Combination therapies and patient stratification with bispecific Anti-EGFR / c-met antibodies

Abstract

To provide a pharmaceutical composition that treats a subject having EGFR or c-Met-expressing cancer.SOLUTION: A pharmaceutical composition is used in a method for inducing trogocytosis to an acceptor macrophage cell or an acceptor monocyte from a donor cancer cell that expresses EGFR, c-Met, or EGFR and c-Met, where the pharmaceutical composition contains bispecific anti-EGFR / c-Met antibodies, and the method includes bringing the donor cancer cell in contact with the bispecific anti-EGFR / c-Met antibodies for a sufficient time for inducing trogocytosis from the donor cancer cell to the acceptor macrophage cell or the acceptor monocyte.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to combination therapy with bispecific anti-EGFR / c-Met antibodies and patient stratification. It does.

[0002] (Sequence Listing) This application includes a sequence listing submitted via EFS-Web, the entire contents of which are incorporated herein by reference. The ASCII text file created on February 13, 2020 is named JBI6051WOPCT1ST25.txt and is 19 kilobytes in size. bytes. It is.

Background Art

[0003] Since the individual roles of both EGFR and c-Met in cancer are well established, these targets have become attractive for combination therapy. Each receptor transmits signals through the same survival and anti-apoptosis pathways (ERK and AKT); therefore inhibiting this pair of pathways together can limit the potential for activation of compensatory pathways and thereby improve overall efficacy.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Recurrence or resistance to existing therapeutic agents is common. Therefore, in order to develop more effective treatments for diseases such as EGFR- or c- Met-positive cancers, improved therapeutic agents or combinations of therapeutic agents and patient stratification biomarkers are needed. It is.

Means for Solving the Problems

[0005] The present disclosure is a method of treating a subject having EGFR- or c-Met-expressing cancer, the pair In combination with an agent that enhances macrophage activity in an elephant, an isolated therapeutically effective amount Bispecific anti-epidermal growth factor receptor (EGFR) / hepatocyte growth factor receptor (c-Met) anti A method is provided that includes administering to a subject.

[0006] The present disclosure also provides a method for diagnosing and treating a subject having EGFR or c-Met-expressing cancer responsive to treatment with a bispecific anti-EGFR / c-Met antibody, the method comprising providing a biological sample from the subject, measuring macrophage or monocyte levels from the biological sample, and when the macrophage or monocyte levels from the biological sample are higher than a threshold, diagnosing that the subject has EGFR or c-Met-expressing cancer responsive to treatment with a bispecific anti-EGFR / c-Met antibody, and administering or providing for administration of a bispecific anti-EGFR / c-Met antibody to a subject diagnosed as responsive to treatment with the anti-EGFR / c-Met antibody. The method also includes. The present disclosure also provides a method for treating a subject suspected of having or having EGFR or c-Met-expressing cancer with a bispecific anti-EGFR / c-Met antibody, the method comprising determining that the subject has macrophage or monocyte levels higher than a threshold, and administering or providing for administration of a bispecific anti-EGFR / c-Met antibody to a subject determined to have macrophage or monocyte levels higher than the threshold.

[0007] The present disclosure also provides a method for predicting the response of a subject having EGFR or c-Met-expressing cancer to treatment with a bispecific anti-EGFR / c-Met antibody, the method comprising providing a biological sample from the subject. higher macrophage or monocyte levels, and administering or providing for administration of a bispecific anti-EGFR / c-Met antibody to a subject determined to have macrophage or monocyte levels higher than the threshold. antibody to a subject determined to have macrophage or monocyte levels higher than the threshold. The method also includes.

[0008] The present disclosure also provides a method for predicting the response of a subject having EGFR or c-Met-expressing cancer to treatment with a bispecific anti-EGFR / c-Met antibody, the method comprising providing a biological sample from the subject. To provide, to measure macrophage or monocyte levels from a biological sample, and when the macrophage or monocyte levels from the biological sample are higher than a threshold, to predict that the subject is a responder. A method is provided that includes predicting that a subject is a responder when macrophage or monocyte levels from a biological sample are higher than a threshold. To provide a method comprising the above steps.

[0009] The present disclosure also provides a method of treating a subject having EGFR or c-Met expressing cancer that responds to treatment with a bispecific anti-EGFR / c-Met antibody, the method comprising providing a biological sample from the subject, measuring macrophage or monocyte levels from the biological sample, and treating the subject with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are higher than a threshold. To provide a method of treating a subject having EGFR or c-Met expressing cancer that responds to treatment with a bispecific anti-EGFR / c-Met antibody, the method comprising providing a biological sample from the subject, measuring macrophage or monocyte levels from the biological sample, and treating the subject with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are higher than a threshold. To provide a biological sample from the subject, measure macrophage or monocyte levels from the biological sample, and treat the subject with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are higher than a threshold. When the macrophage or monocyte levels from the biological sample are higher than a threshold, treat the subject with the bispecific anti-EGFR / c-Met antibody. To provide a method comprising the above steps.

[0010] The present disclosure also provides a method of determining whether a subject having EGFR or c-Met expressing cancer will respond to treatment with a bispecific anti-EGFR / c-Met antibody and of determining whether to treat the subject, the method comprising providing a biological sample from the subject, measuring macrophage or monocyte levels from the biological sample, diagnosing that a subject having EGFR or c-Met expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are higher than a threshold, or diagnosing that a subject having EGFR or c-Met expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are lower than a threshold, and administering the bispecific anti-EGFR / c-Met antibody to the subject diagnosed to respond to treatment with the bispecific anti-EGFR / c-Met antibody, or not administering the bispecific anti-EGFR / c-Met antibody to the subject diagnosed not to respond to treatment with the bispecific anti-EGFR / c-Met antibody. To determine whether a subject having EGFR or c-Met expressing cancer will respond to treatment with a bispecific anti-EGFR / c-Met antibody and of determining whether to treat the subject, the method comprising providing a biological sample from the subject, measuring macrophage or monocyte levels from the biological sample, diagnosing that a subject having EGFR or c-Met expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are higher than a threshold, or diagnosing that a subject having EGFR or c-Met expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are lower than a threshold, and administering the bispecific anti-EGFR / c-Met antibody to the subject diagnosed to respond to treatment with the bispecific anti-EGFR / c-Met antibody, or not administering the bispecific anti-EGFR / c-Met antibody to the subject diagnosed not to respond to treatment with the bispecific anti-EGFR / c-Met antibody. To provide a biological sample from the subject, measure macrophage or monocyte levels from the biological sample, and diagnose that a subject having EGFR or c-Met expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are higher than a threshold, or diagnose that a subject having EGFR or c-Met expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are lower than a threshold, and administer the bispecific anti-EGFR / c-Met antibody to the subject diagnosed to respond to treatment with the bispecific anti-EGFR / c-Met antibody, or not administer the bispecific anti-EGFR / c-Met antibody to the subject diagnosed not to respond to treatment with the bispecific anti-EGFR / c-Met antibody. To measure macrophage or monocyte levels from the biological sample, and diagnose that a subject having EGFR or c-Met expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are higher than a threshold, or diagnose that a subject having EGFR or c-Met expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are lower than a threshold, and administer the bispecific anti-EGFR / c-Met antibody to the subject diagnosed to respond to treatment with the bispecific anti-EGFR / c-Met antibody, or not administer the bispecific anti-EGFR / c-Met antibody to the subject diagnosed not to respond to treatment with the bispecific anti-EGFR / c-Met antibody. When the macrophage or monocyte levels from the biological sample are higher than a threshold, diagnose that a subject having EGFR or c-Met expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody, or when the macrophage or monocyte levels from the biological sample are lower than a threshold, diagnose that a subject having EGFR or c-Met expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody. To diagnose that a subject having EGFR or c-Met expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are higher than a threshold, or diagnose that a subject having EGFR or c-Met expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are lower than a threshold, and administer the bispecific anti-EGFR / c-Met antibody to the subject diagnosed to respond to treatment with the bispecific anti-EGFR / c-Met antibody, or not administer the bispecific anti-EGFR / c-Met antibody to the subject diagnosed not to respond to treatment with the bispecific anti-EGFR / c-Met antibody. When the macrophage or monocyte levels from the biological sample are lower than a threshold, diagnose that a subject having EGFR or c-Met expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody. To diagnose that a subject having EGFR or c-Met expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are lower than a threshold, and administer the bispecific anti-EGFR / c-Met antibody to the subject diagnosed to respond to treatment with the bispecific anti-EGFR / c-Met antibody, or not administer the bispecific anti-EGFR / c-Met antibody to the subject diagnosed not to respond to treatment with the bispecific anti-EGFR / c-Met antibody. To diagnose that a subject having EGFR or c-Met expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte levels from the biological sample are lower than a threshold, and administer the bispecific anti-EGFR / c-Met antibody to the subject diagnosed to respond to treatment with the bispecific anti-EGFR / c-Met antibody, or not administer the bispecific anti-EGFR / c-Met antibody to the subject diagnosed not to respond to treatment with the bispecific anti-EGFR / c-Met antibody. To administer the bispecific anti-EGFR / c-Met antibody to the subject diagnosed to respond to treatment with the bispecific anti-EGFR / c-Met antibody, or not administer the bispecific anti-EGFR / c-Met antibody to the subject diagnosed not to respond to treatment with the bispecific anti-EGFR / c-Met antibody. A method is also provided that includes refraining from administering a bispecific anti-EGFR / c-Met antibody to a subject diagnosed as non-responsive to treatment with an FR / c-Met antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0011]

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Mode for Carrying Out the Invention

[0012] Definitions All publications, including but not limited to patents and patent applications cited herein are hereby incorporated by reference as if fully set forth herein.

[0013] The terms used in this specification are used only for the purpose of describing particular embodiments and are not to be construed as limiting. Unless otherwise specified, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that they are not intended to limit. All technical and scientific terms used herein, unless otherwise defined, shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0014] Any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Exemplary materials and methods are described herein. The following terms are used in the description and claims of the present invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, exemplary materials and methods are described herein. When listing elements, unless otherwise specified, it should be understood that each individual element of the list and all combinations of the list are separate embodiments. For example, a list of embodiments presented as "A, B, or C" should be construed to include the embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0015] When a list is presented, unless otherwise specified, it should be understood that each individual element of the list and all combinations of the list are separate embodiments. For example, a list of embodiments presented as "A, B, or C" should be construed to include the embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C". For example, a list of embodiments presented as "A, B, or C" should be construed to include the embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C". For example, a list of embodiments presented as "A, B, or C" should be construed to include the embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C". For example, a list of embodiments presented as "A, B, or C" should be construed to include the embodiments "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C".

[0016] 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. Accordingly, for example, a reference to "a cell" includes combinations of two or more cells and the like. Accordingly, for example, a reference to "a cell" includes combinations of two or more cells and the like.

[0017] The term "and / or" connecting a plurality of recited elements is understood to include both individual and combined options. For example, when two elements are connected by "and / or", it is understood to include both individual and combined options. For example, when two elements are connected by "and / or", ​​​Thus, when connected, the first option indicates that the first element is applicable without the second element. The second option indicates that the second element is applicable without the first element. The third option indicates that the first and second elements are applicable together. Any one of these options is included in the meaning, and thus, when used herein, it is understood to meet the requirements of the term "and / or". The simultaneous applicability of two or more of the options is also included in the meaning, and thus, it is understood to meet the requirements of the term "and / or".

[0018] The transitional phrases "comprising", "consisting essentially of", and "consisting of" are intended to imply the generally accepted meanings in patent terminology, i.e., (i) "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or non- limiting, and does not exclude other unrecited elements or method steps, (ii) "consisting of" excludes any element, step, or component not specified in the claims, and (iii) "consisting essentially of" limits the scope of the claims to the specified materials or steps and those that do not substantially affect the "basic and novel features (plural available)" of the claimed invention. Embodiments described with respect to the phrase "comprising" (or its equivalent) also provide, as embodiments, those independently described with respect to "consisting of" and "consisting essentially of". ​​

[0019] "Co-administration", "administered together", "administered in combination with", "in combination with", etc. include the administration of a selected therapeutic agent or drug to a single patient, and include treatment regimens in which the therapeutic agent or drug is administered by the same or different routes of administration or at the same or different times. is intended.

[0020] "Isolated" refers to a homogeneous population of molecules (e.g., synthetic polynucleotides, polypeptides, vectors, or viruses) that are substantially separated and / or purified from other components of the system in which a molecule is produced, such as recombinant cells, and also refers to a protein that has been subjected to at least one purification or isolation step. "Isolated" refers to a molecule that is substantially free of other cellular materials and / or chemical substances, and includes molecules that have been isolated to a higher purity, e.g., 80%, 81%, 82%, 83%, 84% %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0021] "Treating", "being treated", or "treatment" of a disease or disorder such as cancer refers to achieving one or more of the following: reducing the severity and / or duration of the disorder, inhibiting the worsening of symptoms characteristic of the disorder being treated, limiting or preventing the recurrence of the disorder in a subject who has previously had the disorder, or limiting or preventing the recurrence of symptoms in a subject who has previously been symptomatic for the disorder.

[0022] "Preventing", "being prevented", "prevention", or "preventive treatment" of a disease or disorder means preventing the occurrence of the disorder in a subject.

[0023] "Diagnosing" or "diagnosis" refers to determining whether a subject is suffering from a given disease or condition, or whether the subject may develop a given disease or condition in the future, or whether the subject is likely to respond to treatment for a pre-diagnosed disease or condition, i.e., a method of stratifying a patient population with respect to the likelihood of responding to treatment. Diagnosis is typically performed by a physician based on general guidelines for the disease being diagnosed, or other criteria indicating that the subject is likely to respond to a particular treatment.

[0024] "Responding", "responsiveness", or "likely to respond" refers to any type of improvement or positive response, whether detectable or undetectable, such as alleviation or recovery of one or more symptoms, reduction in the degree of the disease, a stabilized (i.e., non-worsening) disease state, prevention of the spread of the disease, delay or deceleration of disease progression, recovery or alleviation of the disease state, and remission (partial or complete).

[0025] "Newly diagnosed" refers to a subject who has been diagnosed with EGFR or c-Met expressing cancer but has not yet received treatment for multiple myeloma.

[0026] "Therapeutically effective amount" refers to the amount effective to achieve the desired therapeutic result at the required dosage and duration. The therapeutically effective amount can vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of one or a combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective single therapeutic agent, or combination of therapeutic agents, include, for example, improvement in the health of the patient.

[0027] "Refractory" refers to a disease that does not respond to treatment. A refractory disease may be resistant to treatment before or at the start of treatment, or a refractory disease may become a resistant disease during treatment.

[0028] "Recurrent" refers to the recurrence of a disease or the signs and symptoms of a disease after a period of improvement following prior treatment with a therapeutic agent.

[0029] "Subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, for example, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. The terms "subject" and "patient" are used interchangeably herein.

[0030] "About" means within the acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends to some extent on the method by which the value is measured or determined, i.e., the limitations of the measurement system. Unless otherwise expressly stated elsewhere in the context of a particular assay, result, or embodiment, or in the examples, "about" means within the larger of either one standard deviation or a range of up to 5% according to the practice of the art.

[0031] "Cancer" refers to the abnormal growth of cells that grow in an uncontrolled manner and, in some cases, tend to metastasize (spread) to other areas of the patient's body.

[0032] "EGFR or c-Met expressing cancer" refers to a cancer having a detectable expression of EGFR or c-Met, or having a mutation or amplification of EGFR or c-Met. ​​​​​The expression, amplification, and mutation status of EGFR or c-Met can be detected using known methods such as sequencing, fluorescence in situ hybridization, immunohistochemical examination, flow cytometry, or Western blotting.

[0033] "Epidermal growth factor receptor" or "EGFR" refers to human EGFR having the amino acid sequence shown in GenBank accession number NP _005219 (also known as HER1 or Erb B1; Ullrich et al., Nature 309:4 18-425, 1984), as well as its naturally occurring variants.

[0034] "Hepatocyte growth factor receptor" or "c-Met", as used herein, refers to human c-Met having the amino acid sequence shown in GenB ank accession number NP_001120972, as well as its natural variants.

[0035] "Bispecific anti-EGFR / c-Met antibody" or "bispecific EGFR / c-Met anti body" refers to a bispecific antibody having a first domain that specifically binds to EGFR and a second domain that specifically binds to c-Met. The domains that specifically bind to EGFR and c-Met are typically VH / VL pairs, and the bispecific anti-EGFR / c-Met antibody is monovalent with respect to binding to EGFR and c-Met.

[0036] "Specific binding", or "specifically binds", or "specific binding", or "binds " means that an antibody binds to an antigen or an epitope within an antigen with a higher affinity than to other antigens. Typically, an antibody has an affinity of about 5×10 -8Less than M, for example, about 1 × 10 -9 Less than M, about 1 × 10 -10 Less than M, about 1 × 10 -11 Less than M, or about 1 × 10 -12 with an equilibrium dissociation constant (K D ) such that typically its K for binding to a non-specific antigen (e.g., BSA, casein is at least 100-fold lower than its K D for binding to an antigen or an epitope within the antigen. The dissociation constant can be measured using known protocols. However, an antibody that binds to an antigen or an epitope within the antigen may cross-react with the same antigen (homolog) from other species, such as human or monkey, e.g., Macaca fascicularis (cynomolgus monkey, cyno D ), or Pan troglodytes (chimpanzee, chimp). A monospecific antibody binds to one antigen or one epitope, while a bispecific antibody binds to two different antigens or two different epitopes.

[0037]

[0037] "Antibody" is intended in a broad sense and includes monoclonal antibodies, antigen-binding fragments, bispecific, trispecific, tetravalent, etc. multispecific antibodies, dimeric, tetrameric, or multimeric antibodies, single-chain antibodies, antibody domains and any other modified structure of an immunoglobulin molecule containing an antigen-binding site of the required specificity, including immunoglobulin molecules. "Full-length antibody" refers to two heavy chains (HC) and two light chains ( interconnected by disulfide bonds ), light chain, LC), and multimers thereof (e.g., IgM). Each heavy chain consists of a heavy chain variable region (VH), and a heavy chain constant region (composed of domains CH1, hinge, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH region and VL region are interspersed with framework regions (FR) and can be further classified into hypervariable regions called complementarity-determining regions (CDR). Each VH and VL is composed of three CDRs and four FR segments arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The "complementarity-determining region" (CDR) is the antibody region that binds to an antigen. CDR was defined by Kabat (Wu et al. (1970) J Exp Med 132:211-50) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196:901-17), IMGT ( Lefranc et al. (2003) Dev Comp Immunol 27 :55-77), and AbM (Martin and Thornton (1996

[0038] ​​​​​​​​​​​​) It can be defined using various descriptions such as (J Bmol Biol 263:800-15). The correspondence between various descriptions and the numbering of variable regions is described (for example, , see Lefranc et al. (2003) Dev Comp Immunol 2 7:55-77, Honegger and Pluckthun, (2001) J Mol Biol 309:657-70, International ImMuno GeneTics (IMGT) database; web resource, http: / / www_ imgt_org). CDRs can be depicted using available programs such as abYsis by UCL Business PLC. As used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LC DR1", "LCDR2", and "LCDR3" include CDRs defined by any of the Kabat, Chothia, IMGT, or AbM methods described above, unless otherwise explicitly stated herein.

[0039] Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, according to the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subclassified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and Ig G4. Antibody light chains of any vertebrate species can be assigned to one of two distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain.

[0040] "Antigen-binding fragment" refers to a part of an immunoglobulin molecule that binds to an antigen. The antigen-binding fragment may be a synthetic polypeptide, a polypeptide obtainable by an enzyme, or a recombinantly engineered polypeptide, and may be a VH, VL, VH and VL, Fab, F(ab’)2, Fd, and Fv fragment, a domain antibody (dAb) consisting of one VH domain or one VL domain, a shark variable IgNAR domain, a camelized VH domain, the smallest recognition unit consisting of amino acid residues reproducing the CDRs of an antibody, such as an FR3-CDR3-FR4 portion, HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3. The VH and VL domains are linked to each other via a synthetic linker and can form various types of single-chain antibody designs. When the VH and VL domains are expressed by an individual single-chain antibody construct, the VH / VL domains pair intramolecularly or intermolecularly to form a monovalent antigen-binding site, such as a single-chain Fv (scFv) or a bispecific antibody, which are described, for example, in WO 98 / 44001, WO 88 / 01649, WO 94 / 13804, and WO 92 / 01047.

[0041] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibody molecules (i.e., the individual antibodies comprising the population are identical except for possible well-known modifications such as removal of the C-terminal lysine from the antibody heavy chain, or post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation, or asparagine or glutamine deamidation). Monoclonal antibodies typically bind to A monoclonal antibody binds to two different antigenic epitopes. A monoclonal antibody may have heterogeneous glycosylation within the antibody population. A monoclonal antibody may be monospecific, or may be multispecific such as bispecific, and may be monovalent, bivalent, or multivalent. A monoclonal antibody may have heterogeneous glycosylation within the antibody population. A monoclonal antibody may be monospecific, or may be multispecific such as bispecific, and may be monovalent, bivalent, or multivalent.

[0042] A "humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Since the framework of a humanized antibody can include substitutions, the framework may not be an exact copy of the expressed human immunoglobulin or the human immunoglobulin germline gene sequence. A "humanized antibody" refers to an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Since the framework of a humanized antibody can include substitutions, the framework may not be an exact copy of the expressed human immunoglobulin or the human immunoglobulin germline gene sequence. or the human immunoglobulin germline gene sequence. Since the framework of a humanized antibody can include substitutions, the framework may not be an exact copy of the expressed human immunoglobulin or the human immunoglobulin germline gene sequence.

[0043] A "human antibody" refers to an antibody that is optimized to have a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. When a human antibody includes a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. A human antibody includes heavy and light chain variable regions that "are derived from" sequences of human origin when the variable regions of the human antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals that carry the human immunoglobulin locus, such as mice or rats. A "human antibody" is typically different from the system used to obtain the human antibody and the human immunoglobulin locus, and may include the intentional introduction of somatic mutations or substitutions into the framework or CDRs. A "human antibody" refers to an antibody that is optimized to have a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. When a human antibody includes a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. A "human antibody" refers to an antibody that is optimized to have a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. When a human antibody includes a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. A human antibody includes heavy and light chain variable regions that "are derived from" sequences of human origin when the variable regions of the human antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals that carry the human immunoglobulin locus, such as mice or rats. A "human antibody" is typically different from the system used to obtain the human antibody and the human immunoglobulin locus, and may include the intentional introduction of somatic mutations or substitutions into the framework or CDRs. When a human antibody includes a constant region or a portion of a constant region, the constant region is also derived from a human immunoglobulin sequence. A human antibody includes heavy and light chain variable regions that "are derived from" sequences of human origin when the variable regions of the human antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals that carry the human immunoglobulin locus, such as mice or rats. A "human antibody" is typically different from the system used to obtain the human antibody and the human immunoglobulin locus, and may include the intentional introduction of somatic mutations or substitutions into the framework or CDRs. Such exemplary systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals that carry the human immunoglobulin locus, such as mice or rats. A "human antibody" is typically different from the system used to obtain the human antibody and the human immunoglobulin locus, and may include the intentional introduction of somatic mutations or substitutions into the framework or CDRs. A "human antibody" is typically different from the system used to obtain the human antibody and the human immunoglobulin locus, and may include the intentional introduction of somatic mutations or substitutions into the framework or CDRs. or the intentional introduction of somatic mutations or substitutions into the framework or CDRs. ​or both, contain amino acid differences when compared to immunoglobulins expressed in humans. Typically, a "human antibody" has an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence encoded by a human germline immunoglobulin or a rearranged immunoglobulin gene. In some cases, a "human antibody" is, for example, a consensus framework sequence obtained from human framework sequence analysis as described in Knappik et al., (2000) J Mol Biol 296:57-86, or, for example, a synthetic HCDR3 incorporated into a phage-displayed human immunoglobulin gene library as described in Shi et al., (2010) J MolBiol 397:385-96 and International Publication No. WO 2009 / 085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of a "human antibody". "Recombinant" refers to DNA, antibodies, and other proteins that are prepared, expressed, produced, or isolated by

[0044] recombinant means when joining segments from different sources to produce recombinant DNA, antibodies, or proteins. "Bispecific" refers to an antibody that specifically binds to two different antigens or two different epitopes in

[0045] the same antigen. Bispecific antibodies may bind to other related antigens, such as human or non-human primate, e.g., Macaca cynomolgus (cynomolgus monkey, cyno) or Pan ​having cross - reactivity against the same antigen of other species (homologs) such as Pan troglodytes, or being able to bind to epitopes shared between two or more different antigens or being able to bind to epitopes shared between two or more different antigens.

[0046] "Multispecificity" refers to an antibody that specifically binds to two or more different antigens or two or more different epitopes within the same antigen. Multispecific antibodies may have cross - reactivity against the same antigen (homolog) from other species such as humans or monkeys, for example, Macaca cynomolgus (cynomolgus monkey, cyno) or Pan troglodytes, or be able to bind to epitopes shared between two or three or more different antigens. or Pan troglodytes, or be able to bind to epitopes shared between two or three or more different antigens. from other species such as humans or monkeys, for example, Macaca cynomolgus (cynomolgus monkey, cyno), or Pan troglodytes, or be able to bind to epitopes shared between two or three or more different antigens. from other species such as humans or monkeys, for example, Macaca cynomolgus (cynomolgus monkey, cyno), or Pan troglodytes, or be able to bind to epitopes shared between two or three or more different antigens. from other species such as humans or monkeys, for example, Macaca cynomolgus (cynomolgus monkey, cyno), or Pan troglodytes, or be able to bind to epitopes shared between two or three or more different antigens.

[0047] "Macrophage" means a cell of bone - marrow origin. Macrophages are large white blood cells that are mainly present in connective tissue and in the bloodstream or are resident in tissues or the tumor microenvironment. It has the ability to ingest foreign substances and infectious microorganisms by phagocytosis and has antigen - presenting ability. Inactivated macrophages derived from precursors undergo specific differentiation according to the local tissue environment. Inactivated macrophages differentiate into different functional phenotypes in response to environmental factors within tissues such as damaged cells, activated lymphocytes, or microbial products. For example, monocytes in the blood can enter tissues during inflammation or invasion and polarize into M1 or M2 phenotypes depending on the local microenvironment. The M1 macrophage phenotype is characterized by the production of high levels of inflammation - promoting cytokines, the ability to mediate resistance to pathogens the ability to mediate resistance to pathogens, strong microbicidal properties, high production of reactive nitrogen and oxygen intermediates the promotion of Th1 responses, and the killing of pathogens and tumor cells. In contrast into M1 or M2 phenotypes depending on the local microenvironment. The M1 macrophage phenotype is characterized by the production of high levels of inflammation - promoting cytokines, the ability to mediate resistance to pathogens the ability to mediate resistance to pathogens, strong microbicidal properties, high production of reactive nitrogen and oxygen intermediates the promotion of Th1 responses, and the killing of pathogens and tumor cells. In contrast the promotion of Th1 responses, and the killing of pathogens and tumor cells. In contrast M2 macrophages are characterized by their involvement in parasite control, tissue remodeling, immune regulation, tumor promotion, and efficient phagocytic activity. M2 macrophages are further classified into four different subtypes called M2a, M2b, M2 c, and M2d. "Macrophage" includes all macrophage subtypes.

[0048] "Monocyte" refers to CD14 + CD34 - mononuclear leukocytes that belong to the types of white blood cells involved in the first-line defense mechanism and are recognized as being capable of differentiating into dendritic cells or macrophage precursors. Monocytes usually move within the blood system. In response to external stimulus signals, monocytes secrete cytokines, which are many immunomodulatory substances, move to the infection site or tumor site in the tissue, and differentiate into macrophages. Specifically, monocytes express elevated levels of the CD14 surface antigen marker and can express at least one biomarker selected from CD64, CD93, CD180, CD328, CD329, or peanut agglutinin protein (PNA).

[0049] "Enhance" or "induce" means enhancing one or more functions or activities of macrophages by 10%, 20%, 30%, 4 0%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 9 7%, 98%, 99%, or more than 100%, or statistically significantly, as compared to a control (e.g., enhancement in the presence or absence of an agent that enhances macrophage activity).

[0050] "Enhancing macrophage activity" means phenotypic changes of monocytes and / or macrophage Inducing differentiation of macrophages into rophages or activating inactivated macrophages Refers to enhancing the activity of one or more macrophages.

[0051] "Macrophage activity" refers to phagocytosis, antigen presentation, production of IL-12, IL-1, TNFα, or production of CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9 , CXCL10, CCL2, CCL3, CCL4, CCL11, CCL17, CCL22 and refers to any macrophage function such as production of inflammatory chemokines.

[0052] "Agents that enhance macrophage activity" can be small molecules, peptides, oligopeptides, poly peptides, proteins, antibodies, synthetic binding molecules, aptamers, RNA molecules, DNA molecules, o ligomers, polymers, lipids, or liposomes. Exemplary agents that enhance macrophage function include cytokines, chemokines, pattern recognition receptor ligands, hormones, adrenergic and cholinergic agonists, fatty acids, phospholipids, immunoglobulins or parts thereof, the Fc domain of immunoglobulins, lipopolysaccharides (LPS), toll-like receptor (TLR) ligands, histamine, and peroxisome proliferator-activated receptor ligands.

[0053] "Troggocytosis" refers to a process characterized by the movement of a part of the cell membrane from a donor cell to an acceptor cell. Typical acceptor cells include macrophages and monocytes. Further acceptor cells include NK cells, dendritic cells, T cells, B cells, and neutrophils. The movement mediated by troggocytosis of a part of the cell membrane is by , for example, membrane proteins such as EGFR or c-Met, or antibodies, may involve the movement of antibody-antigen complexes bound to cell surface molecules. Antibody-mediated trogocytosis can occur when the Fc portion of the antibody binds to the Fcγ receptor (FcγR) expressed on the acceptor cell.

[0054] "Antibody-mediated trogocytosis of anti-EGFR / c-Met antibodies" refers to the trogocytosis of the EGFR and / or c-Met-containing portion of the cell membrane from donor cells to acceptor cells mediated by anti-EGFR / c-Met antibodies bound to EGFR and / or c-Met on the donor cell membrane.

[0055] "Threshold value" refers to the level of macrophages or monocytes that is equal to or higher than the approximately 30th percentile value of macrophages or monocytes observed in biological samples from a population of subjects with EGFR- or c-Met-positive cancer.

[0056] "Agonist" refers to a molecule that, when bound to a cellular protein, induces at least one reaction or activity induced by the natural ligand of the protein. When at least one reaction or activity is induced by at least about 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more than at least one reaction or activity induced in the absence of the agonist (e.g., negative control), or when the induction is statistically significant compared to the induction in the absence of the agonist, the molecule is an agonist.

[0057] "Antagonist" or "inhibitor" refers to a molecule that, when bound to a cellular protein, suppresses at least one reaction or activity induced by the natural ligand of the protein. A molecule is considered an antagonist when it suppresses at least one reaction or activity by at least about 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more than at least one reaction or activity that is suppressed in the absence of the antagonist (e.g., in a negative control), or when the suppression is statistically significant compared to the suppression in the absence of the antagonist. "PD-(L)1 axis inhibitor" refers to a molecule that inhibits PD-1 downstream signaling. A PD-(L)1 axis inhibitor can be a molecule that binds to PD-1, PD-L1, or PD-L2. "Biological sample" refers to a similar fluid, cell, or tissue isolated from a subject, as well as an aggregate of fluids, cells, or tissues present in the body of the subject. Exemplary samples include biological fluids such as blood, serum, and serous fluid, plasma, lymph, urine, saliva, cyst fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleural cavity, pericardial cavity, peritoneal cavity, abdominal cavity, and other body cavity fluids, fluids recovered by bronchial lavage, synovial fluid, cell and organ culture media containing a conditioned medium of a subject or biological origin, such as cells or organs, liquid solutions contacted with a wash solution, etc., tissue biopsy materials, tumor tissue biopsy materials, tumor tissue samples, fine needle aspiration, surgically excised tissues, organ culture fluids, or cell culture fluids.

[0058]

[0059]

[0060] ​​​​​​​​​​​​​​​"Low fucose" or "low fucose content", as used herein, refers to an antibody having a fucose content of about 1% - 15%.

[0061] "Normal fucose" or "normal fucose content", as used herein, refers to an antibody having a fucose content of greater than about 50%, typically greater than about 80%, or greater than 85%.

[0062] The methods of the present disclosure JNJ-61186372 (JNJ-372) is an IgG1 anti-EGFR / c-Met bispecific antibody described in U.S. Patent No. 9,593,164. Previous studies have shown that JNJ-372 inhibits tumor growth and progression by three different mechanisms: inhibition of ligand-induced activation by blocking ligand binding to each receptor, inactivation of the receptor via degradation, and killing of EGFR- and c-Met-expressing tumors mediated by Fc effectors of ADCC and ADCP (Moores et al., Cancer Research 76(13), 2016.; Published online May 23, 2016; DOI: 10.1158 / 0008-5472).

[0063] The present invention is based at least in part on the surprising finding that the JNJ-372 Fc interaction not only mediates ADCC and ADCP, but also enhances the inhibition of JNJ-372-mediated EGFR / c-Met signaling, and that monocytes or macrophages are sufficient and necessary for the anti-tumor effect mediated by JNJ-372 through trogocytosis.

[0064] ​While not wishing to be bound by any theory, based on the surprising results disclosed herein, it can be predicted that the level of monocytes in a patient's blood may show a positive correlation with the level of macrophages in the patient's tumor, from which a better response to JNJ-372 can be predicted. Similarly, tumor tissue samples with increased levels of macrophages or increased levels of FcγRI or FcγRIIIa by IHC or immune gene signature can be predicted to respond better to JNJ-372 by providing more immune cell interactions. Also, based on the results described herein, it can be predicted that treatments that enhance macrophage activity used in combination with JNJ-372 can increase the overall efficacy of JNJ-372. For example, treatment with GM-CSF can drive the differentiation of circulating monocytes into tumor-associated macrophages, whereby the efficacy of JNJ-372 can be increased. Treatment with anti-CD47 therapy can block the negative inhibition of macrophages, whereby macrophages can be activated to enhance JNJ-372 activity. Similarly, inhibition of the PD-(L)1 axis, inhibition of HDAC, or stimulation of CD11b can shift the polarization of tumor-associated macrophages, resulting in higher macrophage activity and thus acting synergistically with JNJ-372 treatment to enhance tumor killing. Based on the surprising results disclosed herein, it can be predicted that the level of monocytes in a patient's blood may show a positive correlation with the level of macrophages in the patient's tumor, from which a better response to JNJ-372 can be predicted. Similarly, tumor tissue samples with increased levels of macrophages or increased levels of FcγRI or FcγRIIIa by IHC or immune gene signature can be predicted to respond better to JNJ-372 by providing more immune cell interactions. Also, based on the results described herein, it can be predicted that treatments that enhance macrophage activity used in combination with JNJ-372 can increase the overall efficacy of JNJ-372. For example, treatment with GM-CSF can drive the differentiation of circulating monocytes into tumor-associated macrophages, whereby the efficacy of JNJ-372 can be increased. Treatment with anti-CD47 therapy can block the negative inhibition of macrophages, whereby macrophages can be activated to enhance JNJ-372 activity. Similarly, inhibition of the PD-(L)1 axis, inhibition of HDAC, or stimulation of CD11b can shift the polarization of tumor-associated macrophages, resulting in higher macrophage activity and thus acting synergistically with JNJ-372 treatment to enhance tumor killing. This identification of a novel mechanism enables the selection of patients who may respond better to JNJ-372 based on the relative or absolute monocyte and / or macrophage amounts in the patient's blood or tumor samples. Also, based on the results described herein, it can be predicted that treatments that enhance macrophage activity used in combination with JNJ-372 can increase the overall efficacy of JNJ-372. For example, treatment with GM-CSF can drive the differentiation of circulating monocytes into tumor-associated macrophages, whereby the efficacy of JNJ-372 can be increased. Treatment with anti-CD47 therapy can block the negative inhibition of macrophages, whereby macrophages can be activated to enhance JNJ-372 activity. Similarly, inhibition of the PD-(L)1 axis, inhibition of HDAC, or stimulation of CD11b can shift the polarization of tumor-associated macrophages, resulting in higher macrophage activity and thus acting synergistically with JNJ-372 treatment to enhance tumor killing. This identification of a novel mechanism enables the selection of patients who may respond better to JNJ-372 based on the relative or absolute monocyte and / or macrophage amounts in the patient's blood or tumor samples. Also, based on the results described herein, it can be predicted that treatments that enhance macrophage activity used in combination with JNJ-372 can increase the overall efficacy of JNJ-372. For example, treatment with GM-CSF can drive the differentiation of circulating monocytes into tumor-associated macrophages, whereby the efficacy of JNJ-372 can be increased. Treatment with anti-CD47 therapy can block the negative inhibition of macrophages, whereby macrophages can be activated to enhance JNJ-372 activity.

[0065] This identification of a novel mechanism enables the selection of patients who may respond better to JNJ-372 based on the relative or absolute monocyte and / or macrophage amounts in the patient's blood or tumor samples. Also, based on the results described herein, it can be predicted that treatments that enhance macrophage activity used in combination with JNJ-372 can increase the overall efficacy of JNJ-372. and a combination therapy using a molecule that enhances macrophage activity in combination with JNJ-372 to provide a criterion for

[0066] The present disclosure provides a method for treating a subject having EGFR- or c-Met-expressing cancer, comprising administering to the subject a therapeutically effective amount of an isolated bispecific epidermal growth factor receptor (EGFR) / hepatocyte growth factor receptor (c-Met) antibody in combination with an agent that enhances macrophage activity in the subject.

[0067] The present disclosure also provides a method for diagnosing and treating a subject having EGFR- or c-Met-expressing cancer responsive to treatment with a bispecific anti-EGFR / c-Met antibody, comprising providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and diagnosing that the subject has EGFR- or c-Met-expressing cancer responsive to treatment with a bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold value, and administering or providing for administration of a bispecific anti-EGFR / c-Met antibody to the subject diagnosed as responsive to treatment with an anti-EGFR / c-Met antibody.

[0068] The present disclosure also provides a method for treating a subject suspected of having or having EGFR- or c-Met-expressing cancer with a bispecific anti-EGFR / c-Met antibody, comprising determining that the subject has a macrophage or monocyte level higher than a threshold value, and administering or providing for administration of a bispecific anti-EGFR / c-Met antibody to the subject determined to have a macrophage or monocyte level higher than the threshold value. ​​​​​​​​​​​​​​

[0069] The present disclosure also provides a method for predicting the response of a subject having EGFR or c-Met-expressing cancer to treatment with a bispecific anti-EGFR / c-Met antibody, the method comprising providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and predicting that the subject is a responder when the macrophage or monocyte level from the biological sample is higher than a threshold. The present disclosure also provides a method for predicting the response of a subject having EGFR or c-Met-expressing cancer to treatment with a bispecific anti-EGFR / c-Met antibody, the method comprising providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and predicting that the subject is a responder when the macrophage or monocyte level from the biological sample is higher than a threshold. providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and predicting that the subject is a responder when the macrophage or monocyte level from the biological sample is higher than a threshold. providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and predicting that the subject is a responder when the macrophage or monocyte level from the biological sample is higher than a threshold. The present disclosure also provides a method for treating a subject having EGFR or c-Met-expressing cancer who responds to treatment with a bispecific anti-EGFR / c-Met antibody, the method comprising providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and treating the subject with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold.

[0070] The present disclosure also provides a method for treating a subject having EGFR or c-Met-expressing cancer who responds to treatment with a bispecific anti-EGFR / c-Met antibody, the method comprising providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and treating the subject with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold. providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and treating the subject with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold. providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and treating the subject with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold. providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and treating the subject with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold. The present disclosure also provides a method for determining whether a subject having EGFR or c-Met-expressing cancer will respond to treatment with a bispecific anti-EGFR / c-Met antibody and for determining whether to treat the subject, the method comprising providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and diagnosing that the subject having EGFR or c-Met-expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold, or diagnosing that the subject having EGFR or c-Met-expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is lower than a threshold.

[0071] The present disclosure also provides a method for determining whether a subject having EGFR or c-Met-expressing cancer will respond to treatment with a bispecific anti-EGFR / c-Met antibody and for determining whether to treat the subject, the method comprising providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and diagnosing that the subject having EGFR or c-Met-expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold, or diagnosing that the subject having EGFR or c-Met-expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is lower than a threshold. The present disclosure also provides a method for determining whether a subject having EGFR or c-Met-expressing cancer will respond to treatment with a bispecific anti-EGFR / c-Met antibody and for determining whether to treat the subject, the method comprising providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and diagnosing that the subject having EGFR or c-Met-expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold, or diagnosing that the subject having EGFR or c-Met-expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is lower than a threshold. providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and diagnosing that the subject having EGFR or c-Met-expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold, or diagnosing that the subject having EGFR or c-Met-expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is lower than a threshold. providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and diagnosing that the subject having EGFR or c-Met-expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold, or diagnosing that the subject having EGFR or c-Met-expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is lower than a threshold. providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and diagnosing that the subject having EGFR or c-Met-expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold, or diagnosing that the subject having EGFR or c-Met-expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is lower than a threshold. providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and diagnosing that the subject having EGFR or c-Met-expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold, or diagnosing that the subject having EGFR or c-Met-expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is lower than a threshold. providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and diagnosing that the subject having EGFR or c-Met-expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold, or diagnosing that the subject having EGFR or c-Met-expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is lower than a threshold. providing a biological sample from the subject, measuring the macrophage or monocyte level from the biological sample, and diagnosing that the subject having EGFR or c-Met-expressing cancer will respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is higher than a threshold, or diagnosing that the subject having EGFR or c-Met-expressing cancer will not respond to treatment with the bispecific anti-EGFR / c-Met antibody when the macrophage or monocyte level from the biological sample is lower than a threshold. Diagnosing as such, and diagnosing as responsive to treatment with a bispecific anti-EGFR / c-Met antibody Administering to a subject diagnosed as being a target for the bispecific anti-EGFR / c-Met antibody, or refraining from administering the bispecific anti-EG FR / c-Met antibody to a subject diagnosed as non-responsive to treatment with the bispecific anti-EGFR / c-Met antibody, and a method comprising the same is also provided.

[0072] The "level" of macrophages or monocytes can be qualitative (e.g., presence or absence) or quantitative (e.g., absolute cell number, relative number, percentage (%) from total cell number, or positive cell % within the field of view). In some embodiments, macrophages or monocytes are not present in the biological sample. In some embodiments, the level of macrophages or monocytes is above the average value of macrophages or monocytes observed in biological samples from healthy subjects. In some embodiments, the level of macrophages or monocytes is the approximately 3 0th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. In some embodiments, the level of macrophages or monocytes is the approximately 35th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. In some embodiments, the level of macrophages or monocytes is the approximately 40th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. In some embodiments, the level of macrophages or monocytes is the approximately 45th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. In some embodiments, the level of macrophages or monocytes is the approximately 45th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. In some embodiments, the level of macrophages or monocytes is the approximately 45th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. 0th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. In some embodiments, the level of macrophages or monocytes is the approximately 35th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. In some embodiments, the level of macrophages or monocytes is the approximately 40th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. In some embodiments, the level of macrophages or monocytes is the approximately 45th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. In some embodiments, the level of macrophages or monocytes is the approximately 45th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. In some embodiments, the level of macrophages or monocytes is the approximately 45th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. In some embodiments, the level of macrophages or monocytes is the approximately 45th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. In some embodiments, the level of macrophages or monocytes is the approximately 45th percentile value of macrophages or monocytes observed in biological samples from subjects having EGFR- or c-Met-positive cancers. It is a percentile value. In some embodiments, the level of macrophages or monocytes is , the approximately 50th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 60th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 65th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 70th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 75th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 80th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 85th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 90th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 70th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 75th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 80th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 85th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 90th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 70th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 75th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 80th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 85th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is , the approximately 90th percentile value of macrophages or monocytes observed in a biological sample from a subject having an EGFR or c-Met positive cancer. In some embodiments, In an embodiment, the level of macrophages or monocytes is about the 95th percentile value of macrophages or monocytes observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments, the level of macrophages or monocytes is about the 100th percentile value of macrophages or monocytes observed in a biological sample from a subject having EGFR or c-Met positive cancer. The level of macrophages or monocytes in a subject having EGFR or c-Met expressing cancer can be compared to the level of macrophages or monocytes in a biological sample from a healthy subject. The increased level of macrophages or monocytes can be, for example, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, or about 10-fold higher when compared to the level of macrophages or monocytes in a biological sample from a healthy subject.

[0073] Macrophages can be identified, for example, by using CD68, iNOS (inducible nitric oxide synthase), and CD163 as markers for pan-macrophages, M1 macrophages, or M2 macrophages, respectively, and evaluating the percentage of the area of positive staining, and comparing it to non-tumor tissue, for example, from a tumor tissue biopsy specimen obtained from a subject having an EGFR or c-Met expressing tumor (e.g., it has been described that the area % of positive staining for CD68 is increased 2-fold relative to non-tumor in adenocarcinoma, squamous cell, or large cell carcinoma, Almatoodi et al., Cancer Micreoenvironment 9:1-11).

[0074] i et al., Cancer Micreoenvironment 9:1-11 , see 2016). Macrophages can be obtained, for example, from tumor tissue biopsy materials from a subject having an EGFR or c-Met expressing tumor using an immune gene signature . .

[0075] Monocytes can be identified from blood samples from a subject having an EGFR or c-Met expressing tumor using the monocyte marker CD14 and fluorescence-activated cell sorting .

[0076] In some embodiments, the biological sample is a blood sample

[0077] In some embodiments, the biological sample is a tumor tissue biopsy material

[0078] Similarly, the levels of FcγRI or FcγRIIIa may be used to predict a patient's response to JNJ-372 by providing more immune cell interactions . .

[0079] In some embodiments, the level of FcγRI or FcγRIIIa is above the mean value of the levels of FcγRI or FcγRIIIa observed in biological samples from healthy subjects . In some embodiments, the level of FcγRI or FcγRIIIa is about the 30th percentile value of the levels of FcγRI or FcγRIIIa observed in biological samples from subjects having EGFR or c-Met positive cancer . In some embodiments, the level of FcγRI or FcγRIIIa is about the 35th percentile value of the levels of FcγRI or FcγRIIIa observed in biological samples from subjects having EGFR or c-Met positive cancer . In some embodiments, the level of FcγRI or FcγRIIIa is ​​​​, the level of FcγR I or FcγRIIIa is about the 40th percentile value observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments , the level of FcγRI or FcγRIIIa is about the 45th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments , the level of FcγRI or FcγRIII a is about the 50th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments, the level of FcγRI or FcγRIIIa is about the 60th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Me t positive cancer. In some embodiments , the level of FcγRI or FcγRIIIa is about the 65th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments , the level of FcγRI or FcγRIIIa is about the 70th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments , the level of FcγRI or FcγRIIIa is about the 75th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments , the level of FcγRI or FcγRIIIa is about the 75th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments , the level of FcγRI or FcγRIIIa is about the 70th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments , the level of FcγRI or FcγRIIIa is about the 65th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments , the level of FcγRI or FcγRIIIa is about the 60th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments , the level of FcγRI or FcγRIIIa is about the 50th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments , the level of FcγRI or FcγRIIIa is about the 45th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments , the level of FcγRI or FcγRIIIa is about the 40th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments RI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. It is about the 80th percentile value of the level of RI or FcγRIIIa. In some embodiments the level of FcγRI or FcγRIIIa is the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer about the 85th percentile value. In some embodiments, the level of FcγRI or FcγRII Ia is about the 90th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-Met positive cancer. In some embodiments, the level of FcγRI or FcγRIIIa is about the 95th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-M et positive cancer. In some embodiments, the level of FcγRI or FcγRIIIa is about the 100th percentile value of the level of FcγRI or FcγRIIIa observed in a biological sample from a subject having EGFR or c-M et positive cancer. The level of FcγRI or FcγRIIIa can be measured using immunohistochemical examination in a tumor tissue sample (such as a fresh frozen or paraffin-embedded tumor tissue section). The Fc γRI or FcγRIIIa level can be expressed as the FcγRI or FcγRIII a cell rate (%) within the microscopic field of view. The level of FcγRI or FcγRIIIa can also be measured at the gene expression level using RNA isolated from a tumor tissue sample as part of an immunogenetic signature panel or as an individual gene.

[0080]

[0081] ​​​​​​In some embodiments, the bispecific anti-EGFR / c-Met antibody is comprised of a first domain that binds to EGFR and that includes the heavy chain complementary determining region 1 (HCDR1) of SEQ ID NO: 1, the HCDR2 of SEQ ID NO: 2, the HCDR3 of SEQ ID NO: 3, the light chain complementary determining region 1 (LCDR1) of SEQ ID NO: 4, the LCDR2 of SEQ ID NO: 5, and the LCDR3 of SEQ ID NO: 6, and a second domain that binds to c- Met and that includes the HCDR1 of SEQ ID NO: 7, the HCDR2 of SEQ ID NO: 8, the HCDR3 of SEQ ID NO: 9, the LCDR1 of SEQ ID NO: 10, the LCDR2 of SEQ ID NO: 11, and the LCDR3 of SEQ ID NO: 12.

[0082] In some embodiments, the first domain that binds to EGFR includes the heavy chain variable region (VH) of SEQ ID NO: 13 and the light chain variable region (VL) of SEQ ID NO: 14, and the second domain that binds to c-Met includes the VH of SEQ ID NO: 15 and the VL of SEQ ID NO: 16.

[0083] In some embodiments, the bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype. There are several diversities within the IgG1 constant domain (e.g., well-known allotypes), and having diversity at positions 214, 356, 358, 422, 431, 435, or 436 (numbering of residues according to the EU numbering; see, e.g., the IMGT Web resource; IMGT Repertoire (IG and TR); Proteins and Alleles; allotypes). The bispecific anti-EGFR / c-Me t antibody can be any IgG1 allotype of G1m17, G1m3, G1m1, G1m2, G1m27, or G1m28.

[0084] In some embodiments, the bispecific anti-EGFR / c-Met antibody is of SEQ ID NO: 17 first heavy chain (HC1), first light chain (LC1) of SEQ ID NO: 18, second heavy chain (HC2) of SEQ ID NO: 19, and second light chain (LC2) of SEQ ID NO: 20.

[0085] In some embodiments, the agent for enhancing macrophage activity is GM-CSF, CD 47 antagonist, anti-CD47 antibody, HDAC inhibitor, PD-(L)1 axis inhibitor, or CD11b agonist.

[0086] In some embodiments, the agent for enhancing macrophage activity is GM-CSF .

[0087] In some embodiments, the agent for enhancing macrophage activity is an anti-CD47 antag onist.

[0088] In some embodiments, the agent for enhancing macrophage activity is an anti-CD47 antibody .

[0089] In some embodiments, the agent for enhancing macrophage activity is an HDAC inhibitor .

[0090] In some embodiments, the agent for enhancing macrophage activity is a PD-(L)1 axis inh ibitor.

[0091] In some embodiments, the agent for enhancing macrophage activity is a CD11b agonis t.

[0092] In some embodiments, the HDAC inhibitor is an HDAC2 inhibitor.

[0093] Exemplary CD47 antagonists include CD47 ligand-Fc fusions, such as TTI 621 and SIRPα-Fc fusions such as anti-CD47 antibodies.

[0094] Exemplary anti-CD47 antibodies are Hu5F9-G4, TI-061, TTI-622, AO-176, IBI-188, AL X-148, SRF-231, CC-90002, and anti-CD47 antibodies, as disclosed in International Publication No. WO 2016 / 081423.

[0095] Exemplary HDAC inhibitors include vorinostat, romidepsin, tidamide, panobinostat belinostat, pracinostat, abexinostat, entinostat, panobinostat, GSK-2879552, ricolinostat, iadademstat, domatinostat, resminostat, AZD-9468, nanatinostat, CG-2 00745, mocetinostat, INCB-59872, IMG-7289, chinostam stin, RDN-929, YM-753, HG-146, NBM-BMX, TAK-41 8, securidemstat, CKD-504, CKD-506, CC-90011, KA- 2507, and citrinostat.

[0096] Exemplary PD-(L)1 axis inhibitors include antibodies that bind to PD-1, such as nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), sintilimab, semaprilimab (LIBTAYO®), tripolizumab, tislelizumab, spartalizumab, camrelizumab, dostarlimab, genolimzumab, or cetrelimab, or antibodies that bind to PD-L1. For example, PD-1 antibodies include enbafosumab Remab, atezolizumab (TECENTRIQ®), durvalumab (IMF INZI®), and avelumab (BAVENCIO®).

[0097] Commercially available antibodies can be purchased through authorized vendors or pharmacies. Small molecule amino acid sequence structures can be found from USAN and / or I NN deposits by companies from the CAS registry.

[0098] In some embodiments, EGFR- or c-Met-expressing cancers are related to wild-type EGFR, EG FR activating mutations, EGFR gene amplifications, elevated levels of circulating HGF, wild-type c-Met, c-Met activating mutations, c-Met gene amplifications, or mutant KRAS.

[0099] Exemplary EGFR activating mutations that may be related to cancer include at least one of the biological activities of EGFR such as increased tyrosine kinase activity , formation of receptor homo- and heterodimers, enhanced ligand binding, point mutations, deletion mutations, insertion mutations, inversions, or gene amplifications that increase at least one of the biological activities of EGFR. The mutations can be located in any part of the EGFR gene or the regulatory region associated with the EGFR gene, and include mutations in exons 18, 19, 20, or 21 or mutations in the kinase domain . Other examples of EGFR activating mutations are known in the art (see, for example, US Patent Application Publication No. 2005 / 0272083 ). Information regarding EGFR and other ErbB receptors, including receptor homo- and heterodimers, receptor ligands, autophosphorylation sites, and signaling molecules involved in signal transduction mediated by ErbB is known in the art (see, for example, Hynes and ). receptors involved in signal transduction mediated by ErbB are known in the art (see, for example, Hynes and in the art (see, for example, Hynes and Lane, Nature Reviews Cancer 5:341-354, 20 See 05).

[0100] In some embodiments, the EGFR activating mutation is L718Q, G719A, G719 X (where X is any amino acid), L861X (where X is any amino acid), L8 58R, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, L858P, or substitution of T790M, deletion of E746 - A750, R7 deletion of 48 - P753, insertion of Ala (A) between M766 and A767, Ser, Val, and Ala (SVA) between S768 and V769, insertion of Asn and Ser (NS) between P772 and H773 insertion of one or more amino acids between D761 and E762, A763 and Y764 , Y764 and Y765, M766 and A767, A767 and V768, S768 and V769 , V769 and D770, D770 and N771, N771 and P772, P772 and H773 , insertion of one or more amino acids between H773 and V774, V774 and C775 insertion, one or more deletions, or one or more insertions in EGFR exon 20 is.

[0101] Exemplary c-Met activating mutations include point mutations, deletion mutations, insertion mutations, inversions, or gene amplifications that increase at least one biological activity of the c-Met protein, such as an increase in tyrosine kinase activity, formation of receptor homodimers and heterodimers, and enhanced ligand binding. The mutation can be located in any part of the c-Met gene or in regulatory regions associated with the gene, such as mutations in the kinase domain of c-Met. Exemplary c-Met activating ​​The mutations are at residue positions N375, V13, V923, R175, V136, L229, S32 3, R988, S1058 / T1010, and E168. Methods for detecting mutations or gene amplifications of EGFR and c-Met are well known.

[0102] In some embodiments, the mutant KRAS has a substitution of G12V, G12C, or G12A.

[0103] In some embodiments, the subject has newly diagnosed EGFR or c-Met expressing cancer.

[0104] In some embodiments, the subject having newly diagnosed EGFR or c-Met expressing cancer has one or more EGFR exon 20 mutations. Exon 20 mutations (insertions of one or more amino acids) are generally resistant to EGFR tyrosine kinase inhibitors (TKIs) (see, e.g., WO 2018 / 094225).

[0105] In some embodiments, the subject is resistant or has acquired resistance to treatment with a previous anti-cancer therapy.

[0106] In some embodiments, the previous anti-cancer therapy is chemotherapy, targeted anti-cancer therapy, or a kinase inhibitor.

[0107] In some embodiments, the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HER3, HER4, VEGFR, or AXL.

[0108] In some embodiments, the kinase inhibitor is erlotinib, gefitinib, lapatinib. B, vandetanib, afatinib, osimertinib, lazertinib, poziotinib, cle otinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib , regorafenib, pazopanib, sorafenib, or sunitinib.

[0109] In some embodiments, the subject is resistant to or has acquired resistance to an EGFR inhibitor. Exemplary EGFR inhibitors to which cancer can acquire resistance are anti-EGFR antibodies such as cetuximab (ERBITUX®), panitumumab (VECTIBI X®), matuzumab, nimotuzumab, the small molecule EGFR inhibitor erlotinib (TARCEVA®), gefitinib (IRESSA®), EK B-569 (peritinib, an irreversible EGFR TKI), the pan-ErbB and other receptor tyrosine kinase inhibitor lapatinib (an EGFR and HER2 inhibitor), peritinib (E GFR and HER2 inhibitor), vandetanib (ZD6474, ZACTIMA™, an EGFR, VEGFR2, and RET TKI), PF00299804 (dacomitinib , an irreversible pan-ErbB TKI), CI-1033 (an irreversible pan-erbB TKI), a fatinib (BIBW2992, an irreversible pan-ErbB TKI), AV-412 (a dual E GFR and ErbB2 inhibitor), EXEL-7647 (an inhibitor of EGFR, ErbB2, GEVG R, and EphB4), CO-1686 (an irreversible mutant-selective EGFR TKI ), AZD9291 (an irreversible mutant-selective EGFR TKI), and HKI-272 (neratinib, an irreversible EGFR / ErbB2 inhibitor).

[0110] Using various qualitative and / or quantitative methods, whether a subject is resistant to treatment by anti-cancer therapy, expressing resistance, or prone to expressing resistance can be determined. Symptoms that may be associated with resistance to anti-cancer therapy include a decrease in the health or steady state of the patient, an increase in tumor size, a halt or deceleration in the reduction of tumor growth, and / or the spread of cancerous cells in the body from one location to other organs, tissues, or cells. Re-establishment or worsening of various cancer-related symptoms such as loss of appetite, cognitive impairment, depression, dyspnea, fatigue, hormonal disturbances, neutropenia, pain, peripheral neuropathy, and sexual dysfunction can also be indicators of whether a subject is expressing or prone to expressing resistance to anti-cancer therapy. Cancer-related symptoms can vary depending on the type of cancer. For example, symptoms associated with cervical cancer include abnormal bleeding, abnormal and excessive vaginal discharge, pelvic pain, bladder pain, or pain during urination not related to the normal menstrual cycle, and bleeding after intercourse, vaginal douching, or pelvic examination during regular menstrual periods. Symptoms associated with lung cancer can include persistent cough, hemoptysis, shortness of breath, stabbing chest pain, loss of appetite, unintentional weight loss, and fatigue. Symptoms of liver cancer can include loss of appetite and weight, abdominal pain, particularly in the upper right part of the abdomen that can radiate to the back and shoulders, nausea and vomiting, general weakness and fatigue, hepatomegaly, abdominal distension (ascites), and yellowing of the skin and sclera (jaundice). Those skilled in oncology can easily identify the symptoms associated with a specific type of cancer. In some embodiments, EGFR- and c-Met-expressing cancers are epithelial cell cancers, breast cancers

[0111] , ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, small cell lung cancer, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharyngeal cancer, nasal cancer , pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer , testicular cancer, gastric cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular cancer (H CC), or sporadic or hereditary papillary renal cell cancer (PRCC).

[0112] In some embodiments, the EGFR- or c-Met-expressing cancer is an epithelial cell cancer. In some embodiments, the EGFR- or c-Met-expressing cancer is breast cancer. In some embodiments, the EGFR- or c-Met-expressing cancer is ovarian cancer. In some embodiments , the EGFR- or c-Met-expressing cancer is lung cancer. In some embodiments , the EGFR- or c-Met-expressing cancer is non-small cell lung cancer (NSCLC). In some embodiments, the EGFR- or c-Met-expressing cancer is lung adenocarcinoma. In some embodiments , the EGFR- or c-Met-expressing cancer is small cell lung cancer. In some embodiments , the EGFR- or c-Met-expressing cancer is colorectal cancer. In some embodiments , the EGFR- or c-Met-expressing cancer is anal cancer. In some embodiments, the EGFR- or c-Met-expressing cancer is prostate cancer. In some embodiments, EG FR- or c-Met-expressing cancer is kidney cancer. In some embodiments, EGFR or c-Met-expressing cancer is bladder cancer. In some embodiments, EGFR or c- Met-expressing cancer is head and neck cancer. In some embodiments, EGFR or c-Me The t-expressing cancer is a pharyngeal cancer. In some embodiments, the EGFR- or c-Met-expressing cancer is a nasal cancer. In some embodiments, the EGFR- or c-Met-expressing cancer is a pancreatic cancer. In some embodiments, the EGFR- or c-Met-expressing cancer is a skin cancer. In some embodiments, the EGFR- or c-Met-expressing cancer is an oral cancer and in some embodiments, the EGFR- or c-Met-expressing cancer is a tongue cancer. In some embodiments, the EGFR- or c-Met-expressing cancer is an esophageal cancer. In some embodiments, the EGFR- or c-Met-expressing cancer is a vaginal cancer. In some embodiments the EGFR- or c-Met-expressing cancer is a cervical cancer. In some embodiments the EGFR- or c-Met-expressing cancer is a spleen cancer. In some embodiments the EGFR- or c-Met-expressing cancer is a testicular cancer. In some embodiments, the EG FR- or c-Met-expressing cancer is a gastric cancer. In some embodiments, the EGFR- or c-Met-expressing cancer is a thymus cancer. In some embodiments, the EGFR- or c- Met-expressing cancer is a colon cancer. In some embodiments, the EGFR- or c-Met expressing cancer is a thyroid cancer. In some embodiments, the EGFR- or c-Met-expressing cancer is a liver cancer. In some embodiments, the EGFR- or c-Met-expressing cancer is hepatocellular carcinoma (HCC). In some embodiments, the EGFR- or c-Met-expressing can cer is a sporadic or hereditary papillary renal cell carcinoma (PRCC).

[0113] In some embodiments, NSCLC includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma Well. In some embodiments, the NSCLC cells have an epithelial phenotype. In some embodiments, NSCLC has acquired resistance to treatment with one or more EGFR inhibitors.

[0114] In NSCLC, specific mutations in the EGFR gene are associated with a high response rate (70 - 80%) to treatment with EGFR tyrosine kinase inhibitors (EGFR TKIs). Five - amino - acid deletions in exon 19 or the point mutation L858R in EGFR are associated with sensitivity to EGFR TKIs (Nakata and Gotoh, Expert Opin Ther Targets 16:771 - 781,2012). These mutations result in ligand - independent activation of EGFR kinase activity. Activation of EGFR mutations occurs in 10 - 30% of NSCLC patients and is significantly more common in East Asians, women, never - smokers, and patients with histological findings of adenocarcinoma (Janne and Johnson Clin Cancer Res 12(14 Suppl):4416s - 4420s,2006). EGFR gene amplification is also strongly correlated with response after EGFR TKI treatment (Cappuzzo et al., J Natl Cancer Inst 97:643 - 55,2005). EGFR exon 20 insertions are associated with EGFR TKI resistance.

[0115] Most NSCLC patients with EGFR mutations initially respond to EGFR TKI therapy, but virtually all acquire resistance that prevents a sustained response. Fifty to sixty percent of patients develop resistance due to a second - site is obtained. In almost all tumors that have become resistant to EGFR tyrosine kinase inhibitors, about 60% show increased c-Met expression, c-Met amplification, or increased HGF, the only known ligand for c-Met (Turke et al., Cancer Cell, 17:77-88, 2010).

[0116] In some embodiments, the subject is homozygous for phenylalanine at position 158 of CD16 or heterozygous for valine and phenylalanine at position 158 of CD16.

[0117] Subjects homozygous for phenylalanine at position 158 of CD16 have the FcγRIIIa-158F / F genotype. Subjects heterozygous for valine and phenylalanine at position 158 of CD16 have the FcγRIIIa-158F / V genotype. CD16 is also known as Fc gamma receptor IIIa (FcγRIIIa) or low-affinity immunoglobulin gamma Fc region receptor III-A isoform. The valine / phenylalanine ( V / F) polymorphism at position 158 of the FcγRIIIa protein residue has been shown to affect the FcγRIIIa affinity for human IgG. Receptors with the FcγRIIIa-158F / F or FcγRIIIa-158F / V polymorphism show reduced Fc binding and thus reduced ADCC when compared to FcγRIIIa-158V / V. The lack or low amount of fucose in human N-linked oligosaccharide improves the ability of the antibody to induce ADCC due to improved binding of the antibody to human FcγRIIIa (CD16) (Shields et al., J Bi ... ... ol, (ol Chem 277:26733~40, 2002).

[0118] In some embodiments, the bispecific anti-EGFR / c-Met antibody has a low fucose content of about 1% to about 10 %. The bispecific anti-EGFR / c-Me t antibody having a low fucose content may be more effective in the treatment of patients having the FcγRIIIa-158F / F or FcγRIIIa-158F / V gene type. Using routine methods, patients can be analyzed for Fcγ RIIIa polymorphism.

[0119] Antibodies having a low fucose content can be controlled by the osmotic pressure of the culture (Konno et al., Cytotechnology 64(:249-65, 2012), application of the variant CHO strain Lec13 as a host cell line (Shields et al., J Biol Chem 277:26733-26740, 2002), application of the variant CHO strain EB66 as a host cell line (Olivier et al., MAbs; 2(4), 2 010; electronic publication prior to printing, PMID: 20562582), application as a host cell line of the rat hybridoma cell line YB2 / 0 (Shinkawa et al., J B iol Chem 278:3466-3473, 2003), introduction of small interfering RNA specific for the α1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-908, 200 4), or introduction of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or kifunensine, a potent α-mannosidase I inhibitor et al., Biotechnol Bioeng 88:901-908, 2004), or introduction of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or kifunensine, a potent α-mannosidase I inhibitor 4), or introduction of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or kifunensine, a potent α-mannosidase I inhibitor 4), or introduction of β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II or kifunensine, a potent α-mannosidase I inhibitor Co-expression (Ferrara et al., J Biol Chem 281:5032- 5036, 2006, Ferrara et al., Biotechnol Bioe ng 93:851-861, 2006, Xhou et al., Biotechnol Bioeng 99;652-65, 2008), etc., can lead to the effective expression of a relatively highly defucosylated antibody having a branched complex-type Fc oligosaccharide and can be obtained using various methods that have been reported.

[0120] In some embodiments, the subject is further administered a third anti-cancer therapy.

[0121] In some embodiments, the third anti-cancer therapy is chemotherapy, targeted anti-cancer therapy, or a kinase inhibitor.

[0122] In some embodiments, the kinase inhibitor is an inhibitor of EGFR, c-Met, HER2, HE R3, HER4, VEGFR, or AXL. In some embodiments, the ki nase inhibitor is an inhibitor of EGFR. In some embodiments, the kinase inhibitor is an inhibitor of c-Met. In some embodiments, the kinase inhibitor is an inhibitor of HER2 . In some embodiments, the kinase inhibitor is an inhibitor of HER3. In some embodiments, the kinase inhibitor is an inhibitor of HER4. In some embodiments, the imple mentation form, the kinase inhibitor is an inhibitor of VEGFR. In some embodiments, the ki nase inhibitor is, or an inhibitor of AXL.

[0123] In some embodiments, the kinase inhibitor is erlotinib, gefitinib, lapatinib B, vandetanib, afatinib, osimertinib, lazertinib, poziotinib, clevo tinib, cabozantinib, capmatinib, axitinib, lenvatinib, nintedanib , regorafenib, pazopanib, sorafenib, or sunitinib.

[0124] In some embodiments, the kinase inhibitor is erlotinib. In some embodiments the kinase inhibitor is gefitinib. In some embodiments, the kinase inhibitor is lapatinib. In some embodiments, the kinase inhibitor is vandetanib . In some embodiments, the kinase inhibitor is afatinib. In some embodiments, the kinase inhibitor is osimertinib. In some embodiments, the ki nase inhibitor is lazertinib. In some embodiments, the kinase inhibitor is po ziotinib. In some embodiments, the kinase inhibitor is clevo tinib. In some embodiments, the kinase inhibitor is cabozantinib. In some embodiments the kinase inhibitor is capmatinib. In some embodiments, the kinase inhibitor is axitinib. In some embodiments, the kinase inhibitor is lenvatinib . In some embodiments, the kinase inhibitor is nintedanib. In some embodiments, the kinase inhibitor is regorafenib. In some embodiments, the kinase inhibitor is pazopanib. In some embodiments, the kinase inhibitor is so rafenib. In some embodiments, the kinase inhibitor is sunitinib.

[0125] Can be administered in combination with a bispecific anti-EGFR / c-Met antibody in the methods of the present disclosure Examples of anticancer therapies include any of the chemotherapeutic agents or other anticancer therapeutic agents known to those skilled in the art One or more of them may be mentioned. Chemotherapeutic agents are chemical compounds useful for the treatment of cancer It includes growth inhibitors or other cytotoxic agents, such as alkylating agents, antimetabolites, antimicrotubule inhibitors, topo Isomerase inhibitors, receptor tyrosine kinase inhibitors, angiogenesis inhibitors, etc. may be mentioned . Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (C YTOXAN (registered trademark), etc.; alkyl sulfonates, such as busulfan, imp Losulfan and piposulfan, etc.; aziridines, such as benzodopa, carbocon, me Turedopa, and uredopa, etc.; altretamine, triethylenemelamine, triethyleneho Sulforamide, triethylenethiophosphoramide and trimethylolmelamine-containing ethyl Renimine and methylamelamine; nitrogen mustard, such as Chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifos Famide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin , phenesterine, prednimustine, trofosfamide, uracil mustard, etc.; Nitrosoureas, such as carmustine, chloroztocin, fotemustine, lomustine, Nimustine, ranimustine, etc.; antibiotics, such as aclacinomycin ), actinomycin, authramycin, azaserine, bleomycin , cactinomycin, calicheamicin, carabicin, calminomycin , cardinophyllin, chromomycin, dactinomycin, daunorubicin, detorubi Cine, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, e sorubicin, idarubicin, marcellomycin, mitomycin, mi cofenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin (potfiromycin), puromycin, quelamycin, rodorubicin (rodorubicin), streptozocin, streptozocin, tubercidin, ubenimex s, dinostatin, zorubicin, etc.; antimetabolites, such as methotrexate and 5-F U, etc.; folic acid analogs, such as denopterin, methotrexate, pteropterin, trime trexate, etc.; purine analogs, such as fludarabine, 6-mercaptopurine, thiami purine, thioguanine, etc.; pyrimidine analogs, such as ancitabine, azacitidine, 6 -azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine, etc.; androgens, such as calusterone, propion acid drostanolone, epithiostanol, mepithiostane, testolactone, etc.; adrenal cortical hormone synthesis inhibitors, such as aminoglutethimide, mitotane, trilostane, etc.; leaf acid replenisher, such as folinic acid, etc.; ace glutathione; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestra busil; bisantrene; edatraxate; defofamine ); dexamethasone; diaziquone; elfornithine; elliptinium Acetate (elliptinium acetate); Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine ; Pentostatin; Phenamet; Pirarubicin; Podophyllinic acid; 2-Ethylhydrazide ; Procarbazine; PSK (registered trademark); Razoxane; Schizophyllan; Spirogermanium ; Tenuazonic acid; Triaziquone; 2,2′,2″-Trichlorotriethylamine ; Urethane; Vinblastine; Dacarbazine; Mannomustine; Mitobronitol ; Mitolactol; Pipobroman; Gacytosine; Arabinoside (「Ara-C」); Cyclophosphamide ; Thiotepa; Taxoid or member of the taxane family, for example paclitaxel (TAXOL (registered trademark)), docetaxel (TAXOTERE (registered trademark)) and its analogs; Chlorambucil; Gemcitabine ; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs, for example cisplatin and carboplatin, etc.; Vinblastine; Platinum ; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone ; Teniposide; Daunomycin; Aminopterin; Xeloda; Ibandronate; CPT-11; Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO); Retinoic acid ; Esperamicin; Capecitabine; Sorafenib (NEXAVAR (registered trademark)), Sunitinib (SUTENT (registered trademark)), Pazopanib (VOTRIENT (trademark)), Toceranib (PALLADIA (trademark)), Vandetanib (ZACTIMA (trademark)) ; ; ; ; ; ; TIMA (trademark), cediranib (RECENTIN (registered trademark)), legorafenib ( BAY73-4506), axitinib (AG013736), lestaurtinib (CE P-701), erlotinib (TARCEVA (registered trademark)), gefitinib (IRES SA (trademark)), afatinib (BIBW 2992), lapatinib (TYKERB (reg istered trademark)), neratinib (HKI-272), etc., including receptor tyrosine kinases and / or inhibitors of angiogenesis, and pharmaceutically acceptable salts, acids, or derivatives of any of the above are included. Anti-hormone agents that act to control or inhibit the hormonal action on tumors , such as tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (FARESTON ( registered trademark)), etc.; anti-estrogen drugs including; and anti-androgen drugs, such as flutamide , nilutamide, bicalutamide, leuprorelin, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above are also included in this definition. Other common cytotoxic compounds as described in Wiemann et al., 1985, Medical Oncology (Calabresi et al., eds.), Chapter 10, McMillan Publishing are also applicable to the method of the present invention. al., 1985, Medical Oncology (Calabresi et al., eds.), Chapter 10, McMillan Publishing are also applicable to the method of the present invention.

[0126] Administration The bispecific anti-EGFR / c-Met antibody and the macrophage activator can be administered to the subject together in a mixture, simultaneously as a single agent, or sequentially as single agents in any order. ​​

[0127] In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered prior to administration of the macrophage activator.

[0128] In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered after administration of the macrophage activator.

[0129] In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered simultaneously with administration of the macrophage activator.

[0130] In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered prior to administration of a third anti-cancer agent .

[0131] In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered after administration of a third anti-cancer agent .

[0132] In some embodiments, the bispecific anti-EGFR / c-Met antibody is administered simultaneously with administration of a third anti-cancer agent .

[0133] The length of time between administration of the bispecific anti-EGFR / c-Met antibody and administration of the macrophage activator or the third anti-cancer therapy can be a few minutes, for example, about 1, 2, 5, 10, 30, or 6 60 minutes, or a few hours, for example, about 2, 4, 6, 10, 12, 24, or 36 hours, or, for example, about 2, 4, 7, 14, 21, 28, 35, 42, 49, 56 days or more obtainable. The bispecific anti-EGFR / c-Met antibody and the macrophage activator or the third anti-cancer agent can be administered in a pharmaceutically acceptable carrier. "Carrier" refers to a substance that is administered together with the antibody of the present invention Refers to a diluent, adjuvant, excipient, or vehicle to be used. Such vehicles include water and oils derived from petroleum, animals, plants, or synthetic substances, such as peanut oil, soybean oil, mineral oil and sesame oil, which may be liquids. For example, 0.4% saline and 0.3% glycine may be used to formulate bispecific anti-EGFR / c-Met antibodies. These solutions are sterilized and generally contain no particulate matter. They can be sterilized by conventional well-known sterilization techniques (e.g., filtration). For oral solid preparations such as powder capsules and tablets, suitable carriers and additives include starch, sugars, diluents, granulating agents, lubricants, binding agents, disintegrants, etc. Oral solid preparations may be coated with substances such as sugar or may be enterically coated to regulate the major absorption site. In the case of parenteral administration, the carrier may contain sterile water and other excipients may be added for increased solubility or preservation if necessary. Injectable suspensions or solutions may also be manufactured using an aqueous carrier with appropriate additives if necessary. Preparations containing suitable vehicles and other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21 st Edition, Troy, D.B. ed., Li pincott Williams and Wilkins, Philadelphia a, PA 2006, Part 5, Pharmaceutical Manufact uring pp691-1092, particularly pp. 958-989 should be referred to.

[0134] The composition may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, For example, it may contain a pH adjuster, a buffer, a stabilizer, a thickener, a lubricant, a colorant, and the like. Concentration of bispecific anti-EGFR / c-Met antibody and macrophage activator in pharmaceutical preparations is from less than about 0.5% by weight, usually at least about 1% by weight, up to a maximum of 15% by weight, 20 % by weight, 30% by weight, 40% by weight, or 50% by weight, and can also be selected mainly based on the required dose, fluid volume, viscosity, etc., according to the specific mode of administration selected. A pharmaceutical composition containing a solid form contains from about 0.1 mg to about 2000 mg, for example, about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 100 mg, about 150 mg, about 200 m g, about 300 mg, about 500 mg, about 600 mg, or about 1000 mg of the active ingredient. It may contain.

[0135] The mode of administration is any suitable route for delivering the antibody to the host using tablets, capsules, solutions, powders, gels, particle formulations, for example, parenteral administration, for example, intradermal, intramuscular, intraperitoneal , intravenous, or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal), and may also be included in syringes, implant devices, osmotic pumps, cartridges, micropumps , or may be included in other means recognized by those skilled in the art and well-known in the art, using formulations for parenteral administration, for example, intradermal, intramuscular, intraperitoneal, intravenous, or subcutaneous, pulmonary, transmucosal administration (oral, intranasal, intravaginal, rectal), etc., any suitable route for delivering the antibody to the host. Site-specific administration is, for example, intratumoral, intra-articular, intratracheal, intra-abdominal, intra-articular capsule, soft bone, intra-cavity, intra-luminal, intra-cerebellar, intraventricular, intra-colonic, endocervical, intra-gastric, intra-hepatic, intramyocardial, intra-bone, pelvic intra, intra-pericardial, intra-abdominal, intra-pleural, intra-prostatic, intra-pulmonary, intra-rectal, intra-renal, intra-retinal, intra-spinal cord, synovial fluid It may be within, etc. Intranodal, intrathoracic, intrauterine, intravascular, intravesical, intralesional, intravaginal, intrarectal, intraoral, sublingual, intranasal or can be achieved by transdermal delivery.

[0136] Preparation of Bispecific Anti-EGFR / c-Met Antibodies Used in the Methods of the Present Disclosure Exemplary anti-EGFR / c-Met antibodies that can be used in the methods of the present disclosure are JNJ-372. JNJ-273 is characterized by the following amino acid sequences: EGFR binding arm >SEQ ID NO: 1 (HCDR1, EGFR binding arm) TYGMH >SEQ ID NO: 2 (HCDR2, EGFR binding arm) VIWDDGSYKYYGDSVKG >SEQ ID NO: 3 (HCDR3, EGFR binding arm) DGITMVRGVMKDYFDY >SEQ ID NO: 4 (LCDR1, EGFR binding arm) RASQDISSALV >SEQ ID NO: 5 (LCDR2, EGFR binding arm) DASSLES >SEQ ID NO: 6 (LCDR3, EGFR binding arm) QQFNSYPLT >SEQ ID NO: 7 (HCDR1, c-Met binding arm) SYGIS >SEQ ID NO: 8 (HCDR2, c-Met binding arm) WISAYNGYTNYAQKLQG >SEQ ID NO: 9 (HCDR3, c-Met binding arm) DLRGTNYFDY >SEQ ID NO: 10 (LCDR1, c-Met binding arm) RASQGISNWLA >SEQ ID NO: 11 (LCDR2, c-Met binding arm) AASSLLS >SEQ ID NO: 12 (LCDR3, c-Met binding arm) QQANSFPIT >SEQ ID NO: 13 (VH, EGFR binding arm) QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQ APGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGT LVTVSS >SEQ ID NO: 14 (VL, EGFR binding arm) AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQK PGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQ PEDFATYYCQQFNSYPLTFGGGTKVEIK >SEQ ID NO: 15 (VH, c-Met binding arm) QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISWVRQ APGHGLEWMGWISAYNGYTNYAQKLQGRVTMTTDTSTSTA YMELRSLRSDDTAVYYCARDLRGTNYFDYWGQGTLVTVSS >SEQ ID NO: 16 (VL, c-Met binding arm) DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWFQHK PGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQANSFPITFGQGTRLEIK >SEQ ID NO: 17 HC1 QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQ APGKGLEWVAVIWDDGSYKYYGDSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCARDGITMVRGVMKDYFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK >SEQ ID NO:18 LC1 AIQLTQSPSSLSASVGDRVTITCRASQDISSALVWYQQK PGKAPKLLIYDASSLESGVPSRFSGSESGTDFTLTISSLQ PEDFATYYCQQFNSYPLTFGGGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC >SEQ ID NO:19 HC2 QVQLVQSGAEVKKPGASVKVSCETSGYTFTSYGISWVRQ APGHGLEWMGWISAYNGYTNYAQKLQGRVTMTTDTSTSTA YMELRSLRSDDTAVYYCARDLRGTNYFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK > Sequence number 20 LC2 DIQMTQSPSSVSASVGDRVTITCRASQGISNWLAWFQHK PGKAPKLLIYAASSLLSGVPSRFSGSGSGTDFTLTISSLQ PEDFATYYCQQANSFPITFGQGTRLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

[0137] Similar characteristics when compared with JNJ-372 described in US Patent No. 9,593,164 As long as they show such, other publicly available bispecific anti-EGFR / c-Met antibodies may be used in the method of the present disclosure. The bispecific anti-E GFR / c-Met antibodies that can be used in the method of the present disclosure may also be obtained by combining publicly available EGFR-binding VH / VL domains and c-Met-binding VH / VL domains, and testing the resulting bispecific antibodies for the characteristics described in US Patent No. 9,593,164 can also be prepared by testing. It cuts.

[0138] The bispecific anti-EGFR / c-Met antibody used in the method of the present disclosure can be, for example, in a cell-free environment in vitro, or using co-expression, substitutions are introduced at the heavy chain CH3 interface in each half-molecule so as to facilitate the formation of a heterodimer of two antibody half- molecules having different specificities, and it can be prepared using Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies. The Fab arm exchange reaction is the result of a disulfide bond isomerization reaction and the dissociation-association of the CH3 domain. The heavy chain disulfide bond in the hinge region of the parental monospecific antibody is reduced. One of the obtained free cysteines of the parental monospecific antibody forms an intramolecular disulfide bond with a cysteine residue of the second parental monospecific antibody molecule, and at the same time, the CH3 domain of the parental antibody is liberated and reformed by dissociation-association . The CH3 domain of the Fab arm may be modified to prefer heterodimer formation over homodimer formation. The resulting product is a bispecific antibody having two Fab arms or half-molecules that bind to different epitopes, namely, an epitope on EGFR and an epitope on c-Met. For example, the bispecific antibody of the present invention can be prepared using the technique described in International Publication . In the case of IgG1 antibodies, mutations F405L in one heavy chain and K409R in the other heavy chain can be used . In IgG2 antibodies, wild-type I gG2 and IgG2 antibodies having F405L and R409K substitutions may be used. In IgG4 antibodies, wild-type IgG4 and IgG4 antibodies having F405L and R409 K substitutions may be used. To produce the bispecific antibody ​​​​ To achieve this, the first monospecific bivalent antibody and the second monospecific bivalent antibody are The mutant was engineered to have the aforementioned mutations, and the cysteines in the hinge region were designed to inhibit disulfide bond isomerization. The antibodies are incubated together under reducing conditions sufficient to allow the antibody to undergo Bispecific antibodies are generated by Fab arm exchange. The incubation conditions are optimally: An exemplary reducing agent that can be used is 2-mercaptoethylamine. 2-mercaptoethylamine (2-MEA), dithiothreitol (DT T), dithioerythritol (DTE), glutathione, tris(2 -Carboxyethyl)phosphine (tris(2-carboxyethyl)phosphine, TCEP), L- cysteine, and β-mercaptoethanol. For example, at a temperature of at least 20° C. , in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreate in the presence of ethanol at pH 5-8, e.g. pH 7.0 or pH 7.4, for at least 90 minutes. Incubation of the cells can be used.

[0139] The bispecific anti-EGFR / c-Met antibodies used in the methods of the disclosure are knob-in-hole (Genentech), CrossMAb (Roche) and electrostatic match (ele ctrostatically-matched)(Chugai,Amgen,Nov oNordisk, Oncomed), LUZ-Y (Genentech), Stran d Exchange Engineered Domain body(SEEDbo dy) (EMD Serono), and Biclonic (Merus) designs. It can also be prepared by using.

[0140] In the "knob-in-hole" strategy (see, for example, WO 2006 / 028936), the selected amino acids that form the interface of the CH3 domain of human IgG can be mutated at positions that affect CH3 domain interactions in order to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of an antibody that specifically binds to a first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of an antibody that specifically binds to a second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction between the heavy chain with "hole" and the heavy chain with "knob". Exemplary CH3 substitution pairs that form knob and hole are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V (expressed as the modification position in the first CH3 domain of the first heavy chain / the modification position in the second CH3 domain of the second heavy chain). In addition to the use of the "knob-in-hole" strategy for the promoter of Fab arm exchange, CrossMAb technology utilizes CH1 / CL domain exchange in one of the half arms to ensure correct light chain pairing of the resulting bispecific antibody (see, for example, US Patent No. 8,242,247).

[0141]

[0142] Using other cross-linking methods, between the heavy and light chains or within the heavy chain of a bispecific antibody, one or both In the arms, a full-length bispecific antibody of the present invention may be prepared by exchanging variable or constant, or both domains. Examples of such exchanges include, for example, VH-CH1 and VL-CL, VH and VL, CH3 and CL, and CH3 and CH1 described in International Patent Publication Nos. 2009 / 080254, 2009 / 080251, 2009 / 018386, and 2009 / 080252. Other strategies, such as using electrostatic interactions by substituting positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface to promote heavy chain heterodimerization, are described in US Patent Application Publication Nos. 2010 / 0015133, 2009 / 0182127, 2010 / 028637, or 2011 / 0123532 and may be used as described therein. In other approaches, heterodimerization can be promoted by the following substitutions: L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, or T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W (represented as the modified position in the first CH3 domain of the first heavy chain / the modified position in the second CH3 domain of the second heavy chain) as described in US Patent Application Publication No. 2012 / 0149876 or US Patent Application Publication No. 2013 / 0195849. 2009 / 080254, 2009 / 080251, 2009 / 01838 6, and 2009 / 080252, such as VH-CH1 and VL-CL, VH and VL, CH3 and CL, and CH3 and CH1.

[0143] In the arms, a full-length bispecific antibody of the present invention may be prepared by exchanging variable or constant, or both domains. The promotion of heavy chain heterodimerization by using electrostatic interactions, for example, by substituting positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface, is described in US Patent Application Publication Nos. 2010 / 0015133, 2009 / 018 2127, 2010 / 028637, or 2011 / 0123532 and may be used as described therein. In other approaches, heterodimerization can be promoted by the following substitutions: L351Y_F405A_Y407V / T394W, 2127, 2010 / 028637, or 2011 / 0123532 and may be used as described therein. In other approaches, heterodimerization can be promoted by the following substitutions: L351Y_F405A_Y407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392 M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K4 09F, L351Y_Y407A / T366V_K409F, Y407A / T366A_ T366I_K392M_T394W / F405A_Y407V, T366L_K392 M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K4 09F, L351Y_Y407A / T366V_K409F, Y407A / T366A_ K409F, or T350V_L351Y_F405A_Y407V / T350V_T3 66L_K392L_T394W (represented as the modified position in the first CH3 domain of the first heavy chain / the modified position in the second CH3 domain of the second heavy chain) can be promoted. That is, it can be promoted.

[0144] The bispecific antibodies of the present invention may be produced using SEEDbody technology. SEE Dbody has selected IgG residues substituted with IgA residues in their constant domains to promote heterodimerization as described in US Patent Application Publication No. 2007 / 0287 170. It has selected IgG residues substituted with IgA residues.

[0145] Mutations are typically made at the DNA level on molecules such as the constant domain of an antibody using standard methods. It is performed at the DNA level.

[0146] Next, the present invention will be described with reference to the following specific non-limiting examples.

[0147] Materials and Methods Isolation of PBMC, and NK cells and monocytes from PBMC Peripheral blood mononuclear cells (PBMC) and isolated immune cells (NK cells and monocytes) were purchased from Hemaca re. PBMC were collected from healthy human donors who had consented to the IRB at the FDA-registered collection center of HemaCare according to cGMP and cG TP collection guidelines and isolated from leukopak. Peripheral blood mononuclear cells (PBMC) were purified by density gradient centrifugation, purchased from HemaCare in a cryopreserved format, and stored in liquid nitrogen until use. For some donors, the leukopak from the same donor was divided into three to isolate PBMC, NK cells, and monocytes. NK cells were isolated using CD56 negative selection and monocytes were isolated using CD14 negative selection. The isolated NK cells and monocytes were

[0148] purchased from HemaCare in a cryopreserved format and stored in liquid nitrogen until use.

[0149] Differentiation of monocytes into M1, M2a, and M2c macrophages Monocytes (purchased from Hemacare) were thawed in XVIVO 15 medium supplemented with 10% FBS and plated in tissue culture-treated T75 flasks. On day 0, monocytes were plated in medium containing 50 ng / mL M-CSF (catalog number 216-MC-025 / CF, purchased from R&D s ystems) to obtain Mo macrophages. To polarize M0 macrophages into M2a macrophages, on day 5, the medium was changed to 5 0 ng / mL M-CSF and 20 ng / mL IL-4 (catalog number 204-IL- 020 / CF, purchased from R&D systems) and 20 ng / mL IL-13 (cat alog number 213-ILB-025 / CF, purchased from R&D systems) and incubated for 48 hours. To polarize M0 macrophages into M2c macrophages on day 5, the medium was changed to 50 ng / mL M-CSF and 20 ng / mL IL- 10 (catalog number 217-IL-025 / CF, purchased from R&D systems) and incubated for 48 hours. To obtain M1 macrophages, on day 6, the medium was changed to 50 ng / mL M-CSF and 100 ng / mL IFN-γ (catalog number 28 5-IF-100 / CF, purchased from R&D systems) and incubated for 24 hours . Then, the polarized M1, M2a, and M2c macrophages were removed from the flasks using Accu tase and used for assays.

[0150] Depletion of NK cells and monocytes from PBMC EasySep Human CD from STEMCELL Technologies ​56 Positive Selection kit II (Catalog number 17855 ) was used to deplete NK cells, and EasySep Human CD14 Positive Selection k it II (Catalog number 17858) from STEMCell Technologies was used to deplete monocytes. PBMC( (purchased from Hemacare) was thawed in X-VIVO-15 medium containing 10% FBS and counted. PBMC (10 million cells per depletion) was resuspended in EasySep buffer at a desired concentration of 100 million cells / mL . Depletion of NK cells and monocytes was performed according to the manufacturer's protocol . Briefly, 50 μl of each antibody selection cocktail was added and incubated at room temperature for 10 minutes. Magnetic particles were vortexed for 30 seconds and 50 μl was added to the PBMC + antibody cocktail. This was incubated at room temperature for 3 minutes and made up to 2.5 mL with Easy Sep buffer. The tube was then placed in an EasySep Mag net (Catalog number 18000, from STEMCell Technologies) and incubated for 3 minutes. The supernatant was then carefully transferred to a new tube. The magnetic separation step was repeated twice to obtain PBMC depleted of NK cells and PBMC depleted of monocytes . Depletion was confirmed using flow cytometry (as described below), and then these PBMC were used in a Simple Western assay to detect EGFR and M et protein levels . Determination of immune cell composition within PBMC

[0151] PBMC (purchased from Hemacare) was thawed in X-VIVO-15 containing 10% FBS Thawed and counted in medium. After counting, plate approximately 300,000 - 400,000 cells / well rated (triplicate), and the plates were spun at 1500 rpm for 3 minutes at 4°C Pinned. The supernatant was discarded, and the cells were washed with 150 μl / well of DPBS. The plates were Spun again as above to pellet. By adding 150 μl of DMSO to the contents of the live / dead stain A stock solution of Near IR-Live / Dead stain (Life Technologies, catalog number L10119) was prepared. Then, A working solution was prepared by adding 50 μl of the stock to 10 mL of DPBS Next, 50 μL of the working solution was added to each well of the plate and resuspended The plates were incubated at room temperature for 30 minutes in the dark (covered with foil). At the end of the incubation The plates were spun at 4°C and 1500 rpm for 5 minutes. Then, The cells were washed with FACS / Stain buffer (BD number 554657) buffer by adding 150 μl / well Antibodies for the multicolor flow cytometry panel were prepared in a cocktail according to the calculation and 25 μl / well were added. Antibodies used in the panel Contained CD19 (FITC), CD56 (BV711), Cd11b (BUV39 5), CD14 (PE-cy7), CD3 (BV605), CD4 (BV785), CD 8 (PerCP-cy5.5), CD25 (PE), and PD-1 (APC). The plates were incubated at room temperature for 30 minutes in the dark. Compensation Compensation beads and single-channel antibodies from the above panel were used to prepare compensation plates according to the calculation and incubated at room temperature for 30 minutes in the dark. All plates were at 4°C And incubated for the appropriate time as described above. After incubation, the plates were spun at 1500 rpm for 5 minutes at 4°C. Then, The supernatant was discarded, and the cells were washed with 150 μl / well of DPBS. The plates were spun again at 1500 rpm for 5 minutes at 4°C to pellet. The supernatant was discarded, and the cells were resuspended in 100 μl / well of FACS buffer And incubated at room temperature for the appropriate time as described above. After incubation, the plates were spun at 1500 rpm for 5 minutes at 4°C. Then, Spun at 1500 rpm for 5 minutes and washed twice with 150 μl of FACS buffer. The assay plate was resuspended in 150 μl of FACS buffer and the compensation plate in 20 0 μl of FACS buffer. The plates were run on a Fortessa and corrections were set using single-channel control values from the compensation beads plate. The assay plate was then run at a flow rate of 1.5 μl / sec with corrections applied. The data was then exported and analyzed in FLOWJo, and appropriate gating was performed to obtain the respective percentages of each individual immune cell population within the PB MC.

[0152] Proliferation and apoptosis assays HCI-H1975 cells (also referred to herein as H1975 cells) were obtained from ATCC and cultured in RPMI (Invitrogen, catalog number 72400-047) supplemented with 10% Hi FBS, 1×NEAA, and 1× sodium pyruvate. For the proliferation and apoptosis assays using Incucyte, H1975 cells were infected with Incucyte NucLight Red lentiviral reagent (catalog number 4476, from Essen Biosciences) and selected with 1 μg / ml puromycin to generate H1975 NucRed cells. These cells were plated for the experiment in RPMI-free medium supplemented with 10 % HI FBS, 1×NEAA, and 1× sodium pyruvate. H1975-NucRed cells were used with Invitrogen Cell Dissociation Buffer (since trypsin degrades cell surface receptors / molecules).

[0153] ​ Dissociated and counted. The cells were centrifuged at 1200 rpm for 5 minutes and the supernatant was removed. Next, the pellet was resuspended in an appropriate volume of phenol red-free medium. NucRed cells (target cells) were plated at 12,500 cells / well in 100 μl of phenol red-free medium in tissue culture-treated black flat-bottom plates and incubated overnight at 37 °C and 5% CO 2 . The next day, PBMCs (purchased from HemaCare) were thawed and counted in X-VIVO-10 medium containing 10% FBS. PBMCs were diluted to a concentration of 125,000 cells / well in 50 μl to obtain an effector:target (E:T) ratio of 10:1. Incucyte Annexin V Green reagent (catalog number 4642, purchased from Essen Biosciences) was resuspended in 100 μl of medium and used to stain 100 wells or one 96-well plate. The diluted annexin reagent was added to PBMCs or medium. 50 μl of PBMC / medium (containing annexin) was added to the appropriate wells of the assay plate. Next, the therapeutic antibody was serially diluted 1:5 and prepared at 4× concentration according to the calculation, and 50 μl of the desired antibody was added to the appropriate wells of the assay plate. Then, the assay plate was placed in the appropriate slot in the Incucyte S3 and equilibrated in the Incucyte for 20 minutes before scanning. The plate was scanned at 4 images / well / scanning every 4 hours up to 120 hours to determine target cell proliferation and apoptosis over time. The fluorescence of the target cells (NucRed) and annexin (Green) was quantified using the process definition, and the total NucRed H1 indicating the proliferation of the target cells was measured. 50 μl of PBMC / medium (containing annexin) was added to the appropriate wells of the assay plate. Next, the therapeutic antibody was serially diluted 1:5 and prepared at 4× concentration according to the calculation, and 50 μl of the desired antibody was added to the appropriate wells of the assay plate. Then, the assay plate was placed in the appropriate slot in the Incucyte S3 and equilibrated in the Incucyte for 20 minutes before scanning. The plate was scanned at 4 images / well / scanning every 4 hours up to 120 hours to determine target cell proliferation and apoptosis over time. The fluorescence of the target cells (NucRed) and annexin (Green) was quantified using the process definition, and the total NucRed H1 indicating the proliferation of the target cells was measured. Next, the assay plate was placed in the appropriate slot in the Incucyte S3 and equilibrated in the Incucyte for 20 minutes before scanning. The plate was scanned at 4 images / well / scanning every 4 hours up to 120 hours to determine target cell proliferation and apoptosis over time. The fluorescence of the target cells (NucRed) and annexin (Green) was quantified using the process definition, and the total NucRed H1 indicating the proliferation of the target cells was measured. The fluorescence of the target cells (NucRed) and annexin (Green) was quantified using the process definition, and the total NucRed H1 indicating the proliferation of the target cells The 975 area (μm2 / well) was calculated. Total Gree indicating apoptosis of target cells n NucRed H1975 area (um2 / well) was also calculated. From this analysis, Gr The area under the curve (AUC) was calculated using Graphpad Prism, and a non-linear dose-response curve was created.

[0154] For detecting EGFR, c-Met, pEGFR, and pMet protein levels Simple Western H1975 cells and SNU-5 cells were obtained from ATCC and cultured in RPMI (Invitrogen, catalog number 72400-047) supplemented with 10% Hi FBS, 1 ×NEAA, and 1×sodium pyruvate. H1975 cells were dissociated using Cell Dissociation Buffer, and each cell suspension was placed in a 50 mL conical tube and counted. In assays using SNU-5 (suspension cell line), the cell suspension was transferred to a 50 mL conical tube and counted. The cells were pelleted at 1300 rpm for 5 minutes at 4°C and resuspended in an appropriate volume of RPMI medium. The target cells were plated in a 6-well plate at a concentration of 100, 000 cells / well and incubated overnight. The next day, PBMCs (purchased from HemeCar e) were thawed and counted in X-VIVO-15 medium containing 10% FBS. The PBMCs were diluted and plated at a concentration of 1,000,000 cells / well to obtain a 10:1 E:T ratio. When using individual immune cells such as NK cells, monocytes, or macrophages, these were diluted and plated at a concentration of 500,000 cells / well to obtain a 5:1 E:T ratio. Then, according to the calculation, 2 ​​​Therapeutic antibodies were prepared at × concentration, and 1.5 mL of the desired antibody was added to the appropriate wells of an assay plate. The plate was incubated for 48 hours (for most assays) or at various time points.

[0155] At the end of the incubation period, 100 μl of freshly prepared lysis buffer was added to each well and incubated on ice for 5 minutes. 10 mL of RIPA buffer (Thermo Fisher; catalog number 89901) was used to prepare the lysis buffer together with 1 tablet of the phosphatase inhibitor Phos STOP (Sigma; catalog number 4906837001) and 1 tablet of the protease inhibitor cOmplete™, Mini, EDTA-free protease inhibitor cocktail ( Sigma; catalog number 04693159001). The lysates were transferred to 2 mL Eppendorf tubes using a plate scraper and incubated on ice for 30 minutes with occasional vortexing. The lysates were centrifuged at 13,200 rpm for 25 minutes at 4 °C, and the supernatant was transferred to a new tube. According to the manufacturer's protocol, the Pierce BCA Protein Assay Kit (catalog number 23227, obtained from ThermoFisher) was used to determine the protein concentration of the lysates. The Pierce Bovine Serum Albumin Sta ndard Pre-Diluted standard (catalog number 23208, obtained from ThermoFi sher) was used to obtain the standard curve for the assay. Briefly, 25 μl of the pre-diluted standard was added in triplicate, and 25 μl of the sample (1:5 dilution) was added in duplicate onto a 96-well flat-bottom plate. The prepared BCA working reagent was added to 1 well and incubated on ice for 5 minutes. 10 mL of RIPA buffer (Thermo Fisher; catalog number 89901) was used to prepare the lysis buffer together with 1 tablet of the phosphatase inhibitor Phos STOP (Sigma; catalog number 4906837001) and 1 tablet of the protease inhibitor cOmplete™, Mini, EDTA-free protease inhibitor cocktail ( Sigma; catalog number 04693159001). The lysates were transferred to 2 mL Eppendorf tubes using a plate scraper and incubated on ice for 30 minutes with occasional vortexing. The lysates were centrifuged at 13,200 rpm for 25 minutes at 4 °C, and the supernatant was transferred to a new tube. According to the manufacturer's protocol, the Pierce BCA Protein Assay Kit (catalog number 23227, obtained from ThermoFisher) was used to determine the protein concentration of the lysates. The Pierce Bovine Serum Albumin Sta ndard Pre-Diluted standard (catalog number 23208, obtained from ThermoFi sher) was used to obtain the standard curve for the assay. Briefly, 25 μl of the pre-diluted standard was added in triplicate, and 25 μl of the sample (1:5 dilution) was added in duplicate onto a 96-well flat-bottom plate. The prepared BCA working reagent was added to 1 well of the assay plate. Briefly, 25 μl of the pre-diluted standard was added in triplicate, and 25 μl of the sample (1:5 dilution) was added in duplicate onto a 96-well flat-bottom plate. The prepared BCA working reagent was added to 1 well of the assay plate. Briefly, 25 μl of the pre-diluted standard was added in triplicate, and 25 μl of the sample (1:5 dilution) was added in duplicate onto a 96-well flat-bottom plate. The prepared BCA working reagent was added to 1 well 200 ul was added per hit. The plate was gently mixed on a plate shaker for 1 minute, covered with foil and incubated at 37 °C for 30 minutes. After incubation, the plate was cooled to room temperature for 5 minutes, and then protein quantification was measured at 562 nm using a SpectraMAX spectrophotometer.

[0156] To perform capillary-based electrophoresis using Peggy Sue, the anti-rabbit detection module (catalog number DM-001) and anti-mouse secondary antibody (catalog number 042-205), both purchased from Protein Simple, were used together with the 12-230 kDa Peggy Sue Separation module (catalog number SM-S001, purchased from Protein Simple) alone. Samples, antibodies, and reagents were prepared and capillary-based electrophoresis was performed according to the manufacturer's protocol. Briefly, two standard packs of components were used to prepare a biotinylated ladder and 5× master mix. Protein lysates were diluted to a concentration of 0.25 mg / mL using 0.1× sample buffer according to calculations using values from the BCA protein assay. Four parts of the prepared lysate were combined with one part of 5× Fluorescent Master Mix to obtain a final concentration of 0.2 mg / mL. Samples and biotinylated ladder were denatured using a PCR thermocycler at 95 °C for 5 minutes. The primary antibodies used in the assay were diluted as follows using an antibody diluent: EGFR (catalog number 2646, manufactured by Cell Signaling Technologies), 1:50; pEGFR (catalog number AF1095, manufactured by R&D Systems), 1:50; c- Met (Catalog No. 3148, Cell Signaling Technologi es), 1:50; pMet (Catalog No. 3077, Cell Signaling Technologies), 1:50, and loading control actin (Catalog No. 4970, Cell Signaling Technologies), 1:2 00 or (Catalog No. 4947, Cell Signaling Technolog ies), 1:100. Ladder, samples, primary and secondary antibodies, separation and stacking mat rices were added to a 384-well Peggy Sue plate according to the plate layout. The plate was spun at 2500 rpm for 5 minutes at RT and then loaded onto the machine. This data was analyzed using Compass for SW software. Peaks were determined based on the molecular weight of the target protein, and the area under the curve (AUC) was calculated for each protein in each sample. Subsequently, the densitometry values of the target protein were normalized to the loading control actin for each sample and then normalized to the untreated control to obtain the relative change due to treatment.

[0157] Confocal imaging trogocytosis assay Differentiated macrophages were collected and plated at 1,00,000 cells / well overnight (Perkin -Elmer; Catalog No. 6055302) in a CellCarrier 96 ultra plate. The assay was performed using both adherent and non-adherent target cells. In the assay using adherent target cells, H1975 NucLight Red cells were plated at 20,000 / well, allowed to adhere for 4 hours, and then incubated at 4°C for 1 hour ​, treated with the labeled Ab cocktail. In assays using non-adherent target cells, only the target cells were stained with AF647-labeled JNJ-372 or the control Ab. All live imaging studies were performed at an E:T ratio of 5:1. The labeled antibody cocktail consisted of anti-CD11b (BD Pharm ingen; catalog number 557701), anti-CD14 (BD Pharmingen; catalog number 562689), and 1:8000 Hoechst33342 (Biot ium; catalog number 40046). Images were obtained at 11-minute intervals on a Perkin-Elmer Phenix Opera using a 60× water immersion objective lens and analyzed using Columbus.

[0158] ADCC assay VI VO 10 medium (Lonza, catalog number 04-380Q) supplemented with 10% heat-inactivated FBS (GIBCO, catalog number 16140). PB MCs were thawed one day before the assay and left to stand overnight under standard incubation conditions (37 °C, 5% CO 2 , 95% humidity). On the assay day, NCI-H1975 target cells were loaded with DELFIA BATD A reagent (PerkinElmer Inc., catalog number C136-100) for 30 minutes, washed three times, and resuspended in RPMI medium. PBMCs and BATDA-loaded target cells were added to 96-well U-bottom plates at a ratio of effector to target cells of 25 :1 with increasing concentrations of the test antibody. RPMI medium or RPMI medium containing 2% Triton X-1 00 (EMD Millipore, catalog number 648463) was added to control wells for the measurement of natural and maximum TDA release, respectively. The plates After incubating for 2 hours, 20 μL of the supernatant was taken out and combined with 200 μL of DELPHI A Europium solution (Perkin Elmer, catalog number C135-100 ). After incubating for 15 minutes at RT, the relative fluorescence units (RFU) were measured using an EnVision 2104 M ultilabel Plate Reader (PerkinElmer, catalog number 2104-0010). The dissolution rate was calculated as ( experimental release - natural release) / (maximum release - natural release) × 100

[0159] Differentiation of monocytes into M1, M2a, and M2c macrophages Monocytes (Hemacare) were thawed in XVIVO-15 medium and differentiated with 50 ng / mL of M-C SF (R&D systems; catalog number 216-MC-025 / CF) for 6 days to obtain M0 macrophages. To obtain M1 macrophages, on day 5, M 0 macrophages were polarized with 50 ng / mL M-CSF and 100 ng / mL IFN-γ (R &D systems; catalog number 285-IF-100 / CF) for 48 hours . To obtain M2 macrophages, on day 5, M0 macrophages were polarized with 20 ng / mL IL-4 (R&D systems; catalog number 204-IL -020 / CF) and IL-13 (R&D systems; catalog number 213-IL B-025 / CF) for M2a or 20 ng / mL IL-10 (R&D systems; catalog number 217-IL-025 / CF) for M2c macrophages for 48 hours . (R&D systems; catalog number 217-IL-025 / CF) for 48 hours .

[0160] In vivo test Six- to eight-week-old female BALB / c nude mice (CAnN.Cg-Foxn1 nu / Crl ), Charles River Laboratories (Wilmington, M A)) were subcutaneously implanted with the H1975 cell line. When the tumors reached an average of 72 ± 8.7 mm 3 , starting 5 days before the start of compound administration to promote macrophage depletion, anti-mCSF-1R antibody (400 μg / mouse) was intraperitoneally administered three times a week over the study period . On day 5 (average tumor volume = 102 ± 36.6 mm 3 ), the mice were treated twice a week by intraperitoneal administration of 10 mg / kg isotype control Ab , JNJ-372, or EGFR / cMet IgG2σ Ab . Tumors were sampled to monitor macrophage infiltration after two administrations of the compound . In the SNU5 tumor model study, 7- to 8-week-old female CB17 / SCID mice (HFK Bio-Technology Co., Ltd. (Beijing, Chi na)) were subcutaneously implanted with SNU5 cells. When the tumors reached an average of 155 ± 21.4 mm 3 , the mice were treated twice a week intraperitoneally for 3 weeks with phosphate-buffered saline (PBS), JNJ-372 (5 mg / kg), or the EGFR-cMet IgG2σ antibody (5 mg / kg). For both studies, tumor measurements and body weights were recorded twice a week. Using the calculation [1 - (T / C)] × 100, tumor growth inhibition (TGI) was calculated on the last day that >80% of the control mice remained in the study . All in vivo experiments were conducted in accordance with the Johnson and J ohnson Institutional Animal Care and Use Committee and the Guide for Care and Use o ​​​Performed in accordance with f Laboratory Animals.

[0161] Flow cytometry-based determination of tumor-associated macrophages Tumors were excised from mice, weighed, sectioned into 2 - 4 mm slices, and placed into C-tubes (Miltenyi, catalog number 130 - 093 - 237) containing 2.5 mL of RPMI and maintained on ice. Following the manufacturer's instructions, the lyophilized enzymes contained in the Human Tumor Dissociation Kit (Miltenyi, catalog number 130 - 095 - 929) were reconstituted to prepare a 2× enzyme cocktail, and the tumor was dissociated using a GentleMACS Octo Dissociator (Miltenyi, catalog number 130 - 095 - 937) with the manufacturing protocol "h _tumor_01", followed by two rounds of incubation at 37°C for 30 minutes. The dissociated cells were washed twice with FACS Stain Buffer (BD Pharmingen, catalog number 554657) and passed through a Falcon 40 μm cell strainer (Corning, catalog number 352340). The cells were incubated in GolgiPlug (BD, catalog number 555029) diluted 1:1000 in FACS buffer, incubated at 37°C for 3 hours, washed twice, and resuspended in 100 μL of antibody staining cocktail. The antibody cocktail consisted of anti-CD45 (catalog number 103138), anti-F4 / 80 (catalog number 1 23137), anti-Ly6G (catalog number 127639), anti-MHCII (catalog number 107612), anti-EpCAM (catalog number 324214), anti-PD1 (catalog number 135231), anti-PD-L1 (catalog number 393606), anti-CD206 (catalog number 393606), anti-CD206 (catalog number 393606), anti-CD206 (catalog number 393606), anti-CD206 (catalog number 393606), anti-CD206 (catalog number 393606), anti-CD206 (catalog 107612), anti-EpCAM (catalog number 324214), anti-PD1 (catalog number 135231), anti-PD-L1 (catalog number 393606), anti-CD206 (catalog Number 141729) (manufactured by BioLegend), anti-CD11b (catalog number 56355 3), and anti-Ly6C (catalog number 561237) (Becton-Dickinson manufactured), anti-iNOS (catalog number 25-5920-82), and Fixable Live / Dead stain (catalog number L10119) (manufactured by Invitrogen). The cells were protected from light and incubated with the external cell surface marker antibody at 4°C for 30 minutes and washed twice with PBS, resuspended in PBS containing Fixable Live / Dead sta in, incubated at 4°C for 30 minutes, and washed twice with FACS buffer a (BD Pharmingen; catalog number 554657). The cells were fixed / permeabilized according to the manufacturer's instructions (Invitrogen, catalog number 88- 8824-00), incubated with the internal target antibody at 4°C for 30 minutes, washed twice with FACS buffer and resuspended in 200 μL for analysis with a BD LSR Fortessa. UltraComp Beads (for antibodies; Invitrogen, catalog number 01-2222-42) and ArC Amine Reactive beads (for Fixable Live / Dead; Invitrogen, catalog number A10 346) were used for calibration. FMO controls were performed for all markers. To determine tumor-associated macrophage depletion, macrophages were defined as CD45 CD11b Ly 6C Ly6G F4 / 80 as defined. + CD11b + Ly 6C - Ly6G - F4 / 80 + as defined.

[0162] MSD multiplex assay and statistical analysis For the PBMC, NK cell, and monocyte experiments, NCI-H1975 cells were plated in 96-well plates and incubated overnight at 37 °C and 5% CO 2 . The next day, PBMC, monocytes, or NK cells were added at ratios of 10:1, 5:1, and 5:1, respectively . For the macrophage experiment, monocytes were differentiated as described above, dissociated using StemPro A cutase (Gibco, catalog number A11105-01), plated in 9 6-well plates, and incubated overnight at 37 °C and 5% CO 2 . The next day, NCI-H1975 cells were added at an E:T ratio of 5:1. Cells were treated with various concentrations of isotype controls, JNJ-61186372, or IgG2 Sigma and incubated at 37 °C and 5% CO for 4 hours, 24 hours, 48 hours, and / or 72 hours. At the indicated times, the plates were spun at 1200 rpm for 10 minutes at room temperature. The supernatants were removed and evaluated using MesoScale 2 Discovery (MSD) U-plex and V-plex formats for each cytokine assay according to the manufacturer's instructions. Briefly, for the U-plex plates, the plates were coated with antibodies and linkers according to the manufacturer's protocol the day before the assay and incubated overnight at 4 °C on a rotating shaker. On the day of the experiment, the U-plex or V-plex plates were washed three times with MSD wash buffer and the supernatants, standards and calibrators were added to the plates and run according to the manufacturer's protocol. The plates were read on an MSD Sector instrument and analyzed using GTS Spotfire to determine the amount of each cytokine using a standard curve . Specifically, for the U-plex plates, the day before the assay, the plates were coated with antibodies and linkers according to the manufacturer's protocol and incubated overnight at 4 °C on a rotating shaker. On the day of the experiment, the U-plex or V-plex plates were washed three times with MSD wash buffer and the supernatants, standards and calibrators were added to the plates and run according to the manufacturer's protocol. The plates were read on an MSD Sector instrument and analyzed using GTS Spotfire to determine the amount of each cytokine using a standard curve . The U-plex or V-plex plates were washed three times with MSD wash buffer and the supernatants, standards and calibrators were added to the plates and run according to the manufacturer's protocol. The plates were read on an MSD Sector instrument and analyzed using GTS Spotfire to determine the amount of each cytokine using a standard curve . The plates were read on an MSD Sector instrument and analyzed using GTS Spotfire to determine the amount of each cytokine using a standard curve The calculated level (pg / ml) for

[0163] From the calculated concentrations, to compare the magnitude of the response, for each treatment, cell type, and incubation time, the area under the curve (AUC) was calculated by the trapezoidal method. If the observed value was below the limit of detection, the response data was excluded, and the AUC was calculated only if at least 6 valid observations of the 8 administered concentrations were present. Then, across all cytokines and conditions, a heatmap was created to show the validity of the data (no data, not enough data, or computable AUC data). Then, using the incubation time, a heatmap of the log-transformed AUC was created and limited to cytokines having at least one measurable AUC in the H197 5+PBMC cell type. All heatmaps were created using the package heatmap.2 in the statistical software R version 3.5.0 (R Core Team 2018; R : A language and environment for statistical computing; http: / / www.R-project.org / ) Finally, the relative changes of JNJ-372 and IgG2σ treatments compared to the isotype were calculated for each condition, and bar graphs or dose curves were created using Graphpad Prism : A language and environment for statistical computing; http: / / www.R-project.org / ) (R Core Team 2018; R ).

[0164] Example 1. Anti-proliferative and apoptotic effects mediated by JNJ-372 on tumor cells are driven by Fc interactions with immune cells To evaluate the effect of Fc interactions on three anti-tumor mechanisms of JNJ-372, the standard Using standard cloning methods, Fc effector silent IgG2 sigma (wild type Compared to IgG2, V234A G237A P238S H268A V309L A IgG2 containing the 330S P331S mutation (Sigma) replaces JNJ-372 wild-type IgG1 We engineered JNJ-372 to be an Fc effector-silent molecule by converting (JNJ-372.IgG2 Sigma). The antibody was designated JNJ-372.IgG2 sigma (or IgG2s in some figures). JNJ-372 enhances binding to FcγRIII / CD16 and ADCC. , produced in cell lines incorporating low concentrations (<9%) of fucose. As a result, JNJ-372 was expressed in CHO cells that had normal fucose levels. This molecule is called JNJ-372.NF (NF: normal fucose). Tumor cell killing assessed using H1975 cells (ATCC Catalog No. CRL-5908) The cell line expresses mutant L858R / T790M EGFR and wild-type c-Met. do.

[0165] NCI-H1975 NucLight Red expressing cells were used as isotype control, JNJ Treatment with JNJ-372, JNJ-372.IgG2s, or JNJ-372.NF at 10:1 After initiation of co-culture, the cells were cultured in the presence or absence of PBMCs at an effector:target ratio of Proliferation of NCI-H1975 cells was assessed over a 5-day period. The presence of PBMCs was associated with a dose-dependent increase in Although JNJ-372 potentiated the ability of the antibody to inhibit tumor cell proliferation over time, NJ-372 did not inhibit proliferation in the absence of PBMCs at the concentrations tested ( Figure 2).

[0166] To confirm that Fc engagement on PBMCs is responsible for the effect, H1975 NucLight Red expressing cells were JNJ-372, JNJ-372.IgG2 Sigma and JNJ-372.NF, and were incubated in the presence or absence of PBMCs at an E:T ratio of 10:1. The cells were cultured in the presence of 500 mg / mL ... The presence of PBMCs was assessed at 24, 48, 72, and 96 hours. Dose-dependent antiproliferative effect and dose-dependent apoptosis induced by JNJ-372 (A The results were enriched for isotype or JNJ-372.IgG2 signaling. No or minimal effects were observed in PBM after 72 hours of culture. Isotype control, JNJ-372, JNJ-372 in the presence or absence of C Inhibition of H1975 cell proliferation mediated by IgG2 Sigma or JNJ-372 NF FIG. 4 shows the dose response in the presence or absence of PBMC after 48 hours of culture. , isotype control, JNJ-372, JNJ-372.IgG2 Sigma, or JNJ- 3 shows the dose response of 372.NF-mediated apoptosis. Partial effect on proliferation (Fig. 3) and apoptosis (Fig. 4) when compared with NJ-372 This is because FcγRIII plays a role, but it is not a mediator of Fc interactions. This suggests that this does not fully explain the effect of intervention.

[0167] The assay consisted of PBMCs from seven different donors at an effector:target ratio of 10:1. IC in the NCI-H1975 proliferation assay for six donors. 50 The values and maximum killing percentage are shown in Table 1. PBMC from a single donor had no effect. . Across PBMC obtained from different donors, variability in IC 50 and maximum killing percentage was observed.

[0168]

Table 1

[0169] Example 2. Downregulation of EGFR and c-Met proteins and their downstream signaling by JNJ-372 in EGFR mutant tumor cell lines is mediated by Fc interaction with immune cells. The presence of immune cells (PBMC) enhanced the downregulation of EGFR and c-Met proteins by JNJ-372 and the inhibition of their phosphorylation.

[0170] NCI-H1975 cells were treated with 10 μg / mL isotype or JNJ-372 and cultured at an E:T ratio of 10:1 for 4, 24, 48, or 72 hours in the presence or absence of PBMC from a single donor, and the amounts of EGFR, c-Met, and pEGFR (pY1173 ) were measured. Actin was used as a loading control. The presence of PBMC enhanced the downregulation of EGFR, c-Met, and pEGFR (pY117 3) by JNJ-372 at all time points tested. Figure 5 shows EGFR and c-Met proteins and pEGFR in samples treated with isotype control or JNJ-372 and cultured in the presence or absence of PBMC, as shown in the figure. in the figure. Rally-based electrophoresis (Simple Western using PeggySue) or Images are shown. Figure 6 shows the relative amounts of EGFR in each sample. Figure 7 shows the pE The relative amount of GFR pY1173 is shown. Figure 8 shows the relative amount of c-Met in each sample Samples were normalized to the amount of loading control actin present in each sample and then Normalized to the control (untreated) sample.

[0171] PBMCs from 7 donors were tested for their ability to enhance the inhibition mediated by JNJ-372 of EGFR and c-Met protein levels and pE GFR. Variation was observed among donor PB MCs. PBMCs from donors 1, 3, 4, and 6 were the most potent in enhancing the effect mediated by JNJ- 372. Figure 9 shows, as shown in the figure, isotype control or EGFR and c-Met proteins and pEGFR in samples cultured in the presence or absence of PBMCs from 7 different donors and treated with JNJ-372 Images from capillary-based electrophoresis (Simple We stern using PeggySue) are shown. Figure 10 shows the relative amount of EGFR in each sample. Figure 11 Shows the relative amount of pEGFR pY1173 in each sample. Figure 12 shows c -The relative amount of Met in each sample. Samples were normalized to the amount of loading control actin present in each sample And then normalized to the control (untreated) sample.

[0172] Example 3. The presence of monocytes or macrophages is sufficient and necessary for the Fc-mediated enhancement of the inhibitory effect of JNJ-372 on EGFR and c-Met signaling The ability of JNJ-372 to down-regulate EGFR and c-Met signaling To understand the variability in the ability of various PBMC samples to enhance the ability of JNJ-372, multi-color flow cytometry was used to evaluate the immune cell composition within PBMCs from 7 different donors used in Example 2. Variability was detected among donors in the percentage of individual immune cells (data not shown). The percentage of individual immune cells in each of the 7 donors was correlated with the ability of each donor's PBMC to mediate down-regulation of EGFR / pEGFR / Met. No correlation was observed between NK cells, B cells, or T cells and the ability of PBMCs to enhance down-modulation of EGFR, pEGFR, or c-Met (data not shown). However, a positive correlation was identified between the percentage of monocytes in PBMCs and the ability of PBMCs to enhance down-modulation of EGFR protein levels (Figure 13), between the percentage of monocytes in PBMCs and the ability of PBMCs to enhance down-modulation of pEGFR (Figure 14), and between the percentage of monocytes in PBMCs and the ability of PBMCs to enhance down-modulation of c-Met protein levels (Figure

[0173] 15). This suggests that a higher percentage of monocytes in PBMCs is required for JNJ-372-mediated signal down-regulation. NK cells or monocytes were depleted from PBMCs from one donor, and the effect of NK- or monocyte-depleted PBMCs on JNJ-372-mediated down-modulation of the The cells were cultured for 48 h at an E:T ratio of 10:1 in the presence or absence of MC. Consistent with these results, the presence of PBMCs significantly increased the expression of EGFR, pEGFR, and EGFR-associated ... and enhanced JNJ-372-mediated downregulation of c-Met. Depletion of monocytes from PBMCs had only a small effect, but depletion of monocytes from PBMCs Ability of PBMCs to enhance JNJ-372-mediated signal down-modulation Figure 16 shows that the force was significantly restored in the presence of JNJ-372 or isotype control as shown in the figure. Presence of NK cell-depleted PBMC or monocyte (mono)-depleted PBMC from one donor EGFR, c-Met, and EGFR expression in NCI-H1975 cells cultured for 48 hours in the presence or absence of EGFR. and capillary-based electrophoresis (Pegg) to detect pEGFR protein levels. Figure 17 shows images from a Simple Western run using ySue. As described above, NK cell depletion from one donor was performed using treatment with JNJ-372 or isotype control. NCs cultured for 48 h in the presence or absence of depleted or monocyte (mono)-depleted PBMCs Figure 18 shows the relative amount of EGFR in JNJ-372 I-H1975 as indicated. or isotype control, and NK cell-depleted PBMC or monocytes (monocytes) from one donor were treated with pE in NCI-H1975 cells cultured for 48 hours in the presence or absence of depleted PBMCs Figure 19 shows the relative amount of GFR (pY1173). Treated with isotype control and NK cell-depleted PBMC or monocytes (mono) from one donor c-Me in NCI-H1975 cells cultured for 48 h in the presence or absence of depleted PBMCs It shows the relative amount of t. The samples were normalized to the amount of loading control actin present in each sample and then normalized to the control (untreated) sample.

[0174] To further evaluate the role of the bone marrow compartment, the effect of monocytes or M1 macrophages isolated from one PBM C donor on EGFR / c-Met downregulation driven by JNJ-372 Fc interaction was evaluated. To evaluate any potential differential effects, monocytes were differentiated into M1 macrophages in two ways using M-CSF and GM-CSF. H1975 cells were treated with 10 μg / mL isotype, JNJ-372, JNJ-372.IgG2 sigma and cultured for 48 hours in the presence or absence of PBMCs from one donor at an E:T ratio of 10:1 (for PBMCs) or 5:1 (for individual immune cells (monocytes, NK cells, MCSF M1, or GMCSF M1 macrophages)). The presence of PBMCs enhanced the JNJ-372-mediated downregulation of EGFR, pEGFR, and c-Met proteins. NK cells had no significant effect, but monocytes or M1 macrophages (differentiated by M-CSF or GM-CSF) isolated from the same PBMC donor significantly enhanced the ability of JNJ-372 to mediate signal downmodulation. This suggested that the myeloid cell lineage is sufficient for the Fc interaction and the PBMC-mediated enhancement in JNJ-372 signal downregulation. Figure 20 shows PBMCs, isolated monocytes, isolated NK cells, MCSF-differentiated M1 macrophages from the same donor, as shown in the figure. In the presence or absence of rophage or GMCSF-differentiated M1 macrophages for 48 hours, EGFR in NCI-H1975 cells treated with JNJ-372 or isotype control, c-Met, and pEGFR protein levels were detected, and images from capillary-based electrophoresis (Simple Western using PeggySue) are shown. Figure 2 1 shows PBMC, NK cells, monocytes, MCSF M1 macrophages, or GMCSF M1 macrophages isolated from the same donor as shown in the figure, in the presence or absence of rophage or GMCSF-differentiated M1 macrophages for 48 hours , and shows the relative amount of EGFR in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control. Figure 22 shows, as shown in the figure, the relative amount of pEGFR (pY1173) in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control in the presence or absence of PBMC, NK cells, monocytes, MCSF M1 macrophages, or GMCSF M1 macrophages isolated from the same donor as shown in the figure for 48 hours. Figure 23 shows, as shown in the figure, the relative amount of c-Met in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control in the presence or absence of PBMC, NK cells, monocytes, MCSF M1 macrophages, or GMCSF M1 macrophages isolated from the same donor as shown in the figure for 48 hours. Samples were normalized to the amount of loading control actin present in each sample and then normalized to the control (untreated) sample. NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control. Figure 22 shows, as shown in the figure, the relative amount of pEGFR (pY1173) in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control in the presence or absence of PBMC, NK cells, monocytes, MCSF M1 macrophages, or GMCSF M1 macrophages isolated from the same donor as shown in the figure for 48 hours. Figure 23 shows, as shown in the figure, the relative amount of c-Met in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control in the presence or absence of PBMC, NK cells, monocytes, MCSF M1 macrophages, or GMCSF M1 macrophages isolated from the same donor as shown in the figure for 48 hours. Samples were normalized to the amount of loading control actin present in each sample and then normalized to the control (untreated) sample. PBMC, NK cells, monocytes, MCSF M1 macrophages, or GM CSF M1 macrophages in the presence or absence of rophage or GMCSF-differentiated M1 macrophages for 48 hours, JNJ-372, JNJ -372.IgG2 sigma, or isotype control. Figure 22 shows, as shown in the figure, the relative amount of pEGFR (pY1173) in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control in the presence or absence of PBMC, NK cells, monocytes, MCSF M1 macrophages, or GMCSF M1 macrophages isolated from the same donor as shown in the figure for 48 hours. Figure 23 shows, as shown in the figure, the relative amount of c-Met in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control in the presence or absence of PBMC, NK cells, monocytes, MCSF M1 macrophages, or GMCSF M1 macrophages isolated from the same donor as shown in the figure for 48 hours. Samples were normalized to the amount of loading control actin present in each sample and then normalized to the control (untreated) sample. NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control. Figure 22 shows, as shown in the figure, the relative amount of pEGFR (pY1173) in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control in the presence or absence of PBMC, NK cells, monocytes, MCSF M1 macrophages, or GMCSF M1 macrophages isolated from the same donor as shown in the figure for 48 hours. Figure 23 shows, as shown in the figure, the relative amount of c-Met in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control in the presence or absence of PBMC, NK cells, monocytes, MCSF M1 macrophages, or GMCSF M1 macrophages isolated from the same donor as shown in the figure for 48 hours. Samples were normalized to the amount of loading control actin present in each sample and then normalized to the control (untreated) sample. PBMC, NK cells, monocytes, MCSF M1 macrophages, or GMCSF M1 macrophages in the presence or absence of rophage or GMCSF-differentiated M1 macrophages for 48 hours, JNJ-372, JNJ-37 2.IgG2 sigma, or isotype control. Figure 22 shows, as shown in the figure, the relative amount of pEGFR (pY1173) in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control in the presence or absence of PBMC, NK cells, monocytes, MCSF M1 macrophages, or GMCSF M1 macrophages isolated from the same donor as shown in the figure for 48 hours. Figure 23 shows, as shown in the figure, the relative amount of c-Met in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control in the presence or absence of PBMC, NK cells, monocytes, MCSF M1 macrophages, or GMCSF M1 macrophages isolated from the same donor as shown in the figure for 48 hours. Samples were normalized to the amount of loading control actin present in each sample and then normalized to the control (untreated) sample. Met in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control. Samples were normalized to the amount of loading control actin present in each sample and then normalized to the control (untreated) sample. Samples were normalized to the amount of loading control actin present in each sample and then normalized to the control (untreated) sample.

[0175] Subsequently, the EGFR / c-Met downregulation driven by the JNJ-372 Fc interaction The effects of various macrophage subtypes in regulation were evaluated. Monocytes from one donor were differentiated into M1, M2a, and M2c macrophages, and the ability to enhance EGFR / c-Met downmodulation mediated by JNJ-372 was evaluated. H1975 cells were treated with 10 μg / mL isotype control, JNJ-372, JNJ-372 .IgG2 sigma, and cultured for 48 hours at a 5:1 E:T ratio in the presence or absence of M1, M2a, and M2c macrophages obtained by differentiating monocytes from one donor. The presence of M1, M2a, and M2c all significantly enhanced the JNJ-372-mediated downregulation of EGFR, pEGFR, and c-Met proteins. Figure 24 shows, as depicted in the figure, the EGFR, c-Me t, and pEGFR protein levels in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control and cultured in the presence of M1 macrophages (M1) or M2a macrophages (M2a), detected by capillary-based electrophoresis (Simple Western using PeggySue). Figure 25 shows, as depicted in the figure, the EGFR, c-Met, and pEGFR levels in NCI-H1975 cells treated with JNJ-372, JNJ-372.IgG2 sigma, or isotype control and cultured for 48 hours in the presence or absence of M2c macrophages (M2c), detected by capillary-based electrophoresis (Simple Western using PeggySue ).

[0176] Example 4. JN of EGFR and c-Met proteins and their downstream signal transduction J-372-mediated downregulation of c-Met expression in c-Met-amplified tumor cell lines Mediated by Fc interactions SNU-5 cells (c-Met amplified cell line) were incubated with 10 μg / mL JNJ-372 or In the presence or absence of PBMCs from one donor, treated with isotype control, The cells were cultured for 48 hours at an E:T ratio of 10:1. The addition of PBMCs was performed to confirm that JNJ-372 inhibits EGFR , enhanced ability to downregulate pEGFR, c-Met, and p-Met Figure 26 shows the results of PBMCs treated with JNJ-372 or isotype control as indicated. EGFR, c-Met, and pEGFR in SNU-5 cells cultured in the presence or absence of Capillary-based electrophoresis (PeggySue) to detect FR protein levels Figure 27 shows images from a Simple Western microscope (SWS) using the J Treated with NJ-372 or isotype control and cultured in the presence or absence of PBMCs Figure 28 shows the relative amount of EGFR in SNU-5 cells. SNU-5 cells were treated with IgG or isotype control and cultured in the presence or absence of PBMCs. Figure 29 shows the relative amount of pEGFR(pY1173) in the cells. SN cultured for 48 h in the presence or absence of 372, isotype control, or PBMCs Figure 30 shows the relative amount of c-Met in U-5 cell culture samples. SNU treated with 372 or isotype control and cultured in the presence or absence of PBMCs The relative amounts of pMet (pY1234 / 1235) in -5 cells are shown. Normalize to the amount of loading control actin present and then compare it with the control (untreated) sample. Normalized against .

[0177] Example 5. In Vivo Inhibition of Tumor Growth Mediated by FcγR and JNJ-372 Fc interaction Next, the H1975 and SNU5 cell line xenograft models were used to evaluate the role and relevance of Fc / FcγR interactions in vivo.

[0178] Six- to eight-week-old female BALB / c nude mice (CAnN.Cg-Foxn1 nu / Crl , Charles River Laboratories (Wilmington, M A)) were subcutaneously implanted with the NCI-H1975 cell line. When the tumors reached an average of 72 ± 8.7 mm 3 and to promote macrophage depletion, anti-mCSF-1R antibody (400 μg / mouse) was intraperitoneally administered three times a week starting 5 days before the start of compound administration over the study period. On day 5, when the tumors reached an average of 102 ± 36.6 mm 3 and the mice were treated twice a week by intraperitoneal administration of isotype control Ab ( 10 mg / kg), JNJ-372 (10 mg / kg), or JNJ-372.IgG2 sigma (10 mg / kg). Tumors were sampled from a cohort of mice to monitor macrophage infiltration after two doses of the compound. Seven- to eight-week-old female CB17 / SCID mice (HFK Bio-Technology Co., Ltd. (Beijing, China)) were subcutaneously implanted with the SNU5 cell line. When the tumors reached an average of 155 ± 21.4 mm and 3 the mice were intraperitoneally treated twice a week for 3 weeks with phosphate-buffered saline (PBS ), JNJ-372 (5 mg / kg), or JNJ-372.IgG2 sigma (5 mg / kg). Over the course of each study, in both studies Subsequently, tumor measurements and body weights were recorded twice a week. The calculation [1-(T / C)]×100 was used to calculate tumor growth inhibition (TGI) on the last day that >80% of the control mice remained in the study All in vivo experiments were conducted in accordance with the Johnson and Johnson Ins titutional Animal Care and Use Committee and the Guide for Care and Use of Laborato ry Animals

[0179] In the H1975 model, treatment with JNJ-372 resulted in 75% tumor growth inhibition (TGI) compared to the isotype control (Figure 31). However, JNJ-372.IgG2 sigma was much less effective, with a TGI of only 30% (Figure 31). Similarly, JNJ-372 treatment was highly effective in reducing tumor growth in the MET-amplified SNU5 model, with a TGI of 96%, but JNJ-372.IgG2 sigma treatment was ineffective (TGI was -17%) (Figure 32). In none of these tumor models was there an effect of antibody treatment on mouse body weight (Figure 32). In the H1975 model, treatment with JNJ-372 resulted in 75% tumor growth inhibition (TGI) compared to the isotype control (Figure 31). However, JNJ-372.IgG2 sigma was much less effective, with a TGI of only 30% (Figure 31). Similarly, JNJ-372 treatment was highly effective in reducing tumor growth in the MET-amplified SNU5 model, with a TGI of 96%, but JNJ-372.IgG2 sigma treatment was ineffective (TGI was -17%) (Figure 32). In none of these tumor models was there an effect of antibody treatment on mouse body weight (Figure 32). In the H1975 model, treatment with JNJ-372 resulted in 75% tumor growth inhibition (TGI) compared to the isotype control (Figure 31). However, JNJ-372.IgG2 sigma was much less effective, with a TGI of only 30% (Figure 31). Similarly, JNJ-372 treatment was highly effective in reducing tumor growth in the MET-amplified SNU5 model, with a TGI of 96%, but JNJ-372.IgG2 sigma treatment was ineffective (TGI was -17%) (Figure 32). In none of these tumor models was there an effect of antibody treatment on mouse body weight (Figure 32). In the H1975 model, treatment with JNJ-372 resulted in 75% tumor growth inhibition (TGI) compared to the isotype control (Figure 31). However, JNJ-372.IgG2 sigma was much less effective, with a TGI of only 30% (Figure 31). Similarly, JNJ-372 treatment was highly effective in reducing tumor growth in the MET-amplified SNU5 model, with a TGI of 96%, but JNJ-372.IgG2 sigma treatment was ineffective (TGI was -17%) (Figure 32). In none of these tumor models was there an effect of antibody treatment on mouse body weight (Figure 32). In the H1975 model, treatment with JNJ-372 resulted in 75% tumor growth inhibition (TGI) compared to the isotype control (Figure 31). However, JNJ-372.IgG2 sigma was much less effective, with a TGI of only 30% (Figure 31). Similarly, JNJ-372 treatment was highly effective in reducing tumor growth in the MET-amplified SNU5 model, with a TGI of 96%, but JNJ-372.IgG2 sigma treatment was ineffective (TGI was -17%) (Figure 32). In none of these tumor models was there an effect of antibody treatment on mouse body weight (Figure 32). In the H1975 model, treatment with JNJ-372 resulted in 75% tumor growth inhibition (TGI) compared to the isotype control (Figure 31). However, JNJ-372.IgG2 sigma was much less effective, with a TGI of only 30% (Figure 31). Similarly, JNJ-372 treatment was highly effective in reducing tumor growth in the MET-amplified SNU5 model, with a TGI of 96%, but JNJ-372.IgG2 sigma treatment was ineffective (TGI was -17%) (Figure 32). In none of these tumor models was there an effect of antibody treatment on mouse body weight (Figure 32). In the H1975 model, treatment with JNJ-372 resulted in 75% tumor growth inhibition (TGI) compared to the isotype control (Figure 31). However, JNJ-372.IgG2 sigma was much less effective, with a TGI of only 30% (Figure 31). Similarly, JNJ-372 treatment was highly effective in reducing tumor growth in the MET-amplified SNU5 model, with a TGI of 96%, but JNJ-372.IgG2 sigma treatment was ineffective (TGI was -17%) (Figure 32). In none of these tumor models was there an effect of antibody treatment on mouse body weight (Figure 32).

[0180] Example 6. Fc interaction induced ADCC mediated by NK cells but did not induce CDC The ability of JNJ-372 to induce various Fc effector functions was investigated. ADCC induced by JNJ-372 was measured using a europium release assay in the presence of PBMC from seven different donors. Antibody-mediated lysis of H1975 cells varied among donors, with some donors showing approximately 60-70% ADCC activity, while other donors The ability of JNJ-372 to induce various Fc effector functions was investigated. ADCC induced by JNJ-372 was measured using a europium release assay in the presence of PBMC from seven different donors. Antibody-mediated lysis of H1975 cells varied among donors, with some donors showing approximately 60-70% ADCC activity, while other donors The ability of JNJ-372 to induce various Fc effector functions was investigated. ADCC induced by JNJ-372 was measured using a europium release assay in the presence of PBMC from seven different donors. Antibody-mediated lysis of H1975 cells varied among donors, with some donors showing approximately 60-70% ADCC activity, while other donors The ability of JNJ-372 to induce various Fc effector functions was investigated. ADCC induced by JNJ-372 was measured using a europium release assay in the presence of PBMC from seven different donors. Antibody-mediated lysis of H1975 cells varied among donors, with some donors showing approximately 60-70% ADCC activity, while other donors The ability of JNJ-372 to induce various Fc effector functions was investigated. ADCC induced by JNJ-372 was measured using a europium release assay in the presence of PBMC from seven different donors. Antibody-mediated lysis of H1975 cells varied among donors, with some donors showing approximately 60-70% ADCC activity, while other donors Ner had no measurable ADCC activity (data not shown). Fc / FcγR To evaluate the contribution of Fc / FcγR engagement, H1975 cells were treated with isotype control, JNJ-372, or JNJ-372.IgG2 sigma in the presence of PBMCs from two donors .. JNJ-372 induced dose-dependent ADCC, but no cell death was observed with isotype control or JNJ-372.IgG2 sigma treatment (data not shown). To determine the immune cell subtypes within PBMCs involved in ADCC induced by JNJ-372, ADCC lysis induced in the presence of PBMCs against isolated NK cells or isolated monocytes from the same donors was evaluated . Both PBMCs and NK cells induced JNJ-372-dependent ADCC lysis, but isolated monocytes did not (Figure 33), indicating that NK cells are involved in the ADCC activity induced by JNJ-372 . No measurable CDC activity towards H1975 and H292 NSCLC cell lines was observed with JNJ-372 or JNJ-372.IgG2 sigma (data not shown), suggesting that JNJ-372 does not induce CDC against these NSCLC cell lines Example 7. Effect of Macrophages on JNJ-372-Mediated Tumor Cell Killing In Vivo To investigate the role of macrophages in vivo, anti-CSF1R antibody was used to deplete tumor-associated macrophages in mice bearing H1975 xenograft tumors, and the efficacy of JNJ-372 was measured . Treatment with anti-CSF1R antibody showed a significant decrease in TAM compared to untreated ( , p < 0.002), with macrophages decreasing from 11 - 15% to Example 7. Effect of Macrophages on JNJ-372-Mediated Tumor Cell Killing In Vivo . No measurable CDC activity towards H1975 and H292 NSCLC cell lines was observed with JNJ-372 or JNJ-372.IgG2 sigma (data not shown), suggesting that JNJ-372 does not induce CDC against these NSCLC cell lines . Treatment with anti-CSF1R antibody showed a significant decrease in TAM compared to untreated ( , p < 0.002), with macrophages decreasing from 11 - 15% to

[0181] Example 7. Effect of Macrophages on JNJ-372-Mediated Tumor Cell Killing In Vivo Effect of Macrophages on JNJ-372-Mediated Tumor Cell Killing In Vivo To investigate the role of macrophages in vivo, anti-CSF1R antibody was used to deplete tumor-associated macrophages in mice bearing H1975 xenograft tumors, and the efficacy of JNJ-372 was measured . Treatment with anti-CSF1R antibody showed a significant decrease in TAM compared to untreated ( , p < 0.002), with macrophages decreasing from 11 - 15% to . Treatment with anti-CSF1R antibody showed a significant decrease in TAM compared to untreated ( ** , p < 0.002), with macrophages decreasing from 11 - 15% to Depleted to approximately 2% (Figure 34). Subsequently, animals were treated with isotype control, JNJ-3 72, or JNJ-372.IgG2 sigma for 3 weeks, but no weight loss was observed in any of the groups (data not shown). As previously shown, treatment with JNJ-3 72 demonstrated significantly higher anti-tumor efficacy compared to isotype control ( ** * , p < 0.0001) or JNJ-372.IgG2 sigma treatment ( ** , p = 0.00 4) in tumors treated with non-CSF1R antibodies (Figure 35). Notably, depletion of tumor-associated macrophages (anti-CS1R treatment) significantly reduced TGI from 72.8% to 38.5% ( *** , p < 0.0001) (Figure 35), suggesting that macrophages play an important role in mediating the anti-tumor efficacy of JNJ-3 72 in vivo.

[0182] These results demonstrated that macrophages are essential for anti-tumor efficacy in vivo.

[0183] Example 8. JNJ-372 Fc interaction with immune cells induces antibody-dependent cytokine and chemokine release (ADCR) The interaction between the Fc region of therapeutic antibodies and FcγRs on immune cells is known to induce the secretion of chemokines and cytokines (ADCR) (Kinder et al., mAbs 7:494-504, 2015). Using a 71-plex MSD cytokine panel, chemokines secreted upon treatment with isotype control, JNJ-372, or JNJ-372.IgG2 sigma in the presence or absence of PBMCs for 4 or 72 hours were measured. Interleukins and cytokines were evaluated. Treatment- and time-dependently, characteristic differences were observed in several secreted cytokines; 32 out of 71 cytokines tested and 42 out of 71 cytokines had reliably measurable responses (AUC) at 4 and 72 hours, respectively. Focusing on cytokines with >1.5-fold differences between treatments further analysis showed that 13 and 7 cytokines, respectively, were upregulated upon treatment with JNJ-372 compared to isotype control or JNJ-372.IgG2 sigma in the co-culture of H1975 + PBMC at 4 hours (Figure 36) and 72 hours (Figure 37). Many of the cytokines that changed belonged to the chemotactic cytokine family (CC chemokines) (Figure 6B - MIP1β, MCP-1, MCP-3, eotaxin, eotaxin-2), which are known to function as chemotactic factors for innate immune cells, monocytes, and macrophages (Graves et al., Crit Rev Oral Biol Med 6:109 - 18, 1995; Uguccioni et al., Eur J Immunol 25:64 - 8, 1995; Balkwill, Nat Rev Cancer 4:540 - 50, 2004). To further evaluate these cytokines and examine the role of individual immune cells in their secretion, 23 cytokines were selected (based on their function and changes upon treatment with JNJ-372) and treated with isotype, JNJ-372, or JNJ-372.IgG2 sigma antibodies in the presence of PBMC against individual immune cells isolated from the same donor. .

[0184] ​ were evaluated upon treatment. Heatmap analysis revealed characteristic changes in cytokine expression patterns by individual immune cells, and CC chemokines were the family most frequently upregulated upon treatment with JNJ-372. Upon more intensive analysis of cytokines with a >1.5-fold difference in the presence of PBMC, a pattern of upregulation specific to PBMC, monocytes, and macrophages but not to NK cells upon treatment with JNJ-372 was revealed. For example, as a result of treatment with JNJ-372, the levels of MCP-1 (Figure 38) and MCP-3 (Figure 39) increased in a dose-dependent manner in the presence of PBMC or monocytes, but did not increase in the presence of isotype or JNJ-372.IgG2 sigma, nor in the presence of NK cells. IL1-RA, which is known to be secreted by monocytes and macrophages in response to activation of stimulation (Janson et al., J Immunol 147:4218-23, 1991; Arend et al., Ann Rheum Dis 59 Suppl 1:i60-4, 2000), increased in a dose-dependent manner upon treatment with JNJ-372 in the presence of PBMC and monocytes, but not in NK cells (Figure 40). Furthermore, a dose-dependent increase in MIP1β levels was observed upon treatment with JNJ-372 in the presence of immune cells, but not in the isotype control or JNJ-372.IgG2 sigma (Figure 41). Also, both MIP1 α and MIP1β, which are MIP family proteins, were upregulated in co-culture with M1 and M2 macrophages upon treatment with JNJ-37 but not in the isotype control or JNJ-372.IgG2 sigma (Figure 41). Furthermore, a dose-dependent increase in MIP1β levels was observed upon treatment with JNJ-372 in the presence of immune cells, but not in the isotype control or JNJ-372.IgG2 sigma (Figure 41). Also, both MIP1 α and MIP1β, which are MIP family proteins, Upon treatment with 2, it is upregulated, and in M2c macrophages, a greater magnitude of fold change of MIP1β is observed compared to . Figure 42 shows the dose - response curve of the level of MIP - 1β in H1975 cells cultured in the presence of M1 macrophages. Figure 43 shows the dose - response curve of the level of MIP - 1β in H1975 cells cultured in the presence of M2 macrophages. Figure 44 shows the dose - response curve of the level of MIP - 1α in H1975 cells cultured in the presence of M1 macrophages. Figure 45 shows the dose - response curve of the level of MIP - 1α in H1975 cells cultured in the presence of M2 macrophages. To evaluate the effect of the secreted chemokines on the down - regulation of EGFR / cMet, conditioned media from H1975 cells treated with isotype control,

[0185] JNJ - 372, or JNJ - 372.IgG2 sigma for 4 hours or 72 hours in the presence or absence of PBMCs was transferred to untreated H1975 cells, and changes in the EGFR and cMet pathways were evaluated. A measurable down - regulation of EGFR, pEGFR, and cMet protein levels was observed at all time points in the presence of the conditioned media (data not shown). This suggests that the secreted cytokines and chemokines were not sufficient to induce enhanced down - regulation of EGFR / cMet, presumably because this requires antibody - mediated direct contact between tumor cells and immune cells. )

[0186] Example 9. JNJ - 372 Fc interaction with monocytes and macrophages induces trogocytosis (ADCT) Another important Fc effector function, trogocytosis (ADCT), was measured in target cells using a flow cytometry-based assay that measures the transfer of labeled antibody bound to target cells to effector cells (macrophages). H1975 NucLight Red cells were opsonized with AF488-labeled isotype, JNJ-372, or JNJ-372.IgG2 sigma and co-cultured with M1 or M2c macrophages, and the percentage of AF48 8+ macrophages was evaluated. Labeled JNJ-372 migrated dose-dependently to both M1 (Figure 46) and M2c (Figure 47) macrophages, but neither the isotype control nor the JNJ-372.IgG2 sigma-labeled antibody was detected in CD11b+ macrophages (data not shown). Recognizable NucLight Red+ macrophages were not detected, indicating a lack of phagocytosis. This suggests that trogocytosis is the major mechanism in this assay.

[0187] To visually confirm macrophage trogocytosis induced by JNJ-372, time-lapse microscopy was performed, and macrophages were visualized with a CD11b / CD14 antibody cocktail and nuclei were visualized with Hoechst staining, and H1975 target cells were identified by their NucLight Red+ nuclei. H1975 target cells were opsonized with AF647-labeled isotype, JNJ-372, or JNJ-372.IgG2 sigma antibody and co-cultured with M1 or M2c macrophages to obtain high-content confocal images. Co-culture with target cells opsonized with labeled JNJ-372 resulted in the observation of distinct accumulations of AF647+ spots (JNJ-372) within M1 and M2 macrophages, but labeled No accumulation was observed with the resulting isotype or JNJ-372.IgG2 sigma treatment. Similar to the previous assay, minimal phagocytosis was also observed in these assays, which indicates that the major mechanism of receptor downregulation by JNJ-372 is trogocytosis. Figure 48 shows representative images from high-content confocal microscopy at 11 minutes of a 44-minute culture. Figure 49 shows representative images from high-content confocal microscopy at 77 minutes of a 110-minute culture.

[0188] Next, the ability of monocytes to perform trogocytosis was examined. Similar to macrophages, monocytes co-cultured with JNJ-372 (AF647-labeled) opsonized target cells, which demonstrated specific transfer of the labeled JNJ-372 antibody to monocytes (data not shown). Finally, to simulate antibody interactions within the tumor microenvironment, co-cultures of M1 or M 2c macrophages and AF647-labeled isotype of H1975 target cells were treated with JNJ-372 or JNJ-372.IgG2 sigma antibody. Under these conditions, the isotype control bound only to M1 and M2c macrophages, JNJ-3 72.IgG2 sigma bound only to target cells, while JNJ-372 bound to both target cells and m acrophages (data not shown), thus confirming the binding specificity of each antibody. JNJ-372-mediated trogocytosis was similarly easily observed in co-culture conditions as measured by the distinct transfer of the labeled JNJ-37 2 antibody to macrophages, but was not observed with the isotype or JNJ-372.IgG2 sigma antibody.

[0189] Collectively, these findings demonstrate that JNJ-372 induces trogocytosis through its interaction with Fcγ receptors on macrophages and monocytes.

[0190] Discussion The experiments described herein demonstrate that JNJ-372, an EGFR / cMet bispecific antibody, has multiple Fc-dependent mechanisms that contribute to its antitumor efficacy. In addition to inducing ADCC mediated by NK cells, the interaction of JNJ-372 with Fcγ receptors on immune cells also mediates downmodulation of receptor tyrosine kinases EGFR and cMet and their phosphorylated forms via trogocytosis. This novel Fc function is promoted by monocytes and macrophages, which is also required for antitumor efficacy in vivo. The Fc interaction of JNJ-372 with immune cells was demonstrated to be required for anti-proliferative and apoptotic effects in vitro and antitumor efficacy in vivo. In vitro, the maximum anti-proliferative effect was observed after 48 hours, and given that ADCC occurs early (approximately 2-4 hours (29)), it was hypothesized that the contribution of ADCC to total tumor cell killing could be minimal. CDC activity was not observed in the cell lines evaluated, although NSCLC cell lines are known to express complement inhibitory proteins, CD46, CD55, and CD59 (Varsano et al., Clin Exp Immunol 113:173-82, 1998). This suggests that JNJ-372 did not induce CDC in the cells tested, but that bispecific antibodies may induce CDC activity in other cell lines or tumor types.

[0191] The Fc interaction of JNJ-372 with immune cells was demonstrated to be required for anti-proliferative and apoptotic effects in vitro and antitumor efficacy in vivo. In vitro, the maximum anti-proliferative effect was observed after 48 hours, and given that ADCC occurs early (approximately 2-4 hours (29)), it was hypothesized that the contribution of ADCC to total tumor cell killing could be minimal. CDC activity was not observed in the cell lines evaluated, although NSCLC cell lines are known to express complement inhibitory proteins, CD46, CD55, and CD59 (Varsano et al., Clin Exp Immunol 113:173-82, 1998). This suggests that JNJ-372 did not induce CDC in the cells tested, but that bispecific antibodies may induce CDC activity in other cell lines or tumor types. duce, but that bispecific antibodies may induce CDC activity in other cell lines or tumor types. suggested that it might be able to direct. Finally, it has already been reported that JNJ-372 induced ADCP in vitro (Moores et al., Cancer Res 76:3942-53,2016), but under the conditions used in the flow and confocal microscopy-based trogocytosis assays in this study, ADCP was minimally to not at all observed. These results suggested that JNJ-372 functions through multiple mechanisms of action and that the contribution of each of these Fc effector functions varies among patients . It has also been demonstrated that JNJ-372 induced ADCR, and from the exploration of the functions of cytokines regulated by JNJ-372, it became clear that most belong to a family of chemotactic cytokines called chemokines, specifically the CC chemokines (Graves et al., Crit Rev Oral Biol 6:109-18,1995; Balkwill, Nat Rev Cancer 4:540-50,2004). CC chemokines are known to function as chemotactic factors for innate immune cells such as monocytes and macrophages and are composed of two major subfamilies, monocyte chemoattractant proteins (MCP) and macrophage inflammatory proteins (MIP) (Uguccioni et al., Eur J Immunol 25:64-8,1995; Loetscher et al., FASEB J 8:1055-60,1994). MCP family members, MCP-1 (CCL2) and MCP-3

[0192] ( ( ( ( ( ( ( ( ( ( ( CCL7 has been shown to increase the recruitment of inflammatory monocytes and CD8+ T lymphocytes (Uguccioni et al., Eur J Immunol 25:64- 8, 1995; Loetscher et al., FASEB J 8:1055-6 0, 1994; Jia et al., J Immunol 180:6846-53, 2008). MCP-1 and MIP1β (CCL4) have also been reported to induce the recruitment of monocytes and macrophages to the tumor microenvironment (TME) of NSCLC (Ugu ccioni et al., Eur J Immunol 25:64-8, 1995 ; Arenberg et al., Cancer Immunol Immunoth er 49:63-70, 2000).

[0193] These cytokines can attract and activate immune cells in the TME, but the requirement for antibody-mediated cell-cell contact and the induction of trogocytosis suggest that the mechanism by which the JNJ- 372 Fc interaction mediates the downmodulation of EGFR and cMet signaling is through trogocytosis mediated by monocytes or macrophages. Some recent reports have shown that therapeutic antibody-opsonized tumor cells can lead to cell death through similar transfer of the Her2 receptor to immune cells such as macrophages and neutrophils by trogocytosis (Velmurugan et al., Mol Cancer Ther 15:1879-89, 2016; Matlung et al., Cell Rep 23:3946-59, 2018). This was previously thought to be the mechanism of resistance to antibodies such as rituximab through trogocytosis. (Velmurugan et al., Mol Cancer Ther 15:1879-89, 2016; Matlung et al., Cell Rep 23:3946-59, 2018). This was previously thought to be the mechanism of resistance to antibodies such as rituximab through trogocytosis. suggested that it may also have a function that mediates an anti-tumor effect as an Fc effector function (Taylor and Lindofer, Blood 125:762-6, 201 5; Pham et al., PLoS One 6:e14498, 2011).

[0194] In conclusion, JNJ-372 was demonstrated to present multiple distinct mechanisms of action with several Fc-dependent and Fc-independent functions that contribute to its anti-tumor activity. In vitro and in vivo models suggest that the interaction of JNJ-372 with FcγRs on monocytes and macrophages is required for the down-modulation of EGFR / Met and anti-tumor efficacy, which may predict the efficacy of JNJ-372 in the clinical setting from the levels of these immune cells. ​​

Claims

1. Treating subjects with EGFR, c-Met, or EGFR and c-Met expressing cancer The method includes administering to the subject a therapeutic agent in combination with an agent that enhances macrophage activity in the subject. A therapeutically effective amount of an isolated bispecific anti-epidermal growth factor receptor (EGFR) / hepatocyte growth factor receptor administering to said subject a c-Met antibody.

2. The bispecific anti-EGFR / c-Met antibody is a) a first domain that binds to EGFR, the first domain being a heavy chain complementarity determining region 1 of SEQ ID NO: 1; (HCDR1), HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3, light chain of SEQ ID NO:4 Complementarity determining region 1 (LCDR1), LCDR2 of SEQ ID NO:5, and LCDR of SEQ ID NO:6 a first domain including: b) a second domain that binds to c-Met, comprising HCDR1 of SEQ ID NO: 7, SEQ ID NO: SEQ ID NO: 8, HCDR2, SEQ ID NO: 9, LCDR1, SEQ ID NO: 10, SEQ ID NO: 11 and a LCDR2 of SEQ ID NO: 12; and a second domain comprising an LCDR3 of SEQ ID NO:

13. The method of claim 1 , comprising:

3. a) the first domain that binds to EGFR comprises a heavy chain variable region (VH) of SEQ ID NO: 13; and a light chain variable region (VL) of SEQ ID NO: 14, b) the second domain that binds to c-Met comprises a VH of SEQ ID NO: 15 and a VH of SEQ ID NO: 1 6, The method of claim 2.

4. The bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype.

4. The method according to any one of 1 to 3.

5. The bispecific anti-EGFR / c-Met antibody comprises a first heavy chain (HC1) of SEQ ID NO: 17 18; a first light chain (LC1) of SEQ ID NO: 18; a second heavy chain (HC2) of SEQ ID NO: 19; and The method according to any one of claims 1 to 4, comprising a second light chain (LC2) of sequence number 20.

6. The drug that enhances macrophage activity is GM-CSF, anti-CD47 antibody, HDAC 2 inhibitor, PD-(L)1 axis inhibitor, or CD11b agonist according to claims 1 to 5.

2. The method according to any one of claims 1 to 11.

7. The EGFR or c-Met expressing cancer is selected from the group consisting of wild-type EGFR, activating EGFR mutations, EGFR FR gene amplification, elevated levels of circulating HGF, wild-type c-Met, c-Met activating mutations , c-Met gene amplification, or mutant KRAS. The method described above.

8. The EGFR activating mutations are L718Q, G719A, G719X (wherein X is any amino acid). L861X (wherein X is any amino acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, L858 P or T790M substitution, E746-A750 deletion, R748-P753 deletion , an insertion of Ala (A) between M766 and A767, an insertion of S between S768 and V769 Insertion of Er, Val, and Ala (SVA), Asn and Insertions of Ser(NS), D761 and E762, A763 and Y764, Y764 and Y765 , M766 and A767, A767 and V768, S768 and V769, V769 and D770 , D770 and N771, N771 and P772, P772 and H773, H773 and V774 , an insertion of one or more amino acids between V774 and C775, an EGFR exon one or more deletions in EGFR exon 20, or one or two in EGFR exon 20 The method of claim 7, comprising the above insertions or any combination thereof.

9. The mutant KRAS has a substitution of G12V, G12C, G12A, or G12D; includes any combination thereof.

10. The subject has newly diagnosed EGFR, c-Met, or EGFR and c-Met tumors. The method of any one of claims 1 to 9, wherein the patient has current cancer.

11. The subject is resistant or has acquired resistance to treatment with a previous anti-cancer therapy. The method according to any one of claims 1 to 9,

12. The previous anti-cancer therapy is a chemotherapy, a targeted anti-cancer therapy, or a kinase inhibitor. The method according to claim 11.

13. The kinase inhibitor is selected from the group consisting of EGFR, c-Met, HER2, HER3, HER4, VEGF, The method of claim 12, which is an inhibitor of GFR or AXL.

14. The kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, alanine, Fatinib, osimertinib, lazertinib, poziotinib, cliotinib, cabozantinib Nib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, paclitaxel 14. The method of claim 13, wherein the agent is zopanib, sorafenib, or sunitinib.

15. the EGFR, c-Met, or EGFR and c-Met expressing cancer is an epithelial cell cancer; Breast cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, small cell lung cancer, Colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharyngeal cancer, nasal cancer cancer, pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, splenic cancer Cancer of the pancreas, testicular cancer, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocyte cancer The patient has a cancer of the type 1 or type 2 that is classified as having hepatic pulmonary cancer (HCC), or sporadic or hereditary papillary renal cell carcinoma (PRCC). Item 15. The method according to any one of items 1 to 14.

16. The method according to any one of claims 1 to 15, further comprising administering to the subject one or more anti-cancer therapies. The method according to any one of claims 1 to 5.

17. The one or more anti-cancer therapies are chemotherapy, targeted anti-cancer therapy, or kinase inhibition. The method of claim 16, further comprising the step of:

18. The kinase inhibitor is selected from the group consisting of EGFR, c-Met, HER2, HER3, HER4, VEGF, 18. The method of claim 17, which is an inhibitor of GFR or AXL.

19. The kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, alanine, Fatinib, osimertinib, lazertinib, poziotinib, cliotinib, cabozantinib Nib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, paclitaxel 19. The method of claim 18, wherein the agent is zopanib, sorafenib, or sunitinib.

20. EGFR, c-Met, and or a method of diagnosing and treating a subject with an EGFR- and c-Met-expressing cancer, comprising: a) providing a biological sample from said subject; b) measuring macrophage or monocyte levels from said biological sample; c) when the level of macrophages or monocytes from the biological sample is greater than a threshold value, The subject responds to treatment with the bispecific anti-EGFR / c-Met antibody. diagnosing the patient as having an EGFR, c-Met, or EGFR and c-Met expressing cancer; d) administering to the subject diagnosed as responding to treatment with the anti-EGFR / c-Met antibody administering or providing for administration said bispecific anti-EGFR / c-Met antibody. Toto The method comprising:

21. Patients suspected of having or who have EGFR, c-Met, or EGFR and c-Met expressing cancer 1. A method of treating a subject with a bispecific anti-EGFR / c-Met antibody comprising: a) determining that the subject has a macrophage or monocyte level above a threshold; And, b) the subject determined to have a macrophage or monocyte level higher than the threshold. and administering or providing for administration of said bispecific anti-EGFR / c-Met antibody to said subject. To The method comprising:

22. Treatment with bispecific anti-EGFR / c-Met antibodies: or a method of predicting the response of a subject with an EGFR and c-Met expressing cancer, comprising: a) providing a biological sample from said subject; b) measuring macrophage or monocyte levels from said biological sample; c) when the level of macrophages or monocytes from the biological sample is greater than a threshold value, Predicting that the subject is a responder The method comprising:

23. EGFR, c-Met, and or a method of treating a subject having an EGFR- and c-Met-expressing cancer, comprising: a) providing a biological sample from said subject; b) measuring macrophage or monocyte levels from said biological sample; c) when the level of macrophages or monocytes from the biological sample is greater than a threshold value, treating the subject with the bispecific anti-EGFR / c-Met antibody. The method comprising:

24. Subjects with EGFR, c-Met, or EGFR and c-Met expressing cancers are and determining whether the subject responds to treatment with a specific anti-EGFR / c-Met antibody. A method for determining whether to a) providing a biological sample from said subject; b) measuring macrophage or monocyte levels from said biological sample; c) when the level of macrophages or monocytes from the biological sample is greater than a threshold value, The subject having an EGFR-, c-Met-, or EGFR- and c-Met-expressing cancer is The bispecific anti-EGFR / c-Met antibody is used to diagnose or predict a patient as being responsive to treatment with the bispecific anti-EGFR / c-Met antibody. When the macrophage or monocyte level from the biological sample is lower than the threshold, The subject having a EGFR-, c-Met-, or EGFR- and c-Met-expressing cancer is diagnosing non-responsiveness to treatment with a bispecific anti-EGFR / c-Met antibody; d) a patient diagnosed as responding to treatment with the bispecific anti-EGFR / c-Met antibody administering the bispecific anti-EGFR / c-Met antibody to the subject, or administering the bispecific anti-EGFR / c-Met antibody to the subject. The subject diagnosed as not responding to treatment with an EGFR / c-Met antibody is administered the bispecific antibody. and refraining from administering anti-EGFR / c-Met antibodies to patients with The method comprising:

25. The bispecific anti-EGFR / c-Met antibody is a) a first domain that binds to EGFR, comprising HCDR1 of SEQ ID NO: 1, SEQ ID NO: HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3, LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5 a first domain comprising a DR2, and an LCDR3 of SEQ ID NO:6; b) a second domain that binds to c-Met, comprising HCDR1 of SEQ ID NO: 7, SEQ ID NO: SEQ ID NO: 8, HCDR2, SEQ ID NO: 9, LCDR1, SEQ ID NO: 10, SEQ ID NO: 11 and a LCDR2 of SEQ ID NO: 12; and a second domain comprising an LCDR3 of SEQ ID NO:

13. The method according to any one of claims 20 to 24, comprising:

26. a) the first domain that binds to EGFR comprises a VH of SEQ ID NO: 13 and a VH of SEQ ID NO: 14 and b) the second domain that binds to c-Met comprises a VH of SEQ ID NO: 15 and a VH of SEQ ID NO: 1 26. The method of claim 25, comprising a VL of 6.

27. The bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype.

27. The method according to any one of claims 20 to 26.

28. The bispecific anti-EGFR / c-Met antibody is selected from the group consisting of HC1 of SEQ ID NO: 17, HC2 of SEQ ID NO: 18 27. The method according to claim 20, comprising the LC1 of SEQ ID NO: 19, the HC2 of SEQ ID NO: 20, and the LC2 of SEQ ID NO:

20.

2. The method according to claim 1 .

29. The threshold value is EGFR, c-Met, or EGFR and c-Met positive cancer. 30 percent of macrophages or monocytes observed in said biological samples from a population of subjects The method of any one of claims 20 to 28, wherein the tile value is greater than or equal to the tile value.

30. The method according to any one of claims 20 to 29, wherein the biological sample is a blood sample.

31. The biological sample according to any one of claims 20 to 29, wherein the biological sample is a tumor tissue biopsy. method.

32. EGFR, c-Met, or EGFR and c-Met expressing cancers are Activating GFR mutations, EGFR gene amplification, elevated levels of circulating HGF, wild-type c-Met , c-Met activating mutations, c-Met gene amplification, or mutant KRAS, or The method according to any one of claims 20 to 31, relating to any combination of:

33. The EGFR activating mutations are L718Q, G719A, G719X (wherein X is any amino acid). L861X (wherein X is any amino acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, L858 P or T790M substitution, E746-A750 deletion, R748-P753 deletion , an insertion of Ala (A) between M766 and A767, an insertion of S between S768 and V769 Insertion of Er, Val, and Ala (SVA), Asn and Insertions of Ser(NS), D761 and E762, A763 and Y764, Y764 and Y765 , M766 and A767, A767 and V768, S768 and V769, V769 and D770 , D770 and N771, N771 and P772, P772 and H773, H773 and V774 , an insertion of one or more amino acids between V774 and C775, an EGFR exon one or more deletions in EGFR exon 20, or one or two in EGFR exon 20 33. The method of claim 32, comprising the above insertions, or any combination thereof.

34. 33. The mutant KRAS comprising a G12V, G12C, or G12A substitution. The method described above.

35. The subject has newly diagnosed EGFR, c-Met, or EGFR and c-Met tumors. The method of any one of claims 20 to 34, wherein the patient is suspected of having or has a current cancer. 。

36. The subject is resistant or has acquired resistance to treatment with a previous anti-cancer therapy. The method according to any one of claims 20 to 34,

37. The previous anti-cancer therapy is a chemotherapy, a targeted anti-cancer therapy, or a kinase inhibitor. The method according to claim 36.

38. The kinase inhibitor is selected from the group consisting of EGFR, c-Met, HER2, HER3, HER4, VEGF, 38. The method of claim 37, which is an inhibitor of GFR or AXL.

39. The kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, alanine, Fatinib, osimertinib, lazertinib, poziotinib, cliotinib, cabozantinib Nib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, paclitaxel 39. The method of claim 38, wherein the method is accompanied by zopanib, sorafenib, or sunitinib.

40. EGFR, c-Met, or EGFR and c-Met expressing cancers are epithelial cell carcinomas, breast ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, small cell lung cancer, colon Rectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharyngeal cancer, nasal cancer pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, splenic cancer Cancer, testicular cancer, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, hepatocellular carcinoma ( 2. The method of claim 1, wherein the patient is a patient with idiopathic pulmonary fibrosis (IPF) or a patient with idiopathic pulmonary fibrosis (PFC).

40. The method of any one of claims 0 to 39.

41. The bispecific anti-EGFR / c-Met antibody inhibits macrophage activity in the subject. The method according to any one of claims 20 to 40, which is administered in combination with an agent that enhances Law.

42. The agent that enhances macrophage activity in the subject is selected from the group consisting of GM-CSF, CD47, antagonist, anti-CD47 antibody, HDAC2 inhibitor, PD-(L)1 axis inhibitor, or C 42. The method of claim 41, which is a D11b agonist.

43. 43. The method of claim 20, further comprising administering to the subject one or more anti-cancer therapies.

2. The method according to any one of claims 1 to 11.

44. The one or more anti-cancer therapies are chemotherapy, targeted anti-cancer therapy, or kinase inhibition.

44. The method of claim 43, comprising the agent.

45. The kinase inhibitor is selected from the group consisting of EGFR, c-Met, HER2, HER3, HER4, VEGF, 45. The method of claim 44, which is an inhibitor of GFR or AXL.

46. The kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, alanine, Fatinib, osimertinib, lazertinib, poziotinib, cliotinib, cabozantinib Nib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, paclitaxel 46. ​​The method of claim 45, which is zopanib, sorafenib, or sunitinib.

47. The bispecific anti-EGFR / c-Met antibody has a fucose content of about 15% or less. The method according to any one of claims 1 to 46.

48. EGFR, or c-Met, or EGFR and A method for inducing trogocytosis of c-Met from the donor cells, comprising: The donor cells are incubated for a period of time sufficient to induce trogocytosis into septa cells. contacting the subject with an isomeric anti-EGFR / c-Met antibody.

49. The donor cells express EGFR, c-Met, or EGFR and c-Met.

49. The method of claim 48, wherein the cell is a cancer cell.

50. 50. The method of claim 48 or 49, wherein the acceptor cell is a macrophage or a monocyte. method.

51. The bispecific anti-EGFR / c-Met antibody is a) a first domain that binds to EGFR, comprising HCDR1 of SEQ ID NO: 1, SEQ ID NO: HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3, light chain complementarity determining region 1 (LCD) of SEQ ID NO:4 R1), an LCDR2 of SEQ ID NO:5, and an LCDR3 of SEQ ID NO:

6. and, b) a second domain that binds to c-Met, comprising HCDR1 of SEQ ID NO: 7, SEQ ID NO: SEQ ID NO: 8, HCDR2, SEQ ID NO: 9, LCDR1, SEQ ID NO: 10, SEQ ID NO: 11 and a LCDR2 of SEQ ID NO: 12; and a second domain comprising an LCDR3 of SEQ ID NO:

13. The method of any one of claims 48 to 50, comprising:

52. a) the first domain that binds to EGFR comprises a VH of SEQ ID NO: 13 and a VH of SEQ ID NO: 14 and b) the second domain that binds to c-Met comprises a VH of SEQ ID NO: 15 and a VH of SEQ ID NO: 1 52. The method of claim 51 , comprising a VL of 6.

53. The bispecific anti-EGFR / c-Met antibody is of the IgG1 isotype.

53. The method according to any one of claims 48 to 52.

54. The bispecific anti-EGFR / c-Met antibody is selected from the group consisting of HC1 of SEQ ID NO: 17, HC2 of SEQ ID NO: 18 LC1 of SEQ ID NO: 19, HC2 of SEQ ID NO: 20, and LC2 of SEQ ID NO:

21.

2. The method according to claim 1 .

55. The cancer cell expressing the EGFR, c-Met, or EGFR and c-Met. However, wild-type EGFR, EGFR activating mutations, EGFR gene amplification, and circulating HGF levels Elevated, wild-type c-Met, c-Met activating mutation, c-Met gene amplification, or mutant K 55. The method according to any one of claims 49 to 54, which is associated with the RAS.

56. The EGFR activating mutations are L718Q, G719A, G719X (wherein X is any amino acid). L861X (wherein X is any amino acid), L858R, E746K, L747S, E749Q, A750P, A755V, V765M, C797S, L858 P or T790M substitution, E746-A750 deletion, R748-P753 deletion , an insertion of Ala (A) between M766 and A767, an insertion of S between S768 and V769 Insertion of Er, Val, and Ala (SVA), Asn and Insertions of Ser(NS), D761 and E762, A763 and Y764, Y764 and Y765 , M766 and A767, A767 and V768, S768 and V769, V769 and D770 , D770 and N771, N771 and P772, P772 and H773, H773 and V774 , an insertion of one or more amino acids between V774 and C775, an EGFR exon one or more deletions in EGFR exon 20, or one or two in EGFR exon 20 56. The method of claim 55, comprising the above insertions, or any combination thereof.

57. 56. The mutant KRAS comprising a G12V, G12C, or G12A substitution. The method described above.

58. The method of any one of claims 48 to 56, wherein the contacting step is carried out in vitro. Law.

59. the contacting step comprising administering the bispecific anti-EGFR / c-Met antibody to a subject. The method of any one of claims 48 to 56, comprising:

60. the subject has an EGFR, c-Met, or EGFR and c-Met expressing cancer; 60. The method of claim 59.

61. The subject has newly diagnosed EGFR, c-Met, or EGFR and c-Met tumors.

61. The method of claim 59 or 60, wherein the patient has a current cancer.

62. The subject is resistant or has acquired resistance to treatment with a previous anti-cancer therapy.

61. The method of claim 59 or 60,

63. The previous anti-cancer therapy is a chemotherapy, a targeted anti-cancer therapy, or a kinase inhibitor. The method of claim 62.

64. The kinase inhibitor is selected from the group consisting of EGFR, c-Met, HER2, HER3, HER4, VEGF, 64. The method of claim 63, which is an inhibitor of GFR or AXL.

65. The kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, alanine, Fatinib, osimertinib, lazertinib, poziotinib, cliotinib, cabozantinib Nib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, paclitaxel 64. The method of claim 63, which is zopanib, sorafenib, or sunitinib.

66. The cancer expressing EGFR, c-Met, or EGFR and c-Met is an epithelial Cell carcinoma, breast cancer, ovarian cancer, lung cancer, NSCLC, lung adenocarcinoma, small cell lung cancer, colorectal Cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, pharyngeal cancer, nasal cancer, Pancreatic cancer, skin cancer, oral cancer, tongue cancer, esophageal cancer, vaginal cancer, cervical cancer, spleen cancer , testicular cancer, stomach cancer, thymus cancer, colon cancer, thyroid cancer, liver cancer, HCC, or sporadic 66. The method according to claim 49, wherein the pulmonary artery disease is caused by a genetic or hereditary papillary renal cell carcinoma (PRCC).

13. The method according to claim 1.

67. The method of any one of claims 59 to 66, further comprising administering to the subject one or more anti-cancer therapies.

2. The method according to any one of claims 1 to 11.

68. The one or more anti-cancer therapies are chemotherapy, targeted anti-cancer therapy, or kinase inhibition.

68. The method of claim 67, further comprising the step of:

69. The kinase inhibitor is selected from the group consisting of EGFR, c-Met, HER2, HER3, HER4, VEGF, 69. The method of claim 68, which is an inhibitor of GFR or AXL.

70. The kinase inhibitor is erlotinib, gefitinib, lapatinib, vandetanib, alanine, Fatinib, osimertinib, lazertinib, poziotinib, cliotinib, cabozantinib Nib, capmatinib, axitinib, lenvatinib, nintedanib, regorafenib, paclitaxel 70. The method of claim 69, which is zopanib, sorafenib, or sunitinib.

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