Combinations relating to epidermal growth factor receptor tyrosine kinase inhibitors for treatment of cancer

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 367,068, filed on June 27, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0003] Reference to sequence listings submitted electronically

[0004] This application incorporates by reference a sequence listing in computer readable form (CRF) filed as a text file with this application, which sequence listing is named "EGFCM-400-WO-PCT", created on June 05, 2023, and is 75,711 bytes in size. Technical Field

[0005] The present disclosure relates to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) for use in treating cancer (eg, non-small cell lung cancer [NSCLC]), wherein the EGFR TKI is used in combination with an anti-EGFR / cMET antibody molecule. Background Art

[0006] There are a variety of drugs for epidermal growth factor receptor (EGFR) that have been approved or are in clinical development. For example, there are currently two first-generation (erlotinib and gefitinib), two second-generation (afatinib and dacomitinib) and one third-generation (osimertinib) tyrosine kinase inhibitors (TKIs) available for the management of EGFR mutation-positive non-small cell lung cancer (NSCLC). All of these TKIs are effective in NSCLC patients whose tumors carry in-frame deletions in exon 19 of EGFR and L858R point mutations in exon 21. 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 or absence of T790M mutations). However, even patients treated with osimertinib eventually progress, primarily due to the development of acquired resistance due to other resistance mechanisms. Therefore, there remains a need to develop new therapies for the treatment of cancer, especially for patients who have experienced disease progression after treatment with third-generation EGFR TKIs.

[0007] cMET (gene product of the proto-oncogene MET) is a receptor tyrosine kinase expressed primarily on the surface of epithelial cells. Aberrant expression and dysregulation of the cMET 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 (Organ, 2011; Birchmeier, 2003; Mo, 2017; Sierra, 2011). There is a large and growing body of literature demonstrating that crosstalk and direct interactions exist between the EGFR and cMET signaling pathways, and that this crosstalk functionally translates into clinical resistance to EGFR and cMET targeted therapies (McDermott, 2010; Moores, 2016; Suda, 2010).

[0008] Antibody drug conjugates (ADCs) have been studied as a means to overcome the limitations associated with treating cancers expressing cMET and EGFR. An EGFR-guided ADC (depatuxizumab mafodotin (ABT-414)) is in Phase III clinical development for glioblastoma by AbbVie (Phillips, 2016). ABT-414 was previously tested in Phase II trials for a variety of other solid tumor indications (ClinicalTrials.gov: NCT01741727). ADCs have shown limited efficacy at tolerated doses in these indications, and worrisome ocular toxicity is often observed in treated patients (Tolcher, 2014). The second generation EGFR ADC (ABBV-221) is in clinical development, but was interrupted due to safety issues (Phillips, 2018; Calvo, 2017). There is a cMET-targeted ADC (Telisotuzumab Vedotin, ABBV-399) that has entered Phase II clinical development in patients with non-small cell lung cancer (NSCLC) whose tumors express high levels of cMET as a monotherapy and in combination with the EGFR inhibitor erlotinib (Angevin, 2017; Wang, 2017). In Phase I trials, the cMET ADC + EGFR TKI combination showed clinical activity in this selected patient population, with peripheral neuropathy and rash being the most common treatment-related adverse events (Angevin, 2017; Calvo, 2017).

[0009] Bispecific antibodies targeting EGFR and cMET have also been developed and are being studied clinically as monotherapy and in combination with third-generation EGFR TKIs in patients with advanced NSCLC (ClinicalTrials.gov: NCT02609776).

[0010] The nature of bispecific antibodies allows fine-tuning of the interaction between each target to influence the overall properties of the molecule, which can result in ADCs with acceptable therapeutic windows (Comer, 2018). This concept has been tested in vitro against EGFR and cMET, but researchers have yet to demonstrate in vivo proof of concept with improved safety or efficacy compared to the above EGFR and cMET ADCs (Sellmann, 2016).

[0011] Therefore, new therapeutic strategies for targeting cancers expressing EGFR and cMET that show efficacy as well as an acceptable safety profile are needed. Summary of the invention

[0012] The inventors recognized that developing an anti-EGFR / cMET antibody molecule under development that binds to EGFR with low affinity (e.g., binds to human EGFR with a dissociation constant (Kd) equal to or greater than 10 nM) could be used in combination with known EGFR TKIs as an effective treatment for cancer (e.g., NSCLC).

[0013] As demonstrated herein, anti-EGFR / cMET antibody molecules comprising such a low affinity EGFR binding domain conjugated to a drug (antibody drug conjugate ("ADC")) exhibit reduced on-target toxicity, such as skin toxicity, in normal tissues, compared to conjugates comprising an EGFR antigen binding domain that binds to human EGFR with higher affinity, and are thus demonstrated to exhibit an improved safety profile.

[0014] In addition, ADCs containing this low-affinity EGFR binding domain were shown to be effective in treating a range of EGFR mutant cancer models in combination with the third-generation TKI osimertinib, including cancer models that have become resistant to osimertinib. Therefore, it is believed that the combination of the antibody molecules disclosed herein and EGFR TKIs can provide safe and effective therapies for EGFR-related cancers, such as in patients who have become resistant to EGFR TKIs.

[0015] 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, wherein the EGFR binding domain comprises:

[0016] a. A heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs):

[0017] i. HCDR1 having the amino acid sequence of SEQ ID NO: 1

[0018] ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2

[0019] iii. a HCDR3 having the amino acid sequence of SEQ ID NO: 3,

[0020] or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid; and

[0021] b. a light chain variable (VL) region comprising the following CDRs:

[0022] i. LCDR1 having the amino acid sequence of SEQ ID NO: 4

[0023] ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5

[0024] iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6,

[0025] or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid.

[0026] In another aspect, provided herein is an anti-EGFR / cMET antibody molecule for use in treating cancer in a human patient, wherein the anti-EGFR / cMET antibody molecule is administered in combination with an EGFR TKI, wherein the anti-EGFR / cMET antibody molecule comprises an EGFR binding domain and a cMET binding domain, wherein the EGFR binding domain comprises:

[0027] a. A heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs):

[0028] i. HCDR1 having the amino acid sequence of SEQ ID NO: 1

[0029] ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2

[0030] iii. a HCDR3 having the amino acid sequence of SEQ ID NO: 3,

[0031] or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid; and

[0032] b. a light chain variable (VL) region comprising the following CDRs:

[0033] i. LCDR1 having the amino acid sequence of SEQ ID NO: 4

[0034] ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5

[0035] iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6,

[0036] or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid.

[0037] In another aspect, provided herein is a method of treating cancer in a human patient, the method comprising administering an anti-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, wherein the EGFR binding domain comprises:

[0038] a. A heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs):

[0039] i. HCDR1 having the amino acid sequence of SEQ ID NO: 1

[0040] ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2

[0041] iii. a HCDR3 having the amino acid sequence of SEQ ID NO: 3,

[0042] or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid; and

[0043] b. a light chain variable (VL) region comprising the following CDRs:

[0044] i. LCDR1 having the amino acid sequence of SEQ ID NO: 4

[0045] ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5

[0046] iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6,

[0047] or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid.

[0048] 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, wherein the EGFR binding domain comprises:

[0049] a. A heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs):

[0050] i. HCDR1 having the amino acid sequence of SEQ ID NO: 1

[0051] ii. HCDR2 having the amino acid sequence of SEQ ID NO: 2

[0052] iii. a HCDR3 having the amino acid sequence of SEQ ID NO: 3,

[0053] or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid; and

[0054] b. a light chain variable (VL) region comprising the following CDRs:

[0055] i. LCDR1 having the amino acid sequence of SEQ ID NO: 4

[0056] ii. LCDR2 having the amino acid sequence of SEQ ID NO: 5

[0057] iii. LCDR3 having the amino acid sequence of SEQ ID NO: 6,

[0058] or a variant thereof, wherein one or two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are substituted with another amino acid.

[0059] In some examples, the EGFR TKI and the anti-EGFR / cMET antibody molecule are administered separately, sequentially or simultaneously.

[0060] Examples of suitable EGFR TKIs for use in the claimed combination therapy are described. In some examples, the EGFR TKI is osimertinib or a pharmaceutically acceptable salt thereof.

[0061] In some examples, the anti-EGFR binding domain comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 1, a HCDR2 having an amino acid sequence of SEQ ID NO: 2, a HCDR3 having an amino acid sequence of SEQ ID NO: 3, a LCDR1 having an amino acid sequence of SEQ ID NO: 4, a LCDR2 having an amino acid sequence of SEQ ID NO: 5, and a LCDR3 having an 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 with 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 with the amino acid sequence of SEQ ID NO: 20.

[0062] In some examples, the anti-cMET binding domain comprises:

[0063] a. A heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs):

[0064] i. HCDR1 having the amino acid sequence of SEQ ID NO: 24

[0065] ii. HCDR2 having the amino acid sequence of SEQ ID NO: 25

[0066] iii. a HCDR3 having the amino acid sequence of SEQ ID NO: 26,

[0067] or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid; and

[0068] b. a light chain variable (VL) region comprising the following CDRs:

[0069] i. LCDR1 having the amino acid sequence of SEQ ID NO: 27

[0070] ii. LCDR2 having the amino acid sequence of SEQ ID NO: 28

[0071] iii. LCDR3 having the amino acid sequence of SEQ ID NO: 29,

[0072] or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid.

[0073] In some examples, the anti-cMET binding domain comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 24, a HCDR2 having an amino acid sequence of SEQ ID NO: 25, a HCDR3 having an amino acid sequence of SEQ ID NO: 26, a LCDR1 having an amino acid sequence of SEQ ID NO: 27, a LCDR2 having an amino acid sequence of SEQ ID NO: 28, and a LCDR3 having an 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 an 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 an amino acid sequence of SEQ ID NO: 40.

[0074] In some instances, the antibody molecule is conjugated to a drug. The drug may include 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, a DNA, an RNA, a siRNA, an RNAi, a microRNA, a photoactive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide, a lipid, a carbohydrate, a chelating agent, or a combination thereof. In some instances, the drug is a topoisomerase I inhibitor, as further described herein.

[0075] In some instances, cancer is non-small cell lung cancer (NSCLC). In some instances, non-small cell lung cancer is EGFR mutation-positive NSCLC. EGFR mutation-positive NSCLC includes those comprising activating mutations (e.g., L858R mutations in the EGFR gene and / or one or more deletions in exon 19), and NSCLCs with mutations associated with EGFR TKI resistance (e.g., insertions in exon 20 in the EGFR gene). As demonstrated herein, the combination of EGFR-cMET conjugates and osimertinib shows efficacy in a range of EGFR mutant cancers (including those classified as osimertinib resistance). BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Examples and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying drawings, in which:

[0077] Figure 1A Graphical depiction of RAA22 / B09-57 DuetMab. Shown are the anti-EGFR RAA22 Fab, the anti-cMETB09-57 Fab, and the Hole and Knob heavy chains. The structural drawings are composites of the individual domain structures.

[0078] Figure 1B . Graphical depiction of QD6 / B09-57DuetMab. Shown are the anti-EGFRQD6 Fab, the anti-cMET B09-57 Fab, and the Hole and Knob heavy chains. The structural drawing is a synthesis of the single domain structures.

[0079] Figure 2 .Simultaneous binding studies using antigen capture format were performed by Octet analysis. Sensors loaded with human cMET antigen were subjected to sequential association and dissociation interactions first with antibodies and then with human EGFR antigen. Ass = association; Diss = dissociation; NI-NTA = nickel-nitrilotriacetic acid.

[0080] Figure 3A ELISA results show EGFR and cMet species cross-reactivity. High affinity monospecific EGFRIgG QD6 and monovalent bispecific EGFR / c-Met DuetMAb QD6 / B09 bind to human, cynomolgus monkey and mouse EGFR. The reduced affinity monospecific EGFR IgG RAA22 binds weaker to human, cynomolgus monkey and mouse EGFR than QD6 and the corresponding monovalent bispecific EGFR / c-Met DuetMAb RAA22 / B09, showing still weaker binding to human and cynomolgus monkey EGFR relative to the bivalent parental IgG RAA22, and showing nominal binding to mouse EGFR. Monospecific c-Met IgG B09 and all bispecific variants show comparable binding to human and cynomolgus monkey c-Met, but no detectable binding to mouse c-Met.

[0081] Figure 3B The ELISA results showed EGFR and cMet family specificity. None of the antibodies tested showed any appreciable binding to any EGFR HER family protein (HER2, HER3 or HER4) or any c-Met family member (Ron (CD136) or Semaphorin3a).

[0082] Figure 4A Internalization curves of QD6 / B09 DuetMab and their respective single control antibodies. Internalization curves are shown by the time course of membrane and cytoplasmic signals for each construct. The QD6 / B09 group was acquired using an Opera confocal fluorescence microscope.

[0083] Figure 4BInternalization curves of RAA22 / B09DuetMab and their respective single control antibodies. The internalization curves are shown by the time course of membrane and cytoplasmic signals for each construct. This panel was acquired using a Zeiss spinning disk confocal fluorescence microscope.

[0084] The same graphs for QD6 / B09 and QD6 / IgG indicate an EGFR-group-driven internalization pattern for QD6 / B09 DuetMab, whereas RAA22 / B09 DuetMab requires engagement of both EGFR and c-MET groups for efficient internalization.

[0085] Figure 5A Internalization curves of RAA22 / B09-AZD1508 ADC in cells expressing medium and high target c-MET and EGFR cell surface receptors. Shown are membrane, cytoplasmic and total signals of RAA22 / B09-AF647 ADC in H1975 cells. One representative experiment of 2 is shown. H1975 cells show a simultaneous decrease in total intensity and cell membrane intensity, indicating dissociation of the antibody from the cell surface.

[0086] Figure 5B Equivalent to Figure 8 A, but not in HCC827 cells. HCC827 cells have a stable total signal over the entire experimental time course. The decrease in membrane signal is derived from antibody internalization.

[0087] Fig. 6A Internalization of RAA22 / B09-AZD1508 ADC single-arm control antibody in HCC827 cells. Due to the weaker binding to EGFR by single-arm binding, the intensity curve of RAA22 / IgG single-arm is about 10-fold lower than that of RAA22 / B09.

[0088] Figure 6B The B09 / IgG group dissociated from the cell membrane, which was manifested by the decrease of total signal and membrane signal over time.

[0089] Figure 7.Analyze the relative contribution of each antibody group to the cytotoxic activity of the bispecific ADC. NCI H1975 cells were pretreated with an excess of ungrouped parental antibodies to block EGFR or cMET. Next, EGFR-cMET ADC (RAA22 / B09-AZ1508) was added to the cells in a 4X serial dilution series with a final concentration of 67nM to 0.0009nM. The treated cells were cultured in a humidified incubator at 37°C and 5% CO2 for 72 hours. Metabolic activity was determined using the CellTiter-Glo luminescent activity assay (Promega). The data were plotted as a percentage of metabolic activity relative to the untreated control. Using GraphPad Prism software, the IC50 value was determined using a logistic nonlinear regression analysis between maximum activity (untreated cells) and maximum response (peak inhibition).

[0090] Figure 8 .The activity of the individual antibody groups on the bispecific ADC was further evaluated. Monospecific monovalent ADCs were constructed by pairing each binding group with a non-binding isotype control group (R347) to generate EGFR ADC (RAA22 / R347-AZ1508) and anti-cMET ADC (B09 / R347-AZ1508). The ADCs were added to NCIH1975 cells in a 4X serial dilution series with a final concentration of 67nM to 0.0009nM. Figure 7 The percentage of metabolic activity is determined.

[0091] Fig. 9A . Mouse PDX assays were performed to determine the efficacy of high affinity (QD6 / B09-AZ1508) and low affinity (RAA22 / B09-AZ1508) EGFR-cMET ADCs in a large number of human cancer patient-derived xenograft (PDX) models in immunodeficient mice. For each PDX model representing a different human tumor, each compound was tested at a single dose level of 3 mg / kg in a single mouse. The percentage of tumor growth (%T / C) was calculated for tumors that grew larger than the initial volume relative to untreated control tumors, and the percentage of tumor regression was calculated for tumors that showed a reduction in size compared to the initial tumor volume. (A) shows a direct comparison of the high affinity and low affinity ADCs in each model, and

[0092] Fig. 9B A waterfall plot of high affinity ADCs ranked by efficacy is shown.

[0093] Fig. 9C A waterfall plot of low affinity ADCs ranked by efficacy is shown.

[0094] Fig.10.Dose range discovery in PDX models In vivo efficacy studies were conducted in athymic nude mice unilaterally implanted with tumor fragments collected from host animals. As shown in the figure, the high affinity EGFR-cMET ADC, QD6 / B09-AZ1508, was tested at dose levels of 1 and 2 mg / kg, and the variant RAA22 / B09-AZ1508 with lower affinity for EGFR was tested at 1, 2 and 3 mg / kg. Tumor volume measurements were taken twice a week after the start of dosing and plotted as a line graph of tumor volume over time. The error bars represent the standard error of the mean (SEM), and the inset shows immunohistochemical staining of EGFR and cMET for each model, which is from tumor tissue taken from an early passage of the model.

[0095] Fig.11A In vivo efficacy of the EGFR-cMET bispecific ADC in subcutaneous and orthotopic pancreatic PDX models. A) In vivo efficacy of the high-affinity QD6 / B09 ADC in the subcutaneous MEDI-PANC-08 PDX model, ●-untreated, ■-R347-AZ1508 (3 mg / kg–Q1Wx4), ▲-QD6 / B09-1508 (1 mg / kg–Q1Wx4), ▼-QD6 / B09-1508 (2 mg / kg–Q1Wx4), and - In vivo efficacy in QD6 / B09-1508 (3 mg / kg - Q1Wx4). Treatment days are indicated by the scale on the x-axis and tumor volume was measured twice a week.

[0096] Fig. 11B In vivo efficacy of low-affinity RAA2 / B09 ADC in the subcutaneous MEDI-PANC-08 PDX model. ●-untreated, ■-R347-AZ1508 (3 mg / kg–Q1Wx4), ▲-RAA2 / B09-1508 (1 mg / kg–Q1Wx4), ▼-RAA2 / B09-1508 (2 mg / kg–Q1Wx4) and -RAA2 / B09-1508 (3 mg / kg – Q1Wx4). Treatment days are indicated by the scale on the x-axis, and tumor volume was measured twice a week.

[0097] Fig. 11C In-situ MEDI-PANC-08 LUC Luciferase imaging of (luciferase expression) PDX models. Mice were imaged weekly using the IVIS Spectrum in vivo imaging system. Images for all groups and time points were normalized using a radiance scale (mean radiance [p / s / cm2 / sr]) set between maximum signal (control Gp at day 21) and background.

[0098] Fig.11DIn vivo efficacy of low-affinity RAA2 / B09 ADC in the subcutaneous MEDI-PANC-08 PDX model. ●-untreated, ■-gemcitabine (75 mg / kg–Q3 / 4Dx5), ▲R347-AZ1508 (3 mg / kg–Q1Wx4), ▼-RAA2 / B09-1508 (2 mg / kg–Q1Wx4) and -RAA2 / B09-1508 (3 mg / kg–Q1Wx4). Treatment days are indicated by arrows, and tumor volumes were measured twice a week. Fig.11A and Fig. 11B The data shown are the mean tumor volume (mm 3 ) ± SEM, in Fig.11D where is the group average radiance [p / s / cm 2 / sr]±SEM.

[0099] Fig. 12A Mean concentration-time profiles and mean NCA PK parameters of RAA22 / B09-57-AZ1508 in mice. Target compound concentrations and total antibody concentrations were measured using an immunocapture LC-MS / MS assay.

[0100] Fig. 12B Mean concentration-time profiles of QD6 / B09-57-AZ1508 and mean NCAPK parameters in mice. Target compound concentrations and total antibody concentrations were measured using an immunocapture LC-MS / MS assay.

[0101] Fig.13A Mean concentration-time profiles and mean NCA PK parameters of RAA22 / B09-57-AZ1508 in monkeys.

[0102] Fig. 13B Mean concentration-time profiles and mean NCA PK parameters of QD6 / B09-57-AZ1508 in monkeys.

[0103] The PK profile and NCA PK parameters of 3 mg / kg QD6 / B09-57-AZ1508 in 20067312 were based on the PK data after the second dose.

[0104] Fig. 13C is a table summarizing the PK parameters of a molecule

[0105] Fig.14As a PDX test, the EGFR-cMET Maia Topoi ADC was evaluated in a patient-derived xenograft (PDX) model representing a variety of types of human cancers in immunodeficient mice. For each PDX model representing a unique human tumor, the compound was tested at a dose level of 10 mg / kg in a single mouse. The percentage of tumor growth (%T / C) of tumors that grew larger than the initial volume relative to untreated control tumors was calculated (according to the following formula, tumor growth inhibition (%TGI) was defined as the percentage of tumor growth relative to day 0 between the treatment (TX) group and the control (C) group: %TGI=1-(TX final-TX initial) / (C final-C initial)). And the percentage of tumor regression of tumors showing a reduction in size compared to the initial tumor volume was calculated (according to the following formula, the percentage of tumor regression is defined as the percentage of tumor reduction in the tumor in the treated animal calculated according to the following formula at the end of the study relative to the tumor volume on day 0 (initial dose day): % regression=(TX final average-TX initial average) / (TX initial average)×100).

[0106] Fig.15 As a PDX test, EGFR-cMETTopoi TMADC was evaluated in patient-derived xenograft (PDX) models representing various types of human cancers in immunodeficient mice. For each PDX model representing a unique human tumor, the compound was tested at a dose level of 5 mg / kg in a single mouse. The percentage of tumor growth (%T / C) of tumors that grew larger than the initial volume relative to untreated control tumors was calculated (according to the following formula, tumor growth inhibition (%TGI) was defined as the percentage of tumor growth relative to day 0 between the treatment (TX) group and the control (C) group: %TGI=1-(TX final-TX initial) / (C final-C initial)). And the percentage of tumor regression of tumors showing a reduction in size compared to the initial tumor volume was calculated (according to the following formula, the percentage of tumor regression is defined as the percentage of tumor reduction in the tumor in the treated animal calculated according to the following formula at the end of the study relative to the tumor volume on day 0 (initial dose day): % regression=(TX final average-TX initial average) / (TX initial average)×100).

[0107] Fig.16A The comparability of two different EGFR-cMET ADCs with different IgG Fc formats (Maia and TM) was evaluated in the PDX model SQHN-02. The ADCs were tested at 3 dose levels: 2.5, 5, and 10 mg / kg, and tumor growth was compared to untreated control animals. A total of 10 animals were treated in each treatment group and control group.

[0108] Fig. 16BThe comparability of two different EGFR-cMET ADCs with different IgG Fc formats (Maia and TM) was evaluated in the PDX model Panc-08. The ADCs were tested at 3 dose levels: 2.5, 5, and 10 mg / kg, and tumor growth was compared to untreated control animals. A total of 10 animals were treated in each treatment group and control group.

[0109] Fig.17 Describes the above Fig.14 Results for the NSCLC PDX model of non-small cell lung cancer highlight known EGFR mutation status and histology.

[0110] Fig.18 The pharmacokinetic profiles of the EGFR-cMET bispecific antibodies INT-009 (RAA22 / B09-Maia naked mAb) and INT-009-SG3932DAR8 ADC ("MAIA ADC") were compared with those of B09 / RAA2-IgG1-TM mirror mAb (INT-017) and TM-mirror-SG3932DAR6 ADC ("TM ADC") in NOD-SCID mice at a therapeutic dose of 5 mg / kg.

[0111] Fig.19A Depicted are results of the efficacy of the ADC in an EGFR mutant PDX model 'LUN487' containing the L858R EGFR mutation.

[0112] Fig.19B Results are described for the efficacy of the ADC in combination with the third generation TKI osimertinib ('Osi') in an EGFR mutant PDX model 'LUN487' containing the L858R EGFR mutation.

[0113] Fig.19C Described are results of the efficacy of the ADC in the EGFR mutant PDX model 'LUN439' containing the L858R EGFR mutation.

[0114] Fig.19D Results are described for the efficacy of the ADC in combination with the third generation TKI osimertinib ('Osi') in an EGFR mutant PDX model 'LUN439' containing the L858R EGFR mutation.

[0115] Fig. 20A Results are described for the efficacy of the ADC in an EGFR mutant PDX model 'CTG-2992' (primarily osimertinib-resistant) containing an EGFR exon 20 insertion.

[0116] Fig. 20BResults are described for the efficacy of the ADC in combination with the third-generation TKI osimertinib in an EGFR mutant PDX model 'CTG-2992' containing an EGFR exon 20 insertion (primary osimertinib resistance).

[0117] Fig.21A Results are described for the efficacy of the ADC in the EGFR mutant PDX model 'CTG-2803' (acquired osimertinib resistance).

[0118] Fig. 21B Results are described for the efficacy of the ADC in combination with the third-generation TKI osimertinib in the EGFR mutant PDX model 'CTG-2803' (acquired osimertinib resistance).

[0119] Fig.22A A waterfall plot showing the response of multiple EGFRmut NSCLC patient-derived xenograft models to treatment with 25 mg / kg osimertinib is shown. The x-axis depicts the best response from baseline during the study. The y-axis intercept line shows 30% regression from baseline, which defines a response.

[0120] Fig. 22B A waterfall plot showing the response of multiple EGFRmut NSCLC patient-derived xenograft models to treatment with 2 mg / kg EGFR-cMET TMADC. The x-axis depicts the best response from baseline during the study. The y-axis intercept line shows 30% regression from baseline, which defines a response.

[0121] Fig. 22C Shown is a waterfall plot of the response of multiple EGFRmut NSCLC patient-derived xenograft models to combination treatment with 25 mg / kg osimertinib and 2 mg / kg EGFR-cMET™ ADC. The x-axis depicts the best response from baseline during the study. The y-axis intercept line shows 30% regression from baseline, which defines a response. DETAILED DESCRIPTION

[0122] Various aspects and examples of the present disclosure will now be discussed with reference to the accompanying drawings. Other aspects and disclosures will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0123] Target

[0124] EGFR

[0125] 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) produces four isoforms: isoform 1 (UniProt: P00533-1, v2 (last sequence update: November 1, 1997)); isoform 2 (UniProt: P00533-2, v1), which contains the substitutions F404L and L405S relative to isoform 1, and which lacks position 406 corresponding to isoform 1; to 1210 amino acid sequence; isoform 3 (UniProt: P00533-3, v1), which comprises substitutions at positions 628 to 705 of isoform 1, and which lacks the amino acid sequence corresponding to positions 706 to 1210 of isoform 1; and isoform 4 (UniProt: P00533-4), which comprises the substitution C628S relative to isoform 1, and which lacks the amino acid sequence corresponding to positions 629 to 1210 of isoform 1.

[0126] The structure and function of EGFR are reviewed in, for example, Ferguson, Annu Rev Biophys. [Annual Review of Biophysics] (2008) 37: 353-373. EGFR is a transmembrane protein and a receptor for members of the epidermal growth factor family (EGF family). The receptor comprises 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.

[0127] Aberrant EGFR expression / activity has been implicated in numerous diseases, including neurological disorders and many cancers.

[0128] In the present specification, "EGFR" refers to EGFR from any species and includes EGFR isoforms, fragments, variants or homologs from any species.

[0129] cMET

[0130] Human cMET (also known as c-Met, 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 produces three isoforms: isoform 1 (UniProt: P08581-1, v4 (last sequence update: July 7, 2009)); isoform 2 (UniProt: P08581-2), in which the amino acid sequence "STWWKEPLNIVSFLFCFAS" is inserted at position 755 of isoform 1; and isoform 3 (UniProt: P08581-3) also known as soluble met variant 4, in which the amino acid sequence corresponding to positions 755 to 764 of isoform 1 is replaced by "RHVNIALIQR", and it further lacks the amino acid sequence corresponding to positions 765 to 1390 of isoform 1.

[0131] The structure of cMET is reviewed in, e.g., Gherardi, 2003, which is incorporated herein by reference in its entirety. cMET is a heterodimer consisting of an α chain (50 kDa) and a β chain (145 kDa) linked by a disulfide bond. cMET comprises an N-terminal Sema domain and an intracellular kinase domain that mediates binding to hepatocyte growth factor (HGF). Ligand binding at the cell surface induces autophosphorylation of cMET on its intracellular domain, which provides docking sites for downstream signaling molecules and activates several signaling cascades.

[0132] cMET is expressed in normal tissues on the surface of epithelial cells. Overexpression of cMET has been observed in many human tumors and cancers, which is usually associated with a metastatic phenotype and a 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 esophageal-gastric cancer. In these cancers, co-expression of EGFR and cMET is often observed.

[0133] Antibody molecules

[0134] The present disclosure provides antibody molecules.Antibody molecules according to the present disclosure may be provided in an isolated form, in the sense of being free of contaminants, such as antibodies capable of binding to other polypeptides and / or serum components.

[0135] The term "antibody molecule" describes an immunoglobulin, whether natural or partially or wholly synthetically produced. Antibody molecules may be human or humanized. Antibody molecules may be monoclonal antibody molecules. Examples of antibodies are immunoglobulin isotypes, such as immunoglobulin G (IgG), and their isotype subclasses, such as IgG1, IgG2, IgG3, and IgG4, and fragments thereof.

[0136] As used herein, the term "antibody molecule" therefore includes antibody fragments as long as they show binding to the relevant target molecule. Examples of antibody fragments include Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabodies, triabodies, scFv-Fc, miniantibodies and single domain antibodies (e.g., VhH), etc.). Unless the context requires otherwise, as used herein, the term "antibody molecule" is therefore equivalent to "antibody molecule or its fragment".

[0137] Antibody molecules and methods of construction and use thereof are well known in the art and are described, for example, in Holliger & Hudson, Nature Biotechnology 23(9): 1126-1136 (2005). Monoclonal and other antibody molecules can be used and recombinant DNA techniques can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques can involve introducing the CDR or variable region of one antibody molecule into a different antibody molecule (EP-A-184187, GB 2188638A and EP-A-239400).

[0138] In view of the current technology related to monoclonal antibody technology, antibody molecules can be prepared for most antigens. The antigen binding domain can be a part of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single-chain Fv fragment (ScFv)). Suitable monoclonal antibodies for selected antigens can be prepared by known techniques, such as those disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Applications", J GR Hurrell (CRC Press, 1982). Neuberger, 1988 discussed chimeric antibodies.

[0139] Antibody molecules according to the present disclosure include 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 all or part of a target antigen. When the antigen is large, the antibody may only bind to a specific portion of the antigen, which is called an epitope. The antibody antigen binding site may be provided by one or more antibody variable domains. The antibody antigen binding site optionally includes a variable light chain (VL) region and a variable heavy chain (VH) region. The VH region and VL region of the antigen binding domain together constitute the Fv region.

[0140] An antigen binding domain typically 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, which is the portion of the antigen binding domain that binds to the target antigen.

[0141] The VH region and the VL region include a framework region (FR) on either side of each CDR, which provides a scaffold for the CDR. From N-terminus to C-terminus, the VH region includes the following structure: N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-C-terminus; the VL region includes the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus.

[0142] There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat, 1991, Chothia, 1987; IMGT numbering as described in LeFranc, 2015; and VBASE2 as 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).

[0143] Antibody molecules comprising at least two antigen binding domains, wherein each antigen binding domain 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 cMET) 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.

[0144] Anti-EGFR binding domain

[0145] The binding domain that binds to EGFR (anti-EGFR binding domain) generally comprises the CDR of the antibody molecule that can bind to EGFR. In some instances, the binding domain that binds to EGFR additionally comprises the FR of the antibody molecule that can bind to EGFR. That is, in some instances, the binding domain that binds to EGFR comprises the VH region and the VL region of the antibody molecule that can bind to EGFR.

[0146] 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.

[0147] In some examples, the binding domain that binds to EGFR comprises the anti-EGFR antibody clones RAA22 or QD6, optionally three HCDRs or three LCDRs of RAA22, optionally three VH CDRs and three VL CDRs. The VH and VL domain sequences of antibodies RAA22 and QD6 are described herein, so the three VH and three VL domain CDRs of the antibodies can be determined from the sequences.

[0148] In some examples, the binding domain that binds to EGFR comprises a VH region according to (1) or (2) below:

[0149] (1) A VH region comprising the following CDRs:

[0150] HCDR1 having the amino acid sequence of SEQ ID NO: 1

[0151] HCDR2 having the amino acid sequence of SEQ ID NO: 2

[0152] a HCDR3 having the amino acid sequence of SEQ ID NO: 3,

[0153] or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid, or

[0154] (2) A VH region comprising the following CDRs:

[0155] HCDR1 having the amino acid sequence of SEQ ID NO: 1

[0156] HCDR2 having the amino acid sequence of SEQ ID NO: 7

[0157] a HCDR3 having the amino acid sequence of SEQ ID NO: 3,

[0158] or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid.

[0159] In some examples, the binding domain that binds to EGFR comprises a VH region according to (1) above.

[0160] In some examples, the binding domain that binds to EGFR comprises a VH region according to (1) or (2) above, wherein the VH region further comprises a FR according to (3) below:

[0161] (3) HFR1 having the amino acid sequence of SEQ ID NO: 8

[0162] HFR2 having the amino acid sequence of SEQ ID NO:9

[0163] HFR3 having the amino acid sequence of SEQ ID NO: 10

[0164] HFR4 having the amino acid sequence of SEQ ID NO: 11,

[0165] or a variant thereof, wherein one or two or three amino acids in one or more of HFR1, HFR2, HFR3 or HFR4 are substituted with another amino acid.

[0166] In some examples, the binding domain that binds to EGFR comprises a VH region comprising CDRs according to (1) or (2) above and FRs according to (3) above.

[0167] In some examples, the binding domain that binds to EGFR comprises a VH region according to (4) or (5) below:

[0168] (4) a VH region comprising the CDRs according to (1) and the FRs according to (3),

[0169] (5) A VH region comprising the CDR according to (2) and the FR according to (3).

[0170] In some examples, the binding domain that binds to EGFR comprises a VH region according to (4) above.

[0171] In some examples, the binding domain that binds to EGFR comprises a VH region according to (6) or (7) below:

[0172] (6) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 16, 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.

[0173] (7) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 18, 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.

[0174] In some examples, the binding domain that binds to EGFR comprises a VH region according to (6) above.

[0175] In some examples, the binding domain that binds to EGFR comprises a VL region according to (8) or (9) below:

[0176] (8) A VL region comprising the following CDRs:

[0177] LCDR1 having the amino acid sequence of SEQ ID NO: 4

[0178] LCDR2 having the amino acid sequence of SEQ ID NO: 5

[0179] LCDR3 having the amino acid sequence of SEQ ID NO:6,

[0180] or a variant thereof, wherein one or two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are substituted with another amino acid.

[0181] (9) A VL region comprising the following CDRs:

[0182] LCDR1 having the amino acid sequence of SEQ ID NO: 4

[0183] LCDR2 having the amino acid sequence of SEQ ID NO: 66

[0184] LCDR3 having the amino acid sequence of SEQ ID NO:67,

[0185] or a variant thereof, wherein one or two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are substituted with another amino acid.

[0186] In some examples, the binding domain that binds to EGFR comprises a VL region according to (8) above.

[0187] In some examples, the binding domain that binds to EGFR comprises a VL region according to (8) or (9) above, wherein the VL region further comprises a FR according to (10) below:

[0188] (10) LFR1 having the amino acid sequence of SEQ ID NO: 12

[0189] LFR2 having the amino acid sequence of SEQ ID NO: 13

[0190] LFR3 having the amino acid sequence of SEQ ID NO: 14

[0191] LFR4 having the amino acid sequence of SEQ ID NO: 15,

[0192] or a variant thereof, wherein one or two or three amino acids in one or more of LFR1, LFR2, LFR3 or LFR4 are substituted with another amino acid.

[0193] In some examples, the binding domain that binds to EGFR comprises a VL region comprising a CDR according to (8) or (9) above and a FR according to (10) above.

[0194] In some examples, the binding domain that binds to EGFR comprises a VL region according to (11) or (12) below:

[0195] (11) A VL region comprising the CDR according to (8) and the FR according to (10).

[0196] (12) A VL region comprising the CDR according to (9) and the FR according to (10).

[0197] In some examples, the binding domain that binds to EGFR comprises a VL region according to (11) above.

[0198] In some examples, the binding domain that binds to EGFR comprises a VL region according to (13) or (14) below:

[0199] (13) A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:20, 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.

[0200] (14) A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:22, 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.

[0201] In some examples, the binding domain that binds to EGFR comprises a VL region according to (13) above.

[0202] In some examples, the binding domain that binds to EGFR comprises a VH region according to any one of (1) to (7) above and a VL region according to any one of (8) to (14) above. In some examples, the binding domain comprises a VH region according to any one of (1), (4) and (6) and a VL region according to any one of (8), (11) and (13). In other examples, the binding domain comprises a VH region according to any one of (2), (5) and (7) and a VL region according to any one of (9), (12) and (14).

[0203] Anti-cMET binding domain

[0204] The binding domain that binds to cMET (anti-cMET binding domain) generally comprises the CDR of an antibody molecule capable of binding to cMET. In some examples, the binding domain that binds to cMET further comprises the FR of an antibody molecule capable of binding to cMET. That is, in some examples, the binding domain that binds to cMET comprises the VH region and the VL region of an antibody molecule capable of binding to cMET.

[0205] In some examples, the binding domain that binds cMET comprises a VH region and a VL region that are or are derived from the VH / VL regions of a cMET binding antibody clone described herein (ie, anti-cMET antibody clone B09-GL).

[0206] In some examples, the binding domain that binds cMET comprises three HCDRs or three LCDRs, optionally three VH CDRs and three VLCDRs 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 and three VL domain CDRs of the antibody can be determined from the sequences.

[0207] In some examples, the binding domain that binds cMET comprises a VH region according to (15) below:

[0208] (15) A VH region comprising the following CDRs:

[0209] HCDR1 having the amino acid sequence of SEQ ID NO: 24

[0210] HCDR2 having the amino acid sequence of SEQ ID NO: 25

[0211] a HCDR3 having the amino acid sequence of SEQ ID NO: 26,

[0212] or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid.

[0213] In some examples, the binding domain that binds to cMET comprises a VH region according to (15) above, wherein the VH region further comprises a FR according to (16) below:

[0214] (16) HFR1 having the amino acid sequence of SEQ ID NO: 30

[0215] HFR2 having the amino acid sequence of SEQ ID NO:31

[0216] HFR3 having the amino acid sequence of SEQ ID NO: 32

[0217] HFR4 having the amino acid sequence of SEQ ID NO:33,

[0218] or a variant thereof, wherein one or two or three amino acids in one or more of HFR1, HFR2, HFR3 or HFR4 are substituted with another amino acid.

[0219] In some examples, the binding domain that binds cMET comprises a VH region according to (17) below:

[0220] (17) A VH region comprising the CDR according to (15) and the FR according to (16).

[0221] In some examples, the binding domain that binds cMET comprises a VH region according to (18) below:

[0222] (18) A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:38, 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.

[0223] In some examples, the binding domain that binds cMET comprises a VL region according to (19) below:

[0224] (19) A VL region comprising the following CDRs:

[0225] LCDR1 having the amino acid sequence of SEQ ID NO: 27

[0226] LCDR2 having the amino acid sequence of SEQ ID NO: 28

[0227] LCDR3 having the amino acid sequence of SEQ ID NO: 29,

[0228] or a variant thereof, wherein one or two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are substituted with another amino acid.

[0229] In some examples, the binding domain that binds cMET comprises a VL region according to (19) above, wherein the VL region further comprises a FR according to (20) below:

[0230] (20) LFR1 having the amino acid sequence of SEQ ID NO: 34

[0231] LFR2 having the amino acid sequence of SEQ ID NO:35

[0232] LFR3 having the amino acid sequence of SEQ ID NO: 36

[0233] LFR4 having the amino acid sequence of SEQ ID NO:37,

[0234] or a variant thereof, wherein one or two or three amino acids in one or more of LFR1, LFR2, LFR3 or LFR4 are substituted with another amino acid.

[0235] In some examples, the binding domain that binds cMET comprises a VL region according to (21) below:

[0236] (21) A VL region comprising the CDRs according to (19) and the FRs according to (20).

[0237] In some examples, the binding domain that binds cMET comprises a VL region according to (22) below:

[0238] (22) A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:40, 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.

[0239] In some examples, the binding domain that binds to cMET comprises a VH region according to any one of (15) to (18) above and a VL region according to any one of (19) to (22) above.

[0240] CDR substitution

[0241] In examples according to the present disclosure, one or more amino acids (eg, one, two or three) are substituted with another amino acid.

[0242] Naturally occurring residues can be classified based on common side-chain properties:

[0243] 1) Non-polar, aliphatic: glycine (G), methionine (M), alanine (A), valine (V), leucine (L), isoleucine (I);

[0244] 2) Polar, uncharged: cysteine ​​(C), serine (S), threonine (T), asparagine (N), glutamine (Q), proline (P);

[0245] 3) Acidic (negatively charged): aspartic acid (D), glutamic acid (E);

[0246] 4) Basic (positive charge): histidine (H), lysine (K), arginine (R);

[0247] 5) Aromatic: Tryptophan (W), Tyrosine (Y), Phenylalanine (F).

[0248] Amino acid substitutions can be conservative amino acid substitutions. Conservative amino acid substitutions can involve the exchange of one member of one of these classes with another member of the same class. For example, a conservative amino acid substitution can be the substitution of the acidic amino acid glutamic acid (E) for the acidic amino acid aspartic acid (D).

[0249] In some instances, one or more substitutions may be functionally conservative. That is, in some instances, a substitution may not affect (or may not substantially affect) one or more functional properties (e.g., binding affinity) of the antigen-binding domain comprising the substitution, compared to an equivalent unsubstituted antigen-binding domain.

[0250] Constant region

[0251] In some instances, the antibody molecules described herein comprise an immunoglobulin heavy chain constant (CH) region. In some instances, CH is or is derived from a heavy chain constant region sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM.

[0252] In some examples, the CH region is a human immunoglobulin G1 constant region (IGHG1; UniProt: P01857-1, v1; SEQ ID NO: 42) or a fragment thereof.

[0253] In some examples, the CH region comprises an amino acid sequence having at least 70% sequence identity, such as 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, such as 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.

[0254] In some examples, the antibody molecule comprises a heavy chain comprising or consisting of a VH region as described herein and a CH region as described herein.

[0255] 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 listed 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 listed in SEQ ID NO: 49 or SEQ ID NO: 65. In some examples, the antibody molecule comprises: a first CL region that is or is derived from a kappa CL region listed in SEQ ID NO: 47 or 48; and a second CL region that is or is derived from a lambda CL region listed in SEQ ID NO: 49 or 65.

[0256] In some examples, the antibody molecules described herein comprise:

[0257] a first heavy chain, wherein the first heavy chain comprises the VH region of the anti-EGFR binding domain, and a first heavy chain constant (CH) region or a fragment thereof;

[0258] a first light chain, wherein the first light chain comprises the VL region of the anti-EGFR binding domain, and a first light chain constant (CL) region or a fragment thereof;

[0259] a second heavy chain, wherein the second heavy chain comprises the VH region of the anti-cMET binding domain, and a second heavy chain constant (CH) region or a fragment thereof; and

[0260] A second light chain, wherein the second light chain comprises the VL region of the anti-cMET binding domain, and a second light chain constant (CL) region or a fragment thereof.

[0261] The first CH region and the second CH region may be the same or different. In other words, the first CH region and the second CH region may form a homodimer or a heterodimer. For example, an asymmetric bispecific antibody molecule has a different first CH region and a different second CH region, as described in more detail below. The first CL region and the second CL region may be the same or different. In some instances, the first CL region is or is derived from the kappa CL region listed in SEQ ID NO:47 or 48; and the second CL region is or is derived from the lambda CL region listed in SEQ ID NO:49 or 65.

[0262] It should 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 connected to each other. Similarly, the second VH region and the second CH region form the second heavy chain of the antibody molecule; the first VL region and the first CL region form the first light chain of the antibody molecule; and the second VL region and the second CL region form the second light chain of the antibody molecule.

[0263] In some examples, the antibody molecule comprises a heavy chain having 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 listed in SEQ ID NO:50, the QD6 heavy chain listed in SEQ ID NO:53, the RAA22 heavy chain listed in SEQ ID NO:56, the heavy chain listed in SEQ ID NO:59, or the heavy chain listed in SEQ ID NO:60.

[0264] In some examples, the antibody molecule comprises a first heavy chain and a second heavy chain, wherein

[0265] (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; and

[0266] (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.

[0267] In some examples, the antibody molecule comprises a first heavy chain and a second heavy chain, wherein

[0268] (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; and

[0269] (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.

[0270] In some examples, the antibody molecules described herein comprise a light chain comprising or consisting of a VL region as described herein and a CL region as described herein.

[0271] In some examples, the antibody molecules described herein comprise a light chain having 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 listed in SEQ ID NO:52, the QD6 light chain listed in SEQ ID NO:55, the RAA22 light chain listed in SEQ ID NO:58, the light chain listed in SEQ ID NO:61, or the light chain listed in SEQ ID NO:62.

[0272] In some examples, the antibody molecules described herein comprise a first light chain and a second light chain, wherein

[0273] (i) the first light chain comprises an amino acid sequence having at least 70% sequence identity, such as 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; and

[0274] (ii) the second light chain comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of the B-09-GL light chain set forth in SEQ ID NO:52, 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.

[0275] In some examples, the antibody molecules described herein comprise a first light chain and a second light chain, wherein

[0276] (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; and

[0277] (ii) the second light chain comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of the light chain set forth in SEQ ID NO:62, 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.

[0278] The CH, CL, heavy chain and / or light chain of the antibody molecules described herein may comprise one or more modifications, such as eliminating or reducing Fc effector functions, promoting the formation of heterodimeric antibody molecules, increasing the efficacy of homologous heavy chain and light chain pairing, and / or facilitating conjugate formation, as described in more detail below. The modified CH, CL, heavy chain and light chain may be referred to as modified CH, CL, heavy chain and light chain, respectively.

[0279] The antibody molecule may include mutations in the CH region of the heavy chain to reduce or eliminate the binding of the antibody molecule to one or more Fcγ receptors such as FcγRI, FcγRIIa, FcγRIIb, FcγRIII and / or to complement. This mutation eliminates or reduces Fc effector function. Mutations for reducing or eliminating the binding of antibody molecules to one or more Fcγ receptors and complement are known, and include, for example, "triple mutations" or "TMs" of L234F / L235E / P331S described in Organesyan, 2008. Other mutations known to regulate antibody effector functions are described, for example, in Wang, 2018.

[0280] Therefore, in some examples, the first heavy chain and / or the second heavy chain include phenylalanine (F) at position 234, glutamic acid (E) at position 235, and serine (S) at position 331, wherein numbering is according to the EU index. For example, one or both of the first heavy chain and the second heavy chain may include a CH region having an amino acid sequence with at least 70%, at least 80%, at least 90%, at least 95% sequence identity to the sequence listed in SEQ ID NO: 42, and include phenylalanine (F) at position 234, glutamic acid (E) at position 235, and serine (S) at position 331, wherein numbering is according to the EU index. As demonstrated in examples (e.g., example 12), inclusion of TM in the heavy chain has been shown to improve the pharmacokinetic properties of exemplary antibody molecules and ADCs.

[0281] Examples of CH regions comprising triple mutations are SEQ ID NOs: 63 and 64. Thus, in some examples, one of the first heavy chain and the second 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: 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 (preferably both) of the CH regions comprise a phenylalanine at position 234, a glutamic acid at position 235, and a serine at position 331, wherein numbering is according to the EU index.

[0282] Examples of heavy chains comprising a CH region comprising a triple mutation are SEQ ID NOs: 59 and 60. Thus, in some examples, one of the first heavy chain and the second 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: 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 comprises a phenylalanine at position 234, a glutamic acid at position 235, and a serine at position 331, wherein numbering is according to the EU index.

[0283] The VL region and CL region of the antibody molecule, as well as the VH region and CH1 region, together constitute the Fab region, and the rest of the antibody molecule constitutes the Fc region.

[0284] Unless otherwise indicated, amino acid residue positions in the constant domains (including positions of amino acid sequences, substitutions, deletions and insertions as described herein) are numbered according to EU numbering (Edelman, 2007).

[0285] Bispecific formats

[0286] The bispecific antibody molecules may be provided in any suitable form. Suitable forms of the bispecific antibody molecules described herein and methods for their production 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 Kontermann MAbs 2012, 4(2):182-197 for details. Figure 2 .

[0287] Bispecific antibody molecules can also be generated from existing antibodies by chemical conjugation. For example, two IgG molecules or two Fab' fragments can be coupled using homologous or heterologous bifunctional coupling reagents (e.g., as described in Graziano and Guptill, Methods Mol Biol. 2004; 283: 71-85).

[0288] In some examples, the bispecific antibody molecule can be an immunoglobulin G-like (IgG-like) bispecific antibody molecule. An IgG-like bispecific antibody molecule can include an Fv region, a Fab region, or an sVD specific for one antigen, an Fv / Fab / sVD specific for another antigen, and an Fc region. An IgG-like bispecific antibody molecule can be symmetrical or asymmetrical. In one example, the bispecific antibody molecule is asymmetrical.

[0289] Symmetrical IgG-like bispecific antibody molecules generally contain an antigen binding domain, for example in the form of a scFv fragment or a variable single domain, fused to the N-terminus or C-terminus of the heavy or light chain of the IgG molecule. The unique property of these symmetrical IgG-like bispecific antibody molecules is that they contain two identical heavy chains. In addition, symmetrical IgG-like bispecific antibody molecules are generally bivalent for each epitope. Valency as used herein refers to the number of antigen binding regions in an antibody molecule that can bind to a single epitope. Monoclonal monospecific IgG antibody molecules are bivalent for a single epitope—they contain two antigen binding domains, each of which can bind to an epitope on a single target molecule. Symmetrical IgG-like bispecific antibody molecules are bivalent for each epitope—they generally contain four antigen binding domains, two of which can bind to a first epitope on a target molecule, and two of which can bind to a second epitope on a target molecule.

[0290] Examples of symmetrical 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 These can be formed by methods known in the art, such as chemical cross-linking, somatic cell hybridization or redox methods.

[0291] In contrast, asymmetric IgG-like bispecific antibody molecules are typically monovalent for each target. As described, for example, in Klein 2012, the concept of monovalent bispecific IgGs is believed to have unique therapeutic advantages because they (i) do not cause receptor homodimerization, (ii) potentially have reduced toxicity to non-target tissues due to loss of affinity for each antigen, and (iii) have better selectivity when both antigens are selectively restricted or expressed in large quantities on target cells. Therefore, in some examples, the antibody molecule is an asymmetric IgG-like bispecific antibody molecule.

[0292] Asymmetric IgG-like bispecific antibody molecules involve the heterodimerization of two different heavy chains and the correct pairing of homologous light chains and heavy chains. The heterodimerization of heavy chains can be solved by several techniques, such as knobs-into-holes, electrostatic steering of CH3, engineered domains of CH3 chain exchange, and leucine zippers. The correct pairing of light chains and heavy chains can be ensured by using one of these heavy chain heterodimerization techniques together with the use of a common light chain, domain crossing between CH1 and CL, coupling of heavy and light chains to joints, in vitro assembly of heavy chain-light chain dimers from two separate monoclonal clones, interface engineering of the entire Fab domain, or disulfide engineering at the CH1 / CL interface.

[0293] Examples of asymmetric IgG-like bispecific antibody molecules include DuetMab, kih IgG, kihIgG common LC, CrossMab, kihIgG-scFab, mAb-Fv, charge pair and SEED-body.

[0294] In some examples, the antibody molecule comprises 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 above-mentioned DuetMab antibody molecule may further comprise one or more modifications in one or more of the CH1, CH2 and CH3 domains that promote the formation of heterodimeric antibody molecules. This may involve the knob-in-hole (KiH) strategy described in Ridgway 1996 based on the substitution of a single amino acid in the CH3 domain that promotes heavy chain heterodimerization. The small amino acid of the knob variant heavy chain CH3 has been replaced by a larger amino acid, while the large amino acid of the hole variant has been replaced by a smaller amino acid. Additional modifications may also be introduced to stabilize the association between the heavy chains.

[0295] CH3 modifications that enhance heterodimerization include, for example, Y407V / T366S / L368A on one heavy chain and T366W on the other heavy chain; and S354C / T366W on one heavy chain and Y349C / Y407V / T366S / L368A on the other heavy chain, wherein the numbering of the constant regions is according to the EU index.

[0296] Other examples of CH3 modifications that 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.

[0297] In some examples, the antibody molecule comprises a first heavy chain and a second heavy chain that form a heterodimer, wherein one of the first heavy chain and the second heavy chain 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, wherein the numbering of the constant region is according to the EU index. For example, one of the first heavy chain and the second heavy chain may have the sequence set forth in SEQ ID NO: 42, and further comprises 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 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, wherein the numbering of the constant region is according to the EU index.

[0298] In some embodiments, the antibody molecule comprises:

[0299] (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

[0300] (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) at position 366, and an alanine (A) at position 368,

[0301] The numbering of the constant regions follows the EU index.

[0302] A specific exemplary form of an asymmetric IgG-like bispecific antibody molecule is called "DuetMab". DuetMab antibody molecules use KIH technology for heterodimerization of 2 different heavy chains and increase the effectiveness of homologous heavy and light chain pairing by replacing the native disulfide bonds in one of the CH1-CL interfaces with engineered disulfide bonds. Disclosures related to DuetMab can be found in, for example, U.S. Pat. Nos. 9,527,927 and Mazor 2015, both of which are incorporated herein by reference in their entirety.

[0303] In some embodiments, the antibody molecule comprises:

[0304] (a) a modified CH region, wherein the modified heavy chain comprises a substitution of a native non-cysteine ​​amino acid to a cysteine ​​amino acid; and

[0305] (b) a modified corresponding CL region, wherein the modified CL comprises a substitution of a native non-cysteine ​​amino acid to a cysteine ​​amino acid,

[0306] in:

[0307] (i) the first heavy chain comprises the modified CH region and the first light chain comprises the modified corresponding CL region; or

[0308] (ii) the second heavy chain comprises the modified CH region and the second light chain comprises the modified corresponding CL region.

[0309] In some examples, a substituted cysteine ​​of a modified CH region resulting from substitution of a native non-cysteine ​​amino acid to a cysteine ​​amino acid and a substituted cysteine ​​of a modified corresponding CL region resulting from substitution of a native non-cysteine ​​amino acid to a cysteine ​​amino acid can form a disulfide bond.

[0310] In some examples, the modified CH region comprises a substitution of a native non-cysteine ​​amino acid to a cysteine ​​amino acid at position 126; and the modified corresponding CL region comprises a substitution of a native non-cysteine ​​amino acid to cysteine ​​at position 121, wherein the numbering of the constant regions is according to the EU index.

[0311] In some examples, the modified CH region comprises a substitution of a native non-cysteine ​​amino acid to a cysteine ​​amino acid at position 126 and a substitution of a native cysteine ​​amino acid to a non-cysteine ​​amino acid (e.g., to valine) at position 219; and the modified corresponding CL region comprises a substitution of a native non-cysteine ​​amino acid to cysteine ​​at position 121 and a substitution of a native cysteine ​​amino acid to a non-cysteine ​​amino acid (e.g., to valine) at position 214, wherein the numbering of the constant regions is according to the EU index.

[0312] 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 comprise no substitution of a native non-cysteine ​​amino acid to a cysteine ​​amino acid and no substitution of a native cysteine ​​to a non-cysteine ​​amino acid.

[0313] Conjugate

[0314] The antibody molecule can be conjugated to a drug. In this case, the antibody molecule can be referred to as a "conjugate" or "antibody drug conjugate". Such a conjugate can be used to treat and / or diagnose a disease as described herein. As used herein, a drug can be referred to as a "payload" or "warhead".

[0315] In some examples, the drug comprises 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, a DNA, an RNA, a siRNA, an RNAi, a 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.

[0316] Cytotoxin is a compound that can induce targeted cell death. Typically, in the case of an antibody drug conjugate, the cytotoxin is delivered to the cell targeted by the antibody molecule, where it is released into the cell and induces cell death. The use of cytotoxins in antibody drug conjugates is described in, for example, Chalouni and Doll 2018 J Exp Clin Cancer Res. [Experimental and Clinical Cancer Research Journal] 37 (1): 20. In some instances, the cytotoxin is tubulysin, auristatin, maytansinoid alkaloid, topoisomerase inhibitor or pyrrolobenzodiazepine (PBD).

[0317] In a particular instance, the cytotoxin is or comprises tubulysin. Tubulysin is a class of tetrapeptides that inhibit cell growth, which contains isoleucine and three other complex non-natural amino acids Mep (RN-mepipecolic acid), Tuv (tubuvaline) and Tut (tubulyrosine) or Tup (tubuphenylalanine). Tubulysin is an extremely effective cytotoxic molecule and is effective against multi-drug resistant cell lines (Domling, 2005). These compounds show high cytotoxicity tested for a group of cancer cell lines with IC50 values ​​in the low picomolar range; therefore, they are meaningful as anticancer therapeutic agents. See, for example, WO 2012019123. Tubulysin conjugates are disclosed in, for example, U.S. Patent No. 7,776,814. In some instances, tubulysin is tubulysin A with the following chemical structure:

[0318]

[0319] In some examples, the tubulysin is tubulysin 1508, also known as "AZ1508" and described in more detail in WO 2015157594. Tubulysin 1508 has the following chemical structure:

[0320]

[0321] In some instances, the cytotoxin is or comprises a topoisomerase inhibitor. The term "topoisomerase inhibitor" as used herein refers to a cytotoxic agent that inhibits the activity of one or more topoisomerases (topoisomerase I and II), which are enzymes that play an important role in DNA replication and transcription by regulating DNA supercoiling. Therefore, it is expected that antibody drug conjugates comprising topoisomerase inhibitors as cytotoxins interfere with normal processes involving DNA, thus causing cell death. Conjugates containing topoisomerase inhibitors have been shown to be effective and have anticancer activity against a variety of tumor-containing cell lines in clinical trials. See, for example, Ogitani, 2016a; Ogitani, 2016b; Cardillo, 2015; and Bardia, 2017.

[0322] 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, lortotecan, and cinotecan. Representative examples of topoisomerase II inhibitors include, but are not limited to, amsacrine, daunorubicin, doxorubicin, epipodophyllotoxin, ellipticine, epirubicin, etoposide, razoxane, and teniposide.

[0323] An example of a camptothecin chemical structure is as follows:

[0324]

[0325] General examples of suitable topoisomerase I inhibitors are represented by the following compounds:

[0326]

[0327] The compounds are denoted A*.

[0328] In some examples, a compound (e.g., A*) has a linker for attachment to an antibody molecule described herein (which may be referred to as a "ligand unit" or alternatively a "cell binding agent" (CBA)). Suitably, the linker is cleavably attached (e.g., conjugated) to an amino residue, e.g., an amino acid of an antibody molecule described herein.

[0329] The design and selection of linkers for conjugates is known in the art and described, for example, in Beck, 2017. The linker used herein may be any linker described in Beck, 2017.

[0330] More particularly, examples of suitable topoisomerase I inhibitors are given by the following compounds having formula "I":

[0331]

[0332] and its salts and solvates, wherein R L is a linker for attachment to an antibody molecule described herein, wherein the linker is optionally selected from:

[0333] (ia):

[0334]

[0335] in

[0336] Q is:

[0337] Where Q X such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;

[0338] X is:

[0339]

[0340] 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, wherein 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);

[0341] G L is a linker for attachment to an antibody or antigen-binding fragment thereof described herein (eg, a Ligand unit or a cell-binding agent); or

[0342] (ib):

[0343]

[0344] Where R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropene or cyclobutene group; and

[0345] e is 0 or 1.

[0346] In-style In , the superscripts C(=0) and NH indicate the groups to which the atoms are bound. For example, an NH group is shown bound to a carbonyl group (which is not part of the depicted moiety), and a carbonyl group is shown bound to an NH group (which is not part of the depicted moiety).

[0347] Those skilled in the art will appreciate that more than one such agent (eg, a topoisomerase I inhibitor) may be conjugated to an antibody molecule.

[0348] For example, the conjugates (eg, antibody-drug conjugates) of the present disclosure may have the general formula IV:

[0349] L–(D L ) p (IV)

[0350] or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody molecule described herein (eg, a Ligand unit or a CBA), D L is a drug having a linker (eg, a Drug Linker unit), and p is an integer from 1 to 20.

[0351] In some instances, D L is a topoisomerase I inhibitor having a linker of formula III:

[0352]

[0353] R LL is a linker attached to an antibody molecule described herein (e.g., a Ligand unit), wherein the linker is optionally selected from

[0354] (ia'):

[0355]

[0356] Where Q and X are as defined above and G LLis a linker that is attached to an antibody molecule described herein (e.g., a Ligand unit or a CBA); and

[0357] (ib'):

[0358]

[0359] Where R L1 and R L2 As defined above.

[0360] The drug loading is represented by p, i.e., the number of topoisomerase I inhibitors (e.g., drug units) per antibody molecule (e.g., ligand unit). The drug loading can be in the range of 1 to 20 drug units (D) / ligand units. For compositions, p represents the average drug loading of the conjugate in the composition, and p ranges from 1 to 20. In some instances, 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, an ADC comprising the topoisomerase I inhibitor SG3932 with an average DAR of 6+ / -6% was produced.

[0361] Thus, the disclosure includes conjugates comprising an antibody molecule described herein (e.g., a Ligand unit or a CBA) covalently linked to at least one topoisomerase I inhibitor (e.g., a Drug unit, such as A* as described above). The inhibitor is optionally linked to the topoisomerase I inhibitor via a Linker (e.g., a Linker unit), such as R L and / or R LL The linker is connected to the antibody molecule. In other words, the disclosure includes antibody molecules (such as ligand units or CBA) described herein connected to one or more topoisomerase I inhibitors, optionally via a linker (such as a drug-linker unit). The antibody molecules described more comprehensively above (representing ligand units or CBA) are targeting agents that bind to the target moiety. More specifically, the antibody molecule can, for example, specifically bind to EGFR and cMET on the target cell, thereby delivering the drug unit to the target cell. Therefore, the disclosure also provides methods for treating, for example, various cancers and other obstacles (such as cancers / obstacles related to the presence of cells expressing EGFR and cMET, such as cancer cells) with ADC. These methods are described in more detail below.

[0362] Q X

[0363] In one example, Q is an amino acid residue. The amino acid can be a natural amino acid or a non-natural amino acid. For example, Q can be selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, wherein Cit is citrulline.

[0364] In one example, Q comprises a dipeptide residue. The amino acids in the dipeptide can be any combination of natural amino acids and non-natural amino acids. In some examples, the dipeptide comprises natural amino acids. When the linker is a cathepsin-labile linker, the dipeptide is the action site of cathepsin-mediated cleavage. Then, the dipeptide is the recognition site of cathepsin.

[0365] In one example, Q is selected from:

[0366] NH -Phe-Lys- C＝O ,

[0367] NH -Val-Ala- C＝O ,

[0368] NH -Val-Lys- C＝O ,

[0369] NH -Ala-Lys- C＝O ,

[0370] NH -Val-Cit- C＝O ,

[0371] NH -Phe-Cit- C＝O ,

[0372] NH -Leu-Cit- C＝O ,

[0373] NH -Ile-Cit- C＝O ,

[0374] NH -Phe-Arg- C＝O ,

[0375] NH -Trp-Cit- C＝O ,and

[0376] NH -Gly-Val- C＝O ;

[0377] Where Cit is citrulline.

[0378] In one example, Q is selected from:

[0379] NH -Phe-Lys- C＝O ,

[0380] NH -Val-Ala- C＝O ,

[0381] NH -Val-Lys- C＝O ,

[0382] NH -Ala-Lys- C＝O ,and

[0383] NH -Val-Cit- C＝O .

[0384] In one embodiment, Q is selected from NH -Phe-Lys- C＝O , NH -Val-Cit- C＝O or NH -Val-Ala- C＝O .

[0385] Other suitable dipeptide combinations include:

[0386] NH -Gly-Gly- C＝O ,

[0387] NH -Gly-Val- C＝O

[0388] NH -Pro-Pro- C＝O ,and

[0389] NH -Val-Glu- C＝O .

[0390] Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, which is incorporated herein by reference.

[0391] In some instances, Q is a tripeptide residue. The amino acids in the tripeptide can be any combination of natural amino acids and non-natural amino acids. In some instances, the tripeptide comprises natural amino acids. When the linker is a cathepsin labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. Then, the tripeptide is the recognition site for cathepsin. Particularly interesting tripeptide linkers are:

[0392] NH -Glu-Val-Ala- C＝O

[0393] NH -Glu-Val-Cit- C＝O

[0394] NH -αGlu-Val-Ala- C＝O

[0395] NH -αGlu-Val-Cit- C＝O

[0396] In some instances, Q is a tetrapeptide residue. The amino acids in the tetrapeptide can be any combination of natural amino acids and non-natural amino acids. In some instances, the tetrapeptide comprises natural amino acids. When the linker is a cathepsin labile linker, the tetrapeptide is the site of action for cathepsin-mediated cleavage. Then, the tetrapeptide is the recognition site for cathepsin. Tetrapeptide linkers of particular interest are:

[0397] NH -Gly-Gly-Phe-Gly C＝O ;as well as

[0398] NH -Gly-Phe-Gly-Gly C＝O .

[0399] In some examples, the tetrapeptide is:

[0400] NH -Gly-Gly-Phe-Gly C＝O .

[0401] In the above representation of peptide residues, NH - indicates the N-terminus of the residue, and - C＝O represents the C-terminus of the residue. The C-terminus is bound to the NH of A*.

[0402] Glu represents the residue of glutamic acid, that is:

[0403]

[0404] αGlu represents the residue of glutamic acid when bound via the α chain, i.e.:

[0405]

[0406] In one example, where appropriate, the amino acid side chains are chemically protected. The side chain protecting group can be a group as discussed above. The protected amino acid sequence can be cleaved by an enzyme. For example, a dipeptide sequence comprising a Lys residue protected by a Boc side chain can be cleaved by a cathepsin.

[0407] Protecting groups for amino acid side chains are well known in the art and are described in the Novabiochem catalog and as described above.

[0408] G L

[0409] G L Can be selected from:

[0410]

[0411]

[0412] Where Ar represents C 5-6 An arylene group, such as phenylene, and X' represents C 1-4 alkyl.

[0413] In some instances, G L Selected from G L1-1 and G L1-2 In some of these instances, G L It's G L1-1 .

[0414] G LL

[0415] G LL Can be selected from:

[0416]

[0417]

[0418] Where Ar represents C 5-6 An arylene group, such as phenylene, and X' represents C 1-4 Alkyl. CBA represents a cell binding agent or ligand unit.

[0419] In some instances, G LL Selected from G LL1-1 and G LL1-2 In some of these instances, G LL It's G LL1-1 .

[0420] X

[0421] X is optionally:

[0422]

[0423] Wherein a=0 to 5, b1=0 to 16, b2=0 to 16, c=0 or 1, d=0 to 5, wherein at least b1 or b2=0 and at least c1 or c2=0.

[0424] a can be 0, 1, 2, 3, 4, or 5. In some instances, a is 0 to 3. In some of these instances, a is 0 or 1. In other instances, a is 0.

[0425] 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.

[0426] b2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some instances, b2 is 0 to 12. In some of these instances, b2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8. In some instances, only one of b1 and b2 can be non-zero.

[0427] c1 can be 0 or 1. c2 can be 0 or 1. In some examples, only one of c1 and c2 can be non-zero.

[0428] d can be 0, 1, 2, 3, 4, or 5. In some instances, d is 0 to 3. In some of these instances, d is 1 or 2. In other instances, d is 2. In other instances, d is 5.

[0429] In some instances of X, a is 0, b1 is 0, c1 is 1, c2 is 0 and d is 2, and b2 can be 0 to 8. In some of these instances, b2 is 0, 2, 3, 4, 5, or 8. In some instances of X, a is 1, b2 is 0, c1 is 0, c2 is 0 and d is 0, and b1 can be 0 to 8. In some of these instances, b1 is 0, 2, 3, 4, 5, or 8. In some instances of X, a is 0, b1 is 0, c1 is 0, c2 is 0 and d is 1, and b2 can be 0 to 8. In some of these instances, b2 is 0, 2, 3, 4, 5, or 8. In some instances of X, b1 is 0, b2 is 0, c1 is 0, c2 is 0 and d is 1, and b2 can be 0 to 8. In some of these instances, b2 is 0, 2, 3, 4, 5, or 8. In some instances 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 instances, the other of a and d is 1. In others of these instances, the other of a and d is 5. In some instances 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 instances, b2 is 0, 2, 3, 4, 5, or 8.

[0430] In some instances, R L Has formula Ib. In some examples, R LL It has the formula Ib'.

[0431] RL1 and R L2 The radicals may be independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropene or cyclobutene group.

[0432] In some instances, R L1 and R L2 are H. In some examples, R L1 is H and R L2 is methyl. In some instances, R L1 and R L2 All are methyl.

[0433] In some instances, R L1 and R L2 Together with the carbon atom to which they are bonded, they form a cyclopropene group. L1 and R L2 Together with the carbon atom to which they are bonded they form a cyclobutenyl group.

[0434] In group Ib, in some instances, e is 0. In other instances, e is 1 and the nitro group can be in any available position of the ring. In some of these instances, it is in the ortho position. In other instances of these instances, it is in the para position.

[0435] In some examples where the compounds described herein are provided as single enantiomers or in enantiomerically enriched form, the enantiomerically enriched form has an enantiomeric ratio greater than 60:40, 70:30, 80:20, or 90: 10. In other examples, the enantiomeric ratio is greater than 95:5, 97:3, or 99:1.

[0436] In some instances, R L Selected from:

[0437]

[0438]

[0439] In some instances, R LL is derived from the above R L Group of groups.

[0440] In some embodiments, the compound of formula I has formula I P :

[0441]

[0442] and its salts and solvates, wherein R LP is a linker for attachment to an antibody or antigen-binding fragment thereof described herein, wherein the linker is selected from:

[0443] (ia):

[0444]

[0445] in

[0446] Q P yes:

[0447] Where Q XP Make Q P is an amino acid residue, a dipeptide residue or a tripeptide residue;

[0448] X P yes:

[0449]

[0450] Where aP = 0 to 5, bP = 0 to 16, cP = 0 or 1, dP = 0 to 5;

[0451] G L is a linker for attachment to an antibody or antigen-binding fragment thereof described herein (e.g., a Ligand unit);

[0452] (ib):

[0453]

[0454] Where R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropene or cyclobutene group; and

[0455] e is 0 or 1.

[0456] aP can be 0, 1, 2, 3, 4, or 5. In some instances, aP is 0 to 3. In some of these instances, aP is 0 or 1. In other instances, aP is 0.

[0457] 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.

[0458] cP can be 0 or 1.

[0459] dP can be 0, 1, 2, 3, 4, or 5. In some instances, dP is 0 to 3. In some of these instances, dP is 1 or 2. In other instances, dP is 2.

[0460] In X PIn some examples of, aP is 0, cP is 1 and dP is 2, and bP can be 0 to 8. In some of these examples, bP is 0, 4 or 8.

[0461] For the compound of formula I, Q X The example can be applied to Q XP (e.g. where appropriate).

[0462] The above G for the compound having formula I L , R L1 , R L2 The examples of and e can be applied to compounds having formula I P of compounds.

[0463] In some embodiments, the conjugate of Formula IV has Formula IV P :

[0464] L–(D LP ) p (IV P )

[0465] or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or antigen-binding fragment thereof (eg, a Ligand unit) described herein, and D LP is a topoisomerase I inhibitor (eg, a drug linker unit) and has Formula III P :

[0466]

[0467] R LLP is a linker attached to the antibody or antigen-binding fragment thereof (e.g., a Ligand unit), wherein the linker is selected from

[0468] (ia'):

[0469]

[0470] Where Q P and X P As defined above, and G LL is a linker that is attached to an antibody or antigen-binding fragment thereof described herein (e.g., a Ligand unit); and

[0471] (ib'):

[0472]

[0473] Where R L1 and R L2 as defined above; and

[0474] p is an integer from 1 to 20.

[0475] In some embodiments, the compound of formula I has formula I P2 :

[0476]

[0477] and its salts and solvates, wherein R LP2 is a linker for attachment to an antibody or antigen-binding fragment thereof described herein, wherein the linker is selected from:

[0478] (ia):

[0479]

[0480] in

[0481] Q is:

[0482] Where Q X such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;

[0483] X P2 yes:

[0484]

[0485] Where aP2=0 to 5, b1P2=0 to 16, b2P2=0 to 16, cP2=0 or 1, dP2=0 to 5, wherein at least b1P2 or b2P2=0 (i.e., only one of b1 and b2 may not be 0);

[0486] G L is a linker for attachment to an antibody or antigen-binding fragment thereof described herein (e.g., a Ligand unit);

[0487] (ib):

[0488]

[0489] Where R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropene or cyclobutene group; and

[0490] e is 0 or 1.

[0491] aP2 can be 0, 1, 2, 3, 4, or 5. In some instances, aP2 is 0 to 3. In some of these instances, aP2 is 0 or 1. In other instances, aP2 is 0.

[0492] b1P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some instances, b1P2 is 0 to 12. In some of these instances, b1P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0493] b2P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some instances, b2P2 is 0 to 12. In some of these instances, b2P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0494] In some instances, only one of b1P2 and b2P2 may be non-zero.

[0495] cP2 can be 0 or 1.

[0496] dP2 can be 0, 1, 2, 3, 4, or 5. In some instances, dP2 is 0 to 3. In some of these instances, dP2 is 1 or 2. In other instances, dP2 is 2. In other instances, dP2 is 5.

[0497] In X P2 In some examples of X, aP2 is 0, b1P2 is 0, cP2 is 1 and dP2 is 2, and b2P2 can be 0 to 8. In some of these examples, b2P2 is 0, 2, 3, 4, 5 or 8. P2 In some examples of X, aP2 is 1, b2P2 is 0, cP2 is 0 and 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 of X, aP2 is 0, b1P2 is 0, cP2 is 0 and dP2 is 1, and b2P2 can be 0 to 8. In some of these examples, b2P2 is 0, 2, 3, 4, 5 or 8. P2 In some instances of, b1P2 is 0, b2P2 is 0, cP2 is 0 and one of aP2 and dP2 is 0. The other of aP2 and dP2 is 1 to 5. In some of these instances, the other of aP2 and dP2 is 1. In other of these instances, the other of aP2 and dP2 is 5.

[0498] For the compound of formula I, Q X Examples may be applicable to Formula Ia P2 Q X (e.g. where appropriate).

[0499] The above G for the compound having formula I L , RL1 , R L2 The examples of and e can be applied to compounds having formula I P2 of compounds.

[0500] In some embodiments, the conjugate of Formula IV has Formula IV P2 :

[0501] L–(D LP2 ) p (IV P2 )

[0502] or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or antigen-binding fragment thereof (eg, a Ligand unit) described herein, and D LP2 is a topoisomerase I inhibitor (eg, a drug linker unit) and has Formula III P2 :

[0503]

[0504] R LLP2 is a linker attached to the antibody or antigen-binding fragment thereof (e.g., a Ligand unit), wherein the linker is selected from

[0505] (ia'):

[0506]

[0507] Where Q and X P2 As defined above, and G LL is a linker attached to the antibody or antigen-binding fragment thereof; and

[0508] (ib'):

[0509]

[0510] Where R L1 and R L2 as defined above; and

[0511] p is an integer from 1 to 20.

[0512] Particularly suitable topoisomerase I inhibitors include those having the formula:

[0513]

[0514] and / or

[0515]

[0516] In some examples, the antibody molecules described herein are conjugated to a topoisomerase I inhibitor (eg, SG3932) having the formula:

[0517]

[0518] For the avoidance of doubt, the number "8" specifies that the structure within the square brackets is repeated eight times. Therefore, another representation of SG3932 is:

[0519]

[0520] Another representation of SG4010 is:

[0521]

[0522] Another representation of SG4057 is:

[0523]

[0524] Another representation of SG4052 is:

[0525]

[0526] Any of the antibodies or antigen-binding fragments thereof described herein may be conjugated to one or more of the topoisomerase I inhibitors.

[0527] Synthesis of topoisomerase I inhibitors

[0528] To accomplish this, certain general synthetic routes for preparing one or more exemplary topoisomerase I inhibitors will now be described.

[0529] A compound having formula I (wherein R L The compound having formula Ia) can be prepared from the compound having formula 2:

[0530]

[0531] (where R L* is -QH) by connecting a compound having Formula 3:

[0532]

[0533] or its activated version.

[0534] Such reactions can be carried out under amide coupling conditions.

[0535] The compound having formula 2 can be prepared by a compound having formula 4:

[0536]

[0537] (where R L*prot Yes-Q-Prot N , where Prot N is an amine protecting group).

[0538] The compound having formula 4 can be prepared by a compound having formula 5:

[0539]

[0540] The coupling with compound A3 was synthesized using Friedlander reaction.

[0541] The compound of formula 5 can be prepared from the compound of formula 6:

[0542]

[0543] Synthesized by removal of the trifluoroacetamide protecting group.

[0544] The compound of formula 6 can be synthesized by coupling: R L*prot -OH to compound I7.

[0545] A compound having formula I (wherein R L having formula Ia or Ib) can be obtained from compound I11 via compound R L -OH or its activated form.

[0546] Amine Protecting Groups:

[0547] Amine protecting groups are well known to those skilled in the art. Reference is made in particular to Greene's Protecting Groups in Organic Synthesis, 4th edition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pp. 696-871 for disclosure of suitable protecting groups.

[0548] Drug loading (p) is the average number of drugs (such as tubulysin or topoisomerase inhibitors) per antibody molecule. In the compositions disclosed herein, the range of drug loading is 1 to 20 drugs (D) per antibody molecule. For example, the range of drug loading can be 1 to 10 drugs (D) per antibody molecule, i.e., wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 drugs are covalently attached to the antibody molecule. The composition of the conjugate includes a set of antibody molecules conjugated with 1 to 10 drugs. In the case where the compound disclosed herein is bound to lysine, the range of drug loading can be 1 to 80 drugs (D) per antibody molecule, but the upper limit may preferably be 40, 20, 10 or 8. The composition of the conjugate includes a set of antibody molecules conjugated with 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8 drugs.

[0549] The average number of drugs per antibody in the conjugate preparation from the conjugate reaction can be characterized by conventional methods such as UV, reversed-phase HPLC, HIC, mass spectrometry, ELISA determination and electrophoresis. It is also possible to determine the quantitative distribution of the conjugate in terms of p. By ELISA, the mean value of p in a specific conjugate preparation can be determined (Hamblett, 2004; Sanderson, 2005). However, the distribution of p (drug) values ​​cannot be distinguished by antibody-antigen binding and detection limits of ELISA. In addition, the ELISA determination for detecting the conjugate cannot determine the position at which the drug moiety is attached to the antibody molecule, such as heavy chain or light chain fragments, or specific amino acid residues. In some instances, the separation, purification and characterization of homogeneous conjugates (wherein p is a determined value from a conjugate with other drug loadings) can be achieved by, for example, reversed-phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.

[0550] For some conjugates, p may be limited by the number of attachment sites on the antibody molecule. For example, an antibody molecule may have only one or a few cysteine ​​thiol groups, or may have only one or a few sufficiently reactive thiol groups through which a linker may be attached.

[0551] Typically, during the conjugation reaction, the drug conjugated to the antibody molecule is less than the theoretical maximum. The 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. Moreover, only the most reactive cysteine ​​thiol groups can react with thiol-reactive linker reagents. Typically, the antibody molecule does not contain many (if any) free and reactive cysteine ​​thiol groups that can be connected to the drug moiety. Most of the cysteine ​​thiol residues in the antibody molecule of the conjugate exist in the form of disulfide bridges and must be reduced with a reducing agent (such as dithiothreitol (DTT) or TCEP) under partial or full reducing conditions. The load (drug / antibody ratio) of the conjugate can be controlled in several different ways, including: (i) limiting the drug linker relative to the molar excess of the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reducing conditions for cysteine ​​thiol modification.

[0552] Some antibody molecules have reducible interchain disulfide bridges, i.e., cysteine ​​bridges. By treating with a reducing agent such as DTT (dithiothreitol), antibody molecules can be made reactive to be conjugated with a linker reagent. Therefore, each cysteine ​​bridge will theoretically form two reactive thiol nucleophiles. This process is also referred to as "classical conjugation", and is distinguished from methods such as conjugation occurring at cysteine ​​sites that have been engineered into specific sites in antibody molecules. By reacting lysine with 2-iminothiolane (Traut's reagent), additional nucleophilic groups can be introduced into antibodies, resulting in amine conversion to thiol.

[0553] By classical conjugation, by partially reducing the antibody, then using the desired linker-drug reaction to prepare an ADC with a drug randomly conjugated to a natural cysteine ​​residue. For example, the antibody portion at a concentration of 5 mg / mL can be partially reduced by adding about 3 molar equivalents of DTT at pH 8.0, followed by incubation for about 2 hours at about 37 ° C. The reduction reaction can then be cooled in ice, and excess DTT can be removed, for example, by diafiltration. The linker-drug can then be added with a linker-drug / thiol molar ratio of about 1:10. The conjugation reaction can be carried out in the presence of about 10% v / v DMSO. After conjugation, excess free cysteine ​​(exceeding about 2 times the molar ratio of the linker-drug) can be added to quench unreacted linkers-drugs to produce cysteine-linker-drug adducts. The reaction mixture can then be purified (for example, by hydrophobic interaction chromatography), and the buffer exchanged to PBS. Standard methods (such as hydrophobic interaction chromatography and reduced reverse phase chromatography) can be used to determine the drug loading distribution, as described elsewhere.

[0554] Methods for preparing conjugates using direct conjugation of solvent accessible thiols generated by reduction of interchain disulfide bridges of antibody molecules involving N-alkylmaleimides are known. Other methods use N-hydroxysuccinimide esters to conjugate drugs at the primary amines of lysine. Such methods are reviewed in, for example, Gébleux and Casi, Pharmacol Ther (2016) 167: 48-59, which is incorporated herein by reference in its entirety.

[0555] Separately or in addition to the above-mentioned classical conjugation methods, site-specific conjugation, i.e., methods in which the drug loading and conjugation sites are controlled, can also be used. This can be achieved, for example, by engineering cysteine ​​at specific residues, replacing residues with non-natural amino acids with bioorthogonal reactivity or enzyme ligation methods. One method of site-specific conjugation is described in Dimasi, 2017, which is incorporated herein by reference in its entirety, and involves inserting cysteine ​​into specific positions of antibody molecules.

[0556] Cysteine ​​amino acids can be engineered at reactive sites in antibody molecules and do not form intrachain or intermolecular disulfide bonds (Junutula, 2008; Dornan, 2009; US 7521541; US ​​7723485; WO2009 / 052249). Engineered cysteine ​​thiols can react with linkers or drug-linkers described herein having thiol-reactive electrophilic groups such as maleimide or α-haloamide to form conjugates with cysteine ​​engineered antibody molecules and drugs. Therefore, the position of the drug can be designed, controlled and known. Since the engineered cysteine ​​thiol groups usually react with thiol-reactive linker reagents or drug linker reagents in high yield, the drug loading can be controlled. By replacing a single site on the heavy chain or light chain, IgG antibodies are engineered to introduce cysteine ​​amino acids, giving two new cysteines on symmetrical antibodies. If necessary, a drug loading close to 2 can be achieved, where the conjugated product is close to homogeneity.

[0557] In some examples, the antibody molecule of the conjugate of the present disclosure comprises a CH region and the drug is chemically conjugated at a cysteine ​​amino acid inserted between positions 239 and 240 of the CH region, wherein the numbering of the constant region is according to the EU index. Thus, the connection between the antibody molecule and the drug can be through the inserted cysteine ​​amino acid and the terminal maleimide group on the linker.

[0558] Examples of a CH region comprising a cysteine ​​amino acid inserted between positions 239 and 240 of the CH region are SEQ ID NO: 43 and SEQ ID NO: 45. Examples of a heavy chain comprising a CH region comprising a cysteine ​​amino acid inserted between positions 239 and 240 of the CH region are SEQ ID NO: 50, 53 and 56.

[0559] In other examples, the antibody molecule of the conjugate does not include any amino acid residue inserted into the CH region. In a specific example, the antibody molecule of the conjugate does not include a cysteine ​​amino acid (e.g., between position 239 and 240, wherein the numbering of the constant region is according to the EU index) inserted into the CH region. As demonstrated in the examples (e.g., Example 12), in the case of using classical conjugation to conjugate the drug-linker to the native cysteine, the inserted cysteine ​​is not essential.

[0560] Examples of CH regions that do not include any amino acid residues inserted into the CH region are SEQ ID NOs: 44, 46, 63, and 64. Examples of heavy chains that include a CH region that does not include any amino acid residues inserted into the CH region are SEQ ID NOs: 51, 54, 57, 59, and 60.

[0561] When more than one nucleophilic or electrophilic group of an antibody molecule reacts with a drug-linker intermediate or linker reagent and then reacts with a drug reagent, the resulting product is a mixture of conjugated compounds, in which the drug attached to the antibody is distributed as, for example, 1, 2, 3, etc. Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) can separate compounds in a mixture by drug loading value. Preparations of conjugates with a single drug loading value (p) can be separated, however, these single loading value conjugates may still be heterogeneous mixtures because the drug can be attached to different sites on the antibody molecule via the linker.

[0562] Thus, the conjugate compositions of the present disclosure include mixtures of antibody-drug conjugate compounds, wherein the antibody has one or more drug moieties (e.g., tubulysin or a topoisomerase inhibitor) and wherein the drug moieties may be attached to the antibody molecule at various amino acid residues.

[0563] In some examples, the average number of tubulysin drug moieties per antibody molecule is in the range of 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, such as 1.9 to 2.

[0564] As described above, in some examples, the antibody molecule of the ADC may include one or more mutations in the CH region of the heavy chain to reduce or eliminate the binding of the antibody molecule to one or more Fcγ receptors. Therefore, the first heavy chain and / or the second heavy chain of the ADC described herein may include a phenylalanine (F) at position 234, a glutamic acid (E) at position 235, and a serine (S) at position 331, wherein the numbering is according to the EU index.

[0565] Functional properties of antibody molecules and conjugates

[0566] The antibody molecules and conjugates described herein can be characterized by reference to certain functional properties.

[0567] Binding affinity

[0568] The antibody molecules and conjugates described herein can 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 the antibody molecule to a cognate antigen (e.g., human, mouse, or cynomolgus 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 EGFR and a second antigen binding domain that binds c-Met. Alternatively, binding affinity can be determined using an antibody molecule that has monospecificity for EGFR or c-Met. In some instances, binding affinity is determined using BIACore as described in Example 2.1.

[0569] Binding affinity is usually measured by Kd (equilibrium dissociation constant between an antigen binding domain and its antigen). It is well known that the lower the Kd value, the higher the binding affinity of the antigen binding domain. For example, an antigen binding domain that binds to a target with a Kd of 10 nM will be considered to bind to the target with a higher affinity than an antigen binding domain that binds to the same target with a Kd of 100 nM.

[0570] Reference to human EGFR may refer to a polypeptide comprising the extracellular domain of EGFR, such as a polypeptide having the amino acid sequence set forth in SEQ ID NO:68. Reference to mouse EGFR may refer to a polypeptide produced by the molecule available from SinoBiological under catalog number 51091-M08H. Reference to cynomolgus monkey EGFR may refer to the amino acid sequence set forth in SEQ ID NO:69. Reference to human c-Met may refer to a polypeptide having the amino acid sequence set forth in SEQ ID NO:70. Reference to mouse c-Met may refer to a polypeptide having the amino acid sequence set forth in SEQ ID NO:90 or to a polypeptide produced by the molecule available from SinoBiological under catalog number 50622-M08H. Reference to cynomolgus monkey c-Met may refer to the amino acid sequence set forth in SEQ ID NO:71.

[0571] EGFR affinity

[0572] Antibody molecules and conjugates described herein may include a binding domain that binds to EGFR with low affinity. It is known that EGFR is expressed at low levels in normal tissues such as skin. It is expected that antibody molecules and conjugates that bind to EGFR with low affinity advantageously show reduced target-associated toxicity in normal tissues, while still being able to target tumors that express high levels of EGFR, thereby improving safety characteristics. In addition, as demonstrated herein, conjugates comprising the low-affinity EGFR binding domain are more effective in treating cancer than conjugates comprising a higher affinity EGFR binding domain.

[0573] The binding domain that binds EGFR can bind to human EGFR with an affinity having a Kd equal to or higher than 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, or 40 nM. Alternatively, the binding domain that binds EGFR can bind to human EGFR with a Kd of between 10 and 100 nM, between 20 and 100 nM, between 30 and 100 nM, between 40 and 100 nM, between 10 and 80 nM, between 20 and 80 nM, between 30 and 80 nM, between 40 and 80 nM, between 10 and 70 nM, between 20 and 70 nM, between 30 and 70 nM, between 40 and 70 nM, between 10 and 60 nM, between 20 and 60 nM, between 30 and 60 nM, between 40 and 60 nM, between 10 and 50 nM, between 20 and 50 nM, between 30 and 50 nM, or between 40 and 50 nM.

[0574] The binding domain that binds to EGFR can bind to human EGFR with an affinity lower than that of the binding domain comprising the heavy chain sequence and light chain sequence of the antibody molecule QD6 listed in SEQ ID NOs: 53 and 55, respectively.

[0575] For example, the binding domain that binds to EGFR can bind to human EGFR with an affinity Kd that is at least 2 times, 3 times, 4 times, 5 times, 6 times or 7 times higher than the Kd of the binding domain comprising the heavy chain sequence and light chain sequence of the antibody molecule QD6 listed in SEQ ID NO: 53 and 55, respectively, binding to human EGFR. Alternatively, the binding domain that binds EGFR can bind to human EGFR with an affinity Kd that is between 2 and 10 times higher, between 3 and 10 times higher, between 4 and 10 times higher, between 5 and 10 times higher, between 6 and 10 times higher, between 7 and 10 times higher, between 2 and 9 times higher, between 3 and 9 times higher, between 4 and 9 times higher, between 5 and 9 times higher, between 6 and 9 times higher, between 7 and 9 times higher, between 2 and 8 times higher, between 3 and 8 times higher, between 4 and 8 times higher, between 5 and 8 times higher, between 6 and 8 times higher, and between 7 and 8 times higher than the Kd of the binding domain comprising the heavy chain sequence and light chain sequence of antibody QD6 listed in SEQ ID NOs:53 and 55, respectively, binding to human EGFR.

[0576] The binding domain that binds to EGFR can bind to human EGFR with an affinity similar to that of the binding domain comprising the variable heavy region sequence and the variable light region sequence of the antibody molecule RAA22 listed in SEQ ID NOs: 16 and 20, respectively, binding to human EGFR. For example, the binding domain that binds to EGFR can bind to human EGFR with an affinity Kd that is less than 5 times, less than 4 times, less than 3 times, less than 2 times, less than 1 times, or less than 0.5 times different than the Kd of the binding domain comprising the variable heavy region sequence and the variable light region sequence of the antibody molecule RAA22 listed in SEQ ID NOs: 16 and 20, respectively, binding to human EGFR.

[0577] The binding domain in conjunction with EGFR can also be in conjunction with cynomolgus monkey EGFR.For example, the binding domain in conjunction with EGFR can be in conjunction with cynomolgus monkey EGFR with the affinity of the Kd having less than 700nM, less than 600nM, less than 500nM, less than 400nM, less than 300nM or less than 250nM.Alternately, the antigen binding domain in conjunction with EGFR can be in conjunction with cynomolgus monkey EGFR with the affinity of Kd between 100 and 700nM, between 100 and 600nM, between 100 and 500nM, between 100 and 400nM, between 100 and 300nM, between 150 and 250nM, between 100 and 200nM. The antigen binding domain that binds EGFR can bind to cynomolgus monkey EGFR with a Kd that is less than or equal to 10, 9, 8, 7, 6, 5, 4, or 3 times higher than the Kd of the binding domain that binds to human EGFR.

[0578] The binding domain in conjunction with EGFR can also be in conjunction with mouse EGFR.For example, the binding domain in conjunction with EGFR can be in conjunction with mouse EGFR with an affinity of the Kd having less than 1 μ M, less than 900nM, less than 800nM, less than 700nM, less than 600nM or less than 650nM.Alternately, the binding domain in conjunction with EGFR can be in conjunction with mouse EGFR with a Kd between 100nM and 1 μ M, between 200 and 900nM, between 300 and 800nM, between 400 and 700nM, between 400 and 600nM or between 450 and 550nM.

[0579] In some instances, the binding domain in conjunction with EGFR can be in conjunction with human EGFR and cynomolgus monkey EGFR.This cross-reactivity is advantageous because it allows the dosage and safety testing of antibody molecules and conjugates to be carried out in cynomolgus monkeys during preclinical development.In some instances, the binding domain in conjunction with EGFR can be in conjunction with human EGFR, cynomolgus monkey EGFR and mouse EGFR.For example, the binding domain in conjunction with EGFR can be in conjunction with human EGFR, cynomolgus monkey EGFR and mouse EGFR with Kd value (such as human EGFR Kd between 10 and 100nM, cynomolgus monkey EGFR Kd between 100 and 700nM and mouse EGFR Kd between 100nM and 1 μM) as described above.

[0580] cMET affinity

[0581] The binding domain that binds cMET can bind human cMET with an affinity having a Kd of less than 20 nM, 15 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, or 2.5 nM. Alternatively, the antigen binding domain that binds cMET can bind human c-Met with an affinity having a Kd of between 1 and 20 nM, between 1 and 15 nM, between 1 and 10 nM, between 1 and 9 nM, between 1 and 8 nM, between 1 and 7 nM, between 1 and 6 nM, between 1 and 5 nM, between 1 and 4 nM, between 1 and 3 nM, between 1 and 2.5 nM, or between 2 and 2.5 nM.

[0582] The binding domain that binds cMET can bind to cynomolgus monkey cMET. For example, the binding domain that binds cMET can bind to cynomolgus monkey cMET with an affinity having a Kd of less than 30nM, 25nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, or 2.5nM. Alternatively, the binding domain that binds cMET can bind to cynomolgus monkey cMET with an affinity having a Kd between 1 and 20nM, between 1 and 15nM, between 1 and 10nM, between 1 and 9nM, between 1 and 8nM, between 1 and 7nM, between 1 and 6nM, between 1 and 5nM, between 1 and 4nM, between 1 and 3nM, between 1 and 2.5nM, or between 2 and 2.5nM. The binding domain that binds cMET can bind to cynomolgus monkey cMET, and its Kd is less than or equal to 10, 9, 8, 7, 6, 5, 4, 3, 2, and 1 times higher than the Kd of the antigen binding domain binding to human c-Met.

[0583] In some examples, the binding domain that binds cMET is capable of binding to human cMET and cynomolgus monkey cMET. This cross-reactivity is advantageous because it allows for dosage and safety testing of the antibody molecule in cynomolgus monkeys during preclinical development. For example, the binding domain that binds cMET may be capable of binding to human cMET and cynomolgus monkey cMET at the Kd values ​​described above (e.g., a Kd for human cMET between 1 and 20 nM and a Kd for cynomolgus monkey cMET between 1 and 20 nM).

[0584] Specific binding

[0585] The binding domains described herein can specifically bind to their respective targets (i.e., EGFR and cMET). The term "specificity" may refer to situations where an antigen binding domain will not exhibit any significant binding to molecules other than its specific binding partner (here EGFR or cMET). Such molecules are referred to as "non-target molecules". The term "specificity" also applies to situations where an antibody molecule is specific for a particular epitope, such as an epitope on EGFR or cMET carried by many antigens, in which case the antibody molecule will be able to bind to various antigens carrying the epitope.

[0586] In some examples, an antibody molecule is considered to not exhibit any significant binding to a non-target molecule if the extent of binding to the non-target molecule is less than about 10% of the binding of the binding molecule to the target, as measured, for example, by ELISA, SPR, biomembrane interferometry (BLI), microthermophoresis (MST), or by radioimmunoassay (RIA). Alternatively, binding specificity can be reflected in terms of binding affinity, wherein 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 the affinity for another non-target molecule. In some examples, the antibody molecules of the present disclosure bind to EGFR and / or cMET with an affinity that is at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0 orders of magnitude greater than the affinity for another non-target molecule.

[0587] EGFR is a member of the ErbB family of receptors, which is a subfamily of four closely related receptor tyrosine kinases: EGFR, HER2, HER3, and HER4. The RAA22 antigen binding domain does not show binding to HER2, HER3, and HER4, indicating that the antigen binding domain specifically binds to EGFR. Thus, in specific examples, the antigen binding domain that binds to EGFR does not bind to HER2, HER3, or HER4, or does not show any significant binding to HER2, HER3, or HER4.

[0588] cMET is a member of the receptor tyrosine kinase subfamily, including Ron and Sema 4a. The B09-GL antigen binding domain does not bind to Ron and Sema 4a, indicating that the antigen binding domain specifically binds to cMET. Therefore, in a specific example, the binding domain that binds to cMET does not bind to Ron, Sema 4a, or does not show any significant binding to Ron, Sema 4a.

[0589] Simultaneous Engagement

[0590] The antibody molecules and conjugates described herein are characterized in that the two binding domains are capable of simultaneously engaging their respective EGFR and cMET targets. Antibody molecules and conjugates having simultaneous engagement of EGFR and cMET are expected to be advantageous because many tumors are known to express both EGFR and c-Met simultaneously and can therefore be targeted by the antibody molecules of the present disclosure. Thus, in some instances, the antibody molecule is capable of simultaneously engaging EGFR and cMET.

[0591] For example, simultaneous engagement can be determined by an in vitro cytotoxicity assay using a cell line expressing approximately equal amounts of EGFR and cMET and a conjugate comprising an antibody molecule having EGFR and cMET antigen binding domains. If the individual antigen binding domains in the conjugate function independently to deliver the drug, it is expected that blocking either target in the cell line will only moderately reduce the activity of the conjugate, shifting the IC50 by 2-fold or less, because the targets are present at similar levels. The EGFR and cMET targets can be blocked in this assay by using, for example, a monospecific antibody molecule that binds to the same region on EGFR or cMET but lacks a drug capable of inducing cytotoxicity. For example, the monospecific antibody molecule may contain the same antigen binding domain that binds to EGFR as the conjugate being tested, or the same antigen binding domain that binds to c-Met. On the other hand, if the conjugate requires simultaneous engagement to effectively deliver the conjugate into cells, blocking either target will likely have a greater effect on the activity of the conjugate. That is, an antibody molecule is considered capable of simultaneously engaging two targets if the IC50 shifts by at least 2-fold, at least 5-fold, or at least 10-fold following blocking of either target using this assay.

[0592] Another method to determine simultaneous binding is to compare the activity of the bispecific EGFR-cMET conjugate to a monovalent monospecific control conjugate in an in vitro cytotoxicity assay. The control conjugate comprises one antigen binding domain to either EGFR or c-Met and one non-binding isotype antibody control antigen binding domain. If each antigen binding domain in the bispecific conjugate acts independently, the expected result would be that each monospecific control conjugate would be only moderately less potent than the bispecific conjugate, and the differences would be additive. Alternatively, if the two antigen binding domains of the bispecific conjugate act synergistically by binding simultaneously, the activity of the bispecific conjugate is expected to differ significantly from the monospecific control antibody. That is, if the bispecific conjugate results in a change in IC50 that is greater than the sum of the changes in IC50 observed using the monospecific control conjugates, the antibody molecule is considered to be able to simultaneously bind to two targets.

[0593] Further details of these methods for measuring simultaneous engagement can be found in the Examples.

[0594] Antibody internalization

[0595] The antibody molecules and conjugates described herein can be characterized by their ability to mediate efficient internalization. This is particularly useful for conjugates because it ensures that the conjugate is internalized into the cell and delivered to the lysosomes, where the antibody molecule is subsequently degraded and the drug is released into the cell where it exerts its cellular effect, such as cytotoxicity.

[0596] The internalization of antibody molecules can be analyzed by contacting living cells with antibody molecules and detecting antibody molecules after a time period sufficient for internalization. Internalization can be determined by detecting the location of the antibody molecules. If the antibody molecules remain on the cell surface (e.g., detected on the cell surface, and / or not detected in the cell), it is determined that the antibody molecules are not internalized. If the antibody molecules are detected in the cell (e.g., located in the cytoplasm or organelles), it is determined that the antibody molecules have been internalized.

[0597] The exemplary method that shows whether antibody molecule can mediate effective internalization relates to labeling antibody molecule with pH sensitive dye, and these dyes show fluorescence under acidic pH, and the antibody molecule of these marks is added to the cell. Cell internalization can be detected by monitoring fluorescence. If in a certain period of time (for example 48 hours), the fluorescence observed is greater than the fluorescence of the non-binding control antibody molecule of mark, it is believed that antibody molecule can mediate internalization and be delivered to lysosome. The more detailed information of this method of showing antibody internalization can be found in the example.

[0598] The antibody molecules described herein are characterized in that they are able to mediate more efficient internalization 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 and can minimize the effects of the antibody molecules in normal tissues that do not exhibit significant levels of co-expression.

[0599] In vitro activity

[0600] The antibody molecules described herein may be characterized by their cytotoxic activity, i.e., their ability to kill cells. Cytotoxic activity may be determined using in vitro cell viability assays such as In some instances, the cell is a cell that expresses both EGFR and cMET.

[0601] The potency of an antibody molecule can be expressed as an IC50 value. IC50 is the half-inhibitory concentration of an antibody molecule. In a functional assay, IC50 is the concentration that reduces a biological response by 50% of its maximum value. IC50 can be calculated by any number of means known in the art.

[0602] In some instances, the antibody molecules with cytotoxic activity described herein have an IC50 of less than 4000pM, less than 3500pM, less than 3000pM, less than 2500pM, less than 2000pM, less than 1500pM, less than 1000pM, less than 500pM, less than 400pM, less than 300pM, less than 250pM, less than 200pM, less than 150pM, or less than 100pM when measured using an in vitro cell viability assay. In some instances, the antibody molecules described herein can have an IC50 between 60 and 500pM.

[0603] In some instances, the antibody molecules described herein are capable of increasing the killing of cells (e.g., tumor cells) expressing significant amounts of both EGFR and cMET compared to cells expressing one or the other of low levels of EGFR and cMET. Cells expressing significant amounts of both EGFR and cMET can be determined by measuring the relative receptor density on the cell surface. For example, cells that can express EGFR and cMET at a relative receptor density greater than 15,000 on the cell surface can be considered to be cells expressing significant amounts of both EGFR and cMET, as well as cells expressing one of EGFR and c-Met at a low relative receptor density of 15,000 or less on the cell surface. Relative EGFR and cMET densities can be measured, for example, using the Quantum MESF quantitative FACS assay kit, as described in the examples.

[0604] Examples of cells expressing significant amounts of both EGFR and cMET may include NCI H596, HCC 827GRPool, A549, NCIH1792, NCIH1975, NCIH292, and NCIH358 cell lines. Examples of cells expressing one of EGFR and cMET at low relative receptor density may include A427, NCI H23, and NCI H661 (Ag negative) cell lines. These cell lines may be obtained through ATCC.

[0605] EGFR tyrosine kinase inhibitors

[0606] EGFR TKIs can be characterized as first-generation, second-generation, or third-generation EGFR TKIs, as described below.

[0607] First-generation EGFR TKIs are reversible inhibitors of EGFR with activating mutations that do not significantly inhibit EGFR with the T790M mutation. Examples of first-generation TKIs include gefitinib and erlotinib.

[0608] Second-generation EGFR TKIs are irreversible inhibitors of EGFR with activating mutations, which do not significantly inhibit EGFR with the T790M mutation. Examples of second-generation TKIs include afatinib and dacomitinib.

[0609] The third-generation EGFR TKI is an inhibitor of EGFR with activating mutations, which also significantly inhibits EGFR with T790M mutation but not wild-type EGFR. Examples of third generation TKIs include compounds having formula (V), osimertinib, AZD3759 (zorifertinib), lazertinib, nazartinib (EGF816), CO1686 (rociletinib), HM61713, ASP8273 (naquotinib), PF-06747775 (mavelertinib), avitinib (abivertinib), alflutinib (AST2818) and CX-101 (olafertinib; RX-518), almonertinib (HS-10296; aumolertinib) and BPI-7711 (rezivertinib).

[0610] In one aspect, 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.

[0611] In one aspect, the EGFR TKI is a second generation EGFR TKI. In a further aspect, the second generation EGFR TKI is selected from dacomitinib or a pharmaceutically acceptable salt thereof and afatinib or a pharmaceutically acceptable salt thereof.

[0612] In one aspect, EGFR TKI is a third generation EGFR TKI. In another aspect, the third generation EGFR TKI is a compound having formula (V) as defined below. In another aspect, 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, avitinib 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 and BPI-7711 or a pharmaceutically acceptable salt thereof. In another aspect, the third generation EGFR TKI is osimertinib or a pharmaceutically acceptable salt thereof.

[0613] Compounds having formula (V)

[0614] In one aspect, the EGFR TKI is a compound having formula (V):

[0615]

[0616] in:

[0617] 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;

[0618] R 1 is selected from hydrogen, fluorine, chlorine, methyl and cyano;

[0619] R 2 is selected from the group consisting of methoxy, trifluoromethoxy, ethoxy, 2,2,2-trifluoroethoxy and methyl;

[0620] R 3selected from (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]octan-2-yl, (3aR,6aR)-5-methylhexa Hydrogen-pyrrolo[3,4-b]pyrrol-1(2H)-yl, 1-methyl-1,2,3,6-tetrahydropyridin-4-yl, 4-methylpiperazin-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-aminopropionyl]piperazin-1-yl;

[0621] R 4 is selected from hydrogen, 1-piperidinylmethyl and N,N-dimethylaminomethyl;

[0622] R 5 independently selected from methyl, ethyl, propyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, fluoro, chloro and cyclopropyl;

[0623] X is CH or N; and

[0624] n is 0, 1, or 2;

[0625] or a pharmaceutically acceptable salt thereof.

[0626] In a further aspect, there is provided a compound of formula (V) as defined above, wherein G is selected from indol-3-yl and indazol-1-yl; R 1 is selected from hydrogen, fluorine, chlorine, 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; 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.

[0627] Examples of compounds having 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.

[0628] Osimertinib and its pharmaceutical composition

[0629] Osimertinib has the following chemical structure:

[0630]

[0631] The chemical name of the free base of osimertinib is known: 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 WO2013 / 014448. Osimertinib is also known as AZD9291.

[0632] Osimertinib may be present in the form of a mesylate salt as follows: 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 TM .

[0633] Osimertinib mesylate is currently approved as a once-daily oral tablet formulation at a dose of 80 mg (expressed as free base, equivalent to 95.4 mg osimertinib mesylate) for the treatment of metastatic EGFR T790M mutation-positive NSCLC patients. If the dose needs to be modified, a 40 mg once-daily oral tablet formulation (expressed as free base, equivalent to 47.7 mg osimertinib mesylate) can be used. The core contains a drug diluent (such as mannitol and microcrystalline cellulose), a disintegrant (such as low-substituted hydroxypropyl cellulose) and a lubricant (such as sodium stearyl fumarate). The tablet formulation is described in WO 2015 / 101791.

[0634] Thus, in one aspect, osimertinib or a pharmaceutically acceptable salt thereof is in the form of a methanesulfonate salt, i.e., N-(2-{2-dimethylaminoethyl-methylamino}-4-methoxy-5-{[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino}phenyl)prop-2-enamide methanesulfonate.

[0635] In one aspect, osimertinib or a pharmaceutically acceptable salt thereof is administered once daily. In another aspect, osimertinib mesylate is administered once daily.

[0636] In one aspect, the total daily dose of osimertinib is about 80 mg. In another aspect, the total daily dose of osimertinib mesylate is about 95.4 mg.

[0637] In one aspect, the total daily dose of osimertinib is about 40 mg. In another aspect, the total daily dose of osimertinib mesylate is about 47.7 mg.

[0638] In one aspect, osimertinib or a pharmaceutically acceptable salt thereof is in the form of a tablet.

[0639] In one aspect, 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 another aspect, the composition comprises one or more pharmaceutical diluents (e.g., mannitol and microcrystalline cellulose), one or more pharmaceutical disintegrants (e.g., low-substituted hydroxypropyl cellulose), or one or more pharmaceutical lubricants (e.g., sodium stearyl fumarate).

[0640] In one aspect, the composition is in the form of a tablet, wherein the core comprises: (a) from 2 to 70 parts of osimertinib or a pharmaceutically acceptable salt thereof; (b) from 5 to 96 parts of two or more drug diluents; (c) from 2 to 15 parts of one or more drug disintegrants; and (d) from 0.5 to 3 parts of one or more drug lubricants; and wherein all parts are by weight, and the sum of the parts (a) + (b) + (c) + (d) = 100.

[0641] In one aspect, the composition is in the form of a tablet, wherein the core comprises: (a) from 7 to 25 parts of osimertinib or a pharmaceutically acceptable salt thereof; (b) from 55 to 85 parts of two or more drug diluents, wherein the drug diluents comprise microcrystalline cellulose and mannitol; (c) from 2 to 8 parts of a drug disintegrant, wherein the drug disintegrant comprises low-substituted hydroxypropyl cellulose; (d) from 1.5 to 2.5 parts of a drug lubricant, wherein the drug lubricant comprises sodium stearyl fumarate; and wherein all parts are by weight, and the sum of the parts (a) + (b) + (c) + (d) = 100.

[0642] In one aspect, the composition is in the form of a tablet, wherein the core comprises: (a) about 19 parts of osimertinib mesylate; (b) about 59 parts of mannitol; (c) about 15 parts of microcrystalline cellulose; (d) about 5 parts of low-substituted hydroxypropyl cellulose; and (e) about 2 parts of sodium stearyl fumarate; and wherein all parts are by weight, and the sum of the parts (a) + (b) + (c) + (d) + (e) = 100.

[0643] AZD3759 (Zolitinib)

[0644] AZD3759 has the following chemical structure:

[0645]

[0646] 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.

[0647] In one aspect, AZD3759 or a pharmaceutically acceptable salt thereof is administered twice daily.In a further aspect, AZD3759 is administered twice daily.

[0648] In one aspect, the total daily dose of AZD3759 is about 400 mg. In a further aspect, about 200 mg of AZD3759 is administered twice daily.

[0649] Lazertinib

[0650] Lazertinib has the following chemical structure:

[0651]

[0652] The chemical name of the free base of lazertinib is known as 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.

[0653] In one aspect, lazertinib or a pharmaceutically acceptable salt thereof is administered once daily. In another aspect, lazertinib is administered once daily.

[0654] In one aspect, the total daily dose of lazertinib is about 20 to 320 mg.

[0655] In one aspect, the total daily dose of lazertinib is about 240 mg.

[0656] Avitinib

[0657] Avitinib has the following chemical structure:

[0658]

[0659] The chemical name of the free base of Avitinib is known as: 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 US2014038940. Avitinib is also known as Avitinib.

[0660] In one aspect, avitinib or a pharmaceutically acceptable salt thereof is administered twice daily. In another aspect, avitinib maleate is administered twice daily.

[0661] In one aspect, the total daily dose of avitinib maleate is about 600 mg.

[0662] Afatinib

[0663] Afatinib has the following chemical structure:

[0664]

[0665] The chemical name of the free base of Afatinib is known as: N-{2-{[2-(dimethylamino)ethyl](methyl)amino}-6-(2,2,2-trifluoroethoxy)-5-{[4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl]amino}pyridin-3-yl}acrylamide. Afatinib is disclosed in WO 2016 / 15453. Afatinib is also known as AST2818.

[0666] In one aspect, afatinib or a pharmaceutically acceptable salt thereof is administered once daily. In another aspect, afatinib mesylate is administered once daily.

[0667] In one aspect, the total daily dose of afatinib mesylate is about 80 mg.

[0668] In one aspect, the total daily dose of afatinib mesylate is about 40 mg.

[0669] Afatinib

[0670] Afatinib has the following chemical structure:

[0671]

[0672] The chemical name of the free base of afatinib is known as: 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.

[0673] In one aspect, afatinib or a pharmaceutically acceptable salt thereof is administered once daily. In another aspect, afatinib dimaleate is administered once daily.

[0674] In one aspect, the total daily dose of afatinib dimaleate is about 40 mg.

[0675] In one aspect, the total daily dose of afatinib dimaleate is about 30 mg.

[0676] CX-101 (Olavutinib; RX-518)

[0677] CX-101 has the following chemical structure:

[0678]

[0679] The chemical name of the free base of CX-101 is known as: 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.

[0680] HS-10296 (almonertinib, aumolertinib)

[0681] HS-10296 (almonertinib, aumolertinib) has the following chemical structure:

[0682]

[0683] The chemical name of the free base of HS-10296 is known as: 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.

[0684] In one aspect, the total daily dose of HS-10296 is about 110 mg.

[0685] BPI-7711 (rivulitinib)

[0686] BPI-7711 has the following chemical structure:

[0687]

[0688] The chemical name of the free base of BPI-7711 is known as: 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.

[0689] In one aspect, the total daily dose of BPI-7711 is about 180 mg.

[0690] Dacomitinib

[0691] Dacomitinib has the following chemical structure:

[0692]

[0693] The chemical name of the free form of dacomitinib is known: (2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-7-methoxyquinazolin-6-yl}-4-(piperidin-1-yl)but-2-enamide. Dacomitinib is described in WO 2005 / 107758. Dacomitinib is also known as PF-00299804.

[0694] Dacomitinib can exist in the form of a dacomitinib monohydrate, namely (2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-7-methoxyquinazolin-6-yl}-4-(piperidin-1-yl)but-2-enamide monohydrate.

[0695] In one aspect, dacomitinib or a pharmaceutically acceptable salt thereof is administered once daily. In another aspect, dacomitinib monohydrate is administered once daily.

[0696] In one aspect, the total daily dose of dacomitinib monohydrate is about 45 mg.

[0697] In one aspect, dacomitinib or a pharmaceutically acceptable salt thereof is in the form of a tablet.

[0698] In one aspect, dacomitinib or a pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients. In another aspect, the one or more pharmaceutically acceptable excipients comprise lactose monohydrate, microcrystalline cellulose, sodium starch glycolate, and magnesium stearate.

[0699] Icotinib

[0700] Icotinib has the following chemical structure:

[0701]

[0702] The chemical name of the free base of icotinib is known to be: N-(3-ethynylphenyl)-2,5,8,11-tetraoxa-15,17-diazatricyclo[10.8.0.0 14,19 ] Eicosapenta-1(12),13,15,17,19-pentaen-18-amine. Icotinib is disclosed in WO 2013064128. Icotinib is also known as Conmana.

[0703] In an example, icotinib or a pharmaceutically acceptable salt thereof is administered three times daily. In another example, icotinib hydrochloride is administered three times daily.

[0704] In an example, the total daily dose of icotinib hydrochloride is about 375 mg.

[0705] Gefitinib

[0706] Gefitinib has the following chemical structure:

[0707]

[0708] The chemical name of the free base of gefitinib is known as: 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 TM .

[0709] In an example, gefitinib or a pharmaceutically acceptable salt thereof is administered once daily. In another example, gefitinib is administered once daily.

[0710] In an example, the total daily dose of gefitinib is about 250 mg.

[0711] Erlotinib

[0712] Erlotinib has the following chemical structure:

[0713]

[0714] The chemical name of the free base of erlotinib is known as: N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine. Erlotinib is disclosed in WO 1996 / 030347. Erlotinib is also known as TARCEVA TM .

[0715] In an example, erlotinib or a pharmaceutically acceptable salt thereof is administered once daily. In another example, erlotinib is administered once daily.

[0716] In an example, the total daily dose of erlotinib is about 150 mg.

[0717] In an example, the total daily dose of erlotinib is about 100 mg.

[0718] Combination therapy

[0719] As explained above, co-expression of EGFR and cMET is associated with many cancer types, and antibody molecules targeting both molecules, and conjugates comprising such antibody molecules in particular, provide opportunities for broad clinical benefits in a variety of indications. Therefore, the combination of EGFR TKI and antibody molecules as described herein is expected to be useful for therapeutic applications, particularly in cancer treatment.

[0720] EGFR TKI and / or antibody molecules as described herein can be used in methods of treating the human or animal body. Related aspects of the disclosure provide:

[0721] (i) an EGFR TKI as described herein for use in a method of treating cancer, wherein in the method the EGFR TKI is administered in combination with an antibody molecule as described herein,

[0722] (ii) an antibody molecule as described herein for use in a method for treating cancer, wherein in the method the antibody molecule is administered in combination with an EGFR TKI as described herein,

[0723] (iii) use of an EGFR TKI as described herein in the manufacture of a medicament for treating cancer, wherein in the treatment, the EGFR TKI is administered in combination with an antibody molecule as described herein;

[0724] (iv) use of an antibody molecule described herein in the manufacture of a medicament for treating cancer, wherein in the treatment, the antibody molecule is administered in combination with an EGFR TKI described herein;

[0725] (v) a method of treating cancer in an individual, wherein the method comprises administering to the individual a therapeutically effective amount of an antibody molecule as described herein in combination with a therapeutically effective amount of an EGFR TKI as described herein;

[0726] (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 anti-EGFR / cMET antibody molecule, wherein the first amount and the second amount together constitute a therapeutically effective amount.

[0727] Also provided herein are pharmaceutical combinations of the EGFR / cMET antibody molecules described herein and the EGFR TKI described herein. A pharmaceutical combination refers to a composition comprising a therapeutically effective amount of the EGFR / cMET antibody molecules described herein and a therapeutically effective amount of the EGFR TKI described herein and one or more pharmaceutically acceptable carriers, wherein each active ingredient is intended to be administered to a patient in combination.

[0728] As used herein, "combination therapy" refers to administering to an individual both i) an antibody molecule described herein (which may be conjugated to a drug as described herein); and ii) an EGFR TKI described herein.

[0729] The individual can be a patient. In some instances, the patient is a human patient.

[0730] Treatment can be any treatment or therapy in which some desired therapeutic effect is achieved, e.g., inhibiting or delaying progression of a disorder, and includes reducing the rate of progression, halting the rate of progression, ameliorating a disorder, curing or alleviating a disorder (whether partial or complete), preventing, ameliorating, delaying, alleviating or arresting one or more symptoms and / or signs of a disorder, or prolonging the survival of an individual or patient beyond that expected in the absence of treatment.

[0731] Application

[0732] The antibody molecules, conjugates and EGFR TKI will be administered in the form of a pharmaceutical composition which may comprise at least one component in addition to the active agent.

[0733] Pharmaceutical composition can also include pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials well known to those skilled in the art in addition to antibody molecules, conjugates or EGFR TKI. As used herein, the term "pharmaceutically acceptable" relates to compounds, materials, compositions and / or dosage forms, which are applicable to contact with the tissues of subjects (e.g., people) within the scope of reasonable medical judgment without excessive toxicity, stimulation, allergic reactions or other problems or complications, commensurate with reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with other components of preparation. The exact nature of carrier or other materials will depend on the route of administration, which can be by infusion, injection or any other suitable route, as described below.

[0734] Administration can be a "therapeutically effective amount", which is sufficient to show benefit to the individual. The actual amount administered and the rate and time course of administration will depend on the nature and severity of the substance being treated, the specific individual being treated, the individual's clinical condition, the cause of the disorder, the delivery site of the composition, the type of antibody molecule, the method of administration, the schedule of administration, and other factors known to medical practitioners. The prescription of treatment, such as the decision on dosage, etc., is within the responsibility of ordinary doctors and other doctors, and may depend on the severity and / or progression of the symptoms of the disease being treated. Appropriate doses of EGFR TKI are known in the art, and exemplary doses thereof are described in the above chapters on EGFR TKI. Appropriate doses of antibody molecules are well known in the art (Ledermann, 1991; and Bagshawe, 1991). The specific doses of antibody molecules suitable for administration as indicated in this article or Physician's Desk Reference (2003) can be used. The therapeutically effective amount or suitable dose of the antibody molecule can be determined by comparing the in vitro activity and in vivo activity in animal models. The method of extrapolating the effective dose in mice and other experimental animals to humans is known. The exact dosage will depend on many factors, including the size and location of the area to be treated, and the exact nature of the antibody molecule. The antibody molecule can be administered, for example, daily, weekly, biweekly, or monthly. In some instances, the EGFR TKI is administered as a first amount and the anti-EGFR / cMET antibody molecule is administered as a second amount, wherein the first amount and the second amount together constitute a therapeutically effective amount.

[0735] The EGFR TKI may be administered to the individual simultaneously, sequentially or separately with the administration of the antibody molecule. If 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 preparation.

[0736] The combination therapy (ie, antibody molecule and EGFR TKI) may be administered in combination with a third therapy that is administered to the individual simultaneously, sequentially or separately from the combination therapy. The third therapy may comprise chemotherapy, radiotherapy, another (different) antibody molecule or another (different) EGFR TKI.

[0737] cancer

[0738] The cancer to be treated using the combination therapy as described herein can be selected from the group consisting of lung cancer (such as non-small cell lung cancer (NSCLC)), pancreatic cancer, breast cancer, colorectal cancer, renal cancer, gastric cancer, head and neck cancer, ovarian cancer or glioblastoma.

[0739] The cancer may be one that expresses both EGFR and cMET. Cancer cells may express EGFR and cMET on the cell surface. In one example, it may have been determined that a tumor co-expresses EGFR and cMET. Methods for determining target expression are known in the art and include, for example, immunohistochemistry.

[0740] In some examples, the cancer to be treated using 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 to be treated is non-small cell lung cancer (NSCLC). In one example, the cancer is squamous cell carcinoma of the head and neck (SCCHN).

[0741] In some examples, 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.

[0742] In some instances, the cancer to be treated is an EGFR mutant cancer (also referred to as "EGFR mutation positive"), such as EGFR mutant NSCLC. Exemplary EGFR mutations that may be associated with cancer (such as mutations that activate EGFR) 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 elevated tyrosine kinase activity, formation of receptor homodimers and heterodimers, enhanced ligand binding, etc.). The mutation may be located in any portion of the EGFR gene or in a regulatory region associated with the EGFR gene, and includes mutations in exons 18, 19, 20, or 21. In some instances, an EGFR mutant 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.

[0743] In NSCLC, specific mutations in the EGFR gene are associated with high response rates to EGFR TKIs. The single point mutation leucine-858 to arginine (L858R) in exon 21 and variable deletions of at least three amino acid residues in exon 19 are often collectively referred to as “classical” EGFR-activating mutations and account for the vast majority (85%–90%) of all observed EGFR kinase domain mutations in NSCLC (Vyse and Huang, 2019). Examples of reported EGFR exon 19 deletions include delE746-A750, delE746-T751, delL747-E749, delL747-P753, and delL747-T751. In some instances, an EGFR mutant cancer is a cancer that has an L858R mutation in the EGFR gene and / or one or more deletions in exon 19 (e.g., EGFR mutation-positive NSCLC). In some instances, EGFR mutant cancer is a cancer with one or more deletions and / or L858R mutations in exon 19 in the EGFR gene (e.g., EGFR mutation-positive NSCLC). In some instances, EGFR mutant cancer is a cancer with L858R mutations in the EGFR gene and one or more deletions in exon 19 (e.g., EGFR mutation-positive NSCLC). In some instances, EGFR mutant cancer is a cancer with one or more deletions in exon 19 in the EGFR gene (e.g., EGFR mutation-positive NSCLC). In some instances, EGFR mutant cancer is a cancer with L858R mutations in the EGFR gene (e.g., EGFR mutation-positive NSCLC).

[0744] Although most NSCLC patients with EGFR mutations initially respond to EGFR TKI therapy, almost all patients treated with first-generation EGFR TKIs acquire resistance after a progression-free period of approximately 10 months. A secondary point mutation replacing threonine with methionine at amino acid position 790 (T790M) is a molecular mechanism for generating resistant variants targeting kinases. The T790M mutation is present in approximately half of lung cancer patients with acquired resistance to first- and second-generation EGFR TKIs and is reported to work by increasing the receptor's affinity for adenosine triphosphate relative to its affinity for TKIs (Suda, 2009).

[0745] Not all activating EGFR mutations are intrinsically sensitive to EGFR inhibitors. In-frame base pair insertions in exon 20 also lead to constitutive activation of EGFR, but unlike classical activating EGFR mutations, EGFR exon 20 insertions are associated with denovo resistance to currently clinically available EGFR inhibitors. Low response rates of erlotinib, gefitinib, and the second-generation EGFR inhibitor afatinib between 3%–8% have been reported in patients with EGFR exon 20 insertion mutation 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-M766insX, A767-S768 insX, S768-V769 insX, V769-D770insX, D770-N771 insX, N771-P772insX, P772-H773 insX, H773-V774 insX, V774-C775 insX, where insX indicates an in-frame insertion between 1-7 amino acids.

[0746] As demonstrated herein, combination therapy involving the anti-EGFR / cMET conjugates described herein and osimertinib (third generation EGFR TKI) showed effective tumor inhibition in a range of different mutant EGFR cancer models (including those containing L858R mutations, exon 20 insertions, and exon 19 deletions with T790M mutations). Therefore, it is expected that the combination therapy described herein will be effective in treating a range of different EGFR mutant cancers in humans.

[0747] In some instances, the treated patient has previously been treated with previous anti-cancer therapy (e.g., previous EGFR TKI). In some instances, disease progression has occurred in human patients during or after previous EGFR TKI treatment, that is, the patient has acquired resistance or resistance to the treatment of previous EGFR TKI treatment. In some instances, the treated patient has resistance or acquired resistance to the treatment of erlotinib, gefitinib, lapatinib, vandetanib, afatinib, osimertinib, poziotinib, crizotinib, cabozantinib, canertinib, axitinib, lenvatinib, nintedanib, regorafenib, pazopanib, sorafenib and / or sunitinib. In some instances, disease progression has occurred in human patients during or after previous treatment with different EGFR TKIs, and the cancer treated, for example, is classified as osimertinib-resistant cancer. As described above, mutations associated with EGFR TKI resistance include the T790M mutation and insertions in exon 20. In other examples, the patients treated are EGFR TKI naive patients (ie, they have not been previously treated with an EGFR TKI).

[0748] Therapeutic effects

[0749] In the case 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 of a plurality of indicators indicating that the changes within the cancer are in a more developed form. Therefore, indicators for measuring cancer growth inhibition include a reduction in cancer cell survival, a reduction in tumor volume or morphology (e.g., as determined using computed tomography (CT), ultrasound scanning or other imaging methods), delayed tumor growth, destruction of tumor vasculature, improved performance in delayed hypersensitivity skin tests, an increase in the activity of anti-cancer immune cells or other anti-cancer immune responses, and a reduction in tumor-specific antigen levels. Activating or enhancing an individual's immune response to a cancerous tumor can improve an individual's ability to resist cancer growth (particularly the growth of cancer that already exists in the subject) and / or reduce the tendency of individual cancer growth.

[0750] In some examples, the combination therapies described herein are capable of inhibiting the development or progression of cancer.

[0751] The ability of a given combination therapy to inhibit cancer development or progression can be analyzed, for example, using an in vivo model. For example, an in vivo model can involve measuring tumor growth in a patient-derived xenograft (PDX) model. More details of this exemplary method are described in the Examples.

[0752] Inhibition of cancer development can be inferred by observing slower tumor growth or reduction in tumor size after administration of the antibody molecule, for example by measuring tumor growth inhibition (%TGI). For those subjects administered the antibody molecule, %TGI can be measured by comparing the tumor size measured on day 0 with the tumor size measured at the end of the study period, and comparing it to the tumor growth of subjects administered a control antibody molecule over the same time period. Thus, %TGI can be defined as the percentage of tumor growth between the treatment (TX) group and the control (C) group relative to day 0 according to the following formula: %TGI = (1 – (TX 最终 –TX 初始 ) / (C 最终 –C 初始 ))x 100.

[0753] In some examples, the combination treatments 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%.

[0754] Inhibition of cancer development can be inferred by observing delayed or prevented onset and / or reduced severity of cancer symptoms in response to antibody molecule treatment. Inhibition of cancer progression can be inferred by observing delayed, prevented and / or reduced invasion and / or metastasis in response to antibody molecule treatment.

[0755] The combination therapies described herein may be capable of inhibiting the development or progression of a cancer to less than 100%, e.g., 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 the development / progression of a cancer in the absence of treatment (or with an appropriate control treatment). In some examples, the combination treatments described herein are capable of inhibiting the development or progression of cancer to less than 1-fold, e.g., ≤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, the level of development / progression of cancer in the absence of treatment (or with an appropriate control treatment).

[0756] In some examples, the combination therapy described herein can suppress the development or progress of cancer to a greater extent than using a single agent (i.e., antibody molecule or EGFR TKI) administered alone. The combination therapy described herein can suppress the development or progress of cancer to 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 one of the development / progress of cancer treated with a single antibody molecule, conjugate or EGFR TKI. In some examples, the combination therapy described herein can inhibit the development or progression of cancer to less than 1 times the development / progression level of cancer treated with a single antibody molecule, conjugate or EGFR TKI, for example ≤0.99 times, ≤0.95 times, ≤0.9 times, ≤0.85 times, ≤0.8 times, ≤0.85 times, ≤0.75 times, ≤0.7 times, ≤0.65 times, ≤0.6 times, ≤0.55 times, ≤0.5 times, ≤0.45 times, ≤0.4 times, ≤0.35 times, ≤0.3 times, ≤0.25 times, ≤0.2 times, ≤0.15 times, ≤0.1 times. In some examples, the combination therapy described herein exerts a synergistic effect on inhibiting the development or progression of cancer.

[0757] ***

[0758] The features disclosed in the preceding description or in the following claims or in the accompanying drawings, expressed in their specific form or according to a manner for performing a disclosed function, or a method or process for obtaining a disclosed result, may be used alone or in any combination of such features to implement the present disclosure in its various forms, as the case may be.

[0759] Although the present disclosure has been described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art when the present disclosure is given. Therefore, the exemplary embodiments disclosed above are considered to be illustrative rather than restrictive. Various changes may be made to the described examples without departing from the spirit and scope of the present disclosure.

[0760] For the avoidance of any doubt, any theoretical explanations provided herein are provided to enhance the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0761] Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0762] Unless the context requires otherwise, throughout this specification (including the following claims), the words "comprises" and "comprising" and variations should be understood to imply the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps.

[0763] It must 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 indicates otherwise. Ranges may be expressed herein as "about" one particular value, and / or to "about" another particular value. When expressing such a range, another example includes from the one particular value and / or to the other particular value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it should be understood that the particular value forms another example. The term "about" relative to a numerical value is optional and means, for example, + / - 10%.

[0764] Examples

[0765] Example 1 – RAA22 / B09DuetMab design and construction

[0766] This example describes the generation of a bispecific antibody molecule capable of binding both EGFR and cMET.

[0767] 1.1 Isolation and identification of anti-cMET antibody 0021U3-B09

[0768] cMET-specific scFv antibodies were isolated from a large naive human scFv phage display library in a series of repeated panning selection cycles on recombinant mammalian expressed biotinylated monomeric human cMET (MedImmune) essentially as described (Vaughan, 1996). ScFvs from the second round of selection output were expressed in the bacterial periplasm and screened for their activity against HGF:cMET. The ability of the human cMET receptor to inhibit the binding of the HGF ligand in a (homogeneous time-resolved fluorescence) ligand receptor inhibition binding assay was evaluated. The best hits showing strong inhibition were selected and DNA sequenced. The unique genes were then converted into human immunoglobulin G2 (IgG2) antibodies and produced in mammalian cells essentially as described (Persic; 1997). The HGF:cMET Purified antibodies were ranked by inhibition in the binding assay. The most potent antibody, 0021U3-B09, was selected for further characterization.

[0769] 1.2 Optimization of anti-cMET antibody 0021U3-B09.

[0770] To minimize potential immunogenicity, non-Vernier framework residues (Foote and Winter 1992) in the variable framework region 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 changed to germline residues without loss of activity. 0021U3-B09 was affinity optimized using a hybridization-based mutagenesis approach essentially as described in (Kunkel 1985). A large scFv library derived from the 0021U3-B09 sequence was created using standard molecular biology techniques by oligonucleotide-directed mutagenesis of the VH complementarity determining region 3 (CDR3). The library was subjected to affinity-based solution phase selection to select variants with higher affinity for human and cynomolgus monkey cMET antigens. Screening of crude scFv periplasmic extracts from CDR-targeted selection outputs for enhanced HGF:cMET Inhibitory activity in binding assays. Variants that showed significantly improved inhibition compared to the parent 0021U3-B09 were DNA sequenced and the unique genes were converted to human IgG2. The purified antibodies were then ranked according to their inhibitory effects. The most potent antibody, B09-57, was selected for further characterization.

[0771] 1.3 Isolation and identification of anti-EGFR antibody Tdev-0004.

[0772] Substantially as described in (Vaughan, 1996), EGFR-specific scFv antibodies were isolated from a large natural human scFv phage display library in a series of repeated panning selection cycles on biotinylated monomeric human EGFR (Midi Muni) expressed by recombinant mammals. The ScFv display phages from the 3rd round of selection output were screened for binding to human and cynomolgus monkey EGFR in ELISA. The best hits showing cross-reactivity were selected and DNA sequencing was performed. Substantially as described in (Persic, 1997), the unique gene was then converted into human immunoglobulin G1 (IgG1) antibodies and produced in mammalian cells. The purified antibodies were then sorted by flow cytometry based on the binding of purified antibodies to the cell line A431 expressing EGFR. The antibody Tdev-0004 showing specific cell binding was selected for further characterization.

[0773] 1.4 Optimization of anti-EGFR antibody Tdev-0004.

[0774] Variants RAA22 and QD6 were derived by optimizing the anti-EGFR Tdev-0004 mAb. The VL framework of Tdev-0004 has a 100% match with 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), however, the VH framework has 79% homology with the closest human germline IGHV1-69*01 / JH4 (see https: / / www.ncbi.nlm.nih.gov / projects / igblast / Idlink.cgi?seqname=IGHV 1-69*01&taxid=9606&dbname=IG_DB%2Fimgt.Homo_sapiens.Vforf.p). In order to minimize potential immunogenicity, the VH region was fully germlined by mutating all 13 non-germline framework residues. After germlining, the binding of the fully germlined variant to the cynomolgus monkey EGFR was significantly impaired. In order to restore the binding to the cynomolgus monkey EGFR, four non-germline residues were selectively reversed: K68, I73, R76 and T78. The amino acid residues were numbered by the Kabat numbering system (Kabat and Wu 1991). The resulting partial germline variant (named H4) was used as a template sequence for affinity optimization. According to the manufacturer's instructions, the QuikChangeLightning multi-site directed mutagenesis kit (Agilent) was used to optimize the affinity of the variant H4 by the simple mutagenesis of all six CDRs. The VH and VL libraries of single amino acid mutagenesis were expressed as Fab fragments in bacteria and screened for improved binding to human and cynomolgus monkey EGFR in ELISA. DNA sequencing was performed on the variants that showed improved binding compared to the parent H4, and the unique gene was converted into human IgG1. A variant RAA22 with a single mutation in CDRH3 was identified. To further improve affinity, single positive mutations were combined to create a combinatorial library and screened for variants with enhanced binding to human and cynomolgus EGFR. A variant QD6 with four combined mutations in CDRL2, CDRL3 and CDRH3 was identified.

[0775] 1.5 Generation of monovalent bispecific anti-EGFR / cMETDuetMab antibody.

[0776] The variable domains of anti-cMET mAb B09-57 and anti-EGFR mAbs RAA22 and QD6 were used to construct monovalent bispecific anti-EGFR / cMET antibodies on the backbone of the DuetMab platform (Mazor, 2015). Specifically, the VH gene of anti-cMET B09-57 was inserted into a human γ-1 constant heavy chain carrying a "knob" mutation (T366W) and an alternative interchain cysteine ​​mutation (F126C and C219V). The VL gene of B09-57 was inserted in frame into a human κ constant domain carrying 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 human γ-1 constant heavy chains carrying "hole" mutations (T366S, L368A, and Y407V), while the VL genes of RAA22 and B09-57 were inserted in frame into human λ constant domains designed to pair with the "hole" heavy chain. In addition, two residues in the CH3 domains of the "knob" and "hole" heavy chains were mutated to cysteine ​​(S354C in the "knob" and Y349C in the "hole") to form stable disulfide bridges. The Fc domain was further engineered to carry a cysteine ​​insertion after serine 239 (C239i / "Maia"), which was designed to enable site-specific conjugation of maleimide-carrying cytotoxic drugs (Dimasi, 2017). Amino acid residues are numbered by 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 produced from mammalian cells as previously described (Mazor, 2017).

[0777] The amino acid sequences of the heavy and light chains of DuetMab generated according to this example are provided in the table below:

[0778] RAA22 / B09-57 QD6 / B09-57 EGFR heavy chain 56 53 cMET heavy chain 50 50 EGFR light chain 58 55 c-Met light chain 52 52

[0779] Example 2 – Biochemical and biophysical properties

[0780] This example tests various biochemical and biophysical properties of RAA22, QD6 and B09-57 monoclonal antibodies and RAA22 / B09-57 and QD6 / B09-57 bispecific antibody molecules, including their binding affinity to EGFR and c-Met, respectively, and their ability to bind both antigens simultaneously.

[0781] 2.1 Binding affinity of DuetMab and parental mAb to EGFR and cMET.

[0782] The kinetic rate constants (kon and koff) and equilibrium dissociation constant (Koff) of EGFR-cMETDuetMAb for recombinant human, cynomolgus monkey, and murine EGFR and cMET antigens were determined by SPR at 25°C using an antibody capture assay on a BIAcore T200 instrument (GE Healthcare, Pittsburgh, PA). D ). 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 article antibodies and control article antibodies were prepared at 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), and 3-fold serial dilutions of purified EGFR (0.27–200 nM human, 0.4–900 nM human, 4-1000 nM mouse) or cMET protein (0.27 to 66 nM human and 0.27–22 nM human) in instrument buffer. A sequential approach was used for kinetic measurements. Antibodies were first injected onto the capture surface at a flow rate of 10 μL / min. After the binding of the captured antibody is stabilized, a single concentration of analyte is injected onto the capture surface and the reference surface at a flow rate of 75 μL / min. The resulting binding reaction curve produces association phase data. After the analyte is injected, the flow is then switched back to the instrument buffer for 15 minutes to allow the collection of dissociation phase data, followed by a 1-minute pulse of 10 mM glycine, pH 1.5, to regenerate the antibody capture surface on the chip. The binding reactions for the test article antibody and the control article antibody are recorded from duplicate injections of each concentration of analyte. In addition, several buffer injections are interspersed throughout the injection series. Buffer injections are selected to be used together with reference cell reactions to correct injection artifacts and / or non-specific binding interactions of the raw data set, commonly referred to as "double reference". The corrected binding data are globally fitted to a 1:1 binding model (Biacore T200 Evaluation Software 2.0, General Electric Healthcare, Pittsburgh, Pennsylvania). The calculated kinetic parameters (k on and k off ) and k off / k on Determined K D Shown in Table 1.

[0783] Table 1 Kinetics of DuetMab and parental IgG against EGFR and cMET antigens

[0784]

[0785]

[0786] a ND: Not determined. Kinetic measurements of soluble monomeric forms of EGFR and cMET were performed using a BIACore instrument. D k is calculated from the nonlinear fit of the data off / k on ratio.

[0787] From the above data, it can be seen that compared with QD6 / B09-57, the bispecific antibody molecule QD6 / B09-57 binds to human c-Met with high affinity (about 2nMkD) and binds to human EGFR with high affinity (about 6nMkD), while the bispecific antibody molecule RAA22 / B09-57 binds to human c-Met with similar high affinity (about 2nMkD), but binds to human EGFR with reduced affinity (about 45nM kD).

[0788] 2.2DuetMab binds to EGFR and cMET simultaneously.

[0789] 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 in assay buffer [PBS pH 7.2, 3 mg / mL bovine serum albumin (BSA), 0.05% (v / v) Tween 20] was initially captured on NI-NTA biosensors. After a wash step to remove any unbound protein, the separately loaded biosensors were subjected to sequential association and dissociation interactions, first with 66 nM of antibody and then with 500 nM of EGFR antigen. The data were nonlinearly fit using Octet384 software v.9.0, from which the association and dissociation curves were calculated. Figure 2 As shown, DuetMab exhibited simultaneous binding to both antigens, whereas the parental anti-cMET IgG only exhibited specific binding to cMET, and both anti-EGFR IgGs exhibited no binding to the cMET-loaded sensor.

[0790] 2.3EGFR and c-Met specificity

[0791] The specificity of EGFR and cMET species paralogs and closely related family members was determined by ELISA. Briefly, antigen solutions were prepared at 1 μg / mL in PBS and 50 microliters were coated on half-area ELISA assay plates. The plates were washed and blocked for one hour at room temperature with 1% BSA in PBS containing 0.005% Tween-20 (PBS-T). The wells were washed 4 times in PBS-T. As shown in Figure 3, primary antibodies were used, including: R374 (non-binding IgG1 isotype control antibody), B09 (anti-cMET antibody), QD6 (anti-EGFR antibody), RAA22 (anti-EGFR antibody), QD6 / B09 (bispecific EGFR / c-METDuetMAb), RAA22 / B09 (bispecific EGFR / c-METDuetMAb), PaniX (anti-EGFR antibody), MetMab (anti-cMET antibody) and Mab11311 (anti-HER4 antibody). In addition to the HER4 binding mAb control MAB1131 (the series started at 1 μg / mL), the wells were incubated with 50 microliters of the indicated primary antibodies diluted in PBS-T in a 1:3 dilution series, starting at 10 μg / mL and ending at 0.002 μg / mL. The wells were washed 4 times in PBS-T, and then 50 μl of goat anti-human Fab HRP-labeled secondary antibody diluted 1: 5000 in PBS-T was added to each well and incubated for one hour at room temperature. 50 microliters of TMB substrate solution were added to all wells and incubated at room temperature for 5-30 minutes until a strong signal was observed in the positive control wells. 50 microliters of TMB stop solution were added to all wells and the absorbance was read at 450nm on a SpectraMax M5 microplate reader. Data were analyzed in SoftMaxPro 5 software and plotted using GraphPad Prism 7 plotting software.

[0792] To determine species cross-reactivity, ELISA assays were performed as described above. Figure 3AAs shown, high affinity EGFRIgG QD6 and monovalent bispecific EGFR / cMET DuetMAb QD6 / B09 bind to human, cynomolgus monkey and mouse EGFR and give strong signals in ELISA assays. In contrast, compared with QD6, the EGFR IgG RAA22 with reduced affinity binds weakly to human, cynomolgus monkey and mouse EGFR. The binding to mouse EGFR is weaker, but detectable. The corresponding monovalent bispecific EGFR / cMET DuetMAb RAA22 / B09 shows that the binding to human and cynomolgus monkey EGFR is still weaker relative to the bivalent parent IgG RAA22, and shows nominal binding to mouse EGFR. CMET IgG B09 and all bispecific variants show comparable binding to human and cynomolgus monkey cMET. No antibody binding to mouse cMET was detected. These results are consistent with the binding kinetics determined by surface plasmon resonance on a BIAcore instrument (Table 1 above).

[0793] like Figure 3B As shown, neither the parental IgG nor the derived bispecific antibodies showed measurable binding to any EGFRHER family protein, HER2, HER3 or HER4. Similarly, no antibody showed significant binding to cMET family member Ron (CD136) or Semaphorin3a.

[0794] These results demonstrate that the parental IgG and the resulting bispecific antibodies bind specifically to their cognate targets with no detectable binding to closely related family species.

[0795] 2.4 Internalization of bispecific antibodies

[0796] Internalization kinetics of DuetMab primarily labeled with AlexaFluor647 (AF647): RAA22 / B09 and QD6 / B09 antibodies were evaluated in vitro using the EGFR and c-MET expressing cell line H1975. Each antibody was pre-bound to the cells, and then the translocation of the antibody from the cell surface to the cytoplasm was monitored using live cell confocal fluorescence microscopy. Both antibodies were primarily localized on the cell surface before being subjected to internalization conditions (T=0) and translocated to the cytoplasmic region after 1 hour (data not shown). Kinetic images were taken every 5 minutes during the time course of internalization and processed using a quantitative algorithm to determine the internalization kinetic constant and half-life. The internalization kinetics of QD6 / B09 and RAA22 / B09 were comparable, with half-lives of 37.5±10.6min and 43.2±15.5, respectively.

[0797] To evaluate the mode of antibody internalization and investigate the contribution of each group to the overall internalization of DuetMab, we evaluated the internalization of single-group specific control antibody molecules QD6 / IgG and B09 / IgG against duet QD6 / B09 and RAA22 / IgG and B09 / IgG against duet RAA22 / B09 in H1975 cells expressing EGFR and c-MET. Since only one group was specific for the target receptor, the control antibodies could only be internalized by one receptor, eliminating dual receptor targeting and cross-linking as a mode of internalization.

[0798] QD6 / B09( Figure 4A ) and RAA22 / B09( Figure 4B ) showed a very similar consistent pattern of decrease in membrane mAb-Fl647 signal with an increase in mAb-AF647 signal in the cytoplasm, which is a typical feature of internalization. However, their single group constructs showed very different internalization curves. Single group QD6 / IgG and QD6 / B09 DuetMab had almost the same internalization time course ( Figure 4A , left and center), indicating that internalization of the QD6 / B09 duet is primarily driven by the EGFR-group of the molecule, with minimal contribution from the B09-group. In fact, the B09 / IgG construct showed very little internalization ( Figure 4B , right). The rapid and extensive decrease in membrane signal corresponds to a very modest increase in cytoplasmic signal, likely due to extensive dissociation of pre-bound B09 / IgG from c-MET receptors on the cell surface. Dissociation of the antibody subsequently leads to modest internalization of B09 / IgG. These results suggest that internalization of the QD6 / B09 duet is primarily driven by the EGFR-group of the molecule, with minimal contribution from the B09-group.

[0799] In contrast, RAA22 / B09DuetMab showed a very different internalization profile when compared to its single-group control antibody. Figure 4BAs shown, the cytoplasmic intensity values ​​of RAA22 / B09 DuetMab are 10.98 times and 4.70 times higher than those of RAA22-IgG and B09-IgG, respectively. Although the inefficient internalization of B09 / IgG may be attributed to its obvious dissociation, RAA22 / IgG does undergo rapid internalization. However, due to the lower affinity of the EGFR-group, the number of RAA22 / IgG molecules (based on fluorescence intensity) is 10.98 times less than that of RAA22-B09DuetMab. In contrast to the single-group construct, the significant increase in the amount of duetRAA22 / B09mAb entering the cytoplasm indicates that the two antibody groups must engage with the target receptor to drive internalization. This finding suggests that QD6 / B09 and RAA22 / B09 DuetMab have different internalization mechanisms, in which QD6 / B09 is mainly driven by the EGFR group, but RAA22 / B09 requires both EGFR and c-MET groups for engagement.

[0800] Since binding of the EGFR and c-MET groups to target receptors promotes internalization of RAA22 / B09 receptors in H1975 cells, we examined whether increasing the number of EGFR and c-Met receptors would affect internalization properties. The corresponding receptor levels determined by Western blotting in H1975 cells were approximately 33,000 for EGFR and approximately 50,000 for c-MET, and approximately 790,000 for EGFR and approximately 523,000 for c-MET in HCC827 cells. The internalization curves of RAA22 / B09 in H1975 (medium receptors) and HCC827 (high receptors) cells showed a significant increase in internalization of HCC827 cells (Figure 5).

[0801] As expected, the overall levels of RAA22 / B09, corresponding to EGFR and c-MET, increased 23.9-fold and 10.4-fold, respectively, and binding to HCC827 cells was on average 8.9-fold higher than that to H1975 (T = 0, 3.1 × 10 7 MFI for 3.5×10 6MFI). The internalization level in HCC827 cells (judged by peak cytoplasmic intensity) was 21.7 times higher, indicating that in cells expressing high levels of target receptors, the concentration of antibody entering the cytoplasm was significantly higher. Importantly, in addition to the significantly different intensities, the internalization curves (membrane and cytoplasmic signals over time) between HCC827 and H1975 cells were also significantly different. In high-expressing HCC827 cells, the decrease in RAA22 / B09-AF647 membrane signal corresponded to a mutual increase in RAA22-B09 cytoplasmic signal, in which the total RAA22 / B09 signal was maintained over the time course, indicating a strong dual-group antibody interaction with both receptors and subsequent internalization. In H1975 cells, the total intensity and membrane intensity decreased simultaneously, indicating that some pre-bound antibodies may have dissociated from the cell surface and were unable to be internalized within the cell. Similar features of internalization indication dissociation were observed for RAA22 / IgG and B09 / IgG in HCC827 cells, in which a single set of engagements could not provide effective binding and were easily dissociated ( Fig. 6A and 6B ). The data suggest that there is a mixed mode of receptor interaction (single and double set engagement) when RAA22 / B09 is internalized in H1975 cells. Taken together, these data suggest that target cell receptor expression levels are an important determinant of the extent and efficiency of RAA22 / B09 internalization.

[0802] Example 3 - In vitro potency of ADC

[0803] In this example, the in vitro potency of the EGFR / cMET bispecific ADC was measured in a panel of cancer cell lines.

[0804] 3.1 Site-specific conjugation

[0805] Conjugation of tubulysin drugs to RAA22 / B09 antibody molecules was performed essentially as previously described in Thompson, 2016 and US20150291657A1.

[0806] 3.2 Methods for determining ADC cytotoxic activity

[0807] ADC cytotoxic activity was tested in a variety of cell lines as follows. Cells were plated at a density of 10,000 cells per well of a 96-well plate in a volume of 100 μL in its recommended medium supplemented with 10% fetal bovine serum. Each dose of the antibody to be tested at a 3X concentration was prepared by serially diluting the antibody stock solution in the culture medium. Fifty microliters of each test article was added to the cells in triplicate so that the final antibody concentration ranged from 60nM to 0.0009nM. The treated cells were cultured in a humid incubator at 37°C for 72 hours. Metabolic activity was determined using the CellTiter-Glo luminescent activity assay from Promega according to the manufacturer's instructions. Data were plotted as percentages of metabolic activity relative to untreated controls. Using GraphPadPrism software, IC was determined using a logistic nonlinear regression analysis between maximum activity (untreated cells) and maximum response (peak inhibition). 50 value.

[0808] 3.3 Results – In vitro ADC activity in cell lines

[0809] 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 higher affinity QD6 / B09-AZ1508 and the lower affinity RAA22 / B09-AZ1508 exhibited broad activity in cell lines with a range of target expression levels.

[0810] Table 3 - In vitro activity of EGFR-cMET DuetMAb ADC

[0811]

[0812]

[0813] Overall, ADCs with lower affinity for EGFR showed comparable (slightly reduced) potency in a wide 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 of about 15,000 or less on the cell surface. This effect was more pronounced in the variants with lower affinity, which appeared to be more sensitive to lower levels of cMET.

[0814] Example 4 - Bispecific conjugation of ADC

[0815] To further test the hypothesis that bispecific engagement of affinity-reduced EGFR-cMET antibodies is necessary for optimal ADC delivery, we performed in vitro experiments to determine the relative contribution of individual antibody groups to the activity of the bispecific ADC. In the first experiment, we used excess amounts of the ungrouped parental antibody to block either 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 groups of the bispecific ADC function independently to deliver the ADC, it is expected that blocking either target in this cell line will only modestly reduce the activity of the ADC, resulting in an IC 50 Move two-fold or less because the targets are present at similar levels. On the other hand, if the ADC requires dual target engagement to effectively deliver the ADC into cells, blocking either target will likely have a greater effect on the activity of the bispecific ADC. In a related experiment, we compared the activity of the bispecific EGFR-cMET ADC with a monovalent, monospecific control antibody consisting of an EGFR or cMET binding group and a non-binding isotype antibody control group. Similarly, if each group acts independently, the expected result will be that each monospecific control ADC will be only moderately less potent than the bispecific ADC, and the difference will be additive. Alternatively, if the two groups of the bispecific act synergistically, the activity of the bispecific ADC is expected to be significantly different from that of the monospecific control antibody.

[0816] 4.1 Methods

[0817] The cytotoxic activity of the ADC was tested in the NCI-H1975 cell line as follows. The cells were plated at a density of 10,000 cells per well of a 96-well plate in its recommended medium supplemented with 10% fetal bovine serum, with a volume of 50 μL for blocking experiments and 100 μL for monovalent ADC experiments. For mAb blocking experiments without a group, 50 μL of 300 μg / mL EGFR IgG (RAA22) or cMET IgG (B09) solution was added to the wells and pre-incubated in a humidified incubator for one hour at 37°C. Each dose of the antibody to be tested was prepared at a 3X concentration by serially diluting the antibody stock solution 4X in the culture medium. Fifty microliters of culture medium alone, isotype control IgG ADC (R347-AZ1508) or EGFR-cMET ADC (RAA22 / B09-AZ1508) were added to the cells in triplicate, so that the final antibody concentration ranged from 67nM to 0.0009nM. For monovalent ADC experiments, 50 μL of 3X stock solutions of isotype control ADC (R347-AZ1508), monovalent EGFR ADC (RAA22 / R347-AZ1508), monovalent anti-cMET ADC (B09 / R347-AZ1508), equimolar combinations of monovalent ADCs or EGFR-cMET ADC (RAA22 / B09-AZ1508) were added in triplicate in a 4X dilution series starting at 60nM and ending at 0.009nM. Treated cells were cultured in a humidified incubator at 37°C 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 percentage metabolic activity relative to untreated controls. IC was determined using logistic nonlinear regression analysis between maximum viability (untreated cells) and maximum response (peak inhibition) using GraphPad Prism software. 50 value.

[0818] 4.2 Results and conclusions – In vitro validation of the dual targeting concept (mAb blocking assay and monovalent ADC)

[0819] We performed in vitro experiments to examine the relative contribution of the individual antibody groups to the cytotoxic activity of the bispecific ADCs, as described above. Figure 7 As shown in the representative experiment in Figure 2, pretreatment of NCI H1975 cells with cMET IgG RAA22 resulted in an IC of EGFR-cMET ADC (RAA22 / B09-AZ1508). 50 The shift from approximately 60 pM to 3,480 pM is approximately 60-fold. Treatment with anti-cMET IgG B09 resulted in IC 50Similarly, when NCIH1975 cells were treated with a monovalent monospecific EGFR ADC (RAA22 / R347-AZ1508), the IC 50 The pM of the bispecific EGFR-cMET ADC was about 20,500 pM, which was about 65 times lower than that of the bispecific EGFR-cMET ADC (316 pM). Figure 8 IC of monovalent monospecific anti-cMET ADC (B09 / R347-AZ1508) 50 The concentration of 1g / mL was 2,772pM, which was about 13 times lower than that of the bispecific antibody.

[0820] In summary, these data indicate that EGFR-cMET ADC effectively targets tumor cells that co-express two targets, driven primarily by the bispecific engagement of the ADC. In addition, these results indicate that the binding group with reduced EGFR affinity is insufficient to promote effective ADC delivery in the absence of cMET binding, as demonstrated by the significant reduction in efficacy when the cMET group is blocked by an unset group antibody and the weak cytotoxicity of the monovalent EGFR control ADC, RAA22 / R347-AZ1508. In summary, these results are consistent with the following hypothesis: the reduced affinity of the EGFR binding group of the bispecific ADC (RAA22 / B09-AZ1508) will promote ADC delivery to EGFR and cMET co-expressing tumors, while exhibiting reduced cytotoxicity to cells that mainly express only one target. This effect is most significant when only EGFR is available for engagement, which has an impact on alleviating EGFR-driven toxicity in normal organs such as the skin that express significant levels of EGFR but relatively few cMET.

[0821] Example 5 - In vivo ADC pharmacology in patient-derived xenograft (PDX) models

[0822] Patient-derived xenograft (PDX) models of human cancer have become a mature alternative to tumor xenografts based on tumor cell lines. PDX models are established by implanting primary tumor tissue from patients directly into immunodeficient mice to produce proliferating tumors in mice. The resulting tumors are then propagated in additional mice without in vitro culture to establish a low-passage PDX tumor tissue bank that can be used to implant research mice. A key feature of PDX models is that they maintain histological and genomic heterogeneity to a large extent and retain the gene expression profile of the corresponding original patient tumor. Compared with tumor cell line-based xenograft models that use tumor cell clonal populations that have been adapted to in vitro growth, the characteristics of PDX models are designed to more accurately replicate the characteristics of real human tumors, thereby improving the predictive value of preclinical mouse models. In fact, many studies have shown that the response and resistance characteristics of PDX models to standard of care treatments are closely correlated with clinical data from human subjects with given tumor characteristics.

[0823] Despite the improvements offered by PDX models, there are limitations to standard in vivo pharmacology study designs, even when applied to PDX models. For each tumor model, a typical study tests multiple dose levels of drug treatment and one or more positive or negative control compounds, with enough mice per treatment group to support within-model statistics. The relatively high number of mice used for such study designs and the high cost of PDX models can limit the number of tumor models in which a given compound can be practically tested. An alternative / complementary approach to traditional study designs is the mouse PDX trial, a population-based approach conducted after the design of a human clinical trial. In this approach, for each compound, a single mouse is typically treated at a single dose level established from a previous dose-ranging discovery study, with an optional treatment control group for each model. Because the number of mice required for each model is small, many PDX models can be tested, each representing a unique human tumor. Rather than relying on within-model statistics, responses are assessed across the entire population of tumor models tested. This approach can provide more accurate estimates of response rates for a diverse range of target expression, molecular phenotypes, tumor subtypes, or other clinically relevant features of interest. Furthermore, the large number of unique models that can be tested in PDX trials can enable more meaningful exploratory genomic, transcriptomic, or expression profiling studies to begin early searches for correlates of response or resistance. For the exemplary studies outlined herein, we took an “all-in” approach, testing all available NSCLC models at START Discovery, regardless of target expression level or molecular phenotype.

[0824] 5.1 Methods

[0825] Mouse PDX experiments were performed at South Texas Accelerated Research Therapeutics (START, San Antonio, TX) in San Antonio, Texas. START is accredited by AAALAC International (Association for the Assessment and Accreditation of Laboratories Animal Care International) and complies with AstraZeneca Global Standard on Animal Care and Welfare. All models were developed at START. Patient-derived xenograft (START-PDX) models were established from living human tumor tissue or fluids and have been serially passaged in animals for a limited number of times to maintain tumor heterogeneity. Athymic nude mice (Crl: NU (NCr) -Foxn1nu) / CB-17Scid (CB17 / Icr-Prkdcscid / IcrIcoCrl) were unilaterally implanted with tumor fragments collected from host animals, each transplant from a specific passage batch. Pre-study tumor volume was recorded approximately one week before its estimated start date. When the tumor reaches the appropriate tumor volume initiation (TVI) range (125-250mm 3 ), animals were randomized into treatment and control groups and intravenous (IV) dosing was started (Day 0); animals were followed individually throughout the study. Initial dosing began on Day 0; animals in all groups were dosed intravenously by weight (0.01 ml / g; 10 ml / kg). Drug-treated animals were dosed every 7 days for a total of 4 doses. Starting on Day 0, tumor size was measured by digital caliper and the estimated tumor volume was recorded for each treated and control animal; tumor volume was calculated using the following formula: TV = width 2 × length × 0.52. Tumor growth observation continued for one week after the last administration. Each animal was treated with a 4-week follow-up after reaching the tumor volume (TV) endpoint (tumor volume ≥ 1 cm 3 ) or at the end of the study time of 28 days, whichever comes first. For some PDX models with slow tumor growth, the observation period was extended. Tumor growth inhibition (%TGI) is defined as the percentage of tumor growth between the treatment (TX) group and the control (C) group relative to day 0 according to the following formula: %TGI = 1 – (TX 最终 –TX 初始 ) / (C 最终 –C 初始The percentage of tumor regression was defined as the percentage of tumor reduction in the treated animals at the study endpoint relative to the tumor volume on day 0 (initial dose day) calculated according to the following formula: % regression = (TX 最终平均 –TX 初始平均 ) / (TX 初始平均 )x 100.

[0826] 5.2 Results and Conclusions

[0827] As shown in Figure 9, high affinity EGFR-cMET ADC, QD6 / B09-AZ1508 and variant RAA22 / B09-AZ1508 with reduced affinity for EGFR both induce tumor growth inhibition or regression in numerous PDX models tested. Surprisingly, the ADC with lower affinity showed an overall trend of increased number and depth of observed responses compared to the ADC with higher affinity. For the PDX model with the least response to the reduced affinity ADC, this activity trend was slightly reversed, which was somewhat related to lower cMET expression. These observations increase the possibility that the activity of the lower affinity EGFR-cMET ADC is driven in part by cMET expression levels. The EGFR binding groups of the two bispecific antibodies are derived from the same mouse EGFR cross-reactive antibody (see Example 1). The intrinsic binding affinity of the QD6 / B09 antibody to mouse EGFR is about 6nM, while the affinity of the RAA22 / B09 bispecific antibody is about 575nM. The unexpected improvement in activity of the lower EGFR affinity can be attributed to the effect of reduced EGFR uptake from normal tissues (such as skin), resulting in higher overall circulating exposure of the ADC. Regardless, these data suggest that reducing the affinity of the EGFR-cMET bispecific antibody to EGFR does not compromise the in vivo efficacy of the resulting ADC, but unexpectedly improves activity compared to the higher affinity ADC.

[0828] 5.3 PDX studies at different doses

[0829] Further experiments were performed to test different doses of ADC in PDX models. This experiment was performed as described in Example 5.1, except that individual mice were bred to have tumor volumes ≥ 2 cm 3 Patients were removed from the study and the final measurements were included in the group average until the average reached the volume endpoint or the study reached the time endpoint of 63 days. For the exemplary study, the high affinity EGFR-cMET ADC, QD6 / B09-AZ1508, was tested at dose levels of 1 and 2 mg / kg, and a variant with lower affinity for EGFR, RAA22 / B09-AZ1508, was tested at 1, 2, and 3 mg / kg.

[0830] like Fig.10 As shown, both high-affinity EGFR-cMET ADC, QD6 / B09-AZ1508, and variant RAA22 / B09-AZ1508 with reduced affinity for EGFR induced tumor growth inhibitory activity in the PDX model at the doses tested. According to the results described in Example 5.2 and the results shown in Figure 9, lower affinity ADCs are generally more effective than high affinity ADCs. In all four models tested, lower affinity ADCs induced regression at 2 or 3 mg / kg dose levels, indicating that lower affinity ADCs are effective at moderate doses. These data provide further evidence that reducing the affinity of the EGFR-cMET bispecific antibody to EGFR does not reduce the in vivo efficacy of the resulting ADC, but rather improves the in vivo efficacy compared to higher affinity ADCs.

[0831] Example 6 - ADC Efficacy in an Orthotopic Pancreatic PDX Model

[0832] Subcutaneous in vivo tumor models are the primary means of testing the efficacy of anticancer agents. However, this tumor implantation site is accompanied by many limitations that need to be considered when interpreting in vivo results. These deficiencies include tumor vascularization and the lack of a tissue-specific matrix for tumor growth and response. To address these challenges, we compared the in vivo efficacy of high-affinity and low-affinity EGFR-cMET bispecific antibody-drug conjugates in subcutaneous and orthotopic models of the pancreatic PDX model MEDI-PANC-08. In order to track the growth of this tumor in situ, we developed a PDX variant expressing luciferase (MEDI-PANC-08 LUC ), whose growth can be tracked using imaging.

[0833] 6.1 Methods

[0834] All experiments were performed in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) accredited facility in accordance with the guidelines for humane treatment and care of laboratory animals by the IACUC (Institutional Animal Care and Use Committee) of MediMuni Inc. Animals were monitored daily for morbidity and mortality.

[0835] Subcutaneous PDX Models

[0836] The MEDI-PANC-08 pancreatic PDX model used in this study was obtained from the Internal MedImmune PDX library. The PDX tumors were initially seeded with NSG (NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ) mice to generate sufficient tumor material for inoculation efficacy studies. Once tumors reach 800-1200 mm 3The mice were humanely killed by CO2 asphyxiation. The tumors were isolated under sterile conditions and cut into approximately 2 mm 3 The tumor was subcutaneously implanted into the right flank of individual NSG mice using a No. 11 trocar. When the size of about 150-250 mm3 was reached, the mice were randomly divided into treatment groups (based on tumor volume) and treated with ADC (Q1Wx4). Two EGFR-cMET bispecific ADCs were examined at 1, 2 and 3 mg / kg QD6 / B09 (high affinity) and RAA2 / B09 (low affinity). 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 through the tail vein. Tumor and body weight measurements were collected twice a week, and tumor volume was calculated using the equation (LxW2) / 2, where L and W refer to length and width dimensions, respectively.

[0837] Orthotopic PDX models

[0838] PDX model expressing luciferase (MEDI-PANC-08 LUC ) grown subcutaneously in NSG-seeded mice with a volume of 800-1200mm 3 The tumors were harvested and cut into approximately 2 mm 3 fragments. The tumor fragments were subsequently sutured to the pancreas of NSG mice (day zero). Luciferase signal was determined weekly using the IVIS Spectrum in vivo imaging system. Briefly, 200ul of luciferin dissolved in DPBS (15mg / ml) was injected intraperitoneally (ip) 10 minutes before imaging. The mice were anesthetized under 3% isoflurane, placed in right lateral recumbency and luminescence was measured. Fourteen days after tumor implantation, when luminescence signals were clearly detected, mice were randomized into their respective groups based on luminescence. Mice were treated with isotype control (R347-AZ1508, 3mg / kg–Q1Wx4), gemcitabine (75mg / kg, Q2Dx5) and RAA2 / B09 ADC (2 and 3mg / kg-Q1Wx4). Luminescence was measured weekly. Study endpoints included weight loss, deterioration of physical condition and lethargy. Data were analyzed using Living Image software (Perkin Elmer) and plotted as mean radiance [p / s / cm2 / sr] versus time.

[0839] 6.2 Results and Discussion

[0840] To assist in selecting the appropriate EGFR affinity combination for the EGFR-cMET bispecific ADC, 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 panels A and B of Figure 11, different differences were observed between the two molecules. The high-affinity QD6 / B09 ADC did not show efficacy at any of the three dose levels tested. In contrast, the low-affinity RAA2 / B09 ADC completely regressed the tumor at day 65, followed by tumor regrowth at the 3 mg / kg dose level and tumor growth inhibition at 2 mg / kg.

[0841] Although subcutaneous tumor models have become the main point of in vivo efficacy studies, the main drawback is that the tumor does not grow at the primary site, so any drug response may not truly reflect the patient's response. To address this issue, an orthotopic model of pancreatic cancer was developed using MEDI-PANC-08 tumors that were genetically modified to stably express luciferase. After surgical implantation on the pancreas, tumors were allowed to establish and then randomized based on luminescent signals. Mice were then treated with low-affinity RAA2 / B09 EGFR-cMET ADC, isotype control or gemcitabine (a chemotherapy drug). After treatment, luminescence was measured weekly. As shown in panel C of Figure 11, the luminescence of the untreated control group and the isotype control group increased over time due to poor physical condition and large, palpable abdominal tumors. Gemcitabine showed an initial decrease in the luminescent signal, reaching a minimum around day 21, after which the signal increased over time and the group was removed from the study on day 49. In the orthotopic model, both 2mg / kg and 3mg / kg dose levels of RAA2 / B09 ADC caused a decrease in luminescence signal, reaching near background levels at day 60. The 2mg / kg dose level demonstrated better activity in producing tumor regression compared to the subcutaneous studies. Animals that showed tumors with luminescence signal close to background at necropsy at the end of the study no longer showed visible tumors, thus supporting the correlation between luminescence signal and tumor volume.

[0842] In summary, in the subcutaneous PDX pancreatic PDX model, the low affinity EGFR-cMETRAA2 / B09 ADC showed improved efficacy compared to the high affinity QD6 / B09 ADC, with tumor regression observed at 3 mg / kg. This efficacy was also observed in an orthotopic model using the same PDX tumor (MEDI-PANC-08) engineered to stably express luciferase. Using luminescence as a surrogate for tumor volume, the RAA2 / B09 ADC showed superior efficacy to the subcutaneous model at 2 and 3 mg / kg, producing tumor regression.

[0843] Example 7 – Safety and Pharmacokinetics

[0844] Pharmacokinetic (PK) analysis was performed to compare the plasma PK parameters of low-affinity and high-affinity EGFR-cMET ADCs, including peak and total exposure, clearance, and half-life in mice and cynomolgus monkeys. The key goal was to determine whether reducing affinity for EGFR would affect the circulating exposure of the EGFR-cMET bispecific ADC. PK samples at different dose levels of QD6 / B09-57-AZ1508 and RAA / B09-57-AZ1508 were collected in mice and cynomolgus monkeys. Non-compartmental analysis was performed to estimate the PK parameters of QD6 / B09-57-AZ1508 and RAA22 / B09-57-AZ1508 based on the total ADC concentration of species and dose level.

[0845] In conclusion, in mice and cynomolgus monkeys, RAA22 / B09-57-AZ1508 showed higher exposure and prolonged t1 / 2 compared with QD6 / B09-57-AZ1508, suggesting improved PK in the lower affinity RAA22 / B09-57-AZ1508.

[0846] 7.1 Bioanalysis for Preclinical PK Assays

[0847] The concentrations of target compounds (QD6 / B09-57-AZ1508 and RAA22 / B09-57-AZ1508) and total antibody concentrations were measured using an immunocapture LC-MS / MS assay. Briefly, polyclonal anti-human antibodies were conjugated to magnetic beads. 25 μL of plasma samples were then diluted in PBS and incubated with magnetic beads. After capture, the beads were washed multiple times and then digested with trypsin in the presence of an internal standard. The digestion was quenched by the addition of acid. The liquid contents were then transferred to an injection plate.

[0848] Reverse phase chromatography (RPLC) was used to separate the characteristic tryptic peptides on the Fc region and the cleavage warhead of human antibodies, and then multiple reaction monitoring (MRM) was used for detection. The characteristic peptides on the Fc region were used to calculate the total Ab, and the warheads released by digestion were used to calculate the ADC. The internal standard used in this experiment was an isotope-labeled peptide or protein (SiluMAb, Sigma-Aldrich) or an isotope-labeled warhead. The peak area ratio of the analyte to the internal standard was used to calculate the standard curve.

[0849] Standard curves and QCs were prepared by spiking different levels of target compounds into the same matrix as samples. The quantitation range covered 100 ng / mL-15,000 ng / mL, and dilution QCs covered up to 525,000 ng / mL. The standard curves were fitted with the simplest possible model. The assay accuracy and precision were within 20% for all levels except the lower limit of quantitation (LLOQ) at 25%.

[0850] 7.2 QD6 / B09-57-AZ1508PK in mice

[0851] The mouse studies included in the NCA analysis are summarized in Table 4 .

[0852] Table 4 List of mouse studies of RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508

[0853]

[0854]

[0855] The mean PK concentration-time curves of RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 in mice are shown in FIG12 .

[0856] At the dose levels tested, both RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 exhibited linear PK in mice, with dose-proportional exposures observed at 0.5 mg / kg to 10 mg / kg of RAA22 / B09-57-AZ1508 and 1 mg / kg to 10 mg / kg of QD6 / B09-57-AZ1508, respectively (C max and AUC), comparable CL and t 1 / 2 .

[0857] The PK comparison between RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 was evaluated at the 1, 3, 5, 10 mg / kg dose levels tested for both compounds, and the mean PK parameters based on NCA are summarized in Table 5. The results showed that RAA22 / B09-57-AZ1508 had a slower CL, higher exposure, and t 1 / 2 The mean AUC of RAA22 / B09-57-AZ1508 showed a 2- to 2.91-fold increase compared with QD6 / B09-57-AZ1508; the mean t 1 / 2The t values ​​of RAA22 / B09-57-AZ1508 were 4.24 to 6.38 days, and those of QD6 / B09-57-AZ1508 were 2.57 to 3.33 days, indicating that the t values ​​of RAA22 / B09-57-AZ1508 were 1 / 2 Increased by 1.27 to 2.32 times.

[0858] Table 5 Average NCAPK parameters at dose levels between RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 in mice

[0859]

[0860]

[0861] 7.3 QD6 / B09-57-AZ1508PK in cynomolgus monkeys

[0862] The cynomolgus monkey studies included in the NCA analysis are summarized in Table 6.

[0863] Table 6 List of cynomolgus monkey studies of RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508

[0864]

[0865] The mean PK concentration-time curves of RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 in cynomolgus monkeys are shown in FIG13 .

[0866] RAA22 / B09-57-AZ1508 exhibited linear PK in cynomolgus monkeys at 2 mg / kg to 5 mg / kg, with dose-proportional exposure (C max and AUC), comparable CL and t 1 / 2 .

[0867] QD6 / B09-57-AZ1508 exhibited nonlinear PK in cynomolgus monkeys from 0.67 mg / kg to 3 mg / kg, showing more than dose-proportional exposure (C max and AUC), and faster CL and shorter t were observed at lower dose levels. 1 / 2 .

[0868] The PK comparison between RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 in cynomolgus monkeys was evaluated at the 2 and 3 mg / kg dose levels tested for both compounds, and the mean PK parameters based on NCA are summarized in Table 7. The results showed that RAA22 / B09-57-AZ1508 had a slower CL, higher exposure, and t 1 / 2The mean AUC of RAA22 / B09-57-AZ1508 showed a 1.90- to 2.43-fold increase compared with QD6 / B09-57-AZ1508; the mean t 1 / 2 The t values ​​of RAA22 / B09-57-AZ1508 were 4.30 to 5.90 days, and those of QD6 / B09-57-AZ1508 were 0.969 to 1.07 days, indicating that the t values ​​of RAA22 / B09-57-AZ1508 were 1 / 2 Increased by 4.44 to 5.51 times.

[0869] Table 7 Mean NCAPK parameters at dose levels between RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 in monkeys

[0870]

[0871] In summary, these data indicate that low-affinity RAA22 / B09-57-AZ1508 exhibits higher exposure and longer circulating half-life than high-affinity QD6 / B09-57-AZ1508 in mice and cynomolgus monkeys. These data are consistent with the hypothesis that reduced affinity for EGFR reduces binding to EGFR present in normal tissues, thereby mitigating the effects of normal tissue uptake and improving plasma PK parameters.

[0872] Example 8 - ADCs with Topoisomerase I Inhibitors as Payloads

[0873] An experiment was designed to test the efficacy and safety of the RAA22 / B09-57 bispecific molecule conjugated to a different payload (a topoisomerase I inhibitor instead of tubulysin as used in previous examples).

[0874] The DuetMab RAA22 / B09 (with a "Maia" cysteine ​​insertion after serine 239) bispecific antibody produced according to Example 1 was conjugated to the topoisomerase inhibitor SG3932 via "classical" conjugation to the native cysteine ​​in the bispecific antibody.

[0875]

[0876] The efficacy of the EGFR / cMET topoisomerase I inhibitor ADC was investigated using PDX assays. PDX assays were performed essentially as described in Example 5 above using various different PDX models derived from pancreatic cancer, colon cancer, NSCLC, and squamous head and neck cancer (SQHN) tumors. Animals were injected with a single dose of 10 mg / kg of EGFR-cMET Maia Topo ADC. The results of the PDX assays using the EGFR-cMET Maia Topo ADC are reported in Fig.14 middle.

[0877] like Fig.14 As shown, the EGFR-cMET Maia Topo ADC induced tumor growth inhibition or regression in many of the PDX models tested. Thus, these results demonstrate that the RAA22 / B09-57 ADC containing a topoisomerase I inhibitor is effective in PDX models representing a variety of tumor types.

[0878] Example 9 - Mutations that improve the PK of ADCs

[0879] The ADC with a topoisomerase I inhibitor generated and tested in Example 8 used a RAA22 / B09 bispecific antibody containing a "Maia" cysteine ​​insertion after serine 239. However, given that SG3932 is conjugated to a native cysteine, the Maia cysteine ​​insertion is not necessary. Therefore, we attempted to modify the RAA22 / B09 Maia Topo ADC generated in Example 8 to remove this cysteine ​​insertion.

[0880] It is also recognized that if the effector function of the Fc backbone is reduced or removed, immunotoxicity may be alleviated and pharmacokinetics improved. Therefore, we also introduced a "triple mutant (TM)" of L234F / L235E / P331S (EU numbering), which has previously been shown to reduce Fc effector function in antibody molecules (Organesyan, 2008; Hay, 2016).

[0881] The newly generated "EGFR-cMETTM" molecule, comprising 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:

[0882] EGFR-cMETTM EGFR heavy chain 59 c-Met heavy chain 60 EGFR light chain 61 c-Met light chain 62

[0883] In order to conjugate with SG3932, 50mM tris (2-carboxyethyl) phosphine (TCEP) in phosphate buffered saline pH7.4 (PBS) solution (12.5 molar equivalents / antibody) was added to the reducing buffer solution containing PBS and 1mM ethylenediaminetetraacetic acid (EDTA) of the EGFR-cMET TM bispecific antibody, and the final antibody concentration was about 3mg / mL. The reduction mixture was incubated for 2 hours at 37°C in an orbital shaker with gentle (60rpm) oscillation. The reaction mixture was cooled to room temperature for 45 minutes. Then SG3932 was added as a DMSO solution (12.5 molar equivalents / antibody), and the final DMSO concentration was 10% (v / v). The solution was incubated at room temperature for 2 hours, then quenched by adding N-acetyl cysteine ​​(5 micromoles / SG3932), and incubated at room temperature for 15 minutes. The reaction mixture was filtered using a 0.2uM sterile filter and then stored overnight at 2°C-8°C. Through a tangential flow filtration unit (TFF), using a surface area of ​​375 cm 2 mPES 30kDa fiber filter removed into a buffer containing 30mM histidine, 30mM arginine, pH 6.8. The extent of free drug removal was monitored by UHPLC-RP using pure conjugate. After complete removal of free drug, the ADC was buffer exchanged. The ADC was filtered using a 0.22μm filter under a sterile atmosphere, and polysorbate-80 was then added to a final concentration of 0.02% (w / v).

[0884] UHPLC analysis of the ADC reduced sample on a Shimadzu Recience system using a Thermo Scientific MAbPac 50 mm x 2.1 mm column with gradient elution at 214 nm and 330 nm (SG3932 specific) with water and acetonitrile revealed a drug / antibody ratio (DAR) of 6.0 SG3932 molecules per antibody.

[0885] The efficacy of the EGFR cMET™ ADC was investigated using PDX assays. The PDX assays were performed essentially as described in Example 5 above using a range of different PDX models derived from pancreatic cancer, colon cancer, NSCLC and SQHN tumors. Animals were injected with a single dose of 5 mg / kg of the EGFR-cMET™ ADC. The results of the PDX assays using the EGFR-cMET™ ADC are reported in Fig.15 The results of this experiment demonstrated that the EGFR-cMETTM ADC was able to induce tumor growth inhibition or regression in many of the PDX models tested.

[0886] The efficacy of EGFR-cMET™ ADC ("TM ADC") was compared with the EGFR-cMETMaiaTopoADC ("Maia ADC") produced in Example 8 in 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 untreated controls ("untreated") and animals dosed with unconjugated EGFR-cMET™ (TM mAb). The results are shown in Figure 16.

[0887] The results demonstrated that EGFR-cMETTM ADC and EGFR-cMETMaiaTopo ADC were equally effective ("equivalent") in reducing tumor growth / inducing tumor regression at the dose range tested. This suggests that the use of triple mutants to remove the S239i mutation and eliminate Fc effector function does not negatively affect efficacy in these tumor models.

[0888] The EGFR-cMET™ ADC was also shown to be effective in reducing the growth of NSCLC tumors expressing wild-type or mutant EGFR. The results showed that the EGFR-cMET™ ADC was active in both wild-type and mutant EGFR PDX models, such as Fig.17 This is advantageous as it suggests that the ADC will be able to provide benefit in multiple treatment settings and across a range of different EGFR genotypes.

[0889] In addition, a pharmacokinetic (PK) study was performed in NOD-SCID mice to compare the EGFR-cMET TM ADC (“TMADC”) and EGFR-cMET Maia Topo ADC (“Maia ADC”) produced in Example 8. The experiment was performed essentially as described in Example 7.

[0890] Representative results from these PK studies are presented in Fig.18 As reported in the figure, when compared with EGFR-cMET MaiaTopo ADC (t 1 / 2 =3.0 days; CL=39.7 ml / day / kg), EGFR-cMET™ ADC exhibited a longer half-life (t 1 / 2=5.0 days) and decreased drug clearance (CL=14.8 ml / day / kg). Thus, the results indicate that the EGFR-cMET™ ADC described herein exhibits improved PK compared to the EGFR-cMET Maia Topo ADC produced in Example 7. Similar improvements in PK were also observed when comparing unconjugated EGFR-cMET™ antibody ("TMmAb") and unconjugated EGFR-cMETMaia ("MaiaADC").

[0891] Example 10 - Efficacy of ADC in combination with osimertinib

[0892] This example uses a PDX assay to compare the efficacy of an ADC with that of the third generation tyrosine kinase inhibitor TKI osimertinib ('Osi') in various EGFR mutant tumor models.

[0893] 5.1 Methods

[0894] Preclinical efficacy studies were conducted in immunocompromised mice or carrying patient-derived xenografts (PDX) at Champions Oncology (athymic Foxn1nu nude mice) and Genendesign (Balb / C nude mice). All studies met AstraZeneca global standards for animal care and welfare. Models are established from living human tumor tissue or fluids and have been serially passaged in animals for a limited number of times to maintain tumor heterogeneity. Tumor fragments harvested from host animals are implanted unilaterally into the flank of mice, with each fragment implanted from a specific passage batch. Pre-study tumor volume is recorded starting approximately one week prior to its estimated start date. When tumors reach the appropriate initial tumor volume (TVI) range (150-300mm 3 ), the animals were randomly divided into a treatment group and a control group.

[0895] Treatment-Animals receiving the EGFR-cMETTop1i antibody drug conjugate (ADC) or control EGFR-cMETmAb described in Example 9 were administered a single intravenous (IV) dose at the indicated dose level on day 0; animals were administered IV by weight (at a dose volume of 5 ml / kg). Starting on day 0, animals in the osimertinib treatment group received the drug formulated in an oral dosing solution at 2.5 mg / ml in a vehicle (0.5% w / v HPMC (hydroxypropyl methylcellulose) in deionized water); animals were orally administered by weight at a dosing volume of 10 mL / kg to reach 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 EGFR-cMETADC and osimertinib, and each treatment was administered according to the monotherapy dosing schedule described above. All animals were followed up individually throughout the study.

[0896] Tumor growth inhibition - Starting from day 0, tumor size was measured twice a week by digital caliper, and data including individual and mean estimated tumor volumes (mean TV ± SEM) were recorded for each group; tumor volume was calculated using the formula (1): TV = width 2 × length × 0.52. At the end of the study, the initial (i) and final (f) tumor measurements of each treatment group (T) and control group (C) were calculated and reported as percent tumor growth inhibition (%TGI) values: %TGI = 1-(Tf-Ti) / (Cf-Ci). Individual mice reporting two consecutive measurements of tumor volume less than or equal to 30% of the measurement on day 0 were considered partial responders (PR). Individual mice lacking palpable tumors (two consecutive measurements of 0.00 mm 3 ) were classified as complete responders (CR); CRs that persisted until study completion were considered tumor-free survivors (TFS). Tumor doubling time (DT) for the vehicle-treated group was determined using the formula DT = (Df–Di)*log2 / (logTVf–logTVi), where D = days and TV = tumor volume. Tumor growth was observed in the untreated control group until the mean tumor volume of that group (uncensored) reached 1500 mm 3 The humane endpoint was reached at 1500 mm or until day 60, whichever came first. Tumor growth in the treatment groups was observed until day 60; if the tumor of an individual mouse in the treatment group reached 1500 mm 3 If a humane endpoint was reached, the animal was euthanized and other treated animals were observed. Some animals that showed sustained responses were observed for more than 60 days.

[0897] result

[0898] The results are provided in Figures 19-21.

[0899] Fig.19A and 19C Results are shown for EGFR mutant models 'LUN487' and 'LUN439' containing the EGFR L858R mutation representing first-line EGFRm non-small cell lung cancer (NSCLC). EGFR-cMET™ ADC was dosed at 2, 4, and 8 MPK (mg / kg). One group was also dosed with either a mAb-only control (EGFR-cMET mAb) at 8 MPK or osimertinib-only at 25 MPK. Results show that EGFR-cMET™ ADC monotherapy exhibited a dose-dependent response. The PDX model responded to osimertinib. No treatment response was observed for the mAb-only control group.

[0900] Fig.19B and 19D Results are shown for EGFR mutant models 'LUN487' and 'LUN439', where EGFR-cMETTM ADC was administered at 2 or 4 MPK alone or in combination with osimertinib (25 MPK). The combination of EGFR-cMETTM ADC and osimertinib showed improved tumor growth inhibition compared to either drug (EGFR-cMETTM ADC or osimertinib) administered alone.

[0901] Fig. 20A Results are shown for the exon 20 insertion EGFR mutant model 'CTG-2992' that represents primary resistance to osimertinib. EGFR-cMETTMADC was dosed at 2, 4, and 8 MPK (mg / kg). One group was also dosed with 8mPK of mAb-only control (EGFR-cMETmAb) or 25MPK of osimertinib-only. The results show that EGFR-cMETTMADC monotherapy exhibits a dose-dependent response. The PDX model responded to osimertinib.

[0902] Fig. 20B Results are shown for the EGFR mutant model 'CTG-2992', where EGFR-cMETTM ADC and osimertinib were administered in combination. EGFR-cMETTM ADC was dosed at 2 or 4 MPK. The combination of EGFR-cMET™ ADC and osimertinib showed improved tumor growth inhibition compared to either drug (EGFR-cMET™ ADC or osimertinib) administered alone.

[0903] Fig.21AResults are shown for the EGFR mutant model 'CTG-2803' representing acquired resistance to osimertinib. EGFR-cMETTM ADC was dosed at 2, 4, and 8 MPK (mg / kg). One group was also dosed with either 8 mPK of mAb-only control (EGFR-cMETmAb) or 25 MPK of osimertinib-only. Results show that EGFR-cMET™ ADC monotherapy exhibited a dose-dependent response. The PDX model did not respond to osimertinib or mAb-only monotherapy.

[0904] Fig. 21B Results are shown for the EGFR mutant model 'CTG-2803', where EGFR-cMETTM ADC and osimertinib were administered in combination. EGFR-cMETTM ADC was dosed at 2 or 4 MPK. The combination of EGFR-cMET™ ADC and osimertinib showed improved tumor growth inhibition compared to either drug (EGFR-cMET™ ADC or osimertinib) administered alone.

[0905] Taken together, these results demonstrate that the combination of EGFR-cMET™ ADC and osimertinib shows efficacy in a range of EGFR mutant cancers, including those classified as osimertinib-resistant.

[0906] Example 11 - Response of EGFRmutNSCLCPDX to the combination of ADC and osimertinib

[0907] This example aims to further evaluate the combined efficacy of osimertinib and EGFR-cMETTMADC in the NSCLC PDX model with mutant EGFR. Additional 23 NSCLC PDX models covering various EGFR mutations were recruited. The study was mainly carried out as described in Section 5.1 of Example 10.

[0908] Each PDX mouse was typically divided into three groups and received osimertinib 25 mg / kg, EGFR-cMET™ ADC 2 mg / kg or osimertinib 25 mg / kg and EGFR-cMET™ ADC 2 mg / kg daily for 21 days. Experiments were performed with isotype ADC R347 control at a dose of 8 mg / kg and / or naked EGFR-cMET™ mAb control at a dose of 8 mg / kg.

[0909] Fig.22A , 22B and 22C show the responses of each of the models for each of the three treatment groups.

[0910] The results showed that responses were observed in 14 / 23 (61%) models of the combination group, while osimertinib and EGFR-cMETTMADC monotherapy groups showed response rates in 8 / 23 (34.8%) and 7 / 23 (30.4%) models, respectively. Response was defined as a 30% regression of tumor volume from baseline. Regression was analyzed starting one week after dosing. The reported value is the best response observed during the study.

[0911] The table below provides more detailed response data as well as information on EGFR mutation status.

[0912] The best overall response (R for response and NR for no response) for each study group for each model is shown in Table 9. The % change represents the change in tumor volume compared to baseline.

[0913]

[0914]

[0915]

[0916] References

[0917] Many publications are cited above to more fully describe and disclose the disclosure and the state of the art in the field to which the disclosure belongs. The complete citations of these references are provided below. The full text of each of these references is incorporated herein.

[0918]

[0919]

[0920]

[0921]

[0922]

[0923]

[0924] sequence

[0925] CDR sequences of low affinity anti-EGFR binding group (RAA22)

[0926] HCDR1–DNDFS (SEQ ID NO: 1)

[0927] HCDR2–AIVAVFRTETYAQKFQD(SEQ ID NO:2)

[0928] HCDR3–RLMSAISGPGAPLLM(SEQ ID NO:3)

[0929] LCDR1–TGTSSDVGGYNYVS(SEQ ID NO:4)

[0930] LCDR2–DVSKRPS (SEQ ID NO:5)

[0931] LCDR3–SSYTSSDTLEI(SEQ ID NO:6)

[0932] CDR sequences of high affinity anti-EGFR binding group (QD6)

[0933] HCDR1–DNDFS (SEQ ID NO: 1)

[0934] HCDR2–AIVAVVRTETYAQKFQD(SEQ ID NO:7)

[0935] HCDR3–RLMSAISGPGAPLLM(SEQ ID NO:3)

[0936] LCDR1–TGTSSDVGGYNYVS(SEQ ID NO:4)

[0937] LCDR2–DVSERPS (SEQ ID NO:66)

[0938] LCDR3–FSYTSSDTLEI(SEQ ID NO:67)

[0939] FR sequences of RAA22 and QD6 in the anti-EGFR binding group

[0940] HFR1–QVQLVQSGAEVKKPGSSVKVSCKASGGTFS(SEQ ID NO:8)

[0941] HFR2–WVRQAPGQGLEWMG(SEQ ID NO:9)

[0942] HFR3–RVKITADISTRTTYMELSSLRSEDTAVYYCAR(SEQ ID NO:10)

[0943] HFR4–WGQGTLVTVSS(SEQ ID NO:11)

[0944] LFR1–QSALTQPRSVSGSPGQSVTISC (SEQ ID NO:12)

[0945] LFR2–WYQQHPGKAPKLMIY(SEQ ID NO:13)

[0946] LFR3–GVPDRFSGSSKSGNTASLTISGLQAEDEADYYC(SEQ ID NO:14)

[0947] LFR4–FGGGTKLTVL(SEQ ID NO:15)

[0948] Amino acid sequence of the variable heavy (VH) region of the low affinity anti-EGFR binding group (RAA22) (SEQ ID NO: 16)

[0949] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNDFSWVRQAPGQGLEWMGAIVAVFRTETYAQKFQDRVKITADISTRTTYMELSSLRSEDTAVYYCARRLMSAISGPGAPLLMWGQGTLVTVSS

[0950] Nucleic acid sequence of the VH region of the low affinity anti-EGFR binding group (RAA22) (SEQ ID NO: 17):

[0951] CAGGTGCAGCTGGTGCAGTCTGGGGCCGAAGTGAAGAAACCCGGCAGCAGCGTGAAGGTGTCCTGTAAAGCCAGCGGCGGCACCTTCAGCGACAACGACTTTAGCTGGGTCCGACAGGCCCCTGGACAGGGCCTGGAATGGATGGGAGCCATCGTGGCCGTGTTCCGGACAGAGACATACGCCCAG AAATTCCAGGACAGAGTGAAAATCACCGCCGACATCAGCACCAGAACCACCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGCCAGACGGCTGATGTCTGCCATCTCTGGACCTGGCGCTCCTCTGCTCATGTGGGGACAGGGAACACTGGTCACCGTGTCCAGC

[0952] The amino acid sequence of the VH region of the anti-EGFR antibody clone QD6 (SEQ ID NO: 18):

[0953] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNDFSWVRQAPGQGLEWMGAIVAVVRTETYAQKFQDRVKITADISTRTTYMELSSLRSEDTAVYYCARRLMSAISGPGAPLLMWGQGTLVTVSS

[0954] Nucleic acid sequence of the VH region of the anti-EGFR antibody clone QD6 (SEQ ID NO: 19):

[0955] CAGGTGCAGCTGGTGCAGTCTGGGGCCGAAGTGAAGAAACCCGGCAGCAGCGTGAAGGTGTCCTGTAAAGCCAGCGGCGGCACCTTCAGCGACAACGACTTTAGCTGGGTCCGACAGGCCCCTGGACAGGGCCTGGAATGGATGGGAGCCATCGTGGCCGTGGTCCGGACAGAGACATACGCCCAG AAATTCCAGGACAGAGTGAAAATCACCGCCGACATCAGCACCAGAACCACCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGCCAGACGGCTGATGTCTGCCATCTCTGGACCTGGCGCTCCTCTGCTCATGTGGGGACAGGGAACACTGGTCACCGTGTCCAGC

[0956] The amino acid sequence of the variable light chain (VL) region of the anti-EGFR antibody clone RAA22 (SEQ ID NO: 20):

[0957] QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSSGNTASLTISGLQAEDEADYYCSSYTSSDTLEIFGGGTKLTVL

[0958] Nucleic acid sequence of the VL region of the anti-EGFR antibody clone RAA22 (SEQ ID NO: 21):

[0959] CAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGTTCATATACAAGCAGCGACACTCTCGAAATATTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0960] Amino acid sequence (SEQ ID NO:22) of the VL region of the high-affinity anti-EGFR binding group (QD6):

[0961] QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSERPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCFSYTSSDTLEIFGGGTKLTVL

[0962] Nucleic acid sequence (SEQ ID NO:23) of the VL region of the high-affinity anti-EGFR binding group (QD6):

[0963] CAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTGAACGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTTCTCATATACAAGCAGCGACACTCTCGAAATATTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0964] CDR sequences of anti-cMet binding group B09-GL

[0965] HCDR1–DYYIH (SEQ ID NO: 24)

[0966] HCDR2–WMNPNSGNTGYAQKFQG(SEQ ID NO:25)

[0967] HCDR3-GQGYTHS (SEQ ID NO:26)

[0968] LCDR1–RASEGIYHWLA(SEQ ID NO:27)

[0969] LCDR2–KASSLAS (SEQ ID NO:28)

[0970] LCDR3–QQYSNYPPT(SEQ ID NO:29)

[0971] FR sequence of anti-cMet binding group B09-GL

[0972] HFR1–QVQLVQSGAEVKKPGASVKVSCKASGYTFT(SEQ ID NO:30)

[0973] HFR2–WVRQATGQGLEWMG(SEQ ID NO:31)

[0974] HFR3–RVTMTRDTSISTAYMELSSLRSEDTAVYYCAR(SEQ ID NO:32)

[0975] HFR4–WGQGTMVTVSS(SEQ ID NO:33)

[0976] LFR1–DIQMTQSPSTLSASSVGDRVTITC(SEQ ID NO:34)

[0977] LFR2–WYQQKPGKAPKLLIY(SEQ ID NO:35)

[0978] LFR3–GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(SEQ ID NO:36)

[0979] LFR4–FGGGTKLEIK(SEQ ID NO:37)

[0980] The amino acid sequence of the variable heavy (VH) region of the anti-cMet binding group B09-GL (SEQ ID NO: 38):

[0981] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRSEDTAVYYCARGQGYTHSWGQGTMVTVSS

[0982] Nucleic acid sequence of the VH region of the anti-cMet binding group B09-GL (SEQ ID NO: 39):

[0983] CAAGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCAGCGTGAAGGTCAGCTGCAAGGCCAGCGGCTACACCTTCACCGACTACATCCACTGGGTCCGCCAGGCCACAGGCCAGGGACTGGAATGGATGGGCTGGATGAACCCCAACAGCGGCAACACC GGCTACGCCCAGAAATTCCAGGGCAGAGTGACCATGACCCGGGACACCAGCATCAGCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCCAGAGGCCAGGGCTACACCCACAGCTGGGGCCAGGGCACCATGGTCACAGTGTCCAGC

[0984] The amino acid sequence of the variable light (VL) region of the anti-cMet binding group B09-GL (SEQ ID NO: 40):

[0985] DIQMTQSPSTLSASVGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSNYPPTFGGGTKLEIK

[0986] Nucleic acid sequence of the VL region of the anti-cMet binding group B09-GL (SEQ ID NO: 41):

[0987] GACATCCAGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTCGGCGACAGAGTGACCATCACCTGTCGGGCCAGCGAGGGCATCTACCACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAAGGCCAGCAGCC TGGCCAGCGGAGTCCCTAGCAGATTTTCTGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGCCAGCAGTACAGCAACTACCCCCCCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG

[0988] The amino acid sequence of the human immunoglobulin G1 heavy chain constant (CH) region (SEQ ID NO: 42):

[0989] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0990] Amino acid sequence of a human immunoglobulin G1 CH region modified to include a "knob" mutation, an interchain cysteine ​​mutation, a cysteine ​​to form a stabilizing disulfide bridge, and having a cysteine ​​insertion (SEQ ID NO: 43):

[0991] The following substitutions are underlined:

[0992] "Knob" mutation (T366W); interchain cysteine ​​mutations (F126C and C219V); stabilizing cysteine ​​mutation (S354C); and cysteine ​​insertion (C239i), where numbering of residues is according to the EU index.

[0993] ASTKGPSV C PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS V DKTHTCPPCPAPELLGGPS C VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C REEMTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0994] Amino acid sequence of a human immunoglobulin G1 CH region modified to include a "knob" mutation, an interchain cysteine ​​mutation, a cysteine ​​to form a stabilizing disulfide bridge, and no cysteine ​​insertion (SEQ ID NO: 44):

[0995] The following substitutions are underlined:

[0996] "Knob" mutation (T366W); interchain cysteine ​​mutations (F126C and C219V); and a stabilizing cysteine ​​mutation (S354C), where numbering of residues is according to the EU index.

[0997] ASTKGPSV C PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS V DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C REEMTKNQVSL WCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0998] Amino acid sequence of a 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):

[0999] The following substitutions are underlined:

[1000] "Hole" mutations (T366S, L368A, and Y407V); stabilizing cysteine ​​mutation (Y349C); and cysteine ​​insertion (C239i), where the numbering of residues is according to the EU index.

[1001] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS C VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1002] Amino acid sequence of a 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):

[1003] The following substitutions are underlined:

[1004] "Hole" mutations (T366S, L368A and Y407V); and a stabilizing cysteine ​​mutation (Y349C), wherein the numbering of residues is according to the EU index.

[1005] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1006] Amino acid sequence of wild-type human immunoglobulin kappa constant region (SEQ ID NO: 47):

[1007] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1008] Amino acid sequence of a human immunoglobulin kappa constant region modified to include S121C and C214V substitutions (SEQ ID NO: 48):

[1009] The following substitutions are underlined:

[1010] S121C and C214V, where the numbering is according to the EU index

[1011] RTVAAPSVFIFPP C DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE V

[1012] Amino acid sequence of human immunoglobulin lambda constant region (SEQ ID NO: 49):

[1013] GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[1014] Amino acid sequence of the heavy chain of the anti-cMet binding group B09-GL with cysteine ​​insertion (SEQ ID NO: 50):

[1015] The following substitutions are underlined:

[1016] "Knob" mutation (T366W); interchain cysteine ​​mutations (F126C and C219V); stabilizing cysteine ​​mutation (S354C); and cysteine ​​insertion (C239i), where numbering of residues is according to the EU index.

[1017] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRSEDTAVYYCARGQGYTHSWGQGTMVTVSSASTKGPSV C PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS V DKTHTCPPCPAPELLGGPS C VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C REEMTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[1018] Amino acid sequence of the heavy chain of the anti-cMet binding group B09-GL without cysteine ​​insertion (SEQ ID NO: 51):

[1019] The following substitutions are underlined:

[1020] "Knob" mutation (T366W); interchain cysteine ​​mutations (F126C and C219V); and a stabilizing cysteine ​​mutation (S354C), where numbering of residues is according to the EU index.

[1021] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRSEDTAVYYCARGQGYTHSWGQGTMVTVSSASTKGPSV C PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS V DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP C REEMTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[1022] The amino acid sequence of the light chain of the anti-cMet binding group B09-GL (SEQ ID NO: 52):

[1023] The following substitutions are underlined:

[1024] S121C and C214V, where the numbering is according to the EU index

[1025] DIQMTQSPSTLSASSVGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSNYPPTFGGGTKLEIKRTVAAPSVFIFPP C DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE V

[1026] Amino acid sequence of the heavy chain of the high affinity anti-EGFR binding group (QD6) with cysteine ​​insertion (SEQ ID NO: 53):

[1027] The following substitutions are underlined:

[1028] "Hole" mutations (T366S, L368A, and Y407V); stabilizing cysteine ​​mutation (Y349C); and cysteine ​​insertion (C239i), where the numbering of residues is according to the EU index.

[1029] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNDFSWVRQAPGQGLEWMGAIVAVVRTETYAQKFQDRVKITADISTRTTYMELSSLRSEDTAVYYCARRLMSAISGPGAPLLMWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS C VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1030] Amino acid sequence of the heavy chain of the high affinity anti-EGFR binding group (QD6) without cysteine ​​insertion (SEQ ID NO: 54):

[1031] The following substitutions are underlined:

[1032] "Hole" mutations (T366S, L368A and Y407V); and a stabilizing cysteine ​​mutation (Y349C), wherein the numbering of residues is according to the EU index.

[1033] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNDFSWVRQAPGQGLEWMGAIVAVVRTETYAQKFQDRVKITADISTRTTYMELSSLRSEDTAVYYCARRLMSAISGPGAPLLMWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1034] The amino acid sequence of the light chain of the high affinity anti-EGFR binding group (QD6) (SEQ ID NO: 55):

[1035] QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSERPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCFSYTSSDTLEIFGGGTKL TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[1036] Amino acid sequence of the heavy chain of the low affinity anti-EGFR binding group (RAA22) with cysteine ​​insertion (SEQ ID NO: 56):

[1037] The following substitutions are underlined:

[1038] "Hole" mutations (T366S, L368A, and Y407V); stabilizing cysteine ​​mutation (Y349C); and cysteine ​​insertion (C239i), where the numbering of residues is according to the EU index.

[1039] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNDFSWVRQAPGQGLEWMGAIVAVFRTETYAQKFQDRVKITADISTRTTYMELSSLRSEDTAVYYCARRLMSAISGPGAPLLMWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS C VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1040] Amino acid sequence of the heavy chain of the low affinity anti-EGFR binding group (RAA22) without cysteine ​​insertion (SEQ ID NO: 57):

[1041] The following substitutions are underlined:

[1042] "Hole" mutations (T366S, L368A and Y407V); and a stabilizing cysteine ​​mutation (Y349C), wherein the numbering of residues is according to the EU index.

[1043] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNDFSWVRQAPGQGLEWMGAIVAVFRTETYAQKFQDRVKITADISTRTTYMELSSLRSEDTAVYYCARRLMSAISGPGAPLLMWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS C VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1044] The amino acid sequence of the light chain of the low affinity anti-EGFR binding group (RAA22) (SEQ ID NO: 58):

[1045] QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSDTLEIFGGGTKL TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[1046] Amino acid sequence of the heavy chain of the low affinity anti-EGFR binding group (RAA22) with triple mutation (TM) (SEQ ID NO: 59):

[1047] The following substitutions are underlined:

[1048] Triple mutation (TM; L234F, L235E and P331S); "knob" mutation (T366W); interchain cysteine ​​mutations (F126C and C219V); stabilizing cysteine ​​mutation (S354C), where the numbering of residues is according to the EU index.

[1049] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNDFSWVRQAPGQGLEWMGAIVAVFRTETYAQKFQDRVKITADISTRTTYMELSSLRSEDTAVYYCARRLMSAISGPGAPLLMWGQGTLVTVSSASTKGPSV C PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS V DKTHTCPPCPAPE FE GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA S IEKTISKAKGQPREPQVYTLPP C REEMTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1050] Amino acid sequence of the heavy chain of the anti-cMet binding group with triple mutation (TM) (SEQ ID NO: 60):

[1051] The following substitutions are underlined:

[1052] Triple mutation (TM; L234F, L235E and P331S); "hole" mutation (T366S, L368A and Y407V); and stabilizing cysteine ​​mutation (Y349C), where the numbering of residues is according to the EU index.

[1053] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRSEDTAVYYCARGQGYTHSWGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE FE GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA S IEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1054] Amino acid sequence of the light chain of the low affinity anti-EGFR binding group (RAA22) in the "EGFR-cMET TM" antibody (SEQ ID NO: 61):

[1055] The following substitutions are underlined:

[1056] S121C and C214V, where the numbering is according to the EU index

[1057] QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSSKNTASLTISGLQAEDEADYYCSSYTSSDTLEIFGGGTKLTVLGQPKAAPSVTLFPP C SEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTE V S

[1058] The amino acid sequence of the light chain of the anti-cMet binding group in the "EGFR-cMETTM" antibody (SEQ ID NO: 62):

[1059] DIQMTQSPSTLSASVGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSNYPPTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1060] Amino acid sequence of a human immunoglobulin G1 CH region modified to include a "knob" mutation, a cysteine ​​to form a stabilizing disulfide bridge, no cysteine ​​insertion, and with a TM (SEQ ID NO: 63):

[1061] The following substitutions are underlined:

[1062] Triple mutation (TM; L234F, L235E and P331S); "knob" mutation (T366W); interchain cysteine ​​mutations (F126C and C219V); stabilizing cysteine ​​mutation (S354C), where the numbering of residues is according to the EU index.

[1063] ASTKGPSV C PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS V DKTHTCPPCPAPE FE GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA S IEKTISKAKGQPREPQVYTLPP C REEMTKNQVSL W CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1064] Amino acid sequence of a human immunoglobulin G1 CH region modified to include a "hole" mutation, a cysteine ​​to form a stabilizing disulfide bridge, no cysteine ​​insertion, and with a TM (SEQ ID NO: 64):

[1065] The following substitutions are underlined:

[1066] Triple mutation (TM; L234F, L235E and P331S); "hole" mutation (T366S, L368A and Y407V); and stabilizing cysteine ​​mutation (Y349C), where the numbering of residues is according to the EU index.

[1067] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE FE GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA S IEKTISKAKGQPREPQV C TLPPSREEMTKNQVSL S C A VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL V SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1068] Amino acid sequence of a human immunoglobulin lambda constant region modified to include S121C and C214V substitutions (SEQ ID NO: 65):

[1069] GQPKAAPSVTLFPP C SEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTE V S

[1070] Amino acid sequence of human EGFR extracellular domain (SEQ ID NO: 68):

[1071] LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

[1072] Amino acid sequence of cynomolgus monkey EGFR extracellular domain (SEQ ID NO:69):

[1073] LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSEFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDTLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSSQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCQNVSRGRECVDKCNILEGEPREFVENSECIQCHPECLPQVMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCARNGPKIPS

[1074] Amino acid sequence of human cMET extracellular domain (SEQ ID NO:70):

[1075] ECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFT

[1076] Amino acid sequence of cynomolgus macaque cMET extracellular domain (SEQ ID NO:71):

[1077] ECKEALAKSEMNVNMKYQLPNFTAETAIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPNQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPHHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYIHAFESNNFIYFLTVQRETLNAQTFHTRIIRFCSLNSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRAEFTTALQRVDLFMGQFSEVLLTSISTFVKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHPLNQNGYTLVVTGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECPSGTWTQQICLPAIYKVFPTSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPIITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLHSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFT

Claims

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, wherein the anti-EGFR / cMET antibody molecule comprises an EGFR binding domain and a cMET binding domain, wherein the EGFR binding domain comprises: a. A heavy chain variable (VH) region comprising 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. a HCDR3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid; and b. a light chain variable (VL) region comprising 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 a variant thereof, wherein one or two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are substituted with another amino acid.

2. An anti-EGFR / cMET antibody molecule for use in treating cancer in a human patient, wherein the anti-EGFR / cMET antibody molecule is administered in combination with an EGFR TKI, wherein the anti-EGFR / cMET antibody molecule comprises an EGFR binding domain and a cMET binding domain, wherein the EGFR binding domain comprises: a. A heavy chain variable (VH) region comprising 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. a HCDR3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid; and b. a light chain variable (VL) region comprising 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 a variant thereof, wherein one or two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are substituted with another amino acid.

3. The EGFR TKI for use according to claim 1, or the anti-EGFR / cMET antibody molecule for use according to claim 2, wherein the EGFR TKI and the anti-EGFR / cMET antibody molecule are administered separately, sequentially or simultaneously.

4. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to any one of the preceding claims, wherein the EGFR TKI is a compound having formula (V): in: 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, fluorine, chlorine, methyl and cyano; R 2 is selected from the group consisting of methoxy, trifluoromethoxy, ethoxy, 2,2,2-trifluoroethoxy and methyl; R 3 selected from (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]octan-2-yl, (3aR,6aR)-5-methylhexa Hydrogen-pyrrolo[3,4-b]pyrrol-1(2H)-yl, 1-methyl-1,2,3,6-tetrahydropyridin-4-yl, 4-methylpiperazin-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-aminopropionyl]piperazin-1-yl; R 4 is selected from hydrogen, 1-piperidinylmethyl and N,N-dimethylaminomethyl; R 5 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.

5. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to claim 4, wherein G is selected from indol-3-yl and indazol-1-yl; R 1 is selected from hydrogen, fluorine, chlorine, 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; 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 . The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to claim 5 , wherein the EGFR TKI is osimertinib or a pharmaceutically acceptable salt thereof.

7. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to any of the preceding claims, 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, avitinib 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. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to any one of the preceding claims, 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, afatinib or a pharmaceutically acceptable salt thereof, HS-10296 or a pharmaceutically acceptable salt thereof, and lazertinib or a pharmaceutically acceptable salt thereof.

9. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to any one of the preceding claims, wherein the anti-EGFR binding domain comprises: a. A heavy chain variable (VH) region comprising 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. a HCDR3 having the amino acid sequence of SEQ ID NO: 3; and b. a light chain variable (VL) region comprising 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.

10. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to any one of the preceding claims, 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 with 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 with the amino acid sequence of SEQ ID NO:

20.

11. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to any one of the preceding claims, wherein the anti-cMET binding domain comprises: a. A heavy chain variable (VH) region comprising 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. a HCDR3 having the amino acid sequence of SEQ ID NO: 26, or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid; and b. a light chain variable (VL) region comprising 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 a variant thereof, wherein one or two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are substituted with another amino acid.

12. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to any one of the preceding claims, wherein the anti-cMET binding domain comprises: a. A heavy chain variable (VH) region comprising 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. a HCDR3 having the amino acid sequence of SEQ ID NO: 26; and b. a light chain variable (VL) region comprising 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.

13. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to any of the preceding claims, 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 with 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 with the amino acid sequence of SEQ ID NO:

40.

14. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to any one of the preceding claims, wherein the antibody molecule comprises: a. a first heavy chain, wherein the first heavy chain comprises 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, wherein the first light chain comprises 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, wherein the second heavy chain comprises the VH region of the anti-cMET binding domain, and a second heavy chain constant (CH) region or a fragment thereof; and d. a second light chain, wherein the second light chain comprises the VL region of the anti-cMET binding domain, and a second light chain constant (CL) region or a fragment thereof.

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

42.

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

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

18. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to any one of claims 14 to 17, wherein 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 listed 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 listed in SEQ ID NO: 64, and 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 listed in SEQ ID NO: The sequence listed in ID NO:65 has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and the second CL region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence listed in SEQ ID NO:

47.

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

62.

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

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

22. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to claim 21, wherein the drug comprises 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, a DNA, a RNA, a siRNA, a RNAi, a microRNA, a photoactive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide, a lipid, a carbohydrate, a chelating agent or a combination thereof.

23. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to claim 22, wherein the drug is a topoisomerase I inhibitor having the formula A* 24. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to claim 23, wherein the topoisomerase I inhibitor has the formula or a salt or solvate thereof, wherein R L is a linker for connecting the antibody molecule, optionally wherein the linker is selected from: (ia): in Q is: Where Q X such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; X is: 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, wherein 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); G L is a linker used to connect the antibody molecule; or (ib): Where R L1 and R L2 are independently selected from H and methyl, or together with the carbon atoms to which they are attached form a cyclopropene or cyclobutene group; and e is 0 or 1.

25. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to claim 21, wherein the antibody is conjugated to a topoisomerase I inhibitor having the formula:

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

27. The EGFR TKI for use or the 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. The EGFR TKI for use or the 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 and / or L858R mutations in exon 19 in the EGFR gene.

29. The EGFR TKI for use or the 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 in the EGFR gene, a mutation in exon 20, or both.

30. The EGFR TKI for use or the anti-EGFR / cMET antibody molecule for use according to any preceding claim, wherein the human patient is an EGFR TKI naive human patient.

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

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

33. 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, wherein the anti-EGFR / cMET antibody molecule comprises an EGFR binding domain and a cMET binding domain, wherein the EGFR binding domain comprises: a. A heavy chain variable (VH) region comprising 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. a HCDR3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid; and b. a light chain variable (VL) region comprising 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 a variant thereof, wherein one or two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are substituted with another amino acid.

34. A method of treating cancer in a human patient in need of such treatment, the method 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, wherein the anti-EGFR / cMET antibody molecule comprises an EGFR binding domain and a cMET binding domain, wherein the EGFR binding domain comprises: a. A heavy chain variable (VH) region comprising 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. a HCDR3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid; and b. a light chain variable (VL) region comprising 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 a variant thereof, wherein one or two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are substituted with another amino acid.

35. A method of treating cancer in a human patient in need of such treatment, the method 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 constitute a therapeutically effective amount, wherein the anti-EGFR / cMET antibody molecule comprises an EGFR binding domain and a cMET binding domain, wherein the EGFR binding domain comprises: a. A heavy chain variable (VH) region comprising 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. a HCDR3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid; and b. a light chain variable (VL) region comprising 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 a variant thereof, wherein one or two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are substituted with another amino acid.

36. A drug 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, wherein the EGFR binding domain comprises: a. A heavy chain variable (VH) region comprising 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. a HCDR3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof, wherein one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are substituted with another amino acid; and b. a light chain variable (VL) region comprising 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 a variant thereof, wherein one or two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are substituted with another amino acid.

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