Antibody molecules and conjugates

Abstract

The present disclosure relates to antibody molecules that bind to epidermal growth factor receptor (EGFR) and / or c-Met, and conjugates containing these antibody molecules. The antibody molecules and conjugates have application, for example, in the treatment of cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 263,835, filed November 10, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing Reference This application incorporates by reference the Sequence Listing submitted herewith as a text file of size 95 kilobytes, created on October 17, 2022, entitled "EGFCM-100 Sequence Listing."

[0003] The present disclosure relates to antibody molecules that bind to epidermal growth factor receptor (EGFR) and / or c-Met, and conjugates containing these antibody molecules. The antibody molecules and conjugates have application, for example, in the treatment of cancer. [Background technology]

[0004] Overcoming drug resistance is a major challenge facing targeted cancer therapy. Although multiple EGFR and c-MET-directed drugs have been approved or are in clinical development, de novo and acquired resistance limit their long-term efficacy (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6; Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10). Mechanisms of resistance include secondary mutations, activation of oncogenic downstream signaling modules such as KRAS, ligand upregulation, and amplification of alternative growth factor receptors. For example, MET amplification or protein overexpression is well established as an important mechanism of clinical resistance to EGFR inhibitors. Similarly, emerging evidence suggests that EGFR pathway activation may confer resistance to c-MET-targeted inhibitors (Non-Patent Document 11; Non-Patent Document 6; Non-Patent Document 7; Non-Patent Document 8; Non-Patent Document 9). Antibody-drug conjugates (ADCs) are a growing field of targeted therapy designed to selectively deliver cytotoxic drugs directly to tumor cells (Non-Patent Document 12; Non-Patent Document 13; Non-Patent Document 14; Non-Patent Document 15; Non-Patent Document 16; Non-Patent Document 17). In this context, the antigen targeted by the antibody is primarily used as a handle to deliver the cytotoxic drug, and the mechanism of killing is generally different from the biology of the target. Thus, ADCs have the potential to largely avoid resistance due to activation of alternative receptors or downstream signaling pathways, if a target is present. However, adverse events due to engagement of targets on normal cells may limit the therapeutic index of ADCs (Non-Patent Document 18; Non-Patent Document 19; Non-Patent Document 16). Bispecific antibody technology confers additional specificity to ADCs by engaging two different targets on tumor cells, and may effectively deliver a cytotoxic payload if two targets are present (Non-Patent Document 20; Non-Patent Document 21; Non-Patent Document 22; Non-Patent Document 23; Non-Patent Document 24; Non-Patent Document 25; Non-Patent Document 26). However, simply targeting two antigens does not guarantee selectivity for tumor cells compared to normal non-target cells, and multiple factors influence the properties of a given bispecific antibody (Non-Patent Document 26; Non-Patent Document 27).It is therefore desirable to develop bispecific ADCs that improve targeting of tumor cells while sparing normal tissues. Such ADCs have the potential to overcome resistance to targeted therapy due to pathway alterations while maintaining a therapeutic window by taking advantage of the selective targeting afforded by bispecific antibodies.

[0005] Epidermal growth factor receptor (EGFR, HER1, Erbb1) is the founding member of the human epidermal growth factor receptor (HER) tyrosine kinase family, which also includes HER2 / Erbb2, HER3 / Erbb3, and HER4 / Erbb4 (Non-Patent Document 28; Non-Patent Document 29; Non-Patent Document 30; Non-Patent Document 31; Non-Patent Document 32). Numerous therapies against EGFR have been approved, both in the biologic and small molecule tyrosine kinase inhibitor (TKI) classes (Non-Patent Document 33; Non-Patent Document 5; Non-Patent Document 34; Non-Patent Document 35; Non-Patent Document 36). Despite these successes, the clinical benefits realized by these therapies fall short of those expected based on the widespread expression profile of EGFR in many cancer types. Several factors are responsible for the observed clinical limitations, which mainly involve mechanisms of both intrinsic and acquired resistance due to alterations in signaling pathways (Non-Patent Document 5). For example, in colorectal cancer (CRC), EGFR is overexpressed in more than 65-75% of patients, but in unselected patient populations, the objective response rate of anti-EGFR antibodies such as cetuximab and panitumumab administered as monotherapy was only in the range of about 10% (Non-Patent Document 37; Non-Patent Document 38). These antibodies are only effective in patients whose tumors express wild-type KRAS (about 50-55% of patients), which defines the treatable patient population (Non-Patent Document 39; Non-Patent Document 40). Furthermore, due to additional resistance mechanisms, only about half of eligible patients respond to treatment, and all ultimately fail treatment. Treatment of patients with EGFR-targeted agents is often accompanied by moderate to severe skin toxicities, occurring in 65-90% of patients (Non-Patent Document 41). These skin toxicities may be grade 3-4 in severity (NCI-CTCAE criteria) and may lead to dose modification or cessation of treatment. Thus, EGFR, although highly expressed in a wide range of tumor types, is a challenging target for ADC approaches, potentially exacerbating these observed toxicities. The development of ADCs that can effectively target EGFR-expressing tumors without inducing unacceptable EGFR-associated toxicity would have the potential to treat a broader range of EGFR-positive patient populations compared to existing therapies.

[0006] c-MET is the gene product of the proto-oncogene MET, encoded on chromosome 7. The c-MET protein is a receptor tyrosine kinase expressed primarily on the surface of epithelial cells, which recognizes only one known ligand, hepatocyte growth factor (HGF), also known as scatter factor (Non-Patent Document 42; Non-Patent Document 43). The c-MET / HGF signaling axis plays an essential role in regulating proliferation, differentiation, motility, and morphogenesis in many normal processes, including wound healing, tissue regeneration, and organogenesis during development (Non-Patent Document 44). Aberrant expression and dysregulation of the c-MET pathway have been reported in a wide variety of human cancers, including non-small cell lung cancer, colon cancer, gastrointestinal cancer, head and neck cancer, pancreatic cancer, renal cancer, and hepatocellular carcinoma, among others (Non-Patent Document 44; Non-Patent Document 45; Non-Patent Document 46; Non-Patent Document 47; Non-Patent Document 48; Non-Patent Document 49; Non-Patent Document 50). In many of these cancer conditions, c-MET overexpression is negatively associated with prognosis and survival (Non-Patent Document 51; Non-Patent Document 52; Non-Patent Document 53; Non-Patent Document 54; Non-Patent Document 55; Non-Patent Document 56; Non-Patent Document 57; Non-Patent Document 58; Non-Patent Document 57; Non-Patent Document 59; Non-Patent Document 60; Non-Patent Document 61; Non-Patent Document 62; Non-Patent Document 63; Non-Patent Document 64). Furthermore, in some cancers, c-MET is associated with a cancer stem cell phenotype that is involved in resistance to EGFR-targeted therapy (Non-Patent Document 55; Non-Patent Document 2; Non-Patent Document 65; Non-Patent Document 66; Non-Patent Document 67). There is a large body of literature demonstrating that crosstalk and direct interactions exist between the EGFR and c-MET signaling pathways, and that this crosstalk functionally results in resistance to EGFR and c-MET-targeted therapy in the clinic. (Non-Patent Document 68; Non-Patent Document 69; Non-Patent Document 70; Non-Patent Document 71; Non-Patent Document 72; Non-Patent Document 73; Non-Patent Document 2; Non-Patent Document 74; Non-Patent Document 75; Non-Patent Document 76; Non-Patent Document 77; Non-Patent Document 49; Non-Patent Document 3; Non-Patent Document 66; Non-Patent Document 78; Non-Patent Document 79; Non-Patent Document 80; Non-Patent Document 11; Non-Patent Document 81; Non-Patent Document 9). Based on ample evidence of the role of c-MET as an independent negative prognostic indicator in many tumor types and its implication as a mechanism of resistance to EGFR inhibitors, many efforts have been made to develop c-MET inhibitors as cancer therapeutics.Nevertheless, despite the high frequency of c-MET expression in multiple tumor types, the development of effective c-MET inhibitors has faced significant challenges and setbacks in the clinic (Non-Patent Document 82; Non-Patent Document 83; Non-Patent Document 84; Non-Patent Document 85; Non-Patent Document 86). For example, crizotinib, a dual inhibitor of c-MET and ALK, has been approved for the treatment of non-small cell lung cancer, but other c-MET-targeting drugs, such as the one-arm c-MET antibody MetMAb (Onartuzumab) and the small molecule inhibitor, tivantinib, have failed in late-stage Phase III clinical trials due to lack of efficacy (Non-Patent Document 46; Non-Patent Document 82; Non-Patent Document 83; Company press release). A plausible hypothesis for the discrepancy between the widespread c-MET expression profile and the limited clinical response of c-MET inhibitors is that only a portion of these tumors are driven by the c-MET pathway, and therefore many c-MET-expressing tumors are insensitive to inhibitors that block c-MET signaling activity. Clinical development of several c-MET inhibitors is underway, and some of these programs are attempting to employ biomarker-driven strategies to identify the proportion of patients whose tumors are dependent on c-MET (Non-Patent Document 87; Non-Patent Document 88; Non-Patent Document 89). Clinical experience to date suggests that, similar to EGFR inhibitors, successful development of novel c-MET signal blocking inhibitors will benefit only a portion of the total population of patients with c-MET-expressing tumors. The development of effective c-MET-directed ADCs may overcome some of the limitations of signal blocking c-MET inhibitors.

[0007] The concept of antibody-drug conjugates (ADCs) is simple, aiming to generate drugs with a broad therapeutic window by using the excellent specificity of antibodies to precisely deliver a cytotoxic warhead to cancer cells while minimizing damage to normal tissues (Non-Patent Document 20; Non-Patent Document 15; Non-Patent Document 16; Non-Patent Document 90; Non-Patent Document 91; Non-Patent Document 13; Non-Patent Document 14). Although the concept is simple, achieving the ideal combination of ADC properties has proven difficult, as reflected by the limited number of ADCs approved to date (Non-Patent Document 92). In recent years, it has witnessed a growing field, with clinical-stage ADCs currently having over 70 drug candidates in development. Despite the successes to date and the prospect of new ADCs reaching patients in the future, many challenges remain and there is considerable room for improvement. Ultimately, a key challenge in developing an ADC is to balance its efficacy with safety. One EGFR-directed ADC, depatukizumab mafodotin (ABT-414), is currently in Phase III clinical development by AbbVie for glioblastoma (Non-Patent Document 17). ABT-414 was previously tested in Phase II trials for several additional solid tumor indications (ClinicalTrials.gov:NCT01741727). The ADC showed limited efficacy for these indications at tolerated doses, and troublesome ocular toxicity was frequently observed in treated patients (Non-Patent Document 93). ABBV-221, a second-generation EGFR ADC, was in clinical development but was discontinued due to safety concerns (Non-Patent Document 94; Non-Patent Document 95; Company announcement). Currently, there is one c-MET-targeted ADC, telisotuzumab vedotin (ABBV-399), in phase II clinical development in patients with non-small cell lung cancer (NSCLC) whose tumors express high levels of c-MET, both as monotherapy and in combination with the EGFR inhibitor erlotinib (NPL 12; NPL 96). The combination of a c-MET ADC+EGFR TKI showed clinical activity in phase I trials in this selected patient population, with peripheral neuropathy and skin rash being the most frequent treatment-related adverse events (NPL 12; NPL 95).The nature of bispecific antibodies allows fine-tuning the interaction between each target to affect the overall properties of the molecule, potentially generating ADCs with acceptable therapeutic windows (Non-Patent Document 20). This concept has been tested in vitro for EGFR and c-MET, but researchers have yet to demonstrate in vivo the concept of improved safety or efficacy compared to the above-mentioned EGFR and c-MET ADCs (Non-Patent Document 97). Therefore, it would be beneficial to utilize a bispecific antibody strategy to develop EGFR-cMET ADCs that exhibit both efficacy and an acceptable safety profile.

[0008] The present disclosure has been devised in light of the above considerations. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Ou, S.-HI, N. Agarwal, and SMAli, High MET amplification level as a resistance mechanism to osimertinib(AZD9291) in a patient that symptomatically responded to crizotinib treatment post-osimertinib progression. Lung Cancer, 2016.98(Supplement C):p.59-61. [Non-Patent Document 2] Boccaccio, C., P. Luraghi, and PMComoglio, MET-Mediated Resistance to EGFR Inhibitors: An Old Liaison Rooted in Colorectal Cancer Stem Cells.2014.p.3647-3651. [Non-Patent Document 3] Karamouzis, MV,PAKonstantinopoulos,and AGPapavassiliou,Targeting MET as a strategy to overcome crosstalk-related resistance to EGFR inhibitors.The Lancet Oncology,2009.10(7):p.709-717.

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[0010] The present disclosure provides antibody molecules and conjugates that have a combination of desirable biophysical and / or functional properties compared to antibody molecules disclosed in the prior art.

[0011] Aspects of the present disclosure relate to antibody molecules capable of binding to both EGFR and c-Met. EGFR and c-Met are co-expressed in many cancer types, and antibody molecules targeting both molecules ("bispecific antibody molecules") offer the opportunity for broad clinical benefit across multiple indications. This antibody molecule represents an improved therapy for cancer compared to prior art monospecific EGFR or c-Met antibodies, since the bispecific antibody molecules described herein can bind both targets simultaneously, thus increasing selectivity for tumors that co-express EGFR and c-MET compared to normal tissues.

[0012] Thus, in one aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: a first antigen-binding domain that binds to the epidermal growth factor receptor (EGFR); and A second antigen-binding domain that binds to c-Met Including, The first antigen-binding domain comprises (i) a heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs): HCDR1 having the amino acid sequence of SEQ ID NO:1 HCDR2 having the amino acid sequence of SEQ ID NO:2 HCDR3 having the amino acid sequence of SEQ ID NO:3, or a variant thereof in which one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are replaced by another amino acid; and (ii) a light chain variable (VL) region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO:4 LCDR2 having the amino acid sequence of SEQ ID NO:5 LCDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof in which one, two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are replaced by another amino acid. The present invention provides an antibody molecule comprising:

[0013] In particular, conjugates containing this antibody molecule can selectively deliver drugs (also called "payloads" or "warheads") to tumors that co-express these targets and treat these tumors with high efficacy. The examples demonstrate that different conjugates containing alternative warheads (e.g., tubulysin or topoisomerase I inhibitors) exhibit potent cytotoxic activity in both in vitro cytotoxicity assays and in in vivo models of cancer treatment.

[0014] Thus, in another aspect, the present disclosure provides a conjugate comprising an antibody molecule described herein conjugated to a drug. In some examples, the drug is a topoisomerase I inhibitor, such as a topoisomerase inhibitor having the formula A*. [ka]

[0015] In some examples, the antibody molecule is conjugated to a topoisomerase I inhibitor having the following formula: [ka]

[0016] In some examples, the antibody molecule comprises a first antigen-binding domain that binds to human EGFR with "low affinity". As used herein, "low affinity" refers to a first antigen-binding domain that binds to human EGFR with a dissociation constant (Kd) of 10 nM or greater. As shown herein, the antibodies and conjugates disclosed herein that comprise such low affinity EGFR antigen-binding domains exhibit reduced on-target toxicity in normal tissues, such as skin toxicity, and thus have an improved safety profile compared to conjugates that comprise an EGFR antigen-binding domain that binds to human EGFR with "higher affinity". As used herein, "higher affinity" or "high affinity" refers to a first antigen-binding domain that binds to human EGFR with a Kd of less than 10 nM. Furthermore, the present disclosure shows that conjugates that comprise low affinity EGFR antigen-binding domains are more effective in treating cancer compared to conjugates that comprise higher affinity EGFR antigen-binding domains (e.g., FIG. 12).

[0017] The present disclosure also provides antibody molecules comprising a first antigen-binding domain that binds to EGFR, and a second antigen-binding domain that binds to c-Met as defined herein.

[0018] Furthermore, the present disclosure provides a pharmaceutical composition comprising an antibody molecule as defined herein or a conjugate as defined herein.

[0019] The present disclosure provides antibody molecules, conjugates and pharmaceutical compositions, all as defined herein, for use in a method of treatment of the human or animal body, such as a method of treating cancer. The present disclosure provides a method of treating cancer, comprising administering an antibody molecule, conjugate or pharmaceutical composition as defined herein. In particular, the present disclosure provides a method of treating a cancer selected from pancreatic cancer, colon cancer, non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (SQHN), comprising administering an antibody molecule, conjugate or pharmaceutical composition as defined herein. In some examples, the cancer to be treated is NSCLC.

[0020] The present disclosure also provides the nucleic acids, vectors and host cells defined herein. Additionally, the present disclosure provides methods of producing the antibody molecules defined herein.

[0021] The present disclosure includes combinations of the described aspects and preferred features unless such combinations are clearly contraindicated or explicitly avoided.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present disclosure will now be described with reference to the accompanying drawings. [Brief description of the drawings]

[0023] [Figure 1A] Diagrammatic representation of RAA22 / B09-57. The anti-EGFR RAA22 Fab, anti-cMET B09-57 Fab, and the hole and knob heavy chains are shown. The structural drawing is a composite of the individual domain structures. [Figure 1B] Diagrammatic representation of QD06 / B09-57. The anti-EGFR QD06 Fab, anti-cMET B09-57 Fab, and the hole and knob heavy chains are shown. The structural drawing is a composite of the individual domain structures. [Diagram 2] Simultaneous binding studies with antigen capture format were performed by Octet analysis. Human cMET antigen-loaded sensors were exposed to sequential association and dissociation interactions, first with the antibody and then with the human EGFR antigen. Ass = association; Diss = dissociation; NI-NTA = nickel-nitrilotriacetic acid. [Figure 3A]ELISA results showing EGFR and c-Met species cross-reactivity. The high affinity monospecific EGFR IgG (QD6) and the monovalent bispecific EGFR / cMET DuetMAb (QD6 / B09) bound to human, cynomolgus and mouse EGFR. The low affinity monospecific EGFR IgG (RAA22) bound weaker to human, cynomolgus and mouse EGFR compared to QD6, and the corresponding monovalent bispecific EGFR / c-Met DuetMAb (RAA22 / B09) showed even weaker binding to human and cynomolgus EGFR compared to the bivalent parent IgG (RAA22), with only minor binding to mouse EGFR. The monospecific c-Met IgG (B09) and all bispecific variants showed comparable binding to human and cynomolgus c-Met, but no detectable binding to mouse c-Met. [Figure 3B] ELISA results showing EGFR and c-Met family specificity. The antibodies tested did not show detectable binding to any of the EGFR HER family proteins (HER2, HER3, or HER4), nor to any of the c-Met family members (Ron (CD136) or Semaphorin 3a). [Figure 4] Internalization and trafficking of RAA22 / B09 bispecific mAb to acidified intracellular compartments was visualized using antibodies labeled with pHAb pH-sensitive dye (Promega). Control antibodies included R347 isotype control and monovalent bispecific control antibodies anti-EGFR RAA22 / R347 and anti-cMET B09 / R347. pHAb-labeled antibodies were incubated with NCI-H1975 lung cancer cells at a concentration of 1.25ug / mL in a humidified incubator at 37°C and 5% CO2. Fluorescence images were captured at the indicated time points using a Cy3 filter on an Operetta High Content imaging system. An increase in cell fluorescence intensity over time, as measured by pH-sensitive fluorescence, was taken as evidence of internalization and trafficking to acidic intracellular compartments. [Diagram 5]As in Figure 4, internalization and trafficking of RAA22 / B09 bispecific mAb to acidified intracellular compartments was visualized using antibody labeled with pHAb pH-sensitive dye, but cells were treated with a lower concentration of antibody, 0.625 μg / mL. [Figure 6A] Kinetics of QD6 / B09 and RAA22 / B09 monoclonal antibody (mAb) internalization in H1975 cells. (a) Image overlays at the initiation of internalization and 1 hour after labeling cells with CellTracker Blue CMAC for the cytoplasm (blue) and 2.5 μg / mL QD6 / B09-AlexaFluor-647 (magenta, top panel) or 2.5 μg / mL RAA22 / B09 AlexaFluor-647 (magenta, bottom panel). Cells were labeled with CellTracker Blue CMAC and then bound with mAbs-AlexaFluor6457 at 2-8 °C and subjected to internalization conditions: (37 °C, 70% humidity, 5% CO2). [Figure 6B] Kinetics of QD6 / B09 and RAA22 / B09 monoclonal antibody (mAb) internalization in H1975 cells. The time course of mAb-AlexaFluor647 internalization was determined by quantitative analysis of dynamic images using an algorithm (Materials and Methods). Dynamic images taken at 5 min intervals were processed using an algorithm (Vainshtein, 2015) to determine antibody accumulation in the cytoplasm. The antibody signal in the cytoplasm (cytoplasmic fraction) normalized to intracellular antibody fluorescence for QD6 / B09-AlexaFluor647 (red) and RAA22 / B09-AlexaFluor647 (blue) is shown for one of three independent experiments. The internalization rate constant (kint) was calculated from the internalization time course using curve fitting with the equation Fcyt(t)=(1-ek int -t)·Fmax,cyt, where Fmax,cyt is the maximum ratio of cytoplasmic intensity per cell to total intensity per cell. The T1 / 2 calculated from kint was 37.5±10.6 min for QD6 / B09 and 43.2±15.5 min for RAA22 / B09 (from n=3). [Figure 7A]Internalization profile of QD6 / B09 DuetMab and its respective single-arm control antibody. The internalization profile is shown by the time course of the respective membrane and cytoplasmic signals of each construct. The QD6 / B09 set was acquired using an Opera confocal fluorescence microscope. [Figure 7B] Internalization profiles of the RAA22 / B09 DuetMab and its respective single-arm control antibody. The internalization profiles are shown by the time course of the respective membrane and cytoplasmic signals of each construct. Sets were acquired using a Zeiss spinning disk confocal fluorescence microscope. Identical profiles of QD6 / B09 and QD6 / IgG indicate an internalization mode driven by the EGFR arm of the QD6 / B09 DuetMab, whereas the RAA22 / B09 DuetMab requires engagement of both the EGFR and c-MET arms for efficient internalization. [Figure 8A] Internalization profile of RAA22 / B09-AZD1508 ADC in cells expressing moderate and high target c-MET and EGFR cell surface receptors. Membrane, cytoplasmic and total signal of RAA22 / B09-AF647 ADC in H1975 cells are shown. One representative experiment out of 2 is shown. H1975 cells show a concomitant drop in total and membrane intensity indicating dissociation of the antibody from the cell surface. [Figure 8B] Figure 8B is the same as Figure 8A, but for HCC827 cells. HCC827 cells show a stable total signal over the entire time of the experiment. The decrease in membrane signal is due to antibody internalization. [Figure 9A] Internalization of RAA22 / B09-AZD1508 ADC single-arm control antibody in HCC827 cells. The intensity profile of RAA22 / IgG single-arm is approximately 10-fold lower than RAA22 / B09 due to weaker binding to EGFR via the single-arm conjugate. [Figure 9B] The B09 / IgG single arm dissociates from the cell membrane as shown by a concomitant decrease in total and membrane signals over time. [Figure 10]Blockade of EGFR or cMETR reduces bispecific ADC activity in vitro. NCI H1975 cells were pretreated with excess unarmed parental antibody to block EGFR or cMET. EGFR-cMET ADC (RAA22 / B09-AZ1508) was then added to the cells in a 4x serial dilution series with final concentrations ranging from 67 nM to 0.0009 nM. Treated cells were cultured for 72 hours in a humidified incubator at 37°C and 5% CO2. Metabolic activity was determined using a CellTiter-Glo luminescent viability assay (Promega). Data are plotted as percent metabolic activity relative to untreated controls. IC50 values ​​were determined using logistic nonlinear regression analysis between maximum viability (untreated cells) and maximum response (peak of inhibition) using GraphPad Prism software. [Figure 11] Monovalent ADCs against EGFR or cMET have reduced activity in vitro compared to bispecific EGFR / cMET ADCs. Monovalent ADCs were constructed by pairing each binding arm with a non-binding isotype control arm (R347) to generate EGFR ADCs (RAA22 / R347-AZ1508) and anti-cMET ADCs (B09 / R347-AZ1508). ADCs were added to NCI H1975 cells in a 4x serial dilution series with final concentrations ranging from 67 nM to 0.0009 nM. Percent metabolic activity was measured as described in Figure 9.

[0024] Figure 1 shows the results of mouse PDX studies conducted to determine the efficacy of high affinity (QD6 / B09-AZ1508) and low affinity (RAA22 / B09-AZ1508) EGFR-cMET ADCs in patient tumor xenograft (PDX) models of multiple human cancers in immunocompromised mice. Each compound was tested at a single dose level of 3 mg / kg in a single mouse for each PDX model representing a different human tumor. Tumor growth rates relative to untreated control tumors (T / C%) were calculated for tumors that grew larger than the initial volume, and tumor regression rates were calculated for tumors that showed a reduction in size compared to the initial tumor volume. (A) shows a direct comparison of high and low affinity ADCs in each model.

[0025] FIG. 12B shows a waterfall plot of QD6 / B09-AZ1508 in the rank order of efficacy.

[0026] FIG. 12C shows a waterfall plot of RAA22 / B09-AZ1508 in the rank order of efficacy. [Figure 13] Dose-ranging in vivo efficacy studies in PDX models were performed in athymic nude mice unilaterally implanted with tumor fragments harvested from the host animals in the flank. The high affinity EGFR-cMET ADC (QD6 / B09-AZ1508) was tested at dose levels of 1 mg / kg and 2 mg / kg, and the variant with reduced affinity for EGFR (RAA22 / B09-AZ1508) was tested at 1 mg / kg, 2 mg / kg, and 3 mg / kg, as indicated. Tumor volume measurements were taken twice weekly after dosing initiation and plotted as line graphs of tumor volume over time. Error bars indicate standard error of the mean (SEM), and inset images show immunohistochemical staining of EGFR and cMET for each model from tumor tissue harvested from early passages of the model.

[0027] In vivo efficacy of EGFR-cMET bispecific ADC in subcutaneous and orthotopic pancreatic PDX models. A) In vivo efficacy of high affinity QD6 / B09 ADC in subcutaneous MEDI-PANC-08 PDX model, ●-naive, ■-R347-AZ1508 (3mg / kg-Q1W x 4), ▲-QD6 / B09-1508 (1mg / kg-Q1W x 4), ▼-QD6 / B09-1508 (2mg / kg-Q1W x 4) and -QD6 / B09-1508 (3mg / kg-Q1W x 4). Tumor volumes were measured twice weekly.

[0028] In vivo efficacy of low affinity RAA2 / B09 ADCs in subcutaneous MEDI-PANC-08 PDX model: ●-Untreated, ■-R347-AZ1508 (3mg / kg-Q1W x 4), ▲-RAA2 / B09-1508 (1mg / kg-Q1W x 4), ▼-RAA2 / B09-1508 (2mg / kg-Q1W x 4) and -RAA2 / B09-1508 (3mg / kg-Q1W x 4). Tumor volumes were measured twice weekly.

[0029] Orthotopic MEDI-PANC-08 LUC Luminescence imaging of (luciferase-expressing) PDX models. Mice were imaged weekly using an IVIS Spectrum in vivo imaging system. Images are normalized across groups and time points using a radiance scale (mean radiance [p / s / cm2 / sr]) set between maximum signal (control Gp on day 21) and background.

[0030] In vivo efficacy of low affinity RAA2 / B09 ADC in subcutaneous MEDI-PANC-08 PDX model. ●-Untreated, ■-Gemcitabine (75mg / kg-Q3 / 4D x 5), ▲-R347-AZ1508 (3mg / kg-Q1W x 4), ▼-RAA2 / B09-1508 (2mg / kg-Q1W x 4) and -RAA2 / B09-1508 (3mg / kg-Q1W x 4). Treatment days are indicated by arrows and tumor volumes were measured twice weekly. Data shown in panels A and B represent group mean tumor volumes (mm 3 ) ± SEM, and panel D shows group mean radiance [p / s / cm 2 / sr] ± SEM.

[0031] 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.

[0032] Mean concentration-time profile and mean NCA PK parameters of QD6 / B09-57-AZ1508 in mice. Target compound and total antibody concentrations were measured using an immunocapture LC-MS / MS assay. Signature tryptic peptides and cleaved warheads on human antibody Fc regions were separated using reversed-phase chromatography (RPLC) and then detected using multiple reaction monitoring (MRM). Signature peptides on Fc regions (VVSVLTVLHQDWLNGK) were used to calculate total Abs, while digestion-released warheads were used to calculate RAA22 / B09 ADCs. The internal standards used in this experiment are isotopically labeled peptides or proteins (SiluMAb, Sigma-Aldrich) or isotopically labeled warheads. The peak area ratios of the analytes to the internal standards were used to calculate against a standard curve. Standard curves and QCs are prepared by spiking different levels of RAA22 / B09 ADCs into the same matrix as the samples. The quantification range includes 100 ng / mL to 15,000 ng / mL, with diluted QC up to 525,000 ng / mL. Standard curves were fitted using the simplest possible model. The accuracy and precision of the assay are within 20% at all levels except the LLOQ, which is 25%.

[0033] Mean concentration-time profile of RAA22 / B09-57-AZ1508 in monkeys. Cynomolgus monkey plasma samples were collected and processed using an immunocapture LC-MS / MS assay and non-compartmental PK analysis as described in FIG.

[0034] Mean concentration-time profile of QD6 / B09-57-AZ1508 in monkeys. Cynomolgus monkey plasma samples were collected and processed using an immunocapture LC-MS / MS assay and non-compartmental PK analysis as described in FIG.

[0035] Mean NCA PK parameters of QD6 / B09-57-AZ1508 in monkeys are shown. NCA PK parameters for QD6 / B09-57-AZ1508 (20067312), 3 mg / kg are based on PK data after the second dose. All other results were based on PK data after the first dose. Cynomolgus monkey plasma samples were collected and processed using an immunocapture LC-MS / MS assay and noncompartmental PK analysis as described in FIG. 15. [Figure 17] As a PDX study, EGFR-cMET Maia Topoi ADC was evaluated in patient tumor xenograft (PDX) models representing multiple types of human cancer in immunocompromised mice. Compounds were tested at a dose level of 10 mg / kg in a single mouse for each PDX model representing a unique human tumor. Tumor growth rate (T / C%) relative to untreated control tumors was calculated for tumors that grew larger than the initial volume (tumor growth inhibition rate (TGI%) was defined as the tumor growth rate relative to day 0 between the treated (TX) group and the control (C) group, according to the following formula: TGI%=1-(TXfinal-TXinitial) / (Cfinal-Cinitial)), and tumor regression rate was calculated for tumors that showed a reduction in size compared to the initial tumor volume (tumor regression rate was defined as the tumor reduction rate of tumors of treated animals relative to the tumor volume on day 0 (the day of initial administration), at the end point of the study, according to the following formula: regression %=(TXfinalmean-TXinitialmean) / (TXinitialmean)×100). [Figure 18]For PDX studies, EGFR-cMET Topoi™ ADC was evaluated in patient tumor xenograft (PDX) models representing multiple types of human cancer in immunocompromised mice. Compounds were tested at a dose level of 5 mg / kg in a single mouse for each PDX model representing a unique human tumor. Tumor growth rate (T / C%) relative to untreated control tumors was calculated for tumors that grew larger than the initial volume (tumor growth inhibition rate (TGI%) was defined as the tumor growth rate relative to day 0 between the treated (TX) and control (C) groups according to the following formula: TGI%=1-(TXfinal-TXinitial) / (Cfinal-Cinitial)), and tumor regression rate was calculated for tumors that showed a reduction in size compared to the initial tumor volume (tumor regression rate was defined as the tumor reduction rate of tumors in treated animals relative to the tumor volume on day 0 (the day of initial administration) and calculated at the study endpoint according to the following formula: regression %=(TXfinalmean-TXinitialmean) / (TXinitialmean)×100).

[0036] Two different EGFR-cMET ADCs with different IgG Fc formats (Maia and TM) were evaluated for comparability in the SQHN-02 PDX model. The ADCs were tested at three dose levels: 2.5 mg / kg, 5 mg / kg, and 10 mg / kg, and tumor growth was compared to untreated control animals. A total of 10 animals were treated in each of the treatment and control groups.

[0037] Two different EGFR-cMET ADCs with different IgG Fc formats (Maia and TM) were evaluated for comparability in the Panc-08 PDX model. The ADCs were tested at three dose levels: 2.5 mg / kg, 5 mg / kg, and 10 mg / kg, and tumor growth was compared to untreated control animals. A total of 10 animals were treated in each of the treatment and control groups. [Figure 20] Results from non-small cell lung cancer NSCLC PDX models from Figure 17 above are shown, highlighting EGFR mutation status and histology (where known). [Figure 21]The pharmacokinetic profiles of EGFR-cMET bispecific antibody INT-009 (RAA22 / B09-Maia naked mAb) and INT-009-SG3932 DAR8 ADC ("Maia ADC") were compared to B09 / RAA2-IgG1-TM mirror mAb (INT-017) and TM-mirror-SG3932 DAR6 ADC ("TM ADC") at a therapeutic dose of 5 mg / kg in NOD-SCID mice. [Figure 22] Shows a comparison of EGFR and cMet receptor degradation following treatment with EGFR-cMET Topoi™ ADC versus amivantamab DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Aspects and examples of the present disclosure will now be described with reference to the accompanying drawings. Further aspects and disclosures will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0039] target EGFR Human EGFR (also known as proto-oncogene c-ErbB-1, receptor tyrosine protein kinase erbB-1 and EC 2.7.10.1) is a protein identified by UniProt P00533. Alternative splicing of the mRNA encoded by the human EGFR gene (also known as ERBB, ERBB1 and HER1) results in four isoforms: isoform 1 (UniProt: P00533-1, v2 (sequence last updated: November 1, 1997)); isoform 2 (UniProt: P00533-2, v1) (containing substitutions F404L and L405S relative to isoform 1 and lacking the amino acid sequence corresponding to positions 406 to 1210 of isoform 1); isoform 3 (UniProt: P00533-3, v1) (containing substitutions at positions 628 to 705 of isoform 1 and lacking the amino acid sequence corresponding to positions 706 to 1210 of isoform 1); and isoform 4 (UniProt: P00533-4) (containing substitution C628S relative to isoform 1 and lacking the amino acid sequence corresponding to positions 629 to 1210 of isoform 1).

[0040] The structure and function of EGFR are reviewed, for example, in Ferguson, Annu Rev Biophys. (2008) 37:353-373. EGFR is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family). The receptor contains a large extracellular region, a single transmembrane domain, an intracellular juxtamembrane domain, a tyrosine kinase domain, and a C-terminal regulatory region. Binding of EGFR to a ligand induces receptor dimerization and autophosphorylation of several tyrosine residues (Y992, Y1045, Y1068, Y1148, and Y1173) in the C-terminal regulatory region of EGFR.

[0041] Aberrant EGFR expression / activity has been implicated in many diseases, including nervous system disorders and many cancers.

[0042] As used herein, "EGFR" refers to EGFR from any species, including EGFR isoforms, fragments, variants, or homologs from any species.

[0043] c-Met Human c-Met, also known as hepatocyte growth factor receptor (HGFR) or tyrosine protein kinase Met, is a protein identified by UniProt P08581. Alternative splicing of the mRNA encoded by the human MET gene results in three isoforms: isoform 1 (UniProt: P08581-1, v4 (sequence last updated: July 7, 2009)); isoform 2 (UniProt: P08581-2) (inserted with the amino acid sequence "STWWKEPLNIVSFLFCFAS" at position 755 of isoform 1); and isoform 3 (UniProt: P08581-3), also known as soluble met variant 4, (inserted with the amino acids corresponding to positions 755-764 of isoform 1 by "RHVNIALIQR" and lacking the amino acid sequence corresponding to positions 765-1390 of isoform 1).

[0044] The structure of c-Met is reviewed, for example, in Gherardi, 2003, which is incorporated herein by reference in its entirety. c-Met is a heterodimer consisting of a disulfide-linked alpha (50 kDa) and beta (145 kDa) chain. c-Met contains an N-terminal sema domain that mediates binding to hepatocyte growth factor (HGF) and an intracellular kinase domain. Ligand binding at the cell surface induces autophosphorylation of c-Met on its intracellular domain, providing docking sites for downstream signaling molecules and activation of several signaling cascades.

[0045] c-Met is expressed in normal tissues on the surface of epithelial cells. c-Met overexpression is observed in many human tumors and cancers, and is often associated with a metastatic phenotype and poor prognosis. Examples of cancers in which high levels of c-Met expression have been observed include non-small cell lung cancer, pancreatic cancer, colon cancer, head and neck squamous cell carcinoma, breast cancer, and esophagogastric cancer. In these cancers, co-expression of EGFR and c-Met is often observed.

[0046] antibody molecule The present disclosure provides antibody molecules. The antibody molecules according to the present disclosure may be provided in isolated form, meaning that they are free of contaminants such as antibodies capable of binding to other polypeptides and / or serum components.

[0047] The term "antibody molecule" describes an immunoglobulin, whether naturally produced or partially or wholly synthetically produced. The antibody molecule may be a human or humanized antibody molecule. The antibody molecule is preferably a monoclonal antibody molecule. Examples of antibodies include immunoglobulin isotypes such as immunoglobulin G (IgG), their isotypic subclasses such as IgG1, IgG2, IgG3 and IgG4, and fragments thereof.

[0048] The term "antibody molecule" as used herein includes antibody fragments, provided that they are shown to bind to the relevant target molecule. Examples of antibody fragments include Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabodies, triabodies, scFv-Fc, minibodies and single domain antibodies (e.g., VhH). Unless the context requires otherwise, the term "antibody molecule" as used herein is therefore equivalent to "antibody molecule or fragment thereof."

[0049] Antibody molecules and methods for constructing and using them are well known in the art and are described, for example, in Holliger & Hudson, Nature Biotechnology 23(9):1126-1136 (2005). It is possible to take monoclonal and other antibody molecules and use techniques of recombinant DNA technology to generate other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve introducing the CDRs or variable regions of one antibody molecule into a different antibody molecule (EP-A-184187, GB-A-2188638A, and EP-A-239400).

[0050] In view of current techniques related to monoclonal antibody technology, antibody molecules can be prepared for most antigens. The antigen-binding domain may be a portion of an antibody (e.g., a Fab fragment) or a synthetic antibody fragment (e.g., a single-chain Fv fragment (scFv)). Monoclonal antibodies suitable for a selected antigen may be prepared by known techniques, for example those described in "Monoclonal Antibodies; A manual of techniques", H Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies; Techniques and Applications", JGR Hurrell (CRC Press, 1982). Chimeric antibodies are described in Neuberger, 1988.

[0051] The antibody molecule according to the present disclosure comprises an antigen-binding domain. An "antigen-binding domain" refers to the part of a molecule that binds to all or part of a target antigen and is complementary to it. If the antigen is large, the antibody may only bind to a specific part of the antigen, which part is called an epitope. An antibody antigen-binding site may be provided by one or more antibody variable domains. An antibody antigen-binding site preferably comprises a variable light (VL) region and a variable heavy (VH) region. The VH and VL regions of an antigen-binding domain together constitute an Fv region.

[0052] An antibody molecule domain generally comprises six complementarity determining regions (CDRs): three in the variable heavy (VH) region (HCDR1, HCDR2 and HCDR3) and three in the variable light (VL) region (LCDR1, LCDR2 and LCDR3). The six CDRs together define the paratope of the antigen binding domain, which is the part of the antigen binding domain that binds to the target antigen.

[0053] The VH and VL regions comprise framework regions (FRs) on either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, the VH region comprises the following structure: N-terminus-[HFR1]-[HCDR1]-[HFR2]-[HCDR2]-[HFR3]-[HCDR3]-[HFR4]-C-terminus, and the VL region comprises the following structure: N-terminus-[LFR1]-[LCDR1]-[LFR2]-[LCDR2]-[LFR3]-[LCDR3]-[LFR4]-C-terminus.

[0054] There are several different conventions for defining antibody CDRs and FRs, as described, for example, in Kabat, 1991, Chothia, 1987, the IMGT numbering (described in LeFranc, 2015), and VBASE2 (described in Retter, 2005). The CDRs and FRs of the VH and VL regions of the antibody molecules described herein are defined according to Kabat (Kabat, 1991).

[0055] An antibody molecule that comprises at least two antigen-binding domains, each of which is capable of binding to a different target, may be referred to as a "bispecific antibody molecule." In contrast, an antibody molecule that binds only to a single target (e.g., EGFR or c-Met) is referred to as a "monospecific antibody molecule." The present disclosure provides bispecific antibody molecules that comprise a first antigen-binding domain that binds to EGFR and a second antigen-binding domain that binds to c-Met.

[0056] Anti-EGFR antigen-binding domain The antigen-binding domain that binds to EGFR comprises the CDRs of an antibody molecule capable of binding to EGFR. In some examples, the antigen-binding domain that binds to EGFR further comprises the FRs of an antibody molecule capable of binding to EGFR. That is, in some examples, the antigen-binding domain that binds to EGFR comprises the VH region and the VL region of an antibody molecule capable of binding to EGFR.

[0057] In some examples, the antigen-binding domain that binds to EGFR comprises a VH region and a VL region that are or are derived from the VH / VL region of an EGFR-binding antibody clone described herein (i.e., anti-EGFR antibody clone RAA22 or QD6). Preferably, the antigen-binding domain that binds to EGFR comprises a VH region and a VL region that are or are derived from the VH / VL region of RAA22.

[0058] In some examples, the antigen binding domain that binds to EGFR comprises three HCDRs or three LCDRs, preferably three VH CDRs and three VL CDRs, of the anti-EGFR antibody clone RAA22 or QD6, preferably RAA22. The VH and VL domain sequences of antibodies RAA22 and QD6 are described herein, and thus the three VH domain CDRs and three VL domain CDRs of said antibodies can be determined from said sequences.

[0059] In some examples, the antigen-binding domain that binds to EGFR comprises a VH region set forth in (1) or (2) below: (1) a VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO:1 HCDR2 having the amino acid sequence of SEQ ID NO:2 HCDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof in which one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are replaced by another amino acid; or (2) a VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO:1 HCDR2 having the amino acid sequence of SEQ ID NO:7 HCDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof in which one, two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are replaced by another amino acid.

[0060] Preferably, the antigen-binding domain that binds to EGFR comprises the VH region described in (1) above.

[0061] In some examples, the antigen-binding domain that binds to EGFR comprises a VH region described in (1) or (2) above, and this VH region further comprises a FR described in (3) below: (3) HFR1 having the amino acid sequence of SEQ ID NO:8 HFR2 having the amino acid sequence of SEQ ID NO:9 HFR3 having the amino acid sequence of SEQ ID NO: 10 HFR4 having the amino acid sequence of SEQ ID NO: 11 or a variant thereof in which one or two or three amino acids in one or more of HFR1, HFR2, HFR3 or HFR4 are replaced with another amino acid.

[0062] In some examples, the antigen-binding domain that binds to EGFR comprises a VH region comprising the CDR described in (1) or (2) above and the FR described in (3) above.

[0063] In some examples, the antigen-binding domain that binds to EGFR comprises a VH region described in (4) or (5) below: (4) A VH region comprising the CDR according to (1) and the FR according to (3); (5) A VH region comprising the CDR described in (2) and the FR described in (3).

[0064] Preferably, the antigen-binding domain that binds to EGFR comprises the VH region described in (4) above.

[0065] In some examples, the antigen-binding domain that binds to EGFR comprises a VH region described in (6) or (7) below: (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, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. (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, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0066] Preferably, the antigen-binding domain that binds to EGFR comprises the VH region described in (6) above.

[0067] In some examples, the antigen-binding domain that binds to EGFR comprises a VL region described in (8) or (9) below: (8) a VL region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO:4 LCDR2 having the amino acid sequence of SEQ ID NO:5 LCDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof in which one, two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are replaced by another amino acid. (9) a VL region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO:4 LCDR2 having the amino acid sequence of SEQ ID NO:66 LCDR3 having the amino acid sequence of SEQ ID NO:67 or a variant thereof in which one, two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are replaced by another amino acid.

[0068] Preferably, the antigen-binding domain that binds to EGFR comprises the VL region described in (8) above.

[0069] In some examples, the antigen-binding domain that binds to EGFR comprises a VL region described in (8) or (9) above, and this VL region further comprises a FR described in (10) below: (10) LFR1 having the amino acid sequence of SEQ ID NO: 12 LFR2 having the amino acid sequence of SEQ ID NO: 13 LFR3 having the amino acid sequence of SEQ ID NO: 14 LFR4 having the amino acid sequence of SEQ ID NO: 15 or a variant thereof in which one or two or three amino acids in one or more of LFR1, LFR2, LFR3 or LFR4 are replaced by another amino acid.

[0070] In some examples, the antigen-binding domain that binds to EGFR comprises a VL region comprising the CDR described in (8) or (9) above and the FR described in (10) above.

[0071] In some examples, the antigen-binding domain that binds to EGFR comprises a VL region described in (11) or (12) below: (11) A VL region comprising the CDR described in (8) and the FR described in (10). (12) A VL region comprising the CDR described in (9) and the FR described in (10).

[0072] Preferably, the antigen-binding domain that binds to EGFR comprises the VL region described in (11) above.

[0073] In some examples, the antigen-binding domain that binds to EGFR comprises a VL region described in (13) or (14) below: (13) A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 20, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. (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, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0074] Preferably, the antigen-binding domain that binds to EGFR comprises the VL region described in (13) above.

[0075] In some examples, the antigen-binding domain that binds to EGFR comprises a VH region described in any one of (1) to (7) above, and a VL region described in any one of (8) to (14) above. In some preferred examples, the antigen-binding domain comprises a VH region described in any one of (1), (4), and (6), and a VL region described in any one of (8), (11), and (13). In some preferred examples, the antigen-binding domain comprises a VH region described in any one of (2), (5), and (7), and a VL region described in any one of (9), (12), and (14).

[0076] Anti-c-Met antigen binding domain The antigen-binding domain that binds to c-Met comprises the CDRs of an antibody molecule capable of binding to c-Met. In some examples, the antigen-binding domain that binds to c-Met further comprises the FRs of an antibody molecule capable of binding to c-Met. That is, in some examples, the antigen-binding domain that binds to c-Met comprises the VH region and the VL region of an antibody molecule capable of binding to c-Met.

[0077] In some examples, the antigen-binding domain that binds to c-Met comprises a VH region and a VL region that are or are derived from the VH / VL region of a c-Met binding antibody clone described herein (i.e., anti-c-Met antibody clone B09-GL).

[0078] In some examples, an antigen-binding domain that binds to c-Met comprises the three HCDRs or three LCDRs, preferably the three VH CDRs and three VL CDRs, of the c-Met 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 said sequences.

[0079] In some examples, the antigen binding domain that binds to c-Met comprises a VH region set forth in (15) below: (15) A VH region comprising the following CDRs: HCDR1 having the amino acid sequence of SEQ ID NO: 24 HCDR2 having the amino acid sequence of SEQ ID NO: 25 HCDR3 having the amino acid sequence of SEQ ID NO: 26, or a variant thereof in which one, two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are replaced by another amino acid.

[0080] In some examples, the antigen-binding domain that binds to c-Met comprises a VH region as described in (15) above, and the VH region further comprises a FR as described in (16) below: (16) HFR1 having the amino acid sequence of SEQ ID NO: 30 HFR2 having the amino acid sequence of SEQ ID NO:31 HFR3 having the amino acid sequence of SEQ ID NO: 32 HFR4 having the amino acid sequence of SEQ ID NO: 33 or a variant thereof in which one or two or three amino acids in one or more of HFR1, HFR2, HFR3 or HFR4 are replaced with another amino acid.

[0081] In some examples, an antigen binding domain that binds to c-Met comprises a VH region set forth in (17) below: (17) A VH region comprising the CDR according to (15) and the FR according to (16).

[0082] In some examples, an antigen binding domain that binds to c-Met comprises a VH region set forth in (18) below: (18) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 38.

[0083] In some examples, the antigen binding domain that binds to c-Met comprises a VL region set forth in (19) below: (19) A VL region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO: 27 LCDR2 having the amino acid sequence of SEQ ID NO:28 LCDR3 having the amino acid sequence of SEQ ID NO: 29, or a variant thereof in which one, two or three amino acids in one or more of LCDR1, LCDR2 or LCDR3 are replaced by another amino acid.

[0084] In some examples, the antigen-binding domain that binds to c-Met comprises a VL region as described in (19) above, and the VL region further comprises a FR as described in (20) below: (20) LFR1 having the amino acid sequence of SEQ ID NO: 34 LFR2 having the amino acid sequence of SEQ ID NO: 35 LFR3 having the amino acid sequence of SEQ ID NO: 36 LFR4 having the amino acid sequence of SEQ ID NO: 37, or a variant thereof in which one or two or three amino acids in one or more of LFR1, LFR2, LFR3 or LFR4 are replaced by another amino acid.

[0085] In some examples, the antigen binding domain that binds to c-Met comprises a VL region set forth in (21) below: (21) A VL region comprising the CDRs described in (19) and the FRs described in (20).

[0086] In some examples, the antigen binding domain that binds to c-Met comprises a VL region set forth in (22) below: (22) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0087] In some examples, the antigen-binding domain that binds to c-Met comprises a VH region described in any one of (15) to (18) above and a VL region described in any one of (19) to (22) above.

[0088] Antigen-binding domains of bispecific antibody molecules The present invention provides antibody molecules (i.e., bispecific antibody molecules) comprising a first antigen-binding domain comprising the CDRs of an antigen-binding domain capable of binding to EGFR and a second antigen-binding domain comprising the CDRs of an antigen-binding domain capable of binding to c-Met. In some examples, the first antigen-binding domain comprises the CDRs and FRs of an antigen-binding domain capable of binding to EGFR, and the second antigen-binding domain comprises the CDRs and FRs of an antigen-binding domain capable of binding to c-Met. That is, in some examples, the antibody molecule comprises a first antigen-binding domain comprising the VH and VL regions of an antigen-binding domain capable of binding to EGFR, and a second antigen-binding domain comprising the VH and VL regions of an antigen-binding domain capable of binding to c-Met.

[0089] In some examples, the first antigen-binding domain that binds EGFR comprises a VL region and a VH region that are or are derived from the VH / VL region of an EGFR-binding antibody clone described herein (e.g., anti-EGFR antibody clones RAA22 or QD6, preferably RAA22), and the second antigen-binding domain that binds c-Met comprises a VL region and a VH region that are or are derived from the VH / VL region of a c-Met-binding antibody clone described herein (e.g., anti-c-Met antibody clone B09-GL). A bispecific antibody comprising a first antigen-binding domain that binds EGFR and comprises a VH region and a VL region that are or are derived from the VH / VL region of EGFR-binding antibody clone RAA22, and a second antigen-binding domain that binds c-Met and comprises a VH region and a VL region that are or are derived from the VH / VL region of c-Met-binding antibody clone B09-GL may be referred to as "RAA22 / B09" or a "RAA22 / B09 bispecific antibody molecule."

[0090] In some examples, the first antigen-binding domain comprises: A VH domain according to any one of (1) to (7) above and a VL domain according to any one of (8) to (14) above. Includes; The second antigen-binding domain comprises A VH domain according to any one of (15) to (18) above, and a VL domain according to any one of (19) to (22) above. Includes.

[0091] In some examples, the first antigen-binding domain comprises: A VH domain according to any one of (1), (4), and (6) above, and a VL domain according to any one of (8), (11), and (13) above. Includes; The second antigen-binding domain comprises The VH domain according to any one of (15) to (18) above and the VL domain according to any one of (19) to (22) above Includes.

[0092] For example, in a preferred embodiment, the antibody molecule of the present disclosure comprises: a first antigen-binding domain that binds to the epidermal growth factor receptor (EGFR); a second antigen-binding domain that binds to c-Met; Including, The first antigen-binding domain comprises (i) a heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs): HCDR1 having the amino acid sequence of SEQ ID NO:1 HCDR2 having the amino acid sequence of SEQ ID NO:2 HCDR3 having the amino acid sequence of SEQ ID NO:3, or a variant thereof in which one or two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are replaced by another amino acid; and (ii) a light chain variable (VL) region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO:4 LCDR2 having the amino acid sequence of SEQ ID NO:5 LCDR3 having the amino acid sequence of SEQ ID NO:6, or a variant thereof in which one, two or three amino acids in one or more of HCDR1, HCDR2 or HCDR3 are replaced by another amino acid. Includes.

[0093] In a further preferred embodiment, the first antigen-binding domain comprises: A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 16; and A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 20. Including, and / or the second antigen-binding domain a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 38; and A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 40. Includes.

[0094] CDR replacement In examples according to the present disclosure where one or more amino acids are replaced with another amino acid, the substitutions may be conservative substitutions, for example according to the following table: In some examples, amino acids in the same block in the middle column are replaced, i.e., a non-polar amino acid is replaced with another non-polar amino acid. In some examples, amino acids in the same row in the right-most column are replaced, i.e., a G is replaced with an A or a P.

[0095] [Table 1]

[0096] In some embodiments, substitutions may be functionally conservative, i.e., in some embodiments, substitutions may not affect (or not substantially affect) one or more functional properties (e.g., binding affinity) of the antigen-binding domain that comprises the substitution, compared to the comparable unsubstituted antigen-binding domain.

[0097] Constant region In some examples, the antibody molecules described herein comprise an immunoglobulin heavy chain constant (CH) region. In some examples, the CH is or is derived from a heavy chain constant sequence of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM.

[0098] In some examples, the CH region is the constant of human immunoglobulin G1 (IGHG1; UniProt: P01857-1 (v1); SEQ ID NO: 42) or a fragment thereof.

[0099] In some examples, the CH region comprises an amino acid sequence having at least 70% sequence identity, more preferably 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 preferred examples, the CH region comprises an amino acid sequence having at least 70% sequence identity, more preferably 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.

[0100] In some examples, the antibody molecule comprises a heavy chain that comprises, or consists of, a VH region as described herein and a CH region as described herein.

[0101] In some examples, the antibody molecules described herein comprise an immunoglobulin light chain constant (CL) region or a fragment thereof. In some examples, the CL region is or is derived from a kappa CL region set forth in SEQ ID NO: 47 or SEQ ID NO: 48. In some examples, the CL region is or is derived from a lambda CL region set forth in SEQ ID NO: 49 or SEQ ID NO: 65. In some examples, the antibody molecules comprise a first CL region that is or is derived from a kappa CL region set forth in SEQ ID NO: 47 or 48, and a second CL region that is or is derived from a lambda CL region set forth in SEQ ID NO: 49 or 65.

[0102] In some examples, the antibody molecules described herein include a first heavy chain, wherein the first heavy chain comprises a VH region of a first antigen-binding domain and a first heavy chain constant (CH) region or a fragment thereof; a first light chain, wherein the first light chain comprises a VL region of a first antigen-binding domain and a first light chain constant (CL) region or a fragment thereof; a second heavy chain, wherein the second heavy chain comprises a VH region of a second antigen-binding domain and a second heavy chain constant (CH) region or a fragment thereof; and a second light chain, wherein the second light chain comprises a VL region of a second antigen-binding domain and a second light chain constant (CL) region or a fragment thereof; Includes.

[0103] The first and second CH regions may be the same or different. In other words, the first and second CH regions may form homodimers or heterodimers. For example, an asymmetric bispecific antibody molecule has different first and second CH regions, as described in more detail below. The first and second CL regions may be the same or different. In some examples, the first CL region is or is derived from the kappa CL region set forth in SEQ ID NO: 47 or 48, and the second CL region is or is derived from the lambda CL region set forth in SEQ ID NO: 49 or 65.

[0104] It will be understood that when an antibody molecule comprises a first VH region and a first CH region, these regions together form a first heavy chain of the antibody molecule, i.e., the first VH region and the first CH region are linked to each other. Similarly, a second VH region and a second CH region form a second heavy chain of the antibody molecule, a first VL region and a first CL region form a first light chain of the antibody molecule, and a second VL region and a second CL region form a second light chain of the antibody molecule.

[0105] In some examples, the antibody molecule comprises a heavy chain having an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the B-09-GL heavy chain set forth in SEQ ID NO:50, the QD6 heavy chain set forth in SEQ ID NO:53, the RAA22 heavy chain set forth in SEQ ID NO:56, the heavy chain set forth in SEQ ID NO:59, or the heavy chain set forth in SEQ ID NO:60.

[0106] In some instances, the antibody molecule comprises a first and a second heavy chain, (i) the first heavy chain comprises an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the B-09-GL heavy chain set forth in SEQ ID NO:56; (ii) the second heavy chain comprises an amino acid sequence having at least 70% sequence identity, more preferably at least one of 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.

[0107] In some instances, the antibody molecule comprises a first and a second heavy chain, (i) the first heavy chain comprises an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the heavy chain set forth in SEQ ID NO:59; (ii) the second heavy chain comprises an amino acid sequence having at least 70% sequence identity, more preferably at least one of 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.

[0108] In some examples, the antibody molecules described herein comprise a light chain that comprises, or consists of, a VL region as described herein and a CL region as described herein.

[0109] In some examples, the antibody molecule comprises a light chain having an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the B-09-GL light chain set forth in SEQ ID NO:52, the QD6 light chain set forth in SEQ ID NO:55, the RAA22 light chain set forth in SEQ ID NO:58, the light chain set forth in SEQ ID NO:61, or the light chain set forth in SEQ ID NO:62.

[0110] In some examples, the antibody molecules described herein comprise a first and a second heavy chain, (i) the first light chain comprises an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the RAA22 light chain set forth in SEQ ID NO:58; (ii) the second light chain comprises an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the B-09-GL light chain set forth in SEQ ID NO:52.

[0111] In some examples, the antibody molecules described herein comprise a first and a second light chain, (i) the first light chain comprises an amino acid sequence having at least 70% sequence identity, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the light chain set forth in SEQ ID NO: 61; (ii) the second light chain comprises an amino acid sequence having at least 70% sequence identity, and more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the light chain set forth in SEQ ID NO: 62.

[0112] The CH, CL, heavy and / or light chains of the antibody molecules described herein may comprise one or more modifications, e.g., to eliminate or reduce Fc effector function, to promote the formation of heterodimeric antibody molecules, to increase the efficiency of pairing of cognate heavy and light chains, and / or to aid in complex formation, as described in more detail below. Modified CH, CL, heavy and light chains may be referred to as modified CH, modified CL, modified heavy chains and modified light chains, respectively.

[0113] The antibody molecule may comprise a mutation in the CH region of the heavy chain to reduce or abolish binding of the antibody molecule to one or more Fcγ receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIII, and / or complement. Such mutations abolish or reduce Fc effector function. Mutations that reduce or abolish binding of the antibody molecule to one or more Fcγ receptors and complement are known, including, for example, the L234F / L235E / P331S "triple mutation" or "TM" described in Organesyan, 2008. Other mutations known to modulate antibody effector function are described, for example, in Wang, 2018.

[0114] Thus, in some examples, the first and / or second heavy chain (preferably both) comprises 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). For example, one or both of the first and second heavy chains (preferably both) may comprise a CH region having an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95% sequence identity to the sequence set forth in SEQ ID NO: 42, and may comprise a phenylalanine (F) at position 234, a glutamic acid (E) at position 235, and a serine (S) at position 331 (wherein the numbering is according to the EU index). As shown in the Examples (e.g., Example 12), the inclusion of a TM in the heavy chain has been demonstrated to improve the pharmacokinetic properties of exemplary antibody molecules and ADCs.

[0115] Examples of CH regions containing a triple mutation are SEQ ID NOs: 63 and 64. Thus, in some examples, one of the first and second heavy chains comprises a CH region having an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 63, and the other heavy chain comprises a CH region having an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 64, wherein one or both (preferably both) of the CH regions comprises a phenylalanine at position 234, a glutamic acid at position 235, and a serine at position 331 (wherein the numbering is according to the EU index).

[0116] Examples of heavy chains that include a CH region containing a triple mutation are SEQ ID NOs: 59 and 60. Thus, in some examples, one of the first and second heavy chains has an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 59, and the other heavy chain has an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 60, and one or both (preferably both) of these chains comprise a phenylalanine at position 234, a glutamic acid at position 235, and a serine at position 331 (wherein the numbering is according to the EU index).

[0117] The VL and CL regions and the VH and CH1 regions of an antibody molecule together make up the Fab region. The remainder of the antibody molecule makes up the Fc region.

[0118] Unless otherwise specified, amino acid residue positions of constant domains, including positions of substitutions, deletions and insertions, in amino acid sequences described herein are numbered according to the EU numbering system (Edelman, 2007).

[0119] Bispecific construction Bispecific antibody molecules can be provided in any suitable configuration. Suitable configurations for the bispecific antibody molecules described herein, and methods for producing them, are described in Kontermann, MAbs 2012, 4(2):182-197 and Kontermann and Brinkmann 2015, 20(7):838-847, both of which are incorporated herein by reference in their entirety. See in particular Figure 2 in Kontermann MAbs 2012, 4(2):182-19.

[0120] Bispecific antibody molecules can also be generated from existing antibodies by chemical conjugation, e.g., two IgG molecules or two Fab' fragments can be joined using homo- or heterobifunctional coupling reagents as described in Graziano and Guptill, Methods Mol Biol. 2004(283):71-85.

[0121] In some embodiments, the bispecific antibody molecule may be an immunoglobulin G-like (IgG-like) bispecific antibody molecule. An IgG-like bispecific antibody molecule may comprise an Fv region, Fab region or sVD specific for one antigen and an Fv / Fab / sVD and Fc region specific for another antigen. An IgG-like bispecific antibody molecule may be symmetric or asymmetric. A bispecific antibody molecule is preferably asymmetric.

[0122] Symmetric IgG-like bispecific antibody molecules generally contain an antigen-binding domain fused to the N-terminus or C-terminus of the heavy or light chain of an IgG molecule, for example in the form of an scFv fragment or a variable single domain. A distinctive feature of these symmetric IgG-like bispecific antibody molecules is that they contain two identical heavy chains. Furthermore, symmetric IgG-like bispecific antibody molecules are typically bivalent for each epitope. As used herein, valency refers to the number of antigen-binding regions of an antibody molecule that can bind to a single epitope. A monoclonal monospecific IgG antibody molecule is bivalent for a single epitope. That is, it contains two antigen-binding domains, each of which can bind to an epitope on a single target molecule. A symmetric IgG-like bispecific antibody molecule is bivalent for each epitope. That is, it typically contains four antigen-binding domains, two of which can bind to a first epitope on a target molecule and two of which can bind to a second epitope on a target molecule.

[0123] Examples of symmetric IgG-like bispecific antibody molecules include DVD-IgG, IgG-scFv, scFv-IgG, scFv4-Ig, IgG-scFab, scFab-IgG, IgG-sVD, sVD-IgG, 2in1-IgG, mAb 2 , tandemab common LCs. These can be formed by methods known in the art, such as chemical cross-linking, somatic cell hybridization, or redox methods.

[0124] In contrast, asymmetric IgG-like bispecific antibody molecules are typically monovalent for each target. For example, as described in Klein, 2012, the concept of monovalent bispecific IgG is believed to have a unique therapeutic niche in that they (i) do not undergo receptor homodimerization, (ii) potentially reduce toxicity to non-target tissues by losing the avidity of each antigen, and (iii) have better selectivity when both antigens are selectively restricted or abundantly expressed on target cells. Thus, in some embodiments, the antibody molecule is an asymmetric IgG-like bispecific antibody molecule.

[0125] Asymmetric IgG-like bispecific antibody molecules involve the heterodimerization of two different heavy chains and the correct pairing of cognate light and heavy chains. Heavy chain heterodimerization can be addressed by several techniques, such as knobs-into-holes, electrostatic steering of CH3, CH3 chains exchanged with modified domains and leucine zippers. Correct light and heavy chain pairing can be ensured using one of these heavy chain heterodimerization techniques together with the use of a common light chain, domain crossover between CH1 and CL, linking of heavy and light chains with a linker, in vitro assembly of heavy-light chain dimers from two separate monoclonals, interface modification of the entire Fab domain, or disulfide modification of the CH1 / CL interface.

[0126] Examples of asymmetric IgG-like bispecific antibody molecules include DuetMab, kih IgG, kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pairing, and SEED bodies.

[0127] 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 DuetMab antibody molecule described above comprises 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 include the knob-into-hole (KiH) strategy, based on single amino acid substitutions in the CH3 domain to promote heavy chain heterodimerization, as described in Ridgway, 1996. In the knob variant heavy chain CH3, a small amino acid is replaced with a larger amino acid, and in the hole variant, a large amino acid is replaced with a small amino acid. Further modifications may also be introduced to stabilize the association between the heavy chains.

[0128] CH3 modifications that enhance heterodimerization include, for example, Y407V / T366S / L368A on one heavy chain and T366W on the other heavy chain; S354C / T366W on one heavy chain and Y349C / Y407V / T366S / L368A on the other heavy chain (wherein the numbering of the constant region is according to the EU index).

[0129] Other examples of CH3 modifications to enhance heterodimerization are described, for example, in Table 1 of Brinkmann and Kontermann, 2017 MABS 9(2), 182-212, which is specifically incorporated herein by reference.

[0130] In some examples, the antibody molecule comprises first and second heavy chains that form a heterodimer, where one of the first and second heavy chains comprises a cysteine ​​(C) residue at position 354, 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 and second heavy chains may have the sequence set forth in SEQ ID NO:42 and further comprise a cysteine ​​(C) residue at position 354 and a tryptophan (W) residue at position 366, and the other heavy chain may have the sequence set forth in SEQ ID NO:42 and further comprise a cysteine ​​(C) residue at position 349, a valine (V) residue at position 407, a serine (S) at position 366 and an alanine (A) at position 368 (wherein the numbering of the constant regions is according to the EU index).

[0131] In some instances, the antibody molecule comprises: (i) a first heavy chain comprising a first modified CH3 region, wherein the first modified CH3 region comprises a cysteine ​​(C) residue at position 354 and a tryptophan (W) residue at position 366; and (ii) a second heavy chain comprising a second modified CH3 region, wherein the second modified CH3 region comprises a cysteine ​​(C) residue at position 349, a valine (V) residue at position 407, a serine (S) at position 366, and an alanine (A) at position 368. Including, Here, the numbering of the constant regions is according to the EU index.

[0132] A particular representative configuration of asymmetric IgG-like bispecific antibody molecules is called "DuetMab". DuetMab antibody molecules use KIH technology to heterodimerize two different heavy chains, and increase the efficiency of pairing cognate heavy and light chains by replacing one natural disulfide bond in the CH1-CL interface with an engineered disulfide bond. Disclosures regarding DuetMab can be found, for example, in U.S. Pat. No. 9,527,927 and Mazor, 2015, which are incorporated herein by reference in their entirety.

[0133] In some instances, the antibody molecule comprises: (a) a modified CH region, wherein the modified heavy chain comprises a substitution of a naturally occurring non-cysteine ​​amino acid with a cysteine ​​amino acid; and (b) a corresponding modified CL region, where the modified CL comprises a substitution of a native non-cysteine ​​amino acid with a cysteine ​​amino acid. Including, During the ceremony, (i) the first heavy chain comprises a modified CH region and the first light chain comprises a corresponding modified CL region; or (ii) the second heavy chain comprises a modified CH region and the second light chain comprises a modified corresponding CL region.

[0134] In some examples, a substituted cysteine ​​in a modified CH region resulting from the substitution of a native non-cysteine ​​amino acid with a cysteine ​​amino acid and a corresponding substituted cysteine ​​in a modified CL region resulting from the substitution of a native non-cysteine ​​amino acid with a cysteine ​​amino acid can form a disulfide bond.

[0135] In some examples, the modified CH region comprises a substitution of the native non-cysteine ​​amino acid at position 126 with a cysteine ​​amino acid, and the corresponding modified CL region comprises a substitution of the native non-cysteine ​​amino acid at position 121 with a cysteine ​​(wherein constant region numbering is according to the EU index).

[0136] In some examples, the modified CH region comprises a substitution of the native non-cysteine ​​amino acid at position 126 with a cysteine ​​amino acid, and a substitution of the native cysteine ​​amino acid at position 219 with a non-cysteine ​​amino acid, such as with valine; the corresponding modified CL region comprises a substitution of the native non-cysteine ​​amino acid at position 121 with a cysteine, and a substitution of the native cysteine ​​amino acid at position 214 with a non-cysteine ​​amino acid, such as with valine (wherein the numbering of the constant regions is according to the EU index).

[0137] In some examples, the antibody molecule comprises a second CH region and a second corresponding light chain, wherein the second CH region and the second corresponding CL do not contain substitutions of naturally occurring non-cysteine ​​amino acids with cysteine ​​amino acids and do not contain substitutions of naturally occurring cysteines with non-cysteine ​​amino acids.

[0138] Complex The antibody molecule may be conjugated to a drug. In this case, the antibody molecule may be referred to as a "conjugate" or an "antibody drug conjugate". Such conjugates have application in the treatment and / or diagnosis of diseases as described herein. As used herein, the drug may be referred to as a "payload" or a "warhead".

[0139] In some examples, the drug comprises a cytotoxin, a radioisotope, an immunomodulatory agent, a cytokine, a lymphokine, a chemokine, a growth factor, a tumor necrosis factor, a hormone, a hormone antagonist, an enzyme, an oligonucleotide, DNA, RNA, siRNA, RNAi, microRNA, a photoactive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide, a lipid, a carbohydrate, a chelator, or a combination thereof.

[0140] A cytotoxin is a compound that can induce the death of a targeted cell. In the context of an antibody-drug conjugate, typically, the cytotoxin is delivered to the targeted cell by the antibody molecule, where it is released intracellularly and induces cell death. The use of cytotoxins in antibody-drug conjugates is described, for example, in Chalouni and Doll 2018 J Exp Clin Cancer Res. 37(1):20. In some examples, the cytotoxin is a tubulysin, an auristatin, a maytansinoid, a topoisomerase inhibitor, or a pyrrolobenzodiazepine (PBD).

[0141] In certain examples, the cytotoxin is or includes tubulysin. Tubulysins are a class of cytostatic tetrapeptides that contain isoleucine and three other complex unnatural amino acids Mep (RN-mepipecolic acid), Tuv (tubavalin), and Tut (tubulyosin) or Tup (tubuphenylalanine). Tubulysins are extremely potent cytotoxic molecules and are potent against multidrug resistant cell lines (Domling, 2005). These compounds have shown high cytotoxicity in tests against a panel of cancer cell lines, with IC50 values ​​in the low picomolar range, making them interesting as anticancer therapeutics. See, for example, WO 2012 / 019123. Tubulysin complexes are disclosed, for example, in U.S. Pat. No. 7,776,814. In some examples, the tubulysin is tubulysin A, which has the following chemical structure: [ka]

[0142] In some examples, the tubulysin is tubulysin 1508, also known as "AZ1508," and described in more detail in WO 2015 / 157594. Tubulysin 1508 has the following chemical structure: [ka]

[0143] Preferably, the cytotoxin is or comprises a topoisomerase inhibitor. As used herein, the term "topoisomerase inhibitor" refers to a cytotoxic agent that inhibits one or more activities of topoisomerase enzymes (topoisomerase I and II), enzymes that play a key role in DNA replication and transcription by regulating DNA supercoiling. Thus, antibody-drug conjugates that contain topoisomerase inhibitors as cytotoxins are expected to interfere with normal processes involving DNA, thus resulting in cell death. Conjugates containing topoisomerase inhibitors have been demonstrated to be effective against various tumor-containing cell lines and to have anti-cancer activity in clinical trials. See, for example, Ogitani, 2016a; Ogitani, 2016b; Cardillo, 2015; and Bardia, 2017.

[0144] The antibody molecule is preferably conjugated to a topoisomerase I inhibitor.Representative examples of topoisomerase I inhibitors include, but are not limited to, camptothecin and its analogs topotecan, irinotecan, belotecan, exatecan, lurotecan and sinotecan.Representative examples of topoisomerase II inhibitors include, but are not limited to, amsacrine, daunorubicin, doxorubicin, epipodophyllotoxin, ellipticine, epirubicin, etoposide, razoxane and teniposide.

[0145] An example of the chemical structure of camptothecin is as follows: [ka]

[0146] General examples of suitable topoisomerase I inhibitors are illustrated by the following compounds: [ka] The compound is designated as A*.

[0147] The compound (e.g., A*) is preferably provided with a linker (which may be referred to as a "ligand unit" or "cell binding agent" (CBA)) for linking (preferably conjugating) to an antibody molecule described herein. Suitably, the linker is cleavably linked (e.g., conjugated) to an amino residue, e.g., an amino acid of an antibody molecule described herein.

[0148] The design and selection of linkers for use in conjugates is known in the art and is described, for example, in Beck, 2017. The linkers used herein can be any of the linkers described in Beck, 2017.

[0149] More particularly, examples of suitable topoisomerase I inhibitors are the following compounds having formula "I": [ka] and salts and solvates thereof, wherein R L is a linker for linking to an antibody molecule as described herein, said linker being preferably selected from the following: (ia): [ka] [In the formula, Q is, [ka] (In the formula, Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue. and; X is [ka] (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, and at least b1 or b2=0 (i.e., only one of b1 and b2 may not be 0), and at least c1 or c2=0 (i.e., only one of c1 and c2 may not be 0)). and; G L is a linker (e.g., a ligand unit or cell binding agent) for linking to an antibody or antigen-binding fragment thereof described herein; or (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1).

[0150] It will be appreciated by those skilled in the art that more than one of the above agents (eg, topoisomerase I inhibitors) may be conjugated to the antibody molecule.

[0151] For example, a conjugate (e.g., an antibody-drug conjugate) of the disclosure may be represented by the general formula IV: L-(D L ) p (IV) or or a pharma- ceutically acceptable salt or solvate thereof, in which L is an antibody molecule (e.g., a Ligand unit or CBA) as described herein, and D L is a drug having a linker (eg, a drug linker unit), and p is an integer from 1 to 20.

[0152] Preferably, D L is a topoisomerase I inhibitor with a linker of formula III: [ka] RLL is a linker (e.g., a ligand unit) that is linked to an antibody molecule as described herein, said linker preferably being (ia'): [ka] (wherein Q and X are as defined above; G LL is a linker (e.g., a Ligand unit or CBA) that is linked to an antibody molecule described herein; and (ib'): [ka] (In the formula, R L1 and R L2 is as defined above) is selected from.

[0153] The drug loading is represented by p, which is the number of topoisomerase I inhibitors (e.g., drug units) per antibody molecule (e.g., ligand unit). The drug loading can range from 1 to 20 drug units (D) per ligand unit. In the case of a composition, p represents the average drug loading of the complexes in the composition, and p ranges from 1 to 20. In some examples, when the drug is a topoisomerase inhibitor, the range of p is selected from 2 to 8, preferably 4 to 8, even more preferably 5 to 7, and even more preferably 5.5 to 6.5. As described in the Examples, ADCs containing the topoisomerase I inhibitor SG3932 were produced with an average DAR of 6+ / -6%.

[0154] Thus, the present disclosure encompasses conjugates comprising an antibody molecule (e.g., a Ligand unit or CBA) as described herein covalently attached to at least one topoisomerase I inhibitor (e.g., a Drug unit such as A* as exemplified above). The inhibitor preferably includes a linker (e.g., a Linker unit), such as R L and / or R LLThe antibody molecule is linked to the antibody molecule by a linker such as those described above. In other words, the present disclosure encompasses the antibody molecules described herein (e.g., Ligand Units or CBAs) to which one or more topoisomerase I inhibitors are attached (e.g., Drug-Linker Units), preferably via a linker. The antibody molecules (representing Ligand Units or CBAs) detailed above are targeting agents that bind to a targeting moiety. More specifically, the antibody molecules can specifically bind, for example, to EGFR and cMET on a target cell, thereby delivering the Drug Unit to the target cell. Thus, the present invention also provides methods of treating, for example, various cancers and other disorders (e.g., cancers / disorders associated with the presence of cells, preferably cancerous cells, expressing EGFR and cMET) with ADCs. Such methods are described in more detail below.

[0155] Certain properties of the above-mentioned topoisomerase I inhibitors are particularly preferred and may be defined in more detail as set out below. X Preferred examples are outlined below (eg, within the linker of 1a above).

[0156] The following preferences may apply to all aspects of the present disclosure described above or may relate to a single aspect. The preferences may be combined together in any combination.

[0157] Q X In one example, Q is an amino acid residue. The amino acid may be a natural amino acid or a non-natural amino acid. For example, Q may be selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, where Cit is citrulline.

[0158] In one example, Q comprises a dipeptide residue. The amino acids in the dipeptide can be any combination of natural and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. In that case, the dipeptide is the recognition site for cathepsin.

[0159] In one example, Q is NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , NH -Val-Cit- C=O , NH -Phe-Cit- C=O , NH -Leu-Cit- C=O , NH -Ile-Cit- C=O , NH -Phe-Arg- C=O , NH -Trp-Cit- C=O and NH -Gly-Val- C=O Selected from; Here, Cit is citrulline.

[0160] Preferably, Q is NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O and NH -Val-Cit- C=O is selected from.

[0161] More preferably, Q is NH -Phe-Lys- C=O ,NH -Val-Cit- C=O or NH -Val-Ala- C=O is selected from.

[0162] Other suitable dipeptide combinations include: NH -Gly-Gly- C=O , NH -Gly-Val- C=O , NH -Pro-Pro- C=O and NH -Val-Glu- C=O Examples include:

[0163] Other dipeptide combinations may be used, such as those described in Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, which is incorporated herein by reference.

[0164] In some examples, Q is a tripeptide residue. The amino acids in the tripeptide can be any combination of natural and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. In that case, the tripeptide is the recognition site for cathepsin. Particularly interesting tripeptide linkers are: NH -Glu-Val-Ala- C=O NH -Glu-Val-Cit- C=O NH -αGlu-Val-Ala- C=O NH -αGlu-Val-Cit- C=O It is.

[0165] In some examples, Q is a tetrapeptide residue. The amino acids in the tetrapeptide can be any combination of natural and non-natural amino acids. In some embodiments, the tetrapeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the tetrapeptide is the site of action for cathepsin-mediated cleavage. In that case, the tetrapeptide is the recognition site for the cathepsin. Tetrapeptide linkers of particular interest are: NH -Gly-Gly-Phe-Gly C=O and NH -Gly-Phe-Gly-Gly C=O It is.

[0166] In some examples, the tetrapeptide is NH -Gly-Gly-Phe-Gly C=O It is.

[0167] In the above representation of peptide residues: NH - represents the N-terminus of the residue, - C=O represents the C-terminus of the residue. The C-terminus is bound to the NH of A*.

[0168] Glu is a glutamic acid residue, i.e. [ka] Represents.

[0169] αGlu is a glutamic acid residue when bound via the α chain, i.e. [ka] Represents.

[0170] In one example, where appropriate, amino acid side chains are chemically protected. Side chain protecting groups can be as described above. Protected amino acid sequences can be cleaved by enzymes. For example, dipeptide sequences containing Boc side chain protected Lys residues can be cleaved by cathepsin.

[0171] Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem catalogue, as discussed above.

[0172] G L G L may be selected from the following:

[0173] [Table 2]

[0174] [Table 3]

[0175] In the formula, Ar is C 5~6 represents an arylene group, such as phenylene; X′ is C 1~4 Represents alkyl.

[0176] In some embodiments, G L is G L1-1 and G L1-2 In some of these examples, G L is G L1-1 It is.

[0177] G LL G LL may be selected from the following:

[0178] [Table 4]

[0179] In the formula, Ar is C 5~6 represents an arylene group, such as phenylene; X′ is C 1~4 CBA represents a cell binding agent or ligand unit.

[0180] In some instances, G LL is G LL1-1 and G LL1-2 In some of these examples, G LL is G LL1-1 It is.

[0181] X X is preferably [ka] (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c=0 or 1, and d=0 to 5, where at least b1 or b2=0, and at least c1 or c2=0.) It is.

[0182] a can be 0, 1, 2, 3, 4, or 5. In some examples, a is 0 to 3. In some of these examples, a is 0 or 1. In a further example, a is 0.

[0183] 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.

[0184] b2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, b2 is 0 to 12. In some of these examples, b2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8. Preferably, only one of b1 and b2 may not be 0.

[0185] c1 can be 0 or 1. c2 can be 0 or 1. Preferably, only one of c1 and c2 may be non-0.

[0186] d can be 0, 1, 2, 3, 4, or 5. In some examples, d is 0-3. In some of these examples, d is 1 or 2. In a further example, d is 2. In a further example, d is 5.

[0187] In some examples of X, a is 0, b1 is 0, c1 is 1, c2 is 0, d is 2, and b2 can be 0 to 8. In some of these examples, b2 is 0, 2, 3, 4, 5, or 8. In some examples of X, a is 1, b2 is 0, c1 is 0, c2 is 0, d is 0, and b1 can be 0 to 8. In some of these examples, b1 is 0, 2, 3, 4, 5, or 8. In some examples of X, a is 0, b1 is 0, c1 is 0, c2 is 0, d is 1, and b2 can be 0 to 8. In some of these examples, b2 is 0, 2, 3, 4, 5, or 8. In some examples of X, b1 is 0, b2 is 0, c1 is 0, c2 is 0, and one of a and d is 0. The other of a and d is 1 to 5. In some of these examples, the other of a and d is 1. In others of these examples, the other of a and d is 5. In some examples of X, a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 can be 0-8. In some of these examples, b2 is 0, 2, 3, 4, 5, or 8.

[0188] In some instances, R L is of formula Ib. In some examples, R LL is of formula Ib'.

[0189] R L1 and R L2 are independently selected from H and methyl, or may be taken together with the carbon atom to which they are attached to form a cyclopropylene or cyclobutylene group.

[0190] In some instances, R L1 and R L2 Both are H. In some instances, R L1 is H and R L2 is methyl. In some instances, R L1 and R L2 Both are methyl.

[0191] In some instances, R L1 and R L2 together with the carbon atom to which they are attached form a cyclopropylene group. In some examples, R L1 and R L2 together with the carbon atom to which they are attached form a cyclobutylene group.

[0192] In group Ib, in some instances, e is 0. In other instances, e is 1 and the nitro group can be at any available position on the ring. In some of these instances, it is at the ortho position. In others of these instances, it is at the para position.

[0193] In some examples where the compounds described herein are provided in a single enantiomer or in an enantiomerically enriched form, the enantiomerically enriched form has a high enantiomeric ratio of greater than 60:40, 70:30; 80:20, or 90: 10. In further examples, the enantiomeric ratio is greater than 95:5, greater than 97:3, or greater than 99:1.

[0194] In some instances, R L is selected from the following:

[0195] [Table 5]

[0196] [Table 6]

[0197] In some instances, R LL is the above R L It is a group derived from the group.

[0198] Having outlined these preferences above, formulas for certain preferred Topoisomerase I-linkers (eg, Drug Linker units) are now set forth.

[0199] In some examples, the compound of formula I is P : [ka] and salts and solvates thereof, wherein R LP is a linker for linking to an antibody or antigen-binding fragment thereof described herein, said linker being selected from: (ia): [ka] [In the formula, Q P teeth, [ka] (In the formula, Q XP Q P is an amino acid residue, a dipeptide residue or a tripeptide residue. and; X P teeth, [ka] (In the formula, aP=0 to 5, bP=0 to 16, cP=0 or 1, and dP=0 to 5). and; G L is a linker (e.g., a ligand unit) for linking to an antibody or antigen-binding fragment thereof described herein; (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1).

[0200] aP can be 0, 1, 2, 3, 4, or 5. In some examples, aP is 0 to 3. In some of these examples, aP is 0 or 1. In a further example, aP is 0.

[0201] 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.

[0202] cP can be 0 or 1.

[0203] dP can be 0, 1, 2, 3, 4, or 5. In some examples, dP is 0 to 3. In some of these examples, dP is 1 or 2. In a further example, dP is 2.

[0204] X P In some examples, aP is 0, cP is 1, dP is 2, and bP can be 0 to 8. In some of these examples, bP is 0, 4, or 8.

[0205] The above Q of the compound of formula I X Priorities for Q XP can be applied to.

[0206] The compound of formula I L , R L1 , R L2 and e are of formula I P The present invention can be applied to compounds of the above formula (I).

[0207] In some examples, the conjugate of formula IV isP : L-(D LP ) p (IV P ) or or a pharma- ceutically acceptable salt or solvate thereof, wherein L is an antibody or an antigen-binding fragment thereof (e.g., a ligand unit) as described herein; LP is represented by formula III P [ka] a topoisomerase I inhibitor (e.g., drug linker unit) which is R LLP is a linker (e.g., a ligand unit) that is linked to the antibody or antigen-binding fragment thereof, said linker comprising: (ia'): [ka] (In the formula, Q P and X P is as defined above, and G LL is a linker (e.g., a ligand unit) that is linked to an antibody or antigen-binding fragment thereof described herein; and (ib'): [ka] (In the formula, R L1 and R L2 is as defined above) Selected from; p is an integer from 1 to 20.

[0208] In some examples, the compound of formula I is P2 : [ka] and salts and solvates thereof, wherein R LP2is a linker for linking to an antibody or antigen-binding fragment thereof described herein, said linker being selected from: (ia): [ka] [In the formula, Q is, [ka] (In the formula, Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue. and; X P2 teeth, [ka] (Wherein, 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); G L is a linker (e.g., a ligand unit) for linking to an antibody or antigen-binding fragment thereof described herein; (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1).

[0209] aP2 can be 0, 1, 2, 3, 4, or 5. In some examples, aP2 is 0 to 3. In some of these examples, aP2 is 0 or 1. In further examples, aP2 is 0.

[0210] b1P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, b1P2 is 0 to 12. In some of these examples, b1P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0211] b2P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some examples, b2P2 is between 0 and 12. In some of these examples, b2P2 is between 0 and 8, and can be 0, 2, 3, 4, 5, or 8.

[0212] Preferably, only one of b1P2 and b2P2 may be non-zero.

[0213] cP2 can be 0 or 1.

[0214] dP2 can be 0, 1, 2, 3, 4, or 5. In some examples, dP2 is 0-3. In some of these examples, dP2 is 1 or 2. In a further example, dP2 is 2. In a further example, dP2 is 5.

[0215] X P2 In some examples, aP2 is 0, b1P2 is 0, cP2 is 1, dP2 is 2, and b2P2 can be 0 to 8. In some of these examples, b2P2 is 0, 2, 3, 4, 5, or 8. P2 In some examples, aP2 is 1, b2P2 is 0, cP2 is 0, dP2 is 0, and b1P2 can be 0 to 8. In some of these examples, b1P2 is 0, 2, 3, 4, 5, or 8. P2 In some examples, aP2 is 0, b1P2 is 0, cP2 is 0, dP2 is 1, and b2P2 can be 0 to 8. In some of these examples, b2P2 is 0, 2, 3, 4, 5, or 8. P2In some examples, b1P2 is 0, b2P2 is 0, cP2 is 0, and one of aP2 and dP2 is 0. The other of aP2 and d is 1 to 5. In some of these examples, the other of aP2 and d is 1. In others of these examples, the other of aP2 and dP2 is 5.

[0216] The above Q of the compound of formula I X Preference is given to (e.g., where appropriate) P2 Q X can be applied to.

[0217] The compound of formula I L , R L1 , R L2 and e are of formula I P2 The present invention can be applied to compounds of the above formula (I).

[0218] In some examples, the conjugate of formula IV is P2 : L-(D LP2 ) p (IV P2 ) or or a pharma- ceutically acceptable salt or solvate thereof, wherein L is an antibody or an antigen-binding fragment thereof (e.g., a ligand unit) as described herein; LP2 is represented by formula III P2 [ka] a topoisomerase I inhibitor (e.g., drug linker unit) which is R LLP2 is a linker (e.g., a ligand unit) that is linked to the antibody or antigen-binding fragment thereof, said linker comprising: (ia'): [ka] (Wherein, Q and X P2 is as defined above, and G LLis a linker attached to the antibody or antigen-binding fragment thereof; and (ib'): [ka] (In the formula, R L1 and R L2 is as defined above) Selected from; p is an integer from 1 to 20.

[0219] Particularly suitable topoisomerase I inhibitors include those having the formula: [ka]

[0220] SG3932 is particularly preferred. Thus, in a preferred example, the antibody molecules described herein are conjugated to a topoisomerase I inhibitor (e.g. SG3932) having the following formula: [ka]

[0221] For the avoidance of doubt, the number "8" indicates that the structure within the enclosed brackets is repeated eight times. Thus, an alternative representation of SG3932 is: [ka] It is.

[0222] Another way to describe the SG4010 is: [ka] It is.

[0223] Another expression for SG4057 is [ka] It is.

[0224] Another way to describe the SG4052 is: [ka] It is.

[0225] Any of the antibodies or antigen-binding fragments thereof described herein may be conjugated to one or more of the above topoisomerase I inhibitors.

[0226] Synthesis of Topoisomerase I Inhibitors Towards completion, one general synthetic route for the preparation of preferred Topoisomerase I inhibitors is now described.

[0227] R L However, the compounds of formula I which are of formula Ia are of formula 2: [ka] (In the formula, R L* is -QH) to obtain a compound of formula 3: [ka] or its activated form.

[0228] Such reactions may be carried out under amide coupling conditions.

[0229] The compound of formula 2 may be represented by formula 4: [ka] (In the formula, R L*prot -Q-Prot N (In the formula, Prot N can be synthesized by deprotection of a compound of formula (I) (wherein R is an amine protecting group).

[0230] Compounds of formula 4 can be converted to compounds of formula 5: [ka] with compound A3.

[0231] The compound of formula 5 may be prepared according to formula 6: [ka] by removing the trifluoroacetamide protecting group.

[0232] The compound of formula 6 can be coupled to compound I7: L*prot It can be synthesized by -OH.

[0233] Compounds of formula I, where R L is of formula Ia or Ib) can be prepared from compound I11 to compound R L It can be synthesized by coupling the -OH or an activated form thereof.

[0234] Amine Protecting Groups: Amine protecting groups are well known to those skilled in the art. In particular, reference is made to the disclosure of suitable protecting groups in Greene's Protecting Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pages 696-871.

[0235] Drug loading (p) is the average number of drugs (e.g., tubulysin or topoisomerase inhibitors) per antibody molecule. In the compositions of the present disclosure, drug loading ranges from 1 to 20 drugs (D) per antibody molecule. For example, drug loading can range from 1 to 10 drugs (D) per antibody molecule, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drugs are covalently attached to the antibody molecule. The conjugate composition includes a population of antibody molecules conjugated with drugs in the range of 1 to 10. When the compounds of the present disclosure are conjugated to lysine, drug loading can range from 1 to 80 drugs (D) per antibody molecule, with preferred upper limits of 40, 20, 10, or 8. The conjugate composition includes a population of antibody molecules conjugated with drugs in the range of 1 to 80, 1 to 40, 1 to 20, 1 to 10, or 1 to 8.

[0236] The average number of drugs per antibody molecule when preparing conjugates from the conjugation reaction can be characterized by conventional methods such as UV, reversed-phase HPLC, HIC, mass spectrometry, ELISA assay, and electrophoresis. The quantitative distribution of the conjugates in terms of p can also be determined. The average value of p in a particular preparation of conjugates can be determined by ELISA (Hamblett, 2004; Sanderson, 2005). However, the distribution of p(drug) values ​​cannot be distinguished due to the antibody-antigen binding and detection limits of ELISA. Also, ELISA assays for detecting conjugates do not reveal where the drug moiety is attached on the antibody, e.g., to the heavy or light chain fragment, or to a particular amino acid residue. In some instances, separation, purification, and characterization of homogeneous conjugates with a particular value of p from conjugates with different drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.

[0237] In some conjugates, p may be limited by the number of binding sites on the antibody molecule: for example, an antibody molecule may have only one or several cysteine ​​thiol groups, and may have only one or several sufficiently reactive thiol groups to which a linker may be attached.

[0238] Typically, fewer drugs than the theoretical maximum are conjugated to an antibody molecule during the conjugation reaction. An antibody molecule may contain, for example, many lysine residues that do not react with the linker (L). Only the most reactive lysine groups can react with amine-reactive linker reagents. Also, only the most reactive cysteine ​​thiol groups can react with thiol-reactive linker reagents. In general, an antibody molecule does not contain many, if any, free reactive cysteine ​​thiol groups that can be linked to a drug moiety. Most cysteine ​​thiol residues in the antibody molecule of the conjugate exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP under partial or total reducing conditions. The loading capacity (drug / antibody ratio) of the conjugate can be controlled in several different ways, including: (i) limiting the molar excess of drug linker relative to antibody, (ii) limiting the time or temperature of the conjugation reaction, and (iii) partial or limited reducing conditions of cysteine ​​thiol modification.

[0239] Certain antibody molecules have reducible interchain disulfides, i.e. cysteine ​​bridges. Antibody molecules can be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Thus, in theory, each cysteine ​​bridge becomes two reactive thiol nucleophiles. This process is also called "classical conjugation" to distinguish it from methods such as conjugation occurring at engineered cysteines at specific sites in the antibody molecule. Additional nucleophilic groups can be introduced into antibodies by converting amines to thiols by reaction of lysines with 2-iminothiolane (Traut's reagent).

[0240] ADCs in which drugs are randomly conjugated to natural cysteine ​​residues are prepared by classical conjugation by partial reduction of the antibody followed by reaction with the desired linker-drug. For example, the antibody is partially reduced by adding about 3 molar equivalents of DTT at pH 8.0 to an antibody at a concentration of 5 mg / mL, followed by incubation at about 37° C. for about 2 hours. The reduction reaction is then cooled in ice and excess DTT is removed, for example, by diafiltration. The linker-drug can then be added at about a 1:10 molar ratio of linker-drug / thiol. The conjugation reaction can be carried out in the presence of about 10% v / v DMSO. After conjugation, excess free cysteine ​​(about 2-fold molar ratio to linker-drug) can be added to quench unreacted linker-drug to generate cysteine-linker-drug adducts. The reaction mixture is then purified (e.g., by hydrophobic interaction chromatography) and subjected to buffer exchange with PBS. Drug loading distribution can be determined using standard methods such as hydrophobic interaction chromatography and reduced reversed-phase chromatography, as described elsewhere.

[0241] Methods are known for preparing conjugates using direct conjugation with solvent accessible thiols generated by reduction of antibody interchain disulfide bridges containing N-alkylmaleimides. Another method conjugates drugs with primary amines of lysines using N-hydroxysuccinimide esters. Such methods are reviewed, for example, in Gebleux and Casi, Pharmacol Ther (2016) 167:48-59, which is incorporated herein by reference in its entirety.

[0242] Alternatively or in addition to the above classical conjugation methods, it is also possible to use site-specific conjugation, where the drug loading and conjugation site are controlled. This can be achieved, for example, by engineering cysteines at specific residues, replacing residues with non-natural amino acids with bioorthogonal reactivity, or by enzymatic ligation approaches. One method of site-specific conjugation is described in Dimasi, 2017, which is incorporated herein by reference in its entirety, and involves inserting cysteines into antibody molecules at specific positions.

[0243] Cysteine ​​amino acids can be engineered at reactive sites of antibody molecules and do not form interchain or intermolecular disulfide bonds (Junutula, 2008; Dornan, 2009; US Pat. No. 7,521,541; US ​​Pat. No. 7,723,485; WO 2009 / 052249). The engineered cysteine ​​thiols can react with the drug-linkers of the present invention that have thiol-reactive electrophilic groups (such as maleimides or α-haloamides) to form conjugates with cysteine ​​engineered antibody molecules and drugs. Thus, the placement of the drug can be designed, controlled, and known. The engineered cysteine ​​thiol groups react with thiol-reactive linker reagents or drug linker reagents, typically in high yields, so that the drug loading can be controlled. IgG antibodies are engineered to introduce cysteine ​​amino acids by substitution at a single site on the heavy or light chain, providing two new cysteines on a symmetric antibody. If desired, drug loadings approaching 2 can be achieved with nearly uniform complexation products.

[0244] 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 in the CH region between positions 239 and 240 (where the numbering of the constant region is according to the EU index). Thus, the linkage between the antibody molecule and the drug can be made via this inserted cysteine ​​amino acid and the terminal maleimide group on the linker.

[0245] Examples of CH regions containing 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 heavy chains containing a CH region containing a cysteine ​​amino acid inserted between positions 239 and 240 of the CH region are SEQ ID NO: 50, 53 and 56.

[0246] In other examples, the conjugated antibody molecule does not contain any amino acid residues inserted into the CH region. In certain examples, the conjugated antibody molecule does not contain a cysteine ​​amino acid inserted into the CH region (e.g., between positions 239 and 240 (where the numbering of the constant region is according to the EU index)). As demonstrated in the examples (e.g., Example 12), when classical conjugation is used to conjugate the drug-linker to a native cysteine, an inserted cysteine ​​is not required.

[0247] Examples of CH regions that do not have any amino acid residues inserted therein are SEQ ID NOs: 44, 46, 63, and 64. Examples of heavy chains that include a CH region that does not have any amino acid residues inserted therein are SEQ ID NOs: 51, 54, 57, 59, and 60.

[0248] When two or more nucleophilic or electrophilic groups of an antibody molecule react with a drug-linker intermediate, or a linker reagent followed by a drug reagent, the resulting product is a mixture of conjugate compounds with a distribution of drug units attached to the antibody (e.g., 1, 2, 3, etc.). Liquid chromatography methods such as polymeric reversed phase (PLRP) and hydrophobic interaction (HIC) can separate compounds in a mixture by drug loading value. Although preparations of conjugates with a single drug loading value (p) can be isolated, even these single drug loading value conjugates can still be heterogeneous mixtures because the drugs can be attached to different sites on the antibody molecule via the linker.

[0249] Thus, the conjugate compositions of the present disclosure include a mixture of antibody-drug conjugate compounds in which the antibody bears one or more drug moieties (e.g., tubulysin or topoisomerase inhibitors), which may be attached to the antibody molecule at various amino acid residues.

[0250] In some examples, the average number of tubulysin drug moieties per antibody molecule ranges from 1 to 8. In some examples, the range is selected from 1 to 6, 1 to 4, 1 to 3, preferably 1 to 2, more preferably 1.5 to 2, even more preferably 1.8 to 2, and even more preferably 1.9 to 2.

[0251] As already described above, in some examples, the antibody molecules of the ADCs may comprise one or more mutations in the CH region of the heavy chain to reduce or abolish binding of the antibody molecule to one or more Fcγ receptors. Thus, the first and / or second heavy chains of the ADCs described herein comprise a phenylalanine (F) at position 234, a glutamic acid (E) at position 235, and a serine (S) at position 331 (wherein the numbering is according to the EU index).

[0252] Functional properties of antibody molecules and conjugates The antibody molecules and conjugates described herein may be characterized by reference to particular functional properties.

[0253] binding affinity The antibody molecules and conjugates described herein may be characterized by an antigen binding domain that binds EGFR with a particular affinity for EGFR and / or an antigen binding domain that binds c-Met with a particular affinity for c-Met. The binding affinity of the antibody molecule or conjugate to a cognate antigen, such as human, mouse or cynomolgus monkey EGFR or c-Met, can be determined by surface plasmon resonance (SPR), for example, using Biacore. The binding affinity can be determined using the antibody molecule, for example, as part of a bispecific antibody molecule that includes a first antigen binding domain that binds EGFR and a second antigen binding domain that binds c-Met. Alternatively, the binding affinity can be determined using an antibody molecule that is monospecific for EGFR or c-Met. In some examples, the binding affinity is determined using BIACore, as described in Example 2.1.

[0254] Binding affinity is typically measured by Kd (the equilibrium dissociation constant between antigen-binding domain and its antigen).As is well understood, the lower the Kd value, the higher the binding affinity of antigen-binding domain.For example, an antigen-binding domain that binds to a target with Kd of 10nM will be considered to bind to said target with higher affinity than an antigen-binding domain that binds to the same target with Kd of 100nM.

[0255] Reference to human EGFR may refer to a polypeptide comprising the extracellular domain of EGFR, e.g., having the amino acid sequence set forth in SEQ ID NO: 68. Reference to mouse EGFR may refer to a polypeptide produced from the molecule available from SinoBiological with catalog number 51091-M08H. Reference to cynomolgus 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 produced from the molecule available from SinoBiological with catalog number 50622-M08H. Reference to cynomolgus c-Met may refer to the amino acid sequence set forth in SEQ ID NO: 71.

[0256] EGFR affinity The antibody molecules and conjugates described herein may comprise an antigen-binding domain that binds to EGFR with low affinity. EGFR is known to be expressed at low levels in normal tissues, such as skin. Antibody molecules and conjugates that bind to EGFR with low affinity are advantageously expected to exhibit low on-target toxicity in normal tissues, while still being able to target tumors that express EGFR at high levels, resulting in an improved safety profile. Moreover, as demonstrated herein, conjugates that comprise this low-affinity EGFR antigen-binding domain are more effective in treating cancer than conjugates that comprise EGFR antigen-binding domains with higher affinities.

[0257] The antigen-binding domain that binds to EGFR may bind to human EGFR with an affinity having a Kd of 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, or 40 nM or more. Alternatively, the antigen-binding domain that binds to EGFR may bind to human EGFR with a Kd of 10-100 nM, 20-100 nM, 30-100 nM, 40-100 nM, 10-80 nM, 20-80 nM, 30-80 nM, 40-80 nM, 10-70 nM, 20-70 nM, 30-70 nM, 40-70 nM, 10-60 nM, 20-60 nM, 30-60 nM, 40-60 nM, 10-50 nM, 20-50 nM, 30-50 nM, or 40-50 nM.

[0258] The antigen-binding domain that binds to EGFR may bind to human EGFR with an affinity lower than the affinity of an antigen-binding domain comprising the heavy and light chain sequences of the antibody molecule QD6 set forth in SEQ ID NOs: 53 and 55, respectively.

[0259] For example, an antigen-binding domain that binds to EGFR may bind to human EGFR with an affinity having a Kd that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or 7-fold higher than the Kd with which an antigen-binding domain comprising the heavy chain sequence and light chain sequence of the antibody molecule QD6 set forth in SEQ ID NOs: 53 and 55 binds to human EGFR. Alternatively, an antigen-binding domain that binds to EGFR may bind to human EGFR with an affinity having a Kd that is 2- to 10-fold higher, 3- to 10-fold higher, 4- to 10-fold higher, 5- to 10-fold higher, 6- to 10-fold higher, 7- to 10-fold higher, 2- to 9-fold higher, 3- to 9-fold higher, 4- to 9-fold higher, 5- to 9-fold higher, 6- to 9-fold higher, 7- to 9-fold higher, 2- to 8-fold higher, 3- to 8-fold higher, 4- to 8-fold higher, 5- to 8-fold higher, 6- to 8-fold higher, or 7- to 8-fold higher than the Kd with which an antigen-binding domain comprising the heavy chain sequence and light chain sequence of the antibody QD6 set forth in SEQ ID NOs: 53 and 55 binds to human EGFR.

[0260] An antigen-binding domain that binds to EGFR may bind to human EGFR with an affinity similar to that of an antigen-binding domain comprising the variable heavy and light region sequences of the antibody molecule RAA22 set forth in SEQ ID NOs: 16 and 20, respectively. For example, an antigen-binding domain that binds to EGFR may bind to human EGFR with an affinity having a Kd that differs less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, less than 1-fold, or less than 0.5-fold from the Kd for binding to EGFR by an antigen-binding domain comprising the variable heavy and light region sequences of the antibody molecule RAA22 set forth in SEQ ID NOs: 16 and 20, respectively.

[0261] The antigen-binding domain that binds to EGFR may also bind to cynomolgus monkey EGFR. For example, the antigen-binding domain that binds to EGFR may bind to cynomolgus monkey EGFR with an affinity having a Kd of less than 700 nM, less than 600 nM, less than 500 nM, less than 400 nM, less than 300 nM, or less than 250 nM. Alternatively, the antigen-binding domain that binds to EGFR may bind to cynomolgus monkey EGFR with an affinity having a Kd of 100 to 700 nM, 100 to 600 nM, 100 to 500 nM, 100 to 400 nM, 100 to 300 nM, 150 to 250 nM, or 100 to 200 nM. The antigen-binding domain that binds to EGFR may bind to cynomolgus EGFR with a Kd that is 10-fold or less, 9-fold or less, 8-fold or less, 7-fold or less, 6-fold or less, 5-fold or less, 4-fold or less, or 3-fold or less than the Kd of the antigen-binding domain that binds to human EGFR.

[0262] The antigen-binding domain that binds to EGFR may also bind to mouse EGFR. For example, the antigen-binding domain that binds to EGFR may bind to mouse EGFR with an affinity having a Kd of less than 1 μM, less than 900 nM, less than 800 nM, less than 700 nM, less than 600 nM, or less than 650 nM. Alternatively, the antigen-binding domain that binds to EGFR may bind to mouse EGFR with a Kd of 100 nM to 1 μM, 200 to 900 nM, 300 to 800 nM, 400 to 700 nM, 400 to 600 nM, or 450 to 550 nM.

[0263] Preferably, the antigen-binding domain that binds to EGFR can bind to human EGFR and cynomolgus EGFR. This cross-reactivity is advantageous since it allows for dosing and safety testing of antibody molecules and conjugates in cynomolgus monkeys in preclinical development. Even more preferably, the antigen-binding domain that binds to EGFR can bind to human EGFR, cynomolgus EGFR, and mouse EGFR. For example, the antigen-binding domain that binds to EGFR can bind to human EGFR, cynomolgus EGFR, and mouse EGFR with the above Kd values. (For example, human EGFR with a Kd of 10 to 100 nM, cynomolgus EGFR with a Kd of 100 to 700 nM, and mouse EGFR with a Kd of 100 nM to 1 μM).

[0264] c-Met affinity An antigen-binding domain that binds to c-Met may bind to human c-Met with an affinity having a Kd of less than 20 nM, less than 15 nM, less than 12 nM, less than 11 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, or less than 2.5 nM. Alternatively, an antigen-binding domain that binds to c-Met may bind to human c-Met with an affinity having a Kd of 1 to 20 nM, 1 to 15 nM, 1 to 10 nM, 1 to 9 nM, 1 to 8 nM, 1 to 7 nM, 1 to 6 nM, 1 to 5 nM, 1 to 4 nM, 1 to 3 nM, 1 to 2.5 nM, or 2 to 2.5 nM.

[0265] The antigen-binding domain that binds to c-Met may bind to cynomolgus c-Met. For example, the antigen-binding domain that binds to c-Met may bind to cynomolgus c-Met with an affinity having a Kd of less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, or less than 2.5 nM. Alternatively, the antigen-binding domain that binds to c-Met may bind to cynomolgus c-Met with an affinity having a Kd of 1 to 20 nM, 1 to 15 nM, 1 to 10 nM, 1 to 9 nM, 1 to 8 nM, 1 to 7 nM, 1 to 6 nM, 1 to 5 nM, 1 to 4 nM, 1 to 3 nM, 1 to 2.5 nM, or 2 to 2.5 nM. An antigen-binding domain that binds c-Met may bind to cynomolgus c-Met with an affinity having a Kd that is 10-fold or less, 9-fold or less, 8-fold or less, 7-fold or less, 6-fold or less, 5-fold or less, 4-fold or less, 3-fold or less, 2-fold or less, or 1-fold or less than the Kd of an antigen-binding domain that binds to human c-Met.

[0266] Preferably, the antigen-binding domain that binds to c-Met is capable of binding to human c-Met and cynomolgus c-Met. This cross-reactivity is advantageous since it allows for dosing and safety testing of the antibody molecule in cynomolgus monkeys during preclinical development. For example, the antigen-binding domain that binds to c-Met is capable of binding to human c-Met and cynomolgus c-Met with Kd values ​​as described above (e.g., human c-Met with a Kd of 1-20 nM, and cynomolgus c-Met with a Kd of 1-20 nM).

[0267] specific binding The antibody molecules and conjugates described herein may comprise an antigen binding domain that binds to EGFR, which is an antigen binding domain that specifically binds to EGFR. The antibody molecules and conjugates described herein may comprise an antigen binding domain that binds to c-Met, which is an antigen binding domain that specifically binds to c-Met. The antibody molecules and conjugates described herein may comprise a first antigen binding domain that binds to EGFR, which is a first antigen binding domain that specifically binds to EGFR, and a second antigen binding domain that binds to c-Met, which is a first antigen binding domain that specifically binds to c-Met.

[0268] The term "specific" may refer to the situation where an antigen-binding domain does not exhibit significant binding to molecules other than its specific binding partner. Such molecules are called "non-target molecules." The term "specific" is also applicable when an antibody molecule is specific for a particular epitope, such as an epitope on EGFR or c-Met carried by several antigens, in which case the antibody molecule can bind to various antigens carrying the epitope.

[0269] In some instances, an antibody molecule or conjugate is considered to not exhibit significant binding to a non-target molecule if the extent of binding to the non-target molecule is less than about 10% of the extent of binding of the antibody to the target, for example, as measured by ELISA, SPR, biolayer interferometry (BLI), microscale thermophoresis (MST), or by radioimmunoassay (RIA). Alternatively, binding specificity may be reflected in terms of binding affinity, where the antibody molecules or conjugates described herein bind to EGFR and / or c-Met with an affinity that is at least 0.1 orders of magnitude greater than their affinity to another non-target molecule. In some instances, the antibody molecules or conjugates of the present disclosure bind to EGFR and / or c-Met with an affinity that is at least one of 0.2 orders of magnitude, 0.3 orders of magnitude, 0.4 orders of magnitude, 0.5 orders of magnitude, 0.6 orders of magnitude, 0.7 orders of magnitude, 0.8 orders of magnitude, 0.9 orders of magnitude, 1.0 orders of magnitude, 1.5 orders of magnitude, or 2.0 orders of magnitude greater than their affinity to another non-target molecule.

[0270] EGFR is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases (EGFR, HER2, HER3 and HER4). It has been demonstrated that the RAA22 antigen-binding domain does not bind to HER2, HER3 and HER4, and that this antigen-binding domain specifically binds to EGFR. Thus, in a preferred embodiment, the antigen-binding domain that binds to EGFR does not bind or does not show significant binding to HER2, HER3 or HER4.

[0271] c-Met is a member of the subfamily of receptor tyrosine kinases that includes Ron and Sema 4a. The B09-GL antigen-binding domain does not show binding to Ron and Sema 4a, demonstrating that this antigen-binding domain specifically binds to c-Met. Thus, in a preferred embodiment, the antigen-binding domain that binds to c-Met does not bind to Ron, Sema 4a or does not show significant binding.

[0272] Simultaneous binding The antibody molecules and conjugates comprising a first antigen binding domain that binds to EGFR and a second antigen binding domain that binds to c-Met described herein may be characterized by the ability of both antigen binding domains to simultaneously bind to their respective EGFR and c-Met targets. Antibody molecules and conjugates that have the ability to simultaneously bind to EGFR and c-Met are expected to be advantageous, as many tumors are known to co-express both EGFR and c-Met and therefore may be targeted by the antibody molecules of the present disclosure. Thus, in some instances, the antibody molecules or conjugates are capable of simultaneously binding to EGFR and c-Met.

[0273] Simultaneous binding can be determined, for example, by an in vitro cytotoxicity assay using a cell line expressing approximately equal amounts of EGFR and c-Met, and a complex containing an antibody molecule having an EGFR antigen-binding domain and a c-Met antigen-binding domain. If the individual antigen-binding domains in the complex function independently to deliver the drug, blocking either target in the cell line would only slightly reduce the activity of the complex, since the targets are present at comparable levels, and would be expected to shift the IC50 by no more than 2-fold. The EGFR and c-Met targets can be blocked in this assay, for example, by using a monospecific antibody molecule that binds to the same region of either EGFR or c-Met, but lacks a drug capable of inducing cytotoxicity. For example, the monospecific antibody molecule may contain the same antigen-binding domain that binds EGFR or c-Met as the complex being tested. On the other hand, if the complex requires simultaneous binding to effectively deliver the complex into cells, blocking either target is likely to have a greater effect on the activity of the complex. That is, when using this assay, an antibody molecule is considered to be capable of binding both targets simultaneously if there is a shift in IC50 of at least 2-fold, preferably at least 5-fold, and even more preferably at least 10-fold after blocking either target.

[0274] A further method for determining simultaneous binding is to compare the activity of the bispecific EGFR-c-Met complex with a monovalent monospecific control complex in an in vitro cytotoxicity assay. The control complex contains one antigen-binding domain for either EGFR or c-Met and one non-binding isotype antibody control antigen-binding domain. If each antigen-binding domain in the bispecific complex functions independently, the expected result would be that each monospecific control complex would only be slightly less potent than the bispecific complex, and the differences would be additive. Alternatively, if the two antigen-binding domains of the bispecific complex function synergistically through simultaneous binding, the difference in activity of the bispecific complex compared to the monospecific control antibody would be expected to be greater. That is, if the bispecific complex results in a shift in IC50 that is greater than the sum of the shifts in IC50 observed using the monospecific control complex, the antibody molecule is considered to be capable of binding both targets simultaneously.

[0275] Further details of these methods for measuring simultaneous binding can be found in the Examples.

[0276] Antibody internalization The antibody molecules and conjugates described herein can be characterized by their ability to mediate efficient intracellular internalization. This is particularly useful for conjugates, which ensure that the conjugates are internalized and delivered to lysosomes. The antibody molecule is then degraded, releasing the drug into the cell and exerting its cellular effect, e.g., cytotoxicity.

[0277] The internalization of an antibody molecule or complex by a cell can be analyzed by contacting a living cell with an antibody molecule and detecting the antibody molecule or complex after a period of time sufficient for internalization. Internalization can be determined by detecting the localization of the antibody molecule or complex. If the antibody molecule or complex remains on the surface of the cell (e.g., is detected on the cell surface and / or is not detected inside the cell), the antibody molecule or complex is determined to not be internalized. If the antibody molecule or complex is detected inside the cell (e.g., is localized in the cytoplasm or organelles), the antibody molecule or complex is determined to be internalized.

[0278] An exemplary method for visualizing whether an antibody molecule or complex can mediate efficient internalization includes labeling the antibody molecule with a pH-sensitive dye that exhibits fluorescence at acidic pH and adding these labeled antibody molecules or complexes to cells. Internalization can be detected by monitoring fluorescence. An antibody molecule or complex is deemed to be capable of mediating internalization and delivery to lysosomes if the observed fluorescence is greater than that of a labeled, unbound control antibody molecule or complex over a period of time, e.g., 48 hours. Further details of this method for visualizing antibody internalization can be found in the Examples.

[0279] Antibody molecules or conjugates comprising a first antigen-binding domain that binds EGFR and a second antigen-binding domain that binds c-Met may be characterized by their ability to mediate more efficient internalization compared to EGFR or c-Met monospecific controls. Antibody molecules and conjugates that exhibit this property are expected to exhibit greater selectivity for tumor cells that co-express both targets, which is expected to be advantageous as it can minimize the effect of the antibody molecule in normal tissues that do not exhibit significant levels of co-expression.

[0280] In vitro activity The antibody molecules or conjugates described herein may be characterized by their cytotoxic activity, i.e., their ability to kill cells. Cytotoxic activity can be measured, for example, using an in vitro cell viability assay such as the CellTiter-Glo® (Promega) assay. In some examples, the cells are cells that express both EGFR and c-Met.

[0281] The potency of an antibody molecule can be expressed as an IC50 value. IC50 is the median inhibitory concentration of an antibody molecule. In functional assays, IC50 is the concentration that reduces a biological response by 50% of its maximum. IC50 can be calculated by a number of means known in the art.

[0282] In some examples, antibody molecules or conjugates described herein that have cytotoxic activity have an IC50 of less than 4000 pM, less than 3500 pM, less than 3000 pM, less than 2500 pM, less than 2000 pM, less than 1500 pM, less than 1000 pM, less than 500 pM, less than 400 pM, less than 300 pM, less than 250 pM, less than 200 pM, less than 150 pM, or less than 100 pM, as measured using an in vitro cell viability assay. In some examples, antibody molecules or conjugates described herein may have an IC50 of 60-500 pM.

[0283] In some examples, the antibody molecules or conjugates described herein can increase the killing of cells, e.g., tumor cells, that express both EGFR and c-Met in significant amounts, compared to cells that express either EGFR or c-Met at low levels. Cells that express both EGFR and c-Met in significant amounts can be determined by measuring the relative receptor density on the cell surface. For example, cells that express EGFR and c-Met at a relative receptor density of more than 15,000 on the cell surface can be considered as cells that express both EGFR and c-Met in significant amounts, as well as cells that express one of EGFR and c-Met at a low relative receptor density of 15,000 or less on the cell surface. The relative density of EGFR and c-Met can be measured, for example, using the Quantum MESF quantitative FACS assay kit, as described in the Examples.

[0284] Examples of cells expressing significant amounts of both EGFR and c-Met include NCI H596, HCC 827 GR pool, A549, NCI H1792, NCI H1975, NCI H292 and NCI H358 cell lines. Examples of cells expressing one of EGFR and c-Met at low relative receptor density include A427, NCI H23 and NCI H661 (Ag negative) cell lines. These cell lines are available through ATCC.

[0285] In some examples, the antibody molecules or conjugates described herein may have cytotoxic activity in cells that are resistant to tyrosine kinase inhibitors (TKIs). Examples of TKI-resistant cells may include NCI H1975 (EGFR mutations: T790M, L858R) and HCC827 GR pool (EGFR Δ746-750 deletion + MET amplification).

[0286] In vivo activity In some instances, the antibody molecules or conjugates described herein may inhibit the development or progression of cancer.

[0287] The cancer can be a cancer that expresses both EGFR and c-Met. Cells of the cancer can express EGFR and c-Met on the cell surface. The cancer can be, for example, lung cancer (e.g., non-small cell lung cancer (NSCLC)), pancreatic cancer, breast cancer, colon cancer, gastric cancer, head and neck cancer, ovarian cancer, or glioblastoma.

[0288] The ability of a given antibody molecule or complex to inhibit the development or progression of cancer can be analyzed, for example, using an in vivo model.For example, the in vivo model can include measuring tumor growth in a patient-derived xenograft (PDX) model.Further details of this exemplary method are described in the Examples.

[0289] Inhibition of cancer development can be inferred by observing a slowing of tumor growth rate or a decrease in tumor size following administration of the antibody molecule or conjugate, for example by measuring the tumor growth inhibition rate (TGI%). TGI% can be measured by comparing the size of the tumor measured on day 0 in subjects administered the antibody molecule or conjugate with the size of the tumor measured at the end of the study period and comparing this to the tumor growth over the same period in subjects administered a control antibody molecule or conjugate. In this method, TGI% can be defined as the tumor growth rate relative to day 0 between the treatment (TX) group and the control (C) group according to the following formula: TGI%=(1-(TX final -TX initial ) / (C final -C initial )) x 100.

[0290] In some examples, the antibody molecules or conjugates 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%.

[0291] Inhibition of cancer development can be inferred by observing a delay or prevention of the onset of cancer symptoms and / or a reduction in the severity of cancer symptoms in response to treatment with the antibody molecule. Inhibition of cancer progression can be inferred by observing a delay, prevention and / or reduction in invasion and / or metastasis in response to treatment with the antibody molecule.

[0292] The antibody molecules described herein may be capable of inhibiting the occurrence or progression of cancer by less than 100%, such as 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 occurrence / progression of cancer in the absence of treatment with the antibody molecule (or treatment with a suitable control). In some examples, the antibody molecules described herein are capable of inhibiting cancer development or progression to less than 1-fold the level of cancer development / progression in the absence of treatment with the antibody molecule (or treatment with a suitable control), such as one of: <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.85-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold.

[0293] In some instances, the cancer comprises cancer cells that express high levels of EGFR and / or high levels of c-Met on the cell surface. Methods for measuring levels of EGFR and c-Met are known in the art and include, for example, immunohistochemistry.

[0294] In some examples, the antibody molecules or conjugates described herein may increase inhibition of cancer development or progression in cells that express high levels of both EGFR and c-Met compared to cells that express low levels of either one of these.

[0295] Nucleic Acids, Vectors, Host Cells, Expression and Purification The present disclosure provides nucleic acid molecules encoding the antibody molecules described herein. In some examples, the nucleic acid molecules comprise a polynucleotide sequence encoding a heavy chain variable region of an antibody molecule disclosed herein, a polynucleotide sequence encoding a light chain variable region of an antibody molecule disclosed herein, or both.

[0296] In some instances, the nucleic acid molecule is purified or isolated, for example, from other nucleic acids or naturally occurring biological materials. A person skilled in the art would have no difficulty in preparing such a nucleic acid molecule using methods known in the art.

[0297] The nucleic acid molecule may encode the VH and / or VL domains, preferably the VH and VL domains, of the following: anti-EGFR antibody clone RAA22, anti-EGFR antibody clone QD6, anti-c-Met antibody clone B09-GL, preferably anti-EGFR antibody clone RAA22, or anti-c-Met antibody clone B09-GL. The VH and VL domain sequences of these antibodies are described herein. When a nucleic acid encodes the VH and VL domains, or the heavy and light chains, of an antibody molecule of the present disclosure, the two domains or chains may be encoded on two separate nucleic acid molecules.

[0298] For example, the nucleic acid molecule may be (i) the VH domain amino acid sequence of RAA22 set forth in SEQ ID NO: 17, and / or the VL domain amino acid sequence of RAA22 set forth in SEQ ID NO: 21; (ii) the VH domain nucleic acid sequence of QD6 set forth in SEQ ID NO: 19, and / or the VL domain nucleic acid sequence of QD6 set forth in SEQ ID NO: 23; or (iii) the nucleic acid sequence of the VH domain of B09-GL set forth in SEQ ID NO: 39, and / or the nucleic acid sequence of the VL domain of B09-GL set forth in SEQ ID NO: 41 may include.

[0299] The nucleic acid molecule may encode the heavy and / or light chains, preferably the heavy and light chains, of the following: anti-EGFR antibody clone RAA22, anti-EGFR antibody clone QD6, anti-c-Met antibody clone B09-GL, preferably anti-EGFR antibody clone RAA22, or anti-c-Met antibody clone B09-GL. The sequences of the heavy and light chains of these antibody molecules are described herein.

[0300] The present disclosure also provides vectors comprising a nucleic acid molecule encoding the antibody molecules described herein.

[0301] The isolated nucleic acid molecule can be used to express the antibody molecule of the present disclosure. The nucleic acid is generally provided in the form of a recombinant vector for expression. Thus, another aspect of the present disclosure provides a vector comprising the above-mentioned nucleic acid. A suitable vector can be selected or constructed, containing appropriate regulatory sequences, such as promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as needed. The vector preferably contains suitable regulatory sequences to induce expression of the nucleic acid in a host cell. The vector can be a plasmid, a virus, such as a phage, or a phagemid as needed.

[0302] The nucleic acid molecules or vectors described herein can be introduced into a host cell. The techniques for introducing nucleic acids or vectors into a host cell are well established in the art, and any suitable technique can be used. Various host cells suitable for producing recombinant antibody molecules are known in the art, and include bacterial, yeast, insect or mammalian host cells. Preferred host cells are mammalian cells, such as CHO, NS0, or HEK cells, e.g., HEK293 cells.

[0303] Another aspect of the present disclosure provides a method of producing an antibody molecule of the present disclosure, comprising expressing a nucleic acid encoding the antibody molecule in a host cell and, optionally, isolating and / or purifying the antibody molecule so produced. Methods of culturing host cells are well known in the art. The method may further comprise isolating and / or purifying the antibody molecule. Techniques for purifying recombinant antibody molecules are well known in the art and include, for example, HPLC, FPLC, or affinity chromatography using Protein A or Protein L. In some examples, purification may be performed using an affinity tag on the antibody molecule. The method may also comprise formulating the antibody molecule into a pharmaceutical composition, optionally with a pharma- ceutically acceptable excipient or other substances as described below.

[0304] treatment As explained above, co-expression of EGFR and c-Met is associated with many cancer types, and antibody molecules that target both molecules, and particularly conjugates containing such antibody molecules, offer the opportunity for broad clinical benefit across multiple indications.

[0305] The antibody molecules or conjugates described herein may therefore be useful in therapeutic applications, particularly in the treatment of cancer.

[0306] The antibody molecules, conjugates or pharmaceutical compositions described herein may be used in methods of treatment of the human or animal body. (i) an antibody molecule or conjugate as described herein for use as a medicament; (ii) an antibody molecule or conjugate as described herein for use in a method for the treatment of a disease or disorder; (iii) an antibody molecule or conjugate as described herein in the manufacture of a medicament for use in the treatment of a disease or disorder, and (iv) A method of treating a disease or disorder in an individual, comprising administering to the individual a therapeutically effective amount of an antibody molecule or conjugate described herein.

[0307] The individual may be a patient, preferably a human patient.

[0308] Treatment can be any treatment or therapy that achieves any desired therapeutic effect, such as arresting or slowing the progression of the condition, including slowing the rate of progression, halting the rate of progression, ameliorating the condition, curing or remission (whether partially or totally) the condition, preventing, ameliorating, slowing, reducing or halting one or more symptoms and / or signs of the condition, or prolonging the survival of an individual or patient beyond that expected if not receiving treatment.

[0309] The described therapeutic methods may include administering at least one additional treatment to an individual in addition to the antibody molecule or conjugate. Thus, the antibody molecule or conjugate described herein may be administered to an individual alone or in combination with one or more other treatments. When the antibody molecule or conjugate is administered to an individual in combination with another treatment, the additional treatment may be administered to the individual simultaneously, sequentially, or separately from the administration of the antibody molecule or conjugate. When the additional treatment is administered simultaneously with the antibody molecule or conjugate, the antibody molecule and the additional treatment may be administered to the individual as a combined preparation. For example, the additional treatment may be a known therapy or therapeutic agent for the disease being treated.

[0310] Although the antibody molecule may be administered alone, the antibody molecule or conjugate is usually administered in the form of a pharmaceutical composition that may contain at least one component in addition to the antibody molecule or conjugate. Accordingly, another aspect of the present disclosure provides a pharmaceutical composition comprising the antibody molecule or conjugate described herein. Also provided is a method comprising formulating the antibody molecule or conjugate into a pharmaceutical composition.

[0311] A pharmaceutical composition may include, in addition to the antibody molecule or conjugate, pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. The term "pharmacologically acceptable", as used herein, refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable risk / benefit ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The precise nature of the carrier or other material will depend on the route of administration, as explained below. The route of administration may be by infusion, injection, or any other suitable route.

[0312] Administration may be in a "therapeutically effective amount", which is sufficient to provide benefit to the individual. The actual amount administered and the rate and time course of administration will depend on the nature and severity of what is being treated, the particular individual being treated, the individual's clinical condition, the cause of the disorder, the site of delivery of the composition, the type of antibody molecule, the method of administration, the administration schedule, and other factors known to medical practitioners. Prescribing treatment, such as determining dosage, is within the scope of the duties of general practitioners and other physicians, and may depend on the severity and / or progression of the symptoms of the disease being treated. Appropriate dosages of antibody molecules are well known in the art (Ledermann, 1991; and Bagshawe, 1991). Depending on the antibody being administered, specific dosages as set forth herein or in the Physician's Desk Reference (2003) may be used. Therapeutically effective amounts or suitable dosages of antibody molecules can be determined by comparing in vitro and in vivo activity in animal models. Extrapolation of effective dosages in mice and other test animals to humans is known. The exact dose will depend on many factors, including the size and location of the area to be treated, and the precise nature of the antibody molecule.

[0313] In a preferred example, the antibody molecules or conjugates described herein may be for use in a method of treating cancer.

[0314] Cancer can be characterized by the abnormal proliferation of malignant cancer cells. When a specific type of cancer is mentioned, such as breast cancer, this refers to the abnormal proliferation of malignant cells in related tissues, such as breast tissue. A secondary cancer that is located in the breast but is the result of the abnormal proliferation of malignant cells in another tissue, such as ovarian tissue, is not breast cancer as referred to herein, but ovarian cancer.

[0315] The cancer may be a primary or secondary cancer. Thus, the antibody molecules or conjugates described herein may be for use in a method of treating cancer in an individual where the cancer is a primary tumor and / or tumor metastasis.

[0316] The cancer tumors to be treated with the antibody molecules or conjugates described herein may co-express EGFR and c-Met. In one example, the tumor may be one that has been determined to co-express EGFR and c-Met. Methods for determining target expression are known in the art and include, for example, immunohistochemistry.

[0317] For example, the cancer to be treated using the antibody molecules or conjugates described herein may be selected from the group consisting of lung cancer (e.g. non-small cell lung cancer (NSCLC)), pancreatic cancer, breast cancer, colon cancer, renal cancer, gastric cancer, head and neck cancer, ovarian cancer or glioblastoma.

[0318] In a preferred example, the cancer to be treated using the antibody molecules or conjugates 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).

[0319] In the context of cancer, treatment can include inhibiting cancer growth (including complete cancer remission) and / or inhibiting cancer metastasis, as well as inhibiting cancer recurrence. Cancer growth generally refers to any one of a number of indicators that indicate changes in cancer to more advanced forms. Thus, indicators for measuring inhibition of cancer growth include reduced cancer cell viability, reduced tumor volume or morphology (e.g., determined using computed tomography (CT), ultrasound, or other imaging methods), delayed tumor growth, destruction of tumor vasculature, improved performance in delayed hypersensitivity skin tests, increased activity of anti-cancer immune cells or other anti-cancer immune responses, and reduced levels of tumor-specific antigens. Activating or enhancing the immune response against cancerous tumors in an individual can improve the individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and / or reduce the tendency of cancer growth in the individual.

[0320] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, are expressed in their specific form, or in terms of means for performing a disclosed function, or methods or processes for obtaining a disclosed result, and such features can be used, individually or in any combination as appropriate, to realize the disclosure in its diverse forms.

[0321] Although the present disclosure has been described with the illustrative examples set forth above, many equivalent modifications and variations will become apparent to those skilled in the art upon receipt of this disclosure. Accordingly, the illustrative examples of the present disclosure set forth above are considered to be illustrative and not limiting. Various modifications can be made to the described examples without departing from the spirit and scope of the present disclosure.

[0322] For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purpose of enhancing the understanding of the reader, and the inventors do not wish to be bound by any of these theoretical explanations.

[0323] All section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0324] Throughout this specification, including the claims which follow, unless the context otherwise requires, the words "comprise" and "include", as well as variations such as "comprises", "comprising" and "including", will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of other integers or steps or group of integers or steps.

[0325] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another example includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that the particular value constitutes another example by using "about." The term "about" in connection with numerical values ​​is optional and means, for example, ±10%. EXAMPLES

[0326] Example 1 - Design and construction of RAA22 / B09 DuetMab This example describes the generation of a bispecific antibody molecule capable of binding to both EGFR and c-Met.

[0327] 1.1 Isolation and identification of anti-cMET antibody 0021U3-B09 cMET-specific scFv antibodies were isolated from a large naive human scFv phage display library in a series of iterative panning selection cycles on recombinant mammalian-expressed biotinylated monomeric human cMET (MedImmune) essentially as described (Vaughan, 1996). ScFvs from round 2 of the selection output were expressed in bacterial periplasm and screened for their ability to inhibit the binding of human cMET receptor to HGF ligand in an HGF:cMET HTRF® (homogeneous time-resolved fluorescence) ligand-receptor inhibition binding assay. Top hits showing strong inhibitory effects were selected and subjected to DNA sequencing. The unique genes were then converted into human immunoglobulin G2 (IgG2) antibodies and produced in mammalian cells essentially as described (Persic; 1997). The purified antibodies were then ranked based on their inhibitory effects in the HGF:cMET HTRF® binding assay. The most potent antibody, 0021U3-B09, was selected for further characterization.

[0328] 1.2 Optimization of anti-cMET antibody 0021U3-B09. To minimize potential immunogenicity, non-Vernier framework residues (Foote and Winter 1992) in the variable framework regions of 0021U3-B09 were specifically targeted and modified 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. The affinity of 0021U3-B09 was optimized using hybridization-based mutagenesis essentially as described (Kunkel 1985). A large scFv library derived from the 0021U3-B09 sequence was generated by oligonucleotide-directed mutagenesis of the VH complementarity determining region 3 (CDR3) using standard molecular biology techniques. The library was subjected to affinity-based solution-phase selection to select variants with higher affinity to human and cynomolgus cMET antigens. Crude scFv-containing periplasmic extracts from the CDR-targeted selection output were screened for improved inhibitory activity in the HGF:cMET HTRF® binding assay. Variants with significantly improved inhibitory effects compared to the parent 0021U3-B09 were subjected to DNA sequencing and unique genes were converted to human IgG2. The purified antibodies were then ranked based on their inhibitory effects. The most potent antibody, B09-57, was selected for further characterization.

[0329] 1.3 Isolation and characterization of the anti-EGFR antibody Tdev-0004. EGFR-specific scFv antibodies were isolated from a large naive human scFv phage display library in a series of iterative panning selection cycles on recombinant mammalian-expressed biotinylated monomeric human EGFR (MedImmune) essentially as described (Vaughan, 1996). ScFv-displaying phages from round 3 of the selection output were screened for their binding to human and cynomolgus EGFR in ELISA. Top hits showing cross-reactivity were selected and subjected to DNA sequencing. The unique genes were then converted to human immunoglobulin G1 (IgG1) antibodies and produced in mammalian cells essentially as described (Persic, 1997). Purified antibodies were then ranked based on their binding to the EGFR-expressing cell line A431 by flow cytometry. Antibody Tdev-0004 showing specific cell binding was selected for further characterization.

[0330] 1.4 Optimization of the anti-EGFR antibody Tdev-0004. Variants RAA22 and QD6 were derived by optimizing the anti-EGFR Tdev-0004 mAb. The VL framework of Tdev-0004 was 100% identical to the reference human germline sequence IGLV2-11*01 / IGLJ2 ​​(see https: / / www.ncbi.nlm.nih.gov / projects / igblast / Idlink.cgi?seqname=IGLV2-11*01&taxid=9606&dbname=IG_DB%2Fimgt.Homo_sapiens.Vforf.p), whereas the VH framework was 79% identical to the closest human germline IGHV1-69*01 / JH4 (see https: / / www.ncbi.nlm.nih.gov / projects / igblast / Idlink.cgi?seqname=IGHV1-69*01&taxid=9606&dbname=IG_DB%2Fimgt.Homo_sapiens.Vforf.p). To minimize potential immunogenicity, the VH region was first fully germlined by mutating all 13 non-germline framework residues. Germlined binding of the fully germlined variant to cynomolgus monkey EGFR was largely impaired. Four non-germline residues K68, I73, R76, and T78 were selectively backmutated to restore binding to cynomolgus monkey EGFR. Amino acid residues are numbered according to the Kabat numbering system (Kabat and Wu 1991). The resulting partially germlined variant (designated H4) was used as the template sequence for affinity optimization. The affinity of variant H4 was optimized by parsimonious mutagenesis of all six CDRs using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent) according to the manufacturer's instructions. Single amino acid mutagenized VH and VL libraries were expressed in bacteria as Fab fragments and screened for improved binding to human and cynomolgus EGFR in ELISA.Variants with improved binding compared to parent H4 were subjected to DNA sequencing and unique genes were converted to human IgG1. Mutant RAA22 was identified with a single mutation in CDRH3. To further improve affinity, individual positive mutations were combined to generate a combinatorial library that was screened for variants with enhanced binding to human and cynomolgus EGFR. Variant QD6 was identified with four combined mutations in CDRL2, CDRL3 and CDRH3.

[0331] 1.5 Generation of monovalent bispecific anti-EGFR / cMET DuetMab antibodies. A monovalent bispecific anti-EGFR / cMET antibody was constructed on the backbone of the DuetMab platform using the variable domains of anti-cMET mAb B09-57 and anti-EGFR mAbs RAA22 and QD6 (Mazor, 2015). Specifically, the VH gene of anti-cMET B09-57 was inserted into a human gamma-1 constant heavy chain with a "knob" mutation (T366W) and alternative interchain cysteine ​​mutations (F126C and C219V). The VL gene of B09-57 was inserted in frame into a human kappa constant domain with the corresponding alternative interchain cysteine ​​mutations (S121C and C214V) designed to pair with the "knob" heavy chain. Similarly, the VH genes of anti-EGFR RAA22 and affinity-optimized QD6 were inserted into a human gamma-1 constant heavy chain with "hole" mutations (T366S, L368A, and Y407V), while the VL genes of RAA22 and B09-57 were inserted in frame into a human lambda constant domain designed to pair with the "hole" heavy chain. Additionally, two residues in the CH3 domains of the "knob" and "hole" heavy chains were mutated to cysteines (S354C in "knob" and Y349C in "hole") to form a stabilizing disulfide bridge. The Fc domain was further engineered to carry a cysteine ​​insertion after serine 239 (C239i / "Maia") designed to allow site-specific conjugation of maleimide-bearing cytotoxic drugs (Dimasi, 2017). Amino acid residues are numbered according to the Kabat numbering system (Kabat and Wu 1991). The assembled monovalent bispecific anti-EGFR / cMET DuetMab antibodies were named RAA22 / B09-57 and QD6 / B09-57 (Figure 1). DuetMab antibodies were generated from mammalian cells as previously described (Mazor, 2017).

[0332] The amino acid sequences of the heavy and light chains of DuetMabs generated according to this example are provided in the table below.

[0333] [Table 7]

[0334] Example 2 - Biochemical and biophysical properties This example examines various biochemical and physiophysical properties of the RAA22, QD6 and B09-57 monoclonal antibodies, and the RAA22 / B09-57 and QD6 / B09-57 bispecific antibody molecules, such as their binding affinities to EGFR and c-Met, respectively, and their ability to bind both antigens simultaneously.

[0335] 2.1 Binding affinity of DuetMab and parental mAbs to EGFR and cMET. The kinetic constants (k and k) and equilibrium dissociation constants (K D) was determined by SPR using an antibody capture assay on a BIAcore T200 instrument (GE Healthcare, Pittsburgh, PA) at 25°C. Mouse anti-human IgG was immobilized on a CM4 sensor chip with a final surface density of approximately 2000 resonance units (RU). A reference flow cell surface was also prepared on this sensor chip using the same immobilization protocol. Test and control antibodies were prepared at 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) along with 3-fold serial dilutions of purified EGFR protein (human 0.27-200 nM, cynomolgus monkey 0.4-900 nM, and mouse 4-1000 nM) or cMET protein (human 0.27-66 nM, and cynomolgus monkey 0.27-22 nM) in instrument buffer. A sequential approach was used for kinetic measurements. Antibodies were first injected over the capture surface at a flow rate of 10 μL / min. Once the binding of the captured antibodies had stabilized, a single concentration of analyte was injected over both the capture and reference surfaces at a flow rate of 75 μL / min. The resulting binding response curves were used to obtain association phase data. After injection of the analyte, the flow was switched to instrument buffer for 15 min to allow collection of dissociation phase data, followed by a 1 min pulse of 10 mM glycine, pH 1.5 to regenerate the antibody capture surface on the chip. Binding responses to test and control antibodies were recorded from duplicate injections of each concentration of analyte. In addition, several buffer injections were distributed throughout the injection series. Selected buffer injections were used along with reference cell responses to correct the raw data set for injection artifacts and / or non-specific binding interactions, commonly referred to as "double referencing". The corrected binding data were globally fit to a 1:1 binding model (Biacore T200 Evaluation software 2.0, GE Healthcare, Pittsburgh, PA). Calculated kinetic parameters (k on and k off ), and k off / k on K was determined as D are shown in Table 1.

[0336] [Table 8]

[0337] As can be seen from the above data, the bispecific antibody molecule QD6 / B09-57 binds to human c-Met with high affinity (kD of approximately 2 nM) and binds to human EGFR with high affinity (kD of approximately 6 nM), whereas the bispecific antibody molecule RAA22 / B09-57 also binds to human c-Met with high affinity (kD of approximately 2 nM) but binds to human EGFR with lower affinity (kD of approximately 45 nM) compared to QD6 / B09-57.

[0338] 2.2 Simultaneous binding of DuetMab to EGFR and cMET. Simultaneous binding studies to recombinant human EGFR and cMET proteins were measured by biolayer interferometry on an Octet384 instrument essentially as described (Mazor, 2015). Briefly, His-tagged cMET antigen was first captured on an NI-NTA biosensor at 5 μg / mL in assay buffer [PBS (pH 7.2), 3 mg / mL bovine serum albumin (BSA), 0.05% (v / v) Tween 20]. Following a washing step to remove unbound protein, each loaded biosensor was subjected to sequential association and dissociation interactions, first with 66 nM antibody and then with 500 nM EGFR antigen. Association and dissociation curves were calculated from nonlinear fits of the data using Octet384 software v.9.0. As shown in Figure 2, the DuetMab showed simultaneous binding to both antigens, whereas the parental anti-cMET IgG showed specific binding only to cMET, and the two anti-EGFR IgGs showed no binding to the cMET load sensor.

[0339] 2.3 Specificity of EGFR and c-Met Specificity for EGFR and cMET species paralogs and closely related family members was determined by ELISA. Briefly, 1 μg / mL antigen solution was prepared in PBS and 50 microliters was coated onto half the area of ​​an ELISA assay plate. Plates were washed and blocked with 1% BSA in PBS containing 0.005% Tween-20 (PBS-T) for 1 hour at room temperature. Wells were washed 4 times with PBS-T. As shown in Figure 3, the primary antibodies used were R374 (non-binding IgG1 isotype control antibody), B09 (anti-cMET antibody), QD6 (anti-EGFR antibody), RAA22 (anti-EGFR antibody), QD6 / B09 (bispecific EGFR / c-MET DuetMAb), RAA22 / B09 (bispecific EGFR / c-MET DuetMAb), PaniX (anti-EGFR antibody), MetMab (anti-cMET antibody), and Mab11311 (anti-HER4 antibody). Wells were incubated with 50 microliters of the indicated primary antibody diluted in PBS-T in a 1:3 dilution series starting at 10 μg / mL and ending at 0.002 μg / mL, except for the HER4-binding mAb control MAB1131, which started the dilution series at 1 μg / mL. Wells were washed four times with PBS-T, 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 1 hour at room temperature. Fifty microliters of TMB substrate solution was added to all wells and incubated for 5 to 30 minutes at room temperature until a strong signal was observed in the positive control wells. Fifty microliters of TMB stop solution was added to all wells and absorbance was read at 450 nm on a SpectraMax M5 microplate reader. Data were analyzed with SoftMax Pro 5 software and plotted using GraphPad Prism 7 graphing software.

[0340] To determine species cross-reactivity, ELISA assays were performed as described above. As shown in Figure 3A, the high affinity EGFR IgG (QD6) and the monovalent bispecific EGFR / cMET DuetMAb (QD6 / B09) bound to human, cynomolgus and mouse EGFR, giving strong signals in the ELISA assay. In contrast, the low affinity EGFR IgG (RAA22) bound weaker to human, cynomolgus and mouse EGFR compared to QD6. Binding to mouse EGFR was weaker but detectable. The corresponding monovalent bispecific EGFR / cMET DuetMAb (RAA22 / B09) showed even weaker binding to human and cynomolgus EGFR and only negligible binding to mouse EGFR compared to the bivalent parental IgG (RAA22), whereas cMET IgG (B09) and all bispecific variants showed comparable binding to human and cynomolgus cMET. No binding to mouse cMET was detected for either antibody. These results are consistent with the binding kinetics determined by surface plasmon resonance measurements on a BIAcore instrument (Table 1 above).

[0341] As shown in Figure 3B, neither the parental IgG nor the derived bispecific antibody showed significant binding to any of the EGFR HER family proteins (HER2, HER3, or HER4). Similarly, none of the antibodies showed significant binding to cMET family members (Ron (CD136) or Semaphorin 3a).

[0342] These results demonstrate that the parent IgG and the resulting bispecific antibodies bind specifically to their cognate targets, with no detectable binding to closely related family members.

[0343] Example 3 - Monitoring antibody internalization and trafficking to acidified compartments using pH-sensitive dyes The efficacy of antibody drug conjugates (ADCs) depends in part on the ability of the antibody to mediate efficient internalization and delivery to lysosomes where the antibody is subsequently degraded. This lysosomal degradation allows the cytotoxic warhead to be released and exert its cellular effect on the target tumor cells. Meanwhile, target expression in normal non-tumor tissues can result in toxicity that reduces the therapeutic window of the ADC. The intent of the design of the bispecific antibodies of the present disclosure is to maximize the delivery of the ADC to tumor cells that co-express the two targets while minimizing the effect of the ADC in normal tissues that show little or no co-expression of the targets. To assess whether dual targeting using the bispecific antibody RAA22 / B09 with low EGFR affinity provides selectivity benefits over single target binding, we performed studies using pH-sensitive dye-labeled antibodies to compare the internalization efficiency of the bispecific mAb and the monovalent parent antibody that contains the bispecific.

[0344] Antibodies labeled with pHAb pH-sensitive dye (Promega) were used to facilitate visualization of antibody internalization and trafficking to acidified intracellular compartments such as lysosomes and endosomes. This dye exhibits very low fluorescence at pH>7 but strong fluorescence at acidic pH, reaching a maximum at approximately pH5. Briefly, antibodies were labeled with pHAb amine-reactive dye according to the manufacturer's recommendations. The labeled antibodies were the R347 IgG1 isotype control, monovalent bispecific control antibodies anti-EGFR antibodies RAA22 / R347 and anti-cMET B09 / R347, and the EGFR / cMET monovalent bispecific antibody RAA22 / B09. NCI-H1975 lung cancer cells, which co-express moderate levels of EGFR (approximately 33,000 relative receptor density) and cMET (approximately 50,000 relative receptor density), were cultured in RPMI growth medium supplemented with 10% fetal bovine serum in a volume of 100 microliters at 2 × 10 5Cells were plated at a density of 10 ...

[0345] A representative internalization experiment using 1.25 μg / mL of pHAb-labeled mAb to treat NCI H1975 cells is shown in Figure 4. The non-binding IgG1 isotype control antibody R347 showed no detectable fluorescence at any time point. The EGFR / cMET bispecific antibody RAA22 / B09 showed intracellular fluorescence by 3 hours, and the fluorescence intensity continued to increase up to 48 hours after treatment. The monovalent EGFR binding control antibody RAA22 / R347 showed very weak fluorescence from 24 hours, which did not increase dramatically up to 48 hours. The monovalent monospecific cMET binding control antibody B09 / R347 showed slight fluorescence at 24 hours, which further increased up to 48 hours. Nevertheless, the intensity of the fluorescent signal of the monospecific cMET antibody was weaker compared to the bispecific RAA22 / B09, suggesting that the bispecific antibody has a greater internalization efficiency than the monospecific parent antibody in the dual target expressing cell line tested here. When cells were treated with 0.625 μg / mL of pHAb-labeled mAb, the difference between the bispecific antibody and the monospecific control was even more striking (Figure 5). While the monospecific antibody showed very little fluorescence even at 48 hours, the bispecific RAA22 / B09 antibody showed intracellular fluorescence again by 3 hours, and the fluorescence intensity continued to increase up to 48 hours after treatment. These results are consistent with the hypothesis that dual-targeting bispecific antibodies mediate efficient internalization into cells that co-express both EGFR and cMET. At the same time, the monospecific parent antibody shows weak uptake and fluorescence intensity compared to the bispecific antibody. A logical extension of these conclusions is to suggest that bispecific antibodies may behave like monospecific antibodies in tissues that express only one, but not both, of EGFR and cMET, which is generally the case. Of particular note, the pHAb-labeled low affinity EGFR control mAb RAA22 / R347 showed negligible uptake and fluorescence. This weak binding to EGFR may minimize the effect of the ADC in normal tissues such as skin, which express significant levels of EGFR but little or no cMET.

[0346] Example 4 - Monitoring antibody internalization using confocal microscopy The intracellular internalization kinetics of labeled DuetMab:RAA22 / B09 and QD6 / B09 antibodies were assessed in vitro using live cell confocal fluorescence microscopy.

[0347] 4.1 Materials and Methods H1975 and HCC827 cells were from ATCC. RAA22 / B09, QD6 / B09, and single arm derivatives were obtained from MedImmune. QD6 / B09, and single arm specific controls QD6 / IgG and B09 / IgG with IgG Fab arms from non-specific human IgG1 NMGC were obtained from MedImmune. RAA22 / B09, and single arm specific controls RAA22 / IgG and B09 / IgG from non-specific human IgG1 R347 were obtained from MedImmune. RPMI (11875-093), HEPES (15630106), sodium pyruvate (11360070), AlexaFluor® 647 (A-20186) monoclonal antibody labeling kit, Zeba™ spin desalting columns (87767), and CellTracker™ Blue CMAC (C2110) were obtained from Life Technologies (Carlsbad, CA). Accutase cell detachment solution (423201) was obtained from BioLegend (San Diego, CA). HyClone heat inactivated fetal bovine serum (SH30071.03HI) was obtained from GE Life Sciences (Marlborough, MA). PBS (21-040) was obtained from Corning Incorporated (Corning, NY). FcR blocking reagent (130-059-901) was obtained from Miltenyi Biotec Inc (Auburn, CA). Polypropylene round-bottom tubes (352063) were obtained from BD Biosciences (San Jose, CA). CellCarrier 384-well microplates were obtained from PerkinElmer Inc (catalog number 6007550, Waltham, MA).

[0348] Preparation of AlexaFluor conjugates Monoclonal antibodies were conjugated with AlexaFluor-647 dye using an antibody labeling kit according to the manufacturer's instructions. Briefly, 50-100 micrograms of antibody in sodium bicarbonate buffer (pH=8.3) was incubated with reactive dye reagent for 1 hour at room temperature with gentle agitation. Unincorporated dye was removed by size-exclusion chromatography using Zeba™ spin desalting columns with 40K MWCO equilibrated with 1×PBS according to the manufacturer's instructions.

[0349] Cultivation and preparation of cells for staining Adherent H1975 or HCC827 cells were cultured in T-75 flasks with medium RPMI-1640 containing 10% fetal bovine serum (FBS) in a CO2 incubator until 80-90% confluent from the initial seeding. On the day of the experiment, the adherent monolayer grown in the T-75 flask was detached into a cell suspension using Accutase. The detached cells were washed twice with 1x PBS using centrifugation at 300 x g for 5 min. The cells were then resuspended in phenol-free RPMI at a concentration of 2 x 106 cells / mL and used for staining.

[0350] Cell staining for imaging A cell suspension of 2x106 cells / mL was incubated with 1 μM CellTracker™ Blue CMAC prepared in phenol-free RPMI for 30 minutes at 37°C in a CO2 incubator. Unincorporated CellTracker™ Blue CMAC dye was removed by washing twice with phenol-free RPMI using centrifugation at 300xg for 5 minutes at 4°C. Cells were then cooled on ice and blocked with 10ul FcR blocking reagent per 1x106 cells for 15 minutes. 2x10 5Cells were dispensed into 5 mL round-bottom tubes and incubated with fluorescent antibodies at a final concentration of 2.5 μg / mL. After removing unbound fluorescent reagent by centrifugation at 4° C., cells were resuspended in phenol-free RPMI containing 100 mM HEPES, 1 mM sodium pyruvate, and 1% FBS. Cells were transferred to multiple wells of a 384-well imaging plate at a density of 50.00 cells per well and briefly centrifuged at 2,200 rpm for 2 min at 4° C. before image acquisition.

[0351] Acquiring cell images using a confocal fluorescence microscope Stained cells in imaging plates (384-well format) were imaged with the Opera confocal fluorescence imaging system as previously described (Vainshtein, 2015) or transferred to a Zeiss Axio Observer Z1 inverted microscope (Carl Zeiss Microscopy, Thornwood, NY) with a 40X / 1.2NA LCIPlan Apo objective. For experiments with the Zeiss microscope, the imaging environment was kept at 37°C, 5% CO2 and 70% humidity using an incubator XLmulti S DARK (PeCon Gmbh, Erbach, Germany). Samples were illuminated with 405, 488, 561 and 63 nm solid-state lasers (Carl Zeiss Microscopy, Thornwood, NY). At the indicated times, a series of images was acquired using a Yokogawa CSU-X1 spinning disk unit (Yokogawa Electric Corporation, Tokyo, Japan) equipped with an Evolve 512 EMCCD (Photometrics, Tuscon, AZ). Prior to image acquisition, exposure parameters such as laser power, exposure time, and camera gain were determined using an aliquot of stained cells. Images were processed using ZEN 2.3 (Carl Zeiss Microscopy, Thornwood, NY) and analyzed using Columbus software (PerkinElmer, Waltham, MA).

[0352] Algorithms for image analysis of intracellular trafficking The algorithm used to quantify antibody internalization has been described previously (Vainsthein, 2015) with the following updates and modifications: The reference channel used for iterative image processing is generated from CellTracker™ Blue CMAC (CTB) staining of the cells. The signal channel further originates from the antibody-AlexaFluor-647 channel of the imager. Images were processed by the algorithm using algorithm-defined parameters, which were initially set as default values ​​and then optimized for each cell type and experiment. The CTB staining of the images was used to identify cells using a threshold to detect regions of the image with higher intensity than the surroundings, excluding regions with fluorescence intensity signal below the threshold. The remaining identified cell objects were designated as "whole cells". Cells were further selected by filtering for morphological characteristics area (objects between 120 and 600 μm2) and circularity (>0.5). "Membrane regions" and "cytoplasmic regions" in acceptable cells were then constructed around the boundaries of the objects using algorithm-defined parameters. The fluorescence intensity in each region was used to monitor the antibody-associated AlexaFluor-647 signal. The fluorescence intensity of each region was reported as the average of the sum of all pixels in the allowed cells.

[0353] The kinetics of antibody internalization was quantified by the accumulation of antibody-associated fluorescence in the cytoplasm. To ensure comparability of results due to variations in cell staining and fluorescence intensity, the cytoplasmic signal was normalized by the total cell signal at each time point and designated as the internalized fraction using the following formula: Internalized fraction = Intensity(cytoplasm) / (Intensity(cytoplasm)+Intensity(membrane)). The internalization rate constant k was calculated from the time course of internalization by curve fitting of the data using the following formula: F(t)=(1-e-k·t)·Fmax,cyt, where Fmax,cyt is the maximum ratio of cytoplasmic intensity per cell to total intensity per cell. Curve fitting of the data was performed using Graphpad Prism (GraphPad Software, La Jolla, CA). The half-life of internalization (T1 / 2) was calculated as the ratio of ln(2) to k·t.

[0354] 4.2 In vitro internalization of RAA22 / B09 and QD6 / B09 The internalization kinetics of AlexaFluor647 (AF647) primary labeled DuetMab: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 cells and then the translocation of the antibodies from the cell surface to the cytoplasm was monitored using live cell confocal fluorescence microscopy. Figure 6 shows that both antibodies were primarily localized to the cell surface before being subjected to internalization conditions (T=0) and translocated to the cytoplasmic region (blue) after 1 h (T=1h). Throughout the internalization process, kinetic images were taken every 5 min and processed by a quantification algorithm (see above) to determine the rate constants and half-lives of internalization. Figure 6b shows that QD6 / B09 and RAA22 / B09 have very similar internalization kinetics with half-lives of 37.5±10.6 min and 43.2±15.5 min, respectively.

[0355] To evaluate the mode of antibody internalization and examine the contribution of each arm to the overall internalization of DuetMab, we evaluated the internalization of single arm specific control antibody molecules QD6 / IgG and B09 / IgG for duetQD6 / B09, and RAA22 / IgG and B09 / IgG for duetRAA22 / B09 in H1975 cells expressing both EGFR and c-MET. Since only one arm is specific for the target receptor, the control antibody can only be internalized via one receptor, ruling out dual receptor targeting and cross-linking as a mode of internalization.

[0356] The internalization profiles of QD6 / B09 (Figure 7A) and RAA22 / B09 (Figure 7B) showed very similar patterns of a concordant decrease in membrane mAb-Fl647 signal and a respective increase in cytoplasmic mAb-AF647 signal, which is a typical profile of internalization. However, their single-arm constructs showed significantly different internalization profiles. The single-arm QD6 / IgG had almost the same internalization time course as the QD6 / B09 DuetMab (Figure 7A, left and middle), indicating that the internalization of QD6 / B09 duet was mainly driven by the EGFR arm of the molecule, with a very small contribution from the B09 arm. Indeed, the B09 / IgG construct showed a very low level of internalization (Figure 7B, right). The rapid and widespread decrease in membrane signal corresponded to a very gradual increase in cytoplasmic signal, which was probably due to the widespread dissociation of pre-bound B09 / IgG from the cell surface c-MET receptor. Dissociation of the antibody subsequently resulted in little internalization of B09 / IgG. These results revealed that internalization of QD6 / B09 duet was mainly driven by the EGFR arm of the molecule, with only a very small contribution from the B09 arm.

[0357] In contrast, the RAA22 / B09 DuetMab showed a very different internalization profile when compared to its single-arm control antibody. As seen in Figure 7B, the cytoplasmic intensity values ​​of the RAA22 / B09DuetMab were 10.98- and 4.70-fold higher than RAA22-IgG and B09-IgG, respectively. While the inefficient internalization of B09 / IgG could be attributed to its significant dissociation (above), RAA22 / IgG underwent rapid internalization. However, due to the lower affinity of the EGFR arm, the number of RAA22 / IgG molecules was 10.98-fold lower (based on fluorescence intensity) than for the RAA22-B09 DuetMab. In contrast to the single-arm construct, the significant increase in the amount of duet RAA22 / B09 mAb that entered the cytoplasm demonstrated that both antibody arms must engage the target receptor to drive internalization. This finding indicates that the QD6 / B09 and RAA22 / B09 DuetMabs have different internalization mechanisms, with QD6 / B09 being driven primarily by the EGFR arm, whereas RAA22 / B09 requires both the EGFR and c-MET arms for engagement.

[0358] 4.3 Internalization of RAA22 / B09 in cell lines with different levels of target receptor Since binding of both EGFR and c-MET arms to the target receptor promoted RAA22 / B09 receptor internalization in H1975 cells, we investigated whether increasing the number of EGFR and c-MET receptors affected the internalization properties. The respective receptor levels measured by Western blotting were about 33,000 for EGFR and about 50,000 for c-MET in H1975 cells, and about 790,000 for EGFR and about 523,000 for c-MET in HCC827 cells. The internalization profile of RAA22 / B09 in H1975 (medium receptor) and HCC827 (high receptor) cells shows a significant increase in internalization in HCC827 cells (Figure 8).

[0359] As expected corresponding to a 23.9-fold and 10.4-fold increase in overall levels of EGFR and c-MET RAA22 / B09, respectively, binding to HCC827 cells was on average 8.9-fold higher than H1975 (T = 0, 3.1 × 10 7 MFI vs. 3.5×10 6 MFI). The internalization level (judged by the peak of cytoplasmic intensity) was 21.7-fold higher in HCC827 cells, suggesting that significantly higher concentrations of antibody enter the cytoplasm in cells expressing high levels of the target receptor. Importantly, in addition to the large difference in intensity, the internalization profiles (membrane and cytoplasmic signals over time) also differed significantly between HCC827 and H1975 cells. In high-expressing HCC827 cells, the decrease in RAA22 / B09-AF647 membrane signal corresponded to a reciprocal increase in RAA22-B09 cytoplasmic signal, while the total signal of RAA22 / B09 was maintained over time, indicating strong dual-arm antibody interaction with both receptors and subsequent internalization. In H1975 cells, the total and membrane intensities decreased simultaneously, indicating that a portion of the pre-bound antibody may have dissociated from the cell surface and not been internalized inside the cell. In HCC827 cells, similar dissociation profiles were observed for RAA22 / IgG and B09 / IgG internalization, with single-arm engagement not resulting in effective binding and tending to dissociate (Figures 9A and B). This data suggested that a mixed mode of receptor interaction (single-arm and double-arm engagement) exists when RAA22 / B09 is subjected to internalization in H1975 cells. Collectively, these data suggest that the target cell receptor expression level is an important determinant of the extent and efficiency of RAA22 / B09 internalization.

[0360] Example 5 - Conjugation with Tubulysin AZ1508 5.1 Site-specific conjugation Conjugation of the tubulysin drug to the RAA22 / B09 antibody molecule was performed essentially as previously described in Thompson, 2016 and US Patent Publication No. 2015 / 0291657A1.

[0361] 5.2 Development feasibility assessment The feasibility of both an unconjugated DuetMAb intermediate (cMet-EGFR DuetMAb, RAA22-B09) and an antibody-drug conjugate (ADC) consisting of DuetMAb conjugated to the bimolecular tubulysin AZ1508 was evaluated. The feasibility evaluation of the tubulysin EGFR-cMET ADC included evaluation of sequence defects, stability, biochemical / biophysical properties and manufacturability.

[0362] Sequence Defects The amino acid sequence of RAA22-B09 was evaluated for the presence of known defects, including deamidation sites, oxidation sites, and glycosylation sites introduced into the CDR regions. The analysis did not reveal any sequence defects deemed high risk.

[0363] stability Stability studies were performed for the mAb intermediate RAA22-B09 at a concentration of 50 mg / ml in 20 mM histidine, 240 mM sucrose, pH 6.0, and for the conjugated ADC at 5 mg / ml in 20 mM histidine, 7% sucrose, pH 6.0. Materials were filled into HDPE bottles.

[0364] The materials were tested after three freeze-thaw cycles (-80°C to room temperature) and after incubation at 5°C, 25°C and 40°C.

[0365] Samples were analysed after one month, and materials stored at 5°C and 25°C were also analysed after two months.

[0366] Visible particles were observed in the mAb intermediates; however, these particles are considered a low risk for development and can be easily removed by filtration prior to conjugation; the ADCs remain virtually free of visible particles.

[0367] No changes were observed during the course of the ADC stability studies at 5° C., 25° C. and 40° C. No increase in free drug content was observed.

[0368] Minimal changes in monomer purity were observed after storage at 5°C and 25°C for both the mAb intermediate and the ADC.

[0369] The ADCs retained their cytotoxic activity after 4 weeks of incubation at the indicated temperatures. We conclude that the ADCs exhibit good thermal stability.

[0370] Biochemical / biophysical properties The thermal melting temperatures of the mAb intermediate and ADC were determined by differential scanning calorimetry (DSC). It was found that conjugation did not result in a significant decrease in melting temperature. After conjugation, a homogenous population was observed.

[0371] In conclusion, the cMET-EGFR mAb intermediate (RAA22-B09-Maia) and the corresponding ADC with AZ1508 payload were considered low risk for potential developability.

[0372] 5.3 Ex vivo stability analysis of RAA22 / B09-AZ1508 in serum of rats, mice and cynomolgus monkeys RAA22 / B09-AZ1508 was diluted to a final concentration of 200 μg / mL in 0.2 μM filtered rat, mouse and cynomolgus monkey serum and incubated at 37C for 0, 1, 3 and 7 days. NHS-activated sepharose beads (GE Healthcare) were washed three times with 1× phosphate-buffered saline (PBS; pH 7.4) and centrifuged at 1000 g for 1 min to remove the isopropanol stock solution. One-tenth the volume of sodium bicarbonate (1 M [v / v]) was added to the recombinant EGFR-ECD protein (2.12 mg / mL Lot No.: C20202OCT14C) and 6 mL of this mixture was incubated with 3 mL of washed NHS-activated sepharose beads. The bead mixture was stirred overnight at room temperature. The beads were washed three times with 1× PBS (pH 7.4) to remove unbound EGFR. The immobilization efficiency was determined to be 93% by comparing the amount of binding to the starting amount of EGFR using an Agilent 1200 series HPLC and a Tosoh Bioscience TSKgel G3000 size exclusion chromatography column according to the manufacturer's recommendations. RAA22 / B09-AZ1508 was recovered from serum by incubating 100 μL of the RAA22 / B09-AZ1508-serum incubation mixture with 50 μL of EGFR beads at room temperature for 30 min, followed by three successive washes with 1×PBS buffer and elution with 100 μL of antibody elution buffer (IgG elution buffer; Thermoscientific). The eluate was analyzed by rLCMS to calculate the amount of remaining intact ADC. Reduced reversed-phase mass spectrometry (rLCMS) was used on an Agilent 1290 series HPLC coupled with an Agilent 6520 Accurate-Mass TOF LC / MS with an electrospray ionization source, and the remaining ADC was calculated using peak height intensities obtained using Agilent MassHunter data collection and chromatogram processing software.Approximately 2 μg (35 μL) of the reduced eluent was loaded onto a Poroshell 300SB-C3 column (2.1×75 mm, 255 Agilent) and eluted at a flow rate of 0.4 mL / min using a step gradient 256 of 60% B after 6 minutes (solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile). The peak height intensities of the intact conjugated and unconjugated or modified ADC species were used to calculate the percent ADC remaining by dividing the intact conjugated peak height intensity by the total ADC species peak height intensity. The results are shown in Table 2 below.

[0373] [Table 9]

[0374] These data demonstrate ex vivo deconjugation of the entire payload from the ADC and metabolism of the ADC by liberation of O-acetyl groups from the tubulysin warhead in mouse, rat, and cynomolgus monkey serum. The results show that there is a loss of less than 10% of the conjugated payload after 1 week at 37° C. when incubated with serum from all three species. The initial rates of de-O-acetylation of the tubulysin warhead from the ADC appear to differ slightly between the three species, but the amount of de-O-acetylation at day 7 is comparable in all three species at approximately 40%.

[0375] Example 6 - In vitro ADC cytotoxicity In this example, the in vitro efficacy of EGFR / cMET bispecific ADCs was measured in a panel of cancer cell lines.

[0376] 6.1 Quantitative FACS characterization of cell line panels FACS was performed to quantitate the relative densities of EGFR and c-Met in the cell lines tested.

[0377] Antibody labeling PanIX, anti-EGFR-cMet and R347 purified antibodies were labeled using Alexa Fluor647 monoclonal antibody labeling kit according to the manufacturer's instructions (Invitrogen catalogue no. A20186). Antibody concentrations and fluorescent dye-to-protein (F:P) ratios were calculated using a NanoDrop ND-1000 spectrophotometer.

[0378] Flow cytometry Cells were harvested and washed to a single cell suspension. Cell count and viability (≧90%) were determined by a Vi-Cell XLR analyzer (Beckman Coulter). Cell concentration was adjusted to 5×106 cells / ml in ice-cold FACS buffer (phosphate buffered saline (pH 7.4), 5% FBS, 0.1% sodium azide) and plated at 250,000 cells / well in 96-well u-bottom polystyrene plates. To obtain binding curves, Alexa 647-conjugated antibodies were serially diluted to various concentrations (0.15-40 μg / ml) in cold FACS buffer. Cells and primary antibodies were incubated on wet ice for 20 min in the dark. Cells were washed twice with cold FACS buffer and fixed in 200 μL of ice-cold 2% paraformaldehyde (PFA). For data acquisition, the excitation and emission of Alexa 647 was at 650 / 668 nm with a Red Laser 640 nm. 20,000 events were collected from each sample by a Becton-Dickenson LSR II machine equipped with FACSDiva™ software. Results were analyzed using FlowJo software.

[0379] Analysis of EGFR, EGFR-cMet; Quantum MESF (soluble fluorescent dye equivalents) The amount of EGFR, EGFR-cMet on cells was assessed using Quantum Alexa Fluor 647 MESF beads (catalog no. 647C, Bangs Laboratories) and 10,000 events per sample were acquired (LSRII) using the same flow cytometry settings as the samples listed above. A standard curve was established using the QuickCal program (www.bangslabs.com) relating channel values ​​to fluorescence intensity in MESF. The mean fluorescence intensity (MFI) is directly proportional to the amount of fluorochrome present to calculate the number of molecules per cell. The calculated MESF from the Quickcal program was divided by the antibody F:P ratio to obtain the corrected ABC (antibody binding capacity).

[0380] 6.2 Methods for determining ADC cytotoxicity ADC cytotoxic activity was tested in multiple cell lines as follows: Cells were plated in a volume of 100 μL at a density of 10,000 cells per well of a 96-well plate in the recommended culture medium supplemented with 10% fetal bovine serum. Three-fold concentrations of antibody at each dose to be tested were prepared by serially diluting the antibody stock in culture medium. Fifty microliters of each test substance was added to the cells in triplicate such that the final antibody concentrations ranged from 60 nM to 0.0009 nM. Treated cells were cultured for 72 hours at 37°C in a humidified incubator. Metabolic activity was determined using the CellTiter-Glo Luminescent Viability Assay from Promega, following the manufacturer's instructions. Data were plotted as percent metabolic activity relative to untreated controls. IC 50 Values ​​were determined using logistic nonlinear regression analysis between maximum viability (untreated cells) and maximum response (peak of inhibition) using GraphPad Prism software.

[0381] 6.3 Results – In vitro ADC activity in a panel of cell lines The in vitro potency of the EGFR / cMET bispecific ADCs was measured in a panel of cancer cell lines using the CellTiter-Glo luminescent viability assay. As shown in Table 3, both the high affinity QD6 / B09-AZ1508 and the low affinity RAA22 / B09-AZ1508 demonstrated broad activity across cell lines with various target expression levels.

[0382] [Table 10]

[0383] Overall, the low affinity ADCs for EGFR showed comparable (albeit somewhat reduced) potency in a broad range of cell lines that co-express both EGFR and cMET in significant amounts. In general, both ADCs showed reduced potency when one or the other target had a low relative receptor density at the cell surface below about 15,000. This effect was more pronounced with the low affinity variants, which appeared to be more sensitive to low levels of cMET.

[0384] Example 7 - ADC cytotoxicity in in vitro proof-of-concept (POC) experiments To further test the hypothesis that bispecific engagement of low affinity EGFR-cMET antibodies is required for optimal ADC delivery, we performed in vitro experiments to determine the relative contribution of each antibody arm to the activity of the bispecific ADC. In the first experiment, we used an excess of unarmed parental antibodies to block EGFR or cMET and then measured the activity of the bispecific EGFR-cMET ADC in an in vitro cytotoxicity assay. For this experiment, we used a cell line that expresses moderate levels of EGFR and cMET in approximately equal amounts. If the individual arms of the bispecific ADC function independently in the delivery of the ADC, blocking either target in this cell line would be expected to only slightly decrease the activity of the ADC, and since the targets are present at comparable levels, the IC 50The shift in activity is less than two-fold. On the other hand, if the ADC requires simultaneous binding to effectively deliver the ADC into cells, blocking either target is likely to have a greater impact on the activity of the bispecific ADC. In a related experiment, we compared the activity of the bispecific EGFR-cMET ADC with a monovalent monospecific control antibody that contains one arm that binds to EGFR or cMET and one non-binding isotype antibody control arm. Similarly, if each arm functions independently, the expected result is that each monospecific control ADC is only slightly less potent than the bispecific ADC, and the difference is additive. Alternatively, if the two arms of the bispecific function synergistically, a larger difference in the activity of the bispecific ADC is expected compared to the monospecific control antibody.

[0385] 7.1 Method The cytotoxic activity of the ADCs was tested in the NCI-H1975 cell line as follows: Cells were plated at a density of 10,000 cells per well of a 96-well plate in the recommended culture medium supplemented with 10% fetal bovine serum in a volume of 50 μL for blocking experiments and 100 μL for monovalent ADC experiments. For disarmed mAb blocking experiments, 50 μL of a 300 μg / mL solution of EGFR IgG (RAA22) or cMET IgG (B09) was added to the wells and pre-incubated for 1 h at 37° C. in a humidified incubator. Three-fold concentrations of each dose of antibody to be tested were prepared by serial 4× dilutions of the antibody stock in culture medium. Fifty microliters of media alone, isotype control IgG ADC (R347-AZ1508), or EGFR-cMET ADC (RAA22 / B09-AZ1508) were added to cells in triplicate for final antibody concentrations ranging from 67 nM to 0.0009 nM. For monovalent ADC experiments, 50 μL of a 3× stock of either the isotype control ADC (R347-AZ1508), monovalent EGFR ADC (RAA22 / R347-AZ1508), monovalent anti-cMET ADC (B09 / R347-AZ1508), an equimolar combination of monovalent ADCs, or EGFR-cMET ADC (RAA22 / B09-AZ1508) was added in triplicate in a 4-fold dilution series starting at 60 nM and ending at 0.009 nM. Treated cells were cultured for 72 hours at 37°C in a humidified incubator. Metabolic activity was determined using the CellTiter-Glo luminescent viability assay from Promega according to the manufacturer's instructions. Data were plotted as percent metabolic activity relative to untreated controls. IC 50 Values ​​were determined using logistic nonlinear regression analysis between maximum viability (untreated cells) and maximum response (peak of inhibition) using GraphPad Prism software.

[0386] 7.2 Results and Conclusions - In Vitro Proof of Concept for Dual Targeting (mAb Blocking Experiments and Monovalent ADC) We performed in vitro experiments, as outlined above, to examine the relative contribution of the individual antibody arms to the cytotoxic activity of the bispecific ADC. As shown in a representative experiment in Figure 10, pretreatment of NCI H1975 cells with cMET IgG RAA22 significantly increased the IC 50 Treatment with anti-cMET IgG B09 significantly reduced the IC 50 to 680 pM, resulting in a difference of more than 11-fold. Similarly, when NCI H1975 cells were treated with a monovalent, single-specific EGFR ADC (RAA22 / R347-AZ1508), the IC 50 The IC of the monovalent, monospecific anti-cMET ADC (B09 / R347-AZ1508) was approximately 20,500 pM compared to 316 pM for the bispecific EGFR-cMET ADC, a difference of approximately 65-fold (Figure 11). 50 The activity of the antibody was 2,772 pM, which was approximately 13-fold higher than that of the bispecific antibody.

[0387] Taken together, these data suggest that efficient targeting of EGFR-cMET ADCs to tumor cells that co-express both targets is driven primarily by the bispecific binding of the ADC. Furthermore, these results indicate that the EGFR low affinity binding arm is insufficient to promote efficient ADC delivery in the absence of cMET binding, as shown by the dramatic drop in potency when the cMET arm is blocked by an unarmed antibody, and the weak cytotoxicity of the monovalent EGFR control ADC, RAA22 / R347-AZ1508. Taken together, these results are consistent with the hypothesis that the low affinity of the EGFR binding arm of the bispecific ADC (RAA22 / B09-AZ1508) promotes ADC delivery to tumors that co-express EGFR and cMET, while exhibiting weak cytotoxicity against cells that primarily express only one of the targets. This effect was most pronounced when only EGFR was available for engagement, which has implications for mitigating EGFR-driven toxicity in normal organs such as skin, where EGFR is expressed at significant levels but cMET expression is relatively low.

[0388] Example 8 - ADC in vivo pharmacology in patient-derived xenograft (PDX) models Patient-derived xenograft (PDX) models of human cancer have become a well-established alternative to tumor xenografts based on tumor cell lines. PDX models are established from primary tumor tissues of patients that are directly implanted in immunodeficient mice, resulting in tumors that grow in vivo in mice. The tumors thus obtained are then propagated in additional mice without in vitro culture to establish a bank of low-passage PDX tumor tissues that can be used to implant in research mice. One important feature of PDX models is that they largely maintain the histological and genomic heterogeneity and preserve the gene expression profile of the corresponding original patient tumor. Compared to tumor cell line-based xenograft models, which use clonal populations of tumor cells adapted to grow in vitro, the properties of PDX models aim to more accurately recapitulate the characteristics of real human tumors, which improves the predictive value of preclinical mouse models. Indeed, many studies have shown that the response and resistance profile of PDX models to standard therapeutic treatments closely correlates with clinical data in human subjects with a given tumor profile.

[0389] Despite the improvements offered by PDX models, standard in vivo pharmacology study designs have limitations, even when applied to PDX models. For each tumor model, a typical study tests drug treatments at multiple dose levels, along with one or more positive or negative control compounds, with enough mice per treatment group to support within-model statistics. Such study designs use a relatively large number of mice, and the high cost of PDX models can limit the number of tumor models that can actually be tested for a given compound. An alternative / complementary approach to traditional study designs is mouse PDX studies, which are population-based approaches that mimic human clinical trial designs. In this approach, for each compound, typically one mouse is treated at a single dose level established in a previous dose-ranging study, and each model includes an optional treatment control group. Because fewer mice are required for each model, many PDX models can be tested, each representing a unique human tumor. Responses are assessed across the entire population of tumor models tested, without relying on within-model statistics. This approach allows for more accurate estimation of response rates across a range of target expression, molecular phenotypes, tumor subtypes, or other clinically relevant features of interest. Furthermore, the large number of unique models that can be tested in PDX studies allows for more meaningful exploratory genomics, transcriptomics or expression profiling studies, and allows for early exploration of correlations of response or resistance to begin. For the exemplary studies outlined herein, we have taken an "all comers" approach to test all available NSCLC models in START Discovery, regardless of target expression level or molecular phenotype.

[0390] 8.1 Method Mouse PDX studies were performed at South Texas Accelerated Research Therapeutics (START, San Antonio, TX). START is accredited by AAALAC International (Association for Assessment and Accreditation of Laboratory Animal Care International) and complies with the AstraZeneca Global Standard on Animal Care and Welfare. All models were developed at START. Patient-derived xenograft (START-PDX) models are established from viable human tumor tissue or tumor tissue and serially passaged in animals a limited number of times to maintain tumor heterogeneity. Athymic nude (Crl:NU(NCr)-Foxn1nu) / CB-17 Scid (CB17 / Icr-Prkdcscid / IcrIcoCrl) mice were implanted in the flank with tumor fragments taken from host animals from each specific passage lot. Pre-study tumor volumes were recorded approximately 1 week prior to the planned study initiation date. Tumors were selected to be in the appropriate tumor volume initiation (TVI) range (125–250 mm). 3 When tumor size reached 100 mm, animals were randomized into treatment and control groups, intravenous (IV) dosing was initiated (day 0), and animals were followed individually throughout the study. The first dose was administered intravenously to animals in all groups according to body weight (0.01 ml per gram; 10 ml / kg) beginning on day 0. Drug-treated animals were dosed every 7 days for a total of 4 doses. Beginning on day 0, tumor dimensions were measured by digital calipers and estimated tumor volumes were recorded for each treated and control animal, and tumor volume was calculated using the formula: TV=width 2 × length × 0.52. Observation of tumor growth continued for one week after the final administration. Each animal was assessed for tumor volume (TV) endpoint (tumor volume ≥ 1 cm 3Each animal was sacrificed when it reached the end of the study period of 28 days or 28 days, whichever came first. For some PDX models with slow-growing tumors, the observation period was extended. The tumor growth inhibition rate (TGI%) was defined as the percentage of tumor growth relative to day 0 between the treatment (TX) group and the control (C) group according to the following formula: TGI%=1-(TX 最終 -TX 初期 ) / (C 最終 -C 初期 Tumor regression rate was defined as the percentage of tumor reduction in treated animals relative to the tumor volume on day 0 (day of first administration), calculated at the study endpoint according to the following formula: % regression = (TX 最終平均値 -TX 初期平均値 ) / (TX 初期平均値 ) x 100.

[0391] 8.2 Results and Conclusions As shown in FIG. 12, both the high affinity EGFR-cMET ADC QD6 / B09-AZ1508 and its lower affinity variant for EGFR RAA22 / B09-AZ1508 induced tumor growth inhibition or regression in multiple PDX models tested. Surprisingly, the low affinity ADCs showed an overall trend towards increased number and depth of observed responses compared to the high affinity ADCs. This activity trend was slightly reversed in the PDX models least responsive to the low affinity ADCs, which correlated somewhat with reduced cMET expression. These observations suggest that the activity of the low affinity EGFR-cMET ADCs may be partially dependent on cMET expression levels. The EGFR binding arms of both bispecific antibodies were derived from the same murine EGFR cross-reactive antibody (see Example 1). The intrinsic binding affinity of the QD6 / B09 antibody for murine EGFR was about 6 nM, whereas the affinity of the RAA22 / B09 bispecific antibody was about 575 nM. The unexpected improvement in activity of the low EGFR affinity is likely due to a reduced EGFR sink effect in normal tissues such as the skin, increasing the overall circulating exposure of the ADC. Nonetheless, these data demonstrate that lowering the affinity of the EGFR-cMET bispecific antibody for EGFR does not compromise the in vivo efficacy of the resulting ADC, but rather unexpectedly improves activity compared to the high affinity ADC.

[0392] 8.3 Different dose PDX studies Further experiments were performed to test different doses of ADC in PDX models. 3 The study was performed as described in Example 8.1, except that individual mice that reached a tumor volume of 100 mg / kg were removed from the study and final measurements were included in the group averages until either the mean volume reached the volumetric endpoint or the study reached the duration endpoint of 63 days. The high affinity EGFR-cMET ADC (QD6 / B09-AZ1508) was tested at dose levels of 1 mg / kg and 2 mg / kg, and a variant with reduced affinity for EGFR (RAA22 / B09-AZ1508) was tested at 1 mg / kg, 2 mg / kg, and 3 mg / kg.

[0393] As shown in Figure 13, both the high affinity EGFR-cMET ADC (QD6 / B09-AZ1508) and the variant with reduced affinity for EGFR (RAA22 / B09-AZ1508) induced tumor growth inhibitory activity in PDX models at the doses tested. The results described in Example 8.2 and shown in Figure 12 indicate that the reduced affinity ADCs were generally more effective than the high affinity ADCs. In all four models tested, the low affinity ADCs induced regressions at dose levels of 2 or 3 mg / kg, indicating that the low affinity ADCs are effective at moderate doses. These data provide further evidence that reducing the affinity of an EGFR-cMET bispecific antibody for EGFR does not reduce the in vivo efficacy of the resulting ADCs, but rather improves their in vivo efficacy compared to high affinity ADCs.

[0394] Example 9 - Efficacy of ADC in orthotopic pancreatic PDX models Subcutaneous in vivo tumor models are the mainstay for testing the efficacy of anticancer drugs. However, this tumor implantation site comes with many limitations that need to be considered when interpreting in vivo results. These defects include tumor vascularization and the lack of tissue-specific stroma in tumor growth and response. To address these challenges, we compared the in vivo efficacy of high- and low-affinity EGFR-cMET bispecific antibody-drug conjugates in both subcutaneous and orthotopic models of the pancreatic PDX model MEDI-PANC-08. To track the growth of this tumor orthotopically, we used a luciferase-expressing PDX variant (MEDI-PANC-08) whose growth can be tracked using imaging. LUC ) was developed.

[0395] 9.1 Method All experiments were performed in AAALAC (Association for Assessment and Accreditation of Laboratory Animal) accredited facilities and in accordance with MedImmune IACUC (Institutional Animal care and Use Committee) guidelines for the humane treatment and care of laboratory animals. Animals were monitored daily for morbidity and mortality.

[0396] Subcutaneous PDX model The MEDI-PANC-08 pancreatic PDX model used in this study was derived from the Internal MedImmune PDX library. PDX tumors were initially seeded with NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl Tumors were grown in 100% sham / SzJ mice to generate sufficient tumor material to seed efficacy studies. Tumors were 800-1200 mm 3 Mice were humanely euthanized by CO2 asphyxiation when tumors reached approximately 2 mm 3 Tumors were cut into pieces and implanted subcutaneously into the right flank of individual NSG mice using an 11-gauge trocar needle. Once the size reached approximately 150-250 mm3, mice were randomized (based on tumor volume) into treatment groups and treated with ADCs (Q1Wx4). Two EGFR-cMET bispecific ADCs, QD6 / B09 (high affinity) and RAA2 / B09 (low affinity), were tested at 1, 2 and 3 mg / kg. An isotype control ADC (R347-AZ1508) was also tested at 3 mg / kg. Immediately prior to use, all antibody-drug conjugates were diluted in buffer (25 mM histidine, 7% sucrose, 0.02% PS80, pH 6.0) and administered iv into the tail vein. Tumor and body weight measurements were collected twice weekly and tumor volume was calculated using the equation (LxW2) / 2, where L and W represent the length and width dimensions, respectively.

[0397] Orthotopic PDX models Luciferase-expressing PDX model (MEDI-PANC-08 LUC) were grown subcutaneously in NSG seed mice and grown to 800–1200 mm 3 Tumors were harvested with a volume of approximately 2 mm 3 The tumors were cut into pieces of 0.01 mg / ml each. The tumor fragments were then sutured to the pancreas of NSG mice (day 0). Luciferase signals were determined weekly using an IVIS Spectrum in vivo imaging system. Briefly, 10 min before imaging, 200 μl of luciferin (15 mg / ml) dissolved in DPBS was injected intraperitoneally (ip). Mice were anesthetized with 3% isoflurane, laid on their right side, and luminescence was measured. 14 days after tumor implantation, when the luminescence signal was clearly detectable, mice were randomized into respective groups based on luminescence. Mice were treated with isotype control (R347-AZ1508, 3 mg / kg-Q1W×4), gemcitabine (75 mg / kg, Q2D×5), and RAA2 / B09 ADC (2 and 3 mg / kg-Q1W×4). Luminescence was measured weekly. Study endpoints included weight loss, deterioration in condition, and lethargy. Data were analyzed using Living Image software (Perkin Elmer) and plotted as mean radiance [p / s / cm2 / sr] against time.

[0398] 9.2 Results and Discussion To aid in the selection of suitable EGFR affinity combinations for EGFR-cMET bispecific ADCs, high and low affinity EGFR-cMET bispecific ADCs were compared in an in vivo efficacy study using the MEDI-PANC-08 pancreatic PDX model. As shown in Figure 14, panels A and B, significant differences were observed between the two molecules. The high affinity QD6 / B09 ADC showed no efficacy at any of the three dose levels tested. Conversely, the low affinity RAA2 / B09 ADC caused complete tumor regression by day 65, followed by tumor regrowth at the 3 mg / kg dose level and tumor growth inhibition at 2 mg / kg.

[0399] While subcutaneous tumor models have become the mainstay of in vivo efficacy testing, a major drawback is that tumors do not grow at the site of origin, and therefore drug responses may not truly reflect patient responses. To address this concern, an orthotopic model of pancreatic cancer was developed using MEDI-PANC-08 tumors genetically engineered to stably express luciferase. After surgical implantation into the pancreas, tumors were allowed to establish and were subsequently randomized based on luminescence signal. Mice were then treated with low affinity RAA2 / B09EGFR-cMET ADC, isotype control, or gemcitabine (a chemotherapy drug). Luminescence was measured weekly after treatment. As shown in panel C of Figure 14, luminescence in the untreated and isotype control groups increased over time, and animals were removed from the study due to poor physical condition and large palpable abdominal tumors. Gemcitabine showed an initial decrease in luminescence signal, reaching a nadir around day 21, after which the signal increased over time, leading to removal of the groups from the study at day 49. In the orthotopic model, both the 2 mg / kg and 3 mg / kg dose levels of the RAA2 / B09 ADC reduced the luminescence signal to near background levels by day 60. Compared to the subcutaneous study, the 2 mg / kg dose level showed better activity, resulting in tumor regression. Necropsy of animals that showed tumors with near background luminescence signals at the end of the study no longer showed visible tumors, thus supporting the correlation between luminescence signal and tumor volume.

[0400] In conclusion, the low affinity EGFR-cMET RAA2 / B09 ADC showed improved efficacy compared to the high affinity QD6 / B09 ADC in a subcutaneous pancreatic PDX model, with tumor regression observed at 3 mg / kg. This efficacy was also observed in an orthotopic model using the same PDX tumors (MEDI-PANC-08) engineered to stably express luciferase. Using luminescence as a surrogate for tumor volume, the RAA2 / B09 ADC showed improved efficacy over the subcutaneous model, with tumor regression observed at both 2 mg / kg and 3 mg / kg.

[0401] Example 10 - Safety and Pharmacokinetics Pharmacokinetic (PK) analysis was performed to compare plasma PK parameters of low and high affinity EGFR-cMET ADCs, including peak and total exposure, clearance, and half-life in mice and cynomolgus monkeys. The primary objective was to determine whether the reduced affinity for EGFR affects circulating exposure of EGFR-cMET bispecific ADCs. PK samples were collected from mice and cynomolgus monkeys at various dose levels for both QD6 / B09-57-AZ1508 and RAA / B09-57-AZ1508. Non-compartmental analysis was performed to estimate PK parameters of QD6 / B09-57-AZ1508 and RAA22 / B09-57-AZ1508 based on total ADC concentrations across species and dose levels. Overall, RAA22 / B09-57-AZ1508 showed higher exposure and longer t1 / 2 compared to QD6 / B09-57-AZ1508 in both mice and cynomolgus monkeys, suggesting improved PK in the lower affinity RAA22 / B09-57-AZ1508.

[0402] 10.1 Bioanalysis of Preclinical PK Assays Target compound (QD6 / B09-57-AZ1508 and RAA22 / B09-57-AZ1508) concentrations and total antibody concentrations were measured in a single immunocapture LC-MS / MS assay. Briefly, polyclonal anti-human antibodies were conjugated to magnetic beads. 25 μL of plasma samples were then diluted with PBS and incubated with the magnetic beads. After capture, the magnetic beads were washed multiple times and then digested with trypsin in the presence of an internal standard. Acid was added to quench the digestion. The liquid contents were then transferred to an injection plate.

[0403] Signature tryptic peptides on human antibody Fc regions and cleaved warheads were separated using reversed-phase chromatography (RPLC) and then detected using multiple reaction monitoring (MRM). Signature peptides on Fc regions were used to calculate total Ab, while digestion-released warheads were used to calculate ADC. The internal standards used in this experiment are isotope-labeled peptides or proteins (SiluMAb, Sigma-Aldrich) or isotope-labeled warheads. Peak area ratios of analytes to internal standards were used to calculate against a standard curve.

[0404] Standard curves and QCs are prepared by spiking different levels of the target compound into the same matrix as the samples. The quantification range includes 100ng / mL to 15,000ng / mL, with diluted QCs up to 525,000ng / mL. The standard curves were fitted using the simplest possible model. The precision and accuracy of the assay are within 20% at all levels, except for the lower limit of quantification (LLOQ), which is 25%.

[0405] 10.2 QD6 / B09-57-AZ1508PK in mice The mouse studies included in the NCA analysis are summarized in Table 4.

[0406] [Table 11]

[0407] The mean PK concentration-time profiles in mice of RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 are shown in FIG.

[0408] RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 demonstrated linear PK in mice at the dose levels tested, with dose-proportional exposure (C max and AUC), equivalent CL and t 1 / 2were observed at 0.5 mg / kg to 10 mg / kg for RAA22 / B09-57-AZ1508 and 1 mg / kg to 10 mg / kg for QD6 / B09-57-AZ1508, respectively. PK comparisons between RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 were evaluated at the 1, 3, 5, and 10 mg / kg dose levels tested for both compounds, and mean PK parameters based on NCA are summarized in Table 5. Results showed slow CL, high exposure, and long t 1 / 2 The mean AUC of RAA22 / B09-57-AZ1508 showed a 2- to 2.91-fold increase compared to QD6 / B09-57-AZ1508, and the mean t 1 / 2 The t of RAA22 / B09-57-AZ1508 was in the range of 4.24 to 6.38 days, and that of QD6 / B09-57-AZ1508 was in the range of 2.57 to 3.33 days. 1 / 2 showed a 1.27-2.32-fold increase.

[0409] [Table 12]

[0410] 10.3 PK of QD6 / B09-57-AZ1508 in Cynomolgus Monkeys The cynomolgus monkey studies included in the NCA analysis are summarized in Table 6.

[0411] [Table 13]

[0412] The mean PK concentration-time profiles of RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 in cynomolgus monkeys are shown in FIG.

[0413] RAA22 / B09-57-AZ1508 showed linear PK at 2 mg / kg to 5 mg / kg in cynomolgus monkeys, with dose-proportional exposure (C maxand AUC), equivalent CL and t 1 / 2 was observed.

[0414] QD6 / B09-57-AZ1508 showed nonlinear PK at 0.67 mg / kg to 3 mg / kg in cynomolgus monkeys, with more than dose-proportional exposure (C max and AUC) were shown, with faster CL and shorter t at lower dose levels. 1 / 2 was observed.

[0415] PK comparisons between RAA22 / B09-57-AZ1508 and QD6 / B09-57-AZ1508 in cynomolgus monkeys were evaluated at the 2 mg / kg and 3 mg / kg dose levels tested for both compounds, and mean PK parameters based on NCA are summarized in Table 7. Results showed slow CL, high exposure, and long t 1 / 2 The mean AUC of RAA22 / B09-57-AZ1508 was increased by 1.90-2.43-fold compared to QD6 / B09-57-AZ1508, and the mean t 1 / 2 The t of RAA22 / B09-57-AZ1508 was in the range of 4.30 to 5.90 days, and that of QD6 / B09-57-AZ1508 was in the range of 0.969 to 1.07 days. 1 / 2 showed a 4.44-5.51-fold increase.

[0416] [Table 14]

[0417] Taken together, these data indicate that the low affinity RAA22 / B09-57-AZ1508 has a higher exposure and longer circulating half-life compared to the high affinity QD6 / B09-57-AZ1508 in both mice and cynomolgus monkeys. These data are consistent with the hypothesis that decreasing affinity for EGFR reduces binding to EGFR present in normal tissues, thereby mitigating the effect of normal tissue sink and improving plasma PK parameters.

[0418] Example 11 - ADCs with Topoisomerase I Inhibitors as Payload Experiments were designed to test the efficacy and safety of RAA22 / B09-57 bispecific molecules conjugated to a different payload - a topoisomerase I inhibitor, rather than tubulysin used in the previous examples.

[0419] DuetMab RAA22 / B09 (inserted "Maia" cysteine ​​after serine 239) bispecific antibody produced according to Example 1 was conjugated to the topoisomerase inhibitor SG3932 by "classical" conjugation to a natural cysteine ​​in the bispecific antibody. [ka]

[0420] The efficacy of EGFR / cMET topoisomerase I inhibitor ADC was investigated using PDX studies. PDX studies were essentially performed as described above in Example 8 using a variety of different PDX models derived from pancreatic, colon, NSCLC and head and neck squamous cell carcinoma (SQHN) tumors. Animals were injected with a single dose of 10 mg / kg EGFR-cMET Maia Topo ADC. The results of the PDX studies using EGFR-cMET Maia Topo ADC are reported in Figure 17.

[0421] As shown in Figure 17, the EGFR-cMET Maia Topo ADC induced tumor growth inhibition or regression in multiple PDX models tested. Thus, these results demonstrate that the RAA22 / B09-57 ADC containing a topoisomerase I inhibitor was effective in PDX models representing multiple tumor types.

[0422] Example 12 - Mutations to improve PK of ADCs The ADC with a topoisomerase I inhibitor generated and tested in Example 11 used the RAA22 / B09 bispecific antibody containing a "Maia" cysteine ​​insertion after serine 239. However, given that SG3932 conjugates to a natural cysteine, the insertion of the Maia cysteine ​​is not necessary. Therefore, we sought to modify the RAA22 / B09 Maia Topo ADC generated in Example 11 to remove this cysteine ​​insertion.

[0423] It was also recognized that reducing or eliminating effector functions of the Fc backbone may reduce immunotoxicity and improve pharmacokinetics. Therefore, the inventors also introduced the L234F / L235E / P331S (EU numbering) "triple mutant (TM)" (Organesyan, 2008; Hay, 2016), which was previously shown to reduce Fc effector function in antibody molecules.

[0424] The newly generated "EGFR-cMET TM" molecule, containing 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:

[0425] [Table 15]

[0426] For conjugation to SG3932, a 50 mM solution of tris(2-carboxyethyl)phosphine (TCEP) (12.5 molar equivalents / antibody) dissolved in phosphate buffered saline (pH 7.4) (PBS) was added to the EGFR-cMET TM bispecific antibody solution dissolved in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) to give a final antibody concentration of approximately 3 mg / mL. The reduction mixture was incubated at 37° C. for 2 hours with gentle shaking (60 rpm) on an orbital shaker. The reaction mixture was allowed to cool to room temperature for 45 minutes. SG3932 was then added as a DMSO solution (12.5 molar equivalents / antibody) to give a final DMSO concentration of 10% (v / v). The solution was incubated at room temperature for 2 hours and then quenched by the addition of N-acetylcysteine ​​(5 micromoles / SG3932) and incubated at room temperature for 15 minutes. The reaction mixture was filtered through a 0.2 μM sterile filter and then stored overnight at 2–8 °C. Surface area: 375 cm 2 Excess free drug was removed by tangential flow filtration unit (TFF) using 1000 mL of mPES, MidiKros® 30 kDa fiber filter into a buffer solution (pH 6.8) containing 30 mM histidine, 30 mM arginine. The extent of free drug removal was monitored by UHPLC-RP using neat conjugate. After complete removal of free drug, the ADC was buffer exchanged. The ADC was filtered using a 0.22 μm filter under a sterile atmosphere, and then polysorbate-80 was added to a final concentration of 0.02% (w / v).

[0427] UHPLC analysis of the reduced ADC sample was performed on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50mm x 2.1mm column eluted with a gradient of water and acetonitrile at 214nm and 330nm (specific for SG3932), revealing a drug-antibody ratio (DAR) of 6.0 molecules of SG3932 per antibody.

[0428] The efficacy of EGFR / cMET™ ADC was investigated using PDX studies. PDX studies were performed essentially as described in Example 8 above using a variety of different PDX models derived from pancreatic, colon, NSCLC and SQHN tumors. Animals were injected with a single dose of 5 mg / kg of EGFR-cMET™ ADC. The results of the PDX study with EGFR-cMET™ ADC are reported in Figure 18. The results of this experiment show that EGFR-cMET™ ADC was able to induce tumor growth inhibition or regression in multiple PDX models tested.

[0429] The efficacy of the EGFR-cMET™ ADC ("TM ADC") was compared to the EGFR-cMET Maia Topo ADC ("Maia ADC") generated in Example 11 in PDX models SQHN-02 and PANC-08. Animals were dosed with 2.5 mg / kg, 5 mg / kg, or 10 mg / kg of each ADC and tumor growth was monitored. The experiment also included an untreated control ("Naive") and animals dosed with unconjugated EGFR-cMET TM (TM mAb). The results are shown in Figure 19.

[0430] The results show that both the EGFR-cMET™ ADC and the EGFR-cMET Maia Topo ADC are equally effective at reducing tumor growth / inducing tumor regression ("equipotent") across the dose range tested, suggesting that elimination of the S239i mutation and abrogation of Fc effector function by the triple mutant does not negatively impact efficacy in these tumor models.

[0431] The EGFR-cMET™ ADC was also shown to be effective in reducing tumor growth in NSCLC tumors expressing wild-type or mutated EGFR. Results show that the EGFR-cMET™ ADC is active in both wild-type and mutated EGFR PDX models (Figure 20). This is advantageous as it shows that the ADC can provide benefit in multiple treatment settings and across a range of different EGFR genotypes.

[0432] Finally, a pharmacokinetic (PK) study comparing the EGFR-cMET™ ADC ("TM ADC") produced in Example 11 and the EGFR-cMET Maia Topo ADC ("Maia ADC") was performed in NOD-SCID mice. The experiment was performed essentially as described in Example 10.

[0433] Representative results of these PK studies are shown in Figure 21. As reported in this figure, the EGFR-cMET™ ADC exhibited a PK response similar to that of the EGFR-cMET Maia Topo ADC (t 1 / 2 = 3.0 days; CL = 39.7 ml / day / kg) compared with a longer half-life (t 1 / 2 = 5.0 days) and reduced drug clearance (CL = 14.8 ml / day / kg). Thus, the results demonstrate that the EGFR-cMET™ ADC described herein has improved PK compared to the EGFR-cMET Maia Topo ADC produced in Example 10. Similar improvements in PK were observed when unconjugated EGFR-cMET TM antibody ("TM mAb") was compared to unconjugated EGFR-cMET Maia ("Maia ADC").

[0434] Example 13 - Degradation of EGFR and cMet receptors following treatment with EGFR-CMET TOP1i™ ADC Antibody-mediated receptor degradation was determined by Western blot analysis as follows.

[0435] HCC827 GR pooled cells were cultured in 12-well culture plates (Corning / Costar) at 6 × 10 5Cells were plated at 1000 x 1000 cells / well. Cells were allowed to attach and grow overnight at 37°C in a 5% CO2 incubator. The next morning, the growth medium was carefully aspirated off and the cells were treated with 1 mL of fresh growth medium containing the test mAbs identified in Table 9 at a final concentration of 10 micrograms / mL each. The cells were incubated for 24 hours at 37°C in a humidified growth chamber with 5% CO2. The medium was then carefully aspirated off and the cells were washed once with 2 mL of calcium- and magnesium-free Dulbecco's Phosphate Buffered Saline (DPBS). Cells were lysed by adding 150 microliters of M-per mammalian protein extraction reagent (Thermo Scientific) containing Complete Protease Inhibitor Cocktail (Roche) and PhosStop Phosphatase Inhibitor Cocktail (Roche). To facilitate more complete lysis, the plates were transferred to -80°C and subjected to one freeze-thaw cycle. The resulting lysates were transferred to microcentrifuge tubes and centrifuged at maximum speed (14,000 RPM) in a microcentrifuge at 4°C for 10 minutes to remove cellular debris, and the supernatants were transferred to clean microcentrifuge tubes and stored at -80°C prior to analysis. Protein concentrations were measured using the Pierce BCA Protein Assay Kit (Thermo Scientific).

[0436] For Western blot analysis, samples were prepared using 4× LDS (Invitrogen) and 10× sample reducing agent (Invitrogen) to reach a final protein concentration of 10 micrograms of protein (1× LDS and 1× sample reducing agent) in a 16 microliter volume. Samples were heated at 95°C for 10 min, then cooled to room temperature and briefly centrifuged. Ten micrograms of protein per sample were loaded onto a 4-12% Bis-Tris gel (NuPage, Invitrogen) in MOPS buffer and subjected to electrophoresis at a constant voltage of 200 V for 1 h. Separated proteins were transferred onto a membrane [polyvinylidene difluoride (PDVF) using an iBlot transfer apparatus (Invitrogen) according to the manufacturer's protocol. Blots were blocked with Pierce protein-free T20 (PBS) blocking buffer (Thermo Scientific) for 1 h at room temperature. Then, the cells were incubated overnight at 4°C in blocking buffer with primary specific antibodies [(total cMET at 1:4,500 dilution; total EGFR at 1:2,500 dilution and β-actin at 1:15,000] (Cell Signaling Technologies). Specifically bound antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Immunoresearch) using SuperSignal West Dura ECL reagent (Pierce / Thermo Scientific). Images were captured on a GE Image Quant LAS 4000.

[0437] [Table 16]

[0438] The results shown in Figure 22 indicate that the R347 dummy arm mAb or their combination do not induce significant receptor downregulation, whereas the bispecific mAb does. This is consistent with the hypothesis that mAb internalization is optimal when both EGFR and cMet targets are engaged. Furthermore, the EGFR-cMET mAb appears to induce some degradation of cMet and a comparable amount of EGFR degradation to the EGFR-cMet TOP1i™ ADC. Furthermore, the EGFR-cMet TOP1i™ ADC appears to induce more cMet degradation than amivantamab, a recently approved EGFR-cMet bispecific antibody that has been reported to induce receptor degradation.

[0439] References A number of publications have been cited above in order to more fully describe and disclose this disclosure and the state of the art to which this disclosure pertains. Full citations for these references are set forth below. Each of these references is incorporated herein in its entirety.

[0440] [Table 17]

[0441] [Table 18]

[0442] [Table 19]

[0443] [Table 20]

[0444] [Table 21]

[0445] [Table 22]

[0446] [Table 23]

[0447] For standard molecular biology techniques, see Sambrook, J., Russell, DW Molecular Cloning, A Laboratory Manual. 3 ed. 2001, Cold Spring Harbor, New York; Cold Spring Harbor Laboratory Press.

[0448] Attachment of sequence CDR sequence of anti-EGFR antibody clone RAA22 HCDR1-DNDFS (SEQ ID NO: 1) HCDR2 - AIVAVFRTETYAQKFQD (SEQ ID NO: 2) HCDR3 - RLMSAISGPGAPLLM (SEQ ID NO: 3) LCDR1-TGTSSDVGGYNYVS (SEQ ID NO: 4) LCDR2-DVSKRPS (SEQ ID NO:5) LCDR3-SSYTSSDTLEI (SEQ ID NO: 6) CDR sequence of anti-EGFR antibody clone QD6 HCDR1-DNDFS (SEQ ID NO: 1) HCDR2 - AIVAVVRTETYAQKFQD (SEQ ID NO: 7) HCDR3 - RLMSAISGPGAPLLM (SEQ ID NO: 3) LCDR1-TGTSSDVGGYNYVS (SEQ ID NO: 4) LCDR2-DVSERPS (SEQ ID NO: 66) LCDR3-FSYTSSDTLEI (SEQ ID NO: 67) FR sequences of anti-EGFR antibody clones RAA22 and QD6 HFR1-QVQLVQSGAEVKKPGSSVKVSCKASGGTFS (SEQ ID NO: 8) HFR2-WVRQAPGQGLEWMG (SEQ ID NO: 9) HFR3-RVKITADISTRTTYMELSSLRSEDTAVYYCAR (SEQ ID NO: 10) HFR4-WGQGTLVTVSS (SEQ ID NO: 11) LFR1-QSALTQPRSVSGSPGQSVTISC (SEQ ID NO: 12) LFR2-WYQQHPGKAPKLMIY (SEQ ID NO: 13) LFR3-GVPDRFSGSKSGNTASLTISGLQAEDEADYYC (SEQ ID NO: 14) LFR4-FGGGTKLTVL (SEQ ID NO: 15) Amino acid sequence of the variable heavy (VH) region of anti-EGFR antibody clone RAA22 (SEQ ID NO: 16) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNDFSWVRQAPGQGLEWMGAIVAVFRTETYAQKFQDRVKITADISTRTTYMELSSLRSEDTAVYYCARRLMSAISGPGAPLLMWGQGTLVTVSS Nucleic acid sequence of the VH region of anti-EGFR antibody clone RAA22 (SEQ ID NO:17): [ka] Amino acid sequence of the VH region of anti-EGFR antibody clone QD6 (SEQ ID NO:18): QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDNDFSWVRQAPGQGLEWMGAIVAVVRTETYAQKFQDRVKITADISTRTTYMELSSLRSEDTAVYYCARRLMSAISGPGAPLLMWGQGTLVTVSS Nucleic acid sequence of the VH region of anti-EGFR antibody clone QD6 (SEQ ID NO:19): [ka] Amino acid sequence of the variable light (VL) region of anti-EGFR antibody clone RAA22 (SEQ ID NO:20): QSALTQPRSVGSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSDTLEIFGGGTKLTVL Nucleic acid sequence of the VL region of anti-EGFR antibody clone RAA22 (SEQ ID NO:21): [ka] Amino acid sequence of the VL region of anti-EGFR antibody clone QD6 (SEQ ID NO:22): QSALTQPRSVGSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSERPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCFSYTSSDTLEIFGGGTKLTVL Nucleic acid sequence of the VL region of anti-EGFR antibody clone QD6 (SEQ ID NO:23): [ka] CDR sequence of anti-c-Met antibody clone B09-GL HCDR1-DYYIH (SEQ ID NO: 24) HCDR2 - WMNPNSGNTGYAQKFQG (SEQ ID NO: 25) HCDR3-GQGYTHS (SEQ ID NO: 26) LCDR1-RASEGIYHWLA (SEQ ID NO: 27) LCDR2-KASSLAS (SEQ ID NO: 28) LCDR3-QQYSNYPPT (SEQ ID NO: 29) FR sequence of anti-c-Met antibody clone B09-GL HFR1-QVQLVQSGAEVKKPGASVKVSCKASGYTFT (SEQ ID NO: 30) HFR2-WVRQATGQGLEWMG (SEQ ID NO: 31) HFR3-RVTMTRDTSISTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 32) HFR4-WGQGTMVTVSS (SEQ ID NO: 33) LFR1-DIQMTQSPSTLSASVGDRVTITC (SEQ ID NO: 34) LFR2-WYQQKPGKAPKLLIY (SEQ ID NO: 35) LFR3-GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ ID NO: 36) LFR4-FGGGTKLEIK (SEQ ID NO: 37) Amino acid sequence of the variable heavy (VH) region of anti-c-Met antibody clone B09-GL (SEQ ID NO:38): QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYIHWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRDTSISTAYMELSSLRSEDTAVYYCARGQGYTHSWGQGTMVTVSS The nucleic acid sequence of the VH region of anti-c-Met antibody clone B09-GL (SEQ ID NO:39): [ka] Amino acid sequence of the variable light (VL) region of anti-c-Met antibody clone B09-GL (SEQ ID NO:40): DIQMTQSPSTLSASVGDRVTITCRASEGIYHWLAWYQQKPGKAPKLLIYKASSLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSNYPPTFGGGTKLEIK The nucleic acid sequence of the VL region of anti-c-Met antibody clone B09-GL (SEQ ID NO:41): [ka] Amino acid sequence of human immunoglobulin G1 heavy chain constant (CH) region (SEQ ID NO:42): [ka] Amino acid sequence of human immunoglobulin G1 CH region modified to include "knob" mutations, interchain cysteine ​​mutations, cysteines to form stabilizing disulfide bridges, and cysteine ​​insertions (SEQ ID NO:43): The following substitutions are underlined: a "knob" mutation (T366W); interchain cysteine ​​mutations (F126C and C219V); a stabilizing cysteine ​​mutation (S354C); and a cysteine ​​insertion (C239i) (residue numbering according to the EU index). [ka] Amino acid sequence of human immunoglobulin G1 CH region modified to include "knob" mutations, interchain cysteine ​​mutations, cysteines to form stabilizing disulfide bridges, and no cysteine ​​insertions (SEQ ID NO:44): The following substitutions are underlined: a "knob" mutation (T366W); interchain cysteine ​​mutations (F126C and C219V); and a stabilizing cysteine ​​mutation (S354C) (residue numbering according to the EU index). [ka] Amino acid sequence of human immunoglobulin G1 CH region modified to include a "hole" mutation, a cysteine ​​to form a stabilizing disulfide bridge, and a cysteine ​​insertion (SEQ ID NO:45): The following substitutions are underlined: "Hole" mutations (T366S, L368A, and Y407V); a stabilizing cysteine ​​mutation (Y349C); and a cysteine ​​insertion (C239i) (residue numbering according to the EU index). [ka] Amino acid sequence of human immunoglobulin G1 CH region modified to include a "hole" mutation, a cysteine ​​for forming a stabilizing disulfide bridge, and no cysteine ​​insertion (SEQ ID NO:46): The following substitutions are underlined: "Hole" mutations (T366S, L368A, and Y407V); and a stabilizing cysteine ​​mutation (Y349C) (residue numbering according to the EU index). [ka] Amino acid sequence of the wild-type human immunoglobulin kappa constant region (SEQ ID NO:47): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Amino acid sequence of a human immunoglobulin kappa constant region modified to include S121C and C214V substitutions (SEQ ID NO:48): The following substitutions are underlined: S121C and C214V (numbering according to the EU index) [ka] Amino acid sequence of the human immunoglobulin lambda constant region (SEQ ID NO:49): GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Amino acid sequence of the heavy chain of anti-c-Met antibody clone B09-GL with a cysteine ​​insertion (SEQ ID NO:50): The following substitutions are underlined: a "knob" mutation (T366W); interchain cysteine ​​mutations (F126C and C219V); a stabilizing cysteine ​​mutation (S354C); and a cysteine ​​insertion (C239i) (residue numbering according to the EU index). [ka] Amino acid sequence of the heavy chain of anti-c-Met antibody clone B09-GL without a cysteine ​​insertion (SEQ ID NO:51): The following substitutions are underlined: a "knob" mutation (T366W); interchain cysteine ​​mutations (F126C and C219V); and a stabilizing cysteine ​​mutation (S354C) (residue numbering according to the EU index). [ka] Amino acid sequence of the light chain of anti-c-Met antibody clone B09-GL (SEQ ID NO:52): The following substitutions are underlined: S121C and C214V (numbering according to the EU index) [ka] Amino acid sequence of the heavy chain of anti-EGFR antibody clone QD6 with a cysteine ​​insertion (SEQ ID NO:53): The following substitutions are underlined: "Hole" mutations (T366S, L368A, and Y407V); a stabilizing cysteine ​​mutation (Y349C); and a cysteine ​​insertion (C239i) (residue numbering according to the EU index). [ka] Amino acid sequence of the heavy chain of EGFR antibody clone QD6 without the cysteine ​​insertion (SEQ ID NO:54): The following substitutions are underlined: "Hole" mutations (T366S, L368A, and Y407V); a stabilizing cysteine ​​mutation (Y349C) (residue numbering according to the EU index). [ka] Amino acid sequence of the light chain of anti-EGFR antibody clone QD6 (SEQ ID NO:55): [ka] Amino acid sequence of the heavy chain of anti-EGFR antibody clone RAA22 with a cysteine ​​insertion (SEQ ID NO:56): The following substitutions are underlined: "Hole" mutations (T366S, L368A, and Y407V); a stabilizing cysteine ​​mutation (Y349C); and a cysteine ​​insertion (C239i) (residue numbering according to the EU index). [ka] Amino acid sequence of the heavy chain of EGFR antibody clone RAA22 without the cysteine ​​insertion (SEQ ID NO:57): The following substitutions are underlined: "Hole" mutations (T366S, L368A, and Y407V); and a stabilizing cysteine ​​mutation (Y349C) (residue numbering according to the EU index). [ka] Amino acid sequence of the light chain of anti-EGFR antibody clone RAA22 (SEQ ID NO:58): [ka] Amino acid sequence of the anti-EGFR heavy chain in the "EGFR-cMET TM" antibody (SEQ ID NO:59): The following substitutions are underlined: The triple mutation (TM; L234F, L235E and P331S); the "knob" mutation (T366W); the interchain cysteine ​​mutations (F126C and C219V); the stabilizing cysteine ​​mutation (S354C) (residue numbering according to the EU index). [ka] Amino acid sequence of the heavy chain of anti-c-Met in the "EGFR-cMET TM" antibody (SEQ ID NO:60): The following substitutions are underlined: The triple mutation (TM; L234F, L235E and P331S); the "hole" mutation (T366S, L368A and Y407V); and the stabilizing cysteine ​​mutation (Y349C) (residue numbering according to the EU index). [ka] Amino acid sequence of the anti-EGFR light chain in the "EGFR-cMET TM" antibody (SEQ ID NO:61): The following substitutions are underlined: S121C and C214V (numbering according to the EU index) [ka] Amino acid sequence of the light chain of anti-c-Met in the "EGFR-cMET TM" antibody (SEQ ID NO:62): [ka] Amino acid sequence of human immunoglobulin G1 CH region modified to have a "knob" mutation, a cysteine ​​to form a stabilizing disulfide bridge, no cysteine ​​insertion, and a TM (SEQ ID NO:63): The following substitutions are underlined: The triple mutation (TM; L234F, L235E and P331S); the "knob" mutation (T366W); the interchain cysteine ​​mutations (F126C and C219V); the stabilizing cysteine ​​mutation (S354C) (residue numbering according to the EU index). [ka] Amino acid sequence of human immunoglobulin G1 CH region modified to have a "hole" mutation, a cysteine ​​for forming a stabilizing disulfide bridge, no cysteine ​​insertion, and a TM (SEQ ID NO:64): The following substitutions are underlined: The triple mutation (TM; L234F, L235E and P331S); the "hole" mutation (T366S, L368A and Y407V); and the stabilizing cysteine ​​mutation (Y349C) (residue numbering according to the EU index). [ka] Amino acid sequence of a human immunoglobulin lambda constant region modified to include S121C and C214V substitutions (SEQ ID NO:65): [ka] Amino acid sequence of human EGFR extracellular domain (SEQ ID NO:68): [ka] Amino acid sequence of the cynomolgus monkey EGFR extracellular domain (SEQ ID NO:69): [ka] Amino acid sequence of human c-Met extracellular domain (SEQ ID NO:70): [ka] Amino acid sequence of cynomolgus c-Met extracellular domain (SEQ ID NO:71): [ka]

Claims

1. a first antigen-binding domain that binds to the epidermal growth factor receptor (EGFR); and A second antigen-binding domain that binds to c-Met Including, The first antigen-binding domain comprises: (i) a heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs): HCDR1 having the amino acid sequence of SEQ ID NO: 1 HCDR2 having the amino acid sequence of SEQ ID NO:2 HCDR3 having the amino acid sequence of SEQ ID NO: 3; and (ii) a light chain variable (VL) region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO: 4 LCDR2 having the amino acid sequence of SEQ ID NO:5 LCDR3 having the amino acid sequence of SEQ ID NO:6 and The second antigen-binding domain comprises: (i) a heavy chain variable (VH) region comprising the following complementarity determining regions (CDRs): HCDR1 having the amino acid sequence of SEQ ID NO: 24 HCDR2 having the amino acid sequence of SEQ ID NO: 25 HCDR3 having the amino acid sequence of SEQ ID NO: 26; and (ii) a light chain variable (VL) region comprising the following CDRs: LCDR1 having the amino acid sequence of SEQ ID NO: 27 LCDR2 having the amino acid sequence of SEQ ID NO: 28 LCDR3 having the amino acid sequence of SEQ ID NO: 29 and antibody molecule.

2. The first antigen-binding domain comprises: a VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 16; and A VL region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

20. The antibody molecule of claim 1, comprising:

3. The second antigen-binding domain comprises: a VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 38; and A VL region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

40. The antibody molecule of claim 1, comprising:

4. The antibody molecule is a first heavy chain comprising a VH region of the first antigen-binding domain and a first heavy chain constant (CH) region or a fragment thereof; a first light chain comprising a VL region of the first antigen-binding domain and a first light chain constant (CL) region or a fragment thereof; a second heavy chain comprising a VH region of the second antigen-binding domain and a second heavy chain constant (CH) region or a fragment thereof; and a second light chain comprising a VL region of the second antigen-binding domain and a second light chain constant (CL) region or a fragment thereof; The antibody molecule of claim 1, comprising:

5. The antibody molecule of claim 4, wherein the first and second CH regions comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

42.

6. 5. The antibody molecule of claim 4, wherein the first and second heavy chains form a heterodimer, and optionally one of the first and second heavy chains comprises a cysteine ​​(C) residue at position 354 and a tryptophan (W) residue at position 366, and the other heavy chain comprises a cysteine ​​(C) residue at position 349, a valine (V) residue at position 407, a serine (S) at position 366, and an alanine (A) at position 368, wherein the numbering of the constant regions is according to the EU index.

7. The antibody molecule (a) a modified CH region, the modified CH region comprising a substitution of a naturally occurring non-cysteine ​​amino acid with a cysteine ​​amino acid; and (b) a modified corresponding CL region, wherein the modified CL comprises a substitution of a naturally occurring non-cysteine ​​amino acid with a cysteine ​​amino acid. Including, (i) the first heavy chain comprises the modified CH region and the first light chain comprises the modified corresponding CL region; or (ii) the second heavy chain comprises the modified CH region and the second light chain comprises the modified corresponding CL region; and The antibody molecule of claim 4, wherein the substituted cysteine ​​in the modified CH region and the substituted cysteine ​​in the modified corresponding light chain can form a disulfide bond.

8. 8. The antibody molecule of claim 7, wherein the modified CH region comprises a substitution of a natural non-cysteine ​​amino acid at position 126 with a cysteine ​​amino acid, and the corresponding modified CL region comprises a substitution of a natural non-cysteine ​​amino acid at position 121 with a cysteine ​​amino acid, wherein the numbering of the constant regions is according to the EU index.

9. The antibody molecule of claim 4, wherein the first and / or second CH region comprises a mutation that reduces or abolishes binding of the antibody molecule to one or more Fcγ receptors.

10. 5. The antibody molecule of claim 4, wherein the first and / or second CH region comprises a phenylalanine at position 234, a glutamic acid at position 235, and a serine at position 331, wherein the numbering of the constant regions is according to the EU index.

11. the first CH region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 45, 46 or 63; the second CH region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 43, 44 or 64, and optionally the first CL region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 49 or 65; The antibody molecule of claim 4, wherein 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 to the sequence set forth in SEQ ID NO: 47 or 48.

12. the first CH region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:63; the second CH region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:64, and the first CL region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:65; 12. The antibody molecule of claim 11, wherein 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 to the sequence set forth in SEQ ID NO:

47.

13. the first heavy chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 56, 57 or 59; the second heavy chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 50, 51 or 60, and optionally the first light chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 58 or 61; 12. The antibody molecule of claim 11 , wherein the second light chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 52 or 62.

14. 13. The antibody molecule of claim 12, wherein the first heavy chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO:59; the second heavy chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO:60; the first light chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO:61; and the second light chain comprises an amino acid sequence having the sequence set forth in SEQ ID NO:

62.

15. A conjugate comprising the antibody molecule of claim 1 conjugated to a drug.

16. 16. The conjugate of claim 15, wherein the drug comprises a cytotoxin, a radioisotope, an immunomodulatory agent, a cytokine, a lymphokine, a chemokine, a growth factor, a tumor necrosis factor, a hormone, a hormone antagonist, an enzyme, an oligonucleotide, DNA, RNA, siRNA, RNAi, microRNA, a photoactive therapeutic agent, an anti-angiogenic agent, a pro-apoptotic agent, a peptide, a lipid, a carbohydrate, a chelator, or a combination thereof.

17. 16. The conjugate of claim 15, wherein the drug is a topoisomerase I inhibitor having the formula A*. 【Chemical 1】

18. The topoisomerase I inhibitor has Formula I: 【Chemistry 2】 or a salt or solvate thereof [wherein, R Ｌ is a linker for linking to said antibody molecule, optionally said linker comprising: (ia): 【Chemistry 3】 (In the formula, Q is 【Chemistry 4】 (In the formula, Q Ｘ is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue. and X is 【Chemistry 5】 (wherein a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, and 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)) and G Ｌ is a linker for linking to the antibody molecule; or (ib): 【Chemistry 6】 (In the formula, R Ｌ１ and R Ｌ２ are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1.

18. The complex of claim 17, wherein

19. Formula IV: L-(D Ｌ ) ) ｐ (96) or a pharmaceutically acceptable salt or solvate thereof (In the formula, L is the antibody molecule; D Ｌ is a drug with a linker; p is an integer from 1 to 20.

16. The complex of claim 15, having the formula:

20. 20. The complex of claim 19, wherein p is selected from 2 to 8, 3 to 7, 4 to 7, or 5 to 7.

21. D Ｌ is represented by formula III: 【Chemistry 7】 [R ＬＬ is a linker linked to the antibody molecule, the linker comprising: (ia'): 【Chemistry 8】 (Wherein Q is 【Chemistry 9】 wherein Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue. and X is 【Chemistry 10】 (wherein a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, and 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)) and G ＬＬ is a linker that connects the antibody molecule; and (ib'): 【Chemistry 11】 (In the formula, R Ｌ１ and R Ｌ２ are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group. Selected from 20. The conjugate of claim 19, which is a topoisomerase I inhibitor having a linker of

22. The antibody molecule has the following formula: 【Chemistry 12】 16. The conjugate of claim 15, conjugated to a topoisomerase I inhibitor having the formula:

23. A conjugate comprising an antibody molecule, wherein the antibody molecule has a first antigen-binding domain that binds to an epidermal growth factor receptor (EGFR); and A second antigen-binding domain that binds to c-Met Including, The first antigen-binding domain comprises: (i) a heavy chain variable (VH) region having the sequence of SEQ ID NO: 16; (ii) a light chain variable (VL) region having the sequence of SEQ ID NO: 20 Including, The second antigen-binding domain comprises: (i) a heavy chain variable (VH) region having the sequence of SEQ ID NO: 38; (ii) a light chain variable (VL) region having the sequence of SEQ ID NO: 40 Including, wherein the antibody molecule has the following formula: 【Chemistry 13】 A conjugate comprising:

24. The conjugate of claim 23 having a drug-antibody ratio of 6.

25. A pharmaceutical composition comprising an antibody molecule according to any one of claims 1 to 14, or a conjugate according to any one of claims 15 to 24, and a pharmaceutically acceptable carrier.

26. 26. A pharmaceutical composition according to claim 25 for the treatment of the human or animal body.

27. 26. The pharmaceutical composition of claim 25 for use in a method for the treatment of cancer.

28. 28. The pharmaceutical composition of claim 27, wherein the cancer is lung cancer (such as non-small cell lung cancer (NSCLC)), pancreatic cancer, breast cancer, colorectal cancer, gastric cancer, squamous cell carcinoma of the head and neck (SCCHN or SQHN), ovarian cancer, or glioblastoma.

29. 28. The pharmaceutical composition of claim 27, wherein the cancer is lung cancer (such as non-small cell lung cancer (NSCLC)) or squamous cell carcinoma of the head and neck (SCCHN or SQHN).

30. 30. Use of the pharmaceutical composition of claim 27 in the manufacture of a medicament for the treatment of cancer.

31. 31. The use of claim 30, wherein the cancer is lung cancer (such as non-small cell lung cancer (NSCLC)), pancreatic cancer, breast cancer, colon cancer, gastric cancer, squamous cell carcinoma of the head and neck (SCCHN or SQHN), ovarian cancer or glioblastoma.

32. 31. The use of claim 30, wherein the cancer is lung cancer (such as non-small cell lung cancer (NSCLC)) or squamous cell carcinoma of the head and neck (SCCHN or SQHN).

33. Encoding the antibody molecule of claim 1 A nucleic acid or nucleic acids, optionally isolated.

34. 34. An expression vector or vectors comprising the nucleic acid or nucleic acids of claim 33.

35. 35. A recombinant host cell comprising the nucleic acid or nucleic acids of claim 33, or the expression vector or expression vectors of claim 34.

36. 36. A method of producing the antibody molecule of claim 1, comprising culturing the recombinant host cell of claim 35 under conditions to produce the antibody molecule.

37. 37. The method of claim 36, further comprising the step of isolating and / or purifying the antibody molecule.

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