Treatment of cancer with high EGFR expression treated with immune checkpoint inhibitors, using antibodies that bind to at least EGFR.

Abstract

The present disclosure relates to means and methods in the treatment of cancer. In particular, the present disclosure relates to methods of treating cancer in an individual with an antibody that binds at least EGFR. The present invention further relates to the use in such methods and in the manufacture of a medicament for the treatment of cancers with specific EGFR levels. Such antibodies are particularly useful in the treatment of cancers such as gastric cancer, esophageal cancer, gastroesophageal junction cancer, or head and neck cancer.

Description

Technical Field

[0001] The present disclosure relates to means and methods in the treatment of cancer. The present disclosure particularly relates to methods of treating cancer in an individual with an antibody that binds at least to EGFR. The invention further relates to use in such methods and use in the manufacture of a medicament for the treatment of cancer having a particular EGFR level. Such antibodies are particularly useful in the treatment of cancers such as gastric cancer, esophageal cancer, gastroesophageal junction cancer, or head and neck cancer.

Background Art

[0002] Conventionally, the discovery of most cancer drugs has focused on agents that block essential cell functions and kill dividing cells via chemotherapy. However, chemotherapy rarely leads to a complete cure. In most cases, tumors in patients only stop growing or shrink temporarily and, again, in some cases, start growing more rapidly and become increasingly difficult to treat.

[0003] Cancer remains a major cause of death worldwide, despite many advances made in the treatment of the disease and the increased knowledge of the molecular events leading to cancer.

[0004] In the United States, head and neck cancers, particularly those affecting the oral cavity and pharynx, already account for 3% of all malignant tumors. Approximately 53,000 Americans develop this type of cancer each year, and 10,800 die from it (Siegel et al., CA Cancer J Clin. 2020;70(1):7. Epub 2020 Jan 8.). Furthermore, head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and the 5-year overall survival rate for patients with HNSCC is reported to be approximately 40-50% (Head and Neck Cancer, Union for International Cancer Control, 2014 Review of Cancer Medicines on the WHO List of Essential Medicines).

[0005] A meta-analysis of locally advanced head and neck squamous cell carcinoma (LA-HNSCC) reported that the addition of anti-EGFR agents to radiotherapy or chemoradiotherapy did not improve clinical outcomes in patients with LA-HNSCC (Oncotarget.2017;8(60):102371-102380). Furthermore, the addition of anti-EGFR agents was reported to increase the risk of skin toxicity and mucositis.

[0006] Furthermore, gastric cancer is the fifth most commonly diagnosed cancer and the third most deadly cancer worldwide. In 2018, an estimated 783,000 people died from gastric cancer. Esophageal cancer is the ninth most common cancer and the sixth most common cause of cancer death. Epidermal growth factor receptor (EGFR) has been reported to be overexpressed in more than 30% of gastric adenocarcinoma (GAC) and esophageal adenocarcinoma (EAC) cases. However, a review of six different studies concluded that adding anti-EGFR agents to chemotherapy did not significantly improve overall survival or progression-free survival in patients with advanced / metastatic EAC, GAC, or gastroesophageal junction adenocarcinoma (GEJAC) (Kim et al. 2017 Oncotarget. 2017 Nov 17;8(58):99033-99040).

[0007] Therefore, there is a need for improvement in cancer treatment, particularly for gastric cancer, esophageal cancer, and head and neck cancer. [Overview of the Initiative]

[0008] This disclosure provides the following preferred embodiments; however, the present invention is not limited thereto.

[0009] This disclosure provides an antibody, or a functional moiety, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of cancer in a subject, wherein the cancer expresses EGFR, or EGFR and LGR5.

[0010] This disclosure provides an antibody, or a functional moiety, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of cancer in a subject, wherein the cancer in the subject has progressed after prior treatment with an immune checkpoint inhibitor, and the cancer expresses EGFR, or EGFR and LGR5.

[0011] The disclosure also provides the use of an antibody, or a functional portion thereof, derivative, and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR, in the manufacture of a drug for treating cancer in a subject, wherein the cancer in the subject has progressed after prior treatment with an immune checkpoint inhibitor, and the cancer expresses EGFR, or EGFR and LGR5.

[0012] The disclosure also provides a method for treating a subject having EGFR-expressing cancer, wherein the subject has progressed after prior treatment with an immune checkpoint inhibitor, and the method comprises providing the subject with an effective amount of an antibody, or a functional portion, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR.

[0013] In certain embodiments, the cancers of this disclosure are, in particular, gastric cancer, esophageal cancer, gastroesophageal junction cancer, or head and neck cancer. Head and neck cancer is, in particular, head and neck squamous cell carcinoma (HNSCC). Gastric cancer, esophageal cancer, and gastroesophageal junction cancer are, in particular, adenocarcinomas. Such esophageal cancer may also be squamous cell carcinoma.

[0014] In certain embodiments, the cancer of the Disclosure is, in particular, gastric cancer, esophageal cancer, or gastroesophageal junction cancer having EGFR expression characterized by an IHC score of 3+. In certain embodiments, the cancer of the Disclosure is gastric cancer, esophageal cancer, or gastroesophageal junction cancer having EGFR expression characterized by an H score of more than 200 for EGFR.

[0015] The Disclosure also provides an antibody, or a functional moiety, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a subject, wherein the cancer expresses EGFR characterized by an IHC score of 3+. The Disclosure also provides an antibody, or a functional moiety, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a subject, wherein the cancer expresses EGFR characterized by an H score of more than 200 for EGFR.

[0016] The disclosure also provides antibodies, functional moieties, derivatives, and / or analogs thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of head and neck cancer, gastric cancer, esophageal cancer, or gastroesophageal junction cancer in subjects, wherein the cancer is characterized by comprising EGFR gene amplification. Such EGFR gene amplification is, in certain embodiments, characterized by an EGFR copy number of 8 or more, or a level of circulating tumor DNA (ctDNA) of at least 2.14 or at least 2.5.

[0017] In certain embodiments, amplification of EGFR mRNA is defined as eligible by an EGFR copy number of 8 or more (as defined by next-generation sequencing, for example), or by at least 2.14 or at least 2.5 ctDNA.

[0018] In some embodiments, the subjects have progressed after prior treatment with an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor includes a PD-L1, PD-1, CTLA-4, B7-1, or B7-2 inhibitor. In certain embodiments, such inhibitor includes an antibody targeting a PD-L1, PD-1, CTLA-4, B7-1, or B7-2 inhibitor. In certain embodiments, the immune checkpoint inhibitor includes durvalumab, retifanlimab, semiprimab, pembrolizumab, ipilimumab, nivolumab, or atezolizumab.

[0019] In certain embodiments, the subjects of this disclosure have not received prior treatment with anti-EGFR agents. In certain embodiments, the subjects have not received prior treatment with EGFR-targeting antibodies, or the subjects have not received prior treatment with cetuximab.

[0020] In certain embodiments, the gastric cancer, esophageal cancer, or gastroesophageal junction cancer of the Disclosure expresses EGFR characterized by an H score greater than 200 and less than or equal to 300. In certain embodiments, the H score for said EGFR is determined using immunohistochemistry (IHC).

[0021] In certain aspects, the subject matter of this disclosure is mammalian subjects, such as human subjects.

[0022] In certain embodiments, the treatment of the Disclosure includes providing a subject with an effective amount of the antibody, or its functional portion, derivative, and / or analog. In certain embodiments, the treatment includes providing a uniform dose of 500 mg to 2000 mg. In certain embodiments, the dose is 1100 mg to 1800 mg. In certain embodiments, the dose is 1100 mg to 1500 mg. In certain embodiments, the treatment includes a uniform dose of 1500 mg of the antibody, or its functional portion, derivative, and / or analog to the subject. In certain embodiments, the antibody, or its functional portion, derivative, and / or analog, is provided intravenously to the subject. In certain embodiments, the antibody, or its functional portion, derivative, and / or analog, is provided weekly, bi-weekly, or monthly. In certain embodiments, the antibody, or its functional portion, derivative, and / or analog, is provided bi-weekly.

[0023] In certain embodiments, the antibody, or its functional portion, derivatives, and / or analogs, is ADCC-enhanced. In other embodiments, the antibody, or its functional portion, derivatives, and / or analogs, is afucosylated.

[0024] In certain embodiments, the antibodies of this disclosure, or their functional parts, derivatives, and / or analogs, are multispecific antibodies. In certain embodiments, the antibodies of this disclosure, or their functional parts, derivatives, and / or analogs, are bispecific antibodies that bind to at least EGFR. In certain embodiments, the antibody includes a second variable domain that does not bind to EGFR. In certain embodiments, the antibody includes a second variable domain that binds to LGR5.

[0025] An antibody comprising a first variable domain that binds to the extracellular portion of EGFR of the present disclosure, or a functional portion, derivative, and / or analog thereof is also referred to herein as a therapeutic agent.

Brief Description of the Drawings

[0026] [Figure 1] The human LGR5 sequence is SEQ ID NO: 1. [Figure 2] The human EGFR sequence is SEQ ID NO: 2. [Figure 3a)-1] (a) The amino acid sequences (SEQ ID NOs: 3 to 15) of the heavy chain variable regions that form variable domains that bind to LGR5 and EGFR, together with a common light chain variable region such as the variable region of human kappa light chain IgVκ1 39*01 / IGJκ1*01. The CDR and framework regions are shown in Figure 3b. The respective DNA sequences are shown in Figure 3c. [Figure 3a)-2] (a) The amino acid sequences (SEQ ID NOs: 3 to 15) of the heavy chain variable regions that form variable domains that bind to LGR5 and EGFR, together with a common light chain variable region such as the variable region of human kappa light chain IgVκ1 39*01 / IGJκ1*01. The CDR and framework regions are shown in Figure 3b. The respective DNA sequences are shown in Figure 3c. [Figure 3b)-1] (a) The amino acid sequences (SEQ ID NOs: 3 to 15) of the heavy chain variable regions that form variable domains that bind to LGR5 and EGFR, together with a common light chain variable region such as the variable region of human kappa light chain IgVκ1 39*01 / IGJκ1*01. The CDR and framework regions are shown in Figure 3b. The respective DNA sequences are shown in Figure 3c. [Figure 3b)-2] (a) The amino acid sequences (SEQ ID NOs: 3 to 15) of the heavy chain variable regions that form variable domains that bind to LGR5 and EGFR, together with a common light chain variable region such as the variable region of human kappa light chain IgVκ1 39*01 / IGJκ1*01. The CDR and framework regions are shown in Figure 3b. The respective DNA sequences are shown in Figure 3c. [Figure 3c)-1](a) These are the amino acid sequences (SEQ ID NOs: 3-15) of the heavy chain variable region, which forms variable domains that bind to LGR5 and EGFR, along with common light chain variable regions such as the variable region of the human kappa light chain IgVκ1 39*01 / IGJκ1*01. The CDR and framework region are shown in Figure 3b. The respective DNA sequences are shown in Figure 3c. [Figure 3c)-2] (a) These are the amino acid sequences (SEQ ID NOs: 3-15) of the heavy chain variable region, which forms variable domains that bind to LGR5 and EGFR, along with common light chain variable regions such as the variable region of the human kappa light chain IgVκ1 39*01 / IGJκ1*01. The CDR and framework region are shown in Figure 3b. The respective DNA sequences are shown in Figure 3c. [Figure 3c)-3] (a) These are the amino acid sequences (SEQ ID NOs: 3-15) of the heavy chain variable region, which forms variable domains that bind to LGR5 and EGFR, along with common light chain variable regions such as the variable region of the human kappa light chain IgVκ1 39*01 / IGJκ1*01. The CDR and framework region are shown in Figure 3b. The respective DNA sequences are shown in Figure 3c. [Figure 3c)-4] (a) These are the amino acid sequences (SEQ ID NOs: 3-15) of the heavy chain variable region, which forms variable domains that bind to LGR5 and EGFR, along with common light chain variable regions such as the variable region of the human kappa light chain IgVκ1 39*01 / IGJκ1*01. The CDR and framework region are shown in Figure 3b. The respective DNA sequences are shown in Figure 3c. [Figure 4-1] a) The amino acid sequence of the common light chain amino acid sequence. b) The common light chain variable region DNA sequence and translation (IGKV1-39 / jk1). c) The light chain constant region DNA sequence and translation. d) The V region IGKV1-39A, and e) the common light chain CDR1, CDR2, and CDR3 according to IMGT numbering. [Figure 4-2] a) The amino acid sequence of the common light chain amino acid sequence. b) The common light chain variable region DNA sequence and translation (IGKV1-39 / jk1). c) The light chain constant region DNA sequence and translation. d) The V region IGKV1-39A, and e) the common light chain CDR1, CDR2, and CDR3 according to IMGT numbering. [Figure 5-1] This is an IgG heavy chain for the generation of a bispecific molecule. a) CH1 region DNA sequence and translation. b) Hinge region DNA sequence and translation. c) CH2 region DNA sequence and translation. d) CH3 domain containing mutant L351K and T366K (KK) DNA sequences and translation. e) CH3 domain containing mutant L351D and L368E (DE) DNA sequences and translation. Residue positions are assigned according to EU numbering. [Figure 5-2] This is an IgG heavy chain for the generation of a bispecific molecule. a) CH1 region DNA sequence and translation. b) Hinge region DNA sequence and translation. c) CH2 region DNA sequence and translation. d) CH3 domain containing mutant L351K and T366K (KK) DNA sequences and translation. e) CH3 domain containing mutant L351D and L368E (DE) DNA sequences and translation. Residue positions are assigned according to EU numbering. [Modes for carrying out the invention]

[0027] To facilitate understanding of this description, certain terms are defined first. Additional definitions may be provided throughout the detailed description where deemed necessary. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, and conventional methods of immunology, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are used.

[0028] As used herein, the singular forms "a," "an," and "the" include plural referents. The use of the terms "comprising," "having," and "including," as well as other forms such as "comprise," "comprises," "comprised," "has," "have," "had," "include," "includes," and "included," is not limited.

[0029] As used herein, the term “antibody” means a protein molecule belonging to the immunoglobulin class of proteins, containing one or more domains that bind to an epitope on an antigen, wherein such domains derive from or share sequence homology having a variable region of the antibody. Antibodies typically consist of basic structural units, each consisting of two heavy chains and two light chains. Antibodies according to the present invention are not limited to any particular form or method of production thereof.

[0030] A “bispecific antibody” is an antibody described herein in which one domain of the antibody binds to a first antigen while a second domain of the antibody binds to a second antigen, and the first and second antigens are not identical, or one domain binds to a first epitope on the antigen while the second domain binds to a second epitope on the antigen. The term “bispecific antibody” also encompasses antibodies in which one heavy chain variable region / light chain variable region (VH / VL) combination binds to a first antigen or epitope on the antigen and to a second VH / VL combination that binds to a second antigen or epitope on the antigen. The term further includes antibodies in which VH can specifically recognize the first antigen, and VL paired with VH in the immunoglobulin variable region can specifically recognize the second antigen. The resulting VH / VL pair binds to either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO2008 / 027236, WO2010 / 108127, and Schaefer et al (Cancer Cell 20, 472-486, October 2011). The bispecific antibodies according to the present invention are not limited to any particular bispecific form or method of production thereof.

[0031] As used herein, the term “common light chain” refers to two light chains (or their VL portions) in a bispecific antibody. The two light chains (or their VL portions) may be identical or may have some amino acid sequence differences, but the binding specificity of the full-length antibody is not affected. The terms “common light chain,” “common VL,” “single light chain,” and “single VL” are all used interchangeably herein, with or without the addition of the term “rearranged.” “Common” also refers to a functional equivalent of a light chain whose amino acid sequences are not identical. Many variants exist of the light chain in which mutations (deletions, substitutions, insertions, and / or additions) exist that do not substantially affect the formation of the functional binding region. In certain embodiments, the light chain of the present invention may also be a light chain as defined herein, having 0 to 10 amino acid insertions, deletions, substitutions, additions, or combinations thereof. In certain embodiments, the light chain of the present invention may also be a light chain as defined herein, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. For example, preparing or finding variable light chains that are not identical but are still functionally equivalent by introducing conservative amino acid changes, such as changes in amino acids in regions that do not contribute to or only partially contribute to binding specificity when paired with a heavy chain, falls within the definition of a common light chain as used herein.

[0032] As used herein, “contains” and its conjugations are used in their non-restrictive sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “consistes of” may be replaced with “essentially consists of,” meaning that the compounds or auxiliary compounds defined herein may include additional components other than those specifically identified, and such additional components do not alter the inherent characteristics of the invention.

[0033] The terms "full-length IgG" or "full-length antibody" according to the present invention are defined as containing essentially complete IgG, but not necessarily possessing all the functions of intact IgG. To avoid misunderstanding, full-length IgG contains two heavy chains and two light chains. Each chain contains a constant (C) region and a variable (V) region, which can be classified into domains designated as CH1, CH2, CH3, VH, and CL, VL. IgG antibodies bind to antigens via the variable region domain contained in the Fab portion, and after binding, they can interact with molecules and cells of the immune system via the constant domain, mainly via the Fc portion. Full-length antibodies according to the present invention encompass IgG molecules in which mutations may exist that provide desired characteristics. Full-length IgG should not have deletions of substantial portions of any region. However, IgG molecules with one or more amino acid residues deleted without essentially altering the binding properties of the resulting IgG molecule are included in the term "full-length IgG." For example, such an IgG molecule may have 1 to 10 amino acid residues deleted, preferably within a non-CDR region, and the deleted amino acids are not essential for the antigen-binding specificity of IgG. In certain embodiments, such an IgG molecule may have 1 to 10 amino acid residues deleted within the non-CDR region, and the deleted amino acids are not essential for the antigen-binding specificity of IgG.

[0034] An “antibody derivative” is a protein that deviates from the amino acid sequence of the native antibody by up to 20 amino acids, excluding the CDR region. The antibody derivatives disclosed herein are antibodies that deviate from the amino acid sequence by up to 20 amino acids. Functional moieties, derivatives, and / or analogs maintain the (bispecific) binding specificity of the antibody. An “antibody analog” is a protein that may differ in structure, form, or origin, but maintains the binding specificity of its analog antibody.

[0035] In this specification, “identity percentage (%)” for nucleic acid sequences or amino acid sequences is defined as the percentage of residues in a candidate sequence that are identical to residues in a selected sequence after the sequences have been aligned for optimal comparison purposes. The sequence identity percentage for comparing nucleic acid sequences is determined using the AlignX application of Vector NTI Advance® 11.5.2 software with default settings using the modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, (1994) Nuc. Acid Res. 22(22):4673-4680), a swgapdnamt score matrix, a gap opening penalty of 15, and a gap elongation penalty of 6.66. The amino acid sequence is determined using the AlignX application of Vector NTI Advance® 11.5.2 software with default settings that employ a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, (1994) Nuc. Acid Res. 22(22):4673-4680), a bloomum62mt2 score matrix, a gap opening penalty of 10, and a gap stretching penalty of 0.1.

[0036] Antibodies typically recognize epitopes on antigens, and since such epitopes may also be present on other compounds, an antibody according to the present invention that "specifically recognizes" an antigen, such as EGFR or LGR5, may also recognize other compounds if those other compounds contain the same type of epitope. Therefore, the term "specifically recognizes" in relation to the interaction between an antigen and an antibody does not preclude the binding of the antibody to other compounds containing the same type of epitope.

[0037] An "epitope" or "antigen determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from adjacent or non-adjacent amino acids juxtaposed by tertiary folding of proteins (so-called linear epitopes and conformational epitopes). Epitopes formed from adjacent linear amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding typically lose their conformation upon treatment with denaturing solvents. Epitopes can typically contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids within a specific spatial conformation.

[0038] As used herein, the terms “subject” and “patient” are interchangeable and refer to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cattle, horses, and pigs (for example, patients such as human patients with cancer).

[0039] The terms “to treat,” “to treat,” and “treatment” as used herein refer to any type of intervention or process that involves administering an active agent or combination of active agents to a subject for the purpose of reversing, alleviating, improving, inhibiting, delaying, or preventing the progression, onset, worsening, or recurrence of any symptom, complication, condition, or biochemical sign associated with a disease.

[0040] As used herein, “effective treatment” or “positive treatment response” means a treatment that results in a beneficial effect, for example, improvement of at least one symptom of a disease or disorder, such as cancer. A beneficial effect can take the form of improvement above baseline, including improvement beyond measurements or observations made before initiating treatment according to the method. For example, a beneficial effect can take the form of delaying, stabilizing, stopping, or reversing the progression of cancer in a subject at any clinical stage, as demonstrated by a reduction or elimination of clinical or diagnostic symptoms of the disease or cancer markers. An effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, delay or stop tumor growth, and / or prevent or delay tumor recurrence or relapse.

[0041] The terms “effective dose” or “therapeutic effective dose” refer to the amount of an agent or combination of agents that provides a desired biological, therapeutic, and / or prophylactic outcome. The outcome may be a reduction, improvement, remission, mitigation, delay, and / or relief of one or more signs, symptoms, or causes of a disease, or any other desired change in the biological system. With respect to tumor development, an effective dose is sufficient to delay tumor development. With respect to tumor recurrence, an effective dose is sufficient to prevent or delay tumor recurrence. An effective dose may be administered in one or more doses. An effective dose of an agent or composition may (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, block, to some extent delay, and halt the invasion of cancer cells into peripheral organs, (iv) inhibit tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay tumor development and / or recurrence, and / or (vii) to some extent alleviate one or more symptoms associated with cancer. In one embodiment, “effective dose” is the amount of an antibody disclosed herein as a therapeutic agent that affects the reduction of cancer (e.g., a reduction in the number of cancer cells), the delay of cancer progression, or the prevention of cancer regrowth or recurrence. As previously stated herein, antibodies or functional portions thereof, derivatives, and / or analogs that bind to EGFR or to EGFR and LGR5 are also referred to herein as “therapeutic agents.” In a particular embodiment, the effective dose herein is a uniform dose of 1500 mg administered bi-weekly to a subject with the cancer disclosed herein.

[0042] In this specification, the term “uniform dose” refers to a dosing regimen in which a subject is administered a fixed amount of therapeutic agent over multiple doses, regardless of the subject’s body weight. Uniform dosing is typically abbreviated as qnw, where n is an integer representing the interval and w is a week. For example, a q2w uniform dose dosing regimen of 1500 mg antibody means that a fixed amount of 1500 mg of antibody is administered every two weeks. In certain embodiments, the therapeutic agent is an antibody that binds to EGFR, or to EGFR and LGR5, administered in a 1500 mg q2w dosing regimen. In certain embodiments, the subject is administered in uniform dosing doses of 1500 mg for at least 3 q2w. In certain embodiments, the dosing is at least 4 doses or more and may continue until the patient shows sufficient clinical or radiological progress.

[0043] A uniform dose may be premedicated, meaning that a drug is administered to the subject before the administration of the antibody of the present invention. In certain embodiments, a uniform dose of 1500 mg of antibody may be premedicated with an antihistamine, an analgesic, an antipyretic, and / or an anti-inflammatory drug.

[0044] The term "H-score," sometimes also referred to as "HIST" in the art, refers to a reproducible, standardized scoring methodology that can be used to semi-quantitatively calculate the expression of a target gene in a tumor sample according to a protocol based on immunohistochemistry (IHC) or in-situ hybridization techniques (ISH) well known to those skilled in the art. The ASCO April 10 publication contains the method for calculating the H-score. thSee also Hirsch FR, Varella-Garcia M, Bunn PA Jr, et al: Epidermal growth factor receptor in non-small-cell lung carcinomas: Correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 21:3798-3807, 2003, and John T, Liu G, Tsao MS: Overview of molecular testing in non-small-cell lung cancer: Mutational analysis, gene copy number, protein expression and other biomarkers of EGFR for the prediction of response to tyrosine kinase inhibitors. Oncogene 28:S14-S23, 2009. Relevant teachings from these references are incorporated herein by reference.

[0045] In the context of H scoring for EGFR, the term "determined using IHC" refers to a method that uses IHC as a basis for subsequently determining the H score, or a method that includes IHC, as opposed to an alternative to IHC.

[0046] In some embodiments, the Disclosure provides an antibody, or a functional moiety, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of cancer in a subject, wherein the cancer in the subject has progressed after prior treatment with an immune checkpoint inhibitor, and the cancer expresses EGFR.

[0047] In some embodiments, the cancer is selected from gastric cancer, esophageal cancer, gastroesophageal junction cancer, or head and neck cancer, particularly head and neck squamous cell carcinoma (SCCHN).

[0048] In certain embodiments, the cancer is gastric, esophageal, or gastroesophageal junction cancer with EGFR expression characterized by an IHC score of 3+. In certain embodiments, the cancer has an H score for EGFR greater than 200. In certain embodiments, the IHC is the tumor membrane score.

[0049] Furthermore, this disclosure provides an antibody, or a functional moiety, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of cancer in a subject, wherein the cancer expresses EGFR characterized by an IHC score of 3+, and the variable domain comprises an amino acid further disclosed herein.

[0050] Furthermore, this disclosure provides an antibody, or a functional moiety, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of cancer in a subject, wherein the cancer expresses EGFR characterized by an H score of more than 200 EGFRs, and the variable domain comprises amino acids further disclosed herein.

[0051] Furthermore, this disclosure provides the use of an antibody, or a functional portion thereof, derivative, and / or analog thereof, containing a variable domain that binds to the extracellular portion of EGFR, in the manufacture of a drug for treating cancer in a subject, wherein the cancer in the subject has progressed after prior treatment with an immune checkpoint inhibitor and the cancer expresses EGFR.

[0052] Furthermore, the disclosure provides a method for treating a subject having EGFR-expressing cancer, wherein the subject has progressed after prior treatment with an immune checkpoint inhibitor, and the method comprises providing the subject with an effective amount of an antibody, or a functional portion, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR.

[0053] Furthermore, the Disclosure provides an antibody, or a functional moiety, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of gastric cancer, esophageal cancer, or gastroesophageal junction cancer in the subject, wherein the cancer expresses EGFR characterized by an IHC score of 3+.

[0054] Furthermore, the Disclosure provides an antibody, or a functional moiety, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of gastric cancer, esophageal cancer, or gastroesophageal junction cancer in the subject, wherein the cancer expresses EGFR characterized by an H score of more than 200 EGFRs.

[0055] The terms cancer and tumor are used herein unless otherwise specified, and generally both refer to cancer.

[0056] The epidermal growth factor (EGF) receptor (EGFR, ErbB1, or HER1) is a member of a family of four receptor tyrosine kinases (RTKs) named Her- or cErbB-1, -2, -3, and -4. EGFR is known by various synonyms, the most common being EGFR. EGFR has an extracellular domain (ECD) consisting of four subdomains, two of which are involved in ligand binding, and two of which are involved in homodimerization and heterodimerization. EGFR integrates extracellular signals from various ligands, resulting in diverse intracellular responses. The major signaling pathway activated by EGFR consists of the Ras-mitogen-activated protein kinase (MAPK) pro-mitotic signaling cascade. Activation of this pathway is initiated by the recruitment of Grb2 to tyrosine-phosphorylated EGFR. This leads to activation of Ras by the Grb2-bound Ras-guanine nucleotide exchange factor Son of Sevenless (SOS). In addition, the PI3-kinase-Akt signaling pathway is also activated by EGFR, but this activation is considerably stronger in the presence of co-expression of ErbB-3 (HER3). EGFR is involved in several human epithelial malignancies, particularly breast, bladder, non-small cell lung cancer, colon, ovarian, head and neck, and brain cancers. Activating mutations in the gene, as well as overexpression of the receptor and its ligand, have been found, creating an autocrine activation loop. Therefore, this RTK is widely used as a target in cancer therapy. Both small molecule inhibitors targeting the RTK and monoclonal antibodies (mAbs) directed at the extracellular ligand-binding domain have been developed, and some clinical success has been demonstrated, although mostly in selected patient groups. The database acceptance number for the human EGFR protein and the gene encoding it is GenBank NM_005228.3. Acceptance numbers are primarily assigned to provide a further method for identifying the EGFR protein as a target, as the actual sequence of the EGFR protein bound by the antibody may change due to mutations in the coding gene, such as mutations that occur in certain cancers.

[0057] Where EGFR is referred to herein, unless otherwise stated, the reference refers to human EGFR. The variable domain antigen-binding site that binds to EGFR binds to EGFR and its various variants, for example, variants expressed on some EGFR-positive tumors.

[0058] The term "LGR" refers to a family of proteins known as leucine-rich repeat-containing G protein coupling receptors. Several members of this family, focusing on LGR4, LGR5, and LGR6, are known to be involved in the WNT signaling pathway.

[0059] LGR5 is a leucine-rich repeat containing G protein-coupled receptor 5. Alternative names for the gene or protein include leucine-rich repeat containing G protein-coupled receptor 5, leucine-rich repeat containing G protein-coupled receptor 5, G protein-coupled receptor HG38, G protein-coupled receptor 49, G protein-coupled receptor 67, GPR67, GPR49, orphan G protein-coupled receptor HG38, G protein-coupled receptor 49, GPR49, HG38, and FEX. The proteins or antibodies of the present invention that bind to LGR5 bind to human LGR5. LGR5-binding proteins or antibodies may, but not necessarily, bind to other mammalian orthologs due to sequence and tertiary structure similarities between humans and other mammalian orthologs. The database acceptance numbers for the human LGR5 protein and the gene encoding it are (NC_000012.12, NT_029419.13, NC_018923.2, NP_001264155.1, NP_001264156.1, NP_003658.1). These acceptance numbers are primarily provided to offer further methods for identifying LGR5 as a target, and the actual sequence of the bound LGR5 protein may vary due to mutations in the encoding gene, such as those occurring in certain cancers. The LGR5 antigen-binding site binds to LGR5 and its various variants, such as those expressed by certain LGR5-positive tumor cells.

[0060] In particular, cancer refers to gastric cancer, esophageal cancer, or gastroesophageal junction cancer. Gastric cancer (also called stomach cancer) is cancer that originates from the inner wall of the stomach, especially from the mucin-producing glandular cells found therein. Because such cancer originates from the inner lining of the stomach, it is also called adenocarcinoma, or in this case gastric adenocarcinoma. Therefore, in particular, cancer refers to gastric adenocarcinoma or cancer that originates from the inner lining of the stomach, as used interchangeably herein. Esophageal cancer is cancer that originates from the esophagus. The two main subtypes are ESCC (esophageal squamous cell carcinoma) and EAC (esophageal adenocarcinoma). Gastric junction cancer (also known as gastroesophageal junction adenocarcinoma) originates from the gastroesophageal junction.

[0061] Cancers collectively known as head and neck cancers typically originate from squamous cells lining the moist mucous surfaces of the inner surfaces of the head and neck, such as the mouth, nose, and throat. These squamous cell carcinomas are often referred to as head and neck squamous cell carcinomas, and such cancers are treated in certain aspects of this disclosure. Less frequently, head and neck cancers can also occur in the salivary glands. Specifically, head and neck cancers can occur in the oral cavity. This includes the lips, the anterior two-thirds of the tongue, the gums, the inner surfaces of the cheeks and lips, the floor of the mouth under the tongue, the hard palate, and small areas of the gums behind the wisdom teeth.

[0062] Therefore, specifically, head and neck cancers are squamous cell carcinomas and include nasopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, oral cancer, oropharyngeal cancer, or salivary gland cancer. More specifically, the present invention relates to the treatment of cancers including squamous cell head and neck cancers, such as those located in the oropharynx, hypopharynx, larynx, oral cavity, or tongue.

[0063] Furthermore, head and neck cancers, in particular, include squamous cell carcinoma of unknown primary origin (also referred to as cancer of unknown primary origin or CUP in this technical field).

[0064] In this disclosure, cancer expresses EGFR, or EGFR and LGR5.

[0065] As used herein, cancer expresses EGFR if it contains cells that express EGFR. Cells that express EGFR contain detectable levels of RNA that encodes EGFR. In certain embodiments, EGFR expression is determined by ISH.

[0066] In certain embodiments, EGFR protein expression is detected by IHC. In certain embodiments, EGFR expression is determined by IHC using a commercially available EGFR detection kit, such as the EGFR pharmDx® kit for Dako automated staining systems (Agilent) using the manufacturer's recommendations, or a commercially available IHC EGFR detection kit based on EGFR clone 113 that binds to the extracellular domain of EGFR (Leica, https: / / shop.leicabiosystems.com / us / ihc-ish / ihc-primary-antibodies / pid-epidermal-growth-factor-receptor). Alternatively, EGFR expression is determined using Novocastra® liquid mouse monoclonal antibody epidermal growth factor receptor (product code: NCL-L-EGFR, epidermal growth factor receptor-IHC primary antibody from leicabiosystems.com) based on clone EGFR.113.

[0067] In short, the commercially available EGFR pharmDx™ IHC kit system contains the reagents necessary to complete the IHC staining procedure for paraffin-embedded specimens fixed according to the instructions. Following incubation with primary, non-Her2, Her3, and Her4 cross-reactive monoclonal antibodies (clone 2-18C9) against human EGFR protein, the kit utilizes a readily available visualization reagent based on dextran technology. This reagent consists of both a secondary goat anti-mouse antibody molecule and a horseradish peroxidase molecule linked to a common dextran polymer backbone. Enzymatic conversion of the subsequently added chromogen results in the formation of a visible reaction product at the antigen site. The results are routinely evaluated using a light microscope. Control slides containing two formalin-fixed paraffin-embedded human cell lines with staining intensity scores of 2+ and 0 are provided for quality control of the kit reagent performance.

[0068] Stain intensity is determined as follows: 3+ (strong staining): visible at low magnification with a 5x objective lens, visible at a high level if necessary; 2+ (moderate staining): visible at medium magnification with a 10x or 20x objective lens; 1+ (weak staining): reliably visible only at high magnification with a 40x objective lens; 0 (no staining): no visible staining at high magnification.

[0069] In certain embodiments, EGFR expression is determined using immunohistochemistry (IHC), and the cancer is IHC-positive for EGFR. In certain embodiments, the cancer is gastric cancer, esophageal cancer, or gastroesophageal junction cancer, characterized by an EGFR IHC score of 3+.

[0070] In certain embodiments, EGFR expression is determined using immunohistochemistry (IHC), followed by the assignment of an H score for EGFR using a scale of 0 to 300. In certain embodiments, the cancers of the Disclosure are gastric cancer, esophageal cancer, and gastroesophageal junction cancer characterized by an H score for EGFR greater than 200 on a scale of 0 to 300. Thus, in certain embodiments, the EGFRH score is greater than 200 and less than or equal to 300. In certain embodiments, the cancers of the Disclosure are characterized by an H score for EGFR greater than 50 on a scale of 0 to 300. In certain embodiments, the cancers of the Disclosure are head and neck cancers characterized by an H score for EGFR greater than 50 on a scale of 0 to 300. In certain embodiments, the cancers of the Disclosure are characterized by an H score for EGFR greater than 80 on a scale of 0 to 300. In a particular embodiment, the cancer of the present disclosure is a head and neck cancer characterized by an H score for EGFR greater than 80 on a scale of 0 to 300.

[0071] In another embodiment, the cancer is a head and neck cancer characterized by an EGFR IHC score of 2+ or 3+.

[0072] In this specification, determining the H score to assign EGFR expression status involves a first step of establishing the intensity of membrane staining (resulting in a scoring of 0, 1+, 2+, or 3+) determined for each cell in a given field, as described herein. Subsequently, the percentage of cells at each staining intensity level is calculated, and finally, the H score is assigned using the following formula: [1 × (1+ cells with staining%) + 2 × (2+ cells with staining%) + 3 × (3+ cells with staining%)], resulting in an H score for EGFR between 0 and 300. As a result, the H score gives more relative weight to higher intensities or amounts of staining in a given tumor sample.

[0073] In certain embodiments, the cancer of the Disclosure is characterized by including EGFR gene amplification. In certain embodiments, the cancer is gastric cancer. In certain embodiments, the cancer is gastroesophageal junction adenocarcinoma. The EGFR gene amplification is characterized in certain embodiments by an EGFR copy number of 8 or more solid tissue samples, or at least 2.14 or at least 2.5, but in certain embodiments by an EGFR amplification score (also known as copy number change (CNA)) based on 5 or fewer circulating tumor DNA (ctDNA).

[0074] In certain embodiments, EGFR amplification is defined as an EGFR copy number of 8 or more (in particular, as a ploidy of 8 or more copies based on solid tissue amplification). In certain embodiments, the EGFR copy number is established by next-generation sequencing of formalin-fixed paraffin-embedded (FFPE) tissue samples.

[0075] In certain embodiments, the EGFR gene copy number is established using next-generation sequencing (NGS). In certain embodiments, the NGS is performed on solid tissue samples or liquid samples such as blood or plasma. In certain embodiments, the EGFR amplification score is established by next-generation sequencing of ctDNA, resulting in a score of at least 2.14 or at least 2.5. In certain embodiments, the ctDNA score is 5 or less. The copy number assessment may be based on blood-derived cfDNA. As an example, the determination of the EGFR copy number may be performed as described in Kato et al. 2019 (Revisiting Epidermal Growth Factor Receptor (EGFR) Amplification as a Target for Anti-EGFR Therapy: Analysis of Cell-Free Circulating Tumor DNA in Patients With Advanced Malignancies. JCO Precis Oncol 3: PO.18.00180).

[0076] In this specification, the term "ctDNA" (circulating tumor DNA) is used interchangeably with "cfDNA" (cell-free tumor DNA).

[0077] In certain embodiments, the EGFR gene copy number is established using FISH. In certain embodiments, the cancer is characterized by an EGFR / CEP7 ratio of at least 2.0. Establishing the EGFR / CEP7 ratio is standard practice in the art, but may be established, for example, using a commercially available kit, or performed as described in Maron, et al., 2018 (Targeted Therapies for Targeted Populations: Anti-EGFR Treatment for EGFR-Amplified Gastroesophageal Adenocarcinoma. Cancer Discov 8:696-713). The EGFR FISH test is designed to detect amplification of the EGFR locus (located on chromosome 7p11.2). In this methodology, FISH is performed on formalin-fixed, paraffin-embedded tumor tissue sections. Slides are prepared according to a standard protocol, and 100 interphase cells are scored. The cutoff for amplification is then set at an EGFR:CEP7 ratio of 2.0 or higher.

[0078] Optionally, treatment with an antibody, or a functional portion thereof, derivative, and / or analogue thereof, includes (or, in certain embodiments, follows) a step of diagnosing the subject for EGFR status. In certain embodiments, subjects with gastric cancer, esophageal cancer, or gastroesophageal junction cancer characterized by an IHC score of 3+, or whose cancer is characterized by an H score for EGFR greater than 200 on a scale of 0 to 300, are selected for treatment. In certain embodiments, treatment of a subject follows a step of diagnosing the subject with gastric cancer, esophageal cancer, or gastroesophageal junction cancer characterized by an H score for EGFR greater than 200 on a scale of 0 to 300.

[0079] In certain embodiments, subjects having gastric cancer, esophageal cancer, or gastroesophageal junction cancer characterized by EGFR gene amplification including an EGFR / CEP7 ratio of at least 2.0, an EGFR copy number of 8 or higher, or an EGFR ctDNA score of at least 2.14 or at least 2.5 are selected for treatment. In certain embodiments, treatment of subjects follows the step of diagnosing subjects having gastric cancer, esophageal cancer, or gastroesophageal junction cancer characterized by EGFR gene amplification including an EGFR / CEP7 ratio of at least 2.0, an EGFR copy number of 8 or higher, or an EGFR ctDNA score of at least 2.14 or at least 2.5.

[0080] In particular, the Disclosure provides an antibody, or a functional moiety, derivative, and / or analog thereof, for use in the treatment of gastric cancer, esophageal cancer, and gastroesophageal junction cancer, comprising a variable domain that binds to the extracellular portion of EGFR and potentially having a second variable domain that binds to the extracellular portion of LGR5, wherein the cancer has progressed after prior treatment with an immune checkpoint inhibitor, and the subject has a Her2 state selected from Her2-positive, Her2-high, Her2-3+, Her2-2+, Her2-1+, Her2-0, or Her2-negative subject. In certain embodiments, the subject is Her2-negative. The Disclosure further provides a method for treating such cancer in a Her2-negative subject, comprising providing the antibody, or a functional moiety, derivative, and / or analog thereof, to a subject in need of such treatment. In certain embodiments, such use involves providing a subject with a uniform dose of 1500 mg of antibody, or a functional portion thereof, derivative, and / or analogue thereof. In certain embodiments, the therapeutic agent may be administered to Her2-negative subjects weekly, bi-weekly, or monthly. In certain embodiments, the therapeutic agent is administered once every two weeks. Preferred variable domains that bind to the extracellular portion of EGFR and preferred variable domains that bind to the extracellular portion of LGR5 are disclosed herein. In certain embodiments, the first variable domain comprises at least a CDR3 sequence of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or at least CDR1, CDR2, and CDR3 sequences. In a particular embodiment, the second variable domain includes at least a CDR3 sequence of an LGR5-specific heavy chain variable region selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3, or at least CDR1, CDR2, and CDR3 sequences.

[0081] Methods for determining the expression of human epidermal growth factor receptor 2 (HER2) in a subject are well known in the art. For example, Her2 expression levels can be established using immunohistochemistry (IHC) or (fluorescent) in-situ hybridization (ISH), which allows for the identification of Her2 status, including the identification of Her2-negative subjects. IHC or ISH are both well-defined and standard procedures routinely used to establish Her2 status in human subjects. This specification refers, for example, to the ASCO / CAP guidelines by Bartley et al., (HER2 Testing and Clinical Decision Making in Gastroesophageal Adenocarcinoma. Arch Pathol Lab Med. 2016;140:1345-1363). For example, the use of an anti-HER-2 / neu antibody (clone 4B5) enables semi-quantitative detection of the HER-2 antigen in sections of FFPE gastric cancer, esophageal cancer, gastroesophageal junction cancer, or head and neck cancer using IHC. Staining and scoring are performed according to consensus guidelines for this type of cancer. Such HC tests, which measure the amount of HER2 receptor protein on the surface of cells in cancer tissue samples, typically yield scores ranging from 0 to 3+. Based on the IHC score, a patient may be classified as Her2-negative, for example, when a score of 0 or 1+ is measured. When Her2 expression is established using ISH tests, such as using HER2 probes (17q11.2-q12) and centromere 17 probes (Cen17), the diagnosis may be either "positive" or "negative," and sometimes reported as "zero" for HER2. In certain embodiments, the treatment methods of the present disclosure include subjects who are Her2-negative as established by IHC and / or ISH.

[0082] In this specification, a Her2-negative subject means a subject having cancer, cancer cells, or a tumor, i.e., a subject that is Her2-negative. The Her2 status may be determined according to the IHC and / or ISH described above.

[0083] In certain embodiments, treatment with an antibody, or a functional portion thereof, a derivative, and / or analog thereof, follows a step of diagnosing the subject for Her2 status. In certain embodiments, subjects having a Her2-negative status are selected for treatment. In certain embodiments, treatment of a subject follows a step of diagnosing a subject having Her2-negative gastric cancer, esophageal cancer, or gastroesophageal junction cancer. Such cancers treated by the methods of the Disclosure include gastric adenocarcinoma and esophageal cancer with a squamous cell carcinoma histological diagnosis.

[0084] In certain embodiments, the Her2-negative diagnosis includes an ISH or IHC test for Her2 status.

[0085] In certain embodiments, treatment of Her2-negative subjects follows a step of screening for subjects with Her2-negative gastric cancer, esophageal cancer, or gastroesophageal junction cancer. Such cancers are, in particular, adenocarcinomas. In certain embodiments, such screening includes ISH or IHC testing for Her2 status.

[0086] In certain embodiments, the subject has not been previously treated with an anti-EGFR agent. In certain embodiments, the subject has not been treated with an EGFR-targeting antibody. In certain embodiments, the subject has not been treated with cetuximab. Such subjects are also referred to as cetuximab-naive or anti-EGFR-naive subjects. In other words, the cancer of the subject has not been previously treated with an anti-EGFR agent. In certain embodiments, the cancer of the subject has not been treated with an EGFR-targeting antibody. In certain embodiments, the cancer of the subject has not been treated with cetuximab. Such subjects are also referred to as cetuximab-naive or anti-EGFR-naive subjects.

[0087] The subjects of this disclosure are those who have received prior treatment with an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor includes durvalumab, pembrolizumab, ipilimumab, nivolumab, atezolizumab, retifanlimab, cemiplimab, or other approved or developmental anti-PD1, anti-PD-L1 antibodies. In certain embodiments, the immune checkpoint inhibitor includes durvalumab or pembrolizumab.

[0088] Durvalumab (brand name: Imfinzi®) is an FDA-approved immune checkpoint inhibitor for the treatment of cancers such as bladder cancer and lung cancer. It is a human immunoglobulin G1 kappa (IgG1κ) monoclonal antibody that blocks the interaction between programmed cell death ligand 1 (PD-L1) and PD-1 (CD279). Durvalumab is an immune checkpoint inhibitor, or sometimes referred to as an immune checkpoint inhibitor drug. Clinically relevant responses have been observed in patients who have received prior treatment with durvalumab as an immune checkpoint inhibitor, as shown in the Examples section.

[0089] Pembrolizumab (brand name Keytruda®) is a humanized antibody used in cancer immunotherapy to treat a variety of cancers, including melanoma, lung cancer, and Hodgkin lymphoma, and functions as an immune checkpoint inhibitor. It is an IgG4 isotype antibody that targets the programmed cell death protein 1 (PD-1) receptor on lymphocytes. Pembrolizumab was approved for medical use in the United States in 2014. In 2017, the U.S. Food and Drug Administration (FDA) approved it for any unresectable or metastatic solid tumor with certain genetic abnormalities. It is listed on the World Health Organization's List of Essential Medicines. Clinically relevant responses have been observed in patients who have received prior treatment with pembrolizumab as an immune checkpoint inhibitor, as shown in the Examples section.

[0090] Ipilimumab (marketed under the brand name Yervoy®) is a monoclonal antibody and immune checkpoint inhibitor that works to activate the immune system by targeting CTLA-4, a protein receptor that downmodulates the immune system. Ipilimumab was approved by the U.S. Food and Drug Administration (FDA) in March 2011 for the treatment of melanoma.

[0091] Nivolumab (brand name Opdivo®) is an immune checkpoint inhibitor used to treat many cancers, including melanoma, lung cancer, malignant pleural mesothelioma, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, urothelial carcinoma, colon cancer, esophageal squamous cell carcinoma, liver cancer, gastric cancer, and esophageal or gastroesophageal junction (GEJ) cancer. Nivolumab is a human IgG4 monoclonal antibody that blocks PD-1. Nivolumab was approved for medical use in the United States in 2014. It is listed on the World Health Organization's list of essential medicines. Nivolumab is the second FDA-approved systemic therapy for mesothelioma and the first FDA-approved immunotherapy for first-line treatment of gastric cancer.

[0092] Atezolizumab (brand name Tecentriq®) is a monoclonal antibody drug used to treat urothelial carcinoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), small cell lung cancer (SCLC), and hepatocellular carcinoma (HCC). It is a humanized monoclonal antibody of the IgG1 isotype that targets programmed cell death ligand 1 (PD-L1). Atezolizumab is the first PD-L1 inhibitor approved by the U.S. Food and Drug Administration.

[0093] Retifan-limab (formerly known as MGA012) is a humanized anti-PD-1 monoclonal antibody being developed for use as a monotherapy and in combination with other cancer treatments. Retifan-limab is currently undergoing clinical trials (NCT04472429 and NCT04205812) as a monotherapy for patients with highly microsatellite-instability endometrial cancer, Merkel cell carcinoma, and squamous cell carcinoma of the anal canal (SCAC), and in combination with platinum-based chemotherapy for patients with non-small cell lung cancer and SCAC. Retifan-limab has received orphan drug designation from the FDA for the treatment of anal cancer.

[0094] Cemiplimab (brand name Ributayo®) is a monoclonal antibody drug for the treatment of squamous cell carcinoma. Cemiplimab belongs to a class of drugs that bind to programmed death receptor 1 (PD-1) and block the PD-1 / PD-L1 pathway. It was approved by the FDA in September 2018 for the treatment of patients with metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC who are not candidates for curative surgery or curative radiotherapy. It was approved for medical use in the European Union in June 2019.

[0095] Furthermore, prior treatment with immune checkpoint inhibitors described herein is intended to target immune checkpoint proteins including PD-L1, PD-1, CTLA-4, B7-1, or B7-2. Accordingly, prior treatment with immune checkpoint inhibitors of this disclosure targets immune checkpoint proteins selected from PD-L1, PD-1, CTLA-4, B7-1, or B7-2.

[0096] Programmed death ligand 1 (PD-L1, CD274, or B7 homolog 1 (B7-H1), HGNC:17635, NCBI, Entrez gene:29126, UniProtKB / Swiss-Prot:Q9NZQ7) is a protein encoded by the CD274 gene in humans. This gene encodes an immunosuppressive receptor ligand expressed by hematopoietic and non-hematopoietic cells, including T cells and B cells, as well as various types of tumor cells. The encoded protein is a type I transmembrane protein with immunoglobulin V-like and C-like domains. The interaction between this ligand and its receptor inhibits T cell activation and cytokine production. During infection or inflammation of normal tissues, this interaction is important for preventing autoimmunity by maintaining homeostasis of the immune response. In the tumor microenvironment, this interaction provides immune evasion to tumor cells via cytotoxic T cell inactivation.

[0097] Programmed cell death protein 1 (PD-1 or CD279, HGNC:8760, NCBI Entrez gene:5133, UniProtKB / Swiss-Prot:Q15116) is an immunosuppressive receptor expressed on activated T cells and is involved in regulating T cell function, including the function of effector CD8+ T cells. In addition, this protein may also promote the differentiation of CD4+ T cells into T regulatory cells. It is expressed in many types of tumors, including melanoma, and has been demonstrated to play a role in antitumor immunity. Furthermore, while this protein has been shown to be involved in protection against autoimmunity, it may also contribute to the inhibition of effective antitumor and antimicrobial immunity.

[0098] Cytotoxic T lymphocyte-associated protein 4 (CTLA-4 or CD152, HGNC:2505, NCBI Entrez gene:1493, UniProtKB / Swiss-Prot:P16410) is a member of the immunoglobulin superfamily and encodes a protein that transmits repressive signals to T cells. The protein contains a V domain, a transmembrane domain, and a cytoplasmic tail. Alternative transcriptional splice variants encoding different isoforms have been characterized. The membrane-bound isoform functions as a homodimer interconnected by disulfide bonds, while the soluble isoform functions as a monomer. Mutations in this gene have been associated with insulin-dependent diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated orbitopathy, and other autoimmune diseases.

[0099] B7-1, or differentiated cluster 80 (CD80, HGNC:1700, NCBI Entrez gene 941, UniProtKB / Swiss-Prot:P33681), is a type I membrane protein, part of the immunoglobulin superfamily, possessing an extracellular immunoglobulin constant-like domain and a variable-like domain necessary for receptor binding. The protein encoded by this gene is a membrane receptor activated by the binding of CD28 or CTLA-4. Its functions in biological systems include the induction of T cell proliferation and cytokine production. It is also involved in the co-stimulatory signaling essential for T lymphocyte activation. T cell proliferation and cytokine production are induced by CD28 binding, while binding to CTLA-4 has the opposite effect, inhibiting T cell activation. It is closely related to another B7 protein, CD86, and often functions in conjunction with it. Both CD80 and CD86 interact with the co-stimulatory receptors CD28 and CTLA-4 (CD152).

[0100] B7-2, or differentiated cluster 86 (CD86, HGNC:1705 NCBI Entrez gene:942 UniProtKB / Swiss-Prot:P42081), is a protein constitutively expressed on dendritic cells, Langerhans cells, macrophages, B cells (including memory B cells), and other antigen-presenting cells. Together with CD80, CD86 provides costimulatory signals necessary for T cell activation and survival. Depending on ligand binding, CD86 can signal for autoregulation and cell-cell association, or for regulatory attenuation and cell-cell dissociation. The CD86 gene encodes a type I membrane protein, a member of the immunoglobulin superfamily. Alternative splicing results in two transcriptional variants encoding different isoforms.

[0101] The subjects may also have previously been treated with one or more standard-approved therapies or standard care. Surgery or radiotherapy may be preferred for most patients with early or localized disease and may be considered for locally advanced disease, but may not be applicable to all patients, for example, due to the anatomical location of the cancer. In certain embodiments, standard-approved therapies or standard care as used herein include treatment with the administration of chemotherapeutic agents such as platinum compounds (e.g., cisplatin, carboplatin), antitumor compounds (e.g., methotrexate), fluoropyrimidines (e.g., fluorouracil, 5-FU, capecitabine), taxanes (e.g., docetaxel or paclitaxel), nucleoside analogs (e.g., gemcitabine), or any combination thereof.

[0102] Accordingly, in certain embodiments, the subject of the Disclosure has received prior treatment with a chemotherapeutic agent. In certain embodiments, the chemotherapeutic agent includes platinum compounds (e.g., cisplatin, carboplatin), antitumor compounds (e.g., methotrexate), fluoropyrimidines (e.g., fluorouracil, 5-FU, capecitabine), taxanes (e.g., docetaxel or paclitaxel), nucleoside analogs (e.g., gemcitabine), or any combination thereof.

[0103] According to this disclosure, in certain embodiments, cancer and / or subjects having such cancer are wild-type SMAD4. SMAD4 (HGNC:6770, NCBI Entrez gene:4089, UniProtKB / Swiss-Prot:Q13485) belongs to the SMAD family of signaling proteins. SMAD proteins are phosphorylated and activated by transmembrane serine / threonine receptor kinases in response to transforming growth factor (TGF)-beta signaling. The product of this gene forms homomeric complexes and heteromeric complexes with other activated SMAD proteins, which then accumulate in the nucleus and regulate the transcription of target genes. This protein binds to DNA and recognizes an 8 bp palindromic sequence called the SMAD-binding element (GTCTAGAC). This protein acts as a tumor suppressor, inhibiting epithelial cell proliferation. It may also have inhibitory effects on tumors by reducing angiogenesis and increasing vascular permeability. The encoded protein is an important component of the bone morphogenesis protein signaling pathway. SMAD proteins are subject to complex regulation through post-translational modifications. Mutations or deletions in this gene have been shown to result in pancreatic cancer, juvenile polyposis syndrome, and hereditary hemorrhagic telangiectasia syndrome. Notwithstanding these previously reported implications of mutations occurring in SMAD4, the cancers and / or subjects having such cancers described herein are wild-type SMAD4. In certain embodiments, such patients or cancers do not contain any mutations across the SMAD4 protein information referred to herein.

[0104] Optionally, treatment with an antibody, or a functional portion thereof, derivative, and / or analog thereof, includes a step of diagnosing the subject for SMAD status. In certain embodiments, treatment follows the diagnostic step. In certain embodiments, subjects with gastric cancer, esophageal cancer, or gastroesophageal junction cancer having the wild-type SMAD4 gene and / or protein are selected for treatment. In certain embodiments, treatment of the subject follows a step of diagnosing the subject with gastric cancer, esophageal cancer, or gastroesophageal junction cancer characterized by the wild-type SMAD4 gene or gene product.

[0105] Cancers such as gastric cancer, esophageal cancer, gastroesophageal junction cancer, or head and neck cancer may be associated with the presence of mutations. Such mutations include those in known oncogenes such as PIK3CA, KRAS, BRAF, HRAS, MAP2K1, and NOTCH1. Oncogenic mutations are generally described as activating mutations or mutations that impart new function. Another type of cancer mutation involves tumor suppressor genes such as TP53, MLH1, CDKN2A, and PTEN. Mutations in tumor suppressor genes are generally inactivating.

[0106] In certain embodiments, the cancer has mutations in one or more EGFR signaling pathway genes. In certain embodiments, the mutations are present in genes whose expression products are active downstream of EGFR in the EGFR signaling pathway. In certain embodiments, the cancer has mutations in genes selected from AKT1, KRAS, MAP2K1, NRAS, HRAS, PIK3CA, PTEN, EGFR, and / or PLCG2, and the protein products that encode them. In certain embodiments, the cancer has mutations in the gene encoding HRAS. In certain embodiments, the cancer does not have activating mutations in KRAS and / or BRAF.

[0107] In certain embodiments, cancer has mutations in one or more WNT signaling pathway genes, in certain embodiments, in APC, CREPPB, CUL1, EP300, SOX17, and / or TP53.

[0108] In certain embodiments, mutations in the HRAS gene include missense mutations, somatic mutations, and / or oncogenic driver mutations. In certain embodiments, HRAS includes the G12S mutation in its protein sequence, or a G>A missense mutation leading to a G>S amino acid change. In certain embodiments, the missense mutation G34A in the coding sequence (CDS) of the codon GGC in the HRAS gene. In certain embodiments, the cancer is oral squamous cell carcinoma or buccal mucosal squamous cell carcinoma and includes the G12S missense mutation in HRAS.

[0109] Cancer may have mutations in the gene encoding MAP2K1. In certain embodiments, mutations in the MAP2K1 gene are missense mutations, somatic mutations, and / or oncogenic driver mutations. In certain embodiments, MAP2K1 includes mutations in its protein sequence, such as L375R, or T>G missense mutations that lead to L>R amino acid changes. In certain embodiments, the missense mutation is T1124G in the coding sequence (CDS) of the codon CTC of the MAP2K1 gene.

[0110] TP53 encodes a transcription factor that regulates several activities, including stress response and cell proliferation. Mutations in TP53 are associated with various cancers and are estimated to occur in more than 50% of human cancers, including gastric and esophageal cancer. In particular, the TP53 R248Q mutation has been shown to be associated with cancers, including gastric and esophageal cancer (Pitolli et al. Int.J.Mol.Sci.2019 20:6241). Nonsense mutations at positions R196 and R342 have been identified in breast and esophagus, as well as in several tumors including ovarian, prostate, breast, pancreatic, stomach, colorectal, lung, esophageal, and bone tumors, respectively (Priestly et al. Nature 2019 575:210-216). In particular, the therapeutic agents disclosed herein are useful for treating cancers with TP53 mutations, especially mutations that result in reduced TP53 expression or activity.

[0111] MLH1 (MutL homolog 1) is a known tumor suppressor gene that encodes a protein involved in DNA mismatch repair. Mutations in MLH1 are associated with a variety of cancers, including gastrointestinal cancers. Low levels of MLH1 are also associated with esophageal cancer patients with a family history of esophageal cancer (Chang et al. Oncol Lett. 2015 9:430-436), and MLH1 is mutated in 1.39% of patients with malignant esophageal tumors (The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6). In particular, the MLH1 V384D mutation has been shown to be associated with cancer, such as colorectal cancer (Ohsawa et al. Molecular Medicine Reports 2009 2:887-891). In certain embodiments, the therapeutic agents disclosed herein are useful for treating cancers having MLH1 mutations, particularly mutations that result in reduced MLH1 expression or activity.

[0112] PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) encodes the 110 kDa catalytic subunit of PI3K (phosphatidylinositol 3-kinase). Mutations in PIK3CA are associated with various cancers, including gastrointestinal cancers. According to the American Association for Cancer Research, PIK3CA is mutated in 12.75% of patients with malignant solid tumors. Specifically, the PIK3CA H1047R mutation is present in 2.91% of all malignant solid tumor patients, and the PIK3CA E545K mutation is present in 2.55% of all malignant solid tumor patients (see The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6). In certain embodiments, the therapeutic agents disclosed herein are useful for treating cancers having PIK3CA mutations, particularly oncogenic mutations in PIK2CA or PIK3CA.

[0113] CDKN2A (cyclin-dependent kinase inhibitor 2A) encodes a protein that inhibits CDK4 and ARF. According to the American Association for Cancer Research, CDKN2A is mutated in 22.21% of esophageal cancer patients, 28.7% of esophageal squamous cell carcinoma patients, and 6.08% of gastric adenocarcinoma patients. Specifically, the CDKN2A W110Ter mutation is present in approximately 0.11% of cancer patients. (The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6). In certain embodiments, the therapeutic agents disclosed herein are useful for treating cancers having CDKN2A mutations, particularly mutations that result in reduced CDKN2A expression or activity.

[0114] PTEN (phosphatase and tensin homolog) encodes phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase. According to the American Association for Cancer Research, PTEN is mutated in 6.28% of cancer patients, 3.41% of gastric adenocarcinoma patients, 2.37% of esophageal cancer patients, and 2.22% of esophageal adenocarcinoma patients. In particular, the PTEN R130Ter mutation (Ter refers to a stop / stop codon) is present in 0.21% of all colorectal cancer patients (The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6). In certain embodiments, the therapeutic agents disclosed herein are useful for treating cancers having PTEN mutations, particularly mutations that result in reduced PTEN expression or activity.

[0115] BRAF encodes serine / threonine protein kinase B-Raf, which is involved in growth signaling. According to the American Association for Cancer Research, BRAF is mutated in 1.91% of gastric cancer patients and 1.93% of gastric adenocarcinoma patients. Specifically, the BRAF V600E mutation is present in 2.72% of cancer patients (see The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6). In certain embodiments, the therapeutic agents disclosed herein are useful for treating cancers with BRAF mutations, particularly oncogenic mutations in BRAF. However, in certain embodiments, the therapeutic agents disclosed herein are useful for treating gastric cancers that do not have the BRAF V600E mutation.

[0116] KRAS (Kirsten RAt sarcoma) encodes a protein that is part of the RAS / MAPK pathway. According to the American Association for Cancer Research, KRAS is mutated in 14.7% of patients with malignant solid tumors, and KRAS G12C is present in 2.28% of patients with malignant solid tumors (see The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6.). In certain embodiments, the therapeutic agents disclosed herein are useful for treating cancers with KRAS mutations, particularly oncogenic mutations in KRAS.

[0117] UGT1A1 (uridine diphosphateglucuronosyltransferase 1A1) and UGT1A8 (uridine diphosphateglucuronosyltransferase 1A8) encode enzymes in the glucuronidation pathway. Several polymorphisms that reduce enzyme activity are known to affect the metabolism and effects of irinotecan. For example, the UGT1A1*6 allele (G71R polymorphism) and the UGT1A1*28 allele (dinucleotide repeat polymorphism in the TATA sequence of the promoter region), which have an allele frequency of approximately 0.13% in Chinese, Korean, and Japanese populations, are risk factors for irinotecan-induced neutropenia. In certain embodiments, the therapeutic agents disclosed herein are useful for treating cancers having UGT1A1 and / or UGT1A8 mutations, particularly mutations that result in reduced expression or activity of UGT1A1 and / or UGT1A8.

[0118] ATM (Ataxia Telangiectaisa Mutated) is a member of the serine / threonine kinase family that modulates cellular responses to DNA damage by activating different DNA repair and signaling pathways. ATM germline mutations are associated with ataxia telangiectasia, while ATM somatic mutations are commonly observed in endometrial, colon, pancreatic, breast, and urothelial carcinomas.

[0119] Notch1 (NOTCH1), also known as AOS5, hN1, AOVD1, and TAN1, is a gene encoding a transmembrane protein that functions in multiple developmental processes and interactions between adjacent cells. The transmembrane protein also functions as a receptor for membrane-bound ligands. Fusions, missense mutations, nonsense mutations, silent mutations, frameshift deletions and insertions, and intraframe deletions and insertions are observed in cancers such as esophageal cancer, hematopoietic and lymphoid cancers, and gastric cancer. NOTCH1 alters in 4.48% of all cancers, including colorectal adenocarcinoma, lung adenocarcinoma, invasive ductal carcinoma of the breast, endometrioid adenocarcinoma, and cutaneous squamous cell carcinoma, which have the highest prevalence alterations. In head and neck squamous cell carcinoma, NOTCH1 alters in approximately 16% of patients (The AACR Project GENIE Consortium. Cancer Discovery. 2017;7(8):818-831).

[0120] The HRAS (HGNC ID: 5173) gene product is involved in the activation of Ras protein signaling. The Ras protein binds to GDP / GTP and possesses intrinsic GTPase activity. Somatic mutations in the HRAS proto-oncogene have been shown to be associated with bladder cancer, thyroid cancer, salivary ductal cancer, epithelial-myoepithelial cancer, and renal cancer (Chiosea et al., in Am.J.of Surg.Path.39(6):744-52; Chiosea et al., in Head and Neck Path.2014.8(2):146-50). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers with HRAS mutations, particularly oncogenic mutations in HRAS such as HRAS mutation G12S. Specifically, the cancers are oral or buccal mucosal HNSCC.

[0121] MAP2K1 (HGNC ID: 6840) belongs to the group of mitogen-activated protein kinase kinases. It is active in MAP kinase signaling and encodes protein bispecific mitogen-activated protein kinase kinase 1. As part of the MAP kinase pathway, MAP2K1 is involved in many cellular processes, including cell proliferation, differentiation, and transcriptional regulation. MAP2K1 is altered in 1.05% of all cancers, including cutaneous melanoma, lung adenocarcinoma, colon adenocarcinoma, melanoma, and invasive ductal carcinoma of the breast, which have the highest prevalence alterations (AACR Project GENIE Consortium. Cancer Discovery. 2017; 7(8): 818-831. Dataset Version 8). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having MAP2K1 mutations, specifically the MAP2K1 mutation L375R.

[0122] In certain embodiments, the Disclosure provides a method for treating cancer having mutations in genes encoding TP53, MLH1, PIK3CA, CDKN2A, UGT1A, UGT1A8, BRAF, PTEN, and KRAS. In certain embodiments, the cancer has one or more mutations selected from TP53 R196T, TP53 R342T, TP53 R248Q, MLH1 V384D, PIK3CA H1047R, PIK3CA E545K, CDKN2A W110T, UGT1A1 G71R, UGT1A8 G71R, and KRAS G12C. In certain embodiments, the cancer is wild-type for KRAS. Alternatively, the Disclosure provides a method for treating cancer having mutations in genes encoding ATM, particularly the W57T mutation. Specifically, this disclosure provides a method for treating esophageal cancer, particularly ESCC, having mutations in the gene encoding ATM, in particular the W57T mutation.

[0123] In certain embodiments, the cancer has a mutation in the gene encoding TP53, such as R342T, and the cancer has a mutation in the gene encoding MLH1, such as V384D. In certain embodiments, the cancer has a mutation in the gene encoding TP53, and in certain embodiments, the mutation is R248Q. In certain embodiments, the cancer has a mutation in the gene encoding PIK3CA, and in certain embodiments, the mutation is H1047R. In certain embodiments, the cancer has a mutation in the gene encoding CDKN2A, and in certain embodiments, the mutation is W110T. In certain embodiments, the cancer has a mutation in the gene encoding UGT1A1, and in certain embodiments, the mutation is G71R, and the cancer has a mutation in the gene encoding UGT1A8, and in certain embodiments, the mutation is G71R. In certain embodiments, the cancer is esophageal cancer. In certain embodiments, the cancer is esophageal squamous cell carcinoma (ESCC).

[0124] In certain embodiments, the cancer has a mutation in the gene encoding BRAF. However, in certain embodiments, the cancer does not have the V600E mutation in the gene encoding BRAF, and in certain embodiments, it does not have the R130Ter mutation in the gene encoding PTEN. In certain embodiments, the cancer has a mutation in the gene encoding KRAS, and in certain embodiments, the mutation is G12C. The cancer has a mutation in the gene encoding UGT1A1, and in certain embodiments, the mutation is G71R. The cancer has a mutation in the gene encoding UGT1A8, and in certain embodiments, the mutation is G71R. In certain embodiments, the cancer has a mutation in the gene encoding UGT1A1, and in certain embodiments, the mutation is G71R. The cancer has a mutation in the gene encoding UGT1A8, and in certain embodiments, the mutation is G71R. In certain embodiments, the cancer has a mutation in PIK3CA, and in certain embodiments, the mutation is E545K. In a particular context, cancer is stomach cancer.

[0125] In some embodiments, the antibodies, or functional moieties, derivatives, and / or analogs thereof disclosed herein are multispecific antibodies. In certain embodiments, the antibodies are bispecific antibodies. In certain embodiments, the multispecific or bispecific antibodies, or their functional moieties, derivatives, and / or analogs, include a first variable domain that binds to the extracellular portion of the epidermal growth factor (EGF) receptor, and in certain embodiments, a second variable domain that does not bind to EGFR. In certain embodiments, the antibodies, or their functional moieties, derivatives, and / or analogs, bind to EGFR in a monovalent manner. In certain embodiments, the multispecific or bispecific antibodies, or their functional moieties, derivatives, and / or analogs, include a second variable domain that binds to LGR5.

[0126] In certain embodiments, EGFR is human EGFR. The EGFR conjugated by the antibody of this disclosure, or its functional portion, derivatives, and / or analogs, includes wild-type EGFR and EGFR having an oncogenic driver mutation. In certain embodiments, the oncogenic driver mutation is an activating EGFR mutation. In certain embodiments, such mutation does not structurally alter the epitope conjugated by the antibody of this disclosure. In certain embodiments, the EGFR mutations of this disclosure include exon 18 mutations, including G719A, G719C, 2E709_T710D, E709A, and G719S; exon 19 deletion mutations, including LREA or VAIKEL deletions; exon 19 point mutations, including G735S, P753L, L747S, and D761Y; in-frame exon 20 insertion mutations of 1-7 amino acids, and exon 20 mutations, including V765A, T783A, V774A, S784P, V769M, and T790M; and exon 21 mutations, including L858R, T854A, A871E, L861A, L861C, L861S, V843I, or P848L. The antibodies of this disclosure bind to epitopes that are not located adjacent to such mutations. In particular, the EGFR mutation is S492R, which results in a loss of cetuximab binding to EGFR. The antibody of this disclosure binds to an epitope different from the epitope recognized by cetuximab.

[0127] While not bound by any theory, the amino acid residues I462, G465, K489, I491, N493, and C499 shown in Figure 2 are thought to be involved in the binding of the antibodies of this disclosure to the epitope. In certain embodiments, involvement in binding is determined by observing a reduction in the binding of a variable domain to EGFR having one or more amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A, and C499A.

[0128] In one embodiment, the variable domain that binds to an epitope on the extracellular portion of human EGFR is a variable domain that binds to an epitope located within amino acid residues 420-480 of the sequence shown in Figure 2. In a particular embodiment, the binding of the variable domain to EGFR is reduced by one or more of the following amino acid residue substitutions in EGFR: I462A, G465A, K489A, I491A, N493A, and C499A. In a particular embodiment, the binding of the antibody to human EGFR prevents the binding of EGF to its receptor. In a particular embodiment, the epitope on EGFR is a structural epitope. In one embodiment, the epitope is located within amino acid residues 420-480 of the sequence shown in Figure 2, or within 430-480 of the sequence shown in Figure 2. In a particular embodiment, the epitope is located within 438-469 of the sequence shown in Figure 2.

[0129] While not bound by theory, it is considered highly probable that the epitope contact residues, i.e., the sites where the variable domain contacts human EGFR, are I462, K489, I491, and N493. Amino acid residues G465 and C499 are likely to be indirectly involved in antibody binding to EGFR.

[0130] In certain embodiments, the second variable domain binds to LGR5. In certain embodiments, LGR5 is human LGR5. The multispecific or bispecific antibodies described herein, or their functional parts, derivatives, and / or analogs, include a variable domain that binds to the extracellular portion of the human epidermal growth factor (EGF) receptor and, in certain embodiments, a variable domain that binds to human LGR5.

[0131] In certain embodiments, the antibodies described herein, or functional portions, derivatives, and / or analogs thereof, include a variable domain that binds to the extracellular portion of the epidermal growth factor (EGF) receptor and prevents EGF from binding to the receptor, and a variable domain that binds to LGR5, such that the interaction between the antibody and LGR5 on LGR5-expressing cells does not block the binding of Rspondin (RSPO) to LGR5. A method for determining whether or not an antibody blocks the binding of Rspondin to LGR5 is described in WO2017 / 069528, which is incorporated herein by reference.

[0132] In this specification, protein / gene acceptance numbers or alternative names are given, primarily to provide specific further methods for the proteins described as targets, and the actual sequence of the antibody-bound target protein may be altered due to mutations and / or alternative splicing in the coding gene, such as those occurring in some cancers. The target protein is bound by an antibody insofar as the epitope is present within the protein and the epitope is accessible to the antibody.

[0133] In certain embodiments, the antibodies, or functional moieties, derivatives, and / or analogs thereof described herein, interfere with the binding of EGFR ligands to EGFR. As used herein, “interfering with binding” means that the binding of the antibody, or functional moiety, derivative, and / or analog thereof, to EGFR competes with the ligand for binding to the EGF receptor. The antibody, or functional moiety, derivative, and / or analog thereof, may weaken ligand binding, or, if it is already bound to the EGF receptor, may migrate the ligand, or this may at least partially prevent the ligand from binding to the EGF receptor, for example, via steric hindrance.

[0134] In certain embodiments, the EGFR antibodies disclosed herein inhibit the respective EGFR ligand-induced signaling, which is measured as ligand-induced proliferation of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058), or as ligand-induced cell death of A431 cells (ATCC CRL-1555). EGFR can bind to several ligands and stimulate the proliferation of the described BxPC3 or BxPC3-luc2 cells. Proliferation of BxPC3 or BxPC3-luc2 cells is stimulated in the presence of an EGFR ligand. EGFR ligand-induced proliferation of BxPC3 cells can be measured by comparing cell proliferation in the absence and presence of the ligand. A preferred EGFR ligand for measuring EGFR ligand-induced proliferation of BxPC3 or BxPC3-luc2 cells is EGF. In certain embodiments, ligand-induced proliferation is measured using a saturated amount of the ligand. In certain embodiments, EGF is used in a culture medium at a concentration of 100 ng / ml. In certain embodiments, the EGF is the EGF R&D System, catalog numbers 396-HB and 236-EG (see also WO2017 / 069628, which is incorporated herein by reference).

[0135] In certain embodiments, the EGFR antibodies disclosed herein inhibit EGFR ligand-induced proliferation of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058). EGFR can bind to several ligands and stimulate the proliferation of the described BxPC3 or BxPC3-luc2 cells. In the presence of a ligand, the proliferation of BxPC3 or BxPC3-luc2 cells is stimulated. EGFR ligand-induced proliferation of BxPC3 cells can be measured by comparing cell proliferation in the absence and presence of the ligand. In certain embodiments, the EGFR ligand for measuring EGFR ligand-induced proliferation of BxPC3 or BxPC3-luc2 cells is EGF. In certain embodiments, ligand-induced proliferation is measured using a saturated amount of ligand. In certain embodiments, EGF is used in an amount of 100 ng / ml of culture medium. In certain embodiments, the EGF is the EGF of the R&D system, catalog numbers 396-HB and 236-EG (see also WO2017 / 069628, which is incorporated herein by reference).

[0136] To avoid misunderstanding, as used herein, references to cell proliferation refer to a change in the number of cells. Inhibition of proliferation refers to a reduction in the number of cells that could have otherwise been obtained. Increased proliferation refers to an increase in the number of cells that could have otherwise been obtained. Cell growth usually refers to cell proliferation.

[0137] In certain embodiments, whether the antibodies described herein inhibit signal transduction or proliferation in a multispecific form is determined by the method described herein using monospecific monovalent or monospecific bivalent versions of the antibody. In certain embodiments, such antibodies have a binding site to a receptor on which signal transduction is determined. Monospecific monovalent antibodies may have a variable domain having irrelevant binding specificity, such as tetanus toxoid specificity. In certain embodiments, the antibody is a bivalent monospecific antibody in which the antigen-binding variable domain consists of a variable domain that binds to a member of the EGF receptor family.

[0138] In its Biclonics® antibody program, Merus developed multispecific antibodies targeting EGFR and LGR5 (leucine-rich repeats containing G protein-coupled receptors). The efficacy of these multispecific antibodies has been evaluated in vitro and in vivo using patient-derived CRC organoids and mouse PDX models, respectively (see, for example, WO2017 / 069628, which is incorporated herein by reference). The multispecific antibodies targeting EGFR and LGR5 have been shown to inhibit tumor growth. The efficacy of these inhibitory antibodies has been shown to correlate with the level of LGR5 RNA expression in cancer-derived cells. In certain embodiments, the multispecific antibodies targeting EGFR and LGR5 are those described in WO2017 / 069628.

[0139] The antibodies described herein, or their functional portions, derivatives, and / or analogs, include a variable domain that binds to the extracellular portion of LGR5. In certain embodiments, the variable domain that binds to the extracellular portion of LGR5 binds to an epitope located within amino acid residues 21-118 of the sequence in Figure 1, in which amino acid residues D43, G44, M46, F67, R90, and F91 are involved in the binding of the antibody to the epitope.

[0140] In a particular embodiment, the LGR5 variable domain is a variable domain in which one or more amino acid residue substitutions in LGR5, D43A, G44A, M46A, F67A, R90A, and F91A, reduce the binding of the variable domain to LGR5.

[0141] In a particular embodiment, the epitope on the extracellular portion of LGR5 is located within amino acid residues 21-118 of the sequence in Figure 1. In a particular embodiment, it is an epitope in which the binding of the LGR5 variable domain to LGR5 is reduced by one or more of the following amino acid residue substitutions in LGR5: D43A, G44A, M46A, F67A, R90A, and F91A.

[0142] This disclosure further provides an antibody having a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, wherein the LGR5 variable domain binds to an epitope on LGR5 located within amino acid residues 21-118 of the sequence shown in Figure 1.

[0143] In certain embodiments, the epitope on LGR5 is a structural epitope. In certain embodiments, the epitope is located within amino acid residues 40-95 of the sequence in Figure 1. In certain embodiments, antibody binding to LGR5 is reduced by one or more of the following amino acid residue substitutions: D43A, G44A, M46A, F67A, R90A, and F91A.

[0144] While not bound by theory, M46, F67, R90, and F91 of LGR5 shown in Figure 1 are considered to be contact residues for the antigen-binding site of the variable domain, i.e., the variable domain that binds to the LGR5 epitope, as described herein. The reduction in antibody binding by amino acid substitutions D43A and G44A may be due to them also being contact residues, but it is also possible that these amino acid substitutions induce a (slight) modification of the conformation of a portion of LGR5 with one or more of the other contact residues (i.e., at positions 46, 67, 90, or 91), and that the conformational change is such that antibody binding is reduced. Epitopes are characterized by the aforementioned amino acid substitutions. Whether an antibody binds to the same epitope can be determined in various ways. In an exemplary method, CHO cells express LGR5 on the cell membrane, or alanine-substituted mutants such as mutants containing one or more of the substitutions M46A, F67A, R90A, or F91A. The test antibody is brought into contact with CHO cells, and the binding of the antibody to the cells is compared. The test antibody binds to the epitope if it binds to LGR5 and to LGR5 having the M46A, F67A, R90A, or F91A substitutions, to a lesser extent. It is preferable to compare binding to a panel of mutants, each containing a single alanine residue substitution. Such binding tests are well known in the art. Often, the panel includes single alanine substitution mutants covering substantially all amino acid residues. In the case of LGR5, the panel should cover only the portion that ensures the extracellular portion of the protein and its association with the cell membrane when the cells are used. Expression of a particular mutant may be impaired, which is readily detectable by one or more LGR5 antibodies that bind to different regions. If expression is also reduced with these control antibodies, the level or folding of the protein on the membrane will be impaired for this particular mutant. The binding characteristics of the test antibody to the panel allow for easy identification of whether the test antibody exhibits reduced binding to variants having M46A, F67A, R90A, or F91A substitutions, and therefore whether the test antibody is the antibody of the present invention.Reduced binding to mutants with M46A, F67A, R90A, or F91A substitutions also identifies epitopes located within amino acid residues 21–118 of the sequence in Figure 1. In certain embodiments, the panel includes both D43A and G44A substitution mutants. Antibodies with the VH sequence of MF5816 show reduced binding to these substitution mutants.

[0145] While not bound by any theory, the amino acid residues I462, G465, K489, I491, N493, and C499 shown in Figure 2 are thought to be involved in the binding of antibodies containing the variable domains shown above to the epitope. In certain embodiments, involvement in binding is determined by observing a reduction in the binding of the variable domain to EGFR having one or more amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A, and C499A. In an exemplary method, CHO cells express EGFR on the cell membrane, or alanine-substituted mutants such as those containing one or more of the substitutions I462A, G465A, K489A, I491A, N493A, and C499A. The test antibody is contacted with the CHO cells, and the binding of the antibody to the cells is compared. The test antibody binds to the epitope when it binds to EGFR and, to a lesser extent, to EGFR with the I462A, G465A, K489A, I491A, N493A, and C499A substitutions. It is preferable to compare binding to a panel of mutants, each containing a single alanine residue substitution. Such binding tests are well known in the art. Often, the panel includes single alanine substitution mutants covering substantially all amino acid residues. In the case of EGFR, the panel should, of course, cover only the extracellular portion of the protein and the portion that ensures its relevance to the cell membrane, if cells are used. Expression of a particular mutant may be impaired, which is readily detectable by one or more EGFR antibodies that bind to different regions. If expression is also reduced by these control antibodies, the level or folding of the protein on the membrane will be impaired for this particular mutant. The binding characteristics of the test antibody to the panel make it easy to determine whether the test antibody exhibits reduced binding to variants with I462A, G465A, K489A, I491A, N493A, and C499A substitutions.

[0146] In one embodiment, the variable domain that binds to an epitope on the extracellular portion of human EGFR is a variable domain that binds to an epitope located within amino acid residues 420-480 of the sequence shown in Figure 2. In a particular embodiment, the binding of the variable domain to EGFR is reduced by one or more of the following amino acid residue substitutions in EGFR: I462A, G465A, K489A, I491A, N493A, and C499A. In a particular embodiment, the binding of an antibody to human EGFR prevents the binding of EGF to its receptor. In a particular embodiment, the epitope on EGFR is a structural epitope. In one embodiment, the epitope is located within amino acid residues 420-480 of the sequence shown in Figure 2, such as within 430-480 of the sequence shown in Figure 2. In a particular embodiment, the epitope is located within 438-469 of the sequence shown in Figure 2.

[0147] While not bound by theory, it is considered highly probable that the epitope contact residues, i.e., the sites where the variable domain contacts human EGFR, are I462, K489, I491, and N493. Amino acid residues G465 and C499 are likely to be indirectly involved in antibody binding to EGFR.

[0148] In certain embodiments, the variable domain that binds to human EGFR is a variable domain having a heavy chain variable region that includes at least the VH CDR3 sequence of MF3755 shown in Figure 3, or the VH CDR3 sequence of MF3755 shown in Figure 3 and a CDR3 sequence that differs by at least three, at least two, or one or fewer amino acids.

[0149] In a particular embodiment, the variable domain that binds to human EGFR is a variable domain having a heavy chain variable region that includes the CDR1, CDR2, and CDR3 sequences of MF3755 VH shown in Figure 3, or at least three, at least two, or at least one amino acid substitution.

[0150] In a particular embodiment, the variable domain that binds to human EGFR is a variable domain having a heavy chain variable region containing the VH chain amino acid sequence of MF3755 shown in Figure 3, or at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof with respect to the VH chain of MF3755 shown in Figure 3.

[0151] In certain embodiments, the present disclosure relates to an antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, The present invention provides an antibody in which the heavy chain variable region of the variable domain includes at least one CDR3 sequence of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or the heavy chain variable region of the variable domain includes a heavy chain CDR3 sequence that differs from a VH CDR3 sequence selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3 by at least three, at most two, or one or fewer amino acids. In a particular embodiment, the variable domain includes a heavy chain variable region containing at least one CDR3 sequence of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3.

[0152] In a particular embodiment, the variable domain includes a heavy chain variable region containing CDR1, CDR2, and CDR3 sequences of an EGFR-specific heavy chain variable region selected from at least the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or a heavy chain variable region containing CDR1, CDR2, and CDR3 sequences of an EGFR-specific heavy chain variable region selected from at least the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, and CDR1, CDR2, and CDR3 sequences that differ by at least three, at least two, or at least one amino acid. In a particular embodiment, the variable domain includes a heavy chain variable region containing CDR1, CDR2, and CDR3 sequences of at least MF3370, MF3755, MF4280, or MF4289 shown in Figure 3. In a particular embodiment, the heavy chain variable region is MF3755. In a particular embodiment, the heavy chain variable region is MF4280.

[0153] In a particular embodiment, an antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, wherein the EGFR-binding variable domain having the CDR3, CDR1, CDR2, and CDR3 and / or VH sequences shown herein is at least MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5 An antibody having a variable domain that binds to LGR5, comprising a CDR3 sequence of an LGR5-specific heavy chain variable region selected from the group consisting of 818, or a VH CDR3 sequence selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3, and a heavy chain CDR3 sequence that differs by at least three, at most two, or one or fewer amino acids. In a particular embodiment, the variable domain comprises a heavy chain variable region containing at least the CDR3 sequences of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3.

[0154] In a particular embodiment, the LGR5 variable domain includes at least the CDR1, CDR2, and CDR3 sequences of an LGR5-specific heavy chain variable region selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3, or a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences of an LGR5-specific heavy chain variable region selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3, and heavy chain CDR1, CDR2, and CDR3 sequences that differ by at least three, at least two, or at least one amino acid. In certain embodiments, the variable domain includes a heavy chain variable region containing at least the CDR1, CDR2, and CDR3 sequences of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 as shown in Figure 3. In certain embodiments, the heavy chain variable region is MF5790, MF5803, MF5814, MF5816, MF5817, or MF5818. In certain embodiments, the heavy chain variable region is MF5790, MF5814, MF5816, and MF5818. In certain embodiments, the heavy chain variable region is MF5814, MF5818, or MF5816. In certain embodiments, the heavy chain variable region is MF5816. In certain embodiments, the heavy chain variable region is MF5818.

[0155] Antibodies containing one or more variable domains having the heavy chain variable region MF3755 or one or more CDRs thereof have been shown to exhibit better efficacy when used to inhibit the proliferation of EGFR ligand-responsive cancers or cells. In the context of bispecific or multispecific antibodies, an antibody arm containing the heavy chain variable region MF3755 or one or more variable domains having one or more CDRs thereof combines well with an arm containing the heavy chain variable region MF5818 or one or more variable domains having one or more CDRs thereof.

[0156] The VH chain of the variable domain that binds to EGFR or LGR5 may have one or more amino acid substitutions with respect to the sequence shown in Figure 3. In certain embodiments, the VH chain has the amino acid sequence of EGFR or LGR5 VH in Figure 3, having at most 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in certain embodiments, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof with respect to the VH chain sequence in Figure 3.

[0157] The CDR sequence may have one or more amino acid residue substitutions with respect to the CDR sequence shown in the figure. Such one or more substitutions are made for optimization purposes, for example, to improve the antibody binding strength or stability. Optimization is performed by a mutagenesis procedure, for example, after the resulting antibody stability and / or binding affinity is preferably tested and an improved EGFR-specific or LGR5-specific CDR sequence is preferably selected. Those skilled in the art can generate antibody variants containing at least one modified CDR sequence according to the present invention. For example, conservative amino acid substitutions may be applied. Examples of conservative amino acid substitutions include the substitution of one hydrophobic residue, e.g., isoleucine, valine, leucine, or methionine, with another hydrophobic residue, and the substitution of one polar residue with another polar residue, e.g., arginine to lysine, glutamic acid to aspartic acid, or glutamine to asparagine.

[0158] In certain embodiments, at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid substitutions mentioned in the VH or VL as specified herein are conservative amino acid substitutions. In certain embodiments, amino acid insertions, deletions, and substitutions in the VH or VL as specified herein are not present in the CDR3 region. In certain embodiments, the mentioned amino acid insertions, deletions, and substitutions are also not present in the CDR1 and CDR2 regions. In certain embodiments, the mentioned amino acid insertions, deletions, and substitutions are also not present in the FR4 region.

[0159] In certain embodiments, at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid substitutions mentioned are conservative amino acid substitutions. In certain embodiments, insertions, deletions, substitutions, or combinations thereof are not present in the CDR3 region of the VH chain, in certain embodiments, not present in the CDR1, CDR2, or CDR3 region of the VH chain, and in certain embodiments, not present in the FR4 region.

[0160] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in a particular embodiment, a variable domain that binds to the extracellular portion of LGR5, - The amino acid sequence of VH chain MF3755 shown in Figure 3, or -With respect to the VH, the amino acid sequence of the VH chain MF3755 shown in Figure 3 includes at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof, The VH chain of the variable domain that binds to LGR5 is - The amino acid sequence of VH chain MF5790 shown in Figure 3, or -The VH contains the amino acid sequence of the VH chain MF5790 shown in Figure 3, which has at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof.

[0161] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in a particular embodiment, a variable domain that binds to the extracellular portion of LGR5, - The amino acid sequence of VH chain MF3755 shown in Figure 3, or -With respect to the VH, the amino acid sequence of the VH chain MF3755 shown in Figure 3 includes at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof, The VH chain of the variable domain that binds to LGR5 is - The amino acid sequence of VH chain MF5803 shown in Figure 3, or -The VH contains the amino acid sequence of the VH chain MF5803 shown in Figure 3, which has at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof.

[0162] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in a particular embodiment, a variable domain that binds to the extracellular portion of LGR5, - The amino acid sequence of VH chain MF3755 shown in Figure 3, or -With respect to the VH, the amino acid sequence of the VH chain MF3755 shown in Figure 3 includes at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof, The VH chain of the variable domain that binds to LGR5 is - The amino acid sequence of VH chain MF5814 shown in Figure 3, or -The VH contains the amino acid sequence of the VH chain MF5814 shown in Figure 3, which has at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof.

[0163] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in a particular embodiment, a variable domain that binds to the extracellular portion of LGR5, - The amino acid sequence of VH chain MF3755 shown in Figure 3, or -With respect to the VH, the amino acid sequence of the VH chain MF3755 shown in Figure 3 includes at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof, The VH chain of the variable domain that binds to LGR5 is - The amino acid sequence of VH chain MF5816 shown in Figure 3, or -The VH contains the amino acid sequence of the VH chain MF5816 shown in Figure 3, which has at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof.

[0164] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in a particular embodiment, a variable domain that binds to the extracellular portion of LGR5, - The amino acid sequence of VH chain MF3755 shown in Figure 3, or -With respect to the VH, the amino acid sequence of the VH chain MF3755 shown in Figure 3 includes at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof, The VH chain of the variable domain that binds to LGR5 is - The amino acid sequence of VH chain MF5817 shown in Figure 3, or -The VH contains the amino acid sequence of the VH chain MF5817 shown in Figure 3, which has at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof.

[0165] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in a particular embodiment, a variable domain that binds to the extracellular portion of LGR5, - The amino acid sequence of VH chain MF3755 shown in Figure 3, or -With respect to the VH, the amino acid sequence of the VH chain MF3755 shown in Figure 3 includes at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof, The VH chain of the variable domain that binds to LGR5 is - The amino acid sequence of VH chain MF5818 shown in Figure 3, or -The VH contains the amino acid sequence of the VH chain MF5818 shown in Figure 3, which has at most 15 (or in a particular embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in a particular embodiment 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or combinations thereof.

[0166] Variants of the disclosed amino acid sequences that retain EGFR or LGR5 binding can be obtained, for example, from phage display libraries containing rearranged human IGKVl-39 / IGKJl VL regions (De Kruif et al. Biotechnol Bioeng. 2010(106)741-50) and from collections of VH regions incorporating amino acid substitutions into the amino acid sequences of the EGFR or LGR5 VH regions disclosed herein, as previously described (e.g., WO2017 / 069628). Phages encoding Fab regions that bind to EGFR or LGR5 can be selected, analyzed by flow cytometry, and sequenced to identify variants having amino acid substitutions, insertions, deletions, or additions that retain antigen binding.

[0167] The light chain variable regions of the VH / VL EGFR and LGR5 variable domains of an EGFR / LGR5 antibody may be the same or different. In certain embodiments, the VL region of the VH / VL EGFR variable domain of an EGFR / LGR5 antibody is the same as the VL region of the VH / VL LGR5 variable domain. In certain embodiments, the VL regions of the first and second VH / VL variable domains are identical.

[0168] In certain embodiments, one or both light chain variable regions of the VH variable domain / VL variable domain of an EGFR / LGR5 antibody include a common light chain variable region. In certain embodiments, the common light chain variable region of one or both VH variable domains / VL variable domains includes the germline IgVκ1-39 variable region V segment. In certain embodiments, one or both light chain variable regions of the VH variable domain / VL variable domains include the kappa light chain V segment IgVκ1-39*01. IgVκ1-39 is an abbreviation for the immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39, IGKV139, or IGKV1-39. The external ID for this gene is HGNC:5740, Entrez Gene:28930, Ensembl:ENSG00000242371. A suitable amino acid sequence for the V region is provided in Figure 4. The V region can be combined with one of the five J regions. In a particular embodiment, the J regions are jk1 and jk5, and the concatenated sequence is denoted as IGKV1-39 / jk1 and IGKV1-39 / jk5, with alternative names being IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (named by the IMGT database World Wide Web at imgt.org). In a particular embodiment, one or both light chain variable regions of the VH variable domain / VL variable domain contain the kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ1*05 (shown in Figure 4).

[0169] In certain embodiments, one or both light chain variable regions of the VH variable domain / VL variable domain of an EGFR / LGR5 bispecific antibody include LCDR1 (shown in Figure 4) containing the amino acid sequence QSISSY, LCDR2 (shown in Figure 4) containing the amino acid sequence AAS, and LCDR3 (shown in Figure 4) containing the amino acid sequence QQSYSTP (i.e., the CDR of IGKV1-39 by IMGT). In certain embodiments, one or both light chain variable regions of the VH variable domain / VL variable domain of an EGFR / LGR5 antibody include LCDR1 (shown in Figure 4) containing the amino acid sequence QSISSY, LCDR2 (shown in Figure 4) containing the amino acid sequence AASSLQS, and LCDR3 (shown in Figure 4) containing the amino acid sequence QQSYSTP.

[0170] In certain embodiments, the light chain variable regions of one or both of the VH variable domains / VL variable domains of an EGFR / LGR5 bispecific antibody include LCDR1 (shown in Figure 4) containing the amino acid sequence QSISSY, LCDR2 (shown in Figure 4) containing the amino acid sequence AAS, and LCDR3 (shown in Figure 4) containing the amino acid sequence QQSYSTPPT (i.e., the CDRs of IGKV1-39 according to IMGT). In certain embodiments, the light chain variable regions of one or both of the VH variable domains / VL variable domains of an EGFR / LGR5 antibody include LCDR1 (shown in Figure 4) containing the amino acid sequence QSISSY, LCDR2 (shown in Figure 4) containing the amino acid sequence AASSLQS, and LCDR3 (shown in Figure 4) containing the amino acid sequence QQSYSTPPT. The CDR sequences conform to the IMGT numbering system.

[0171] In certain embodiments, one or both of the VH variable domain / VL variable domain of the EGFR / LGR5 antibody include a light chain variable region containing an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence shown in Figure 4.

[0172] For example, one or both of the variable light chains of the VH variable domain / VL variable domain of an EGFR / LGR5 antibody may have 0 to 10, and in certain embodiments 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof with respect to the sequence shown in Figure 4. In certain embodiments, one or both of the light chain variable regions of the VH variable domain / VL variable domain of an EGFR / LGR5 antibody may include 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, in certain embodiments 0 to 3, in certain embodiments 0 to 2, in certain embodiments 0 to 1, and in certain embodiments 0 amino acid insertions, deletions, substitutions, additions, or combinations thereof with respect to the indicated amino acid sequence.

[0173] Furthermore, one or both of the light chain variable regions of the VH variable domain and / or VL variable domain of an EGFR / LGR5 antibody may contain the amino acid sequence shown in Figure 4. In certain embodiments, both the VH variable domain and / or VL variable domain of an EGFR / LGR5 antibody contain the same VL region. In certain embodiments, both VL regions of the VH variable domain and / or VL variable domain of an EGFR / LGR5 bispecific antibody contain the amino acid sequence shown in Figure 4. In certain embodiments, both VL regions of the VH variable domain and / or VL variable domain of an EGFR / LGR5 bispecific antibody contain the amino acid sequence shown in Figure 4.

[0174] In certain embodiments, the EGFR / LGR5 antibodies described herein are bispecific antibodies having two variable domains: one that binds to EGFR and another that binds to LGR5 as described herein. EGFR / LGR5 bispecific antibodies for use in the methods disclosed herein may be provided in several formats. Many different forms of bispecific antibodies are known in the art and have been reviewed by Kontermann (Drug Discov Today, 2015 Jul;20(7):838-47, MAbs, 2012 Mar-Apr;4(2):182-97) and Spiess et al. (Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol.Immunol. (2015) http: / / dx.doi.org / 10.1016 / j.molimm.2015.01.003), which are incorporated herein by reference, respectively. For example, a bispecific antibody format that is not a classical antibody having two VH / VL combinations has at least a variable domain including a heavy chain variable region and a light chain variable region. This variable domain may be linked to a single-chain Fv fragment, a monolayer, a VH, and a Fab fragment that provides a second binding activity.

[0175] In certain embodiments, the EGFR / LGR5 bispecific antibody used in the methods provided herein is generally of a human IgG subclass (e.g., IgG1, IgG2, IgG3, IgG4). In certain embodiments, the antibody is of a human IgG1 subclass. Full-length IgG antibodies are preferred due to their preferred half-life and lower immunogenicity. Therefore, in certain embodiments, the EGFR / LGR5 bispecific antibody is a full-length IgG molecule. In certain embodiments, the EGFR / LGR5 bispecific antibody is a full-length IgG1 molecule.

[0176] Therefore, in certain embodiments, the EGFR / LGR5 bispecific antibody comprises a crystallizable fragment (Fc). In certain embodiments, the Fc of the EGFR / LGR5 bispecific antibody consists of a human constant region. The constant region or Fc of the EGFR / LGR5 bispecific antibody may contain one or more, ten or fewer, or five or fewer amino acid differences from the constant region of a naturally occurring human antibody. For example, each Fab arm of a bispecific antibody may further contain an Fc region containing modifications that promote the formation of the bispecific antibody and enhance stability and / or other characteristics described herein.

[0177] Antibodies are typically produced by cells that express the nucleic acid encoding the antibody. Therefore, in certain embodiments, the bispecific EGFR / LGR5 antibodies disclosed herein are produced by providing cells containing one or more nucleic acids encoding the heavy and light chain variable regions and constant region of the bispecific EGFR / LGR5 antibody. In certain embodiments, the cells are mammalian cells or animal cells such as primate cells, and in certain embodiments, human cells. Preferred cells may contain, and preferably produce, the EGFR / LGR5 bispecific antibody.

[0178] Cells suitable for antibody production are known in the art and include hybridoma cells, Chinese hamster ovary (CHO) cells, NS0 cells, or PER-C6 cells. Various institutions and companies are developing cell lines for large-scale production of antibodies for clinical use, for example. Non-limiting examples of such cell lines are CHO cells, NS0 cells, or PER.C6 cells. In particular, these cells are human cells. Preferably, the cells are transformed with the adenovirus E1 region or its functional equivalent. A preferred example of such cell line is the PER.C6 cell line or its equivalent. In particular, these cells are CHO cells or their variants. Preferably, the variants use a glutamine synthase (GS) vector system for antibody expression. In certain embodiments, the cells are CHO cells.

[0179] In certain embodiments, cells express different light and heavy chains constituting an EGFR / LGR5 bispecific antibody. In certain embodiments, cells express two different heavy chains and at least one light chain. In certain embodiments, cells express a “common light chain” as described herein to reduce the number of different antibody species (combinations of different heavy and light chains). For example, each VH region, in conjunction with a rearranged human IGKV1 39 / IGKJ1 (huVκ1 39) light chain, can be cloned into an expression vector using methods known in the art for the production of bispecific IgG (WO2013 / 157954, incorporated herein by reference), and it has been previously shown that pairing with two or more heavy chains can result in antibodies with diverse specificities, thereby promoting the production of bispecific molecules (De Kruif et al. J.Mol.Biol.2009(387)548 58;WO2009 / 157771).

[0180] Antibody-producing cells expressing a common light chain and equal amounts of two heavy chains typically produce 50% bispecific antibodies and 25% monospecific antibodies (i.e., those with the same heavy chain combination). Several methods have been described to favor the production of bispecific antibodies over the production of each monospecific antibody. This is typically achieved by modifying the constant regions of the heavy chains so that they favor heterodimerization (i.e., dimerization with heavy chains of other heavy / light chain combinations) over homodimerization. In certain embodiments, the bispecific antibodies of the present invention comprise two different immunoglobulin heavy chains having compatible heterodimerization domains. Various compatible heterodimerization domains have been described in the art. In certain embodiments, the compatible heterodimerization domain is a compatible immunoglobulin heavy chain CH3 heterodimerization domain. Various methods have been described in the art that can achieve heterodimerization of such heavy chains.

[0181] A preferred method for producing EGFR / LGR5 bispecific antibodies is disclosed in US9,248,181 and US9,358,286. Specifically, preferred mutations that essentially produce only bispecific full-length IgG molecules are amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain ("KK variant" heavy chain) and amino acid substitutions L351D and L368E in the second domain ("DE variant" heavy chain), or vice versa. As described above, the DE variant and KK variant preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of the DE variant heavy chain (DEDE homodimer) or the KK variant heavy chain (KKKK homodimer) is hardly observed due to strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0182] Therefore, in certain embodiments, a heavy / light chain combination containing a variable domain that binds to EGFR includes a DE variant of the heavy chain. In certain embodiments, a heavy / light chain combination containing a variable domain that binds to LGR5 includes a KK variant of the heavy chain.

[0183] Candidate EGFR / LGR5 IgG bispecific antibodies can be tested for binding using any suitable assay. For example, binding to membrane-expressed EGFR or LGR5 on CHO cells can be evaluated by flow cytometry (by the FACS procedure previously described in WO2017 / 069628). In certain embodiments, binding of candidate EGFR / LGR5 bispecific antibodies to LGR5 on CHO cells is demonstrated by flow cytometry performed according to standard procedures known in the art. Binding to CHO cells is compared to CHO cells that have not been transfected with EGFR and / or LGR5 expression cassettes. Binding of candidate bispecific IgG1 to EGFR is determined using CHO cells transfected with an EGFR expression construct, and LGR5 monospecific antibodies and EGFR monospecific antibodies, as well as unrelated IgG1 isotype control mAbs, are included in the assay as controls (e.g., antibodies that bind to LGR5 and another antigen such as tetanus toxin (TT)).

[0184] The affinity of candidate EGFR / LGR5 bispecific antibodies for a target to LGR5 and EGFR Fab can be measured by surface plasmon resonance (SPR) technology using BIAcore T100. Briefly, an anti-human IgG mouse monoclonal antibody (Becton and Dickinson, catalog no. 555784) is conjugated to the surface of a CM5 sensor chip using free amine chemistry (NHS / EDC). The bsAb is then captured on the sensor surface. Subsequently, recombinant purified antigens of human EGFR (Sino Biological Inc, catalog no. 11896-H07H) and human LGR5 protein are run across the sensor surface in a range of concentrations, and the on and off speeds are measured. After each cycle, the sensor surface is regenerated by a pulse of HCl, and the bsAb is captured again. From the obtained sensorgrams, the on-rate and off-rate and affinity values ​​for binding to human LGR5 and EGFR are determined using the BIAevaluation software previously described for CD3 in US2016 / 0368988.

[0185] The antibodies disclosed herein are typically bispecific full-length antibodies of a human IgG subclass, in certain embodiments. In certain embodiments, the antibody is of the human IgG1 subclass. Such antibodies have excellent ADCC properties, which can be enhanced by techniques known in the art as desired, have a favorable half-life upon in vivo administration to humans, and for which there are CH3 engineering techniques that can provide modified heavy chains that preferentially form heterodimers over homodimers upon co-expression in clonal cells.

[0186] The ADCC activity of an antibody can be improved by modifying the constant region of the antibody, if the antibody itself has low ADCC activity. Another way to improve the ADCC activity of an antibody is by enzymatically interfering with the glycosylation pathway that results in reduced fucose. Several in vitro methods exist to determine the effectiveness of an antibody or effector cell in inducing ADCC. These include chromium-51 [Cr51] release assays, europium [Eu] release assays, and sulfur-35 [S35] release assays. Typically, labeled target cell lines expressing an antigen exposed to a particular surface are incubated with an antibody specific to that antigen. After washing, effector cells expressing the Fc receptor CD16 are co-incubated with antibody-labeled target cells. Subsequently, target cell lysis is measured by the release of intracellular labels using a scintillation counter or spectrophotometric method.

[0187] The bispecific antibodies disclosed herein may be ADCC-enhancing. In certain embodiments, such bispecific antibodies are afucosylated. In certain embodiments, the bispecific antibody involves a reduction in the amount of fucosylation of the N-linked carbohydrate structure in the Fc region compared to the same antibody produced in normal CHO cells. Low fucose levels are associated with increased CD16(FcγRIIIa) binding on NK effector cells, resulting in increased ADCC activity. In certain embodiments, in addition to their direct antitumor activity, the bispecific antibodies disclosed herein can eliminate tumor cells following opsonization and subsequent natural killer (NK) cell-mediated ADCC activity and complement-dependent cytotoxicity (CDC) activity.

[0188] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 may further comprise one or more additional variable domains capable of binding to one or more further targets. In certain embodiments, the further targets are proteins such as membrane proteins that include an extracellular portion. As used herein, membrane proteins are cell membrane proteins such as proteins located in the outer membrane of a cell, which is the membrane that separates the cell from the outside world. Membrane proteins have an extracellular portion. Membrane proteins are at least on the cell if they contain a transmembrane region located within the cell membrane of the cell.

[0189] Antibodies having two or more variable domains are known in the art. For example, it is possible to conjugate additional variable domains. In certain embodiments, antibodies having three or more variable domains are polyvalent multimer antibodies described in PCT / NL2019 / 050199, which are incorporated herein by reference.

[0190] In a particular embodiment, the antibody is a bispecific antibody comprising two variable domains, one of which binds to the extracellular portion of EGFR and the other variable domain binds to the extracellular portion of LGR5. In a particular embodiment, the variable domain is the variable domain described herein.

[0191] The functional portion of the antibody described herein includes at least a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 as described herein. Therefore, it includes the antigen-binding portion of the antibody described herein and typically contains the variable domain of the antibody. The variable domain of the functional portion may be a single-chain Fv fragment or a so-called single-domain antibody fragment. In certain embodiments, the antibody portion or derivative has at least two variable domains of the antibody or its equivalent. Non-limiting examples of such variable domains or their equivalents are F(ab) fragments and single-chain Fv fragments. The functional portion of a bispecific antibody includes the antigen-binding portion of the bispecific antibody, or derivatives and / or analogs of the binding portion. As described herein above, the binding portion of the antibody is contained within the variable domain.

[0192] Also provided herein are antibodies, or functional portions thereof, derivatives, and / or analogues (i.e., therapeutic agents) and pharmaceutically acceptable carriers. Such pharmaceutical compositions are useful in the treatment of cancer, particularly for the treatment of gastric cancer, esophageal cancer, or gastroesophageal junction cancer. As used herein, the term “pharmaceutically acceptable” means approved by a government regulatory body or listed in the United States Pharmacopeia or another generally accepted pharmacopoeia for use in animals, particularly humans, and includes any and all physiologically compatible solvents, salts, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, etc. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle on which the compound is administered. Such pharmaceutically acceptable carriers may be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol ricinoleate, polyethylene glycol, etc. Water or physiological saline, dextrose aqueous solution, and glycerol aqueous solution may be used as carriers, particularly for injectable solutions. Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravesical, intratumoral, intravenous, intraperitoneal, intramuscular, subarachnoid, and subcutaneous. Depending on the route of administration (e.g., intravenous, subcutaneous, intra-articular, etc.), the active compound may be coated with a material to protect it from the action of acids and other natural conditions that can inactivate the compound.

[0193] Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, for example, in a liquid carrier, or for reconstitution into a solution or suspension for intravenous administration. Compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Solid preparations intended to be converted into liquid preparations for either oral or parenteral administration immediately before use are also included. Such liquid forms include solutions, suspensions, and emulsions.

[0194] The disclosed therapeutic agents may be administered according to a preferred dosage and preferred route (e.g., intravenous, intraperitoneal, intramuscular, subarachnoid, or subcutaneous). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. In certain embodiments, a subject is administered a single dose of the antibody disclosed herein, or its functional portion, derivative, and / or analog. In certain embodiments, the therapeutic agent is administered repeatedly over the course of treatment. For example, in certain embodiments, multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the therapeutic agent are administered to a subject in need of treatment. In some embodiments, the therapeutic agent may be administered weekly, bi-weekly, or monthly.

[0195] Clinicians may use a preferred dose deemed appropriate based on the patient's condition. The dose may depend on several factors, including the stage of the disease. Determining a specific dose to be administered based on the presence of one or more such factors is within the scope of the skills of those skilled in the art. Generally, treatment is initiated with a dose lower than the optimal dose of the compound. The dosage is then increased in small increments until the optimal effect is achieved under these circumstances. For convenience, the total daily dose may be divided and administered throughout the day, if necessary. Intermittent therapy (e.g., one week out of three weeks or three weeks out of four weeks) may also be used.

[0196] In certain embodiments, the therapeutic agent is administered in doses of 0.1, 0.3, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg body weight. Alternatively, the therapeutic agent is administered in doses of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg body weight.

[0197] In certain embodiments, the therapeutic agent is provided to a subject using a uniform dose of 1500 mg. A uniform dose offers several advantages over surface or body weight administration, as it reduces preparation time and potential dose calculation errors. In certain embodiments, the therapeutic agent is provided in a dose of at least 500 mg. In certain embodiments, the dose is 1100–2000 mg. In certain embodiments, the dose is 1100–1800 mg. As will be understood by those skilled in the art, the dose may be administered over time. For example, the dose may be administered IV, for example, by infusion over 1–6 hours, preferably 2–4 hours. In certain embodiments, the therapeutic agent is administered once every two weeks. In particular, the uniform dose disclosed herein is suitable for use in adult subjects and / or subjects weighing at least 35 kg. In certain embodiments, the subject has gastric cancer, esophageal cancer, or gastroesophageal junction cancer.

[0198] In certain embodiments, premedication regimens may be used. Such regimens may be useful in reducing the likelihood or severity of infusion-related reactions. Generally, steroids such as dexamethasone and / or antihistamines, such as dexchlorpheniramine, diphenhydramine, or chlorpheniramine, are administered (e.g., orally or intravenously) before antibody treatment.

[0199] The treatment methods described herein are typically continued as long as the clinician supervising the patient's care considers the treatment to be effective, i.e., the patient is responding to the treatment. Non-limiting parameters indicating the effectiveness of a treatment method may include one or more of the following: reduction of tumor cells, inhibition of tumor cell proliferation, elimination of tumor cells, progression-free survival, and an appropriate response to a suitable tumor marker (if applicable).

[0200] Those skilled in the art will be able to determine the appropriate frequency of administration of therapeutic agents. For example, a clinician may decide to administer the therapeutic agent relatively infrequently (e.g., once every two weeks) and gradually shorten the interval between doses that is tolerable for the patient. Examples of exemplary timeframes relating to the course of therapy by the method described in the claims include approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 30 months, 3 years, 4 years, 5 years, and permanent (e.g., continuing maintenance therapy). The aforementioned duration may be associated with one or more rounds / cycles of treatment.

[0201] The effectiveness of the treatment methods provided herein may be evaluated using any preferred means. In certain embodiments, the clinical effectiveness of the treatment is analyzed using a reduction in the number of cancer cells as an objective response criterion. Patients treated according to the methods disclosed herein, e.g., humans, preferably experience improvement in at least one sign of cancer. In certain embodiments, one or more of the following may occur: a reduction in the number of cancer cells, prevention or delay of cancer recurrence, and some degree of relief of one or more of the symptoms associated with cancer. In addition, an in vitro assay to determine T cell-mediated target cell lysis. In certain embodiments, tumor evaluation is based on CT scans and / or MRI scans; see, for example, the RECIST 1.1 guidelines (response evaluation criteria in solid tumors) (Eisenhauer et al., 2009 Eur J Cancer 45:228-247). Such evaluations are generally performed every 4 to 8 weeks after treatment.

[0202] In certain embodiments, tumor cells are no longer detectable after the treatment described herein. In certain embodiments, the subject is in partial or complete remission. In certain embodiments, the subject has an increase in overall survival, median survival, and / or progression-free survival.

[0203] Therapeutic agents (i.e., antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, or functional portions thereof, derivatives, and / or analogs) may also be used in conjunction with other well-known therapies (e.g., chemotherapy or radiotherapy) selected for their specific utility against the cancer being treated.

[0204] Methods for the safe and effective administration of chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in standard literature. For example, the administration of many chemotherapeutic agents is described in the Physicians' Desk Reference (PDR), e.g., the 1996 edition (Medical Economics Company, Montvale, NJ07645-1742, USA), and its disclosure is incorporated herein by reference.

[0205] It will be apparent to those skilled in the art that the administration of chemotherapeutic agents and / or radiotherapy may vary depending on the disease being treated and the known effects of chemotherapeutic agents and / or radiotherapy on that disease. Furthermore, according to the knowledge of those skilled in the art, treatment protocols (e.g., dosage and administration timing) may be modified in consideration of the observed effects of the administered therapeutic agent on the patient and the observed response of the disease to the administered therapeutic agent.

[0206] The compounds and compositions disclosed herein are useful as therapies and in therapeutic procedures, and therefore useful as pharmaceuticals, and can be used in methods for preparing pharmaceuticals.

[0207] All documents and references described herein, including Genbank entries, patents and published patent applications, and websites, are expressly incorporated herein by reference to the same extent as if they were described herein in whole or in part.

[0208] For the purpose of clear and concise description, features are described herein as part of the same or distinct parts of this disclosure, but it will be understood that the scope of the invention may include preferred embodiments having all or some combinations of the described features.

[0209] Herein, the present invention will be described with reference to the following embodiments, which are illustrative and not intended to limit the invention. Although the present invention has been described in detail with reference to its specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope thereof.

[0210] List of clauses 1. An antibody, or a functional portion, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of cancer in a subject, wherein the cancer in the subject has progressed after prior treatment with an immune checkpoint inhibitor, and the cancer expresses EGFR. 2. Use in the manufacture of a drug for treating cancer in a subject, an antibody containing a first variable domain that binds to the extracellular portion of EGFR, or a functional portion thereof, derivative, and / or analog, in which the cancer in the subject has progressed after prior treatment with an immune checkpoint inhibitor, and the cancer expresses EGFR. 3. A method for treating a subject having cancer expressing EGFR, wherein the subject has progressed after prior treatment with an immune checkpoint inhibitor, and the method comprises providing the subject with an effective amount of an antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional portion thereof, derivative, and / or analog. 4. The antibody, functional portion, derivative, and / or analog thereof, or use or method thereof, wherein the cancer is a head and neck cancer, preferably a squamous cell carcinoma of the head and neck (SCCHN), and the cancer expresses EGFR, preferably characterized by an IHC score of 2+ or 3+. 5. An antibody, or functional portion thereof, derivative, and / or analog thereof, or use or method thereof, for which the cancer is gastric cancer, esophageal cancer, or gastroesophageal junction cancer having EGFR expression characterized by an IHC score of 3+. 6. An antibody, functional portion thereof, derivative, and / or analog thereof, or use or method thereof, for any cancer having EGFR expression characterized by an H score for more than 200 EGFRs, such as gastric cancer, esophageal cancer, or gastroesophageal junction cancer. 7. An antibody, or functional portion thereof, derivative, and / or analogue thereof, or use or method thereof, characterized by cancer comprising EGFR gene amplification. 8. The antibody, or functional portion thereof, derivatives and / or analogues, or use or method thereof, characterized in that the EGFR gene amplification is by an EGFR copy number of 8 or more determined by next-generation sequencing of a solid tissue sample, an EGFR score of at least 2.14 or at least 2.5 determined by next-generation sequencing of circulating tumor DNA (ctDNA), or an EGFR / CEP7 ratio of 2 or more based on FISH. 9. An antibody, or a functional moiety, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a subject, wherein the cancer expresses EGFR characterized by an IHC score of 3+. 10. An antibody, functional moiety, derivative, and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR, for use in the treatment of gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a subject, wherein the cancer expresses EGFR characterized by an H score of more than 200 EGFRs. 11. An antibody, or a functional portion, derivative, and / or analog thereof, for use in the treatment of cancer in the subject, comprising a first variable domain that binds to the extracellular portion of EGFR, wherein the first variable domain is - At least one CDR3 sequence of the VH of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or a CDR3 sequence that differs from the VH of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3 by at least three, preferably at least two, preferably one or fewer amino acids. - At least the CDR1, CDR2, and CDR3 sequences of the VH of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or the CDR1, CDR2, and CDR3 sequences of the VH of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3 having at least three, preferably at least two, preferably at least one amino acid substitution, or The sequences of the VH chains of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or An antibody, or a functional portion, derivative, and / or analog thereof, comprising the amino acid sequence of the VH chain of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, having at least 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or combinations thereof, wherein the cancer is a head and neck cancer, preferably a squamous cell carcinoma of the head and neck (SCCHN), and the cancer preferably expresses EGFR characterized by an IHC score of 2+ or 3+, or the cancer is a gastric cancer, esophageal cancer, or gastroesophageal junction cancer having EGFR expression characterized by an IHC score of 3+, or preferably an H score for EGFR greater than 200. 12. The antibody, functional portion thereof, derivative, and / or analogue thereof, or use, or method thereof, of any one of the preceding clauses, provided that the subject has not received prior treatment with an anti-EGFR agent. 13. An antibody, or a functional portion thereof, derivative, and / or analog thereof, or a method of use, that the subject has not received prior treatment with an antibody targeting EGFR, as described in any one of clauses 1 to 11. 14. The subject is not prior to treatment with cetuximab, and the antibody, functional portion thereof, derivative, and / or analogue thereof, or use or method described in any one of the clauses 1 to 11. 15. An antibody, functional portion thereof, derivative, and / or analogue thereof, relating to any one of the clauses 9 to 14, in which the cancer has progressed after prior treatment with an immune checkpoint inhibitor. 16. The antibody, functional portion thereof, derivative, and / or analogue thereof, or use, or method thereof, for which the cancer expresses EGFR characterized by an H score greater than 200 and less than or equal to 300. 17. An antibody, or a functional portion, derivative, and / or analog thereof, whose H score for the EGFR is determined using IHC, as specified in any one of clauses 16. 18. An antibody, or a functional portion thereof, derivative, and / or analog thereof, or a use or method thereof, relating to any one of the preceding clauses, wherein the subject is a mammal, preferably a human. 19. The antibody, functional part, derivative, and / or analog thereof, or use or method of any of the preceding clauses, which includes providing to a subject an effective amount of the antibody, or a functional part thereof, derivative, and / or analog thereof. 20. The antibody, functional part, derivative, and / or analog thereof, or use or method of any of the preceding clauses, wherein the treatment includes providing a subject with a uniform dose of 1500 mg of the antibody, or a functional part, derivative, and / or analog thereof. 29. An antibody, or a functional portion thereof, derivative, and / or analog thereof, provided intravenously to a subject, as described in any of the preceding clauses, or a use or method thereof. 22. An antibody, or a functional portion, derivative, and / or analog thereof, or a use or method thereof, according to any one of the preceding clauses, wherein the antibody, or a functional portion, derivative, and / or analog thereof, is provided weekly, bi-weekly, or monthly, preferably bi-weekly, and more preferably, the subject is provided with at least three or more bi-weekly doses of the antibody, or a functional portion, derivative, and / or analog thereof. 22. An antibody, or a functional portion, derivative, and / or analog thereof, or a method of use, or an antibody that is ADCC-enhanced, as described in any of the preceding clauses. 24. An antibody, or a functional portion thereof, derivative, and / or analog thereof, or a method of use thereof, which is afucosylated, as described in any of the preceding clauses. 25. An antibody, or functional portion thereof, derivative, and / or analog thereof, or use or method thereof, relating to any one of the preceding clauses, wherein the cancer is an adenocarcinoma or squamous cell carcinoma, in particular gastric adenocarcinoma, esophageal adenocarcinoma, or gastroesophageal junction adenocarcinoma, in particular head and neck squamous cell carcinoma (HNSCC). 26. The antibody, functional portion thereof, derivative, and / or analogue thereof, or use or method thereof, wherein the cancer and / or subject is SMAD4 wild-type. 27. An antibody, functional portion thereof, derivative, and / or analog thereof, or use or method thereof, relating to the cancer or subject having a mutation in TP53, preferably an activating TP53 mutation, as described in any one of the preceding clauses. 28. An antibody, or a functional portion, derivative, and / or analog thereof, or use or method thereof, which is Her2-negative for the cancer or subject. 29. An antibody according to any one of the preceding clauses, or a functional portion thereof, derivative, and / or analogue thereof, or a use or method thereof, wherein the antibody is a multispecific antibody, preferably a bispecific antibody. 30. An antibody, or a functional portion thereof, derivative, and / or analog thereof, or a method of use thereof, comprising a second variable domain that does not bind to EGFR, as described in any of the preceding clauses. 31. An antibody, or a functional portion thereof, derivative, and / or analog thereof, or a method of use thereof, comprising a second variable domain that binds to LGR5, as described in any of the preceding clauses. 32. An antibody, functional portion thereof, derivative, and / or analogue thereof, or use or method thereof, which is a monovalent antibody that does not contain a second variable domain, or an antibody that contains the first EGFR-binding variable domain as the sole variable domain, as described in any one of Clauses 1 to 28. 33. The immune checkpoint inhibitor is an antibody, functional portion thereof, derivative, and / or analog thereof, or a method of use thereof, which includes a PD-L1 or PD-1 inhibitor, as described in any one of the preceding clauses. 34. An antibody, or a functional portion thereof, derivative, and / or analogue thereof, or a method of use thereof, according to any one of the preceding clauses, wherein the treatment includes, or follows, a step of diagnosing the subject for EGFR status, SMAD4 status, and / or Her2 status, and the diagnosis for Her2 status is preferably by ISH or IHC. 35. An antibody, or functional portion thereof, derivative, and / or analogue thereof, or use or method thereof, wherein the first variable domain that binds to EGFR binds to an epitope located within amino acid residues 420-480 of the human EGFR sequence shown in Figure 2. 36. An antibody, or functional portion thereof, derivative, and / or analogue thereof, or use or method thereof, wherein the binding of the first variable domain to EGFR is reduced by one or more of the following amino acid residue substitutions in EGFR: I462A, G465A, K489A, I491A, N493A, and C499A, compared to an EGFR protein without such substitutions. 37. An antibody, or a functional portion thereof, derivative, and / or analogue thereof, or a method of use thereof, wherein the variable domain that binds to LGR5 binds to an epitope located within amino acid residues 21-118 of the human LGR5 sequence shown in Figure 1, according to any one of the clauses 29-34. 38. The first variable domain is, - At least one CDR3 sequence of the VH of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or a CDR3 sequence that differs from the VH of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3 by at least three, preferably at least two, preferably one or fewer amino acids. - At least the CDR1, CDR2, and CDR3 sequences of the VH of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or the CDR1, CDR2, and CDR3 sequences of the VH of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3 having at least three, preferably at least two, preferably at least one amino acid substitution, or The sequences of the VH chains of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or An antibody, functional portion thereof, derivative and / or analogue thereof, or use or method thereof, according to any one of the clauses 1 to 10 or 12 to 37, comprising a heavy chain variable region of the amino acid sequence of the VH chain of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, having at least 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or combinations thereof. [Examples]

[0211] As used herein, “MFXXXX,” where X is independently a digit from 0 to 9, refers to a Fab containing a variable domain, and VH has an amino acid sequence identified by the four digits shown in Figure 3. Unless otherwise indicated, the light chain variable region of the variable domain typically has the sequence shown in Figure 4b. The light chain in the examples has the sequence shown in Figure 4a. “MFXXXX VH” refers to the amino acid sequence of VH identified by the four digits. MF further includes a constant region of the light chain and a constant region of the heavy chain that typically interacts with the constant region of the light chain. The VH / variable region of the heavy chain is different, and typically the CH3 region is also different, with one heavy chain having a KK mutation in its CH3 domain and the other having a complementary DE mutation in its CH3 domain (see reference PCT / NL2013 / 050294 (published as WO2013 / 157954), and Figures 5d and 5e). The bispecific antibodies in the examples have an Fc tail containing a KK / DE CH3 heterodimerized domain, a CH2 domain, and a CH1 domain as shown in Figure 5, a common light chain as shown in Figure 4a, and a VH specified by the MF number. For example, the bispecific antibody represented by MF3755×MF5816 has the above general sequence, a variable domain having a VH with the sequence of MF3755, and a variable domain having a VH with the sequence of MF5816.

[0212] The amino acid and nucleic acid sequences of various heavy chain variable regions (VHs) are shown in Figure 3. Among the other LGR5 and EGFR combinations shown in Figure 3, the bispecific antibody EGFR / LGR5, MF3755×MF5816, which includes the heavy chain variable regions MF3755 and MF5816 and a common light chain, and which includes modifications for enhancement of ADCC from afucosylation, has been shown to be effective in WO2017 / 069628.

[0213] Production of bispecific antibodies Bispecific antibodies were generated by transient cotransfection of two plasmids encoding IgG having different VH domains, using proprietary CH3 engineering techniques to ensure efficient heterodimerization and bispecific antibody formation. The common light chain is also cotransfected within the same cell, either on the same plasmid or on a different plasmid. Our applications (e.g., WO2013 / 157954 and WO2013 / 157953, incorporated herein by reference) disclose methods and means for producing bispecific antibodies from a single cell, thereby providing means to favor the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can also be advantageously used in the present invention. Specifically, preferred mutations that essentially produce only bispecific full-length IgG molecules are amino acid substitutions at positions 351 and 366 in the first CH3 domain, e.g., L351K and T366K (numbered according to EU numbering) ("KK variant" heavy chain), and amino acid substitutions at positions 351 and 368 in the second CH3 domain, e.g., L351D and L368E ("DE variant" heavy chain), or vice versa (see Figures 5d and 5e). It was demonstrated in the aforementioned application that the negatively charged DE variant heavy chain and the positively charged KK variant heavy chain preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of the DE variant heavy chain (DE-DE homodimer) or the KK variant heavy chain (KK-KK homodimer) hardly occurs due to strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0214] The VH gene of the variable domain that binds to LGR5, as described above, was cloned into a vector encoding a positively charged CH3 domain. The VH gene of the variable domain that binds to EGFR, such as that disclosed in WO2015 / 130172 (incorporated herein by reference), was cloned into a vector encoding a negatively charged CH3 domain. 293F freestyle cells adapted for suspension growth were cultured in T125 flasks on a shaker plateau until a density of 3.0 × 10⁶ cells / ml. Cells were seeded in each well of a 24-deep-well plate at a density of 0.3–0.5 × 10⁶ live cells / ml. Cells were transiently transfected with a mixture of two plasmids encoding different antibodies and cloned into proprietary vector systems. Seven days after transfection, the cell supernatant was collected and filtered through a 0.22 μM filter (Sartorius). The sterile supernatant was stored at 4°C until the antibodies were purified.

[0215] IgG purification and quantification Purification was performed using Protein A affinity chromatography under sterile conditions on a filter plate. First, the pH of the culture medium was adjusted to pH 8.0, and then the IgG-containing supernatant was incubated with Protein A Sepharose CL-4B beads (50% v / v) (Pierce) on a shaking platform at 600 rpm for 2 hours at 25°C. Next, the beads were collected by filtration. The beads were washed twice with PBS pH 7.4. Then, the bound IgG was eluted with 0.1 M citrate buffer at pH 3.0, and the eluate was immediately neutralized with Tris pH 8.0. Buffer exchange was performed by centrifugation using a Multiscreen Ultracel 10 multiplate (Millipore). The sample was finally collected in PBS pH 7.4. The IgG concentration was measured using Octet. The protein sample was stored at 4°C.

[0216] To determine the amount of purified IgG, the antibody concentration was determined by Octet analysis using a Protein A biosensor (Forte-Bio, as recommended by the supplier), with total human IgG (Sigma Aldrich, catalog number I4506) as the standard.

[0217] The following bispecific antibodies are suitable for use in this embodiment and in the method of the present invention: MF3370×MF5790, MF3370×5803, MF3370×5805, MF3370×5808, MF3370×5809, MF3370×5814, MF3370×5816, MF3370×5817, MF3370×5818, MF3755×MF5790, MF3755×5803, MF3755×5805, MF3755×5808, MF3755×5809, MF3755×5814, MF3755×5816, MF3755×5817, MF3755×5818, M F4280×MF5790, MF4280×5803, MF4280×5805, MF4280×5808, MF4280×5809, MF4280×5814, MF4280×5816, MF4280×5817, MF4280×5818, MF4289×MF5790, MF4289×5803, MF4289×5805, MF4289×5808, MF4289×5809, MF4289×5814, MF4289×5816, MF4289×5817, and MF4289×5818. Each bispecific antibody contains two VHs, each designated by an MF number capable of binding to EGFR and LGR5, and further includes a KK / DE CH3 heterodimerized domain, indicated by SEQ ID NO: 136 (Figure 5d) and SEQ ID NO: 138 (Figure 5e), a CH2 domain, indicated by SEQ ID NO: 134 (Figure 5c), a CH1 domain, indicated by SEQ ID NO: 131 (Figure 5a), and an Fc tail having a common light chain, indicated by SEQ ID NO: 121 (Figure 4).

[0218] Example 1: Dose expansion and efficacy of anti-EGFR × anti-LGR5 antibody in patients with EAC, GAC, GEJAC, or head and neck cancer: Phase 1 dose escalation study in advanced solid tumors Test design A Phase 1 open-label, multicenter trial was conducted using the initial dose escalation portion to determine the recommended Phase 2 dose (RP2D) of an anti-EGFR × anti-LGR5 bispecific antibody for solid tumors in mCRC patients, with a uniform starting dose of 5 mg. Once the RP2D is established, the antibody will be further evaluated in an expanded portion of the trial, including patients diagnosed with head and neck cancers, including EAC, GAC, GEJAC, or head and neck squamous cell carcinoma (SCCHN). The antibody's safety, PK, immunogenicity, and preliminary antitumor activity will be characterized in all patients, and biomarker analyses, including EGFR and LGR5 status, will be performed.

[0219] Selection Criteria Patients must meet all of the following requirements to participate in the trial: 1. I signed informed consent before commencing any of the test procedures. 2. At the time of signing the informed consent form, the person must be 18 years of age or older. 3. Histologically or cytologically confirmed solid tumors with evidence of metastatic or locally advanced disease that are not suitable for standard curative therapy: Expanded cohort: Patients with advanced metastatic EAC, GAC, or GEJAC head and neck squamous cell carcinoma will be sought, regardless of whether they have previously been treated with at least two standard-of-care approved therapies (if applicable). 4. Fresh baseline tumor samples from metastatic or primary sites (FFPE, provided sufficient material has been frozen). 5. Suitable for biopsy. 6. Measurable diseases as defined in RECIST version 1.1 by radiological methods. Performance status of Eastern Cooperative Oncology Group (ECOG) 7.0 or 1. 8. According to researchers, the average life expectancy is ≥12 weeks. 9. Left ventricular ejection fraction (LVEF) ≥ 50% as measured by echocardiography (ECHO) or multi-gated acquisition scan (MUGA). 10. Appropriate organ function: ● Absolute Neutrophil Count (ANC) ≥ 1.5 × 10 9 / L ●Hemoglobin ≥ 9 g / dL ●Platelet≧100x109 / L ● Corrected total serum calcium within the normal range ● Serum magnesium within the normal range (or corrected with supplements) ● Alanine aminotransferase (ALT), aspartate aminotransferase (AST) ≤ 2.5 × upper limit of normal (ULN), total bilirubin ≤ 1.5 × ULN (except in cases of known Gilbert's syndrome, which is excluded if total bilirubin > 3.0 × ULN or direct bilirubin > 1.5 × ULN), in cases of liver disease, when total bilirubin ≤ 3.0 × ULN or direct bilirubin ≤ 1.5 × ULN is acceptable, except in cases of known Gilbert's syndrome, or when total bilirubin < 3 mg / dL is acceptable, except in cases of hepatocellular carcinoma [Child-Pugh class A], ALT / AST ≤ 5 × ULN and total bilirubin ≤ 2 × ULN are acceptable. ● For patients over 65 years of age, serum creatinine ≤ 1.5 × ULN or creatinine clearance ≥ 60 mL / min, calculated according to the Cockroft and Gault formula or the MDRD formula. ● Serum albumin > 3.3 g / dL

[0220] Exclusion criteria If any of the following criteria are met, the patient will be excluded from the trial: 1. Central nervous system metastases that are untreated, symptomatic, or require radiation, surgery, or continuous steroid therapy to control symptoms within 14 days of trial enrollment. 2. Known involvement of the leptomeninges. 3. Participation in any other clinical trial or treatment involving the investigational drug within four weeks prior to trial registration. 4. Any systemic anticancer therapy within 4 weeks of the first dose of the study treatment administration or within 5 half-lives, whichever is longer. A 6-week washout period is required for cytotoxic agents with significant delayed toxicity (e.g., mitomycin C, nitrosourea) or for anticancer immunotherapy. 5. Requirements for immunosuppressants (e.g., methotrexate, cyclophosphamide) 6. Major surgery or radiotherapy within three weeks of the first dose of the study treatment. Patients who have previously received radiotherapy to ≥25% of their bone marrow are ineligible, regardless of when they received it. 7. Clinically significant toxicity of sustained grade > 1 associated with existing anti-cancer therapy (excluding alopecia); stable sensory neuropathy ≤ grade 2 NCI-CTCAE v4.03 is acceptable. 8. A history of hypersensitivity reactions or toxicity of either human protein or excipients that justifies the permanent discontinuation of these drugs. 9. Uncontrolled hypertension with appropriate treatment or unstable angina (systolic > 150 mmHg and / or diastolic > 100 mmHg). 10. A history of congestive heart failure according to Class II-IV New York Heart Association (NYHA) criteria, or severe cardiac arrhythmias requiring treatment (excluding atrial fibrillation and paroxysmal supraventricular tachycardia). 11. A history of myocardial infarction within six months of registration for the trial. 12. A history of previous malignancies, excluding cervical intraepithelial neoplasia or non-melanoma skin cancer, or cancers that have been curatively treated and for which there has been no evidence of disease for at least three years and the risk of recurrence is considered low. 13. Current dyspnea or other condition requiring continuous oxygen therapy in the remainder of either origin. 14. Patients with a history of interstitial lung disease (e.g., pneumonia or pulmonary fibrosis) or evidence of interstitial lung disease (ILD) on baseline chest CT scan. 15. An uncontrolled active infection, a clinically significant pulmonary, metabolic or psychiatric disorder, or any other current serious illness or mental disorder. 16. Active HIV, HBV, or HCV infection requiring treatment. 17. Patients with current cirrhosis of Child-Pugh class B or C, known fibrous lamellar HCC, sarcomatoid HCC, or mixed cholangiocarcinoma and HCC 18. Pregnant or breastfeeding women, and patients of childbearing potential, must use highly effective contraception before trial enrollment, during trial participation, and for six months after the last dose of the antibody.

[0221] Dose-limiting toxicity (DLT) Any of the following clinical toxicity and / or laboratory abnormalities that occur during the first cycle (28 days) and are considered by the principal investigator to be related to antibody therapy will be considered a DLT: ●Hematological toxicity: - Grade 4 neutropenia lasting 7 days or more (absolute neutrophil count [ANC] < 0.5 × 10⁹ cells / L) - Grade 3-4 febrile neutropenia - Grade 4 thrombocytopenia - Grade 3 thrombocytopenia associated with a bleeding episode - Other Grade 4 hematological toxicity ●Grade 3-4 non-hematological AEs and clinical laboratory toxicity, excluding the following: - Grade 3-4 infusion-related reactions - Grade 3 skin toxicity that resolves to Grade 2 or lower within 2 weeks with optimal treatment. - Grade 3 diarrhea, nausea, and / or vomiting that resolve to Grade 1 or below or to baseline within 3 days with optimal treatment. - Grade 3 electrolyte abnormalities that resolve within 48 hours with optimal treatment - Grade 3-4 liver abnormalities within 48 hours ● Any liver function abnormality that satisfies the definition of Hy's Law. ● Any drug-related toxicity lasting 15 days or longer that prevents the next two doses.

[0222] Dosage expansion In the expanded section, bispecific antibodies are administered in a RP2D (Responsible Programme 2 Days) to patients with EAC, GAC, GEJAC, or head and neck cancer, particularly SCCHN. Once the RP2D is defined, additional patients are treated with this dose and schedule to further characterize the safety, tolerability, PK, and immunogenicity of the antibody, and to perform a preliminary evaluation of its antitumor activity and biomarker capabilities.

[0223] Antibody therapy in patients with EAC, GAC, GEJAC, or head and neck cancer, particularly SCCHN, will be explored in 10-20 patients for each indication, with the possibility of expansion to up to 40 patients, for example, conditional on preliminary signs of antitumor activity. The safety of RP2D will be continuously assessed by the Safety Monitoring Committee during the expanded portion of the trial. If the incidence of DLT exceeds a predetermined threshold of 33% for any cohort, enrollment for that cohort will be paused, and a full review of safety, PK, and biomarkers will be conducted by the SMC to determine whether it is safe to continue the occurrence in that cohort. At that point, the overall safety of the drug will also be questioned.

[0224] Investigational Therapies and Regimens The anti-EGFR × anti-LGR5 bispecific antibody is formulated as a clear liquid solution for IV infusion. IV infusions are administered every two weeks using a standard infusion procedure, with an initial dose of 5 mg (uniform dose) and a recommended Phase 2 dose of 1500 mg (uniform dose). Dose escalation was stopped after reaching RP2D. Infusions must be administered over a minimum of 4 hours during Cycle 1. Subsequent infusions after Cycle 1 may be shortened to 2 hours at the discretion of the investigator and in the absence of an IRR. A cycle is considered to be 4 weeks.

[0225] Premedication During Cycle 1, all infusions are administered over at least 4 hours using the following premedication regimen: 8 mg of dexamethasone PO is administered 24 hours prior to the start of the infusion, and 1 hour prior to the start of the infusion, each patient is given dexamethasone 20 mg IV, dexchlorpheniramine 5 mg IV or diphenhydramine 50 mg PO or chlorpheniramine 10 mg IV, ranitidine 50 mg IV or 150 mg PO, and paracetamol 1 g IV or 650 mg PO.

[0226] If the patient tolerates all cycle 1 infusions without IRR and the investigator deems it appropriate, the patient may continue to receive further antibody infusions without dexamethasone premedication, and the duration of infusions may be reduced to 2 hours. In such cases, the duration of infusions may be extended up to approximately 4 hours if deemed appropriate to avoid or reduce the incidence or severity of IRR. For the first antibody infusion (cycle 1 on day 1), each patient is observed for 6 hours from the start of the infusion and for 4 hours from the start of the second infusion. Thereafter, the patient is observed for all subsequent administration periods (minimum 2 hours).

[0227] Treatment period The investigational treatment will be administered until progressive disease (according to RECIST 1.1), unacceptable toxicity, withdrawal of consent, patient non-compliance, investigator's decision (e.g., clinical exacerbation), or antibody discontinuation for more than six consecutive weeks is confirmed. Patients will be followed for safety for at least 30 days after the last antibody infusion and until all related toxicities are resolved or stabilized, as well as for disease progression and survival for 12 months.

[0228] Pre-screening of gastric patients for EGFR amplification or high EGFR protein expression For patients with gastric / gastroesophageal junction adenocarcinoma, clinical trials require documentation of EGFR amplification or high EGFR expression by DNA pre-screening. To be eligible for pre-screening, patients must undergo a histological diagnosis of gastric cancer if no other viable targets exist. Pre-screening tests are performed on-site at Clinical Laboratory Improvement and Amendment (CLIA) accredited laboratories qualified to perform molecular screening for EGFR amplification and tumor gene mutations or EGFR IHC (e.g., the EGFR PharmDx kit or an equivalent validated IVD). EGFR amplification may be tested using FISH, ctDNA analysis, or tissue NGS. If suitable on-site testing options are unavailable, samples may be sent to an appropriately qualified and approved central laboratory.

[0229] For ctDNA analysis, blood is collected in two 10 mL tubes using the tubes provided in the blood collection kit available from Guardant. For Guardant tissue NGS analysis, FFPE slides or tissue blocks can be submitted using the collection kit available from Guardant. The thresholds for EGFR amplification or EGFR protein expression to be defined as eligible are a FISH score EGFR / CEP7 ratio ≥ 2.0, or an NGS EGFR copy ≥ 8, or ctDNA > 2.14, or an EGFR IHC H score ≥ 200 (Maron SB, et al., 2018. Targeted Therapies for Targeted Populations: Anti-EGFR Treatment for EGFR-Amplified Gastroesophageal Adenocarcinoma. Cancer Discov 8:696-713., Kato et al. 2019. Revisiting Epidermal Growth Factor Receptor (EGFR) Amplification as a Target for Anti-EGFR Therapy: Analysis of Cell-Free Circulating Tumor DNA in Patients With Advanced Malignancies. JCO Precis Oncol 3: PO.18.00180). Next, patients with EGFR amplification or an EGFR IHC H score of 200 or higher are eligible to sign the primary study ICF if they are willing to participate in the primary study. At least 10 patients with high EGFR IHC will be enrolled.

[0230] Patients whose EGFR amplification has been documented by ctDNA testing at a local accredited laboratory are eligible to sign the Primary Study ICF without requiring additional pre-screening.

[0231] Effectiveness evaluation Tumor evaluation is based on contrast-enhanced CT / MRI according to RECIST 1.1 (Eisenhauer et al., 2009 Eur J Cancer 45:228-247) every 8 weeks after the start of treatment. Objective response must be confirmed at least 4 weeks after the first observation. Bone scans are performed as clinically directed for patients with bone metastases at baseline or suspected study lesions. Circulating blood tumor markers, including cancer embryo antigen (CEA), are evaluated at screening and on day 1 of each cycle.

[0232] Example 2 A 67-year-old male patient with squamous cell carcinoma of the head and neck located in the larynx was enrolled in the clinical trial of Example 1. The patient had previously been treated with platinum-based chemotherapy (carboplatin), as well as paclitaxel and, importantly, durvalumab as an immune checkpoint inhibitor.

[0233] Observed responses included a -41% PRc after receiving a bispecific antibody characterized by having first and second variable domains represented by MF3755×MF5816. Patients were administered the antibody for more than 6 q2w cycles using a uniform dose of 1500 mg, after which the clinical response was evaluated.

[0234] The patient showed an EGFR IHC tumor membrane staining score of 2+.

[0235] Example 3 A 59-year-old female patient with squamous cell carcinoma of the head and neck located on the tongue was enrolled in the clinical trial of Example 1. The patient had previously been treated with platinum-based chemotherapy (carboplatin), as well as 5-FU and, importantly, pembrolizumab as an immune checkpoint inhibitor.

[0236] The observed responses included an 88% complete response (CR) to a second evaluation of the tumor state after receiving a bispecific antibody characterized by having first and second variable domains as shown by MF3755×MF5816, with an -88% partial response (PR). Patients were given the antibody at a uniform dose of 1500 mg at a cycle of 4q2w, and then the clinical response was evaluated.

[0237] Patients showed a 3+ EGFR IHC tumor membrane staining score.

[0238] Example 4 A 67-year-old male patient with squamous cell carcinoma of the head and neck in the hypopharynx was enrolled in the clinical trial of Example 1. The patient had previously been treated with platinum-based chemotherapy (carboplatin) and importantly, pembrolizumab as an immune checkpoint inhibitor.

[0239] The observed responses included a -40% PRc after receiving a bispecific antibody characterized by having first and second variable domains as shown by MF3755×MF5816. Patients were given the antibody at a uniform dose of 1500 mg at a cycle of 8q2w, and then the clinical response was evaluated.

[0240] Patients showed a 3+ EGFR IHC tumor membrane staining score.

[0241] EGFR H scoring was performed as described in Example 6.

[0242] Example 5 An 80-year-old male patient with gastroesophageal junction cancer was enrolled in the clinical trial of Example 1. The patient had previously been treated with chemotherapy based on oxaliplatin and irinotecan.

[0243] Observed responses included stable disease (SD) following treatment with a bispecific antibody characterized by having first and second variable domains represented by MF3755×MF5816. Patients were administered the antibody in question for 4 q2w cycles using a uniform dose of 1500 mg, after which clinical responses were evaluated.

[0244] The patient presented with an EGFR IHC score of 3+ and an EGFR H score of 300. Genetic profiling revealed that the patient was wild-type for SMAD4.

[0245] EGFR H scoring was performed as described in Example 8.

[0246] Example 6 A 62-year-old male patient with gastric cancer was enrolled in the clinical trial of Example 1. The patient had previously been treated with cisplatin / capecitabine chemotherapy.

[0247] Observed responses included confirmed partial responses (PRc) after receiving a bispecific antibody characterized by having first and second variable domains represented by MF3755×MF5816. Patients were administered the antibody in question for 7 q2w cycles using a uniform dose of 1500 mg, after which clinical responses were evaluated.

[0248] The patient presented with an EGFR IHC score of 3+ and an EGFR H score of 300. Genetic profiling revealed that the patient was wild-type for SMAD4.

[0249] EGFR H scoring was performed as described in Example 8.

[0250] Example 7 The safety profile at the resumed Phase II dose was based on 29 patients with solid tumors treated with RP2D. The most frequent adverse event was infusion-related reaction (IRR), with 72% being of any grade and 7% being grade 3 or higher. Time to onset: First infusion for all patients. IRRs were manageable with prophylactic / long-term infusions. Mild to moderate skin toxicity was observed (with 3% of severe events).

[0251] Infusion-related reactions (IRRs) is a compound term encompassing all adverse events (AEs) considered by the principal investigator as IRRs within 24 hours of infusion.

[0252] Example 8: EGFR scoring via IHC The EGFR pharmDx® assay is a qualitative immunohistochemistry (IHC) kit system for identifying epidermal growth factor receptor (EGFR) expression in normal and tumor tissues fixed according to specifications for histological evaluation. EGFR pharmDx specifically detects the EGFR(HER1) protein in EGFR-expressing cells.

[0253] The EGFR pharmDx™ assay detects the EGFR protein using the EGFR antibody, clone 2-18C9 (2-18C9). Clone 2-18C9 has been tested for reactivity to cell lines expressing EGFR, HER2, HER3, and HER4. In Western blots of SKBR3 and A431 cell lysates, 2-18C9 recognized a 170kD band consistent with the known molecular weight of EGFR. Clone 2-18C9 has also been found to recognize the receptor in the EGFRvIII (145kD) form in immunohistochemistry, flow cytometry, and Western blotting of cell lines transfected with EGFRvIII. In Western blotting experiments, 2-18C9 was unreactive to HER2-positive CAMA-1 cell lysates, HER3-transformed E. coli BL-21 protein extract, and CHO-HER4-transfected cell lysates. Furthermore, Chinese hamster ovary (CHO) transfectants expressing myc (vector tag) were grown alone or co-expressed with one of the HER family members in formalin-fixed and paraffin-embedded chamber slides and stained with anti-myc and 2-18C9. The myc antibody stained all five CHO transfectants, while 2-18C9 stained only CHO cells transfected with HER1.

[0254] EGFR scoring is performed using the Dako EGFR pharmDx® user protocol, in accordance with the manufacturer's instructions and recommendations. See the World Wide Web at agilent.com / cs / library / usermanuals / public / 08052_egfr_pharmdx_interpretation_manual.pdf.

[0255] Specimen preparation Biopsy specimens were processed to preserve tissue for IHC staining. Standard tissue processing methods should be used for all specimens. Specimens preserved in the following fixatives are suitable for testing with EGFR pharmDx: 10% (v / v) neutral buffered formalin, 10% (v / v) unbuffered formalin, 25% (v / v) unbuffered formalin, AFA (formaloacetate alcohol), Richard-Allen Scientific Pen-fix, and Bouin's fixative.

[0256] Paraffin-embedded sections Tissue processed according to regulations and embedded in paraffin is suitable for use. Specimens from biopsies should be blocked to a thickness of 3 or 4 mm and fixed for a suitable period of time with the fixative. The tissue was then dehydrated, cleared with a series of alcohols and xylene, and subsequently impregnated with molten paraffin. The paraffin temperature should not exceed 60°C. When stored in a cool place (15-25°C), properly fixed and embedded tissue blocks expressing EGFR protein can be retained indefinitely before sectioning and slide mounting.

[0257] The tissue specimen needs to be cut into sections of 3 - 5 μm. After sectioning, the tissue needs to be attached to slides and placed in a drying rack. The following slides are recommended for use: Fisher’s SuperFrost Plus, Dako’s Silanized (code S3003), charged or poly-L-lysine coated slides. Tap the slide rack on top of an absorbent towel to remove the moisture trapped under the paraffin and on the glass, and dry at room temperature for 1 hour. Then, the slide rack needs to be placed in an incubator at 56 - 60 °C for 1 hour. After removing from the incubator, the extra water remaining on the slides needs to be removed by tapping the slides on a towel and drying in the incubator for another 1 hour. After removing from the incubator, the slides need to be kept at room temperature until the paraffin hardens and cools. To maintain antigenicity, tissue sections attached to slides (Fisher’s SuperFrost Plus, poly-L-lysine, charged, or Dako’s Silanized slides (code S3003)) need to be stained within 2 months after sectioning when kept at room temperature (20 - 25 °C).

[0258] Slides necessary for the evaluation of EGFR and verification of the presence of tumors need to be prepared simultaneously.

[0259] At least 5 slides are recommended: 1 slide for tumor presence, 2 slides for EGFR protein evaluation (1 slide for the primary antibody and 1 slide for the negative control reagent), and 2 slides for backup.

[0260] Reagent Preparation Prepare the following reagents before staining:

[0261] Washing buffer: For the washing step, prepare a sufficient amount of washing buffer by diluting 10-fold, 1:10, using distilled water or deionized water (reagent-grade water). Discard the buffer if it appears cloudy.

[0262] Substrate-Dye Stock Solution (DAB+): This solution must be thoroughly mixed before use. Any precipitate that forms in the solution does not affect the staining quality. To prepare the DAB+ substrate-dye stock solution, add 11 drops of liquid DAB+ dye stock to one vial of DAB+ substrate buffer and mix. Discard any unused solution. Dilute according to the guidelines above. Adding excess liquid DAB+ dye stock to DAB+ substrate buffer will result in a deterioration of the positive signal.

[0263] Counterstaining. Prepare aqueous ammonia for counterstaining bluing, if necessary.

[0264] Ammonia solution (0.037 mol / L) is prepared by mixing 2.5 (±0.5) mL of 15 mol / L (concentrated) ammonium hydroxide with 1 liter of reagent-grade water. Unused 0.037 mol / L ammonia solution can be stored in a tightly capped bottle at room temperature (20-25°C) for up to 12 months.

[0265] Mounting medium. For aqueous mountings, we recommend mounting media such as Dako's Faramount Aqueous Mounting Medium, Ready-to-use (code S3025) or Dako's Glycergel Mounting Medium (code C0563). Liquefy the glycerol gel by warming it to approximately 40 (±5)°C before use. Non-aqueous permanent mounting media such as Dako's Ultramount (code S1964) are also suitable.

[0266] Dako Automatic Staining Device Staining Procedure Notes on the procedure All reagents must be equilibrated to room temperature (20-25°C) before immunohistochemical staining. Similarly, all incubations must be performed at room temperature.

[0267] Tissue sections should not be allowed to dry during the staining procedure. Dry tissue sections may show increased nonspecific staining.

[0268] Deparaffinization and rehydration. Before staining, tissue slides must be deparaffinized to remove the embedding medium and then rehydrated. Avoid incomplete removal of paraffin. Residual embedding medium will result in increased nonspecific staining.

[0269] Step 1. Place the slide in a xylene bath and incubate for 5 (±1) minutes. Change the bath and repeat once. Step 2. Gently tap off any excess liquid and place the slide in anhydrous ethanol for 3 (±1) minutes. Change the bath and repeat once. Step 3. Gently tap off any excess liquid and place the slide in 95% ethanol for 3 (±1) minutes. Change the bath and repeat once. Step 4. Gently tap off any excess liquid and place the slide in reagent-quality water for 5 (±1) minutes. Step 5. Gently tap off any excess liquid and place the slide in the wash buffer. Begin the staining procedure as outlined in the staining protocol.

[0270] Xylene and alcohol solutions need to be replaced after 40 slides. Toluene or xylene substitutes such as Histoclear can be used instead of xylene. EGFR pharmDx includes pretreatment with a proteolytic enzyme digestion step. Tissue sections may occasionally be over-digested, causing disruption of cell membranes and overall tissue structure. Perform the assay carefully, paying attention to the duration of the proteolytic step.

[0271] Post-fixation procedure 1. Deparaffinize the sections and immerse them in reagent-grade water. 2. Immerse the slide in 10% neutral buffered formalin for 10 minutes. 3. Rinse the slide twice with deionized water or distilled water. 4. Continue with the EGFR pharmDx staining procedure.

[0272] Automated staining protocol Step 1. Select the desired protocol and program for staining. Step 2. Use the automated program to set up the program and start the EGFR pharmDx program. Step 3. Place the reagent vials in the DAKO automated staining system reagent rack according to the computer-generated reagent map. Step 4. Load the slides into the DAKO automated staining system according to the computer-generated slide map. Step 5. Start execution. Step 6. Remove the slide from the DAKO automated staining device.

[0273] Proceed to counterstaining and mounting. After the DAB+ substrate-dye stock solution step, rinse the slide with reagent-quality water. (DAKO automated staining system hardware versions 02 and 03 rinse the slide with reagent-quality water after the substrate-dye stock solution step. DAKO automated staining system hardware version 01 rinses the slide with buffer. Therefore, slides stained with hardware version 01 must be rinsed with reagent-quality water after being removed from the automated staining system).

[0274] Interpretation of staining procedures Slide evaluation must be performed by a pathologist using a light microscope. All evaluations should be performed on the tumor area of ​​the specimen. For immunocytochemical staining and scoring evaluations, a 10x or 20x objective lens is appropriate.

[0275] Use intact cells to interpret staining results. Necrotic or degenerated cells often stain nonspecifically. Positive and negative cell lines are included in each EGFR pharmDx kit, and verify the staining run each time the assay is performed. Appropriate staining of control cell lines provides evidence that the EGFR pharmDx assay is functioning correctly. No membrane staining in the CAMA-1 control cell line (0) and moderate brown complete or incomplete membrane staining in the HT-29 control cell line (2+) indicate a valid staining run. If the staining intensity of the positive control cell line is too weak or too strong, a false negative or false positive result may be obtained, and the test should be repeated. Reference images are available in the EGFR pharmDx interpretation guide.

[0276] EGFR pharmDx primarily stains the cell membrane, exhibiting both complete and incomplete circumferential staining. The immunohistochemical pattern is often heterogeneous, showing varying staining intensities within a single neoplasm. Staining is also observed in the cytoplasm and extracellular space. Cytoplasmic staining is common, but repeated testing may be necessary if significant cytoplasmic staining makes it difficult to distinguish from membrane staining and interpret the results.

[0277] Tumors must be reported as EGFR-positive or EGFR-negative using membrane staining as an evaluable structure. Tumor cells are EGFR-positive if they have any membrane staining above the background, regardless of whether it is perfectly circumferential. Tumors in which no membrane staining above the background is present in any tumor cells are reported as EGFR-negative tumors.

[0278] Depending on the incubation length and the potency of the hematoxylin used, counterstaining will result in a pale to dark blue coloration of the cell nuclei. Excessive or incomplete counterstaining may impair the interpretation of the results.

[0279] Stain intensity is determined as follows: 3+ (strong staining): visible at low magnification with a 5x objective lens, visible at a high level if necessary; 2+ (moderate staining): visible at medium magnification with a 10x or 20x objective lens; 1+ (weak staining): reliably visible only at high magnification with a 40x objective lens; 0 (no staining): no visible staining at high magnification. [Table 1]

[0280] EGFR H scoring The evaluation of membrane staining using IHC classifies samples into four staining intensity categories (0 to 3+). It is noteworthy that only linear intercellular staining of tumor cells is considered positive, while complete and incomplete membrane staining are considered and recorded. Furthermore, for Histo-score calculation, all membrane staining is considered independent of completeness (complete and incomplete membrane staining).

[0281] The H score is assigned using the following formula: [1 × (1 + cells with staining %) + 2 × (2 + cells with staining %) + 3 × (3 + cells with staining %)], resulting in an H score for EGFR between 0 and 300.

Claims

1. A composition comprising an antibody or functional portion thereof for use in the treatment of cancer in a human subject, comprising: a first variable domain that binds to the extracellular portion of EGFR, comprising a heavy chain variable region comprising CDR1 comprising SEQ ID NO: 17, CDR2 comprising SEQ ID NO: 19, and CDR3 comprising SEQ ID NO: 21; and a second variable domain that binds to LGR5, comprising a heavy chain variable region comprising CDR1 comprising SEQ ID NO: 87, CDR2 comprising SEQ ID NO: 89, and CDR3 comprising SEQ ID NO: 91, wherein both variable domains comprise a light chain variable region comprising LCDR1 comprising the amino acid sequence QSISSY, LCDR2 comprising the amino acid sequence AAS, and LCDR3 comprising the amino acid sequence QQSYSTPPT, wherein the cancer in the subject has progressed after prior treatment with an immune checkpoint inhibitor, the immune checkpoint inhibitor comprises an antibody targeting PD-L1 or PD-1, and the cancer is head and neck cancer.

2. The composition according to claim 1, wherein the cancer is squamous cell carcinoma (SCCHN) of the head and neck.

3. The composition according to claim 1, wherein the cancer expresses an EGFR characterized by an IHC score of 2+ or 3+.

4. The first variable domain includes a heavy chain variable region containing the sequence of the VH chain of MF3755 shown in Sequence ID No. 4, The composition according to claim 1, wherein the antibody or functional portion thereof comprises a second variable domain including a heavy chain variable region containing the VH sequence of MF5816 shown in SEQ ID NO:

13.

5. The first variable domain is, - The amino acid sequence of the VH chain of MF3755 as shown in Sequence ID No. 4, or - Amino acid sequence of the VH chain of MF3755 having at least 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, with respect to the VH chain. Includes, The antibody or its functional portion - The amino acid sequence of the VH chain of MF5816 as shown in Sequence ID No. 13, or - Amino acid sequence of the VH chain of MF5816 having at least 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, with respect to the VH chain. Includes a second variable domain which includes The composition according to claim 1, wherein the insertion, deletion, substitution, or combination thereof is not present in the CDR1, CDR2, or CDR3 region of the VH chain.

6. The composition according to claim 1, wherein the subject has not received prior treatment with an anti-EGFR agent.

7. The composition according to claim 1, wherein the cancer expresses an EGFR characterized by an H score greater than 200 and less than or equal to 300.

8. The composition according to claim 7, wherein the H score for the EGFR is determined using IHC.

9. The composition according to claim 1, wherein the treatment comprises providing the subject with an effective amount of the antibody or a functional portion thereof.

10. The composition according to claim 1, wherein the treatment comprises providing the subject with a uniform dose of 1500 mg of the antibody.

11. The composition according to claim 1, wherein the antibody is provided weekly, bi-weekly, or monthly.

12. The composition according to claim 1, wherein the antibody is provided every other week.

13. The composition according to claim 1, wherein the antibody is afucosylated.

14. The composition according to claim 1, wherein the antibody is a bispecific antibody.

15. The composition according to claim 1, wherein the antibody comprises a common light chain.

16. The composition according to claim 1, wherein the first and second variable domains include light chain variable regions containing an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO: 122 by at least 90%, in a particular embodiment at least 95%, in a particular embodiment at least 97%, in a particular embodiment at least 98%, in a particular embodiment at least 99%, or in a particular embodiment 100%, or the first and second variable domains include light chain variable regions containing an amino acid sequence having 0 to 10, in a particular embodiment 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof, with respect to the amino acid sequence of SEQ ID NO:

122.

17. The composition according to claim 4, wherein the first and second variable domains include a light chain variable region having the amino acid sequence of SEQ ID NO:

122.

18. The composition according to claim 1, wherein the immune checkpoint inhibitor comprises durvalumab, retifanlimab, semiprimab, pembrolizumab, nivolumab, or atezolizumab.

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