fold higher, 4- to 8-fold higher, 5- to 8-fold higher, 6- to 8-fold higher, or 7- to 8-fold higher than the Kd for binding to human EGFR of a binding domain comprising the heavy chain and light chain sequences of the antibody QD6 set forth in SEQ ID NOs: 53 and 55, respectively.

[0235] The binding domain that binds to EGFR may bind to human EGFR with an affinity similar to that of a binding domain comprising the variable heavy region sequence and variable light region sequence of the antibody molecule RAA22 set forth in SEQ ID NOs: 16 and 20, respectively. For example, the binding domain that binds to EGFR may bind to human EGFR with an affinity having a Kd that is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, less than 1-fold, or less than 0.5-fold different from the Kd that a binding domain comprising the variable heavy region sequence and variable light region sequence of the antibody molecule RAA22 set forth in SEQ ID NOs: 16 and 20, respectively, binds to human EGFR.

[0236] The binding domain that binds to EGFR may also bind to cynomolgus EGFR. For example, the binding domain that binds to EGFR may bind to cynomolgus EGFR with an affinity having a Kd of less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, or less than 250 nM. Alternatively, the antigen-binding domain that binds to EGFR may bind to cynomolgus EGFR with an affinity having a Kd of 100-700 nM, 100-600 nM, 100-500 nM, 100-400 nM, 100-300 nM, 150-250 nM, or 100-200 nM. The antigen-binding domain that binds to EGFR may bind to cynomolgus EGFR with a Kd that is 10-fold or less, 9-fold or less, 8-fold or less, 7-fold or less, 6-fold or less, 5-fold or less, 4-fold or less, or 3-fold or less than the Kd of the binding domain that binds to human EGFR.

[0237] The binding domain that binds to EGFR may also bind to mouse EGFR. For example, the binding domain that binds to EGFR may bind to mouse EGFR with an affinity having a Kd of less than 1 μM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, or less than 650 nM. Alternatively, the binding domain that binds to EGFR may bind to mouse EGFR with a Kd of 100 nM to 1 μM, 200 to 900 nM, 300 to 800 nM, 400 to 700 nM, 400 to 600 nM, or 450 to 550 nM.

[0238] In some examples, the EGFR-binding binding domain can bind to human EGFR and cynomolgus monkey EGFR. This cross-reactivity is advantageous in preclinical development, allowing dosing and safety testing of antibody molecules and conjugates in cynomolgus monkeys. In some examples, the EGFR-binding binding domain can bind to human EGFR, cynomolgus monkey EGFR, and mouse EGFR. For example, the EGFR-binding binding domain can bind to human EGFR, cynomolgus monkey EGFR, and mouse EGFR with the above-mentioned Kd values (e.g., human EGFR with a Kd of 10 to 100 nM, cynomolgus monkey EGFR with a Kd of 100 to 700 nM, and mouse EGFR with a Kd of 100 nM to 1 μM).

[0239] cMET affinity A binding domain that binds to c-Met may bind to human c-Met with an affinity having a Kd of less than 20 nM, less than 15 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, or less than 2.5 nM. Alternatively, an antigen-binding domain that binds to cMET may bind to human c-Met with an affinity having a Kd of 1 to 20 nM, 1 to 15 nM, 1 to 10 nM, 1 to 9 nM, 1 to 8 nM, 1 to 7 nM, 1 to 6 nM, 1 to 5 nM, 1 to 4 nM, 1 to 3 nM, 1 to 2.5 nM, or 2 to 2.5 nM.

[0240] A binding domain that binds to cMET may also bind to cynomolgus monkey cMET. For example, a binding domain that binds to cMET may bind to cynomolgus monkey cMET with an affinity having a Kd of less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, or less than 2.5 nM. Alternatively, a binding domain that binds to cMET may bind to cynomolgus monkey cMET with an affinity having a Kd of 1 to 20 nM, 1 to 15 nM, 1 to 10 nM, 1 to 9 nM, 1 to 8 nM, 1 to 7 nM, 1 to 6 nM, 1 to 5 nM, 1 to 4 nM, 1 to 3 nM, 1 to 2.5 nM, or 2 to 2.5 nM. A binding domain that binds to cMET may bind to cynomolgus cMET with an affinity having a Kd that is 10-fold or less, 9-fold or less, 8-fold or less, 7-fold or less, 6-fold or less, 5-fold or less, 4-fold or less, 3-fold or less, 2-fold or less, or 1-fold or less than the Kd of a binding domain that binds to human cMet.

[0241] In some examples, the binding domain that binds to cMET can bind to human cMET and cynomolgus monkey cMET. This cross-reactivity is advantageous for enabling dosing and safety testing of antibody molecules in cynomolgus monkeys during preclinical development. For example, the binding domain that binds to cMET may be capable of binding to human cMET and cynomolgus monkey cMET with the Kd values described above (e.g., human cMET with a Kd of 1-20 nM and cynomolgus monkey cMET with a Kd of 1-20 nM).

[0242] specific binding The binding domains described herein can specifically bind to their respective targets (i.e., EGFR and cMET). The term "specific" can refer to a state in which an antigen-binding domain does not exhibit significant binding to molecules other than its specific binding partner (here, EGFR or cMET). Such molecules are called "non-target molecules." The term "specific" can also be applied when an antibody molecule is specific for a particular epitope, such as an epitope on EGFR or cMET carried by several antigens, in which case the antibody molecule can bind to various antigens carrying the epitope.

[0243] In some examples, an antibody molecule is considered to exhibit no significant binding to a non-target molecule if the extent of binding to the non-target molecule is less than about 10% of the extent of binding of the antibody to the target, as measured, for example, by ELISA, SPR, biolayer interferometry (BLI), microscale thermophoresis (MST), or radioimmunoassay (RIA). Alternatively, binding specificity may be reflected in terms of binding affinity, and the antibody molecules described herein bind to EGFR and / or c-Met with an affinity that is at least 0.1 orders of magnitude greater than their affinity to another non-target molecule. In some examples, the antibody molecules of the present disclosure bind to EGFR and / or c-MET with an affinity that is at least 0.2 orders of magnitude, 0.3 orders of magnitude, 0.4 orders of magnitude, 0.5 orders of magnitude, 0.6 orders of magnitude, 0.7 orders of magnitude, 0.8 orders of magnitude, 0.9 orders of magnitude, 1.0 orders of magnitude, 1.5 orders of magnitude, or 2.0 orders of magnitude greater than their affinity to another non-target molecule.

[0244] EGFR is a member of the ErbB receptor family, a subfamily of four closely related receptor tyrosine kinases: EGFR, HER2, HER3, and HER4. The RAA22 antigen-binding domain did not exhibit binding to HER2, HER3, or HER4, demonstrating that this antigen-binding domain specifically binds to EGFR. Thus, in certain examples, an antigen-binding domain that binds to EGFR does not bind or exhibits no significant binding to HER2, HER3, or HER4.

[0245] cMET is a member of the receptor tyrosine kinase subfamily, which also includes Ron and Sema 4a. The B09-GL antigen-binding domain did not exhibit binding to Ron or Sema 4a, indicating that this antigen-binding domain specifically binds to cMET. Thus, in certain instances, a binding domain that binds to cMET does not bind or exhibits significant binding to Ron or Sema 4a.

[0246] Simultaneous binding The antibody molecules and conjugates described herein may be characterized by the ability of both binding domains to simultaneously bind to their respective EGFR and cMET targets. Antibody molecules and conjugates with the ability to simultaneously bind to EGFR and cMET are expected to be advantageous, as many tumors are known to co-express both EGFR and c-Met and therefore may be targeted by the antibody molecules of the present disclosure. Thus, in some instances, the antibody molecule or conjugate is capable of simultaneously binding to EGFR and cMET.

[0247] Simultaneous binding can be determined, for example, by an in vitro cytotoxicity assay using a cell line expressing approximately equal amounts of EGFR and cMET and a conjugate containing an antibody molecule having an EGFR antigen-binding domain and a cMET antigen-binding domain. If the individual antigen-binding domains in the conjugate function independently to deliver the drug, blocking either target in the cell line would only slightly reduce the activity of the conjugate, since the targets are present at comparable levels; the IC50 is expected to shift by no more than two-fold. In this assay, EGFR and cMET targets can be blocked, for example, by using a monospecific antibody molecule that binds to the same region of either EGFR or cMET but lacks a drug capable of inducing cytotoxicity. For example, the monospecific antibody molecule could contain the same antigen-binding domain that binds EGFR or c-Met as the conjugate being tested. On the other hand, if the conjugate requires simultaneous binding to effectively deliver the conjugate into cells, blocking either target is likely to have a greater impact on the activity of the conjugate. That is, using this assay, an antibody molecule is considered to be capable of binding both targets simultaneously if there is at least a 2-fold, at least a 5-fold, or at least a 10-fold shift in IC50 after blocking either target.

[0248] Another method for determining simultaneous binding is to compare the activity of a bispecific EGFR-cMET complex with a monovalent, monospecific control complex in an in vitro cytotoxicity assay. The control complex contains one antigen-binding domain for either EGFR or c-Met and one non-binding isotype antibody control antigen-binding domain. If each antigen-binding domain in the bispecific complex functions independently, the expected result would be that each monospecific control complex would be only slightly less potent than the bispecific complex, and the difference would be additive. Instead, if the two antigen-binding domains of the bispecific complex function synergistically through simultaneous binding, the difference in activity of the bispecific complex compared to the monospecific control antibody would be greater. That is, if the bispecific complex produces an IC50 shift greater than the sum of the IC50 shifts observed using the monospecific control complex, the antibody molecule is considered to be capable of simultaneously binding both targets.

[0249] Further details of these methods for measuring simultaneous binding can be found in the Examples.

[0250] Antibody internalization The antibody molecules and conjugates described herein can be characterized by their ability to mediate efficient cellular internalization. This is particularly useful for conjugates, ensuring that the conjugates are internalized and delivered to lysosomes. The antibody molecule is then degraded, releasing the drug into the cell and exerting its cellular effects, such as cytotoxicity.

[0251] The internalization of antibody molecules by cells can be analyzed by contacting live cells with the antibody molecules and detecting the antibody molecules after a period sufficient for internalization. Internalization can be determined by detecting the localization of the antibody molecules. If the antibody molecules remain on the surface of the cells (e.g., are detected on the cell surface and / or are not detected inside the cells), the antibody molecules or complexes are determined to be not internalized. If the antibody molecules are detected inside the cells (e.g., are localized in the cytoplasm or organelles), the antibody molecules are determined to be internalized.

[0252] An exemplary method for visualizing whether an antibody molecule can mediate efficient internalization involves labeling the antibody molecule with a pH-sensitive dye that exhibits fluorescence at acidic pH and adding these labeled antibody molecules to cells. Internalization into cells can be detected by monitoring fluorescence. An antibody molecule is considered to be capable of mediating internalization and delivery to lysosomes if the observed fluorescence is greater than that of a labeled, unbound control antibody molecule over a period of time, e.g., 48 hours. Further details of this method for visualizing antibody internalization can be found in the Examples.

[0253] The antibody molecules described herein may be characterized by their ability to mediate more efficient internalization when compared to EGFR or cMET monospecific controls. Antibody molecules and conjugates exhibiting this property are expected to be advantageous because they are expected to exhibit greater selectivity for tumor cells that co-express both targets, minimizing the effects of the antibody molecules on normal tissues that do not exhibit significant levels of co-expression.

[0254] In vitro activity The antibody molecules described herein can be characterized by their cytotoxic activity, i.e., their ability to kill cells. Cytotoxic activity can be measured, for example, using an in vitro cell viability assay such as the CellTiter-Glo® (Promega) assay. In some examples, the cells are cells that express both EGFR and cMET.

[0255] The potency of an antibody molecule can be expressed as an IC50 value. IC50 is the median inhibitory concentration of an antibody molecule. In functional assays, IC50 is the concentration that reduces a biological response by 50% of its maximum. IC50 can be calculated by several means known in the art.

[0256] In some examples, antibody molecules described herein that have cytotoxic activity have an IC50 of less than 4000 pM, less than 3500 pM, less than 3000 pM, less than 2500 pM, less than 2000 pM, less than 1500 pM, less than 1000 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 250 pM, less than 200 pM, less than 150 pM, or less than 100 pM, as measured using an in vitro cell viability assay. In some examples, antibody molecules described herein may have an IC50 of 60-500 pM.

[0257] In some examples, the antibody molecules described herein can increase the killing of cells, e.g., tumor cells, that express both EGFR and cMET at significant levels compared to cells that express either EGFR or cMET at low levels. Cells that express significant amounts of both EGFR and cMET can be determined by measuring the relative receptor density on the cell surface. For example, cells that express EGFR and cMET at a relative receptor density of greater than 15,000 on the cell surface can be considered to express significant amounts of both EGFR and cMET, as well as cells that express one of EGFR and c-Met at a low relative receptor density of 15,000 or less on the cell surface. The relative density of EGFR and cMET can be measured, for example, using the Quantum MESF quantitative FACS assay kit, as described in the Examples.

[0258] Examples of cells that express both EGFR and cMET in significant amounts include NCI H596, HCC 827 GR pool, A549, NCI H1792, NCI H1975, NCI H292, and NCI H358 cell lines. Examples of cells that express one of EGFR and cMET at a low relative receptor density include A427, NCI H23, and NCI H661 (Ag negative) cell lines. These cell lines are available through ATCC.

[0259] EGFR tyrosine kinase inhibitors EGFR TKIs can be characterized as first-, second-, or third-generation EGFR TKIs, as described below.

[0260] First-generation EGFR TKIs are reversible inhibitors of EGFR with activating mutations and do not significantly inhibit EGFR with the T790M mutation. Examples of first-generation TKIs include gefitinib and erlotinib.

[0261] Second-generation EGFR TKIs are irreversible inhibitors of EGFR with activating mutations and do not significantly inhibit EGFR with the T790M mutation. Examples of second-generation TKIs include afatinib and dacomitinib.

[0262] Third-generation EGFR TKIs are inhibitors of EGFR with activating mutations, and also significantly inhibit EGFR with T790M mutation, but do not significantly inhibit wild-type EGFR. Examples of third-generation TKIs include the compound of formula (V), osimertinib, AZD3759 (zolifertinib), lazertinib, nazartinib (EGF816), CO1686 (rociletinib), HM61713, ASP8273 (nacotinib), PF-06747775 (mavereltinib), avitinib (abivertinib), alflutinib (AST2818), CX-101 (orafeltinib; RX-518), almonertinib (HS-10296; aumoreltinib) and BPI-7711 (resivertinib).

[0263] In one embodiment, the EGFR TKI is a first-generation EGFR TKI. In a further example, the first-generation EGFR TKI is selected from the group consisting of gefitinib or a pharmaceutically acceptable salt thereof, icotinib or a pharmaceutically acceptable salt thereof, and erlotinib or a pharmaceutically acceptable salt thereof.

[0264] In one embodiment, the EGFR TKI is a second-generation EGFR TKI. In a further embodiment, the second-generation EGFR TKI is selected from dacomitinib or a pharmaceutically acceptable salt thereof and afatinib or a pharmaceutically acceptable salt thereof.

[0265] In one embodiment, the EGFR TKI is a third-generation EGFR TKI. In a further embodiment, the third-generation EGFR TKI is a compound of formula (V) as defined below. In a further embodiment, the third-generation EGFR TKI is selected from the group consisting of osimertinib or a pharmaceutically acceptable salt thereof, AZD3759 or a pharmaceutically acceptable salt thereof, lazertinib or a pharmaceutically acceptable salt thereof, abivertinib or a pharmaceutically acceptable salt thereof, alfurtiniib or a pharmaceutically acceptable salt thereof, CX-101 or a pharmaceutically acceptable salt thereof, HS-10296 or a pharmaceutically acceptable salt thereof, and BPI-7711 or a pharmaceutically acceptable salt thereof. In a further embodiment, the third-generation EGFR TKI is osimertinib or a pharmaceutically acceptable salt thereof.

[0266] Compounds of formula (V): In one embodiment, the EGFR TKI has formula (V): [ka] (In the formula, G is selected from 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl, indol-3-yl, indazol-1-yl, 3,4-dihydro-1H-[1,4]oxazino[4,3-a]indol-10-yl, 6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl, 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl, pyrrolo[3,2-b]pyridin-3-yl and pyrazolo[1,5-a]pyridin-3-yl; R 1 is selected from hydrogen, fluoro, chloro, methyl and cyano; R 2is selected from methoxy, trifluoromethoxy, ethoxy, 2,2,2-trifluoroethoxy and methyl; R 3 are (3R)-3-(dimethylamino)pyrrolidin-1-yl, (3S)-3-(dimethyl-amino)pyrrolidin-1-yl, 3-(dimethylamino)azetidin-1-yl, [2-(dimethylamino)ethyl]-(methyl)amino, [2-(methylamino)ethyl](methyl)amino, 2-(dimethylamino)ethoxy, 2-(methylamino)ethoxy, 5-methyl-2,5-diazaspiro[3.4]oct-2-yl, (3aR,6aR)-5-methylhexa-hydro-pyrrolo[3,4- b] selected from pyrrol-1(2H)-yl, 1-methyl-1,2,3,6-tetrahydropyridin-4-yl, 4-methylpiperidin-1-yl, 4-[2-(dimethylamino)-2-oxoethyl]piperazin-1-yl, methyl[2-(4-methylpiperazin-1-yl)ethyl]amino, methyl[2-(morpholin-4-yl)ethyl]amino, 1-amino-1,2,3,6-tetrahydropyridin-4-yl and 4-[(2S)-2-aminopropanoyl]piperazin-1-yl; R 4 is selected from hydrogen, 1-piperidinomethyl and N,N-dimethylaminomethyl; R 5 is independently selected from methyl, ethyl, propyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, fluoro, chloro, and cyclopropyl; X is CH or N, and n is 0, 1 or 2) or a pharmaceutically acceptable salt thereof.

[0267] In a further embodiment, compounds of formula (V) as defined above, wherein G is selected from indol-3-yl and indazol-1-yl, and R 1 is selected from hydrogen, fluoro, chloro, methyl and cyano; R 2 is selected from methoxy and 2,2,2-trifluoroethoxy; R 3is selected from [2-(dimethylamino)ethyl]-(methyl)amino, [2-(methylamino)ethyl](methyl)amino, 2-(dimethylamino)ethoxy and 2-(methylamino)ethoxy; R 4 is hydrogen and R 5 is selected from methyl, 2,2,2-trifluoroethyl, and cyclopropyl; X is CH or N; and n is 0 or 1; or a pharmaceutically acceptable salt thereof.

[0268] Examples of compounds of formula (V) include those described in WO 2013 / 014448, WO 2015 / 175632, WO 2016 / 054987, WO 2016 / 015453, WO 2016 / 094821, WO 2016 / 070816 and WO 2016 / 173438.

[0269] Osimertinib and its pharmaceutical composition Osimertinib has the following chemical structure: [ka] The free base of osimertinib is known by the chemical name N-(2-{2-dimethylaminoethyl-methylamino}-4-methoxy-5-{[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino}phenyl)prop-2-enamide. Osimertinib is described in WO 2013 / 014448. Osimertinib is also known as AZD9291.

[0270] Osimertinib can be found in the form of a mesylate salt: N-(2-{2-dimethylaminoethyl-methylamino}-4-methoxy-5-{[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino}phenyl)prop-2-enamide mesylate. Osimertinib mesylate is also known as TAGRISSO™.

[0271] Osimertinib mesylate is currently approved for the treatment of patients with metastatic EGFR T790M mutation-positive NSCLC at a dose of 80 mg (expressed as the free base, equivalent to 95.4 mg of osimertinib mesylate) in an oral, once-daily tablet formulation. If a dose adjustment is required, a 40 mg oral, once-daily tablet formulation (expressed as the free base, equivalent to 47.7 mg of osimertinib mesylate) is available. The tablet core contains pharmaceutical diluents (e.g., mannitol and microcrystalline cellulose), disintegrants (e.g., low-substituted hydroxypropyl cellulose), and lubricants (e.g., sodium stearyl fumarate). Tablet formulations are described in WO 2015 / 101791.

[0272] Thus, in one embodiment, osimertinib or a pharmaceutically acceptable salt thereof is in the form of a mesylate salt, i.e., N-(2-{2-dimethylaminoethyl-methylamino}-4-methoxy-5-{[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino}phenyl)prop-2-enamide mesylate salt.

[0273] In one embodiment, osimertinib or a pharmaceutically acceptable salt thereof is administered once daily. In a further embodiment, osimertinib mesylate is administered once daily.

[0274] In one embodiment, the total daily dose of osimertinib is about 80 mg. In a further embodiment, the total daily dose of osimertinib mesylate is about 95.4 mg.

[0275] In one embodiment, the total daily dose of osimertinib is about 40 mg. In a further embodiment, the total daily dose of osimertinib mesylate is about 47.7 mg.

[0276] In one embodiment, the osimertinib or a pharmaceutically acceptable salt thereof is in tablet form.

[0277] In one embodiment, osimertinib or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients (e.g., diluents or carriers). In a further embodiment, the composition comprises one or more pharmaceutical diluents (such as mannitol and microcrystalline cellulose), one or more pharmaceutical disintegrants (such as low-substituted hydroxypropyl cellulose), or one or more pharmaceutical lubricants (such as sodium stearyl fumarate).

[0278] In one aspect, the composition is in the form of a tablet, wherein the tablet core comprises: (a) 2-70 parts osimertinib or a pharmaceutically acceptable salt thereof; (b) 5-96 parts of two or more pharmaceutical diluents; (c) 2-15 parts of one or more pharmaceutical disintegrants; and (d) 0.5-3 parts of one or more pharmaceutical lubricants, wherein all parts are by weight and the total of the parts is (a)+(b)+(c)+(d)=100.

[0279] In one aspect, the composition is in the form of a tablet, wherein the tablet core comprises: (a) 7 to 25 parts osimertinib or a pharmaceutically acceptable salt thereof; (b) 55 to 85 parts of two or more pharmaceutical diluents comprising microcrystalline cellulose and mannitol; (c) 2 to 8 parts of a pharmaceutical disintegrant comprising low-substituted hydroxypropyl cellulose; and (d) 1.5 to 2.5 parts of a pharmaceutical lubricant comprising sodium stearyl fumarate, where all parts are by weight and the total of the parts is (a)+(b)+(c)+(d)=100.

[0280] In one embodiment, the composition is in the form of a tablet, wherein the tablet core comprises (a) about 19 parts osimertinib mesylate, (b) about 59 parts mannitol, (c) about 15 parts microcrystalline cellulose, (d) about 5 parts low-substituted hydroxypropyl cellulose, and (e) about 2 parts sodium stearyl fumarate, where all parts are by weight and the sum of the parts is (a)+(b)+(c)+(d)+(e)=100.

[0281] AZD3759 (zolifertinib) AZD3759 has the following chemical structure: [ka] The free base of AZD3759 is known by the chemical name 4-[(3-chloro-2-fluorophenyl)amino]-7-methoxy-6-quinazolinyl(2R)-2,4-dimethyl-1-piperazinecarboxylate. AZD3759 is described in WO 2014 / 135876.

[0282] In one aspect AZD3759 or a pharmaceutically acceptable salt thereof is administered twice daily, hi a further aspect AZD3759 is administered twice daily.

[0283] In one embodiment the total daily dose of AZD3759 is about 400 mg, hi a further embodiment about 200 mg of AZD3759 is administered twice daily.

[0284] Lazertinib Lazertinib has the following chemical structure: [ka] The free base of lazertinib is known by the chemical name N-{5-[(4-{4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl}-2-pyrimidinyl)amino]-4-methoxy-2-(4-morpholinyl)phenyl}acrylamide. Lazertinib is described in WO 2016 / 060443. Lazertinib is also known as YH25448 and GNS-1480.

[0285] In one embodiment, lazertinib or a pharmaceutically acceptable salt thereof is administered once daily. In a further embodiment, lazertinib is administered once daily.

[0286] In one embodiment, the total daily dose of lazertinib is about 20 to 320 mg.

[0287] In one embodiment, the total daily dose of lazertinib is about 240 mg.

[0288] Avitinib Avitinib has the following chemical structure: [ka] The free base of avitinib is known by the chemical name: N-(3-((2-((3-fluoro-4-(4-methylpiperazin-1-yl)phenyl)amino)-7H-pyrrolo(2,3-d)pyrimidin-4-yl)oxy)phenyl)prop-2-enamide. Avitinib is disclosed in U.S. Patent Application Publication No. 2014038940. Avitinib is also known as abivertinib.

[0289] In one embodiment, avitinib or a pharmaceutically acceptable salt thereof is administered twice daily. In a further embodiment, avitinib maleate is administered twice daily.

[0290] In one embodiment, the total daily dose of avitinib maleate is about 600 mg.

[0291] Alfutinib Alflutinib has the following chemical structure: [ka] The free base of alflutinib is known by the chemical name N-{2-{[2-(dimethylamino)ethyl](methyl)amino}-6-(2,2,2-trifluoroethoxyl)-5-{[4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl]amino}pyridin-3-yl}acrylamide. Alflutinib is disclosed in WO 2016 / 15453. Alflutinib is also known as AST2818.

[0292] In one embodiment, alfurtinib or a pharmaceutically acceptable salt thereof is administered once daily. In a further embodiment, alfurtinib mesylate is administered once daily.

[0293] In one embodiment, the total daily dose of alfurtinib mesylate is about 80 mg.

[0294] In one embodiment, the total daily dose of alfurtinib mesylate is about 40 mg.

[0295] Afatinib Afatinib has the following chemical structure: [ka] The free base of afatinib is known by the chemical name N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)-oxolan-3-yl]oxyquinazolin-6-yl]-4-(dimethylamino)but-2-enamide. Afatinib is disclosed in WO 02 / 50043. Afatinib is also known as Gilotrif.

[0296] In one embodiment, afatinib or a pharmaceutically acceptable salt thereof is administered once daily. In a further embodiment, afatinib dimaleate is administered once daily.

[0297] In one embodiment, the total daily dose of afatinib dimaleate is about 40 mg.

[0298] In one embodiment, the total daily dose of afatinib dimaleate is about 30 mg.

[0299] CX-101 (orafeltinib; RX-518) CX-101 has the following chemical structure: [ka] The free base of CX-101 is known by the chemical name N-(3-(2-((2,3-difluoro-4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)amino)quinazolin-8-yl)phenyl)acrylamide. CX-101 is disclosed in WO 2015 / 027222. CX-101 is also known as RX-518.

[0300] HS-10296 (almonertinib, aumoreltinib) HS-10296 (almonertinib, aumoreltinib) has the following chemical structure: [ka] The free base of HS-10296 is known by the chemical name N-[5-[[4-(1-cyclopropylindol-3-yl)pyrimidin-2-yl]amino]-2-[2-(dimethylamino)ethyl-methyl-amino]-4-methoxy-phenyl]prop-2-enamide. HS-10296 is disclosed in WO 2016 / 054987.

[0301] In one embodiment, the total daily dose of HS-10296 is about 110 mg.

[0302] BPI-7711 (resivertinib) BPI-7711 has the following chemical structure: [ka] The free base of BPI-7711 is known by the chemical name N-[2-[2-(dimethylamino)ethoxy]-4-methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide. BPI-7711 is disclosed in WO 2016 / 94821.

[0303] In one embodiment, the total daily dose of BPI-7711 is about 180 mg.

[0304] Dacomitinib Dacomitinib has the following chemical structure: [ka] The free form of dacomitinib is known by the chemical name (2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-7-methoxyquinazolin-6-yl}-4-(piperidin-1-yl)but-2-enamide. Dacomitinib is described in International Publication No. 2005 / 107758. Dacomitinib is also known as PF-00299804.

[0305] Dacomitinib can be found in the form of dacomitinib monohydrate, i.e. (2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-7-methoxyquinazolin-6-yl}-4-(piperidin-1-yl)but-2-enamide monohydrate.

[0306] In one embodiment, dacomitinib or a pharmaceutically acceptable salt thereof is administered once daily. In a further embodiment, dacomitinib monohydrate is administered once daily.

[0307] In one embodiment, the total daily dose of dacomitinib monohydrate is about 45 mg.

[0308] In one embodiment, the dacomitinib or a pharmaceutically acceptable salt thereof is in tablet form.

[0309] In one embodiment, dacomitinib or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients. In a further embodiment, the one or more pharmaceutically acceptable excipients comprise lactose monohydrate, microcrystalline cellulose, sodium starch glycolate, and magnesium stearate.

[0310] Icotinib Icotinib has the following chemical structure: [ka] The free base of icotinib has the chemical name: N-(3-ethynylphenyl)-2,5,8,11-tetraoxa-15,17-diazatricyclo[10.8.0.0 14,19 Icotinib is known as icosa-1(12),13,15,17,19-pentaen-18-amine. Icotinib is disclosed in International Publication No. 2013064128. Icotinib is also known as Conmana.

[0311] In examples, Icotinib or a pharmaceutically acceptable salt thereof is administered three times daily. In further embodiments, Icotinib hydrochloride is administered three times daily.

[0312] In an example, the total daily dose of icotinib hydrochloride is about 375 mg.

[0313] Gefitinib Gefitinib has the following chemical structure: [ka] The free base of gefitinib is known by the chemical name: N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine. Gefitinib is disclosed in WO 1996 / 033980. Gefitinib is also known as IRESSA (trademark).

[0314] In examples, gefitinib or a pharmaceutically acceptable salt thereof is administered once daily. In further embodiments, gefitinib is administered once daily.

[0315] In an example, the total daily dose of gefitinib is about 250 mg.

[0316] Erlotinib Erlotinib has the following chemical structure: [ka] The free base of erlotinib is known by the chemical name N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine. Erlotinib is disclosed in WO 1996 / 030347. Erlotinib is also known as TARCEVA™.

[0317] In an example, erlotinib or a pharmaceutically acceptable salt thereof is administered once daily. In a further example, erlotinib is administered once daily.

[0318] In an example, the total daily dose of erlotinib is about 150 mg.

[0319] In an example, the total daily dose of erlotinib is about 100 mg.

[0320] Combination therapy As explained above, co-expression of EGFR and cMET is associated with many cancer types, and antibody molecules that target both molecules, particularly conjugates containing such antibody molecules, offer the opportunity for broad clinical benefit across multiple indications. Thus, the combination of an EGFR TKI and an antibody molecule described herein is expected to be useful in therapeutic applications, particularly in the treatment of cancer.

[0321] The EGFR TKI and / or antibody molecules described herein can be used in methods of treatment of the human or animal body. In related aspects of the disclosure, the following are provided: (i) an EGFR TKI as described herein for use in a method of treating cancer, wherein the EGFR TKI is administered in combination with an antibody molecule as described herein; (ii) an antibody molecule as described herein for use in a method for treating cancer, wherein the antibody molecule is administered in combination with an EGFR TKI as described herein; (iii) the use of an EGFR TKI as described herein in the manufacture of a medicament for treating cancer, wherein the EGFR TKI is administered in combination with an antibody molecule as described herein; (iv) use of an antibody molecule as described herein in the manufacture of a medicament for treating cancer, wherein the antibody molecule is administered in combination with an EGFR TKI as described herein; (v) a method of treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of an antibody molecule described herein in combination with a therapeutically effective amount of an EGFR TKI described herein; (vi) A method of treating cancer in an individual, wherein the method comprises administering to the individual a first amount of an EGFR TKI and a second amount of an EGFR / cMET antibody molecule, wherein the first amount and the second amount together constitute a therapeutically effective amount.

[0322] Also provided herein is a pharmaceutical combination of an EGFR / cMET antibody molecule described herein and an EGFR TKI described herein. A pharmaceutical combination refers to a composition comprising a therapeutically effective amount of an EGFR / cMET antibody molecule described herein, a therapeutically effective amount of an EGFR TKI described herein, and one or more pharmaceutically acceptable carriers, wherein the active ingredients are intended to be given to a patient in combination.

[0323] As used herein, "combination therapy" refers to the administration to an individual of both i) an antibody molecule described herein (which may be conjugated to a drug, as described herein) and ii) an EGFR TKI described herein.

[0324] The individual may be a patient. In some examples, the patient is a human patient.

[0325] Treatment can be any treatment or therapy that achieves any desired therapeutic effect, such as preventing or slowing the progression of the condition, including slowing the rate of progression, halting the rate of progression, ameliorating the condition, curing or remission (whether partial or total) of the condition, preventing, ameliorating, delaying, reducing or arresting one or more symptoms and / or signs of the condition, or prolonging the survival of an individual or patient beyond that expected if not receiving treatment.

[0326] Administration The antibody molecules, conjugates and EGFR TKIs are typically administered in the form of a pharmaceutical composition, which may contain at least one component in addition to the active agent.

[0327] In addition to the antibody molecule, conjugate, or EGFR TKI, the pharmaceutical composition may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. The term "pharmaceutically acceptable," as used herein, refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The precise nature of the carrier or other material will depend on the route of administration, as explained below. The route of administration may be by infusion, injection, or any other suitable route.

[0328] Administration may be in a "therapeutically effective amount," which is sufficient to provide benefit to the individual. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated, the particular individual being treated, the individual's clinical condition, the cause of the disorder, the site of delivery of the composition, the type of antibody molecule, the method of administration, the administration schedule, and other factors known to medical practitioners. Prescribing treatment, e.g., determining dosage, is within the scope of the competence of general practitioners and other physicians and may depend on the severity and / or progression of the symptoms of the disease being treated. Appropriate dosages of EGFR TKIs are known in the art, and exemplary dosages are described above in the EGFR TKI section. Appropriate dosages of antibody molecules are well known in the art (Ledermann, 1991 and Bagshawe, 1991). Depending on the antibody being administered, specific dosages set forth herein or in the Physician's Desk Reference (2003) may be used. A therapeutically effective amount or suitable dosage of an antibody molecule can be determined by comparing in vitro and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other test animals to humans are known. The exact dosage depends on many factors, including the size and location of the treatment area and the exact nature of the antibody molecule. The antibody molecule may be administered, for example, once daily, once weekly, once every two weeks, or once monthly. In some examples, an EGFR TKI is administered as a first amount and an anti-EGFR / cMET antibody molecule is administered as a second amount, with the first and second amounts together comprising a therapeutically effective amount.

[0329] The EGFR TKI may be administered to the individual simultaneously with, sequentially with, or separately from the administration of the antibody molecule. When the EGFR TKI is administered simultaneously with the antibody molecule, the antibody molecule and the EGFR TKI may be administered to the individual as a combined formulation.

[0330] The combination therapy (i.e., the antibody molecule and the EGFR TKI) may be administered in combination with a third therapy, which is administered to the individual simultaneously with, sequentially with, or separately from the combination therapy. The third therapy may include chemotherapy, radiation therapy, another (different) antibody molecule, or another (different) EGFR TKI.

[0331] cancer For example, the cancer treated by the combination therapy described herein may be selected from the group consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC)), pancreatic cancer, breast cancer, colorectal cancer, renal cancer, gastric cancer, head and neck cancer, ovarian cancer, or glioblastoma.

[0332] The cancer may be a cancer that expresses both EGFR and cMET. Cells of the cancer may express EGFR and cMET on the cell surface. In one example, the tumor may be one that has been determined to co-express EGFR and cMET. Methods for determining target expression are known in the art and include, for example, immunohistochemistry.

[0333] In some examples, the cancer treated by the combination therapy described herein is selected from the group consisting of lung cancer (such as non-small cell lung cancer (NSCLC)), pancreatic cancer, colon cancer, colorectal cancer, and squamous cell carcinoma of the head and neck (SCCHN or SQHN). In one example, the cancer treated is non-small cell lung cancer (NSCLC). In one example, the cancer is squamous cell carcinoma of the head and neck (SCCHN).

[0334] In some instances, the cancer is a wild-type EGFR cancer, an EGFR mutant cancer, a wild-type cMET cancer, or a cMET mutant cancer. Methods for detecting EGFR and cMET mutant cancers are well known.

[0335] In some examples, the cancer to be treated is an EGFR-mutated cancer (also referred to as "EGFR mutation-positive"), such as an EGFR-mutated NSCLC. Exemplary EGFR mutations, such as EGFR-activating mutations, that may be associated with cancer include point mutations, deletion mutations, insertion mutations, inversions, or gene amplifications that result in an increase in at least one biological activity of EGFR, such as increased tyrosine kinase activity, formation of receptor homodimers and heterodimers, enhanced ligand binding, etc. The mutation can be located in any part of the EGFR gene or a regulatory region associated with the EGFR gene, including mutations in exons 18, 19, 20, or 21. In some examples, the EGFR-mutated cancer is a cancer having an L858R mutation in the EGFR gene, one or more deletions in exon 19 or one or more insertions in exon 20, a T790M mutation, or a combination thereof.

[0336] In NSCLC, certain mutations in the EGFR gene are associated with high response rates to EGFR TKIs. Single point mutations in exon 21 (leucine-858 to arginine (L858R)) and variable deletions of at least three amino acid residues in exon 19 are often collectively referred to as "classic" EGFR activating mutations and represent the majority (85-90%) of all EGFR kinase domain mutations observed in NSCLC (Vyse and Huang, 2019). Reported examples of EGFR exon 19 deletions include delE746-A750, delE746-T751, delL747-E749, delL747-P753, and delL747-T751. In some instances, EGFR-mutated cancers are cancers with the L858R mutation in the EGFR gene and / or one or more deletions in exon 19 (e.g., EGFR mutation-positive NSCLC). In some examples, the EGFR mutant cancer is a cancer with one or more deletions in exon 19 of the EGFR gene and / or an L858R mutation (e.g., EGFR mutation-positive NSCLC). In some examples, the EGFR mutant cancer is a cancer with an L858R mutation and / or one or more deletions in exon 19 of the EGFR gene (e.g., EGFR mutation-positive NSCLC). In some examples, the EGFR mutant cancer is a cancer with one or more deletions in exon 19 of the EGFR gene (e.g., EGFR mutation-positive NSCLC). In some examples, the EGFR mutant cancer is a cancer with an L858R mutation in the EGFR gene (e.g., EGFR mutation-positive NSCLC).

[0337] Although the majority of NSCLC patients with EGFR mutations initially respond to EGFR TKI therapy, virtually all patients treated with first-generation EGFR TKIs acquire resistance after approximately 10 months of progression-free survival. A secondary point mutation (T790M), which results in a methionine-to-threonine substitution at amino acid position 790, is the molecular mechanism that generates drug-resistant variants of the target kinase. The T790M mutation is present in approximately half of lung cancer patients with acquired resistance to first- and second-generation EGFR TKIs and has been reported to act by increasing the receptor's affinity for adenosine triphosphate (ATP) relative to its affinity for the TKI (Suda, 2009).

[0338] Not all activating EGFR mutations are inherently sensitive to EGFR inhibitors. In-frame base pair insertions in exon 20 also result in constitutive activation of EGFR. However, unlike classical activating EGFR mutations, EGFR exon 20 insertions are associated with de novo resistance to currently clinically available EGFR inhibitors. Low response rates of 3–8% to erlotinib, gefitinib, and the second-generation EGFR inhibitor afatinib have been reported in patients with EGFR exon 20 insertion-mutated NSCLC, and therefore, effective treatment options are limited (Vyse and Huang, 2019). Examples of reported EGFR exon 20 insertions include D761-E762 insX, A764-Y764 insX, Y764-V765 insX, V765-M766 insX, A767-S768 insX, S768-V769 insX, V769-D770 insX, D770-N771 insX, N771-P772 insX, P772-H773 insX, H773-V774 insX, and V774-C775 insX, where insX indicates an in-frame insertion of 1 to 7 amino acids.

[0339] As shown herein, combination therapies comprising the anti-EGFR / cMET complex described herein and osimertinib (a third-generation EGFR TKI) demonstrated effective tumor inhibition across a variety of mutant EGFR cancer models, including those containing the L858R mutation, exon 20 insertion, and exon 19 deletion with T790M mutation. Accordingly, the combination therapies described herein are expected to be effective in treating a variety of EGFR mutant cancers in humans.

[0340] In some instances, the patient being treated has previously been treated with a previous anticancer therapy, such as a previous EGFR TKI. In some instances, the human patient's disease has progressed during or after a previous EGFR TKI treatment, i.e., the patient has acquired resistance to or is resistant to a previous EGFR TKI treatment. In some instances, the patient being treated is resistant or has acquired resistance to treatment with erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, poziotinib, criotinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib, and / or sunitinib. In some instances, the human patient's disease has progressed during or after a previous treatment with a different EGFR TKI, for example, the cancer being treated is classified as an osimertinib-resistant cancer. As noted above, mutations associated with EGFR TKI resistance include the T790M mutation and an insertion in exon 20. In other examples, the patient being treated is an EGFR TKI-naive human patient (i.e., the patient has not been previously treated with an EGFR TKI).

[0341] Treatment effect In the context of cancer, treatment can include inhibiting cancer growth (including complete cancer remission) and / or inhibiting cancer metastasis and inhibiting cancer recurrence. Cancer growth generally refers to any one of a number of indicators that indicate a change in cancer to a more advanced form. Thus, indicators for measuring inhibition of cancer growth include a decrease in cancer cell viability, a decrease in tumor volume or morphology (e.g., determined using computed tomography (CT), ultrasound, or other imaging methods), slowing tumor growth, destruction of tumor vasculature, improved performance in delayed hypersensitivity skin tests, increased activity of anti-cancer immune cells or other anti-cancer immune responses, and a decrease in the level of tumor-specific antigens. Activating or enhancing the immune response against cancerous tumors in an individual can improve the individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and / or reduce the individual's propensity for cancer growth.

[0342] In some instances, the combination therapies described herein can inhibit the development or progression of cancer.

[0343] The ability of a given combination therapy to inhibit the development or progression of cancer can be analyzed, for example, using an in vivo model. For example, the in vivo model can include measuring tumor growth in a patient-derived xenograft (PDX) model. Further details of this exemplary method are described in the Examples.

[0344] Inhibition of cancer development can be inferred by observing a decrease in tumor growth rate or tumor size after administration of the antibody molecule, for example by measuring tumor growth inhibition rate (TGI%). TGI% can be measured by comparing the size of the tumor measured on day 0 in subjects administered the antibody molecule with the size of the tumor measured at the end of the study period and comparing this to the tumor growth over the same period in subjects administered a control antibody molecule. In this method, TGI% can be defined as the tumor growth rate relative to day 0 between the treatment (TX) group and the control (C) group according to the following formula: TGI%=(1-(TX 最終 -TX 初期 ) / (C 最終 -C初期 ))×100.

[0345] In some examples, the combination therapies described herein have a TGI% of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0346] Inhibition of cancer development can be inferred by observing a delay or prevention of the onset of cancer symptoms and / or a reduction in the severity of cancer symptoms in response to treatment with the antibody molecule. Inhibition of cancer progression can be inferred by observing a delay, prevention and / or reduction in invasion and / or metastasis in response to treatment with the antibody molecule.

[0347] The combination therapies described herein may be capable of inhibiting cancer development or progression by less than 100%, for example, to one of 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less of cancer development / progression in the absence of treatment (or treatment with a suitable control). In some examples, the combination therapies described herein can inhibit cancer development or progression to less than 1-fold the level of cancer development / progression in the absence of treatment (or treatment with a suitable control), e.g., one of: <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.85-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold.

[0348] In some instances, the combination therapies described herein can inhibit cancer onset or progression to a greater extent than the use of a single agent (i.e., antibody molecule or EGFR TKI) administered individually. The combination therapies described herein may be able to inhibit cancer onset or progression by less than 100%, for example, 99% or less, 95% or less, 90% or less, 85% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less of the onset / progression of cancer treated with the antibody molecule, conjugate, or EGFR TKI alone. In some examples, the combination therapy described herein can inhibit cancer development or progression to less than 1-fold the level of cancer development / progression following treatment with the antibody molecule, conjugate, or EGFR TKI alone, e.g., one of the following: <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.85-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold. In some examples, the combination therapy described herein exerts a synergistic effect in inhibiting cancer development or progression.

[0349] The features disclosed in the foregoing description, or the following claims, or the accompanying drawings are expressed in their specific form or in terms of means for performing a disclosed function or methods or processes for obtaining a disclosed result, and can be used to realize the disclosure in various of its forms, using such features individually or in any combination as appropriate.

[0350] While the present disclosure has been described in conjunction with the illustrative examples set forth above, many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the illustrative examples of the present disclosure set forth above are considered to be illustrative and not limiting. Various modifications can be made to the described examples without departing from the spirit and scope of the present disclosure.

[0351] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0352] Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0353] Throughout this specification, including the claims which follow, unless the context otherwise requires, the words "comprise" and "comprises," "including," and variations such as "comprises," "including," and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of other integers or steps or groups of integers or steps.

[0354] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another example includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, it will be understood that the particular value constitutes another example by the use of "about." The term "about" in connection with numerical values is optional and means, for example, ±10%. [Example]

[0355] Example 1 – Design and construction of the RAA22 / B09 DuetMab This example describes the generation of a bispecific antibody molecule capable of binding to both EGFR and cMET.

[0356] 1.1 Isolation and Characterization of Anti-cMET Antibody 0021U3-B09 cMET-specific scFv antibodies were isolated from a large naive human scFv phage display library through a series of iterative panning selection cycles against recombinant mammalian-expressed biotinylated monomeric human cMET (MedImmune) essentially as described (Vaughan, 1996). scFvs from round 2 of the selection output were expressed in the bacterial periplasm and screened for their ability to inhibit human cMET receptor binding with HGF ligand in an HGF:cMET HTRF® (homogeneous time-resolved fluorescence) ligand-receptor inhibitory binding assay. Top hits showing strong inhibitory effects were selected and subjected to DNA sequencing. The unique genes were then converted into human immunoglobulin G2 (IgG2) antibodies and produced in mammalian cells essentially as described (Persic, 1997). Purified antibodies were then ranked based on their inhibitory effects in the HGF:cMET HTRF® binding assay. The most potent antibody, 0021U3-B09, was selected for further characterization.

[0357] 1.2 Optimization of anti-cMET antibody 0021U3-B09. To minimize potential immunogenicity, non-Vernier framework residues (Foote and Winter 1992) within the variable framework regions of 0021U3-B09 were specifically targeted and altered to match the closest human germline sequence. In the VH region, seven amino acid residues were mutated to match the reference human germline sequence IGHV1-8*01. In the VL region, three residues were mutated to match the reference human germline sequence IGKV1-5*03. All residues in the VH and VL regions were successfully altered to germline residues without loss of activity. 0021U3-B09 was affinity-optimized using hybridization-based mutagenesis, essentially as described (Kunkel 1985). A large-scale scFv library derived from the 0021U3-B09 sequence was generated by oligonucleotide-directed mutagenesis of the VH complementarity-determining region 3 (CDR3) using standard molecular biology techniques. The library was subjected to affinity-based solution-phase selection to select variants with higher affinity for human and cynomolgus monkey cMET antigens. Crude scFv-containing periplasmic extracts from the CDR-targeting selection output were screened for improved inhibitory activity in the HGF:cMET HTRF® binding assay. Variants showing significantly improved inhibitory effects compared to the parent 0021U3-B09 were subjected to DNA sequencing, and the unique genes were converted to human IgG2. The purified antibodies were then ranked based on their inhibitory effects. The most potent antibody, B09-57, was selected for further characterization.

[0358] 1.3 Isolation and characterization of the anti-EGFR antibody Tdev-0004. EGFR-specific scFv antibodies were isolated from a large naive human scFv phage display library through a series of iterative panning selection cycles against recombinant mammalian-expressed biotinylated monomeric human EGFR (MedImmune) essentially as described (Vaughan, 1996). ScFv-displaying phages from round 3 of the selection output were screened for their binding to human and cynomolgus monkey EGFR by ELISA. Top hits showing cross-reactivity were selected and subjected to DNA sequencing. The unique genes were then converted into human immunoglobulin G1 (IgG1) antibodies and produced in mammalian cells essentially as described (Persic, 1997). Purified antibodies were then ranked based on binding to the EGFR-expressing cell line A431 by flow cytometry. Antibody Tdev-0004, showing specific cell binding, was selected for further characterization.

[0359] 1.4 Optimization of the anti-EGFR antibody Tdev-0004. The variants RAA22 and QD6 were obtained by optimizing the anti-EGFR Tdev-0004 mAb. The VL framework of Tdev-0004 was 100% identical to the reference human germline sequence IGLV2-11*01 / IGLJ2 (see https: / / www.ncbi.nlm.nih.gov / projects / igblast / Idlink.cgi?seqname=IGLV2-11*01&taxid=9606&dbname=IG_DB%2Fimgt.Homo_sapiens.Vforf.p), whereas the VH framework was 79% identical to the closest human germline IGHV1-69*01 / JH4 (see https: / / www.ncbi.nlm.nih.gov / projects / igblast / Idlink.cgi?seqname=IGHV1-69*01&taxid=9606&dbname=IG_DB%2Fimgt.Homo_sapiens.Vforf.p). To minimize potential immunogenicity, the VH region was first fully germlined by mutating all 13 non-germline framework residues. Germlined binding of the fully germlined variant to cynomolgus monkey EGFR was significantly impaired. To restore binding to cynomolgus monkey EGFR, four non-germline residues, K68, I73, R76, and T78, were selectively backmutated. Amino acid residues are numbered according to the Kabat numbering system (Kabat and Wu 1991). The resulting partially germlined variant (designated H4) was used as the template sequence for affinity optimization. The affinity of variant H4 was optimized by parsimonious mutagenesis of all six CDRs using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent) according to the manufacturer's instructions. The single-amino acid mutagenized VH and VL libraries were expressed in bacteria as Fab fragments and screened for improved binding to human and cynomolgus monkey EGFR by ELISA. Variants with improved binding compared to the parent H4 were subjected to DNA sequencing and the unique gene converted to human IgG1.Mutant RAA22 was identified with a single mutation in CDRH3. To further improve affinity, individual positive mutations were combined to generate a combinatorial library that was screened for variants with enhanced binding to human and cynomolgus monkey EGFR. Variant QD6 was identified with four combined mutations in CDRL2, CDRL3, and CDRH3.

[0360] 1.5 Generation of monovalent bispecific anti-EGFR / cMET DuetMab antibodies. The variable domains of anti-cMET mAb B09-57 and anti-EGFR mAbs RAA22 and QD6 were utilized to construct a monovalent, bispecific anti-EGFR / cMET antibody on the backbone of the DuetMab platform (Mazor, 2015). Specifically, the VH gene of anti-cMET B09-57 was inserted into a human gamma 1 constant heavy chain carrying a "knob" mutation (T366W) and alternative interchain cysteine mutations (F126C and C219V). The VL gene of B09-57 was inserted in frame into a human kappa constant domain carrying the corresponding alternative interchain cysteine mutations (S121C and C214V) designed to pair with the "knob" heavy chain. Similarly, the VH genes of anti-EGFR RAA22 and affinity-optimized QD6 were inserted into a human gamma 1 constant heavy chain carrying the "hole" mutations (T366S, L368A, and Y407V), and the VL genes of RAA22 and B09-57 were inserted in frame into a human lambda constant domain designed to pair with the "hole" heavy chain. Additionally, two residues in the CH3 domains of the "knob" and "hole" heavy chains were mutated to cysteines (S354C in the "knob" and Y349C in the "hole") to form stabilizing disulfide bridges. The Fc domain was further engineered to include a cysteine insertion after serine 239 (C239i / "Maia") to enable site-specific conjugation of maleimide-containing cytotoxic drugs (Dimasi, 2017). Amino acid residues are numbered according to the Kabat numbering system (Kabat and Wu, 1991). The assembled monovalent bispecific anti-EGFR / cMET DuetMab antibodies were named RAA22 / B09-57 and QD6 / B09-57 (Figure 1). DuetMab antibodies were generated from mammalian cells as previously described (Mazor, 2017).

[0361] The amino acid sequences of the heavy and light chains of the DuetMabs produced according to this example are provided in the table below.

[0362] [Table 8]

[0363] Example 2 – Biochemical and biophysical properties In this example, various biochemical and biophysical properties of the RAA22, QD6, and B09-57 monoclonal antibodies and the RAA22 / B09-57 and QD6 / B09-57 bispecific antibody molecules are examined, including their binding affinity to EGFR and c-Met, respectively, and their ability to bind both antigens simultaneously.

[0364] 2.1 Binding affinity of DuetMab and parental mAbs to EGFR and cMET. The kinetic constants (k and k) and equilibrium dissociation constants (K) of EGFR-cMET DuetMAb against recombinant human, cynomolgus monkey, and mouse EGFR and cMET antigens. D) was determined by SPR using an antibody capture assay on a BIAcore T200 instrument (GE Healthcare, Pittsburgh, PA) at 25°C. Mouse anti-human IgG was immobilized on a CM4 sensor chip with a final surface density of approximately 2000 resonance units (RU). A reference flow cell surface was also prepared on this sensor chip using the same immobilization protocol. Test and control antibodies were prepared at concentrations of 5–20 nM in instrument buffer (HBS-EP buffer; 0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, and 0.005% P-20). Three-fold serial dilutions of purified EGFR protein (human 0.27–200 nM, cynomolgus monkey 0.4–900 nM, and mouse 4–1000 nM) or cMET protein (human 0.27–66 nM and cynomolgus monkey 0.27–22 nM) were also prepared in instrument buffer. Kinetic measurements were performed using a sequential approach. Antibody was first injected over the capture surface at a flow rate of 10 μL / min. Once the binding of the captured antibody stabilized, a single concentration of analyte was injected over both the capture and reference surfaces at a flow rate of 75 μL / min. The resulting binding response curves were used to obtain association phase data. After analyte injection, the flow was switched to instrument buffer for 15 minutes to allow for dissociation phase data collection, followed by a 1-minute pulse of 10 mM glycine (pH 1.5) to regenerate the antibody capture surface on the chip. Binding responses to test and control antibodies were recorded from duplicate injections of each concentration of analyte. Additionally, several buffer injections were distributed throughout the injection series. Select buffer injections were used in conjunction with reference cell responses to correct for injection artifacts and / or nonspecific binding interactions in the raw data set (commonly referred to as "double referencing"). The corrected binding data were globally fit to a 1:1 binding model (Biacore T200 Evaluation software 2.0, GE Healthcare, Pittsburgh, PA). Calculated kinetic parameters (k on and k off ) and k off / k on K was determined as D is shown in Table 1.

[0365] [Table 9]

[0366] [Table 10]

[0367] As can be seen from the above data, the bispecific antibody molecule QD6 / B09-57 binds to human c-Met with high affinity (approximately 2 nM kD) and to human EGFR with high affinity (approximately 6 nM kD), while the bispecific antibody molecule RAA22 / B09-57 also binds to human c-Met with high affinity (approximately 2 nM kD) but binds to human EGFR with lower affinity (approximately 45 nM kD) compared to QD6 / B09-57.

[0368] 2.2 Simultaneous binding of DuetMab to EGFR and cMET. Simultaneous binding studies of recombinant human EGFR and cMET proteins were measured by biolayer interferometry on an Octet384 instrument essentially as described (Mazor, 2015). Briefly, 5 μg / mL of His-tagged cMET antigen was first captured onto an NI-NTA biosensor in assay buffer [PBS (pH 7.2), 3 mg / mL bovine serum albumin (BSA), 0.05% (v / v) Tween 20]. Following a wash step to remove unbound protein, each loaded biosensor was subjected to sequential association and dissociation interactions, first with 66 nM antibody and then with 500 nM EGFR antigen. Association and dissociation curves were calculated from nonlinear fits of the data using Octet384 software v.9.0. As shown in Figure 2, the DuetMab showed simultaneous binding to both antigens, whereas the parent anti-cMET IgG showed specific binding only to cMET, and the two anti-EGFR IgGs showed no binding to the cMET-loaded sensor.

[0369] 2.3 Specificity of EGFR and c-Met Specificity for EGFR and cMET species paralogs and closely related family members was determined by ELISA. Briefly, a 1 μg / mL antigen solution was prepared in PBS, and 50 microliters was coated onto a half-area ELISA assay plate. The plate was washed and blocked with 1% BSA in PBS containing 0.005% Tween-20 (PBS-T) for 1 hour at room temperature. The wells were washed four times with PBS-T. As shown in Figure 3, the primary antibodies used were R374 (non-binding IgG1 isotype control antibody), B09 (anti-cMET antibody), QD6 (anti-EGFR antibody), RAA22 (anti-EGFR antibody), QD6 / B09 (bispecific EGFR / c-MET DuetMAb), RAA22 / B09 (bispecific EGFR / c-MET DuetMAb), PaniX (anti-EGFR antibody), MetMab (anti-cMET antibody), and Mab11311 (anti-HER4 antibody). Wells were incubated with 50 μL of the indicated primary antibody diluted in PBS-T in a 1:3 dilution series starting at 10 μg / mL and ending at 0.002 μg / mL, except for MAb1131, the HER4-binding mAb control, which started at 1 μg / mL. The wells were washed four times with PBS-T, and then 50 μl of goat anti-human Fab HRP-conjugated secondary antibody diluted 1:5000 in PBS-T was added to each well and incubated at room temperature for 1 hour. Fifty microliters of TMB substrate solution was added to all wells and incubated at room temperature for 5 to 30 minutes until a strong signal was observed in the positive control wells. Fifty microliters of TMB stop solution was added to all wells, and absorbance was read at 450 nm on a SpectraMax M5 microplate reader. Data were analyzed using SoftMax Pro 5 software and plotted using GraphPad Prism 7 graphing software.

[0370] To determine species cross-reactivity, ELISA assays were performed as described above. As shown in Figure 3A, the high-affinity EGFR IgG (QD6) and the monovalent bispecific EGFR / cMET DuetMAb (QD6 / B09) bound to human, cynomolgus, and mouse EGFR, giving strong signals in the ELISA assay. In contrast, the low-affinity EGFR IgG (RAA22) bound to human, cynomolgus, and mouse EGFR less strongly than QD6. Binding to mouse EGFR was weaker but detectable. The corresponding monovalent bispecific EGFR / cMET DuetMAb (RAA22 / B09) showed even weaker binding to human and cynomolgus EGFR compared to the bivalent parent IgG (RAA22), and only negligible binding to mouse EGFR. cMET IgG (B09) and all bispecific variants showed comparable binding to human and cynomolgus cMET. No binding of either antibody to mouse cMET was detected. These results are consistent with the binding kinetics determined by surface plasmon resonance measurements on a BIAcore instrument (Table 1 above).

[0371] As shown in Figure 3B, neither the parental IgG nor the derived bispecific antibody showed significant binding to any of the EGFR HER family proteins (HER2, HER3, or HER4). Similarly, none of the antibodies showed significant binding to cMET family members (Ron (CD136) or semaphorin 3a).

[0372] These results demonstrate that the parental IgG and the resulting bispecific antibody bind specifically to their cognate targets, with no detectable binding to closely related family members.

[0373] 2.4 Internalization of bispecific antibodies The internalization kinetics of AlexaFluor647 (AF647)-primary labeled DuetMabs, RAA22 / B09 and QD6 / B09, were evaluated in vitro using the EGFR- and c-MET-expressing cell line H1975. Each antibody was pre-bound to cells, and live-cell confocal fluorescence microscopy was used to monitor antibody translocation from the cell surface to the cytoplasm. Both antibodies were primarily localized to the cell surface before exposure to internalization conditions (T = 0) and translocated to the cytoplasmic region after 1 hour (data not shown). Kinetic images were taken every 5 minutes over the course of internalization and processed using a quantification algorithm to determine the kinetic constants and half-lives of internalization. The internalization kinetics of QD6 / B09 and RAA22 / B09 were similar, with half-lives of 37.5 ± 10.6 minutes and 43.2 ± 15.5 minutes, respectively.

[0374] To assess the mode of antibody internalization and investigate the contribution of each arm to the overall internalization of DuetMab, we evaluated the internalization of single-arm specific control antibody molecules QD6 / IgG and B09 / IgG for DuetQD6 / B09 and RAA22 / IgG and B09 / IgG for DuetRAA22 / B09 in H1975 cells expressing both EGFR and c-MET. Because only one arm is specific for the target receptor, the control antibody can only internalize via one receptor, eliminating dual receptor targeting and crosslinking as modes of internalization.

[0375] The internalization profiles of QD6 / B09 (Figure 4A) and RAA22 / B09 (Figure 4B) showed very similar patterns of a concomitant decrease in membrane mAb-Fl647 signal and an increase in cytoplasmic mAb-AF647 signal, which is typical of internalization. However, their single-arm constructs exhibited significantly different internalization profiles. The single-arm QD6 / IgG had an internalization time course nearly identical to that of the QD6 / B09 DuetMab (Figure 4A, left and center), indicating that internalization of QD6 / B09 Duet was primarily driven by the EGFR arm of the molecule, with only a very small contribution from the B09 arm. Indeed, the B09 / IgG construct exhibited very low levels of internalization (Figure 4B, right). The rapid and widespread decrease in membrane signal corresponded to a very gradual increase in cytoplasmic signal, likely due to extensive dissociation of pre-bound B09 / IgG from the cell surface c-MET receptor. Dissociation of the antibody subsequently resulted in a slight internalization of B09 / IgG. These results revealed that the internalization of QD6 / B09 duet was primarily driven by the EGFR arm of the molecule, with only a minor contribution from the B09 arm.

[0376] In contrast, the RAA22 / B09 DuetMab exhibited a very different internalization profile when compared to its single-arm control antibody. As seen in Figure 4B, the cytoplasmic intensity values of the RAA22 / B09 DuetMab were 10.98- and 4.70-fold higher than those of RAA22-IgG and B09-IgG, respectively. While the inefficient internalization of B09 / IgG may be due to its significant dissociation, RAA22 / IgG was rapidly internalized. However, due to the lower affinity of the EGFR arm, the number of RAA22 / IgG molecules was 10.98-fold lower (based on fluorescence intensity) than for the RAA22-B09 DuetMab. In contrast to the single-arm construct, the significant increase in the amount of duet RAA22 / B09 mAb that entered the cytoplasm demonstrated that both antibody arms must engage the target receptor to drive internalization. This finding indicates that the QD6 / B09 and RAA22 / B09 DuetMabs have different internalization mechanisms, with QD6 / B09 being primarily driven by the EGFR arm, whereas RAA22 / B09 requires both the EGFR and c-MET arms for engagement.

[0377] Because binding of both the EGFR and c-MET arms to the target receptor promoted RAA22 / B09 receptor internalization in H1975 cells, we investigated whether increasing the number of EGFR and c-MET receptors affected the internalization properties. The respective receptor levels, determined by Western blotting, were approximately 33,000 and 50,000, respectively, for EGFR and c-MET in H1975 cells, and approximately 790,000 and 523,000, respectively, for c-MET in HCC827 cells. The internalization profiles of RAA22 / B09 in H1975 (medium receptor) and HCC827 (high receptor) cells show a significant increase in internalization in HCC827 cells (Figure 5).

[0378] As expected corresponding to the 23.9-fold and 10.4-fold increase in overall levels of EGFR and c-MET RAA22 / B09, respectively, binding to HCC827 cells was on average 8.9-fold higher than H1975 (T = 0, 3.1 × 10 7MFI vs. 3.5x10 6 MFI). The internalization level (determined by the peak cytoplasmic intensity) was 21.7-fold higher in HCC827 cells, suggesting that significantly higher concentrations of antibody enter the cytoplasm in cells expressing high levels of the target receptor. Importantly, in addition to the large difference in intensity, the internalization profiles (membrane and cytoplasmic signals over time) also differed significantly between HCC827 and H1975 cells. In high-expressing HCC827 cells, the decrease in RAA22 / B09-AF647 membrane signal corresponded to a reciprocal increase in RAA22-B09 cytoplasmic signal, while the total RAA22 / B09 signal was maintained over time, indicating strong dual-arm antibody interaction with both receptors and subsequent internalization. In H1975 cells, the total and membrane intensities decreased simultaneously, indicating that some of the pre-bound antibody may have dissociated from the cell surface and not been internalized. In HCC827 cells, similar dissociation profiles were observed for the internalization of RAA22 / IgG and B09 / IgG, with single-arm engagement not resulting in effective binding and tending to dissociate (Figures 6A and 6B). This data suggested that a mixed mode of receptor interaction (single-arm and double-arm engagement) exists when RAA22 / B09 is internalized in H1975 cells. Collectively, these data suggest that the target cell receptor expression level is an important determinant of the extent and efficiency of RAA22 / B09 internalization.

[0379] Example 3 – In vitro efficacy of ADCs In this example, the in vitro efficacy of an EGFR / cMET bispecific ADC was measured in a panel of cancer cell lines.

[0380] 3.1 Site-specific conjugation Conjugation of Tubulysin drugs to the RAA22 / B09 antibody molecule was performed essentially as previously described in Thompson, 2016 and US Patent Application Publication No. 2015 / 0291657A1.

[0381] 3.2 Methods for determining ADC cytotoxic activity ADC cytotoxic activity was tested in multiple cell lines as follows. Cells were plated in 100 μL of recommended culture medium supplemented with 10% fetal bovine serum at a density of 10,000 cells per well in 96-well plates. Three-fold concentrations of antibody at each dose to be tested were prepared by serially diluting the antibody stock in culture medium. Fifty microliters of each test substance was added to the cells in triplicate to achieve final antibody concentrations ranging from 60 nM to 0.0009 nM. Treated cells were cultured at 37°C in a humidified incubator for 72 hours. Metabolic activity was determined using the CellTiter-Glo luminescent viability assay from Promega according to the manufacturer's instructions. Data were plotted as percent metabolic activity relative to the untreated control. IC 50 Values were determined using logistic nonlinear regression analysis between maximum viability (untreated cells) and maximum response (peak of inhibition) using GraphPad Prism software.

[0382] 3.3 Results—In vitro ADC activity in a panel of cell lines The in vitro potency of the EGFR / cMET bispecific ADCs was measured in a panel of cancer cell lines using the CellTiter-Glo luminescent viability assay. As shown in Table 3, both the high-affinity QD6 / B09-AZ1508 and the low-affinity RAA22 / B09-AZ1508 demonstrated broad activity across cell lines with varying target expression levels.

[0383] [Table 11]

[0384] Overall, the ADCs with low affinity for EGFR showed comparable (albeit somewhat reduced) potency across a broad range of cell lines that co-expressed significant amounts of both EGFR and cMET. In general, both ADCs showed reduced potency when one or the other target had a low relative receptor density at the cell surface, below approximately 15,000. This effect was more pronounced for the lower affinity variants, which appeared to be more sensitive to low levels of cMET.

[0385] Example 4 – Bispecific conjugation of ADCs To further test the hypothesis that bispecific engagement of low-affinity EGFR-cMET antibodies is necessary for optimal ADC delivery, we performed in vitro experiments to determine the relative contribution of each antibody arm to the activity of the bispecific ADC. In the first experiment, we used excess unarmed parental antibodies to block EGFR or cMET and then measured the activity of the bispecific EGFR-cMET ADC in an in vitro cytotoxicity assay. For this experiment, we used a cell line that expresses moderate levels of EGFR and cMET in approximately equal amounts. If the individual arms of the bispecific ADC function independently in ADC delivery, blockade of either target in this cell line is expected to only slightly reduce the activity of the ADC, and since the targets are present at equivalent levels, the IC 50 The shift in activity is less than twofold. On the other hand, if the ADC requires simultaneous binding to effectively deliver the ADC into cells, blocking either target is likely to have a greater impact on the activity of the bispecific ADC. In related experiments, we compared the activity of the bispecific EGFR-cMET ADC with a monovalent monospecific control antibody containing one arm that binds to EGFR or cMET and one non-binding isotype antibody control arm. Similarly, if each arm functions independently, the expected result would be that each monospecific control ADC would be only slightly less potent than the bispecific ADC, and the difference would be additive. Instead, if the two arms of the bispecific function synergistically, a greater difference in the activity of the bispecific ADC would be expected compared to the monospecific control antibody.

[0386] 4.1 Method The cytotoxic activity of the ADCs was tested in the NCI-H1975 cell line as follows: Cells were plated at a density of 10,000 cells per well of a 96-well plate in the recommended culture medium supplemented with 10% fetal bovine serum in a volume of 50 μL for blocking experiments and 100 μL for monovalent ADC experiments. For disarmed mAb blocking experiments, 50 μL of a 300 μg / mL solution of EGFR IgG (RAA22) or cMET IgG (B09) was added to the well and preincubated for 1 hour at 37°C in a humidified incubator. Three-fold concentrations of antibody for each dose to be tested were prepared by serial 4x dilutions of the antibody stock in culture medium. Fifty microliters of medium alone, isotype control IgG ADC (R347-AZ1508), or EGFR-cMET ADC (RAA22 / B09-AZ1508) were added to cells in triplicate to achieve final antibody concentrations ranging from 67 nM to 0.0009 nM. For monovalent ADC experiments, 50 μL of a 3x stock of either the isotype control ADC (R347-AZ1508), monovalent EGFR ADC (RAA22 / R347-AZ1508), monovalent anti-cMET ADC (B09 / R347-AZ1508), an equimolar combination of monovalent ADCs, or EGFR-cMET ADC (RAA22 / B09-AZ1508) was added in triplicate in a 4-fold dilution series starting at 60 nM and ending at 0.009 nM. Treated cells were cultured for 72 hours at 37°C in a humidified incubator. Metabolic activity was determined using the CellTiter-Glo luminescent viability assay from Promega according to the manufacturer's instructions. Data were plotted as percent metabolic activity relative to untreated controls. IC 50 Values were determined using logistic nonlinear regression analysis between maximum viability (untreated cells) and maximum response (peak of inhibition) using GraphPad Prism software.

[0387] 4.2 Results and Conclusions—In Vitro Proof of Concept for Dual Targeting (mAb Blocking Experiments and Monovalent ADC) We performed in vitro experiments to examine the relative contribution of the individual antibody arms to the cytotoxic activity of the bispecific ADC, as outlined above. As shown in a representative experiment in Figure 7, pretreatment of NCI H1975 cells with cMET IgG RAA22 significantly increased the IC of the EGFR-cMET ADC (RAA22 / B09-AZ1508). 50 Treatment with anti-cMET IgG B09 significantly increased the IC 50 to 680 pM, a difference of over 11-fold. Similarly, when NCI H1975 cells were treated with a monovalent, monospecific EGFR ADC (RAA22 / R347-AZ1508), the IC 50 The IC of the monovalent, monospecific anti-cMET ADC (B09 / R347-AZ1508) was approximately 20,500 pM compared to 316 pM for the bispecific EGFR-cMET ADC, a difference of approximately 65-fold (Figure 8). 50 The activity of the antibody was 2,772 pM, which was approximately 13-fold higher than that of the bispecific antibody.

[0388] Collectively, these data suggest that efficient targeting of EGFR-cMET ADCs to tumor cells co-expressing both targets is primarily driven by the bispecific binding of the ADC. Furthermore, these results demonstrate that the EGFR low-affinity binding arm is insufficient to promote efficient ADC delivery in the absence of cMET binding, as shown by the dramatic drop in potency when the cMET arm is blocked by an unarmed antibody and the weak cytotoxicity of the monovalent EGFR control ADC, RAA22 / R347-AZ1508. Collectively, these results are consistent with the hypothesis that the low affinity of the EGFR-binding arm of the bispecific ADC (RAA22 / B09-AZ1508) promotes ADC delivery to tumors co-expressing EGFR and cMET, while exhibiting weak cytotoxicity against cells primarily expressing only one of the targets. This effect is most pronounced when only EGFR is available for engagement, which has implications for mitigating EGFR-driven toxicity in normal organs such as skin, where EGFR is expressed at significant levels but cMET expression is relatively low.

[0389] Example 5 – ADC in vivo pharmacology in patient-derived xenograft (PDX) models Patient-derived tumor xenograft (PDX) models of human cancer have become a well-established alternative to tumor cell line-based tumor xenografts. PDX models are established from primary patient tumor tissue that is directly implanted into immunocompromised mice, resulting in tumors that grow in vivo in mice. These tumors are then propagated in additional mice without in vitro culture, establishing a bank of low-passage PDX tumor tissues that can be used for transplantation into research mice. One key feature of PDX models is that they largely maintain the histological and genomic heterogeneity and preserve the gene expression profile of the corresponding original patient tumor. Compared to tumor cell line-based xenograft models, which use clonal populations of tumor cells adapted to grow in vitro, the characteristics of PDX models aim to more accurately recapitulate the characteristics of actual human tumors, thereby improving the predictive value of preclinical mouse models. Indeed, numerous studies have shown that the response and resistance profiles of PDX models to standard therapeutic treatments closely correlate with clinical data in human subjects with a given tumor profile.

[0390] Despite the improvements offered by PDX models, standard in vivo pharmacology study designs have limitations, even when applied to PDX models. For each tumor model, a typical study tests drug treatments at multiple dose levels, along with one or more positive or negative control compounds, using enough mice per treatment group to support within-model statistics. The relatively large number of mice required for such study designs, along with the high cost of PDX models, can limit the number of tumor models that can be practically tested for a given compound. An alternative / complementary approach to traditional study designs is the mouse PDX study, a population-based approach mimicking human clinical trial designs. In this approach, for each compound, typically one mouse is treated at a single dose level established in a previous dose-ranging study, and each model includes an optional treatment control group. The small number of mice required for each model allows for the testing of many PDX models, each representing a unique human tumor. Responses are assessed across the entire population of tumor models tested, without relying on within-model statistics. This approach allows for more accurate estimation of response rates across a range of target expression, molecular phenotypes, tumor subtypes, or other clinically relevant characteristics of interest. Furthermore, the large number of unique models that can be tested in a PDX study allows for more meaningful exploratory genomics, transcriptomics, or expression profiling studies, allowing for earlier exploration of correlates of response or resistance. For the exemplary study outlined herein, we adopted an "all-comers" approach, testing all available NSCLC models in START Discovery, regardless of target expression level or molecular phenotype.

[0391] 5.1 Method Mouse PDX studies were conducted at South Texas Accelerated Research Therapeutics (START, San Antonio, TX). START is accredited by AAALAC International (Association for Assessment and Accreditation of Laboratory Animal Care International) and complies with the AstraZeneca Global Standard on Animal Care and Welfare. All models were developed at START. Patient-tumor tissue explant (START-PDX) models are established from viable human tumor tissue or tumor cells and serially passaged in animals a limited number of times to maintain tumor heterogeneity. Athymic nude (Crl:NU(NCr)-Foxn1nu) / CB-17 Scid (CB17 / Icr-Prkdcscid / IcrIcoCrl) mice were implanted in the flank with tumor fragments harvested from host animals from each specific passage lot. Pre-study tumor volumes were recorded approximately 1 week before the planned study initiation date. Tumors were measured to determine whether they were within the appropriate tumor volume initiation (TVI) range (125-250 mm). 3 ), animals were randomized into treatment and control groups, and intravenous (IV) dosing began (day 0) and animals were followed individually throughout the study. The first dose began on day 0 and was administered intravenously to animals in all groups according to body weight (0.01 ml per gram; 10 ml / kg). Drug-treated animals received a total of four doses every seven days. Starting on day 0, tumor dimensions were measured with digital calipers, and estimated tumor volumes were recorded for each treated and control animal, and tumor volume was calculated using the following formula: TV = width 2 × length × 0.52. Tumor growth observation continued for 1 week after the final administration. Tumor volume (TV) endpoint (tumor volume ≥ 1 cm 3 Each animal was sacrificed upon reaching the end point of the study period of 28 days, whichever came first. For some PDX models with slow-growing tumors, the observation period was extended. The tumor growth inhibition rate (TGI%) was defined as the percentage of tumor growth relative to day 0 between the treatment (TX) group and the control (C) group according to the following formula: TGI% = 1 - (TX 最終 -TX 初期 ) / (C 最終 -C初期 The tumor regression rate was defined as the percentage of tumor reduction in treated animals relative to the tumor volume on day 0 (the day of first administration), calculated at the study endpoint according to the following formula: % regression = (TX 最終平均値 -TX 初期平均値 ) / (TX 初期平均値 )×100.

[0392] 5.2 Results and conclusions As shown in Figure 9, both the high-affinity EGFR-cMET ADC QD6 / B09-AZ1508 and its lower-affinity variant for EGFR, RAA22 / B09-AZ1508, induced tumor growth inhibition or regression in multiple PDX models tested. Surprisingly, the low-affinity ADCs showed an overall trend toward increased number and depth of observed responses compared with the high-affinity ADCs. This activity trend was slightly reversed in the PDX models least responsive to the low-affinity ADCs, which correlated somewhat with reduced cMET expression. These observations suggest that the activity of low-affinity EGFR-cMET ADCs may be partially dependent on cMET expression levels. The EGFR-binding arms of both bispecific antibodies were derived from the same murine EGFR-cross-reactive antibody (see Example 1). The intrinsic binding affinity of the QD6 / B09 antibody for murine EGFR was approximately 6 nM, while the affinity of the RAA22 / B09 bispecific antibody was approximately 575 nM. The unexpected improvement in activity of the low EGFR affinity antibody is likely due to a reduced EGFR sink effect in normal tissues such as the skin, increasing overall circulatory exposure of the ADC. Nevertheless, these data demonstrate that reducing the affinity of the EGFR-cMET bispecific antibody for EGFR does not compromise the in vivo efficacy of the resulting ADC, but rather unexpectedly improves its activity compared to the high-affinity ADC.

[0393] 5.3 Different dose PDX studies Further experiments were conducted to test different doses of ADC in PDX models. 3The study was performed as described in Example 5.1, except that individual mice that reached a tumor volume of 0.001 were removed from the study and the final measurement was included in the group average until either the mean volume reached the volume endpoint or the study reached the 63-day duration endpoint. The high-affinity EGFR-cMET ADC (QD6 / B09-AZ1508) was tested at dose levels of 1 mg / kg and 2 mg / kg, and a variant with reduced affinity for EGFR (RAA22 / B09-AZ1508) was tested at 1 mg / kg, 2 mg / kg, and 3 mg / kg.

[0394] As shown in Figure 10, both the high-affinity EGFR-cMET ADC (QD6 / B09-AZ1508) and its variant with reduced affinity for EGFR (RAA22 / B09-AZ1508) induced tumor growth inhibitory activity in PDX models at the doses tested. According to the results described in Example 5.2 and shown in Figure 9, the reduced-affinity ADCs were generally more effective than the high-affinity ADCs. In all four models tested, the low-affinity ADCs induced regressions at dose levels of 2 or 3 mg / kg, indicating that the low-affinity ADCs are effective at moderate doses. These data provide further evidence that reducing the affinity of an EGFR-cMET bispecific antibody for EGFR does not reduce the in vivo efficacy of the resulting ADC, but rather improves its efficacy compared to high-affinity ADCs.

[0395] Example 6 – Efficacy of ADC in an orthotopic pancreatic PDX model Subcutaneous in vivo tumor models are the mainstay for testing the efficacy of anticancer drugs. However, this tumor implantation site carries many limitations that must be considered when interpreting in vivo results. These deficiencies include the lack of tumor vascularization and tissue-specific stromal involvement in tumor growth and response. To address these challenges, we compared the in vivo efficacy of high- and low-affinity EGFR-cMET bispecific antibody-drug conjugates in both subcutaneous and orthotopic models of the pancreatic PDX model MEDI-PANC-08. To track the growth of this tumor orthotopically, we used a luciferase-expressing PDX variant (MEDI-PANC-08) whose growth can be tracked using imaging. LUC ) was developed.

[0396] 6.1 Method All experiments were performed in AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care)-accredited facilities and in accordance with MedImmune's IACUC (Institutional Animal Care and Use Committee) guidelines for the humane treatment and care of laboratory animals. Animals were monitored daily for morbidity and mortality.

[0397] Subcutaneous PDX model The MEDI-PANC-08 pancreatic PDX model used in this study was obtained from the Internal MedImmune PDX library. PDX tumors were initially seeded on NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl The tumors were grown in 100% WT / SzJ mice to generate sufficient tumor material to seed efficacy studies. Tumors were 800-1200 mm 3 Once tumors reached approximately 2 mm, mice were humanely euthanized by CO2 asphyxiation. Tumors were isolated under sterile conditions and measured at approximately 2 mm 3Tumors were cut into pieces and subcutaneously implanted into the right flank of individual NSG mice using an 11-gauge trocar needle. Once tumor size reached approximately 150–250 mm, mice were randomized (based on tumor volume) into treatment groups and treated with ADCs (Q1W × 4). Two EGFR-cMET bispecific ADCs, QD6 / B09 (high affinity) and RAA2 / B09 (low affinity), were tested at 1, 2, and 3 mg / kg. An isotype control ADC (R347-AZ1508) was also tested at 3 mg / kg. Immediately before use, all antibody-drug conjugates were diluted in buffer (25 mM histidine, 7% sucrose, 0.02% PS80, pH 6.0) and administered intravenously via the tail vein. Tumor and body weight measurements were collected twice weekly, and tumor volume was calculated using the equation (L × W2) / 2, where L and W represent the length and width dimensions, respectively.

[0398] Orthotopic PDX models Luciferase-expressing PDX model (MEDI-PANC-08 LUC ) were grown subcutaneously in NSG seed mice, and the resulting cells were grown to 800–1200 mm 3 Tumors were harvested at a volume of approximately 2 mm 3 The tumors were cut into 100 mm pieces. The tumor fragments were then sutured to the pancreas of NSG mice (day 0). Luciferase signals were measured weekly using an IVIS Spectrum in vivo imaging system. Briefly, 10 minutes before imaging, 200 μl of luciferin (15 mg / ml) dissolved in DPBS was injected intraperitoneally (ip). Mice were anesthetized with 3% isoflurane, placed on their right side, and luminescence was measured. 14 days after tumor implantation, when luminescence signals were clearly detectable, mice were randomized into respective groups based on luminescence. Mice were treated with an isotype control (R347-AZ1508, 3 mg / kg Q1W × 4), gemcitabine (75 mg / kg Q2D × 5), and RAA2 / B09 ADC (2 and 3 mg / kg Q1W × 4). Luminescence was measured weekly. Study endpoints included weight loss, deterioration in body condition, and lethargy. Data were analyzed using Living Image software (Perkin Elmer) and plotted as mean radiance [p / s / cm2 / sr] against time.

[0399] 6.2 Results and Discussion To aid in the selection of suitable EGFR affinity combinations for EGFR-cMET bispecific ADCs, high-affinity and low-affinity EGFR-cMET bispecific ADCs were compared in an in vivo efficacy study using the MEDI-PANC-08 pancreatic PDX model. As shown in Figure 11, panels A and B, significant differences were observed between the two molecules. The high-affinity QD6 / B09 ADC showed no efficacy at any of the three dose levels tested. Conversely, the low-affinity RAA2 / B09 ADC resulted in complete tumor regression by day 65, followed by tumor regrowth at the 3 mg / kg dose level and tumor growth inhibition at 2 mg / kg.

[0400] Subcutaneous tumor models have become the mainstay of in vivo efficacy testing, but a major drawback is that tumors do not grow at the site of origin, and therefore drug responses may not truly reflect patient responses. To address this concern, we developed an orthotopic model of pancreatic cancer using MEDI-PANC-08 tumors genetically engineered to stably express luciferase. After surgical implantation into the pancreas, tumors were allowed to establish and then randomized based on luminescence signal. Mice were then treated with the low-affinity RAA2 / B09 EGFR-cMET ADC, an isotype control, or gemcitabine (a chemotherapy drug). Luminescence was measured weekly after treatment. As shown in Figure 11, panel C, luminescence in the untreated and isotype control groups increased over time, and animals were removed from the study due to poor health and large, palpable abdominal tumors. Gemcitabine showed an initial decrease in luminescence signal, reaching a nadir around day 21. Subsequently, signal increased over time, and the group was removed from the study on day 49. In the orthotopic model, the luminescence signal of the RAA2 / B09 ADC at both the 2 mg / kg and 3 mg / kg dose levels decreased to near background levels by day 60. Compared to the subcutaneous study, the 2 mg / kg dose level showed better activity, resulting in tumor regression. Necropsy of animals that exhibited tumors with near background luminescence signals at the end of the study revealed no more visible tumors, thus supporting the correlation between luminescence signal and tumor volume.

[0401] In conclusion, the low-affinity EGFR-cMET RAA2 / B09 ADC demonstrated improved efficacy compared to the high-affinity QD6 / B09 ADC in a subcutaneous pancreatic PDX model, with tumor regression observed at 3 mg / kg. This efficacy was also observed in an orthotopic model using the same PDX tumors (MEDI-PANC-08) engineered to stably express luciferase. Using luminescence as a surrogate for tumor volume, the RAA2 / B09 ADC demonstrated improved efficacy compared to the subcutaneous model, with tumor regression observed at both 2 mg / kg and 3 mg / kg.

[0402] Example 7 – Safety and Pharmacokinetics Pharmacokinetic (PK) analysis was performed to compare plasma PK parameters, such as peak and total exposure, clearance, and half-life, between the low-affinity EGFR-cMET ADC and the high-affinity EGFR-cMET ADC in mice and cynomolgus monkeys. The primary objective was to determine whether reduced affinity for EGFR affects the circulating exposure of the EGFR-cMET bispecific ADC. PK samples were collected from mice and cynomolgus monkeys at various dose levels for both QD6 / B09-57-AZ1508 and RAA / B09-57-AZ1508. Noncompartmental analysis was performed to estimate PK parameters for QD6 / B09-57-AZ1508 and RAA22 / B09-57-AZ1508 based on total ADC concentrations across species and dose levels.

[0403] Overall, RAA22 / B09-57-AZ1508 showed higher exposure and longer t1 / 2 compared to QD6 / B09-57-AZ1508 in both mice and cynomolgus monkeys, suggesting improved PK with the lower affinity RAA22 / B09-57-AZ1508.

[0404] 7.1 Bioanalysis of Preclinical PK Assays Target compound (QD6 / B09-57-AZ1508 and RAA22 / B09-57-AZ1508) and total antibody concentrations were measured in a single immunocapture LC-MS / MS assay. Briefly, polyclonal anti-human antibodies were conjugated to magnetic beads. 25 μL of plasma sample was then diluted with PBS and incubated with the magnetic beads. After capture, the magnetic beads were washed multiple times and then digested with trypsin in the presence of an internal standard. Acid was added to quench the digestion. The liquid contents were then transferred to an injection plate.

[0405] Signature tryptic peptides on the Fc region of human antibodies and cleaved warheads were separated using reversed-phase chromatography (RPLC) and then detected using multiple reaction monitoring (MRM). The signature peptides on the Fc region were used to calculate total Abs, while the digested and released warheads were used to calculate ADCs. The internal standards used in this experiment were isotope-labeled peptides or proteins (SiluMAb, Sigma-Aldrich) or isotope-labeled warheads. The peak area ratios of the analytes to the internal standard were used to calculate against a standard curve.

[0406] Standard curves and QCs were prepared by spiking different levels of the target compound into the same matrix as the samples. The quantification range included 100 ng / mL to 15,000 ng / mL, with diluted QCs up to 525,000 ng / mL. The standard curve was fitted using the simplest possible model. The precision and accuracy of the assay were within 20% at all levels, except for the lower limit of quantitation (LLOQ), which was 25%.

[0407] 7.2 PK of QD6 / B09-57-AZ1508 in mice The mouse studies included in the NCA analysis are summarized in Table 4.

[0408] [Table 12]

[0409] The mean PK concentration-time profiles in mice of RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 are shown in FIG.

[0410] RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 exhibited linear PK in mice at the dose levels tested, with dose-proportional exposure (C max and AUC), equivalent CL and t 1 / 2was observed at 0.5 mg / kg to 10 mg / kg for RAA22 / B09-57-AZ1508 and 1 mg / kg to 10 mg / kg for QD6 / B09-57-AZ1508, respectively.

[0411] PK comparisons between RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 were evaluated at the 1, 3, 5, and 10 mg / kg dose levels tested for both compounds, and mean PK parameters based on NCA are summarized in Table 5. Results showed a slow CL, high exposure, and long t 1 / 2 The mean AUC of RAA22 / B09-57-AZ1508 was increased by 2-2.91 fold compared to QD6 / B09-57-AZ1508, and the mean t 1 / 2 The range of RAA22 / B09-57-AZ1508 was 4.24 to 6.38 days, and the range of QD6 / B09-57-AZ1508 was 2.57 to 3.33 days. 1 / 2 showed a 1.27-2.32 fold increase.

[0412] [Table 13]

[0413] 7.3 PK of QD6 / B09-57-AZ1508 in Cynomolgus Monkeys The cynomolgus monkey studies included in the NCA analysis are summarized in Table 6.

[0414] [Table 14]

[0415] The mean PK concentration-time profiles of RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 in cynomolgus monkeys are shown in FIG.

[0416] RAA22 / B09-57-AZ1508 showed linear PK at 2 mg / kg to 5 mg / kg in cynomolgus monkeys, with dose-proportional exposure (Cmax and AUC), equivalent CL and t 1 / 2 was observed.

[0417] QD6 / B09-57-AZ1508 showed nonlinear PK in cynomolgus monkeys at 0.67 mg / kg to 3 mg / kg, with more than dose-proportional exposure (C max and AUC) were shown, with a faster CL and shorter t at lower dose levels. 1 / 2 was observed.

[0418] PK comparisons between RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 in cynomolgus monkeys were evaluated at the 2 mg / kg and 3 mg / kg dose levels tested for both compounds, and mean PK parameters based on NCA are summarized in Table 7. Results showed a slow CL, high exposure, and long t 1 / 2 The mean AUC of RAA22 / B09-57-AZ1508 was increased by 1.90-2.43 fold compared to QD6 / B09-57-AZ1508, and the mean t 1 / 2 The range of RAA22 / B09-57-AZ1508 was 4.30 to 5.90 days, and the range of QD6 / B09-57-AZ1508 was 0.969 to 1.07 days. 1 / 2 showed a 4.44-5.51-fold increase.

[0419] [Table 15]

[0420] Collectively, these data demonstrate that the low-affinity RAA22 / B09-57-AZ1508 exhibits higher exposure and a longer circulating half-life compared to the high-affinity QD6 / B09-57-AZ1508 in both mice and cynomolgus monkeys. These data are consistent with the hypothesis that lowering affinity for EGFR reduces binding to EGFR present in normal tissues, thereby mitigating the normal tissue sink effect and improving plasma PK parameters.

[0421] Example 8 – ADC with a Topoisomerase I Inhibitor as the Payload Experiments were designed to test the efficacy and safety of RAA22 / B09-57 bispecific molecules conjugated to a different payload - a topoisomerase I inhibitor - rather than tubulysin as used in the previous examples.

[0422] The DuetMab RAA22 / B09 (serine 239 followed by a "Maia" cysteine) bispecific antibody produced according to Example 1 was conjugated to the topoisomerase inhibitor SG3932 by "classical" conjugation to a natural cysteine in the bispecific antibody. [ka]

[0423] The efficacy of the EGFR / cMET topoisomerase I inhibitor ADC was investigated using a PDX study. The PDX study was essentially performed as described above in Example 5 using a variety of different PDX models derived from pancreatic, colon, NSCLC, and squamous cell carcinoma of the head and neck (SQHN) tumors. Animals were injected with a single dose of 10 mg / kg of EGFR-cMET Maia Topo ADC. The results of the PDX study using EGFR-cMET Maia Topo ADC are reported in Figure 14.

[0424] As shown in Figure 14, the EGFR-cMET Maia Topo ADC induced tumor growth inhibition or regression in multiple PDX models tested. Thus, these results demonstrate that the RAA22 / B09-57 ADC, which contains a topoisomerase I inhibitor, was effective in PDX models representing multiple tumor types.

[0425] Example 9 – Mutations to improve PK of ADCs The ADC with a topoisomerase I inhibitor produced and tested in Example 8 used the RAA22 / B09 bispecific antibody, which contains a "Maia" cysteine insertion after serine 239. However, given that SG3932 conjugates to a natural cysteine, the insertion of the Maia cysteine is not necessary. Therefore, we sought to modify the RAA22 / B09 Maia Topo ADC produced in Example 8 to remove this cysteine insertion.

[0426] It was also recognized that reducing or eliminating effector functions of the Fc backbone may reduce immunotoxicity and improve pharmacokinetics. Therefore, the inventors also introduced the L234F / L235E / P331S (EU numbering) "triple mutant (TM)" (Organesyan, 2008; Hay, 2016), which was previously shown to reduce Fc effector function in antibody molecules.

[0427] The newly generated "EGFR-cMET TM" molecule, which contains the variable regions from RAA22 and B09, with the 239i mutation removed and the TM introduced, has the amino acid sequence shown in the table below.

[0428] [Table 16]

[0429] To conjugate SG3932, a 50 mM solution of tris(2-carboxyethyl)phosphine (TCEP) (12.5 molar equivalents / antibody) in phosphate-buffered saline (pH 7.4) (PBS) was added to the EGFR-cMET™ bispecific antibody solution in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) to a final antibody concentration of approximately 3 mg / mL. The reduction mixture was incubated at 37°C for 2 hours with gentle shaking (60 rpm) on an orbital shaker. The reaction mixture was allowed to cool to room temperature for 45 minutes. SG3932 was then added as a DMSO solution (12.5 molar equivalents / antibody) to a final DMSO concentration of 10% (v / v). The solution was incubated at room temperature for 2 hours, then quenched by the addition of N-acetylcysteine (5 micromoles / SG3932) and incubated at room temperature for 15 minutes. The reaction mixture was filtered through a 0.2 μM sterile filter and then stored overnight at 2–8 °C. Surface area: 375 cm 2 Excess free drug was removed by tangential flow filtration (TFF) using a MidiKros® 30 kDa fiber filter containing mPES into a buffer solution (pH 6.8) containing 30 mM histidine and 30 mM arginine. The extent of free drug removal was monitored by UHPLC-RP using the neat conjugate. After complete removal of free drug, the ADC was buffer exchanged. The ADC was filtered under a sterile atmosphere using a 0.22 μm filter, and then polysorbate-80 was added to a final concentration of 0.02% (w / v).

[0430] UHPLC analysis of the reduced ADC sample at 214 nm and 330 nm (specific for SG3932) using a Thermo Scientific MAbPac 50 mm x 2.1 mm column on a Shimadzu Prominence system eluted with a gradient of water and acetonitrile revealed a drug-antibody ratio (DAR) of 6.0 molecules of SG3932 per antibody.

[0431] The efficacy of the EGFR / cMET™ ADC was investigated using PDX studies. PDX studies were performed essentially as described in Example 5 above using a variety of different PDX models derived from pancreatic, colon, NSCLC, and SQHN tumors. Animals were injected with a single dose of 5 mg / kg of EGFR-cMET™ ADC. The results of the PDX studies using the EGFR-cMET™ ADC are reported in Figure 15. The results of this experiment demonstrate that the EGFR-cMET™ ADC was able to induce tumor growth inhibition or regression in multiple PDX models tested.

[0432] The efficacy of the EGFR-cMET™ ADC ("TM ADC") was compared to the EGFR-cMET Maia Topo ADC ("Maia ADC") generated in Example 8 in the PDX models SQHN-02 and PANC-08. Animals were administered 2.5 mg / kg, 5 mg / kg, or 10 mg / kg of each ADC and tumor growth was monitored. The experiment also included an untreated control ("Naive") and animals administered unconjugated EGFR-cMET™ (TM mAb). The results are shown in Figure 16.

[0433] The results show that both the EGFR-cMET™ ADC and the EGFR-cMET Maia Topo ADC are equally effective at reducing tumor growth / inducing tumor regression ("equipotent") across the dose range tested, suggesting that elimination of the S239i mutation and abrogation of Fc effector function by the triple mutant does not negatively impact efficacy in these tumor models.

[0434] The EGFR-cMET™ ADC was also shown to be effective in reducing tumor growth in NSCLC tumors expressing wild-type or mutant EGFR. Results show that the EGFR-cMET™ ADC is active in both wild-type and mutant EGFR PDX models (Figure 17). This advantageously demonstrates that the ADC can provide benefit in multiple treatment settings and across a variety of different EGFR genotypes.

[0435] Additionally, a pharmacokinetic (PK) study was conducted in NOD-SCID mice comparing the EGFR-cMET™ ADC ("TM ADC") and the EGFR-cMET Maia Topo ADC ("Maia ADC") produced in Example 8. The experiment was performed essentially as described in Example 7.

[0436] Representative results of these PK studies are shown in Figure 18. As reported in this figure, the EGFR-cMET™ ADC exhibited a PK response comparable to that of the EGFR-cMET Maia Topo ADC (t 1 / 2 = 3.0 days; CL = 39.7 ml / day / kg) compared to a longer half-life (t 1 / 2 = 5.0 days) and reduced drug clearance (CL = 14.8 ml / day / kg). Thus, the results demonstrate that the EGFR-cMET™ ADC described herein has improved PK compared to the EGFR-cMET Maia Topo ADC produced in Example 7. Similar improvements in PK were observed when unconjugated EGFR-cMET TM antibody ("TM mAb") was compared to unconjugated EGFR-cMET Maia ("Maia ADC").

[0437] Example 10 – Efficacy of ADC in combination with osimertinib This example compares the efficacy of an ADC in combination with the third-generation tyrosine kinase inhibitor TKI osimertinib ("Osi") in various EGFR-mutant tumor models using a PDX study.

[0438] 5.1 Method Preclinical efficacy studies in immunocompromised mice or mice bearing patient tumor xenografts (PDX) were conducted at Champions Oncology (athymic nude-Foxn1 mice) and Genendesign (Balb / C nude mice). All studies complied with the AstraZeneca Global Standard on Animal Care and Welfare. Models were established from viable human tumor tissue or tumor fluid and serially passaged in animals a limited number of times to maintain tumor heterogeneity. Tumor fragments harvested from host animals transplanted from each specific passage lot were implanted into one flank of mice. Pre-study tumor volumes were recorded approximately 1 week before the planned study start date. Tumors were grown to the appropriate tumor volume initiation (TVI) range (150-300 mm). 3 ), animals were randomly divided into treatment and control groups.

[0439] Treatment—Animals receiving the EGFR-cMET Top1i antibody-drug conjugate (ADC) or control EGFR-cMET mAb described in Example 9 received a single intravenous (IV) dose at the indicated dose level on Day 0 (animals were administered IV at a dose volume of 5 ml / kg based on body weight). Beginning on Day 0, animals in the osimertinib-treated group received the drug formulated as an oral dosing solution at 2.5 mg / ml in vehicle (0.5% w / v HPMC (hydroxypropylmethylcellulose) in deionized water) (animals were administered orally at a dose volume of 10 ml / kg based on body weight to obtain a final dose level of 25 mg / kg). Osimertinib-treated animals were dosed daily for the first 21 days of the study. Animals in the combination treatment group received both the EGFR-cMET ADC and osimertinib, with each treatment administered according to the monotherapy dosing schedule described above. All animals were followed individually throughout the study.

[0440] Tumor Growth Inhibition—Starting on day 0, tumor size was measured twice weekly with digital calipers, and data including individual tumor volumes and mean estimated tumor volume (mean TV ± SEM) for each group were recorded (tumor volume was calculated using formula (1): TV = width × length × 0.52). At study completion, tumor growth inhibition (TGI%) values were calculated using the initial (i) and final (f) tumor measurements according to formula (2): %TGI = 1 − (Tf − Ti) / (Cf − Ci). Values were reported for each treatment group (T) versus control (C). Individual mice with two consecutive measurements reporting tumor volumes ≤ 30% of the day 0 measurement were considered partial responders (PR). Individual mice lacking palpable tumors (two consecutive measurements of 0.00 mm) were considered partial responders (PR). 3 ) were classified as complete responders (CR) (CRs sustained to study completion were considered tumor-free survivors (TFS). For the vehicle-treated group, tumor doubling time (DT) was determined using the formula DT = (Df - Di) * log2 / (logTVf - logTVi), where D = days and TV = tumor volume. Observations of tumor growth in the untreated control group were performed until the group's mean tumor volume (uncorrected) reached the humane endpoint of 1500 mm. 3 The tumor growth observations for the treatment groups were continued until the tumors in individual mice in the treatment groups reached 1500 mm or until day 60, whichever came first. 3 If the humane endpoint of 10 days was reached, the animal was euthanized and observation of the other treated animals continued. Some animals that showed sustained responses were observed for more than 60 days.

[0441] result The results are presented in Figures 19-21.

[0442] Figures 19A and C show results for EGFR-mutant models "LUN487" and "LUN439," which contain the EGFR L858R mutation, representing primary EGFRm non-small cell lung cancer (NSCLC). EGFR-cMET™ ADC was administered at 2, 4, and 8 MPK (mg / kg). One group also received a mAb-only control (EGFR-cMET mAb) at 8 MPK or osimertinib alone at 25 MPK. Results show that EGFR-cMET™ ADC monotherapy demonstrated a dose-dependent response. PDX models responded to osimertinib. No treatment response was observed in the mAb-only control.

[0443] Figures 19B and D show results for the EGFR-mutant models "LUN487" and "LUN439" administered with EGFR-cMET™ ADC alone at 2 or 4 MPK or in combination with osimertinib (25 MPK). The combination of EGFR-cMET™ ADC and osimertinib demonstrated improved tumor growth inhibition compared to either agent (EGFR-cMET™ ADC or osimertinib) administered individually.

[0444] Figure 20A shows results for the EGFR mutation model "CTG-2992," which contains an exon 20 insertion that conferred primary resistance to osimertinib. EGFR-cMET™ ADC was administered at 2, 4, and 8 MPK (mg / kg). One group also received a mAb-only control (EGFR-cMET mAb) at 8 MPK or osimertinib alone at 25 MPK. Results show that EGFR-cMET™ ADC monotherapy demonstrated a dose-dependent response. The PDX model responded to osimertinib.

[0445] Figure 20B shows the results of administering a combination of EGFR-cMET™ ADC and osimertinib to the EGFR mutation model "CTG-2992." The EGFR-cMET™ ADC was administered at 2 or 4 MPK. The combination of EGFR-cMET™ ADC and osimertinib demonstrated improved tumor growth inhibition compared to either agent (EGFR-cMET™ ADC or osimertinib) administered individually.

[0446] Figure 21A shows results for the EGFR mutation model "CTG-2803," which exhibits acquired resistance to osimertinib. EGFR-cMET™ ADC was administered at 2, 4, and 8 MPK (mg / kg). One group also received either an 8 MPK mAb control (EGFR-cMET mAb) or 25 MPK osimertinib alone. Results show that EGFR-cMET™ ADC monotherapy demonstrated a dose-dependent response. The PDX model did not respond to osimertinib or mAb-only monotherapy.

[0447] Figure 21B shows the results of administering a combination of EGFR-cMET™ ADC and osimertinib to the EGFR mutation model "CTG-2803." The EGFR-cMET™ ADC was administered at 2 or 4 MPK. The combination of EGFR-cMET™ ADC and osimertinib demonstrated improved tumor growth inhibition compared to either agent (EGFR-cMET™ ADC or osimertinib) administered individually.

[0448] Taken together, these results demonstrate that the combination of EGFR-cMET™ ADC and osimertinib showed efficacy across a range of EGFR-mutant cancers, including those classified as osimertinib-resistant.

[0449] Example 11 – Response of EGFRmut NSCLC PDX to an ADC in combination with osimertinib This example is designed to further evaluate the efficacy of the combination of osimertinib and EGFR-cMET™ ADC in NSCLC PDX models with mutated EGFR. An additional 23 NSCLC PDX models containing various EGFR mutations were enrolled. The study was primarily conducted as described in Section 5.1 of Example 10.

[0450] Mice from each PDX were divided into three groups, typically receiving osimertinib at 25 mg / kg / day for 21 days, EGFR-cMET™ ADC at 2 mg / kg, or osimertinib at 25 mg / kg / day and EGFR-cMET™ ADC at 2 mg / kg. Experiments were performed with an isotype-ADC R347 control at 8 mg / kg and / or a naked EGFR-cMET™ mAb control at 8 mg / kg.

[0451] Figures 22A, 22B and 22C show the respective responses of the model for each of the three treatment groups.

[0452] Results show that responses were observed in 14 / 23 (61%) models in the combination group, while the osimertinib and EGFR-cMET™ ADC monotherapy groups demonstrated response rates of 8 / 23 (34.8%) and 7 / 23 (30.4%) models, respectively. Response was defined as a 30% regression in tumor volume from baseline. Regression was analyzed starting one week after dosing. Reported values are the best responses observed over the study period.

[0453] More detailed response data and information on EGFR mutation status are provided in the table below.

[0454] [Table 17]

[0455] [Table 18]

[0456] References A number of publications have been cited above in order to more fully describe and disclose the present disclosure and the state of the art to which it pertains. Full citations for these references are set forth below. Each of these references is incorporated herein in its entirety.

[0457] [Table 19]

[0458] [Table 20]

[0459] [Table 21]

[0460] [Table 22]

[0461] array CDR sequences of the low-affinity anti-EGFR binding arm (RAA22) HCDR1 - DNDFS (SEQ ID NO: 1) HCDR2 - AIVAVFRTETYAQKFQD (SEQ ID NO: 2) HCDR3 - RLMSAISGPGAPLLM (SEQ ID NO: 3) LCDR1 - TGTSSDVGGYNYVS (SEQ ID NO: 4) LCDR2 - DVSKRPS (SEQ ID NO: 5) LCDR3 - SSYTSSDTLEI (SEQ ID NO: 6) CDR sequences of the high-affinity anti-EGFR binding arm (QD6) HCDR1 - DNDFS (SEQ ID NO: 1) HCDR2 - AIVAVVRTETYAQKFQD (SEQ ID NO: 7) HCDR3 - RLMSAISGPGAPLLM (SEQ ID NO: 3) LCDR1 - TGTSSDVGGYNYVS (SEQ ID NO: 4) LCDR2 - DVSERPS (SEQ ID NO: 66) LCDR3 - FSYTSSDTLEI (SEQ ID NO: 67) FR sequences of anti-EGFR binding arms RAA22 and QD6 HFR1 - QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 8) HFR2 - WVRQAPGQGLEWMG (SEQ ID NO: 9) HFR3 - RVKITADISTRTTYMELSSLRSEDTAVYYCAR (SEQ ID NO: 10) HFR4 - WGQGTLVTVSS (SEQ ID NO: 11) LFR1 - QSALTQPRSVSGSPGQSVTISC (SEQ ID NO: 12) LFR2 - WYQQHPGKAPKLMIY (SEQ ID NO: 13) LFR3 - GVPDRFSGSKSGNTASLTISGLQAEDEADYYC (SEQ ID NO: 14) LFR4 - FGGGTKLTVL (SEQ ID NO: 15) Amino acid sequence of the variable heavy (VH) region of the low affinity anti-EGFR binding arm (RAA22) (SEQ ID NO: 16) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNDFSWVRQAPGQGLEWMGAIVAVFRTETYAQKFQDRVKITADISTRTTYMELSSLRSEDTAVYYCARRLMSAISGPGAPLLMWGQGTLVTVSS Nucleic acid sequence of the VH region of the low affinity anti-EGFR binding arm (RAA22) (SEQ ID NO: 17): [ka] Amino acid sequence of the VH region of anti-EGFR antibody clone QD6 (SEQ ID NO: 18): QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNDFSWVRQAPGQGLEWMGAIVAVVRTETYAQKFQDRVKITADISTRTTYMELSSLRSEDTAVYYCARRLMSAISGPGAPLLMWGQGTLVTVSS Nucleic acid sequence of the VH region of anti-EGFR antibody clone QD6 (SEQ ID NO: 19): [ka] Amino acid sequence of the variable light (VL) region of anti-EGFR antibody clone RAA22 (SEQ ID NO: 20): QSALTQPRSVGSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSDTLEIFGGGTKLTVL Nucleic acid sequence of the VL region of anti-EGFR antibody clone RAA22 (SEQ ID NO: 21): [ka] Amino acid sequence of the VL region of the high affinity anti-EGFR binding arm (QD6) (SEQ ID NO: 22): QSALTQPRSVGSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSERPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCFSYTSSDTLEIFGGGTKLTVL Nucleic acid sequence of the VL region of the high affinity anti-EGFR binding arm (QD6) (SEQ ID NO: 23): [ka] CDR sequence of anti-cMet binding arm B09-GL HCDR1 - DYYIH (SEQ ID NO: 24) HCDR2 - WMNPNSGNTGYAQKFQG (SEQ ID NO: 25) HCDR3 - GQGYTHS (SEQ ID NO: 26) LCDR1 - RASEGIYHWLA (SEQ ID NO: 27) LCDR2 - KASSLAS (SEQ ID NO: 28) LCDR3 - QQYSNYPPT (SEQ ID NO: 29) FR sequence of anti-cMet binding arm B09-GL HFR1 - QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 30) HFR2 - WVRQATGQGLEWMG (SEQ ID NO: 31) HFR3 - RVTMTRDTSISTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 32) HFR4 - WGQGTMVTVSS (SEQ ID NO: 33) LFR1 - DIQMTQSPSTLSASVGDRVTITC (SEQ ID NO: 34) LFR2 - WYQQKPGKAPKLLIY (SEQ ID NO: 35) LFR3 - GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ ID NO: 36) LFR4 - FGGGTKLEIK (SEQ ID NO: 37) Amino acid sequence of the variable heavy (VH) region of anti-cMet binding arm B09-GL (SEQ ID NO: 38) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRSEDTAVYYCARGQGYTHSWGQGTMVTVSS Nucleic acid sequence of the VH region of anti-cMet binding arm B09-GL (SEQ ID NO: 39) [ka] Amino acid sequence of the variable light (VL) region of anti-cMet binding arm B09-GL (SEQ ID NO: 40) DIQMTQSPSTLSASVGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSNYPPTFGGGTKLEIK Nucleic acid sequence of the VL region of anti-cMet binding arm B09-GL (SEQ ID NO: 41) [ka] Amino acid sequence of human immunoglobulin G1 heavy chain constant (CH) region (SEQ ID NO: 42): [ka] Amino acid sequence of human immunoglobulin G1 CH region modified to include "knob" mutations, interchain cysteine mutations, cysteines to form stabilizing disulfide bridges, and cysteine insertions (SEQ ID NO:43): The following substitutions are underlined: "Knob" mutation (T366W), interchain cysteine mutations (F126C and C219V), a stabilizing cysteine mutation (S354C) and a cysteine insertion (C239i) (residue numbering according to the EU index). [ka] Amino acid sequence of human immunoglobulin G1 CH region modified to include "knob" mutations, interchain cysteine mutations, cysteines to form stabilizing disulfide bridges, and no cysteine insertions (SEQ ID NO:44): The following substitutions are underlined: "Knob" mutation (T366W), interchain cysteine mutations (F126C and C219V) and a stabilizing cysteine mutation (S354C) (residue numbering according to the EU index). [ka] Amino acid sequence of human immunoglobulin G1 CH region modified to include a "hole" mutation, a cysteine to form a stabilizing disulfide bridge, and a cysteine insertion (SEQ ID NO:45): The following substitutions are underlined: "Hole" mutations (T366S, L368A and Y407V), a stabilizing cysteine mutation (Y349C) and a cysteine insertion (C239i) (residue numbering according to the EU index). [ka] Amino acid sequence of human immunoglobulin G1 CH region modified to include a "hole" mutation, a cysteine to form a stabilizing disulfide bridge, and no cysteine insertion (SEQ ID NO:46): The following substitutions are underlined: "Hole" mutations (T366S, L368A and Y407V), a stabilizing cysteine mutation (Y349C) (residue numbering according to the EU index). [ka] Amino acid sequence of the wild-type human immunoglobulin kappa constant region (SEQ ID NO:47): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Amino acid sequence of a human immunoglobulin kappa constant region modified to contain S121C and C214V substitutions (SEQ ID NO:48): The following substitutions are underlined: S121C and C214V (numbering according to EU index) [ka] Amino acid sequence of the human immunoglobulin lambda constant region (SEQ ID NO:49): GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Amino acid sequence of the heavy chain of anti-cMet antibody binding arm B09-GL with a cysteine insertion (SEQ ID NO: 50): The following substitutions are underlined: "Knob" mutation (T366W), interchain cysteine mutations (F126C and C219V), a stabilizing cysteine mutation (S354C) and a cysteine insertion (C239i) (residue numbering according to the EU index). [ka] Amino acid sequence of the heavy chain of anti-cMet antibody binding arm B09-GL without the cysteine insertion (SEQ ID NO: 51): The following substitutions are underlined: "Knob" mutation (T366W), interchain cysteine mutations (F126C and C219V) and a stabilizing cysteine mutation (S354C) (residue numbering according to the EU index). [ka] Amino acid sequence of the light chain of anti-cMet binding arm B09-GL (SEQ ID NO: 52) The following substitutions are underlined: S121C and C214V (numbering according to EU index) [ka] Amino acid sequence of the heavy chain of the high affinity anti-EGFR binding arm (QD6) with a cysteine insertion (SEQ ID NO: 53): The following substitutions are underlined: "Hole" mutations (T366S, L368A and Y407V), a stabilizing cysteine mutation (Y349C) and a cysteine insertion (C239i) (residue numbering according to the EU index). [ka] Amino acid sequence of the heavy chain of the high affinity anti-EGFR binding arm (QD6) without cysteine insertion (SEQ ID NO: 54): The following substitutions are underlined: "Hole" mutations (T366S, L368A and Y407V), a stabilizing cysteine mutation (Y349C) (residue numbering according to the EU index). [ka] Amino acid sequence of the light chain of the high affinity anti-EGFR binding arm (QD6) (SEQ ID NO: 55): [ka] Amino acid sequence of the heavy chain of the low affinity anti-EGFR binding arm (RAA22) with a cysteine insertion (SEQ ID NO: 56): The following substitutions are underlined: "Hole" mutations (T366S, L368A and Y407V), a stabilizing cysteine mutation (Y349C) and a cysteine insertion (C239i) (residue numbering according to the EU index). [ka] Amino acid sequence of the heavy chain of the low affinity anti-EGFR binding arm (RAA22) without the cysteine insertion (SEQ ID NO: 57): The following substitutions are underlined: "Hole" mutations (T366S, L368A and Y407V), a stabilizing cysteine mutation (Y349C) (residue numbering according to the EU index). [ka] Amino acid sequence of the light chain of the low affinity anti-EGFR binding arm (RAA22) (SEQ ID NO: 58): [ka] Amino acid sequence of the heavy chain of the low affinity anti-EGFR binding arm (RAA22) with a triple mutation (TM) (SEQ ID NO: 59): The following substitutions are underlined: Triple mutation (TM; L234F, L235E and P331S), "knob" mutation (T366W), interchain cysteine mutations (F126C and C219V), stabilizing cysteine mutation (S354C) (residue numbering according to the EU index). [ka] Amino acid sequence of the heavy chain of the anti-cMet binding arm with the triple mutation (TM) (SEQ ID NO: 60): The following substitutions are underlined: The triple mutation (TM; L234F, L235E and P331S), the "hole" mutation (T366S, L368A and Y407V) and the stabilizing cysteine mutation (Y349C) (residue numbering according to the EU index). [ka] Amino acid sequence of the light chain of the low-affinity anti-EGFR binding arm (RAA22) in the "EGFR-cMET™" antibody (SEQ ID NO: 61): The following substitutions are underlined: S121C and C214V (numbering according to EU index) [ka] Amino acid sequence of the light chain of the anti-cMet binding arm in the "EGFR-cMET™" antibody (SEQ ID NO: 62): [ka] Amino acid sequence of human immunoglobulin G1 CH region modified to have a "knob" mutation, a cysteine to form a stabilizing disulfide bridge, no cysteine insertion, and a TM (SEQ ID NO:63): The following substitutions are underlined: Triple mutation (TM; L234F, L235E and P331S), "knob" mutation (T366W), interchain cysteine mutations (F126C and C219V), stabilizing cysteine mutation (S354C) (residue numbering according to the EU index). [ka] Amino acid sequence of human immunoglobulin G1 CH region modified to have a "hole" mutation, a cysteine to form a stabilizing disulfide bridge, no cysteine insertion, and a TM (SEQ ID NO:64): The following substitutions are underlined: The triple mutation (TM; L234F, L235E and P331S), the "hole" mutation (T366S, L368A and Y407V) and the stabilizing cysteine mutation (Y349C) (residue numbering according to the EU index). [ka] Amino acid sequence of a human immunoglobulin lambda constant region modified to contain S121C and C214V substitutions (SEQ ID NO:65): [ka] Amino acid sequence of human EGFR extracellular domain (SEQ ID NO: 68): [ka] Amino acid sequence of cynomolgus monkey EGFR extracellular domain (SEQ ID NO: 69): [ka] Amino acid sequence of human cMet extracellular domain (SEQ ID NO:70): [ka] Amino acid sequence of cynomolgus monkey cMet extracellular domain (SEQ ID NO:71): [ka]

Claims

1. 1. An EGFR TKI for use in treating cancer in a human patient, wherein the EGFR TKI is administered in combination with an anti-EGFR / cMET antibody molecule, the anti-EGFR / cMET antibody molecule comprising an EGFR binding domain and a cMET binding domain, the EGFR binding domain comprising: a. The following complementarity determining regions (CDRs): i. HCDR1 having the amino acid sequence of SEQ ID NO: 1; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2; iii. HCDR3 having the amino acid sequence of SEQ ID NO: 3, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; a heavy chain variable (VH) region comprising: b. The following CDRs: i. LCDR1 having the amino acid sequence of SEQ ID NO: 4; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6, or variants thereof in which one, two, or three amino acids in one or more of LCDR1, LCDR2, or LCDR3 are replaced with another amino acid; a light chain variable (VL) region comprising EGFR TKIs, including

2. 1. An anti-EGFR / cMET antibody molecule for use in treating cancer in a human patient, the antibody molecule being administered in combination with an EGFR TKI and comprising an EGFR binding domain and a cMET binding domain, the EGFR binding domain comprising: a. The following complementarity determining regions (CDRs): i. HCDR1 having the amino acid sequence of SEQ ID NO: 1; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2; iii. HCDR3 having the amino acid sequence of SEQ ID NO: 3, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; a heavy chain variable (VH) region comprising: b. The following CDRs: i. LCDR1 having the amino acid sequence of SEQ ID NO: 4; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6, or variants thereof in which one, two, or three amino acids in one or more of LCDR1, LCDR2, or LCDR3 are replaced with another amino acid; a light chain variable (VL) region comprising 1. An anti-EGFR / cMET antibody molecule comprising:

3. 3. The EGFR TKI for use of claim 1 or the anti-EGFR / cMET antibody molecule for use of claim 2, wherein the administration of the EGFR TKI and the anti-EGFR / cMET antibody molecule is separate, sequential, or simultaneous.

4. The EGFR TKI has the formula (V): 【Chemical 1】 (In the formula, G is selected from 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl, indol-3-yl, indazol-1-yl, 3,4-dihydro-1H-[1,4]oxazino[4,3-a]indol-10-yl, 6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl, 5,6-dihydro-4H-pyrrolo[3,2,1-ij]quinolin-1-yl, pyrrolo[3,2-b]pyridin-3-yl and pyrazolo[1,5-a]pyridin-3-yl; R １ is selected from hydrogen, fluoro, chloro, methyl and cyano; R ２ is selected from methoxy, trifluoromethoxy, ethoxy, 2,2,2-trifluoroethoxy and methyl; R ３ are (3R)-3-(dimethylamino)pyrrolidin-1-yl, (3S)-3-(dimethyl-amino)pyrrolidin-1-yl, 3-(dimethylamino)azetidin-1-yl, [2-(dimethylamino)ethyl]-(methyl)amino, [2-(methylamino)ethyl](methyl)amino, 2-(dimethylamino)ethoxy, 2-(methylamino)ethoxy, 5-methyl-2,5-diazaspiro[3.4]oct-2-yl, (3aR,6aR)-5-methylhexa-hydro-pyrrolo[3,4- b] selected from pyrrol-1(2H)-yl, 1-methyl-1,2,3,6-tetrahydropyridin-4-yl, 4-methylpiperidin-1-yl, 4-[2-(dimethylamino)-2-oxoethyl]piperazin-1-yl, methyl[2-(4-methylpiperazin-1-yl)ethyl]amino, methyl[2-(morpholin-4-yl)ethyl]amino, 1-amino-1,2,3,6-tetrahydropyridin-4-yl and 4-[(2S)-2-aminopropanoyl]piperazin-1-yl; R ４ is selected from hydrogen, 1-piperidinomethyl and N,N-dimethylaminomethyl; R ５ is independently selected from methyl, ethyl, propyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, fluoro, chloro, and cyclopropyl; X is CH or N, and n is 0, 1 or 2.

4. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 3, which is a compound of the formula:

5. G is selected from indol-3-yl and indazol-1-yl; R １ is selected from hydrogen, fluoro, chloro, methyl and cyano; R ２ is selected from methoxy and 2,2,2-trifluoroethoxy; R ３ is selected from [2-(dimethylamino)ethyl]-(methyl)amino, [2-(methylamino)ethyl](methyl)amino, 2-(dimethylamino)ethoxy and 2-(methylamino)ethoxy; R ４ is hydrogen, and R ５ is selected from methyl, 2,2,2-trifluoroethyl and cyclopropyl, X is CH or N, and n is 0 or 1, or a pharmaceutically acceptable salt thereof.

6. 6. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to claim 5, wherein the EGFR TKI is osimertinib or a pharmaceutically acceptable salt thereof.

7. 7. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 6, wherein the EGFR TKI is selected from the group consisting of osimertinib or a pharmaceutically acceptable salt thereof, AZD3759 or a pharmaceutically acceptable salt thereof, lazertinib or a pharmaceutically acceptable salt thereof, abivertinib or a pharmaceutically acceptable salt thereof, alflutinib or a pharmaceutically acceptable salt thereof, afatinib or a pharmaceutically acceptable salt thereof, CX-101 or a pharmaceutically acceptable salt thereof, HS-10296 or a pharmaceutically acceptable salt thereof, BPI-7711 or a pharmaceutically acceptable salt thereof, dacomitinib or a pharmaceutically acceptable salt thereof, icotinib or a pharmaceutically acceptable salt thereof, gefitinib or a pharmaceutically acceptable salt thereof, and erlotinib or a pharmaceutically acceptable salt thereof.

8. 8. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 7, wherein the EGFR TKI is selected from the group consisting of osimertinib or a pharmaceutically acceptable salt thereof, AZD3759 or a pharmaceutically acceptable salt thereof, alflutinib or a pharmaceutically acceptable salt thereof, HS-10296 or a pharmaceutically acceptable salt thereof, and lazertinib or a pharmaceutically acceptable salt thereof.

9. The anti-EGFR binding domain a. The following complementarity determining regions (CDRs): i. HCDR1 having the amino acid sequence of SEQ ID NO: 1; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2; iii. HCDR3 having the amino acid sequence of SEQ ID NO: 3 a heavy chain variable (VH) region comprising: b. The following CDRs: i. LCDR1 having the amino acid sequence of SEQ ID NO: 4; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6 a light chain variable (VL) region comprising 9. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 8, comprising:

10. 10. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 9, wherein the anti-EGFR binding domain comprises a VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16, and a VL region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

20.

11. The anti-cMET binding domain comprises: a. The following complementarity determining regions (CDRs): i. HCDR1 having the amino acid sequence of SEQ ID NO: 24; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 25; iii. HCDR3 having the amino acid sequence of SEQ ID NO: 26, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; a heavy chain variable (VH) region comprising: b. The following CDRs: i. LCDR1 having the amino acid sequence of SEQ ID NO: 27; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 28; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 29, or variants thereof in which one, two, or three amino acids in one or more of LCDR1, LCDR2, or LCDR3 are replaced with another amino acid; a light chain variable (VL) region comprising 11. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 10, comprising:

12. The anti-cMET binding domain comprises: a. The following complementarity determining regions (CDRs): i. HCDR1 having the amino acid sequence of SEQ ID NO: 24; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 25; iii. HCDR3 having the amino acid sequence of SEQ ID NO: 26 a heavy chain variable (VH) region comprising: b. The following CDRs: i. LCDR1 having the amino acid sequence of SEQ ID NO: 27; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 28; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 29 a light chain variable (VL) region comprising 12. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 11, comprising:

13. 13. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 12, wherein the cMET binding domain comprises a VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 38, and a VL region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

40.

14. The antibody molecule a. a first heavy chain comprising the VH region of the anti-EGFR binding domain and a first heavy chain constant (CH) region or a fragment thereof; b. a first light chain comprising the VL region of the anti-EGFR binding domain and a first light chain constant (CL) region or a fragment thereof; c. a second heavy chain comprising the VH region of the anti-cMET binding domain and a second heavy chain constant (CH) region or fragment thereof; and d. a second light chain comprising the VL region of the anti-cMET binding domain and a second light chain constant (CH) region or fragment thereof.

14. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 13, comprising:

15. 15. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use of claim 14, wherein the first and second CH regions comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

42.

16. 16. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to claim 14 or 15, wherein the first and / or second CH region comprises a mutation that reduces or abolishes binding of the antibody molecule to one or more Fcγ receptors.

17. 17. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to claim 16, wherein the first and / or second CH region comprises phenylalanine at position 234, glutamic acid at position 235, and serine at position 331, and the numbering of the constant regions is according to the EU index.

18. the first CH region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:63, and the second CH region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:64; and the first CL region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:

64.

18. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 14 to 17, wherein the first CL region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:

47.

19. 19. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 14 to 18, wherein the first heavy chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 59, the second heavy chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 60, the first light chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO: 61, and the second light chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO:

62.

20. the anti-EGFR binding domain binds to human EGFR with an affinity having a Kd of 10 nM or greater, 30 nM or greater, 40 nM or greater; and / or 20. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 19, wherein the anti-EGFR binding domain binds to human EGFR with an affinity having a Kd of 10-100 nM, 20-80 nM, 30-75 nM or 35-50 nM.

21. 21. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 20, wherein the antibody molecule is conjugated to a drug.

22. 22. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use of claim 21 , wherein the drug comprises a cytotoxin, a radioisotope, an immunomodulatory agent, a cytokine, a lymphokine, a chemokine, a growth factor, a tumor necrosis factor, a hormone, a hormone antagonist, an enzyme, an oligonucleotide, DNA, RNA, siRNA, RNAi, microRNA, a photoactive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide, a lipid, a carbohydrate, a chelator, or a combination thereof.

23. The drug has the formula A* 【Chemistry 2】 23. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to claim 22, which is a topoisomerase I inhibitor having the formula:

24. The topoisomerase I inhibitor has the formula 【Chemistry 3】 (In the formula, R Ｌ is a linker for linking to said antibody molecule, optionally said linker being (ia): 【Chemistry 4】 (In the formula, Q is, 【Chemistry 5】 (In the formula, Q Ｘ is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue. and X is 【Chemistry 6】 (wherein a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, and at least b1 or b2=0 (i.e., only one of b1 and b2 may not be 0), and at least c1 or c2=0 (i.e., only one of c1 and c2 may not be 0)) and G Ｌ is a linker for linking to the antibody molecule), or (ib): 【Chemistry 7】 (In the formula, R Ｌ１ and R Ｌ２ are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; and e is 0 or 1. (selected from or a salt or solvate thereof.

25. The antibody has the following formula: 【Chemistry 8】 22. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to claim 21 , conjugated to a topoisomerase I inhibitor having the formula:

26. 26. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 25, wherein the cancer is non-small cell lung cancer.

27. 27. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to claim 26, wherein the non-small cell lung cancer is EGFR mutation-positive non-small cell lung cancer.

28. 28. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to claim 27, wherein the EGFR mutation-positive non-small cell lung cancer comprises one or more deletions in exon 19 of the EGFR gene and / or an L858R mutation.

29. 29. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to claim 27 or 28, wherein the EGFR mutation-positive non-small cell lung cancer comprises a T790M mutation, a mutation in exon 20 of the EGFR gene, or both.

30. 30. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 29, wherein the human patient is an EGFR TKI naive human patient.

31. 32. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use according to any one of claims 1 to 31, wherein the human patient's disease has progressed during or after previous EGFR TKI treatment.

32. 34. The EGFR TKI for use or anti-EGFR / cMET antibody molecule for use of claim 33, wherein the EGFR TKI is osimertinib or a pharmaceutically acceptable salt thereof, and the human patient's disease has progressed during or after previous treatment with a different EGFR TKI.

33. 1. Use of an EGFR TKI in the manufacture of a medicament for treating cancer in a human patient, wherein the EGFR TKI is administered in combination with an anti-EGFR / cMET antibody molecule, the anti-EGFR / cMET antibody molecule comprising an EGFR binding domain and a cMET binding domain, the EGFR binding domain comprising: a. The following complementarity determining regions (CDRs): i. HCDR1 having the amino acid sequence of SEQ ID NO: 1; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2; iii. HCDR3 having the amino acid sequence of SEQ ID NO: 3, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; a heavy chain variable (VH) region comprising: b. The following CDRs: i. LCDR1 having the amino acid sequence of SEQ ID NO: 4; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6, or variants thereof in which one, two, or three amino acids in one or more of LCDR1, LCDR2, or LCDR3 are replaced with another amino acid; a light chain variable (VL) region comprising Including, use.

34. 1. A method of treating cancer in a human patient in need thereof, comprising administering to the human patient a therapeutically effective amount of an EGFR TKI, wherein the EGFR TKI is administered in combination with a therapeutically effective amount of an anti-EGFR / cMET antibody molecule, the anti-EGFR / cMET antibody molecule comprising an EGFR binding domain and a cMET binding domain, the EGFR binding domain comprising: a. The following complementarity determining regions (CDRs): i. HCDR1 having the amino acid sequence of SEQ ID NO: 1; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2; iii. HCDR3 having the amino acid sequence of SEQ ID NO: 3, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; a heavy chain variable (VH) region comprising: b. The following CDRs: i. LCDR1 having the amino acid sequence of SEQ ID NO: 4; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6, or variants thereof in which one, two, or three amino acids in one or more of LCDR1, LCDR2, or LCDR3 are replaced with another amino acid; a light chain variable (VL) region comprising A method comprising:

35. 1. A method of treating cancer in a human patient in need thereof, comprising administering to the human patient a first amount of an EGFR TKI and a second amount of an anti-EGFR / cMET antibody molecule, wherein the first amount and the second amount together comprise a therapeutically effective amount, and the anti-EGFR / cMET antibody molecule comprises an EGFR binding domain and a cMET binding domain, and the EGFR binding domain comprises: a. The following complementarity determining regions (CDRs): i. HCDR1 having the amino acid sequence of SEQ ID NO: 1; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2; iii. HCDR3 having the amino acid sequence of SEQ ID NO: 3, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; a heavy chain variable (VH) region comprising: b. The following CDRs: i. LCDR1 having the amino acid sequence of SEQ ID NO: 4; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6, or variants thereof in which one, two, or three amino acids in one or more of LCDR1, LCDR2, or LCDR3 are replaced with another amino acid; a light chain variable (VL) region comprising A method comprising:

36. A pharmaceutical combination of an EGFR / cMET antibody molecule and an EGFR TKI, wherein the anti-EGFR / cMET antibody molecule comprises an EGFR binding domain and a cMET binding domain, and the EGFR binding domain comprises: a. The following complementarity determining regions (CDRs): i. HCDR1 having the amino acid sequence of SEQ ID NO: 1; ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2; iii. HCDR3 having the amino acid sequence of SEQ ID NO: 3, or variants thereof in which one, two, or three amino acids in one or more of HCDR1, HCDR2, or HCDR3 are replaced with another amino acid; a heavy chain variable (VH) region comprising: b. The following CDRs: i. LCDR1 having the amino acid sequence of SEQ ID NO: 4; ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5; iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6, or variants thereof in which one, two, or three amino acids in one or more of LCDR1, LCDR2, or LCDR3 are replaced with another amino acid; a light chain variable (VL) region comprising 10. A pharmaceutical combination comprising:

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