Cancer with high EGFR expression treated with antibodies binding at least EGFR

Abstract

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

Description

[0001] The present invention is a divisional application of the invention patent application with application number 202280067481.0 filed on October 6, 2022, and invention name “Treatment of cancer with high EGFR expression treated with immune checkpoint inhibitors using antibodies that at least bind to EGFR”. Technical Field

[0002] The present disclosure relates to tools and methods for treating cancer. In particular, the present disclosure relates to a method for treating cancer in an individual using an antibody that binds at least to EGFR. The present disclosure further relates to uses in such methods and in the manufacture of medicaments for treating cancers with specific EGFR levels. Such antibodies are particularly useful for treating cancers such as gastric cancer, esophageal cancer, gastroesophageal junction cancer, or head and neck cancer. Background Art

[0003] Traditionally, most cancer drug discovery has focused on agents that block essential cell functions and kill dividing cells through chemotherapy. However, chemotherapy rarely results in a complete cure. In most cases, a patient's tumor stops growing or only shrinks temporarily before starting to grow again, sometimes more rapidly, and becoming increasingly difficult to treat.

[0004] Despite numerous advances in the treatment of the disease and an increased understanding of the molecular events that lead to cancer, cancer remains the leading cause of death worldwide.

[0005] It is reported that in the United States, head and neck cancer, especially oral and pharyngeal cancer, accounts for 3% of malignant tumors, with approximately 53,000 Americans diagnosed with this cancer each year and 10,800 of them dying (Siegel et al., CA Cancer J Clin. 2020; 70(1):7. Epub 2020 Jan 8.). In addition, it is reported that head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world, with a five-year overall survival rate of approximately 40 to 50% for HNSCC patients (see, Head and Neck Cancer, Union for International Cancer Control, 2014 Review of Cancer Medicines on the WHO List of Essential Medicines).

[0006] A comprehensive analysis of locally advanced head and neck squamous cell carcinoma (LA-HNSCC) reported that adding anti-EGFR drugs to radiotherapy or chemoradiotherapy did not improve the clinical outcomes of LA-HNSCC patients (Oncotarget. 2017; 8(60): 102371-102380). Furthermore, adding anti-EGFR agents was reported to increase the risk of skin toxicity and mucositis.

[0007] In addition, 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. It has been reported that epidermal growth factor receptor (EGFR) is overexpressed in more than 30% of gastric adenocarcinoma (GAC) and esophageal adenocarcinoma (EAC) cases. However, an analysis reviewing six different studies concluded that adding anti-EGFR agents to chemotherapy does 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).

[0008] Therefore, there is a need for improved cancer treatments, particularly for gastric, esophageal, and head and neck cancers. Summary of the Invention

[0009] The present disclosure provides the following preferred aspects. However, the present invention is not limited thereto.

[0010] The present disclosure provides an antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative and / or analog thereof, for use in treating cancer in a subject, wherein the cancer expresses EGFR or EGFR and LGR5.

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

[0012] The present disclosure also provides use of an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative and / or analog thereof, in the manufacture of a medicament for treating cancer in a subject, wherein the subject's cancer has progressed after prior treatment with an immune checkpoint inhibitor, and the cancer expresses EGFR or EGFR and LGR5.

[0013] The present disclosure also provides a method of treating a subject having a cancer that expresses EGFR, wherein the subject has progressed after prior treatment with an immune checkpoint inhibitor, the method comprising providing to the subject an effective amount of an antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative and / or analog thereof.

[0014] In certain aspects, the cancer of the present disclosure is particularly gastric cancer, esophageal cancer, gastroesophageal junction cancer, or head and neck cancer. Head and neck cancer is particularly head and neck squamous cell carcinoma (HNSCC). Gastric cancer, esophageal cancer, and gastroesophageal junction cancer are particularly adenocarcinomas. The esophageal cancer may also be squamous cell carcinoma.

[0015] In certain aspects, the cancer of the present disclosure is particularly a gastric cancer, an esophageal cancer, or a gastroesophageal junction cancer characterized by EGFR expression with an IHC score of 3+. In certain aspects, the cancer of the present disclosure is a gastric cancer, an esophageal cancer, or a gastroesophageal junction cancer characterized by EGFR expression with an H-score of greater than 200.

[0016] The present disclosure also provides an antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative and / or analog thereof, for use in treating gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a subject, wherein the cancer expresses EGFR and is characterized by an IHC score of 3+. The present disclosure also provides an antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative and / or analog thereof, for use in treating gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a subject, wherein the cancer expresses EGFR and is characterized by an H-score of EGFR of greater than 200.

[0017] The present disclosure also provides an antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative, and / or analog thereof, for use in treating head and neck cancer, gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a subject, wherein the cancer is characterized by comprising EGFR gene amplification. In certain aspects, the gene amplification of EGFR is characterized by an EGFR copy number of 8 or more, or a circulating tumor DNA (ctDNA) level of at least 2.14, or at least 2.5.

[0018] In certain aspects, EGFR mRNA amplification is qualified by an EGFR copy number of 8 or more (eg, as defined by next generation sequencing), or a ctDNA of at least 2.14 or at least 2.5.

[0019] In some aspects, the subject has progressed after receiving prior treatment with an immune checkpoint inhibitor. In some aspects, the immune checkpoint inhibitor comprises a PD-L1, PD-1, CTLA-4, B7-1, or B7-2 inhibitor. In some aspects, such inhibitors comprise antibodies targeting PD-L1, PD-1, CTLA-4, B7-1, or B7-2 inhibitors. In some aspects, the immune checkpoint inhibitor comprises durvalumab, retifanlimab, cemiplimab, pembrolizumab, ipilimumab, nivolumab, or atezolizumab.

[0020] In certain aspects, the subject of the present disclosure has not received prior treatment with an anti-EGFR agent. In certain aspects, the subject has not received prior treatment with an antibody targeting EGFR, or the subject has not received prior treatment with cetuximab.

[0021] In certain aspects, the gastric cancer, esophageal cancer, or gastroesophageal junction cancer of the present disclosure expresses EGFR and is characterized by an H-score of between greater than 200 and no greater than 300. In certain aspects, the H-score of EGFR is determined using immunohistochemistry (IHC).

[0022] In certain aspects, the subject of the present disclosure is a mammalian subject, such as a human subject.

[0023] In certain aspects, the treatment of the present disclosure includes providing an effective amount of the antibody or its functional part, derivative and / or analog to the subject. In certain aspects, the treatment includes providing a fixed dose (flat dose) between 500mg and 2000mg. In certain aspects, the dose is between 1100mg and 1800mg. In certain aspects, the dose is between 1100mg and 1500mg. In certain aspects, the treatment includes providing a fixed dose of 1500mg of the antibody or its functional part, derivative and / or analog to the subject. In certain aspects, the antibody or its functional part, derivative and / or analog is provided to the subject by intravenous injection. In certain aspects, the antibody or its functional part, derivative and / or analog is provided weekly, biweekly or monthly. In certain aspects, the antibody or its functional part, derivative and / or analog is provided every two weeks.

[0024] In certain aspects, the antibody or its functional part, derivative and / or analog is ADCC enhanced. In certain aspects, the antibody or its functional part, derivative and / or analog is defucosylated.

[0025] In certain aspects, the antibodies of the present disclosure, or their functional portions, derivatives, and / or analogs, are multispecific antibodies. In certain aspects, the antibodies of the present disclosure, or their functional portions, derivatives, and / or analogs, are bispecific antibodies that bind at least to EGFR. In certain aspects, the antibodies comprise a second variable domain that does not bind to EGFR. In certain aspects, the antibodies comprise a second variable domain that binds to LGR5.

[0026] Antibodies comprising a first variable domain that binds to the extracellular portion of EGFR of the present disclosure, or functional parts, derivatives and / or analogs thereof, are also referred to herein as therapeutic agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Human LGR5 sequence; SEQ ID NO: 1.

[0028] Figure 2 Human EGFR sequence; SEQ ID NO: 2.

[0029] Figure 3a ) together with the common light chain variable region (e.g., the variable region of human kappa light chain IgVκ1 39*01 / IGJκ1*01) form the amino acid sequence of the heavy chain variable region that binds to LGR5 and EGFR (SEQ ID No: 3-15). The CDR region and the framework region are as follows Figure 3b ). The corresponding DNA sequence is shown in Figure 3c ) as shown.

[0030] Figure 4 a) Consensus light chain amino acid sequence. b) Consensus light chain variable region DNA sequence and translation (IGKV1-39 / jk1). c) Light chain constant region DNA sequence and translation. d) V region IGKV1-39A; e) Consensus light chain CDR1, CDR2, and CDR3 numbering according to IMGT.

[0031] Figure 5 IgG heavy chain used to generate bispecific molecules. 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 the DNA sequence variants L351K and T366K (KK) and translation. e) CH3 domain containing the DNA sequence variants L351D and L368E (DE) and translation. Residue positions are numbered according to EU. DETAILED DESCRIPTION

[0032] To make this specification easier to understand, certain terms are first defined. Additional definitions may be set forth throughout the embodiments as deemed necessary. Unless otherwise defined herein, all technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the art, and conventional methods of immunology, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are employed.

[0033] As used herein, the singular forms "a," "an," and "the" include plural references. The use of the terms "comprise," "have," "includes," "comprised," "have," "has," "had," "include," "includes," and "included" as well as other forms, for example, "comprise," "comprises," "comprised," "have," "has," "had," "include," "includes," and "included" is non-limiting.

[0034] As used herein, the term "antibody" refers to a protein molecule belonging to the immunoglobulin class that contains one or more domains that bind to an epitope on an antigen, wherein such domains are from or derived from the variable region of an antibody or share sequence homology with the variable region of an antibody. Antibodies are generally composed of basic structural units, each having two heavy chains and two light chains. The antibodies according to the present invention are not limited to any particular form or method of producing the same.

[0035] "Bispecific antibodies" are antibodies as described herein, wherein one domain of the antibody binds to a first antigen and a second domain of the antibody binds to a second antigen, wherein the first and second antigens are different, or wherein one domain binds to a first epitope on an antigen and the second domain binds to a second epitope on an 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 an epitope on an antigen and a second VH / VL combination binds to a second antigen or an epitope on an antigen. The term further includes antibodies in which the VH can specifically recognize the first antigen and the VL (which is paired with the VH in an immunoglobulin variable region) can specifically recognize the second antigen. The resulting VH / VL pairing will bind to either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO 2008 / 027236, WO 2010 / 108127 and Schaefer et al. (Cancer Cell 20, 472-486, October 2011).The bispecific antibodies according to the invention are not restricted to any particular bispecific format or method of producing the same.

[0036] As used herein, the term "common light chain" means the two light chains (or their VL portions) in a bispecific antibody. The two light chains (or their VL portions) may be identical or have some amino acid sequence differences, while the binding specificity of the full-length antibody is unaffected. The terms "common light chain," "common VL," "single light chain," "single VL," whether or not the term "rearrangement" is added, are used interchangeably herein. "Common" also refers to the functional equivalents of light chains whose amino acid sequences are not identical. There are many variants of the light chain, in which there are mutations (deletions, substitutions, insertions, and / or additions) that do not affect the formation of the functional binding region. In some aspects, the light chain of the present invention may also be a light chain as specified herein, having 0 to 10 amino acid insertions, deletions, substitutions, additions, or combinations thereof. In some aspects, the light chain of the present invention may also be a light chain as specified herein, having 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. For example, within the definition of a common light chain as used herein, different but still functionally equivalent light chains are made or found, e.g., by introducing and testing conservative amino acid changes, changes in amino acids in regions that do not contribute or only partially contribute to binding specificity when paired with a heavy chain, etc.

[0037] As used herein, "comprising" and its conjunctions are used in a non-limiting sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb "consisting of" can be replaced with "consisting essentially of...", meaning that the compounds or auxiliary compounds as defined herein may contain additional components in addition to the specifically specified components, which additional components do not change the unique characteristics of the invention.

[0038] According to the present invention, the term "full-length IgG" or "full-length antibody" is defined as comprising a substantially complete IgG, however, it does not necessarily have all the functions of a complete IgG. For the avoidance of doubt, a 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 divided into domains designated as CH1, CH2, CH3, VH and CL, VL. IgG antibodies bind to antigens via the variable region domains contained in the Fab portion, and after binding, can interact with molecules and cells of the immune system through the constant domains, primarily through the Fc portion. The full-length antibodies according to the present invention encompass IgG molecules, wherein there may be variants that provide the desired characteristics. Full-length IgG should not have a substantial portion of any region missing. However, IgG molecules in which one or more amino acid residues are missing and the binding properties of the resulting IgG molecule are substantially unchanged are all included in the term "full-length IgG". For example, such IgG molecules may have a deletion of between 1 and 10 amino acid residues, preferably in a non-CDR region, wherein the deleted amino acids are not required for the antigen-binding specificity of the IgG. In certain aspects, such IgG molecules may have deletions of between 1 and 10 amino acid residues in non-CDR regions, wherein the deleted amino acids are not essential for the antigen-binding specificity of the IgG.

[0039] "Antibody derivatives" are proteins that deviate from the amino acid sequence of a native antibody by up to 20 amino acids, excluding the CDR regions. Antibody derivatives disclosed herein are antibodies that deviate from this amino acid sequence by up to 20 amino acids. Functional parts, derivatives and / or analogs maintain the binding specificity of the (bispecific) antibody. An "antibody analog" is a protein that may not be identical in structure, form or origin, but maintains its antibody binding specificity as an analog.

[0040] The "percent identity (%)" of nucleic acid or amino acid sequences referred to herein is defined as the percentage of residues in a candidate sequence that are identical to the residues in a selected sequence after alignment for optimal comparison purposes. The percent sequence identity of nucleic acid sequences is compared using Vector NTI The results were determined using the AlignX application of 11.5.2 software, using the original settings and a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson T.J., (1994) Nuc. Acid Res. 22(22): 4673-4680), the swgapdnamt scoring matrix, a gap opening penalty of 15 and a gap extension penalty of 6.66. Amino acid sequences were aligned using the AlignX application of 11.5.2 software using native settings and a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, (1994) Nuc. Acid Res. 22(22):4673-4680), a blosum62mt2 scoring matrix, a gap opening penalty of 10, and a gap extension penalty of 0.1.

[0041] Since antibodies generally recognize an epitope of an antigen, and this epitope may also exist in other compounds, if other compounds contain the same type of epitope, then the antibodies of the present invention that "specifically recognize" an antigen (e.g., EGFR or LGR5) may also recognize such other compounds. Therefore, the term "specifically recognizes" with respect to the interaction between an antigen and an antibody does not exclude the binding of an antibody to other compounds containing the same type of epitope.

[0042] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein (so-called linear and conformational epitopes). Epitopes formed by contiguous, linear amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding conformations are generally lost upon treatment with denaturing solvents. An epitope can typically include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

[0043] As used herein, the terms "subject" and "patient" are used interchangeably and refer to mammals, e.g., humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cows, horses, pigs, etc. (e.g., a patient, e.g., a human patient, having cancer).

[0044] As used herein, the terms "treat," "treating," and "treatment" mean any type of intervention or procedure performed on a subject, or the administration of an active agent or combination of active agents to a subject, with the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical markers associated with a disease.

[0045] As used herein, "effective treatment" or "positive therapeutic response" means a treatment that produces a beneficial effect, e.g., an improvement in at least one symptom of a disease or disorder (e.g., cancer). A beneficial effect can be in the form of an improvement over baseline, including improvements that were measured or observed before starting therapy according to the present method. For example, a beneficial effect can be in the form of slowing, stabilizing, halting, or reversing the progression of a cancer in a subject at any clinical stage, as evidenced by a reduction or elimination of clinical or diagnostic symptoms of the disease or markers of the cancer. An effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent metastasis of a tumor, slow or stop tumor growth, and / or prevent or delay tumor recurrence or recurrence.

[0046] The term "effective amount" or "therapeutically effective amount" means the amount of an agent or combination of agents that provides the desired biological, therapeutic and / or preventive result. The result can be one or more of a reduction, improvement, alleviation, reduction, delay and / or alleviation of the signs, symptoms or causes of a disease, or any other desired change in a biological system. In terms of tumor development, an effective amount is an amount sufficient to delay tumor development. In terms of tumor recurrence, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered once or multiple times. An effective amount of an agent or composition can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, postpone, slow down and prevent cancer cell infiltration into surrounding organs to a certain extent; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate one or more symptoms associated with cancer to a certain extent. In one aspect, an "effective amount" is an amount of an antibody disclosed herein as a therapeutic agent to affect cancer reduction (e.g., a reduction in the number of cancer cells); slow the progression of cancer or prevent the regrowth or recurrence of cancer. As previously described herein, the antibodies that bind to EGFR or bind to EGFR and LGR5 of the present disclosure, or their functional portions, derivatives, and / or analogs, are also referred to herein as "therapeutic agents." In certain aspects, the effective amount herein is a fixed dose of 1500 mg administered on a biweekly basis to a subject with a cancer of the present disclosure.

[0047] The term "fixed dose" herein means a dosing regimen in which a fixed amount of a therapeutic substance is administered to a subject over multiple administrations, regardless of the subject's weight. A fixed dose is typically abbreviated as qnw, where n is an integer representing an interval and w is a week. For example, a q2w fixed dose dosing regimen of 1500 mg of an antibody means that a fixed amount of 1500 mg of the antibody is administered every 2 weeks. In certain aspects, the therapeutic substance is an antibody that binds to EGFR or EGFR and LGR5, administered at a q2w dosing regimen of 1500 mg. In certain aspects, the subject has been administered at least 3 q2w fixed doses of 1500 mg. In certain aspects, the administration is at least 4 doses or more and may be continued until the patient shows sufficient clinical or radiological progress.

[0048] A fixed dose can be administered in advance, meaning that the drug is administered to the subject prior to administration of the antibody of the invention. In certain aspects, a fixed dose of 1500 mg of the antibody is administered in advance with an antihistamine, pain reliever, fever reducer, and / or anti-inflammatory drug.

[0049] The term "H-score", sometimes referred to in the art as the "histo" score, refers to a reproducible and standardized scoring method that can be used to calculate the expression of genes of interest in tumor samples in a semi-quantitative manner using procedures based on immunohistochemistry (IHC) or in situ hybridization (ISH), all of which are well known to those skilled in the art and follow the ASCO guidelines for calculating the H-score published on April 10, 2015. See 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. The relevant teachings of these references are hereby incorporated by reference.

[0050] In the context of the H-score for EGFR, the term "determined using IHC" means a method that uses or includes IHC as the basis for the subsequent determination of the H-score, rather than a method that replaces IHC.

[0051] In some aspects, the present disclosure provides an antibody or a functional portion, derivative and / or analog thereof, comprising a first variable domain that binds to the extracellular portion of EGFR and is used to treat cancer in a subject whose cancer has progressed after prior treatment with an immune checkpoint inhibitor and which cancer expresses EGFR.

[0052] In some aspects, the cancer is selected from gastric cancer, esophageal cancer, gastroesophageal junction cancer, or head and neck cancer, particularly squamous cell carcinoma of the head and neck (SCCHN).

[0053] In some aspects, the cancer is a gastric cancer, esophageal cancer, or gastroesophageal junction cancer with EGFR expression characterized by an IHC score of 3+. In some aspects, the cancer has an EGFR H-score greater than 200. In some aspects, the IHC is a tumor membrane score.

[0054] The present disclosure also provides antibodies or functional parts, derivatives and / or analogs thereof, which comprise a first variable domain that binds to the extracellular portion of EGFR and are used to treat cancer in a subject, wherein the cancer expresses EGFR, is characterized by an IHC score of 3+, and wherein the variable domain comprises amino acids as further disclosed herein.

[0055] The present disclosure also provides antibodies or functional parts, derivatives and / or analogs thereof, comprising a first variable domain that binds to the extracellular portion of EGFR for use in treating cancer in a subject, wherein the cancer expresses EGFR, is characterized by an H-score for EGFR of greater than 200, and wherein the variable domain comprises an amino acid as further disclosed herein.

[0056] The present disclosure also provides the use of an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative and / or analog thereof, in the manufacture of a medicament for treating a cancer in a subject whose cancer has progressed after prior treatment with an immune checkpoint inhibitor, and the cancer expresses EGFR.

[0057] The present disclosure also provides a method of treating a subject having a cancer that expresses EGFR, wherein the subject has progressed after prior treatment with an immune checkpoint inhibitor, the method comprising providing to the subject 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.

[0058] The present disclosure also provides an antibody or a functional part, derivative and / or analog thereof, which comprises a first variable domain that binds to the extracellular portion of EGFR and is used to treat gastric cancer, esophageal cancer or gastroesophageal junction cancer in a subject, wherein the cancer expresses EGFR and is characterized by an IHC score of 3+.

[0059] The present disclosure also provides an antibody or a functional part, derivative and / or analog thereof, which comprises a first variable domain that binds to the extracellular portion of EGFR and is used to treat gastric cancer, esophageal cancer or gastroesophageal junction cancer in a subject, wherein the cancer expresses EGFR and is characterized by an H-score of EGFR greater than 200.

[0060] Unless otherwise specified, the terms cancer and tumor used in this article generally refer to cancer.

[0061] The epidermal growth factor (EGF) receptor (EGFR, ErbB1 or HER1) is a member of the four receptor tyrosine kinase (RTK) family, known as Her- or cErbB-1, -2, -3 and -4. EGFR is known by a variety of synonyms, the most common of which is EGFR. EGFR has an extracellular domain (ECD) composed 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 a variety of ligands to produce different intracellular responses. The main signaling pathway activated by EGFR consists of the Ras mitogen-activated protein kinase (MAPK) mitogenic signaling cascade. Activation of this pathway is initiated by recruiting Grb2 to tyrosine-phosphorylated EGFR. This leads to the activation of Ras through the Ras-guanine purine nucleotide exchange factor Son of Sevenless (SOS), which binds to Grb2. In addition, the PI3-kinase-Akt signaling pathway is also activated by EGFR, but this activation is stronger when ErbB-3 (HER3) is co-expressed. EGFR is associated with several human epithelial malignancies, particularly breast cancer, bladder cancer, non-small cell lung cancer, colon cancer, ovarian head and neck cancer, and brain cancer. Activating mutations in the gene, as well as overexpression of the receptor and its ligands, have been found, leading to autocrine activation loops. Therefore, this RTK has been widely used as a target for cancer therapy. Small molecule inhibitors targeting RTKs and monoclonal antibodies (mAbs) directed against the extracellular ligand-binding domain have both been developed and have shown numerous clinical successes to date, but primarily in specific patient groups. The database accession number for the human EGFR protein and its encoding gene is GenBank NM_005228.3. This accession number primarily provides further methods for identifying EGFR protein as a target. The actual sequence of the EGFR protein bound by the antibody may vary, for example, due to mutations in the encoding gene, such as those that occur in some cancers.

[0062] When EGFR is referred to herein, unless otherwise indicated, the reference is to human EGFR.The variable domain antigen binding sites that bind EGFR can bind to EGFR and its various variants, such as those expressed on some EGFR-positive tumors.

[0063] The term "LGR" refers to a family of proteins known as leucine-rich repeat-containing G protein-coupled receptors. Several members of this family are known to participate in the WNT signaling pathway, notably LGR4, LGR5, and LGR6.

[0064] LGR5 is leucine-rich repeat-containing G protein-coupled receptor 5. Alternative names for this gene or protein are 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. Proteins or antibodies of the invention that bind to LGR5 may bind to human LGR5. Due to the sequence and tertiary structure similarities between human and other mammalian xenologs, LGR5-binding proteins or antibodies of the invention may also bind to these xenologs, but this is not required. The database accession numbers for the human LGR5 protein and its encoding gene are (NC_000012.12; NT_029419.13; NC_018923.2; NP_001264155.1; NP_001264156.1; NP_003658.1). The accession numbers are provided primarily for further methods to identify LGR5 as a target. The actual sequence to which the LGR5 protein binds may vary, for example, due to mutations in the encoding gene, such as those that occur in some cancers. The LGR5 antigen binding site binds to LGR5 and various variants thereof, such as those expressed by some LGR5-positive tumor cells.

[0065] In particular, the cancer is gastric cancer, esophageal cancer, or gastroesophageal junction cancer. Gastric cancer (also known as gastric cancer) is a cancer that develops from the lining of the stomach, particularly the mucus-producing glandular cells found in the stomach. This cancer is also known as adenocarcinoma, or in this case, gastric adenocarcinoma, because it develops from the lining of the stomach. In particular, the cancer is therefore gastric adenocarcinoma or a cancer that develops from the lining of the stomach, which are used interchangeably herein. Esophageal cancer is a cancer that develops from the esophagus. The two main subtypes are ESCC (esophageal squamous cell carcinoma) and EAC (esophageal adenocarcinoma). Gastric-esophageal junction cancer (also known as gastroesophageal junction adenocarcinoma) originates from the gastroesophageal junction.

[0066] Cancers collectively known as head and neck cancers typically originate from squamous cells that line the moist, sticky surfaces inside the head and neck, such as the inside of the mouth, nose, and throat. These squamous cell carcinomas are often referred to as head and neck squamous cell carcinomas, and in certain aspects of the present disclosure, they are treated. Although rare, head and neck cancers can also occur in the salivary glands. In particular, head and neck cancers can occur in the oral cavity. This includes the lips, the front two-thirds of the tongue, the gums, the lining of the cheeks and lips, the floor of the mouth under the tongue, the hard palate, and a small area of ​​gums behind the wisdom teeth.

[0067] Thus, in particular, the head and neck cancer is a squamous cell carcinoma and includes nasopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, oral cancer, oropharyngeal cancer or salivary gland cancer. More particularly, the present invention relates to the treatment of cancers including squamous cell head and neck cancer, such as cancers located in the oropharynx, hypopharynx, larynx, oral cavity or tongue.

[0068] Meanwhile, head and neck cancer is particularly squamous cell carcinoma of unknown primary (also known in the art as carcinoma of unknown primary or CUP).

[0069] In the present disclosure, the cancer expresses EGFR or EGFR and LGR5.

[0070] As used herein, a cancer expresses EGFR if it comprises cells that express EGFR. Cells that express EGFR comprise detectable levels of RNA encoding EGFR. In some aspects, EGFR expression is determined by ISH.

[0071] In some aspects, EGFR protein expression is detected by IHC. In some aspects, EGFR expression is determined by IHC using a commercially available EGFR detection kit, such as the EGFR pharmDx® for Dako automated staining instruments (Agilent). TM kit using the manufacturer's recommendations; or a commercially available IHC EGFR detection kit based on EGFR clone 113, which binds to the EGFR extracellular domain (Leica, https: / / shop.leicabiosystems.com / us / ihc-ish / ihc-primary-antibodies / pid-epidermal-growth-factor-receptor). Alternatively, use Novocastra TM EGFR expression was determined using a liquid mouse monoclonal antibody against EGFR based on clone EGFR.113 (product code: NCL-L-EGFR, EGFR-IHC primary antibody, purchased from leicabiosystems.com).

[0072] Briefly, the commercially available EGFR pharmDx TMThe IHC kit system contains the reagents required to complete the IHC staining procedure for routine fixed, paraffin-embedded samples. After incubation with a primary, non-Her2, Her3 and Her4 cross-reactive monoclonal antibody (clone 2-18C9) against the human EGFR protein, this kit uses a ready-to-use visualization reagent based on dextran technology. This reagent consists of a secondary goat anti-mouse antibody molecule and a horseradish peroxidase molecule connected 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 position. The results are routinely evaluated using an optical microscope. Control slides containing two formalin-fixed, paraffin-embedded human cell lines with staining intensity scores of 2+ and 0 are provided for quality management of the kit reagent performance.

[0073] Staining intensity was established as follows: 3+ (strong staining): visible at low magnification, x5 objective, and confirmed at higher levels as needed; 2+ (moderate staining): visible at moderate magnification, x10 or x20 objective; 1+ (weak staining): reliably confirmed only at high magnification, x40 objective; 0 (no staining): no staining visible at high magnification.

[0074] In some aspects, EGFR expression is determined using immunohistochemistry (IHC), and the cancer is IHC positive for EGFR. In some aspects, the cancer is gastric cancer, esophageal cancer, or gastroesophageal junction cancer, characterized by an EGFR IHC score of 3+.

[0075] In certain aspects, EGFR expression is determined using immunohistochemistry (IHC), and EGFR is then assigned an H-score using a scale of 0 to 300. In certain aspects, the cancer of the present disclosure is a gastric cancer, an esophageal cancer, or a gastroesophageal junction cancer, characterized by an H-score for EGFR of greater than 200 on a scale of 0 to 300. In certain aspects, the H-score for EGFR is therefore greater than 200 and up to and including 300. In certain aspects, the cancer of the present disclosure is characterized by an H-score for EGFR of greater than 50 on a scale of 0 to 300. In certain aspects, the cancer of the present disclosure is a head and neck cancer, characterized by an H-score for EGFR of greater than 50 on a scale of 0 to 300. In certain aspects, the cancer of the present disclosure is characterized by an H-score for EGFR of greater than 80 on a scale of 0 to 300. In certain aspects, the cancer of the present disclosure is a head and neck cancer, characterized by an H-score for EGFR of greater than 80 on a scale of 0 to 300.

[0076] In another aspect, the cancer is head and neck cancer characterized by an IHC score of EGFR of 2+ or 3+.

[0077] Herein, determining the H score to assign EGFR expression status involves a first step of establishing membrane staining intensity (resulting in a score of 0, 1+, 2+, or 3+), which is determined for each cell within a predefined range as described herein. Subsequently, the percentage of cells at each staining intensity level is calculated, and ultimately, the H score is assigned using the following formula: [1×(percentage of cells with 1+ staining)+2×(percentage of cells with 2+ staining)+3×(percentage of cells with 3+ staining)], resulting in an H score for EGFR between 0 and 300. Thus, the H score gives more relative weight to higher staining intensities or amounts of staining in a given tumor sample.

[0078] In some aspects, the cancer of the present disclosure is characterized by comprising EGFR gene amplification. In some aspects, the cancer is gastric cancer. In some aspects, the cancer is gastric-esophageal junction adenocarcinoma. In some aspects, the gene amplification of EGFR is characterized by an EGFR copy number of 8 or more based on a solid tissue sample, or an EGFR amplification score (also referred to as copy number alteration (CNA)) based on circulating tumor DNA (ctDNA) of at least 2.14 or at least 2.5, but in some aspects, no more than 5.

[0079] In certain aspects, EGFR amplification is defined as an EGFR copy number of 8 or more (particularly defined as 8 copies or more based on the ploidy of solid tissue amplification). In certain aspects, the EGFR copy number is determined by performing next generation sequencing on formalin-fixed paraffin-embedded (FFPE) tissue samples.

[0080] In some aspects, the EGFR gene copy number is determined using next generation sequencing (NGS). In some aspects, NGS is performed on a solid tissue sample or a liquid sample (e.g., blood or plasma). In some aspects, the EGFR amplification score is determined by performing next generation sequencing on ctDNA, which results in a score of at least 2.14 or at least 2.5. In some aspects, the ctDNA score is not more than 5. The copy number assessment can be based on blood-derived cfDNA. As an example, the determination of the EGFR copy number can be 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).

[0081] Herein, the term “ctDNA” (circulating tumor DNA) is used interchangeably with “cfDNA” (cell-free tumor DNA).

[0082] In some aspects, FISH is used to determine the EGFR gene copy number. In some aspects, the cancer is characterized in that the EGFR / CEP7 ratio is at least 2.0 or higher. Establishing the EGFR / CEP7 ratio is a standard in the art, but can be established, for example, using a commercially available kit, or according to 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 the amplification of the EGFR locus (located at chromosome 7p11.2). In this method, FISH is performed on formalin-fixed, paraffin-embedded tumor tissue sections. Slides are prepared according to standard protocols, and 100 interphase cells are scored. The cutoff value for amplification is then set at an EGFR:CEP7 ratio of ≥2.0.

[0083] Alternatively, treatment with the antibody or its functional portion, derivative and / or analogue comprises (or in some aspects is preceded by) a step of diagnosing the subject's EGFR status. In some aspects, a subject having gastric cancer, esophageal cancer, gastroesophageal junction cancer characterized by an IHC score of 3+, or a subject having an H score of EGFR greater than 200 on a scale of 0 to 300 is selected as a treatment target. In some aspects, treatment of the subject is preceded by a step of diagnosing the subject having gastric cancer, esophageal cancer, gastroesophageal junction cancer characterized by an H score of EGFR greater than 200 on a scale of 0 to 300.

[0084] In certain aspects, subjects with cancer, such as gastric cancer, esophageal cancer, or gastroesophageal junction cancer, are selected for treatment, and the cancer is characterized by EGFR gene amplification, comprising an EGFR / CEP7 ratio of at least 2.0 or higher, an EGFR copy number of 8 or more, or an EGFR ctDNA score of at least 2.14 or at least 2.5. In certain aspects, treatment of the subject is preceded by a step of diagnosing the subject with gastric cancer, esophageal cancer, or gastroesophageal junction cancer, and the cancer is characterized by EGFR gene amplification, comprising an EGFR / CEP7 ratio of at least 2.0 or higher, an EGFR copy number of 8 or more, or an EGFR ctDNA score of at least 2.14 or at least 2.5.

[0085] In particular, the present disclosure provides an antibody or a functional portion, derivative and / or analog thereof, which comprises a first variable domain that binds to the extracellular portion of EGFR and may comprise a second variable domain that binds to the extracellular portion of LGR5, for treating gastric cancer, esophageal cancer, gastroesophageal junction cancer or head and neck cancer, which has progressed after previous treatment with an immune checkpoint inhibitor, wherein the subject has a Her2 status selected from subjects who are Her2 positive, Her2 high, Her2 3+, Her2 2+, Her2 1+, Her2 0 or Her2 negative. In some aspects, the subject is Her2 negative. The present disclosure further provides a method for treating such cancers in Her2 negative subjects, comprising providing the antibody or its functional portion, derivative and / or analog to a subject in need. In some aspects, the use comprises providing a fixed dose of 1500 mg of the antibody or its functional portion, derivative and / or analog to the subject. In certain aspects, the therapeutic agent can be administered weekly, biweekly, or monthly to a Her2-negative subject. In certain aspects, the therapeutic agent is administered biweekly. Disclosed herein are suitable variable domains that bind to the extracellular portion of EGFR and suitable variable domains that bind to the extracellular portion of LGR5. In certain aspects, the first variable domain comprises at least a CDR3 sequence, or at least a CDR1, CDR2, and CDR3 sequence, of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370; MF3755; MF4280; or MF4289, as shown in FIG3 . In certain aspects, the second variable domain comprises at least a CDR3 sequence, or at least a CDR1, CDR2, and 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, as shown in FIG3 .

[0086] Methods for determining the expression of human epidermal growth factor receptor 2 (HER2) of a subject are well known in the art. For example, immunohistochemistry (IHC) or (fluorescence) in situ hybridization (ISH) can be used to determine the expression level of Her2, which allows identification of Her2 status, including identification of Her2 negative subjects. IHC or ISH are both well-defined standard procedures, conventionally used to determine the Her2 status of human subjects. Reference is made herein to, for example, the ASCO / CAP guidelines according to Bartley et al. (HER2 Testing and Clinical Decision Making in Gastroesophageal Adenocarcinoma. Arch Pathol Lab Med. 2016; 140: 1345-1363). For example, anti-HER-2 / neu antibodies (clone 4B5) are used to allow the use of IHC semi-quantitative detection of HER-2 antigens in FFPE gastric cancer, esophageal cancer, gastroesophageal junction cancer or head and neck cancer sections. Staining and scoring are performed according to the consensus guidelines for this cancer type. Such IHC tests typically give a score of 0 to 3+, which measures the amount of HER2 receptor protein on the cell surface in a cancer tissue sample. Based on the IHC score, the patient can be classified as Her2 negative, for example, when the measured score is 0 or 1+. In the case of using an ISH test to determine Her2 expression, for example, using a HER2 probe (17q11.2-q12) and a centromere 17 probe (Cen 17), the diagnosis is "positive" or "negative", sometimes also reported as HER2 being "zero". In certain aspects, the methods of treatment disclosed herein relate to subjects determined to be Her2 negative by IHC and / or ISH.

[0087] Herein, a Her2-negative subject refers to a subject having cancer, cancer cells or tumors (ie, Her2-negative). Her2 status can be determined according to the above-mentioned IHC and / or ISH.

[0088] In certain aspects, there is a step of diagnosing the Her2 status of the subject prior to treatment with the antibody or its functional portion, derivative and / or analog. In certain aspects, subjects with Her2 negative status are selected as treatment targets. In certain aspects, there is a step of diagnosing the subject with Her2 negative gastric cancer, esophageal cancer, gastroesophageal junction cancer or head and neck cancer prior to treatment of the subject. Such cancers treated by the methods of the present disclosure include gastric adenocarcinoma and esophageal cancer with squamous cell carcinoma histology.

[0089] In certain aspects, the Her2-negative diagnosis involves ISH or IHC testing of Her2 status.

[0090] In certain aspects, prior to treating a Her2 negative subject, there is a step of screening for subjects with Her2 negative gastric cancer, esophageal cancer, or gastroesophageal junction cancer. Such cancers are particularly adenocarcinomas. In certain aspects, the screening involves ISH or IHC testing of Her2 status.

[0091] In certain aspects, the subject has not been previously treated with an anti-EGFR agent. In certain aspects, the subject has not been treated with an antibody targeting EGFR. In certain aspects, the subject has not been treated with cetuximab. This subject is also referred to as a cetuximab-naive or anti-EGFR-naive subject. In other words, the subject's cancer has not been previously treated with an anti-EGFR agent. In certain aspects, the subject's cancer has not been treated with an antibody targeting EGFR. In certain aspects, the subject's cancer has not been treated with cetuximab. This subject is also referred to as a cetuximab-naive or anti-EGFR-naive subject.

[0092] The subject of the present disclosure has received prior treatment with an immune checkpoint inhibitor. In certain aspects, the immune checkpoint inhibitor comprises durvalumab, pembrolizumab, ipilimumab, nivolumab, atezolizumab, rivaroxaban, similomab, or other approved or under development anti-PD1 or anti-PD-L1 antibodies. In certain aspects, the immune checkpoint inhibitor comprises durvalumab or pembrolizumab.

[0093] Durvalumab (trade name Imfinzi TM Durvalumab (marketed as durvalumab) is an FDA-approved immune checkpoint inhibitor used to treat cancers such as bladder and lung cancer. It is a human immunoglobulin G1κ (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. As described in the Examples section, clinically relevant responses were observed in patients who had previously received treatment with durvalumab as an immune checkpoint inhibitor.

[0094] Pembrolizumab (trade name Keytruda TMPembrolizumab (trading) is a humanized antibody used in cancer immunotherapy to treat a variety of cancers, including melanoma, lung cancer and Hodgkin's lymphoma, and has the effect of an immune checkpoint inhibitor. It is an IgG4 isotype antibody and targets the programmed cell death protein 1 (PD-1) receptor of 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 use in any unresectable or metastatic solid tumor with a specific genetic abnormality. It is on the World Health Organization's list of essential medicines. As described in the Examples section, clinically relevant responses were observed in patients who had received prior treatment with pembrolizumab as an immune checkpoint inhibitor.

[0095] Ipilimumab (trade name Yervoy TM Ipilimumab (marketed as ipilimumab) is a monoclonal antibody and immune checkpoint inhibitor that activates the immune system by targeting CTLA-4, a protein receptor that downregulates the immune system. Ipilimumab was approved by the U.S. Food and Drug Administration (FDA) in March 2011 for the treatment of melanoma.

[0096] Nivolumab (trade name Opdivo TM Nivolumab (marketed as nivolumab) is an immune checkpoint inhibitor used to treat a variety of cancers, including melanoma, lung cancer, malignant pleural mesothelioma, renal cell carcinoma, Hodgkin's lymphoma, head and neck cancer, urothelial carcinoma, colon cancer, esophageal squamous cell carcinoma, liver cancer, gastric cancer, and cancer of the esophagus or gastroesophageal junction (GEJ). 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 on the World Health Organization's List of Essential Medicines. Nivolumab is the second FDA-approved systemic treatment for mesothelioma and the first FDA-approved immunotherapy for the first-line treatment of gastric cancer.

[0097] Atezolizumab (trade name Tecentriq TM Atezolizumab (marketed as Atezolizumab) is a monoclonal antibody 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.

[0098] Revulimab (formerly known as MGA012) is a humanized anti-PD-1 monoclonal antibody being developed as a monotherapy and in combination with other cancer therapeutics. Revulimab is currently in clinical trials (NCT04472429 and NCT04205812) as a monotherapy for patients with microsatellite instability-high endometrial cancer, Merkel cell carcinoma, and anal squamous cell carcinoma (SCAC); and in combination with platinum-based chemotherapy for patients with non-small cell lung cancer and SCAC. Revulimab has been granted orphan drug designation by the FDA for the treatment of anal cancer.

[0099] Cimetidine (trade name Cimizumab (traded) is a monoclonal antibody used to treat squamous cell skin cancer. Cimizumab belongs to a class of drugs that bind to programmed death receptor-1 (PD-1), blocking the PD-1 / PD-L1 pathway. In September 2018, cimizumab was approved by the FDA for the treatment of patients with metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC who are not candidates for curative surgery or radical radiation therapy. Cimizumab was approved for medical use in the European Union in June 2019.

[0100] At the same time, the previous treatment with immune checkpoint inhibitors mentioned herein is intended to target immune checkpoint proteins, which include PD-L1, PD-1, CTLA-4, B7-1 or B7-2. Therefore, the previous treatment with immune checkpoint inhibitors disclosed herein targets immune checkpoint proteins selected from PD-L1, PD-1, CTLA-4, B7-1 or B7-2.

[0101] 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 that is encoded by the CD274 gene in humans. This gene encodes an immunoinhibitory receptor ligand that is expressed by hematopoietic and non-hematopoietic cells (e.g., 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 of this ligand with 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 the constancy of the immune response. In the tumor microenvironment, this interaction provides tumor cells with immune escape by inactivating cytotoxic T cells.

[0102] Programmed cell death protein 1 (PD-1 or CD279; HGNC: 8760; NCBI Entrez Gene: 5133; UniProtKB / Swiss-Prot: Q15116) is an immunoinhibitory receptor expressed on activated T cells; it is involved in regulating T cell functions, including those of effector CD8+ T cells. Furthermore, this protein promotes the differentiation of CD4+ T cells into T regulatory cells. It is expressed in many tumor types, including melanoma, and has been shown to play a role in anti-tumor immunity. Furthermore, this protein has been shown to be involved in maintaining autoimmunity; however, it may also contribute to the suppression of potent anti-tumor and anti-microbial immunity.

[0103] 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 inhibitory signals to T cells. This protein contains a V domain, a transmembrane domain, and a cytoplasmic tail. Alternative transcript splice variants encoding different isoforms have been identified. 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.

[0104] B7-1, or cluster of differentiation 80 (CD80; HGNC: 1700; NCBI Entrez Gene: 941; UniProtKB / Swiss-Prot: P33681), is a B7, type I membrane protein that is part of the immunoglobulin superfamily and possesses an extracellular immunoglobulin constant-like domain and a variable-like domain required for receptor binding. The protein encoded by this gene is a membrane receptor that is activated by binding to CD28 or CTLA-4. Its functions in biological systems include inducing T cell proliferation and cytokine production. It also participates in the co-stimulatory signals necessary for T lymphocyte activation. Binding to CD28 induces T cell proliferation and cytokine production, but binding to CTLA-4 has the opposite effect and inhibits T cell activation. It is closely associated with another B7 protein, CD86, and often works in concert. Both CD80 and CD86 interact with the co-stimulatory receptors CD28 and CTLA-4, CD152.

[0105] B7-2, or cluster of differentiation 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, it provides the co-stimulatory signals required for T cell activation and survival. Depending on the bound ligand, CD86 can signal autoregulation and cell-cell binding, or attenuation and cell-cell dissociation. The CD86 gene encodes a type I membrane protein that is a member of the immunoglobulin superfamily. Alternative splicing results in two transcript variants encoding different isoforms.

[0106] Subjects may also have previously received one or more standard approved therapies or standard care treatments. Although surgery or radiotherapy may be preferred for most patients with early or localized disease, and may be considered for locally advanced disease, it may not be applicable to all patients, for example, due to the anatomical location of the cancer. In certain aspects, standard approved therapies or standard care herein include the treatment by administering chemotherapeutic agents, such as platinum-based compounds (e.g., cisplatin, carboplatin), anti-malignant tumor compounds (e.g., methotrexate), fluoropyrimidines (e.g., fluorouracil, 5-FU, capecitabine (capecitabine)), taxanes (taxanes) (e.g., docetaxel (docetaxel) or paclitaxel (paclitaxel)), nucleoside analogs (e.g., gemcitabine (gemcitabine)) or one or more of its combination.

[0107] Thus, in certain aspects, the subject of the present disclosure has received prior treatment with a chemotherapeutic agent. In certain aspects, the chemotherapeutic agent comprises a platinum-based compound (e.g., cisplatin, carboplatin), an anti-malignant tumor compound (e.g., methotrexate), a fluoropyrimidine (e.g., fluorouracil, 5-FU, capecitabine), a taxane (e.g., docetaxel or paclitaxel), a nucleoside analog (e.g., gemcitabine), or a combination thereof.

[0108] According to the present disclosure, in certain aspects, the cancer and / or the subject suffering from the cancer is SMAD4 wild-type. 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)-β signaling. The product of this gene forms homo- and hetero-complexes with other activated SMAD proteins, which then accumulate in the cell nucleus and regulate the transcription of target genes. This protein binds to DNA and recognizes an 8-bp palindromic sequence (GTCTAGAC) called the SMAD binding element (SBE). This protein acts as a tumor suppressor and inhibits epithelial cell proliferation. It may also have an inhibitory effect 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 complexly regulated through post-translational modifications. Mutation or deletion of this gene have been shown to cause pancreatic cancer, juvenile polyposis syndrome and hereditary hemorrhagic telangiectasia syndrome. Despite the influence of these previously reported mutations occurring in SMAD4, cancer of the present disclosure and / or the subject suffering from this cancer are SMAD4 wild-type. In some aspects, this patient or cancer do not comprise any mutation in the SMAD4 protein information mentioned herein.

[0109] Alternatively, the step of SMAD states of the subject including diagnosis with the treatment of antibody or its functional part, derivative and / or analog.In some aspects, before treatment, it is the diagnostic step.In some aspects, the subject with gastric cancer, esophageal cancer, gastro-esophageal junction cancer with wild-type SMAD4 gene and / or protein is selected as treatment target.In some aspects, before treatment, there is the step of diagnosing the subject and suffering from gastric cancer, esophageal cancer, gastro-esophageal junction cancer characterized by wild-type SMAD4 gene or gene product.

[0110] Cancers such as gastric cancer, esophageal cancer, gastroesophageal junction cancer, or head and neck cancer can be associated with the presence of mutations. Such mutations include mutations in known oncogenes such as PIK3CA, KRAS, BRAF, HRAS, MAP2K1, and NOTCH1. Oncogene mutations are often described as activating mutations or mutations that result in new functions. Another type of cancer mutation involves tumor suppressor genes such as TP53, MLH1, CDKN2A, and PTEN. Mutations in tumor suppressor genes are often inactivating.

[0111] In some aspects, the cancer has a mutation in one or more EGFR signaling pathway genes. In some aspects, the mutation is present in a gene whose expression product is active downstream of EGFR in the EGFR signaling pathway. In some aspects, the cancer has a mutation in a gene and the protein encoded by it selected from AKT1, KRAS, MAP2K1, NRAS, HRAS, PIK3CA, PTEN, EGFR and / or PLCG2. In some aspects, the cancer has a mutation in a gene encoding HRAS. In some aspects, the cancer does not have an activating mutation in KRAS and / or BRAF.

[0112] In some aspects, the cancer has a mutation in one or more WNT signaling pathway genes, in some aspects, APC, CREPPB, CUL1, EP300, SOX17, and / or TP53.

[0113] In some aspects, the mutation in the HRAS gene is a missense mutation, somatic mutation and / or oncogene driven mutation. In some aspects, HRAS comprises a mutation G12S in its protein sequence, or a G>A missense mutation, which results in a G>S amino acid change. In some aspects, the missense mutation G34A in the coding sequence (CDS) of the codon GGC of the HRAS gene. In some aspects, the cancer is oral squamous cell carcinoma or the squamous cell carcinoma of the buccal mucosa, and comprises the missense mutation G12S in HRAS.

[0114] Cancer can be caused by a mutation in the gene encoding MAP2K1. In certain aspects, the mutation in the MAP2K1 gene is a missense mutation, a somatic mutation, and / or an oncogene-driven mutation. In certain aspects, MAP2K1 comprises a mutation L375R, or a T>G missense mutation in its protein sequence, which results in an L>R amino acid change. In certain aspects, the missense mutation is T1124G in the coding sequence (CDS) of codon CTC of the MAP2K1 gene.

[0115] TP53 encodes a transcription factor that regulates multiple activities, including stress response and cell proliferation. Mutations in TP53 are associated with a variety of 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 many tumors, such as those from the breast and esophagus, respectively; as well as tumors of the ovary, prostate, breast, pancreas, stomach, colon / rectum, lung, esophagus, and bone (Priestly et al., Nature 2019 575:210-216). In particular, the therapeutic agents disclosed herein are suitable for treating cancers with TP53 mutations, particularly mutations that result in decreased TP53 expression or activity.

[0116] MLH1 (MutL homolog 1) encodes a protein involved in DNA mismatch repair and is a known tumor suppressor gene. Mutations in MLH1 are associated with a variety of cancers, including gastrointestinal cancer. 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 mutations occur 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 cancers such as colorectal cancer (Ohsawa et al., Molecular Medicine Reports 2009 2:887-891). In certain aspects, the therapeutic agents disclosed herein are useful for treating cancers harboring MLH1 mutations, particularly mutations that result in decreased MLH1 expression or activity.

[0117] PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α) encodes the 110kDa catalytic subunit of PI3K (phosphatidylinositol 3-kinase). Mutations in PIK3CA are associated with various cancers, including gastrointestinal cancer. As reported by the American Association for Cancer Research, PIK3CA is mutated in 12.75% of patients with malignant solid tumors. In particular, the PIK3CA H1047R mutation is present in 2.91% of all patients with malignant solid tumors, and PIK3CA E545K is present in 2.55% of all patients with malignant solid tumors (see, The AACR Project GENIE Consortium.AACR Project GENIE: powering precision medicine through an international consortium. CancerDiscovery.2017; 7(8): 818-831. Dataset Version 6). In certain aspects, the therapeutic agents disclosed herein are suitable for treating cancers with PIK3CA mutations, particularly oncogene mutations in PIK2CA or PIK3CA.

[0118] CDKN2A (cyclin-dependent kinase inhibitor 2A) encodes a protein that inhibits CDK4 and ARF. As reported by the American Association for Cancer Research, CDKN2A mutations occur in 22.21% of esophageal cancer patients, 28.7% of esophageal squamous cell carcinoma patients, and 6.08% of gastric adenocarcinoma patients. In particular, the CDKN2AW110Ter 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 aspects, the therapeutic agents disclosed herein are suitable for treating cancers having CDKN2A mutations (particularly mutations that result in reduced CDKN2A expression or activity).

[0119] PTEN (phosphatase and tensin homolog) encodes phosphatidylinositol 3,4,5-triphosphate 3-phosphatase. As reported by 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 (wherein Ter means terminal / stop codon) is present in 0.21% of all colorectal cancer patients (The AACR Project GENIEConsortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017; 7(8): 818-831. Dataset Version 6). In certain aspects, the therapeutic agents disclosed herein are suitable for treating cancers having PTEN mutations (particularly mutations that result in decreased PTEN expression or activity).

[0120] BRAF encodes the serine / threonine protein kinase B-Raf, which is involved in growth signal transduction. As reported by the American Association for Cancer Research, BRAF mutations occur in 1.91% of gastric cancer patients and in 1.93% of gastric adenocarcinoma patients. In particular, BRAF V600E mutations are present in 2.72% of cancer patients (see, The AACR Project GENIEConsortium.AACR Project GENIE:powering precision medicine through aninternational consortium.Cancer Discovery.2017;7(8):818-831. Dataset Version 6). In certain aspects, the therapeutic agents disclosed herein are suitable for treating cancers with BRAF mutations (particularly oncogene mutations in BRAF). However, in certain aspects, the therapeutic agents disclosed herein are suitable for treating gastric cancers that do not have the BRAF mutation V600E.

[0121] KRAS (Kirsten RAt Sarcoma) encodes a protein that belongs to the RAS / MAPK pathway. As reported by the American Association for Cancer Research, KRAS mutations occur in 14.7% of patients with malignant solid tumors, of which KRAS G12C is present in 2.28% of all patients with malignant solid tumors (see, The AACR Project GENIEConsortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017; 7(8): 818-831. Dataset Version 6). In certain aspects, the therapeutic agents disclosed herein are suitable for treating cancers having KRAS mutations (particularly oncogenic mutations in KRAS).

[0122] UGT1A1 (uridine diphosphate glucuronosyltransferase 1A1) and UGT1A8 (uridine diphosphate glucuronosyltransferase 1A8) encode enzymes of the glucuronidation pathway. Several polymorphisms that reduce enzyme activity are known to affect the metabolism and action of irinotecan. For example, the UGT1A1*6 allele (G71R polymorphism) has an allele frequency of approximately 0.13% in Chinese, Korean, and Japanese populations, and the UGT1A1*28 allele (a dinucleotide repeat polymorphism in the TATA sequence of the promoter region) is a risk factor for irinotecan-induced neutropenia. In certain aspects, the therapeutic agents disclosed herein are useful for treating cancers harboring UGT1A1 and / or UGT1A8 mutations, particularly mutations that result in reduced expression or activity of UGT1A1 and / or UGT1A8.

[0123] ATM (ataxia telangiectasia mutated protein) is a member of the serine-threonine family and orchestrates the cellular response to DNA damage by activating significant DNA repair and signaling pathways. Germline mutations in ATM are associated with ataxia telangiectasia, and somatic mutations in ATM are commonly observed in endometrial, colorectal, pancreatic, breast, and urothelial cancers.

[0124] Notch 1 (NOTCH1), also known as AOS5, hN1, AOVD1, and TAN1, is a gene that encodes a transmembrane protein that plays a role in multiple developmental processes and interactions between adjacent cells. Transmembrane proteins also serve as receptors for membrane-bound ligands. Fusions, missense mutations, nonsense mutations, silent mutations, frameshift deletions and insertions, and in-frame deletions and insertions have been observed in cancers such as esophageal cancer, hematopoietic and lymphoid carcinomas, and gastric cancer. NOTCH1 is altered in 4.48% of all cancers, with the highest prevalence of alterations in colon adenocarcinoma, lung adenocarcinoma, invasive ductal carcinoma, endometrial endometrioid adenocarcinoma, and skin squamous cell carcinoma. In head and neck squamous cell carcinoma, NOTCH1 is altered in approximately 16% of patients (The AACR Project GENIE Consortium. Cancer Discovery. 2017; 7(8):818-831).

[0125] HRAS (HGNC ID: 5173) gene products are involved in the activation of Ras protein signaling. Ras proteins bind GDP / GTP and have intrinsic GTPase activity. Somatic mutations in proto-oncogenes have been shown to be associated with bladder cancer, thyroid cancer, salivary duct cancer, epithelial-myoepithelial cancer, and renal cancer (Chiosea et al., Am. J. of Surg. Path. 39(6): 744-52; Chiosea et al., Head and Neck Path. 2014. 8(2): 146-50). In some embodiments, the therapeutic compounds disclosed herein are suitable for treating cancers having HRAS mutations (particularly oncogene mutations in HRAS, such as HRAS mutation G12S). The cancer is particularly HNSCC of the oral or buccal mucosa.

[0126] MAP2K1 (HGNC ID: 6840) belongs to the mitogen-activated protein kinases. It is active in MAP kinase signaling and encodes the protein dual-specificity mitogen-activated protein 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, with the highest prevalence of alterations in skin melanoma, lung adenocarcinoma, colon adenocarcinoma, melanoma, and invasive ductal carcinoma (The AACR Project GENIE Consortium. Cancer Discovery. 2017; 7(8): 818-831. Dataset Version 8). In some embodiments, the therapeutic compounds disclosed herein are suitable for treating cancers having MAP2K1 mutations (particularly the MAP2K1 mutation L375R).

[0127] In certain aspects, the present disclosure provides methods for treating cancers that have mutations in genes encoding TP53, MLH1, PIK3CA, CDKN2A, UGT1A, UGT1A8, BRAF, PTEN, and KRAS. In certain aspects, the cancer has one or more mutations selected from the following: TP53 R196T; TP53 R342T; TP53 R248Q; MLH1 V384D; PIK3CA H1047R; PIK3CA E545K; CDKN2A W110T; UGT1A1 G71R; UGT1A8 G71R; and KRAS G12C. In certain aspects, the cancer is KRAS wild type. Alternatively, the present disclosure provides methods for treating cancers that have mutations in the gene encoding ATM, particularly mutation W57T. In particular, the present disclosure provides methods for treating esophageal cancer (particularly ESCC) having a mutation (particularly mutation W57T) in the gene encoding ATM.

[0128] In certain aspects, the cancer has a mutation in the gene encoding TP53, for example, wherein the mutation is R342T, and the cancer has a mutation in the gene encoding MLH1, for example, wherein the mutation is V384D. In certain aspects, the cancer has a mutation in the gene encoding TP53. In certain aspects, the mutation is R248Q. In certain aspects, the cancer has a mutation in the gene encoding PIK3CA. In certain aspects, the mutation is H1047R. In certain aspects, the cancer has a mutation in the gene encoding CDKN2A, in certain aspects, the mutation is W110T. In certain aspects, the cancer has a mutation in the gene encoding UGT1A1, in certain aspects, the mutation is G71R, and the cancer has a mutation in the gene encoding UGT1A8, in certain aspects, the mutation is G71R. In certain aspects, the cancer is esophageal cancer. In certain aspects, the cancer is esophageal squamous cell carcinoma (ESCC).

[0129] In certain aspects, the cancer has a mutation in the gene encoding BRAF. However, in certain aspects, the cancer does not have the V600E mutation in the gene encoding BRAF, and in certain aspects, the cancer does not have the R130T mutation in the gene encoding PTEN. In certain aspects, the cancer has a mutation in the gene encoding KRAS, in certain aspects, the mutation is G12C, in the gene encoding UGT1A1, in certain aspects, the mutation is G71R, and in the gene encoding UGT1A8, in certain aspects, the mutation is G71R. In certain aspects, the cancer has a mutation in the gene encoding UGT1A1, in certain aspects, the mutation is G71R, and in the gene encoding UGT1A8, in certain aspects, the mutation is G71R. In certain aspects, the cancer has a mutation in PIK3CA, in certain aspects, the mutation is E545K. In certain aspects, the cancer is gastric cancer.

[0130] In some aspects, the antibodies disclosed herein, or their functional portions, derivatives, and / or analogs, are multispecific antibodies. In some aspects, the antibody is a bispecific antibody. In some aspects, the multispecific or bispecific antibody, or its functional portion, derivative, and / or analog, comprises a first variable domain that binds to the extracellular portion of an epidermal growth factor (EGF) receptor, and a second variable domain that does not bind to EGFR in some aspects. In some aspects, the antibody, or its functional portion, derivative, and / or analog, binds to EGFR in a monovalent manner. Simultaneously, in some aspects, the multispecific or bispecific antibody, or its functional portion, derivative, and / or analog, comprises a second variable domain that binds to LGR5.

[0131] In some aspects, EGFR is human EGFR. The EGFR bound by the antibodies of the present disclosure or their functional portions, derivatives and / or analogs includes wild-type EGFR and EGFR with oncogene-driven mutations. In some aspects, the oncogene-driven mutation is an activating EGFR mutation. In some aspects, this mutation does not conformationally alter the epitope bound by the antibodies of the present disclosure. In certain aspects, the EGFR mutations disclosed herein include the following mutations: for example, exon 18 mutations including G719A, G719C, 2E709_T710D, E709A, G719S; exon 19 deletion mutations including deletion of LREA or VAIKEL; exon 19 point mutations G735S, P753L, L747S, D761Y; in-frame insertion mutations of 1 to 7 amino acids of exon 20; exon 20 point mutations including V765A, T783A, V774A, S784P, V769M, T790M; exon 21 mutations including L858R, T854A, A871E, L861A, L861C, L861S, V843I or P848L. The antibodies disclosed herein bind to epitopes that are not in close proximity to the mutations. In particular, the EGFR mutation is S492R, which results in loss of binding of cetuximab to EGFR.The antibodies of the present disclosure bind to an epitope that is different from the epitope recognized by cetuximab.

[0132] Without being bound by any theory, it is believed that Figure 2 Amino acid residues I462, G465, K489, I491, N493, and C499 described in are involved in epitope binding by the antibodies of the present disclosure. In certain aspects, involvement in binding is determined by observing a decrease in binding of the variable domain to EGFR having one or more amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A, and C499A.

[0133] In one aspect, 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 at Figure 2 In some aspects, the variable domains of the epitope within amino acid residues 420 to 480 of the sequence shown in . In some aspects, the combination of the variable domains and EGFR is weakened by replacing one or more of the following amino acid residues in EGFR: I462A, G465A, K489A, I491A, N493A, and C499A. In some aspects, the combination of the antibody and human EGFR interferes with the combination of EGF and the receptor. In some aspects, the epitope on EGFR is a conformational epitope. In one aspect, the epitope is located at Figure 2 within amino acid residues 420 to 480 of the sequence shown in, or within Figure 2In certain aspects, the epitope is located within amino acid residues 430 to 480 of the sequence shown in . Figure 2 within amino acid residues 438 to 469 of the sequence shown in .

[0134] Without being bound by any theory, it is believed that the epitope contact residues, i.e., the positions where the variable domain contacts human EGFR, may be I462, K489, I491, and N493. Amino acid residues G465 and C499 may be indirectly involved in the binding of the antibody to EGFR.

[0135] In certain aspects, the second variable domain binds to LGR5. In certain aspects, LGR5 is human LGR5. The multispecific or bispecific antibodies or functional parts, derivatives and / or analogs thereof as described herein comprise a variable domain that binds to the extracellular portion of the human epidermal growth factor (EGF) receptor, and in certain aspects, the variable domain binds to human LGR5.

[0136] In certain aspects, an antibody or functional portion, derivative and / or analog thereof as described herein comprises a variable domain that binds to the extracellular portion of the epidermal growth factor (EGF) receptor and interferes with the binding of EGF to the receptor, and a variable domain that binds to LGR5, wherein the interaction of the antibody with LGR5 on cells expressing LGR5 does not block the binding of R spondin (RSPO) to LGR5. Methods for determining whether an antibody blocks or does not block the binding of R spondin to LGR5 are described in WO2017069528, which is incorporated herein by reference.

[0137] Where accession numbers or alternative names for proteins / genes are given herein, they are primarily given to provide a further means of identifying the protein being referred to as a target. The actual sequence of the target protein to which the antibodies of the invention bind may vary, for example due to mutations and / or alternative splicing in the encoding gene, such as those that occur in some cancers or their analogous diseases. The target protein is bound by the antibody as long as the epitope is present in the protein and the epitope is accessible to the antibody.

[0138] In some aspects, antibodies as described herein or their functional parts, derivatives and / or analogs interfere with the binding of a part to EGFR. As used herein, the term "interference binding" means that the binding of an antibody or its functional part, derivative and / or analog to EGFR competes with the binding of a part to the EGF receptor. An antibody or its functional part, derivative and / or analog can weaken the binding of a part, displace the part when it has been bound to the EGF receptor, or it can, for example, at least partially prevent the part from being bound to the EGF receptor by steric hindrance.

[0139] In some aspects, EGFR antibodies as disclosed herein inhibit EGFR ligand-induced signal transduction, respectively, which is measured as the growth of ligand-induced BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058), or the cell death of ligand-induced A431 cells (ATCC CRL-1555). EGFR can bind to a variety of ligands and stimulate the growth of the BxPC3 cells or BxPC3-luc2 cells mentioned. In the presence of an EGFR ligand, the growth of BxPC3 or BxPC3-luc2 cells is stimulated. The EGFR ligand-induced BxPC3 cell growth can be measured by comparing the cell growth in the absence and presence of a ligand. The preferred EGFR ligand for measuring the EGFR ligand-induced BxPC3 or BxPC3-luc2 cell growth is EGF. In some aspects, the ligand-induced growth is measured using a saturated amount of ligand. In some aspects, EGF is used in an amount of 100 ng / ml culture medium. In certain aspects, the EGF is EGF R&D System, Catalog Nos. 396-HB and 236-EG (see also WO 2017 / 069628; which is incorporated herein by reference).

[0140] In some aspects, EGFR antibodies as disclosed herein inhibit the growth of EGFR ligand-induced BxPC3 cells (ATCCCRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058). EGFR can bind to a variety of ligands and stimulate the growth of the BxPC3 cells or BxPC3-luc2 cells mentioned. In the presence of a ligand, the growth of BxPC3 or BxPC3-luc2 cells is stimulated. The BxPC3 cell growth induced by EGFR ligand can be measured by comparing the cell growth in the absence and presence of a ligand. In some aspects, the EGFR ligand for measuring the BxPC3 or BxPC3-luc2 cell growth induced by EGFR ligand is EGF. In some aspects, the growth of ligand-induced BxPC3 or BxPC3-luc2 cells is measured using a saturated amount of ligand. In some aspects, EGF is used in an amount of 100 ng / ml culture medium. In certain aspects, the EGF is EGF of R&D Systems, Catalog Nos. 396-HB and 236-EG (see also WO 2017 / 069628; which is incorporated herein by reference).

[0141] For the avoidance of doubt, as used herein, reference to cell growth means a change in the number of cells. Growth inhibition means a decrease in the number of cells that would otherwise be obtained. Growth enhancement means an increase in the number of cells that would otherwise be obtained. Cell growth generally means cell proliferation.

[0142] Whether an antibody as described herein inhibits signal transduction or inhibits growth in a multispecific format is determined in certain aspects by methods as described above using a monospecific monovalent or monospecific bivalent form of the antibody. In certain aspects, such antibodies have a binding site for a receptor whose signal transduction is to be determined. A monospecific monovalent antibody can have a variable domain with an unrelated binding specificity, such as a tetanus toxoid specificity. In certain aspects, 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.

[0143] Merus in its Multispecific antibodies targeting EGFR and LGR5 (leucine-rich repeats G protein-coupled receptor) have been developed in the antibody program. The efficacy of such multispecific antibodies has been evaluated in vitro and in vivo using patient-derived CRC organoids and mouse PDX models, respectively (see, e.g., WO2017 / 069628; which is incorporated herein by reference). Multispecific antibodies targeting EGFR and LGR5 have been shown to inhibit tumor growth. The efficacy of such inhibitory antibodies has been shown to be correlated with LGR5 RNA expression levels in cells from cancer. In certain aspects, the multispecific antibodies targeting EGFR and LGR5 are as described in WO2017 / 069628.

[0144] The antibodies or functional parts, derivatives and / or analogs thereof as described herein comprise a variable domain that binds to the extracellular portion of LGR5. In certain aspects, the variable domain that binds to the extracellular portion of LGR5 binds to a region located at Figure 1 An epitope within amino acid residues 21 to 118 of a sequence of , wherein amino acid residues D43, G44, M46, F67, R90 and F91 of the sequence are involved in binding of the antibody to the epitope.

[0145] In certain aspects, the LGR5 variable domain is a variable domain wherein amino acid residue substitutions in LGR5 of one or more of D43A, G44A, M46A, F67A, R90A, and F91A reduce binding of the variable domain to LGR5.

[0146] In certain aspects, an epitope on the extracellular portion of LGR5 is located at Figure 1 In some aspects, the epitope is an epitope wherein the binding of the LGR5 variable domain to LGR5 is attenuated by substitution of one or more of the following amino acid residues in LGR5: D43A, G44A, M46A, F67A, R90A, and F91A.

[0147] The present 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 a region located on LGR5. Figure 1 The epitope is within amino acid residues 21 to 118 of the sequence.

[0148] In some aspects, the epitope on LGR5 is a conformational epitope. In some aspects, the epitope is located at Figure 1 In certain aspects, the binding of the antibody to LGR5 is diminished by substitution of one or more of the following amino acid residues: D43A, G44A, M46A, F67A, R90A, and F91A.

[0149] Without being bound by any theory, it is believed that Figure 1The LGR5 M46, F67, R90, and F91 are contact residues of the variable domain as indicated above, i.e., the antigen binding site of the variable domain that binds the LGR5 epitope. The fact that the amino acid residue substitutions D43A and G44A weaken the binding of the antibody may be because these residues are also contact residues. However, it is also possible that these amino acid residue substitutions induce (slight) modifications in the conformation of the portion of LGR5 that has one or more of the other contact residues (i.e., at positions 46, 67, 90, or 91), and that this conformational change weakens the antibody binding. The epitope is characterized by the amino acid substitutions mentioned. Whether an antibody binds to the same epitope can be determined in various ways. In an exemplary method, CHO cells express LGR5 or an alanine substitution mutant on the cell membrane, e.g., a mutant comprising one or more of the substitutions M46A, F67A, R90A, or F91A. The test antibody is contacted with the CHO cells, and the binding of the antibody to the cells is compared. If the test antibody binds to LGR5 and, to a lesser extent, to LGR5 with an M46A, F67A, R90A, or F91A substitution, then the test antibody binds to the epitope. Binding is preferably compared to a panel of mutants in which each mutant comprises a single alanine residue substitution. Such binding studies are well known in the art. Typically, the panel includes single alanine substitution mutants covering substantially all amino acid residues. For LGR5, the panel need only cover the extracellular portion of the protein and, when using cells, of course, the portion that is guaranteed to associate with the cell membrane. Expression of a particular mutant may be impaired, but this is easily detected using one or more LGR5 antibodies that bind to different regions. If expression is also reduced against these control antibodies, then protein levels or folding on the membrane are impaired for this particular mutant. The binding characteristics of the test antibody to this panel readily identify whether the test antibody exhibits reduced binding to mutants with an M46A, F67A, R90A, or F91A substitution, and therefore whether the test antibody is an antibody of the invention. Reduced binding to mutants with M46A, F67A, R90A, or F91A substitutions also identified an epitope located at Figure 1 MF5816. In certain aspects, the group includes both D43A and G44A substitution mutants. Antibodies with the VH sequence of MF5816 exhibit reduced binding to these substitution mutants.

[0150] Without being bound by any theory, it is believed that Figure 2The amino acid residues I462, G465, K489, I491, N493, and C499 participate in epitope binding by antibodies comprising variable domains as indicated above. In certain aspects, participation in binding is determined by observing reduced 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 or an alanine substitution mutant, such as one or more substitutions selected from I462A, G465A, K489A, I491A, N493A, and C499A, on the cell membrane. A test antibody is contacted with the CHO cells, and binding of the antibody to the cells is compared. If the test antibody binds to EGFR and binds to EGFR with I462A, G465A, K489A, I491A, N493A, and C499A substitutions to a lesser extent, the test antibody binding epitope is obtained. Preferably, the binding of a group of mutants in which each mutant comprises an alanine residue substitution is compared. Such binding studies are well known in the art. Typically, the group comprises single alanine substitution mutants covering substantially all amino acid residues. For EGFR, the group only needs to cover the extracellular portion of the protein, and when using cells, of course, the portion that is guaranteed to associate with the cell membrane is covered. The expression of a specific mutant may be impaired, but this is easily detected by one or more EGFR antibodies that are bound to different regions. If, for these control antibodies, expression is also reduced, then for this specific mutant, the protein level or folding on the membrane is impaired. Characterization of the binding of a test antibody to this panel readily identifies whether a test antibody exhibits reduced binding to mutants having I462A, G465A, K489A, I491A, N493A, and C499A substitutions.

[0151] In one aspect, 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 at Figure 2 In some aspects, the variable domains of the epitope within amino acid residues 420 to 480 of the sequence shown in FIG. In some aspects, the binding of the variable domains to EGFR is weakened by replacing one or more of the following amino acid residues in EGFR: I462A, G465A, K489A, I491A, N493A, and C499A. In some aspects, the binding of the antibody to human EGFR interferes with the binding of EGF to the receptor. In some aspects, the epitope on EGFR is a conformational epitope. In one aspect, the epitope is located at Figure 2 Within amino acid residues 420 to 480 of the sequence shown, e.g. Figure 2 In certain aspects, the epitope is located at Figure 2 Within 438 to 469 of the sequence shown.

[0152] Without being bound by any theory, it is believed that the epitope contact residues, i.e., the positions where the variable domain contacts human EGFR, may be I462, K489, I491, and N493. Amino acid residues G465 and C499 may be indirectly involved in the binding of the antibody to EGFR.

[0153] In certain aspects, the variable domain that binds to human EGFR is a variable domain having a heavy chain variable region that comprises at least the CDR3 sequence of VH of MF3755 as described in Figure 3, or a CDR3 sequence that differs from the CDR3 sequence of VH of MF3755 as described in Figure 3 in at most three, or at most two, or no more than one amino acid.

[0154] In certain aspects, the variable domain that binds to human EGFR is a variable domain having a heavy chain variable region, which comprises at least the CDR1, CDR2 and CDR3 sequences of the VH of MF3755 as shown in Figure 3; or the CDR1, CDR2 and CDR3 sequences of the VH of MF3755 as shown in Figure 3 and has at most three, or at most two, or at most one amino acid substitution.

[0155] In certain aspects, the variable domain that binds to human EGFR is a variable domain having a heavy chain variable region comprising the sequence of the VH chain of MF3755 as shown in Figure 3; or the amino acid sequence of the VH chain of MF3755 as shown in Figure 3, which has up to 15 (or in certain aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in certain aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or a combination thereof relative to the VH chain of MF3755.

[0156] In certain aspects, the present disclosure provides 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 heavy chain variable region of the variable domain comprises a CDR3 sequence of at least an EGFR-specific heavy chain variable region selected from the group consisting of MF3370; MF3755; MF4280, or MF4289 as shown in Figure 3 , or wherein the heavy chain variable region of the variable domain comprises a heavy chain CDR3 sequence that differs from a CDR3 sequence of a VH selected from the group consisting of MF3370; MF3755; MF4280, or MF4289 as shown in Figure 3 in at most three, at most two, or at most one amino acid. In certain aspects, the variable domain comprises a heavy chain variable region comprising a CDR3 sequence of at least MF3370; MF3755; MF4280, or MF4289 as shown in Figure 3 .

[0157] In certain aspects, the variable domain comprises a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 as shown in Figure 3 , or a heavy chain variable region comprising at least CDR1, CDR2, and CDR3 sequences that differ from the CDR1, CDR2, and CDR3 sequences of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 as shown in Figure 3 in at most three, at most two, or at most one amino acid sequence. In certain aspects, the variable domain comprises a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of MF3370, MF3755, MF4280, or MF4289 as shown in Figure 3 . In certain aspects, the heavy chain variable region is MF3755. In certain aspects, the heavy chain variable region is MF4280.

[0158] In certain aspects, 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 has a CDR3, CDR1, CDR2 and CDR3 and / or VH sequence as indicated above, has a variable domain that binds to LGR5, the variable domain comprising at least one selected from the group consisting of MF5790; MF5803; MF5805; MF5808; MF5809; MF5810 as shown in FIG. 4; MF5816; MF5817; or MF5818, or a heavy chain CDR3 sequence that differs from a CDR3 sequence of a VH selected from the group consisting of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in Figure 3 in at most three, or at most two, or at most one amino acid. In certain aspects, the variable domain comprises a heavy chain variable region comprising at least the CDR3 sequence of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as shown in Figure 3 .

[0159] In certain aspects, the LGR5 variable domain comprises a heavy chain variable region comprising 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 as shown in Figure 3, or heavy chain CDR1, CDR2, and CDR3 sequences that differ from 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 as shown in Figure 3 in at most three, or at most two, or at most one amino acid. In certain aspects, the variable domain comprises a heavy chain variable region comprising 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 aspects, the heavy chain variable region is MF5790; MF5803; MF5814; MF5816; MF5817; or MF5818. In certain aspects, the heavy chain variable region is MF5790; MF5814; MF5816; and MF5818. In certain aspects, the heavy chain variable region is MF5814, MF5818, or MF5816. In certain aspects, the heavy chain variable region is MF5816. In certain aspects, the heavy chain variable region is MF5818.

[0160] It has been shown that antibodies comprising one or more variable domains comprising the heavy chain variable region MF3755 or one or more CDRs thereof are more effective when used to inhibit the growth of EGFR ligand-responsive cancers or cells. In the case of bispecific or multispecific antibodies, an antibody arm comprising a variable domain comprising the heavy chain variable region MF3755 or one or more CDRs thereof is well combined with an arm comprising a variable domain comprising the heavy chain variable region MF5818 or one or more CDRs thereof.

[0161] The VH chain of the variable domain that binds to EGFR or LGR5 can have one or more amino acid substitutions relative to the sequence shown in Figure 3. In certain aspects, the VH chain has the amino acid sequence of the EGFR or LGR5 VH of Figure 3, which has up to 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in certain aspects 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or combinations thereof relative to the VH chain sequence of Figure 3.

[0162] The CDR sequence may have one or more amino acid residue replacements relative to the CDR sequence in the figure. Such one or more replacements are, for example, carried out for optimization purposes, for example, in order to improve the binding strength or stability of the antibody. Optimization is, for example, carried out by a mutagenesis program, wherein preferably after testing the stability and / or binding affinity of the resulting antibody, the improved EGFR-specific CDR sequence or LGR5-specific CDR sequence is selected. Those familiar with the art are fully capable of producing antibody variants comprising at least one changed CDR sequence according to the present invention. For example, retained amino acid replacements can be applied. Examples of retained amino acid replacements include replacing a hydrophobic residue (e.g., isoleucine, valine, leucine or methionine) with another hydrophobic residue, and replacing a polar residue with another polar residue, such as replacing arginine with lysine, glutamic acid with aspartic acid or glutamine with asparagine.

[0163] In some aspects, up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in some aspects, 1, 2, 3, 4, or 5) amino acid substitutions mentioned in a VH or VL as specified herein are reserved amino acid substitutions. In some aspects, amino acid insertions, deletions, and substitutions in a VH or VL as specified herein are not present in the CDR3 region. In some aspects, the amino acid insertions, deletions, and substitutions mentioned are also not present in the CDR1 and CDR2 regions. In some aspects, the amino acid insertions, deletions, and substitutions mentioned are also not present in the FR4 region.

[0164] In certain aspects, the up to 15 (or in certain aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain aspects, 1, 2, 3, 4, or 5) amino acid substitutions mentioned are conservative amino acid substitutions. In certain aspects, the insertion, deletion, substitution, or combination thereof is not in the CDR3 region of the VH chain, in certain aspects, is not in the CDR1, CDR2, or CDR3 region of the VH chain, and in certain aspects, is not in the FR4 region.

[0165] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in certain aspects, a variable domain that binds to the extracellular portion of LGR5, comprising

[0166] - the amino acid sequence of the VH chain MF3755 as shown in Figure 3; or

[0167] - the amino acid sequence of the VH chain MF3755 as shown in Figure 3, which has up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in some aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof relative to the VH; and

[0168] The VH chain of the variable domain that binds to LGR5 comprises

[0169] - the amino acid sequence of the VH chain MF5790 as shown in Figure 3; or

[0170] - The amino acid sequence of the VH chain MF5790 as shown in Figure 3, which has up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in some aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof relative to the VH.

[0171] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in certain aspects, a variable domain that binds to the extracellular portion of LGR5, comprising

[0172] - the amino acid sequence of the VH chain MF3755 as shown in Figure 3; or

[0173] - the amino acid sequence of the VH chain MF3755 as shown in Figure 3, which has up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in some aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof relative to the VH; and

[0174] The VH chain of the variable domain that binds to LGR5 comprises

[0175] - the amino acid sequence of the VH chain MF5803 as shown in Figure 3; or

[0176] - The amino acid sequence of the VH chain MF5803 as shown in Figure 3, which has up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in some aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof relative to the VH.

[0177] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in certain aspects, a variable domain that binds to the extracellular portion of LGR5, comprising

[0178] - the amino acid sequence of the VH chain MF3755 as shown in Figure 3; or

[0179] - the amino acid sequence of the VH chain MF3755 as shown in Figure 3, which has up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in some aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof relative to the VH; and

[0180] The VH chain of the variable domain that binds to LGR5 comprises

[0181] - the amino acid sequence of the VH chain MF5814 as shown in Figure 3; or

[0182] - The amino acid sequence of the VH chain MF5814 as shown in Figure 3, which has up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in some aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof relative to the VH.

[0183] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in certain aspects, a variable domain that binds to the extracellular portion of LGR5, comprising

[0184] - the amino acid sequence of the VH chain MF3755 as shown in Figure 3; or

[0185] - the amino acid sequence of the VH chain MF3755 as shown in Figure 3, which has up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in some aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof relative to the VH; and

[0186] The VH chain of the variable domain that binds to LGR5 comprises

[0187] - the amino acid sequence of the VH chain MF5816 as shown in Figure 3; or

[0188] - The amino acid sequence of the VH chain MF5816 as shown in Figure 3, which has up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in some aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof relative to the VH.

[0189] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in certain aspects, a variable domain that binds to the extracellular portion of LGR5, comprising

[0190] - the amino acid sequence of the VH chain MF3755 as shown in Figure 3; or

[0191] - the amino acid sequence of the VH chain MF3755 as shown in Figure 3, which has up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in some aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof relative to the VH; and

[0192] The VH chain of the variable domain that binds to LGR5 comprises

[0193] - the amino acid sequence of the VH chain MF5817 as shown in Figure 3; or

[0194] - The amino acid sequence of the VH chain MF5817 as shown in Figure 3, which has up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in some aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof relative to the VH.

[0195] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and, in certain aspects, a variable domain that binds to the extracellular portion of LGR5, comprising

[0196] - the amino acid sequence of the VH chain MF3755 as shown in Figure 3; or

[0197] - the amino acid sequence of the VH chain MF3755 as shown in Figure 3, which has up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in some aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof relative to the VH; and

[0198] The VH chain of the variable domain that binds to LGR5 comprises

[0199] - the amino acid sequence of the VH chain MF5818 as shown in Figure 3; or

[0200] - The amino acid sequence of the VH chain MF5818 as shown in Figure 3, which has up to 15 (or in some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in some aspects, 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or combinations thereof relative to the VH.

[0201] Additional 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 IGKV1-39 / IGKJ1 VL regions (De Kruif et al., Biotechnol Bioeng. 2010 (106) 741-50), and collections of VH regions that incorporate amino acid substitutions into the amino acid sequences of the EGFR or LGR5 VH regions disclosed herein, as previously described (e.g., WO2017 / 069628). Phage encoding Fab regions that bind to EGFR or LGR5 can be selected and analyzed by flow cytometry and sequenced to identify variants with amino acid substitutions, insertions, deletions, or additions that retain antigen binding.

[0202] The light chain variable regions of the VH / VL EGFR and LGR5 variable domains of the EGFR / LGR5 antibody can be the same or different. In certain aspects, the VL region of the VH / VL EGFR variable domain of the EGFR / LGR5 antibody is similar to the VL region of the VH / VL LGR5 variable domain. In certain aspects, the VL regions in the first and second VH / VL variable domains are the same.

[0203] In certain aspects, the light chain variable region of one or both of the VH / VL variable domains of the EGFR / LGR5 antibody comprises a common light chain variable region. In certain aspects, the common light chain variable region of one or both of the VH / VL variable domains comprises a germline IgVκ1-39 variable region V segment. In certain aspects, the light chain variable region of one or both of the VH / VL variable domains comprises a 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; IGKV1-39. The gene external ID is HGNC: 5740; Entrez gene: 28930; Ensembl: ENSG00000242371. The amino acid sequence of a suitable V region is provided at Figure 4 In certain aspects, the V region can be combined with one of the five J regions. In certain aspects, the J regions are jk1 and jk5, and the junction sequences are indicated as IGKV1-39 / jk1 and IGKV1-39 / jk5; alternative names are IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (named according to the IMGT database global information network imgt.org). In certain aspects, the light chain variable region of one or both of the VH / VL variable domains comprises a kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ1*05 (described in Figure 4 middle).

[0204] In certain aspects, the light chain variable region of one or both of the VH / VL variable domains of the EGFR / LGR5 bispecific antibody comprises a LCDR1 comprising the amino acid sequence QSISSY (described in Figure 4 ), LCDR2 comprising the amino acid sequence AAS (described in Figure 4 ) and LCDR3 comprising the amino acid sequence QQSYSTP (described in Figure 4 In certain aspects, the light chain variable region of one or both of the VH / VL variable domains of the EGFR / LGR5 antibody comprises a LCDR1 comprising the amino acid sequence QSISSY (described in Figure 4), LCDR2 comprising the amino acid sequence AASLQS (described in Figure 4 ) and LCDR3 comprising the amino acid sequence QQSYSTP (described in Figure 4 middle).

[0205] In certain aspects, the light chain variable region of one or both of the VH / VL variable domains of the EGFR / LGR5 bispecific antibody comprises a LCDR1 comprising the amino acid sequence QSISSY (described in Figure 4 ), LCDR2 comprising the amino acid sequence AAS (described in Figure 4 ) and LCDR3 comprising the amino acid sequence QQSYSTPPT (described in Figure 4 In certain aspects, the light chain variable region of one or both of the VH / VL variable domains of the EGFR / LGR5 antibody comprises a LCDR1 comprising the amino acid sequence QSISSY (described in Figure 4 ), LCDR2 comprising the amino acid sequence AASSLQS (described in Figure 4 ) and LCDR3 comprising the amino acid sequence QQSYSTPPT (described in Figure 4 The CDR sequences are numbered according to the IMGT system.

[0206] In certain aspects, one or both of the VH / VL variable domains of the EGFR / LGR5 antibody comprises a light chain variable region comprising Figure 4 In some aspects, one or both of the VH / VL variable domains of the EGFR / LGR5 antibody comprises a light chain variable region comprising an amino acid sequence that is at least 90%, in some aspects at least 95%, in some aspects at least 97%, in some aspects at least 98%, in some aspects at least 99% identical or in some aspects 100% identical to the amino acid sequence set forth in . Figure 4 An amino acid sequence that is at least 90%, in some aspects at least 95%, in some aspects at least 97%, in some aspects at least 98%, in some aspects at least 99% identical, or in some aspects 100% identical to the amino acid sequence set forth in.

[0207] For example, the variable light chain of one or both of the VH / VL variable domains of the EGFR / LGR5 antibody is Figure 4The sequence in can have 0 to 10, or in some aspects 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof. In some aspects, the light chain variable region of one or both of the VH / VL variable domains of the EGFR / LGR5 antibody comprises 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, in some aspects 0 to 3, in some aspects 0 to 2, in some aspects 0 to 1, and in some aspects 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof relative to the specified amino acid sequence.

[0208] At the same time, the light chain variable region of one or both of the VH / VL variable domains of the EGFR / LGR5 antibody may comprise Figure 4 In some aspects, the two VH / VL variable domains of the EGFR / LGR5 antibody comprise the same VL region. In some aspects, the VL of the two VH / VL variable domains of the EGFR / LGR5 bispecific antibody comprises Figure 4 In certain aspects, the VL of the two VH / VL variable domains of the EGFR / LGR5 bispecific antibody comprises Figure 4 The described amino acid sequence.

[0209] In certain aspects, the EGFR / LGR5 antibodies described herein are bispecific antibodies having two variable domains, one that binds EGFR and the other that binds LGR5, as described herein. The EGFR / LGR5 bispecific antibodies for use in the methods disclosed herein can be provided in a variety of formats. Many different formats 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), each of which is incorporated herein by reference. For example, bispecific antibody formats, which are not typical antibodies with two VH / VL combinations, have at least one variable domain comprising both a heavy chain variable region and a light chain variable region. This variable domain can be linked to a single-chain Fv fragment, a monobody, a VH, or a Fab fragment that provides a second binding activity.

[0210] In some aspects, the EGFR / LGR5 bispecific antibodies used in the methods provided herein are generally human IgG subclasses (e.g., IgG1, IgG2, IgG3, IgG4). In some aspects, the antibody is a human IgG1 subclass. Full-length IgG antibodies are preferred due to their favorable half-life and for reasons of low immunogenicity. Therefore, in some aspects, the EGFR / LGR5 bispecific antibodies are full-length IgG molecules. In some aspects, the EGFR / LGR5 bispecific antibodies are full-length IgG1 molecules.

[0211] Therefore, in some aspects, the EGFR / LGR5 bispecific antibody comprises a crystallizable fragment (Fc). In some aspects, the Fc of the EGFR / LGR5 bispecific antibody is composed of a human constant region. The constant region or Fc of the EGFR / LGR5 bispecific antibody may contain one or more, or no more than 10, or no more than 5 amino acid differences from naturally occurring human antibody constant regions. For example, each Fab arm of the bispecific antibody may further include a modified Fc region comprising a modification that promotes bispecific antibody formation, promotes stability and / or other features described herein.

[0212] Bispecific antibodies are typically produced by cells that express nucleic acids encoding the antibodies. Thus, in certain aspects, the bispecific EGFR / LGR5 antibodies disclosed herein are produced by providing cells comprising one or more nucleic acids encoding the heavy and light chain variable regions and constant regions of the bispecific EGFR / LGR5 antibodies. In certain aspects, the cells are animal cells, such as mammalian cells, or primate cells, and in certain aspects human cells. Suitable cells are any cells capable of containing and preferably capable of producing the EGFR / LGR5 bispecific antibodies.

[0213] Cells suitable for antibody production are known in the art and include hybridoma cells, Chinese hamster ovary (CHO) cells, NSO cells or PER-C6 cells. Various institutions and companies have developed cell lines for large-scale production of antibodies, for example for clinical use. Non-limiting examples of such cell lines are CHO cells, NSO cells or PER.C6 cells. In particular, the cells are human cells. Preferably, the cells are transformed with the adenovirus E1 region or its functional equivalent. A preferred example of such cell lines is the PER.C6 cell line or its equivalent. In particular, the cells are CHO cells or variants thereof. Preferably, the variant utilizes a glutamine synthetase (GS) vector system to express the antibody. In certain aspects, the cells are CHO cells.

[0214] In some aspects, cells express different light chains and heavy chains constituting EGFR / LGR5 bispecific antibodies. In some aspects, cells express two different heavy chains and at least one light chain. In some aspects, cells express "common light chains" as described herein to reduce the number of different antibody species (combinations of different heavy and light chains). For example, methods known in the art for producing bispecific IgG (WO2013 / 157954; incorporated herein by reference) are used, and each VH region is cloned into an expression vector together with a rearranged human IGKV1 39 / IGKJ1 (huVκ1 39) light chain, which has previously been shown to be capable of pairing with more than one heavy chain, thereby producing antibodies with diverse specificities, which promotes the production of bispecific molecules (De Kruif et al., J. Mol. Biol. 2009 (387) 548 58; WO2009 / 157771).

[0215] Antibody-producing cells expressing a common light chain and an equal amount of two heavy chains typically produce 50% of each of the bispecific antibodies and 25% of the monospecific antibodies (i.e., having the same heavy-light chain combination). Several methods have been disclosed to prioritize the production of bispecific antibodies over the production of single monospecific antibodies. This is typically achieved by modifying the constant region of the heavy chain so that it favors heterodimerization (i.e., heavy chain dimerization with another heavy / light chain combination) relative to homodimerization. In some aspects, the bispecific antibodies of the present invention comprise two different immunoglobulin heavy chains having a compatibility heterodimerization domain. Various compatibility heterodimerization domains have been described in the art. In some aspects, the compatibility heterodimerization domain is a compatibility immunoglobulin heavy chain CH3 heterodimerization domain. The present technology describes various ways in which such heterodimerization of heavy chains can be achieved.

[0216] A preferred method for producing EGFR / LGR5 bispecific antibodies is disclosed in US 9,248,181 and US 9,358,286. Specifically, the preferred mutation that substantially only produces bispecific full-length IgG molecules is the amino acid substitution L351K and T366K (EU numbering) in the first CH3 domain ("KK variant" heavy chain), and the amino acid substitution L351D and L368E ("DE variant" heavy chain) in the second domain, or vice versa. As previously described, DE variants and KK variants preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of the DE variant heavy chain (DEDE homodimer) or homodimerization of the KK variant heavy chain (KKKK homodimer) is due to the strong repulsion between the charged residues in the CH3-CH3 interface between the same heavy chains.

[0217] Thus, in certain aspects, the heavy chain / light chain combination comprising a variable domain that binds to EGFR comprises a DE variant of the heavy chain.In certain aspects, the heavy chain / light chain combination comprising a variable domain that binds to LGR5 comprises a KK variant of the heavy chain.

[0218] Any suitable assay can be used to test the binding of candidate EGFR / LGR5 IgG bispecific antibodies. For example, binding to membrane-expressed EGFR or LGR5 on CHO cells can be assessed by flow cytometry (according to a FACS procedure as previously described in WO2017 / 069628). In certain aspects, the 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. The binding to CHO cells is compared with CHO cells that have not been transfected with expression cassettes for EGFR and / or LGR5. The binding of candidate bispecific IgG1 to EGFR is determined using CHO cells transfected with an EGFR expression construct; LGR5 monospecific antibodies and EGFR monospecific antibodies are included in the assay, and an unrelated IgG1 isotype control mAb is used as a control (e.g., an antibody that binds to LGR5 and another antigen such as tetanus toxin (TT)).

[0219] The affinity of LGR5 and EGFR Fab of the candidate EGFR / LGR5 bispecific antibody for its target can be measured using BIAcore T100 by surface plasmon resonance (SPR) technology. Briefly, an anti-human IgG mouse monoclonal antibody (Becton and Dickinson, catalog number 555784) was coupled to the surface of a CM5 sensor chip using free amine chemistry (NHS / EDC). Subsequently, the bsAb was captured on the sensor surface. Subsequently, recombinant purified antigen human EGFR (SinoBiological Inc, catalog number 11896-H07H) and human LGR5 protein were flowed on the sensor surface at a certain concentration range and the association and dissociation rates were measured. After each cycle, the sensor surface was regenerated by HCl pulses and the bsAb was captured again. Based on the obtained sensor patterns, the association and dissociation rates and affinity values ​​for binding to human LGR5 and EGFR were determined using BIAevaluation software, as previously described for CD3 in US2016 / 0368988.

[0220] Antibodies as disclosed herein are generally bispecific full-length antibodies, in certain aspects of which are human IgG subclasses. In certain aspects, the antibodies are human IgG1 subclasses. Such antibodies have good ADCC properties that can be enhanced, if necessary, by techniques known in the art, have favorable half-lives when administered to humans in vivo, and CH3 engineering techniques exist that can provide modified heavy chains that preferentially form heterodimers over homodimers when co-expressed in clonal cells.

[0221] When an antibody itself has low ADCC activity, the ADCC activity of the antibody can be improved by modifying the antibody constant region. Another way to improve the ADCC activity of an antibody is to enzymatically interfere with the glycosylation pathway, which leads to a reduction in fucose. There are several in vitro methods for determining the efficacy of antibodies or effector cells in inducing ADCC. Among them are the chromium-51 [Cr51] release assay, the europium [Eu] release assay, and the sulfur-35 [S35] release assay. Typically, a labeled target cell line expressing a surface-exposed antigen is cultured with an antibody specific for the antigen. After washing, effector cells expressing the Fc receptor CD16 are co-cultured with the antibody-labeled target cells. Subsequently, target cell lysis is measured by the release of the intracellular label using a scintillation counter or spectrophotometry.

[0222] Bispecific antibodies as disclosed herein can be enhanced by ADCC. In certain aspects, this bispecific antibody is defucosylated. In certain aspects, when compared to the same antibody produced in normal CHO cells, the bispecific antibody comprises a reduced amount of fucosylation of N-linked carbohydrate structures in the Fc region. Low fucose levels are associated with increased binding of CD16 (FcγRIIIa) on NK effector cells, which leads to increased ADCC activity. In certain aspects, and in addition to its direct anti-tumor activity, the bispecific antibodies of the present disclosure can eliminate tumor cells after opsonization and subsequent natural killer (NK) cell-mediated ADCC activity and complement dependent cytotoxicity (CDC) activity.

[0223] Antibodies comprising variable regions that bind to the extracellular portion of EGFR and variable regions that bind to the extracellular portion of LGR5 may further comprise one or more additional variable regions that can bind to one or more further targets. In certain aspects, the further target is a protein, such as a membrane protein that comprises an extracellular portion. As used herein, a membrane protein is a cell membrane protein, such as a protein that resides in the outer membrane of a cell, i.e., the membrane that separates the cell from the outside world. A membrane protein has an extracellular portion. A membrane protein is at least on a cell if it contains a transmembrane region that resides in the cell membrane of the cell.

[0224] Antibodies with more than two variable domains are known in the art. For example, it is possible to attach additional variable domains. In certain aspects, antibodies with three or more variable domains are multivalent multimeric antibodies as described in PCT / NL2019 / 050199 (incorporated herein by reference).

[0225] In certain aspects, the antibody is a bispecific antibody comprising two variable domains, wherein one variable domain binds to the extracellular portion of EGFR and the other variable domain binds to the extracellular portion of LGR5. In certain aspects, the variable domains are as described herein.

[0226] The functional portion of an antibody as described herein comprises 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. Thus, it comprises the antigen-binding portion of an antibody as described herein, and typically contains the variable domains of an antibody. The variable domains of the functional portion may be a single-chain Fv fragment or a so-called single-domain antibody fragment. In certain aspects, the antibody portion or derivative has at least two variable domains of an 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 comprises the antigen-binding portion of a bispecific antibody, or a derivative and / or analog of the binding portion. As mentioned above, the binding portion of the antibody is encompassed in the variable domain.

[0227] Also provided are antibodies or functional portions, derivatives and / or analogs thereof (i.e., therapeutic agents) as disclosed herein and pharmaceutically acceptable carriers. Such pharmaceutical compositions are suitable for treating cancer, particularly for treating gastric cancer, esophageal cancer, or gastroesophageal junction cancer. As used herein, the term "pharmaceutically acceptable" means approved by a government regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, particularly humans, and includes any and all solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The term "carrier" means a diluent, adjuvant, excipient, or vehicle administered with the compound. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glyceryl ricinoleate, and the like. Water or physiological saline solutions, as well as dextrose and glycerol aqueous solutions, 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, intrathecal, and subcutaneous. Depending on the route of administration (e.g., intravenous, subcutaneous, intraarticular, etc.), the active compound can be coated in a material to protect the compound from the effects of acids and other natural conditions that may inactivate the compound.

[0228] Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, for example, in a liquid carrier or suitable for reconstitution into a liquid solution or suspension for intravenous administration. Compositions can be formulated in unit dosage form for ease of administration and uniformity of dosage. Also included are solid preparations that are intended to be converted into liquid preparations for oral or parenteral administration immediately prior to use. Such liquid forms include solutions, suspensions, and emulsions.

[0229] The disclosed chemotherapeutic agents can be administered according to applicable doses and applicable routes (e.g., intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgent needs of the treatment situation. In certain aspects, the subject is administered a single dose of an antibody as disclosed herein, or its functional portion, derivative, and / or analog. In certain aspects, the therapeutic agent will be administered repeatedly during the course of treatment. For example, in certain embodiments, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of the therapeutic agent are administered to a subject in need of treatment. In some embodiments, the administration of the therapeutic agent can be weekly, biweekly, or monthly.

[0230] The clinician can utilize a preferred dosage that is appropriate for the patient's condition being treated. The dosage may depend on a variety of factors, including the stage of the disease. It is within the skill of the artisan to determine the specific dosage to be administered based on the presence of one or more of these factors. Generally speaking, treatment begins with a smaller dose that is smaller than the optimal dose of the compound. Thereafter, the dosage is increased in small amounts until the optimal effect in the situation is achieved. For convenience, if necessary, the total daily dose may be divided and administered in several portions over the course of a day. Intermittent therapy (e.g., one week out of three or three weeks out of four) may also be used.

[0231] In certain aspects, the therapeutic agent is administered at a dose 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 at a dose of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg body weight.

[0232] In certain aspects, the therapeutic agent is provided to the subject at a fixed dose of 1500 mg. Fixed doses offer several advantages over topical or body weight administration because they shorten preparation time and reduce potential dose calculation errors. In certain aspects, the therapeutic agent is provided at a dose of at least 500 mg. In certain aspects, the dose is between 1100 and 2000 mg. In certain aspects, the dose is between 1100 and 1800 mg. As will be appreciated by those skilled in the art, the dose can be administered over time. For example, the dose can be administered by IV, for example, as an infusion over 1 to 6 hours, preferably over 2 to 4 hours. In certain aspects, the therapeutic agent is administered once every 2 weeks. In particular, the fixed doses disclosed herein are suitable for adults and / or subjects weighing at least 35 kg. In certain aspects, the subject suffers from gastric cancer, esophageal cancer, or gastroesophageal junction cancer.

[0233] In certain aspects, a premedication regimen may be used. Such regimens may be suitable for 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, intravenously) prior to antibody treatment.

[0234] The treatment methods described herein are generally continued as long as the clinician supervising the patient's care deems the treatment to be effective, i.e., the patient is responding to treatment. Non-limiting parameters indicating that the treatment method is effective may include one or more of the following: reduction in tumor cells; inhibition of tumor cell proliferation; elimination of tumor cells; progression-free survival; and appropriate response of appropriate tumor markers (if applicable).

[0235] Regarding the frequency of administering the therapeutic agent, one skilled in the art will be able to determine the appropriate frequency. For example, a clinician may decide to administer the therapeutic agent relatively infrequently (e.g., once every two weeks) and gradually shorten the time period between doses as tolerated by the patient. Exemplary lengths of time associated with a course of therapy according to the claimed methods include: about one week; about two weeks; about three weeks; about four weeks; about five weeks; about six weeks; about seven weeks; about eight weeks; about nine weeks; about ten weeks; about eleven weeks; about twelve weeks; about thirteen weeks; about fourteen weeks; about fifteen weeks; about sixteen weeks; about seventeen weeks; about eighteen weeks; about nineteen weeks; about twenty weeks; about twenty-one weeks; about twenty-two weeks; about twenty-three weeks; about twenty-four weeks; about seven months; about eight months; about nineteen months; about ten months; about eleven months; about twelve months; about thirteen months; about fourteen months; about fifteen months; about sixteen months; about seventeen months; about eighteen months; about nineteen months; about twenty months; about twenty-one months; about twenty-two months; about twenty-three months; about twenty-four months; about thirty months; about three years; about four years; about five years; and permanently (e.g., ongoing maintenance therapy). The foregoing durations may be associated with one or more rounds / cycles of treatment.

[0236] Any suitable means can be used to evaluate the efficacy of the treatment methods provided herein. In certain aspects, a reduction in the number of cancer cells is used as an objective response criterion to analyze the clinical efficacy of the treatment. Patients (e.g., humans) treated according to the methods disclosed herein preferably experience improvement in at least one symptom of cancer. In certain aspects, one or more of the following may occur: the number of cancer cells may be reduced; cancer recurrence is prevented or delayed; one or more symptoms associated with cancer may be alleviated to a certain extent. In addition, in vitro tests are performed to determine T cell-mediated target cell lysis. In certain aspects, tumor assessment is based on CT scans and / or MRI scans, see, for example, RECIST 1.1 guidelines (Response Evaluation Criteria in Solid Tumours) (Eisenhauer et al., 2009 Eur J Cancer 45: 228-247). Such assessments are generally performed every 4 to 8 weeks after treatment.

[0237] In some aspects, the tumor cells are no longer detectable after treatment as described herein. In some aspects, the subject is in partial or complete remission. In some aspects, the subject's overall survival, median survival rate, and / or progression-free survival increase.

[0238] The therapeutic agent (i.e., an antibody comprising a variable region that binds to the extracellular portion of EGFR and a variable region that binds to the extracellular portion of LGR5, or a functional part, derivative and / or analog thereof) can also be used together with other well-known therapies (e.g., chemotherapy or radiation therapy) selected for their specific usefulness against the cancer being treated.

[0239] 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, NJ 07645-1742, USA); the disclosure of which is incorporated herein by reference.

[0240] It will be apparent to those skilled in the art that the administration of chemotherapeutic agents and / or radiation therapy can vary depending on the disease being treated and the known effects of the chemotherapeutic agents and / or radiation therapy on the disease. At the same time, according to the knowledge of the skilled clinician, the treatment regimen (e.g., dosage and administration time) can vary in view of the observed effects of the administered therapeutic agent on the patient and in view of the observed response of the disease to the administered therapeutic agent.

[0241] The compounds and compositions disclosed herein are useful as therapy and for therapeutic treatment and are therefore useful as medicaments and in methods of preparing medicaments.

[0242] All documents and references mentioned herein, including Genbank entries, patents and published patent applications, and websites are each expressly incorporated by reference to the same extent as if fully or partially written into this document.

[0243] For purposes of clarity and concise description, features are described herein as being part of the same or separate parts of the disclosure; however, it is to be understood that the scope of the invention may include preferred aspects having combinations of all or some of the features.

[0244] The present invention will now be described with reference to the following examples, which are illustrative only and are not intended to limit the present invention. Although the present invention has been described in detail with reference to specific aspects thereof, it will be apparent to those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0245] List of items

[0246] 1. An antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative and / or analog thereof, for use in treating cancer in a subject whose cancer has progressed after prior treatment with an immune checkpoint inhibitor, and wherein the cancer expresses EGFR.

[0247] 2. Use of an antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative and / or analog thereof, in the manufacture of a medicament for treating a cancer in a subject, wherein the cancer has progressed after prior treatment with an immune checkpoint inhibitor and the cancer expresses EGFR.

[0248] 3. A method for treating a subject having a cancer that expresses EGFR, wherein the subject has progressed after prior treatment with an immune checkpoint inhibitor, the method comprising providing to the subject an effective amount of an antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative and / or analog thereof.

[0249] 4. The antibody or functional part, derivative and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the cancer is head and neck cancer, preferably head and neck squamous cell carcinoma (SCCHN), and the cancer preferably expresses EGFR and is characterized by an IHC score of 2+ or 3+.

[0250] 5. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding clauses, wherein the cancer is gastric cancer, esophageal cancer or gastroesophageal junction cancer with EGFR expression characterized by an IHC score of 3+.

[0251] 6. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding clauses, wherein the cancer is gastric cancer, esophageal cancer or gastroesophageal junction cancer with EGFR expression characterized by an EGFR H-score of greater than 200.

[0252] 7. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding clauses, wherein the cancer is characterized by EGFR gene amplification.

[0253] 8. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to clause 7, wherein the EGFR gene amplification is characterized by an EGFR copy number of 8 or more, as determined by next generation sequencing of a solid tissue sample; an EGFR score of at least 2.14 or at least 2.5, as determined by next generation sequencing of circulating tumor DNA (ctDNA); or an EGFR / CEP7 ratio of 2 or higher based on FISH.

[0254] 9. An antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative and / or analog thereof, for use in treating gastric cancer, esophageal cancer or gastroesophageal junction cancer in a subject, wherein the cancer expresses EGFR and is characterized by an IHC score of 3+.

[0255] 10. An antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative and / or analog thereof, for use in treating gastric cancer, esophageal cancer or gastroesophageal junction cancer in a subject, wherein the cancer expresses EGFR and is characterized by an H-score for EGFR greater than 200.

[0256] 11. An antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative and / or analogue thereof, for use in treating cancer in a subject, wherein the first variable domain is a heavy chain variable region comprising:

[0257] - a CDR3 sequence of at least the VH of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 , or a CDR3 sequence that differs from the VH CDR3 sequence of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 in at most three, preferably at most two, preferably not more than one amino acid;

[0258] at least the CDR1, CDR2 and CDR3 sequence of the VH of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 ; or the CDR1, CDR2 and CDR3 sequence of the VH of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 and having at most three, preferably at most two, preferably at most one amino acid substitution; or

[0259] 3 ; or the amino acid sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as shown in FIG3 and having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and preferably 1, 2, 3, 4 or 5, amino acid insertions, deletions, substitutions or a combination thereof relative to the VH chain of MF3370; MF3755; MF4280 or MF4289; and wherein the cancer is head and neck cancer, preferably head and neck squamous cell carcinoma (SCCHN), the cancer preferably expresses EGFR, characterized by an IHC score of 2+ or 3+, or wherein the cancer is gastric cancer, esophageal cancer or gastroesophageal junction cancer with EGFR expression characterized by an IHC score of 3+ or preferably an EGFR H-score of greater than 200.

[0260] 12. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding clauses, wherein the subject has not received previous treatment with an anti-EGFR agent.

[0261] 13. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of clauses 1 to 11, wherein the subject has not received previous treatment with an antibody targeting EGFR.

[0262] 14. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of clauses 1 to 11, wherein the subject has not received previous treatment with cetuximab.

[0263] 15. The antibody or functional part, derivative and / or analogue thereof according to any one of clauses 9 to 14, wherein the cancer has progressed following previous treatment with an immune checkpoint inhibitor.

[0264] 16. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding clauses, wherein the cancer expresses EGFR and is characterized by an H-score of greater than 200 and no greater than 300.

[0265] 17. The antibody or functional part, derivative and / or analogue thereof according to any one of clause 16, wherein the H-score of EGFR is determined using IHC.

[0266] 18. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding clauses, wherein the subject is a mammal, preferably a human.

[0267] 19. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any of the preceding clauses, wherein the treatment comprises providing to the subject an effective amount of the antibody or functional part, derivative and / or analogue thereof.

[0268] 20. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding clauses, wherein the treatment comprises providing the subject with a fixed dose of 1500 mg of the antibody or functional part, derivative and / or analogue thereof.

[0269] 29. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding clauses, wherein the antibody or functional part, derivative and / or analogue thereof is provided to the subject by intravenous injection.

[0270] 22. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any of the preceding clauses, wherein the antibody or functional part, derivative and / or analogue thereof is provided weekly, biweekly or monthly, preferably biweekly, more preferably at least 3 or more biweekly doses of the antibody or functional part, derivative and / or analogue thereof are provided to the subject.

[0271] 22. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding clauses, wherein the antibody is ADCC enhanced.

[0272] 24. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any of the preceding clauses, wherein the antibody is defucosylated.

[0273] 25. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding clauses, wherein the cancer is adenocarcinoma or squamous cell carcinoma, in particular gastric adenocarcinoma, esophageal adenocarcinoma or gastro-esophageal junction adenocarcinoma or in particular head and neck squamous cell carcinoma (HNSCC).

[0274] 26. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding clauses, wherein the cancer and / or the subject is SMAD4 wild-type.

[0275] 27. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any of the preceding items, wherein the cancer or subject has a TP53 mutation, preferably an activating TP53 mutation.

[0276] 28. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding items, wherein the cancer or subject is Her2 negative.

[0277] 29. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any of the preceding items, wherein the antibody is a multispecific antibody, preferably a bispecific antibody.

[0278] 30. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding items, wherein the antibody comprises a second variable domain that does not bind to EGFR.

[0279] 31. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding items, wherein the antibody comprises a second variable domain that binds to LGR5.

[0280] 30. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of clauses 1 to 28, wherein the antibody is a monovalent antibody that does not comprise a second variable domain, or wherein the antibody comprises the first EGFR binding variable domain as the only variable domain.

[0281] 33. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding clauses, wherein the immune point inhibitor comprises a PD-L1 or PD-1 inhibitor.

[0282] 34. An antibody or functional part, derivative and / or analogue thereof, or use or method as described in any of the preceding clauses, wherein the treatment comprises or is preceded by a step of diagnosing the subject's EGFR status, SMAD4 status and / or Her2 status, wherein the diagnosis of Her2 status is preferably performed by ISH or IHC.

[0283] 35. The antibody or functional part, derivative and / or analogue thereof, or use or method according to any of the preceding clauses, wherein said first variable domain that binds to EGFR binds to a region located at ( Figure 2 ) is an epitope within amino acid residues 420 to 480 of the human EGFR sequence shown in .

[0284] 36. The antibody or functional part, derivative and / or analogue thereof, or use or method according to any of the preceding clauses, wherein the binding of the first variable domain to EGFR is reduced by substitution of one or more of the following amino acid residues in EGFR: I462A, G465A, K489A, I491A, N493A, and C499A, compared to an EGFR protein not comprising said substitution.

[0285] 37. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of clauses 29 to 34, wherein the variable domain that binds to LGR5 binds to a region located at Figure 1 Epitopes within amino acid residues 21 to 118 of the human LGR5 sequence are shown.

[0286] 38. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of clauses 1 to 10 or 12 to 37, wherein the first variable domain is a heavy chain variable region comprising:

[0287] - a CDR3 sequence of at least the VH of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 , or a CDR3 sequence that differs from the VH CDR3 sequence of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 in at most three, preferably at most two, preferably not more than one amino acid;

[0288] at least the CDR1, CDR2 and CDR3 sequence of the VH of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 ; or the CDR1, CDR2 and CDR3 sequence of the VH of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 and having at most three, preferably at most two, preferably at most one amino acid substitution; or

[0289] The sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3; or the amino acid sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 and having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and preferably 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof relative to the VH chain of MF3370; MF3755; MF4280 or MF4289.

[0290] Examples

[0291] As used herein, "MFXXXX" (wherein X is independently a number 0 to 9) refers to a Fab comprising a variable domain wherein VH has an amino acid sequence identified by the 4-digit number shown in Figure 3. Unless otherwise indicated, the light chain variable region of the variable domain typically has Figure 4 b. The light chain in the example has Figure 4The sequence shown in a. "MFXXXX VH" means the amino acid sequence of the VH identified by the 4-digit number. The MF further comprises the constant region of the light chain and the constant region of the heavy chain that generally interacts with the constant region of the light chain. The VH / variable regions of the heavy chains are different, and generally the CH3 regions are also different, wherein one of the heavy chains has a KK mutation in its CH3 domain and the other has a complementary DE mutation in its CH3 domain (see PCT / NL2013 / 050294 (published as WO2013 / 157954) for reference) and Figure 5 d and Figure 5 e). The bispecific antibody in the example has Figure 5 The indicated Fc tail, CH2 domain and CH1 domain with KK / DE CH3 heterodimerization domain, as shown Figure 4 a Common light chain indicated by a and VH as designated by MF number. For example, the bispecific antibody indicated by MF3755xMF5816 has the common sequence described above and has a variable domain of VH comprising the sequence of MF3755 and a variable domain of VH comprising the sequence of MF5816.

[0292] The amino acid and nucleic acid sequences of the various heavy chain variable regions (VH) are indicated in Figure 3. The bispecific antibody EGFR / LGR5, MF3755xMF5816 (which comprises the heavy chain variable regions MF3755 and MF5816 and a common light chain and includes a modification that enhances ADCC by defucosylation), as well as other LGR5 and EGFR combinations as shown in Figure 3, have been shown to be effective in WO2017 / 069628.

[0293] Generation of bispecific antibodies

[0294] Using dedicated CH3 engineering technology to ensure effective heterodimerization and bispecific antibody formation, bispecific antibodies are produced by transient co-transfection of two plasmids encoding IgG with different VH regions. The common light chain is also co-transfected in the same cell on the same plasmid or another plasmid. In the inventor's application (e.g., WO2013 / 157954 and WO2013 / 157953, incorporated herein by reference), the inventors have disclosed methods and means for producing bispecific antibodies from a single cell, wherein means are provided for giving priority to the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can also be advantageously used in the present invention. In particular, preferred mutations that substantially only generate bispecific full-length IgG molecules are amino acid substitutions at positions 351 and 366 in the first CH3 domain, e.g., L351K and T366K (numbering 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 Figure 5 d and Figure 5 e). Previously, it was demonstrated in the aforementioned application that negatively charged DE variant heavy chains and positively charged KK variant heavy chains preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE variant heavy chains (DE-DE homodimers) or homodimerization of KK variant heavy chains (KK-KK homodimers) rarely occurs due to the strong repulsion between charged residues in the CH3-CH3 interface between the same heavy chains.

[0295] The VH genes of the variable domains binding to LGR5 described above are cloned into a vector encoding a positively charged CH3 domain. The VH genes of the variable domains binding to EGFR, such as those disclosed in WO 2015 / 130172 (incorporated herein by reference), are cloned into a vector encoding a negatively charged CH3 domain. Suspension-adapted 293F Freestyle cells are cultured in a T125 flask on a shaker platform to a density of 3.0 x 10e6 cells / ml. The cells are seeded in each well of a 24-deep-well plate at a density of 0.3-0.5 x 10e6 cells / ml. The cells are transiently transfected with a mixture of two plasmids encoding different antibodies cloned in a dedicated vector system. Seven days after transfection, the cell supernatant is collected and filtered through a 0.22 μM filter (Sartorius). The sterile supernatant is stored at 4 ° C until antibody purification is performed.

[0296] IgG purification and quantification

[0297] Purification was performed using protein A affinity chromatography in a filter dish under aseptic conditions. First, the pH of the culture medium was adjusted to pH 8.0, and subsequently the IgG supernatant was cultured on a rocking platform at 25°C with 600 rpm for 2 hours using protein A sepharose CL-4B beads (50% v / v) (Pierce). The beads were then collected by filtration. The beads were washed twice with PBS pH 7.4. Subsequently, the bound IgG was eluted with 0.1 M citrate buffer at pH 3.0, and then the eluent was neutralized using Tris pH 8.0. The multi-disc (Millipore) multiscreen Ultracel 10 was centrifuged to perform buffer exchange. Finally, the sample was collected in PBS pH 7.4. The IgG concentration was measured using Octet. The protein samples were stored at 4°C.

[0298] To determine the amount of purified IgG, antibody concentration was determined by Octet analysis using a Protein A biosensor (Forte-Bio, according to the supplier's recommendations) using whole human IgG (Sigma Aldrich, catalog number 14506) as a standard.

[0299] The following bispecific antibodies are suitable for use in this example and in the methods of the invention: MF3370xMF5790, MF3370x5803, MF3370x5805, MF3370x5808, MF3370x5809, MF3370x5814, MF3370x5816, MF3370x5817, MF3370x5818, MF3755xMF5790, MF3755x5803, MF3755x5805, MF3755x5808, MF3755x5809, MF3755x5814, MF3755x5816, MF3755x5817 , MF3755x5818, MF4280xMF5790, MF4280x5803, MF4280x5805, MF4280x5808, MF4280x5809, MF4280x5814, MF4280x5816, MF4280x5817, MF4280x5818, MF4289xMF5790, MF4289x5803, MF4289x5805, MF4289x5808, MF4289x5809, MF4289x5814, MF4289x5816, MF4289x5817 and MF4289x5818. Each bispecific antibody comprises two VHs designated by MF numbering capable of binding to EGFR and LGR5, respectively, and further comprises VHs having VHs designated by MF numbering as represented by SEQ ID NO: 136 ( Figure 5d) and SEQ ID NO: 138 ( Figure 5 e) The Fc tail of the indicated KK / DE CH3 heterodimerization domain, as represented by SEQ ID NO: 134 ( Figure 5 c) the CH2 domain indicated by SEQ ID NO: 131 ( Figure 5 a) CH1 domain indicated by SEQ ID NO: 121 ( Figure 4 ) indicated common light chain.

[0300] Example 1: Dose escalation of anti-EGFRx anti-LGR5 antibodies for patients with EAC, GAC, GEJAC, or head and neck cancer Increase and efficacy

[0301] Phase 1 dose-escalation study in advanced solid tumors

[0302] Study Design

[0303] A Phase 1 open-label, multicenter study was conducted with an initial dose-escalation portion to determine the recommended Phase 2 dose (RP2D) of the anti-EGFRx anti-LGR5 bispecific antibody against solid tumors in patients with mCRC, starting at a fixed dose of 5 mg. Once the RP2D was established, the antibody was further evaluated in an expansion portion of the study, including patients diagnosed with EAC, GAC, GEJAC, or head and neck cancer, including squamous cell carcinoma of the head and neck (SCCHN). The safety, PK, immunogenicity, and preliminary anti-tumor activity of the antibody were confirmed in all patients, and biomarker analysis, including EGFR and LGR5 status, was performed.

[0304] Inclusion criteria

[0305] Patients must meet all of the following requirements to enter the study:

[0306] 1. Sign the informed consent form before starting any research procedures.

[0307] 2. Applicants must be ≥18 years old when signing the informed consent form.

[0308] 3. Patients with histologically or cytologically confirmed solid tumors, evidence of metastatic or locally advanced disease, and who are unable to receive standard therapy with curative intent:

[0309] Expansion Group: Explore patients with advanced metastatic EAC, GAC, GEJAC, or head and neck squamous cell carcinoma, regardless of whether they have previously received at least 2 standard approved therapies (if applicable).

[0310] 4. Baseline fresh tumor sample (FFPE and also frozen if sufficient material is available) from metastatic or primary site.

[0311] 5. Suitable for biopsy.

[0312] 6. Measurable disease defined by radiological methods according to RECIST version 1.1.

[0313] 7. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.

[0314] 8. According to researchers, life expectancy is ≥12 weeks.

[0315] 9. Left ventricular ejection fraction (LVEF) ≥ 50% as determined by echocardiography (ECHO) or multichannel cardiac imaging (MUGA).

[0316] 10. Adequate Organ Function:

[0317] Absolute neutrophil count (ANC) ≥ 1.5 × 10 9 / L

[0318] Hemoglobin ≥9 g / dL

[0319] Platelet count ≥100 x 109 / L

[0320] Corrected total serum calcium within normal range

[0321] Serum magnesium within normal range (or corrected with supplementation)

[0322] Alanine aminotransferase (ALT), aspartate aminotransferase (AST) ≤ 2.5 x upper limit of normal (ULN), and total bilirubin ≤ 1.5 x ULN (unless excluded due to known Gilbert's syndrome, in which case total bilirubin > 3.0 x ULN or direct bilirubin > 1.5 x ULN); in cases of liver involvement, ALT / AST ≤ 5 x ULN and total bilirubin ≤ 2 x ULN will be permitted, except in cases of known Gilbert's syndrome, in which case total bilirubin ≤ 3.0 x ULN or direct bilirubin ≤ 1.5 x ULN will be permitted, or in cases of hepatocellular carcinoma [Child-Pugh A], in which case total bilirubin < 3 mg / dL will be permitted

[0323] Serum creatinine ≤1.5x ULN or creatinine clearance ≥60 mL / min, calculated according to the Cockroft and Gault formula or the MDRD formula, and patients >65 years of age

[0324] Serum albumin > 3.3 g / dL (> 3.3 g / dL).

[0325] Exclusion criteria

[0326] Patients with any of the following criteria were excluded from the study:

[0327] 1. Have untreated or symptomatic central nervous system metastases within 14 days of study entry, or require radiation, surgery, or continuous steroid therapy to control symptoms.

[0328] 2. Known leptomeningeal involvement.

[0329] 3. Participated in another clinical trial or received any investigational drug treatment within 4 weeks prior to entering the study.

[0330] 4. Any systemic anticancer therapy within 4 weeks or 5 half-lives, whichever is longer, based on the first dose of study treatment. For cytotoxic agents with major delayed toxicity (e.g., mitomycin C, nitrosoureas) or anticancer immunotherapies, a 6-week washout period is required.

[0331] 5. Requirement of immunosuppressive drugs (e.g., methotrexate, cyclophosphamide).

[0332] 6. Major surgery or radiation therapy within 3 weeks of the first dose of study treatment. Patients who have previously received ≥25% bone marrow radiation therapy are not eligible, regardless of when received.

[0333] 7. Persistent clinically significant toxicity > Grade 1 (excluding alopecia) related to prior anti-malignant tumor therapy; stable sensory neuropathy ≤ Grade 2 NCI-CTCAE v4.03 is allowed.

[0334] 8. History of anaphylactic reaction or any toxicity attributable to human proteins or any excipients requiring permanent discontinuation of these agents.

[0335] 9. Uncontrolled hypertension (systolic blood pressure >150 mmHg and / or diastolic blood pressure >100 mmHg) or unstable angina with appropriate treatment.

[0336] 10. History of congestive heart failure that meets New York Heart Association (NYHA) class II-IV criteria, or severe arrhythmia requiring treatment (excluding atrial fibrillation and paroxysmal supraventricular tachycardia).

[0337] 11. History of myocardial infarction within 6 months of entering the study.

[0338] 12. History of previous malignancy, other than excised cervical intraepithelial neoplasia or non-melanoma skin cancer, or curative cancer treatment considered to be at low risk of recurrence with no evidence of disease for at least 3 years.

[0339] 13. Current dyspnea at rest from any source, current dyspnea from any source, or other conditions requiring continuous oxygen therapy.

[0340] 14. Patients with a history of interstitial lung disease (e.g., pneumonia or pulmonary fibrosis) or evidence of ILD on baseline chest CT scan.

[0341] 15. Current serious illness or medical condition, including but not limited to uncontrolled active infection, clinically significant pulmonary, metabolic or psychiatric disease.

[0342] 16. Active HIV, HBV, or HCV infection requiring treatment.

[0343] 17. Patients with current Child-Pugh B or C cirrhosis; known fibrolamellar HCC, sarcomatoid HCC, or mixed cholangiocarcinoma and HCC.

[0344] 18. Pregnant or lactating women; patients of childbearing potential must use highly effective contraceptive methods before entering the study, during the study, and within 6 months after the last dose of the antibody.

[0345] Dose-limiting toxicity (DLT)

[0346] Any of the following clinical toxicities and / or laboratory abnormalities occurring during the first cycle (28 days) and considered by the investigator to be related to the antibody treatment will be considered a DLT:

[0347] Hematological toxicity:

[0348] - Grade 4 neutropenia (absolute neutrophil count [ANC] <0.5x10 9 cells / L) for up to ≥7 days

[0349] - Grade 3 to 4 febrile neutropenia

[0350] - Grade 4 thrombocytopenia

[0351] - Grade 3 thrombocytopenia associated with bleeding events

[0352] - Other grade 4 hematologic toxicities

[0353] Grade 3 to 4 non-hematologic AEs and laboratory toxicities, except for the following:

[0354] - Grade 3 to 4 infusion-related reactions

[0355] - Grade 3 skin toxicity that recovers to ≤ Grade 2 within 2 weeks due to optimized treatment

[0356] - Grade 3 diarrhea, nausea, and / or vomiting that recovers to ≤ Grade 1 or baseline within 3 days after optimized treatment

[0357] - Grade 3 electrolyte abnormalities that resolve with ideal treatment within 48 hours

[0358] - Grade 3 to 4 liver abnormalities lasting ≤48 hours

[0359] Any abnormal liver function that meets the definition of Hy's law.

[0360] • Any drug-related toxicity lasting ≥15 days that prevents the next two doses.

[0361] Dose expansion

[0362] In the expansion phase, the bispecific antibody will be administered at an RP2D in patients with EAC, GAC, GEJAC, or head and neck cancer (particularly SCCHN). Once the RP2D is determined, additional patients will be treated at this dose and scheduled to further confirm the safety, tolerability, PK, and immunogenicity of the antibody, and to undergo preliminary assessments of anti-tumor activity and biomarker evaluation.

[0363] Explore antibody treatment in patients with EAC, GAC, GEJAC or head and neck cancer (especially SCCHN), for example, 10 to 20 patients for each indication, which may be expanded to 40 patients subject to signs of preliminary anti-tumor activity. During the expansion portion of the study, the safety of the RP2D will be continuously evaluated by the Safety Monitoring Committee. If the DLT incidence exceeds the predefined threshold of 33% for any group, the enrollment of this group will be suspended and a comprehensive review of safety, PK and biomarkers will be conducted by the SMC to determine whether it is safe to continue accumulation in this group. The overall safety of the drug will also be inquired at that time.

[0364] Investigational therapies and regimens

[0365] The anti-EGFR x anti-LGR5 bispecific antibody is formulated into a clear solution for IV infusion. Using a standard infusion procedure, IV infusion is performed every 2 weeks with a starting dose of 5 mg (fixed dose) and a recommended 2-phase dose of 1500 mg (fixed dose). Once the RP2D is reached, the dose escalation is discontinued. During the first cycle, the infusion must be administered over a minimum of 4 hours. At the discretion of the investigator and in the absence of IRR, subsequent infusions after the first cycle can be shortened to 2 hours. One cycle is considered 4 weeks.

[0366] Premedication

[0367] During Cycle 1, all infusions will be administered over a period of at least 4 hours with the following premedication regimen: 24 hours before the start of the infusion, 8 mg of dexamethasone will be administered orally, and 1 hour before the start of the infusion, each patient will receive dexamethasone 20 mg IV, dexclofenamine 5 mg IV or diphenhydramine 50 mg orally, or chlorpheniramine 10 mg IV, ranitidine 50 mg IV or 150 mg orally, and acetaminophen 1 g IV or 650 mg orally.

[0368] If the patient tolerates all 1 cycle infusions without IRR and the investigator deems it appropriate, the patient may continue to receive further antibody infusions without the need for premedication with dexamethasone, and the infusion duration may be reduced to 2 hours. In such cases, the infusion duration may be extended back to approximately 4 hours, if deemed appropriate, to avoid or reduce the incidence or severity of IRR. For the initial antibody infusion (Day 1 of Cycle 1), each patient will be observed for 6 hours from the start of the infusion and for 4 hours from the start of the second infusion. Thereafter, the patient will be observed during all subsequent administrations (minimum 2 hours).

[0369] Duration of treatment

[0370] Study treatment was administered until confirmed progressive disease (according to RECIST 1.1), unacceptable toxicity, withdrawal of consent, patient noncompliance, investigator decision (e.g., clinical worsening), or antibody discontinuation for more than 6 consecutive weeks. After the last antibody infusion, patients were followed for safety for at least 30 days and until all relevant toxicities recovered or stabilized, and for 12 months for disease progression and survival.

[0371] Pre-screening of gastric patients for EGFR amplification or high EGFR protein expression

[0372] For patients with gastric / gastroesophageal junction adenocarcinoma, documentation of EGFR amplification or high EGFR expression through DNA pre-screening is required in clinical trials. To be eligible for pre-screening, patients must have a histological diagnosis of gastric cancer in the absence of other actionable targets. Pre-screening testing will be performed in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory that is qualified to perform molecular screening for EGFR amplification and tumor gene mutations or EGFR IHC (e.g., EGFR PharmDx kit or equivalent validated IVD). EGFR amplification can be tested using FISH testing, ctDNA analysis, or tissue NGS. If appropriate local testing options are not available, samples can be sent to an approved central laboratory with appropriate qualifications.

[0373] For ctDNA analysis, collect two 10 mL tubes of blood using the tubes provided in the Guardant blood collection kit. For Guardant tissue NGS analysis, submit FFPE slides or tissue blocks using the Guardant collection kit. The threshold for eligible EGFR amplification or EGFR protein expression was defined as a FISH score EGFR / CEP7 ratio ≥ 2.0, or NGS EGFR copies ≥ 8, or ctDNA > 2.14, or 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). Subsequently, patients with EGFR amplification or EGFR IHC H score ≥ 200 were eligible to sign the main study ICF if they were willing and able to enter the main study. At least 10 patients with EGFR IHC high will be recruited.

[0374] Patients with documented EGFR amplification by ctDNA testing in a qualified local laboratory were eligible to enroll in the primary study ICF without additional prescreening.

[0375] Efficacy evaluation

[0376] Every 8 weeks after the start of treatment, tumor assessment was based on CT / MRI and compared with RECIST 1.1 (Eisenhauer et al., 2009 Eur J Cancer 45:228-247). Objective responses must be confirmed at least 4 weeks after the first observation. Bone scans were performed on patients with bone metastases or suspected lesions at baseline as clinically indicated. Circulating blood tumor markers, including carcinoembryonic antigen (CEA), were assessed at screening and on day 1 of each cycle.

[0377] Example 2

[0378] A 67-year-old male patient with head and neck squamous cell carcinoma of the larynx was enrolled in the clinical trial of Example 1. The patient had previously been treated with platinum-based chemotherapy (carboplatin) and paclitaxel, and more importantly, durvalumab as an immune checkpoint inhibitor.

[0379] Observed responses included a PRc of -41% after receiving the bispecific antibody, characterized by the first and second variable domains designated MF3755 x MF5816. Patients were given a fixed dose of 1500 mg of the antibody over six cycles every 2 weeks, after which clinical response was assessed.

[0380] The patient showed EGFR IHC tumor membrane staining score of 2+.

[0381] Example 3

[0382] A 59-year-old female patient with head and neck squamous cell carcinoma of the tongue was enrolled in the clinical trial of Example 1. The patient had previously been treated with platinum-based chemotherapy (carboplatin) and 5-FU, and more importantly, pembrolizumab as an immune checkpoint inhibitor.

[0383] Responses observed included a partial response (PR) of -88% after receiving the bispecific antibody, characterized by having first and second variable domains indicated by MF3755xMF5816, and a complete response (CR) at the second assessment of tumor status. Patients were given the antibody at a fixed dose of 1500 mg over four cycles every 2 weeks before clinical response was assessed.

[0384] The patient showed EGFR IHC tumor membrane staining score of 3+.

[0385] Example 4

[0386] A 67-year-old male patient with head and neck squamous cell carcinoma of the oropharynx was enrolled in the clinical trial of Example 1. The patient had previously been treated with platinum-based chemotherapy (cisplatin and carboplatin), importantly with pembrolizumab as an immune checkpoint inhibitor.

[0387] The observed responses included a PRc of -40% after receiving the bispecific antibody characterized by the first and second variable domains indicated by MF3755xMF5816. The patient was given the antibody at a fixed dose of 1500 mg for eight cycles every 2 weeks, after which clinical response was assessed.

[0388] The patient showed EGFR IHC tumor membrane staining score of 3+.

[0389] EGFR H-score was performed as described in Example 6.

[0390] Example 5

[0391] 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 oxaliplatin- and irinotecan-based chemotherapy.

[0392] Observed responses included stable disease (SD) after receiving a bispecific antibody characterized by first and second variable domains designated MF3755xMF5816. Patients were given a fixed dose of 1500 mg of the antibody for four cycles every 2 weeks, after which clinical responses were assessed.

[0393] The patient showed an EGFR IHC score of 3+ and an EGFR H-score of 300. Genetic analysis showed that the patient was SMAD4 wild-type.

[0394] EGFR H-score was performed as described in Example 8.

[0395] Example 6

[0396] 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 chemotherapy of cisplatin / capecitabine.

[0397] The observed responses included a confirmed partial response (PRc) after receiving the bispecific antibody characterized by the first and second variable domains indicated by MF3755xMF5816. The patient was given a fixed dose of 1500 mg of the antibody for seven cycles every 2 weeks, after which the clinical response was assessed.

[0398] The patient showed an EGFR IHC score of 3+ and an EGFR H-score of 300. Genetic analysis showed that the patient was SMAD4 wild-type.

[0399] EGFR H-score was performed as described in Example 8.

[0400] Example 7

[0401] The safety analysis at the proposed Phase 2 dose was based on 29 patients with solid tumors who received RP2D. The most common adverse event was an infusion-related reaction (IRR), which was any grade in 72% of patients and grade ≥3 in 7%. Onset: First infusion in all patients. IRRs were manageable with prophylaxis / extended infusion. Mild to moderate skin toxicity was observed (with 3% severe events).

[0402] Infusion-related reactions are a composite term that includes all AEs considered to be IRRs by the investigator during the 24 hours after infusion.

[0403] Example 8. EGFR Scoring via IHC

[0404] EGFR pharmDx TM The test is a qualitative immunohistochemistry (IHC) kit system designed to differentiate epidermal growth factor receptor (EGFR) expression in routinely fixed normal and tumor tissues for histological evaluation. EGFR pharmDx specifically detects EGFR (HER1) protein in EGFR-expressing cells.

[0405] EGFR pharmDx TM The assay uses an EGFR antibody (clone 2-18C9 (2-18C9)) to detect EGFR protein. Clone 2-18C9 has been tested for reactivity against cell lines expressing EGFR, HER2, HER3, and HER4. In Western blots of SKBR3 and A431 cell lysates, 2-18C9 recognized a 170 kD band, which is consistent with the known molecular weight of EGFR. Clone 2-18C9 has also been found to recognize the EGFRvIII (145 kD) form of the receptor in EGFRvIII-transfected cell lines using immunohistochemistry, flow cytometry, and Western blots. In Western blot experiments, 2-18C9 did not react with HER2-positive CAMA-1 cell lysates, HER3-transfected E. coli BL-21 protein extracts, or CHO-HER4-transfected cell lysates. In addition, Chinese hamster ovary (CHO) cells expressing myc (vector-tagged) alone or co-expressed with a HER family member were grown on formalin-fixed, paraffin-embedded chamber slides and stained with anti-myc and 2-18C9. The myc antibody stained all five CHO transfected cells, while 2-18C9 only stained CHO cells transfected with HER1.

[0406] The Dako EGFR pharmDx was used according to the manufacturer's instructions and recommendations. TM User protocol for EGFR scoring. See the Internet address:

[0407] agilent.com / cs / library / usermanuals / public / 08052_egfr_pharmdx_interpretation_manual.pdf.

[0408] Sample preparation

[0409] Process biopsy specimens 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 the EGFR pharmDx: 10% (v / v) neutral buffered formalin, 10% (v / v) unbuffered formalin, 25% (v / v) unbuffered formalin, AFA (formalin alcohol acetate), Richard-Allen Scientific Pen-fix, and Bouin's fixative.

[0410] paraffin-embedded sections

[0411] Routinely processed and paraffin-embedded tissue is suitable for use. Specimens from biopsies should be cut into 3 or 4 mm thick blocks and fixed for a period of time appropriate for the fixative. The tissue is then dehydrated and cleared in a series of alcohol and xylene, followed by infiltration with molten paraffin. The temperature of the paraffin should not exceed 60°C. If stored in a cool place (15 to 25°C), properly fixed and embedded tissue blocks expressing EGFR protein will be preserved indefinitely before sectioning and slide mounting.

[0412] Tissue samples should be cut into 3 to 5 μm sections. After sectioning, the tissue should be mounted on slides and placed on a drying rack. The following slides are recommended: Fisher's SuperFrost Plus, Dako's Silanized (code S3003), charged or poly-L-lysine coated slides. The slide rack should be tapped on an absorbent towel to remove moisture from the paraffin and glass, then dried at room temperature for one hour. The slide rack should then be placed in an incubator at 56 to 60°C for one hour. After removing the slides from the incubator, any excess moisture remaining on the slides should be removed by tapping the slides on a towel and drying them in the incubator for another hour. After removal from the incubator, the slides should be kept at room temperature until they have cooled and the paraffin has hardened. To preserve antigenicity, tissue sections mounted on glass slides (Fisher's SuperFrost Plus, poly-L-lysine, charged, or Dako's Silanized slides (code S3003)) should be stained within 2 months of sectioning when stored at room temperature (20 to 25°C).

[0413] Slides for EGFR assessment and verification of tumor presence should be prepared simultaneously.

[0414] A minimum of 5 slides is recommended: 1 for tumor visualization, 2 for EGFR protein assessment (one for primary antibody and one for negative control reagent), and 2 spares.

[0415] Reagent preparation

[0416] Prepare the following reagents prior to staining:

[0417] Wash Buffer: Prepare sufficient wash buffer by diluting 10x Wash Buffer 1:10 with distilled or deionized (reagent grade) water for the wash steps. Discard if the buffer appears cloudy.

[0418] Substrate-Chromogen Solution (DAB+): This solution should be mixed thoroughly before use. Any precipitation that occurs in the solution will not affect the quality of the staining. To prepare the DAB+ Substrate-Chromogen Solution, add 11 drops of liquid DAB+ Chromogen to one bottle of DAB+ Substrate Buffer and mix. Discard any unused solution. Dilute according to the above guidelines. Adding excess liquid DAB+ Chromogen to the DAB+ Substrate Buffer will result in a decreased positive signal.

[0419] Counterstain. If necessary, prepare ammonia solution for counterstaining.

[0420] Prepare ammonia water (0.037 mol / L) 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 water can be stored in a sealed bottle at room temperature (20-25°C) for up to 12 months.

[0421] Mounting media. Aqueous mounting media such as Dako's Faramount Aqueous Mounting Medium, Ready-to-Use (Code S3025) or Dako's Glycerol Gel Mounting Medium (Code C0563) are recommended. Warm to approximately 40 (±5)°C before use to liquefy the glycerol gel. Non-aqueous permanent mounts are also suitable, such as Dako's Ultramount (Code S1964).

[0422] Staining procedures for the Dako automated stainer

[0423] Program Description

[0424] All reagents should be equilibrated to room temperature (20-25°C) prior to immunostaining. Similarly, all incubations should be performed at room temperature.

[0425] Do not allow tissue sections to dry out during the staining process. Dry tissue sections may show increased nonspecific staining.

[0426] Dewax and rehydrate. Before staining, tissue slides must be dewaxed to remove the embedding medium and rehydrated. Avoid incomplete paraffin removal. Residual embedding medium can lead to increased nonspecific staining.

[0427] Step 1. Place the slide in a xylene bath and incubate for 5 (± 1) minutes. Change the bath and repeat once.

[0428] Step 2. Tap off excess liquid and place the slide in absolute ethanol for 3 (± 1) minutes. Change the bath and repeat once.

[0429] Step 3. Tap off excess liquid and place the slide in 95% ethanol for 3 (± 1) minutes. Change the bath and repeat once.

[0430] Step 4. Tap off excess liquid and place the slide in reagent-grade water for 5 (± 1) minutes.

[0431] Step 5. Tap off excess liquid and place slides in wash buffer. Begin the staining procedure as outlined in the "Staining Protocol."

[0432] The xylene and alcohol solutions should be replaced after 40 slides. Toluene or a xylene substitute, such as Histoclear, can be used in place of xylene. EGFR pharmDx includes a pretreatment step involving a proteolytic digestion step. Tissue sections can sometimes be over-digested, resulting in disruption of cell membranes and overall tissue architecture. Pay close attention to the duration of the proteolytic digestion step when running the experiment.

[0433] Post-fixation procedures

[0434] 1. Deparaffinize sections and immerse in reagent-grade water.

[0435] 2. Immerse the slides in 10% neutral buffered formalin for 10 minutes.

[0436] 3. Rinse the slides twice with deionized or distilled water.

[0437] 4. Continue with EGFR pharmDx staining procedure.

[0438] Automated staining protocol

[0439] Step 1. Select the desired protocol and program to run the staining.

[0440] Step 2. Use the automated program to set up the program and start the EGFR pharmDx program.

[0441] Step 3. Place the reagent bottles in the reagent rack of the DAKO automatic stainer according to the reagent map generated by the computer.

[0442] Step 4. Load the slides onto the DAKO automatic stainer according to the slide map generated by the computer.

[0443] Step 5. Start running.

[0444] Step 6. Remove the slides from the DAKO automated stainer.

[0445] Proceed with counterstaining and mounting. After the DAB + substrate-chromogen solution step, rinse the slides with reagent-grade water (DAKO autostainer hardware versions 02 and 03 rinse the slides with reagent-grade water after the substrate-chromogen solution step. DAKO autostainer hardware version 01 rinses the slides with buffer. Therefore, slides stained on hardware 01 must be rinsed with reagent-grade water after they are removed from the autostainer).

[0446] Explanation of the staining procedure

[0447] Section evaluation should be performed by a pathologist using a light microscope. All evaluations should be performed on the tumor region of the specimen. For evaluation and scoring of immunocytochemical staining, a 10X or 20X magnification objective is appropriate.

[0448] Interpret staining results using intact cells; necrotic or degenerated cells will often stain nonspecifically. Positive and negative cell lines are included in each EGFR pharmDx kit to verify that the staining run is working properly each time an assay is performed. Appropriate staining of the control cell lines provides evidence that the EGFR pharmDx assay is working properly. No membrane staining (0) for the CAMA-1 control cell line and moderate brown complete or incomplete membrane staining (2+) for the HT-29 control cell line indicate that the staining run is working properly. If the staining intensity of the positive control cell line is too weak or too strong, false negative or false positive results may be obtained and the assay should be repeated. Reference images are available in the EGFR pharmDx Interpretation Guide.

[0449] EGFR pharmDx primarily stains the cell membrane, demonstrating both complete and incomplete circumferential staining. Immunostaining patterns are often heterogeneous, exhibiting varying staining intensities within a single tumor. Staining is also observed in the cytoplasm and extracellular space. Cytoplasmic staining is common; however, if prominent cytoplasmic staining makes it difficult to distinguish membrane staining and interpret the results, the test should be repeated.

[0450] Tumors should be reported as EGFR positive or EGFR negative, using membrane staining as an evaluable structure. If a tumor cell has any membrane staining result above background, then it is EGFR positive regardless of whether it is a complete circumference. If there is no membrane staining result above background in any tumor cell, then the tumor is reported as an EGFR negative tumor.

[0451] Depending on the incubation time and potency of the hematoxylin used, counterstaining will result in a light to dark blue nucleus. Excessive or incomplete counterstaining may affect the interpretation of the results.

[0452] Staining intensity was established as follows: 3+ (strong staining): visible at low magnification, x5 objective, and confirmed at higher levels as needed; 2+ (moderate staining): visible at moderate magnification, x10 or x20 objective; 1+ (weak staining): reliably confirmed only at high magnification, x40 objective; 0 (no staining): no staining visible at high magnification.

[0453]

[0454] EGFR H-score

[0455] Membrane staining assessment using IHC categorizes samples into four staining intensity categories (0 to 3+). Of note, only linear intercellular staining of tumor cells was considered positive, while complete and incomplete membrane staining were taken into account and recorded. Furthermore, for the Histo-score calculation, all membrane staining was considered independent of integrity (complete and incomplete membrane staining).

[0456] The H-score was assigned using the following formula: [1×(percentage of cells with 1+ staining)+2×(percentage of cells with 2+ staining)+3×(percentage of cells with 3+ staining)], resulting in an EGFR H-score between 0 and 300.

Claims

1. An antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative and / or analog thereof, for use in treating cancer in a subject whose cancer has progressed after prior treatment with an immune checkpoint inhibitor, and wherein the cancer expresses EGFR.

2. Use of an antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative and / or analog thereof, in the manufacture of a medicament for treating a cancer in a subject, wherein the cancer has progressed after prior treatment with an immune checkpoint inhibitor and the cancer expresses EGFR.

3. A method for treating a subject having a cancer that expresses EGFR, wherein the subject has progressed after prior treatment with an immune checkpoint inhibitor, the method comprising providing to the subject an effective amount of an antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative and / or analog thereof.

4. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the cancer is head and neck cancer, preferably head and neck squamous cell carcinoma (SCCHN), and the cancer preferably expresses EGFR, characterized in that IHC score was 2+ or 3+.

5. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the cancer is gastric cancer, esophageal cancer or gastroesophageal junction cancer with EGFR expression characterized by an IHC score of 3+.

6. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the cancer is gastric cancer, esophageal cancer or gastroesophageal junction cancer with EGFR expression characterized by an EGFR H-score of greater than 200.

7. An antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative and / or analog thereof, for use in treating gastric cancer, esophageal cancer or gastroesophageal junction cancer in a subject, wherein the cancer expresses EGFR, characterized in that IHC score was 3+.

8. An antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative and / or analog thereof, for use in treating gastric cancer, esophageal cancer or gastroesophageal junction cancer in a subject, wherein the cancer expresses EGFR, characterized in that The H-score of EGFR is greater than 200.

9. An antibody comprising a first variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative and / or analogue thereof, for use in treating cancer in a subject, wherein the first variable domain is a heavy chain variable region comprising: - a CDR3 sequence of at least the VH of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 , or a CDR3 sequence that differs from the VH CDR3 sequence of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 in at most three, preferably at most two, preferably not more than one amino acid; at least the CDR1, CDR2 and CDR3 sequence of the VH of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 ; or the CDR1, CDR2 and CDR3 sequence of the VH of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 and having at most three, preferably at most two, preferably at most one amino acid substitution; or The sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3; or the amino acid sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 and having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and preferably 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof relative to the VH chain of MF3370; MF3755; MF4280 or MF4289; and wherein the cancer is head and neck cancer, preferably head and neck squamous cell carcinoma (SCCHN), the cancer preferably expresses EGFR, characterized in that An IHC score of 2+ or 3+, or wherein the cancer is a gastric cancer, an esophageal cancer, or a gastroesophageal junction cancer with EGFR expression characterized by an IHC score of 3+ or preferably an H-score of EGFR greater than 200.

10. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the subject has not received previous treatment with an anti-EGFR agent.

11. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of claims 1 to 9, wherein the subject has not received previous treatment with an antibody targeting EGFR.

12. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of claims 1 to 9, wherein the subject has not received previous treatment with cetuximab.

13. The antibody or functional part, derivative and / or analogue thereof according to any one of claims 7 to 12, wherein the cancer has progressed after previous treatment with an immune checkpoint inhibitor.

14. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the cancer expresses EGFR, characterized in that The H score is between greater than 200 and no greater than 300.

15. The antibody or functional part, derivative and / or analogue thereof according to claim 14, wherein the H-score of EGFR is determined using IHC.

16. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the subject is a mammal, preferably a human.

17. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the treatment comprises providing an effective amount of the antibody or functional part, derivative and / or analogue thereof to the subject.

18. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the treatment comprises providing the subject with a fixed dose of 1500 mg of the antibody or functional part, derivative and / or analogue thereof.

19. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the antibody or functional part, derivative and / or analogue thereof is provided to the subject by intravenous injection.

20. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the antibody or functional part, derivative and / or analogue thereof is provided weekly, biweekly or monthly, preferably biweekly, more preferably at least 3 or more biweekly doses of the antibody or functional part, derivative and / or analogue thereof are provided to the subject.

21. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the antibody is ADCC enhanced.

22. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the antibody is defucosylated.

23. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the cancer is adenocarcinoma or squamous cell carcinoma, in particular gastric adenocarcinoma, esophageal adenocarcinoma or gastro-esophageal junction adenocarcinoma or in particular head and neck squamous cell carcinoma (HNSCC).

24. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the cancer and / or the subject is SMAD4 wild type.

25. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the cancer or subject has a TP53 mutation, preferably an activating TP53 mutation.

26. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the cancer or subject is Her2 negative.

27. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the antibody is a multispecific antibody, preferably a bispecific antibody.

28. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the antibody comprises a second variable domain that does not bind to EGFR.

29. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the antibody comprises a second variable domain that binds LGR5.

30. The antibody or functional part, derivative and / or analogue thereof, or use or method as described in any one of claims 1 to 26, wherein the antibody is a monovalent antibody that does not comprise a second variable domain, or wherein the antibody comprises the first EGFR binding variable domain as the only variable domain.

31. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of the preceding claims, wherein the immune checkpoint inhibitor comprises a PD-L1 or PD-1 inhibitor.

32. antibodies or their functional part, derivative and / or analog, or purposes or methods as described in any one of the preceding claims, wherein the treatment comprises a step or said step of diagnosing the experimenter's EGFR status, SMAD4 status and / or Her2 status before the treatment, wherein the diagnosis of Her2 status is preferably carried out by ISH or IHC.

33. An antibody or functional part, derivative and / or analogue thereof, or use or method as claimed in any preceding claim, wherein the first variable domain that binds to EGFR binds to an epitope located within amino acid residues 420 to 480 of the human EGFR sequence shown in Figure 2.

34. The antibody or functional part, derivative and / or analogue thereof, or use or method according to any of the preceding claims, wherein the binding of the first variable domain to EGFR is reduced by substitution of one or more of the following amino acid residues in EGFR: I462A, G465A, K489A, I491A, N493A, and C499A, compared to an EGFR protein not comprising said substitution.

35. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to any one of claims 29 to 34, wherein the variable domain that binds to LGR5 binds to an epitope located within amino acid residues 21 to 118 of the human LGR5 sequence shown in Figure 1.

36. The antibody or functional part, derivative and / or analogue thereof, or use or method according to any one of claims 1 to 8 or 10 to 35, wherein the first variable domain is a heavy chain variable region comprising: - a CDR3 sequence of at least the VH of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 , or a CDR3 sequence that differs from the VH CDR3 sequence of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 in at most three, preferably at most two, preferably not more than one amino acid; at least the CDR1, CDR2 and CDR3 sequence of the VH of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 ; or the CDR1, CDR2 and CDR3 sequence of the VH of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 and having at most three, preferably at most two, preferably at most one amino acid substitution; or The sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3; or the amino acid sequence of the VH chain of MF3370; MF3755; MF4280 or MF4289 as shown in Figure 3 and having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and preferably 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof relative to the VH chain of MF3370; MF3755; MF4280 or MF4289.

Images