Cancer treatment with antibodies that bind to LGR5 and EGFR

Abstract

One objective is to provide improved or alternative cancer treatments, particularly for head and neck cancers. [Solution] This disclosure relates to means and methods in the treatment of cancer. This disclosure relates, in particular, to a method for treating cancer in an individual with antibodies that bind to LGR5 and EGFR. The present invention further relates to combinations for use in such a method, and combinations for use in the manufacture of drugs for the treatment of head and neck cancer.

Description

[Technical Field]

[0001] This disclosure relates to means and methods in the treatment of cancer. In particular, this disclosure relates to methods for treating cancer in an individual with antibodies that bind to LGR5 and EGFR. The present invention further relates to combinations for use in such methods and combinations for use in the manufacture of drugs for the treatment of head and neck cancer. Such antibodies are particularly useful in the treatment of head and neck cancer. [Background technology]

[0002] Traditionally, most cancer drug discoveries have focused on drugs that block essential cellular functions and kill dividing cells through chemotherapy. However, chemotherapy rarely results in a complete cure. In most cases, tumors in patients only stop growing or shrink temporarily (known as remission), only to start growing again, and sometimes more rapidly (known as relapse), becoming increasingly difficult to treat. More recently, the focus of cancer drug development has shifted from broad-spectrum cytotoxic chemotherapy to less toxic targeted cell suppression therapies. Targeted therapies that specifically inhibit signaling pathway components have been clinically validated in treating advanced cancers in leukemia. However, targeted approaches have still proven ineffective in most cancers.

[0003] Despite numerous advances in treating the disease and increasing knowledge of the molecular events that lead to cancer, cancer remains a leading cause of death worldwide. In the United States, head and neck cancers, particularly those of the oral cavity and pharynx, already account for 3% of malignant tumors, with approximately 53,000 Americans developing cancer each year and 10,800 dying from such conditions (Siegel et al., CA Cancer J Clin. 2020;70(1):7. Epub 2020 Jan 8.). Furthermore, head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally, with a reported 5-year overall survival rate of approximately 40-50% for patients with HNSCC (Head and Neck Cancer, Union for International Cancer Control, 2014 Review of Cancer Medicines on the WHO List of Essential Medicines).

[0004] A meta-analysis of locally advanced head and neck squamous cell carcinoma (LA-HNSCC) reported that the addition of anti-EGFR agents to radiotherapy or chemoradiotherapy did not improve clinical outcomes in patients with LA-HNSCC (Oncotarget.2017;8(60):102371-102380). Furthermore, the addition of anti-EGFR agents was reported to increase the risk of skin toxicity and mucositis.

[0005] Therefore, there is a need for improved or alternative cancer treatments, particularly for treating head and neck cancers. [Overview of the project]

[0006] This disclosure provides the following preferred embodiments and forms. However, the present invention is not limited thereto.

[0007] This disclosure provides an antibody, or a functional moiety, derivative, and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, for use in the treatment of cancer in a subject, wherein such use includes providing the subject with a uniform dose of 1500 mg of the antibody, or a functional moiety, derivative, and / or analog thereof. The cancer to be treated is preferably head and neck cancer.

[0008] This disclosure further provides a method for treating head and neck cancer, comprising administering an antibody, or a functional portion thereof, a derivative, and / or analog thereof, to a subject in need of treatment for head and neck cancer, the 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.

[0009] Furthermore, the present invention provides an antibody, or a functional portion thereof, derivative, and / or analog thereof, 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 such use includes providing or administering to a subject a uniform dose of 1500 mg of the antibody, or a functional portion thereof, derivative, and / or analog thereof.

[0010] This disclosure provides an antibody, or a functional moiety, derivative, and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, for use in the treatment of head and neck cancer in a subject. This disclosure further provides a method for treating head and neck cancer, comprising providing an antibody, or a functional moiety, derivative, and / or analog thereof, to a subject in need of treatment for head and neck cancer. Preferably, such use comprises providing the subject with a uniform dose of 1500 mg of the antibody, or a functional moiety, derivative, and / or analog thereof.

[0011] Preferably, the antibody, or its functional portion, derivatives, and / or analogs, are delivered intravenously.

[0012] Preferably, the cancer has mutations in one or more EGFR signaling pathway genes, more preferably in HRAS, MAP2K1, and / or PLCG2. More preferably, the mutation is in a gene whose expression product is active downstream of EGFR in the EGFR signaling pathway, most preferably in HRAS.

[0013] Preferably, the cancer has mutations in one or more WNT signaling pathway genes, more preferably in APC, CREPPB, CUL1, EP300, SOX17, and / or TP53.

[0014] Preferably, the cancer has mutations in genes selected from AKT1, KRAS, MAP2K1, NRAS, HRAS, PIK3CA, PTEN, and EGFR. More preferably, the cancer has mutations in genes encoding TP53, PIK3CA, CDKN2A, NOTCH1, HRAS, and / or MAP2K1. Preferably, the cancer has mutations in one or more genes shown in Table 1. Preferably, the cancer has one or more mutations from among those shown in Table 1.

[0015] Specifically, the cancer is head and neck cancer, more specifically squamous cell carcinoma or adenocarcinoma, most specifically head and neck squamous cell carcinoma (HNSCC). Specifically, head and neck cancer can occur in the pharynx, including the nasopharynx, oropharynx, and hypopharynx. Specifically, head and neck cancer can occur in the larynx. Specifically, head and neck cancer can occur in the paranasal sinuses and nasal cavity. Specifically, head and neck cancer can occur in the salivary glands. In the preferred disclosure, the cancer is oropharyngeal HNSCC.

[0016] Therefore, head and neck cancer specifically includes adenocarcinoma, but more preferably squamous cell carcinomas of the head and neck, such as nasopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, oral cancer, oropharyngeal cancer, and salivary gland cancer.

[0017] Preferably, the cancer expresses EGFR and / or LGR5. As used herein, the cancer expresses LGR5 if it includes cells that express LGR5. Cells that express LGR5 contain detectable levels of RNA encoding LGR5. As used herein, the cancer expresses EGFR if it includes cells that express EGFR. Cells that express EGFR contain detectable levels of RNA encoding EGFR. Expression can also be detected by incubating cells with an antibody that binds to LGR5 or EGFR, and by detection using immunohistochemistry against either or both antigens.

[0018] Preferably, cancer expresses LGR5 at a sufficient level for antibodies that bind to the LGR5 protein, particularly antibodies containing a variable domain that binds to LGR5, including the amino acid sequence of the VH chain of MF5816 shown in Figure 3, or an alternative variable domain that binds to LGR5 as described herein. Preferably, cancer expresses EGFR at a sufficient level for antibodies that bind to the EGFR protein, particularly antibodies containing a variable domain that binds to EGFR, including the amino acid sequence of the VH chain of MF3755 shown in Figure 3, or an alternative variable domain that binds to EGFR as described herein.

[0019] Preferably, the VH chain of the variable domain that binds to EGFR includes the amino acid sequence of the VH chain MF3755 shown in Figure 3, or insertions, deletions, substitutions, or combinations thereof to the VH, with a maximum of 15 amino acid modifications, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less. The VH chain of the variable domain that binds to LGR5 includes the amino acid sequence of the VH chain MF5816 shown in Figure 3, or insertions, deletions, substitutions, or combinations thereof to the VH, with a maximum of 15 amino acid modifications, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less.

[0020] Preferably, the variable domain that binds to LGR5 binds to an epitope located within amino acid residues 21-118 of the human LGR5 sequence shown in Figure 1. Preferably, the amino acid residues at positions 43, 44, 46, 67, 90, and 91 of human LGR5 are involved in the binding of the LGR5-binding variable domain to LGR5. Preferably, the LGR5-binding variable domain rarely binds to LGR5 proteins containing one or more amino acid residue mutations selected from 43A, 44A, 46A, 67A, 90A, and 91A.

[0021] Preferably, the EGFR-binding variable domain binds to an epitope located within amino acid residues 420-480 of the human EGFR sequence shown in Figure 2. Preferably, the amino acid residues at positions I462, G465, K489, I491, N493, and C499 of human EGFR are involved in the binding of the EGFR-binding variable domain to EGFR. Preferably, the EGFR-binding variable domain rarely binds to EGFR proteins containing one or more amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A, and C499A.

[0022] Preferably, the antibody is ADCC-enhancing. Preferably, the antibody is defucosylated. Preferably, the subject to which the antibody of the present disclosure is administered has an immune system that enables engagement of the Fc region of the antibody of the present invention. More preferably, the subject includes FcγRIIIa (CD16+) and / or FcγRIIa (CD32+) immune effector cells for engaging the Fc region of the antibody of the present invention. The immune effector cells are preferably natural killer cells (NK cells), macrophages, or neutrophils that include the Fc receptor. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] <000090>Human LGR5 sequence, SEQ ID NO: 1. <000091><000092>Human EGFR sequence; SEQ ID NO: 2. <000093><000094>a). The amino acid sequence (SEQ ID NOs: 3-15) of the heavy chain variable region that forms a variable domain that binds to LGR5 and EGFR, together with a common light chain variable region such as the variable region of human kappa light chain IgVκ1 39*01 / IGJκ1*01. The CDRs and framework regions are shown in FIG. 3b. The respective DNA sequences are shown in FIG. 3c. <000095><000096>a). The amino acid sequence of the common light chain amino acid sequence. b) The common light chain variable region DNA sequence and translation (IGKV1-39 / jk1). c) The light chain constant region DNA sequence and translation. d) Of the V region IGKV1-39A, e) CDR1, CDR2, and CDR3 of the common light chain by IMGT numbering. <000097><000098>IgG heavy chain for the production of 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 mutant L351K and T366K (KK) DNA sequence and translation. e) CH3 domain containing the mutant L351D and L,368E (DE) DNA sequence and translation. The residue positions are by EU numbering. <000099><00001Data showing mean tumor volume in six head and neck PDX models treated with EGFR and LGR5-targeted controls and antibodies, along with relevant error bars based on a two-sided test. Both antibodies and controls were administered once weekly for 6 weeks, indicated by the gray zone. [Modes for carrying out the invention]

[0024] To facilitate understanding of this description, certain terms are defined first. Additional definitions are provided throughout the modes for carrying out the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, and conventional methods of immunology, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology are used.

[0025] As used herein, the singular forms "a," "an," and "the" include plural referents. The use of terms such as "comprise," "comprises," "comprised," "has," "have," "had," "include," "includes," and "included," as well as other forms of "comprising," "having," and "including," is not limited.

[0026] As used herein, the term “antibody” means a protein molecule belonging to the immunoglobulin class of proteins, containing one or more domains that bind to an epitope on an antigen, wherein such domains derive from or share sequence homology having a variable region of the antibody. Antibodies typically consist of basic structural units, each having two heavy chains and two light chains. Antibodies according to the present invention are not limited to any particular form or method of production thereof.

[0027] A “bispecific antibody” is an antibody described herein in which one domain of the antibody binds to a first antigen while a second domain of the antibody binds to a second antigen, and the first and second antigens are not identical, or one domain binds to a first epitope on the antigen while the second domain binds to a second epitope on the antigen. The term “bispecific antibody” also encompasses antibodies in which one heavy chain variable region / light chain variable region (VH / VL) combination binds to a first antigen or epitope on the antigen and to a second VH / VL combination that binds to a second antigen or epitope on the antigen. The term further includes antibodies in which VH can specifically recognize the first antigen and VL can specifically recognize the second antigen by pairing with VH in an immunoglobulin variable domain. The resulting VH / VL pair binds to either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO2008 / 027236, WO2010 / 108127, and Schaefer et al (Cancer Cell 20, 472-486, October 2011). The bispecific antibodies according to the present invention are not limited to any particular bispecific form or method of production thereof.

[0028] As used herein, the term “common light chain” refers to two light chains (or their VL portions) in a bispecific antibody. The two light chains (or their VL portions) may be identical or may have some amino acid sequence differences, but the binding specificity of the full-length antibody is not affected. The terms “common light chain,” “common VL,” “single light chain,” and “single VL” are all used interchangeably herein, with or without the addition of the term “rearranged.” “Common” also refers to a functional equivalent of a light chain whose amino acid sequences are not identical. Many variants of the light chain exist in which mutations (deletions, substitutions, insertions, and / or additions) exist that do not substantially affect the formation of the functional binding region. The light chain of the present invention may also be the light chain described herein having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof. For example, preparing or finding variable light chains that are not identical but are still functionally equivalent by introducing conservative amino acid changes, such as changes in amino acids in regions that do not contribute to or only partially contribute to binding specificity when paired with a heavy chain, falls within the definition of a common light chain as used herein.

[0029] As used herein, “contains” and its conjugations are used in their non-restrictive sense, meaning that the item preceding the word is included, but items not specifically mentioned are not excluded. In addition, the verb “~consist of” may be replaced with “~essentially consist of,” meaning that a compound or auxiliary compound as defined herein may contain additional components other than those specifically identified, and such additional components do not alter the inherent characteristics of the invention.

[0030] The terms "full-length IgG" or "full-length antibody" according to the present invention are defined as containing essentially complete IgG, but not necessarily possessing all the functions of intact IgG. To avoid misunderstanding, full-length IgG contains two heavy chains and two light chains. Each chain contains a constant (C) region and a variable (V) region, which can be classified into domains designated as CH1, CH2, CH3, VH, and CL, VL. IgG antibodies bind to antigens via the variable region domain contained in the Fab portion, and after binding, they can interact with molecules and cells of the immune system via the constant domain, mainly via the Fc portion. Full-length antibodies according to the present invention include IgG molecules in which mutations may exist that provide desired characteristics. Full-length IgG should not have deletions of substantial portions of any region. However, IgG molecules with one or more amino acid residues deleted without essentially altering the binding properties of the resulting IgG molecule are included in the term "full-length IgG." For example, such an IgG molecule may have 1 to 10 amino acid residues deleted, preferably within a non-CDR region, and the deleted amino acids are not essential for the antigen-binding specificity of IgG.

[0031] An "antibody derivative" is a protein that deviates from the amino acid sequence of a natural antibody by at most 20 amino acids, excluding the CDR region. The antibody derivatives disclosed herein are antibodies that deviate from the amino acid sequence by at most 20 amino acids.

[0032] In this specification, "identity percentage (%)" for nucleic acid sequences or amino acid sequences is defined as the percentage of residues in a candidate sequence that are identical to residues in a selected sequence after the sequences have been aligned for optimal comparison purposes. The sequence identity percentage for comparing nucleic acid sequences is determined using the AlignX application of Vector NTI Advance® 11.5.2 software with default settings using the modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, (1994) Nuc. Acid Res. 22(22):4673-4680), a swgapdnamt score matrix, a gap opening penalty of 15, and a gap elongation penalty of 6.66. The amino acid sequence is determined using the AlignX application of Vector NTI Advance® 11.5.2 software with default settings that employ a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, (1994) Nuc. Acid Res. 22(22):4673-4680), a bloomum62mt2 score matrix, a gap opening penalty of 10, and a gap stretching penalty of 0.1.

[0033] Antibodies typically recognize epitopes on antigens, and such epitopes may also be present in other compounds. Therefore, an antibody according to the present invention that "specifically recognizes" an antigen, such as EGFR or LGR5, may also recognize other compounds if those other compounds contain the same type of epitope. Thus, the term "specifically recognizes" in relation to the interaction between an antigen and an antibody does not preclude the binding of the antibody to other compounds containing the same type of epitope.

[0034] An "epitope" or "antigen determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from adjacent or non-adjacent amino acids juxtaposed by tertiary folding of proteins (so-called linear epitopes and conformational epitopes). Epitopes formed from adjacent linear amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding typically lose their conformation upon treatment with denaturing solvents. Epitopes can typically contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids within a specific spatial conformation.

[0035] As used herein, the terms “subject” and “patient” are interchangeable and refer to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cattle, horses, and pigs (for example, patients such as human patients with cancer). Preferably, the subject is a human subject.

[0036] The terms “to treat,” “to treat,” and “treatment” as used herein refer to any type of intervention or process that involves administering an active agent or combination of active agents to a subject for the purpose of reversing, alleviating, improving, inhibiting, delaying, or preventing the progression, onset, worsening, or recurrence of any symptom, complication, condition, or biochemical sign associated with a disease.

[0037] As used herein, “effective treatment” or “positive treatment response” means a treatment that results in a beneficial effect, for example, improvement of at least one symptom of a disease or disorder, such as cancer. A beneficial effect can take the form of improvement above baseline, including improvement beyond measurements or observations made before initiating treatment according to the method. For example, a beneficial effect can take the form of delaying, stabilizing, stopping, or reversing the progression of cancer in a subject at any clinical stage, as demonstrated by a reduction or elimination of clinical or diagnostic symptoms of the disease, or cancer markers. An effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, delay or stop tumor growth, and / or prevent or delay tumor recurrence or relapse.

[0038] The term “effective dose” or “therapeutic effective dose” refers to the amount of an agent or combination of agents that provides a desired biological, therapeutic, and / or preventive outcome. The outcome may be reduction, improvement, remission, lessening, delay, and / or mitigation of one or more signs, symptoms, or causes of a disease, or any other desired change in the biological system. In some embodiments, the effective dose is sufficient to delay tumor development. In some embodiments, the effective dose is sufficient to prevent or delay tumor recurrence. The effective dose may be administered in one or more doses. An effective dose of an agent or composition may (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, delay, to some extent delay, and halt the invasion of cancer cells into peripheral organs, (iv) inhibit tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay tumor development and / or recurrence, and / or (vii) to some extent alleviate one or more symptoms associated with cancer. For example, the "effective dose" is the amount of EGFR / LGR5 antibody that affects the reduction of cancer (e.g., a reduction in the number of cancer cells), slows the progression of cancer, or prevents cancer regrowth or recurrence.

[0039] In this specification, the term “uniform dose” refers to a dosing regimen in which a subject is administered with a fixed amount of therapeutic agent over multiple doses, regardless of the subject’s body weight. Uniform dosing is typically abbreviated as qnw, where n is an integer indicating the interval and w is a week. For example, a q2w uniform dose dosing regimen of 1500 mg antibody means that a fixed amount of 1500 mg of antibody is administered every two weeks. In this specification, the therapeutic agent is preferably antibody-conjugated EGFR and LGR5 administered by a 1500 mg q2w dosing regimen.

[0040] A uniform dose may be premedicated, meaning that a drug is administered to the subject before the antibody of the present invention is administered. Preferably, a uniform dose of 1500 mg of the antibody is premedicated with an antihistamine, analgesic, antipyretic, and / or anti-inflammatory drug.

[0041] This disclosure provides antibodies, functional portions thereof, derivatives, and / or analogs thereof, for use in the treatment of cancer, comprising variable domains that bind to the extracellular portion of EGFR and variable domains that bind to the extracellular portion of LGR5. The terms cancer and tumor are used herein, unless otherwise specified, and usually both refer to cancer.

[0042] The epidermal growth factor (EGF) receptor (EGFR, ErbB1, or HER1) is a member of a family of four receptor tyrosine kinases (RTKs) named Her- or cErbB-1, -2, -3, and -4. EGFR is known by various synonyms, the most common being EGFR. EGFR has an extracellular domain (ECD) consisting of four subdomains, two of which are involved in ligand binding, and two of which are involved in homodimerization and heterodimerization. EGFR integrates extracellular signals from various ligands, resulting in diverse intracellular responses. The major signaling pathway activated by EGFR consists of the Ras-mitogen-activated protein kinase (MAPK) pro-mitotic signaling cascade. Activation of this pathway is initiated by the recruitment of Grb2 to tyrosine-phosphorylated EGFR. This leads to activation of Ras by the Grb2-bound Ras-guanine nucleotide exchange factor Son of Sevenless (SOS). In addition, the PI3-kinase-Akt signaling pathway is also activated by EGFR, but this activation is considerably stronger in the presence of co-expression of ErbB-3 (HER3). EGFR is involved in several human epithelial malignancies, particularly breast, bladder, non-small cell lung cancer, colon, ovarian, and brain cancers. Activating mutations in the gene, as well as overexpression of the receptor and its ligand, have been found, creating an autocrine activation loop. Therefore, this RTK is widely used as a target in cancer therapy. Both small molecule inhibitors targeting the RTK and monoclonal antibodies (mAbs) directed at the extracellular ligand-binding domain have been developed, and some clinical success has been demonstrated, although mostly in selected patient groups. The database acceptance number for the human EGFR protein and the gene encoding it is GenBank NM_005228.3. The acceptance number is primarily assigned to provide a further method for identifying EGFR proteins as targets, as the actual sequence of the antibody-bound EGFR protein may change due to mutations in the coding gene, such as mutations that occur in certain cancers.

[0043] Where EGFR is referred to herein, unless otherwise stated, the reference refers to human EGFR. The variable domain antigen-binding site that binds to EGFR binds to EGFR and its various variants, such as variants expressed on certain EGFR-positive tumors.

[0044] The term "LGR" refers to a family of proteins known as leucine-rich repeat-containing G protein coupling receptors. Several members of this family, focusing on LGR4, LGR5, and LGR6, are known to be involved in the WNT signaling pathway.

[0045] LGR5 is a leucine-rich repeat containing G protein-coupled receptor 5. Alternative names for the gene or protein include leucine-rich repeat containing G protein-coupled receptor 5, leucine-rich repeat containing G protein-coupled receptor 5, G protein-coupled receptor HG38, G protein-coupled receptor 49, G protein-coupled receptor 67, GPR67, GPR49, orphan G protein-coupled receptor HG38, G protein-coupled receptor 49, GPR49, HG38, and FEX. The proteins or antibodies of the present invention that bind to LGR5 bind to human LGR5. LGR5-binding proteins or antibodies may, but not necessarily, bind to other mammalian orthologs due to sequence and tertiary structure similarities between humans and other mammalian orthologs. The database acceptance numbers for the human LGR5 protein and the gene encoding it are (NC_000012.12, NT_029419.13, NC_018923.2, NP_001264155.1, NP_001264156.1, NP_003658.1). These acceptance numbers are primarily provided to offer further identification methods for LGR5 as a target, and the actual sequence of the bound LGR5 protein may vary due to mutations in the encoding gene, such as those occurring in certain cancers. The LGR5 antigen-binding site binds to LGR5 and its various variants, such as those expressed by certain LGR5-positive tumor cells.

[0046] Cancers collectively known as head and neck cancers typically begin in squamous cells lining the moist mucous membranes of the inner surfaces of the head and neck, such as the mouth, nose, and throat. These squamous cell cancers are often referred to as head and neck squamous cell carcinomas. Although rare, head and neck cancers can also occur in the salivary glands. Specifically, head and neck cancers can develop in the oral cavity. This includes the lips, the front two-thirds of the tongue, the gums, the inner surfaces of the cheeks and lips, the floor of the mouth under the tongue, the hard palate, and small areas of the gums behind the wisdom teeth.

[0047] Therefore, specifically, head and neck cancers include nasopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, oral cancer, oropharyngeal cancer, or salivary gland cancer. More specifically, the present invention relates to the treatment of cancers including squamous cell head and neck cancers located in the oropharynx.

[0048] In some embodiments, cancer expresses LGR5 and / or EGFR. As used herein, cancer expresses LGR5 if it includes cells that express LGR5. Cells that express LGR5 contain detectable levels of RNA encoding LGR5. As used herein, cancer expresses EGFR if it includes cells that express EGFR. Cells that express EGFR contain detectable levels of RNA encoding EGFR. Expression can often also be detected by incubating cells with antibodies that bind to LGR5 or EGFR. However, some cells do not express sufficiently high levels of protein for such antibody testing. In such cases, mRNA or other forms of nucleic acid sequence detection are preferred. Preferably, EGFR protein expression and LGR5 mRNA expression are detected. Preferably, EGFR and LGR5 detection is performed by tissue microarray (TMA) staining. LGR5 expression is preferably determined using in-situ hybridization (ISH). Therefore, preferably, cancer is ISH positive for LGR5. ISH positivity preferably means that expression is characterized by an H score of 1 or higher. EGFR expression is preferably determined using immunohistochemistry (IHC). Therefore, preferably, cancer is IHC positive for EGFR. Preferably, cancer has an EGFR IHC score of 0, 1+, 2+, or 3+, more preferably 1+, 2+, or 3+, and even more preferably 2+ or 3+. Techniques for detection and scoring based on TMA, ISH, and IHC are each well known to those skilled in the art and are usually commercially available as standard kits. Preferably, the EGFR score is determined using a commercially available EGFR detection kit, for example, the EGFR pharmDx® kit for Dako autostainer (Agilent).For example, for LGR5, quantifying mRNA levels using ISH and determining expression based on the H score can be performed using commercially available kits such as the RNAscope® kit from Advanced Cell Diagnostics (Hayward, CA, USA) on a staining platform such as the BondRx platform (Leica, Wetzlar, Germany). Typically, the ISH H score is in the range of 0 to 400. Alternatively, LGR5 and EGFR expression were determined by RNA sequencing (RNAseq).

[0049] The subjects may not have been previously treated with anti-EGFR agents. More preferably, the subjects have not been treated with EGFR-targeting antibodies, and most preferably, the subjects have not been treated with cetuximab. Such subjects are also referred to as cetuximab-naive subjects or anti-EGFR-naive subjects.

[0050] Furthermore, the subjects may have previously been treated with one or more standard approved therapies or standard care. Surgery or radiotherapy may be preferred for most patients with early or localized disease and may be considered for locally advanced disease, but may not be applicable to all patients, for example, due to the anatomical location of the cancer. Standard approved therapies or standard care as used herein preferably include treatment with the administration of one or more chemotherapeutic agents, preferably platinum-based compounds (e.g., cisplatin, carboplatin), antitumor compounds (e.g., methotrexate), fluoropyrimidines (e.g., fluorouracil, 5-FU, capecitabine), taxanes (e.g., docetaxel or paclitaxel), nucleoside analogs (e.g., gemcitabine), or any combination thereof.

[0051] Cancers such as head and neck cancer may be associated with the presence of mutations. Such mutations include those in known oncogenes such as PIK3CA, KRAS, BRAF, HRAS, MAP2K1, and NOTCH1. Oncogenic mutations are generally described as activating mutations or mutations that impart new function. Another type of cancer mutation involves tumor suppressor genes such as TP53, MLH1, CDKN2A, and PTEN. Mutations in tumor suppressor genes are generally inactivating.

[0052] Preferably, the cancer has mutations in one or more EGFR signaling pathway genes. Preferably, the mutations are located in genes whose expression products are active downstream of EGFR in the EGFR signaling pathway. More preferably, the cancer has mutations in one or more EGFR signaling pathway genes that are not active downstream of EGFR.

[0053] Preferably, the cancer has mutations in a gene and its encoded protein product selected from AKT1, KRAS, MAP2K1, NRAS, HRAS, PIK3CA, PTEN, and EGFR. More preferably, the cancer has mutations in the gene encoding HRAS and / or PLCG2.

[0054] Mutations in the HRAS gene are preferably missense mutations, somatic mutations, and / or oncogenic driver mutations. More preferably, HRAS includes the G12S mutation in its protein sequence, or a G>A missense mutation leading to a G>S amino acid change, and more preferably, the G34A missense mutation in the coding sequence (CDS) of each GGC codon of the HRAS gene. Preferably, the cancer is oral squamous cell carcinoma or squamous cell carcinoma of the oral buccal mucosa and includes the G12S missense mutation in the gene encoding HRAS.

[0055] Mutations in the PLCG2 gene are preferably the R956H mutation, the G>A missense mutation leading to an R>H amino acid change, and the G2867A missense mutation in the coding sequence (CDS) of the CGC codon of the PLCG2 gene.

[0056] Cancer may have mutations in the gene encoding MAP2K1. Mutations in the MAP2K1 gene are preferably missense mutations, somatic mutations, and / or oncogenic driver mutations. More preferably, MAP2K1 includes the mutation L375R in its protein sequence, or the T>G missense mutation leading to an L>R amino acid change, and more preferably the missense mutation T1124G in the coding sequence (CDS) of each CTC codon in the gene encoding MAP2K1.

[0057] Preferably, the cancer does not have mutations in the genes encoding PIK3C2B and / or PTPN11. Preferably, mutations in PIK3C2B include mutation E1169K in its protein sequence, or a G>A missense mutation leading to an E>K amino acid change, and more preferably, a missense mutation G3505A in the coding sequence (CDS) of each GAG codon of the gene encoding PIK3C2B. Preferably, mutations in PTPN11 include mutation G39E in its protein sequence, or a G>A missense mutation leading to a G>E amino acid change, and more preferably, a missense mutation G116A in the coding sequence (CDS) of each GGA codon of the gene encoding PTPN11.

[0058] Notch1 (HGNC ID7881; NOTCH1), also known as AOS5, hN1, AOVD1, and TAN1, is a gene encoding a transmembrane protein that functions in multiple developmental processes and interactions between adjacent cells. The transmembrane protein also functions as a receptor for membrane-bound ligands. Fusions, missense mutations, nonsense mutations, silent mutations, frameshift deletions and insertions, as well as intraframe deletions and insertions, are observed in cancers such as esophageal cancer, hematopoietic and lymphoid cancers, and gastric cancer. NOTCH1 is altered in 4.48% of all cancers, including colorectal adenocarcinoma, lung adenocarcinoma, invasive ductal carcinoma of the breast, endometrioid adenocarcinoma, and cutaneous squamous cell carcinoma, which has the highest prevalence of alterations. 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).

[0059] TP53 (HGNC ID11998) encodes a transcription factor that regulates several activities, including stress response and cell proliferation. Mutations in TP53 are associated with various cancers and are estimated to occur in more than 50% of human cancers, including gastric and esophageal cancer. In particular, the TP53 R248Q mutation has been shown to be associated with cancers including gastric and esophageal cancer (Pitolli et al. Int.J.Mol.Sci.2019 20:6241). Nonsense mutations at positions R196 and R342 have been identified in breast and esophagus, as well as in several tumors including ovarian, prostate, breast, pancreatic, stomach, colon / rectal, lung, esophagus, and bone, respectively (Priestly et al. Nature 2019 575:210-216). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having TP53 mutations, particularly mutations that result in reduced TP53 expression or activity.

[0060] MLH1 (HGNC ID7127; MutL homolog1) is a known tumor suppressor gene that encodes a protein involved in DNA mismatch repair. Mutations in MLH1 are associated with a variety of cancers, including gastrointestinal cancers. Low levels of MLH1 are also associated with esophageal cancer patients with a family history of esophageal cancer (Chang et al. Oncol Lett. 2015 9:430-436), and MLH1 is mutated in 1.39% of patients with malignant esophageal tumors (The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6). In particular, the MLH1 V384D mutation has been shown to be associated with cancer, such as colorectal cancer (Ohsawa et al. Molecular Medicine Reports 2009 2:887-891). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having MLH1 mutations, particularly mutations that result in reduced expression or activity of MLH1.

[0061] PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) encodes the 110 kDa catalytic subunit of PI3K (phosphatidylinositol 3-kinase). Mutations in PIK3CA are associated with various cancers, including gastrointestinal cancers. According to the American Association for Cancer Research, PIK3CA is mutated in 12.75% of patients with malignant solid tumors. Specifically, the PIK3CA H1047R mutation is present in 2.91% of all malignant solid tumor patients, and the PIK3CA E545K mutation is present in 2.55% of all malignant solid tumor patients (see The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having PIK3CA mutations, specifically oncogenic mutations in PIK2CA.

[0062] PIK3C2B (HGNC:8972, phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 beta) encodes a protein belonging to the phosphoinositide 3-kinase (PI3K) family. PI3 kinases play a role in signaling pathways involved in cell proliferation, oncogenic transformation, cell survival, cell migration, and intracellular protein transport. This protein contains a lipid kinase catalytic domain as well as a C-terminal C2 domain, which is characteristic of class II PI3 kinases. The C2 domain acts as a calcium-dependent phospholipid binding motif that mediates translocation of the protein to the membrane and can also mediate protein-protein interactions.

[0063] CDKN2A (HGNC ID1787; cyclin-dependent kinase inhibitor 2A) encodes a protein that inhibits CDK4 and ARF. According to the American Association for Cancer Research, CDKN2A is mutated in 22.21% of esophageal cancer patients, 28.7% of esophageal squamous cell carcinoma patients, and 6.08% of gastric adenocarcinoma patients. Specifically, the CDKN2A W110Ter mutation is present in approximately 0.11% of cancer patients. (The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having CDKN2A mutations, particularly mutations that result in reduced CDKN2A expression or activity.

[0064] PTEN (HGNC ID9588; phosphatase and tensin homolog) encodes phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase. According to the American Association for Cancer Research, PTEN is mutated in 6.28% of cancer patients, 3.41% of gastric adenocarcinoma patients, 2.37% of esophageal cancer patients, and 2.22% of esophageal adenocarcinoma patients. Specifically, the PTEN R130Ter mutation (Ter refers to a stop / stop codon) is present in 0.21% of all colorectal cancer patients (The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having PTEN mutations, particularly mutations that result in reduced PTEN expression or activity.

[0065] BRAF (HGNC ID: 1097) encodes B-Raf, a serine / threonine protein kinase involved in proliferation signaling. According to the American Association for Cancer Research, BRAF is mutated in 1.91% of gastric cancer patients and 1.93% of gastric adenocarcinoma patients. Specifically, the BRAF V600E mutation is present in 2.72% of cancer patients (see The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers with BRAF mutations, specifically oncogenic mutations in BRAF.

[0066] KRAS (HGNC ID6407; Kirsten RAt Sarcoma) encodes a protein that is a party to the RAS / MAPK pathway. According to the American Association for Cancer Research, KRAS is mutated in 14.7% of patients with malignant solid tumors, and KRAS G12C is present in 2.28% of patients with malignant solid tumors (see The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers with KRAS mutations, specifically oncogenic mutations in KRAS.

[0067] The HRAS (HGNC ID: 5173) gene product is involved in the activation of Ras protein signaling. The Ras protein binds to GDP / GTP and possesses intrinsic GTPase activity. Somatic mutations in the HRAS proto-oncogene have been shown to be associated with bladder cancer, thyroid cancer, salivary ductal cancer, epithelial-myoepithelial carcinoma, and renal cancer (Chiosea et al., in Am.J.of Surg.Path.39(6):744-52; Chiosea et al., in Head and Neck Path.2014.8(2):146-50). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having HRAS mutations, specifically oncogenic mutations in HRAS, more preferably HRAS mutation G12S. The cancers are specifically HNSCC of the oral cavity or buccal mucosa.

[0068] MAP2K1 (HGNC ID: 6840) belongs to the group of mitogen-activated protein kinase kinases. It is active in MAP kinase signaling and encodes protein bispecific mitogen-activated protein kinase kinase 1. As part of the MAP kinase pathway, MAP2K1 is involved in many cellular processes, including cell proliferation, differentiation, and transcriptional regulation. MAP2K1 is altered in 1.05% of all cancers with the highest prevalence of mutations: cutaneous melanoma, lung adenocarcinoma, colon adenocarcinoma, melanoma, and invasive ductal carcinoma (AACR Project GENIE Consortium. Cancer Discovery. 2017; 7(8): 818-831. Dataset Version 8). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having MAP2K1 mutations, specifically the MAP2K1 mutation L375R.

[0069] UGT1A1 (HGNC ID12530; uridine diphosphate glucuronosyltransferase 1A1) and UGT1A8 (uridine diphosphate glucuronosyltransferase 1A8) encode enzymes in the glucuronidation pathway. Several polymorphisms that reduce enzyme activity are known to affect the metabolism and effects of irinotecan. For example, UGT1A1, which has an allele frequency of approximately 0.13% in Chinese, Korean, and Japanese populations, * 6 alleles (G71R polymorphism) and UGT1A1 * 28 alleles (dinucleotide repeat polymorphisms in the TATA sequence of the promoter region) are risk factors for irinotecan-induced neutropenia. In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers having mutations in UGT1A1 and / or UGT1A8, specifically mutations resulting in reduced expression or activity of UGT1A1 and / or UGT1A8.

[0070] In this disclosure, head and neck cancer preferably includes one or more gene mutations present in head and neck models HN2167, HN2590, HN2579, HN5124, HN3164, HN3642, HN3411 and / or HN5125 (see Table 1), more preferably one or more gene mutations present in head and neck models HN2167, HN2590, HN2579, HN5124, HN3642, HN3411 and / or HN5125. The mutations are preferably somatic mutations, missense mutations, frameshift mutations, deletions, or any combination thereof.

[0071] In this disclosure, head and neck cancer has one or more mutations in the LGR5 and / or EGFR pathways present in a model selected from the group consisting of HN5124, HN5125, HN2579, HN2590, HN2167, HN3642, and HN3164 (see Table 1).

[0072] In this disclosure, head and neck cancer preferably comprises one or more oncogenic mutations present in head and neck models HN2167, HN2590, HN2579, HN5124, HN3164, HN3642, HN3411 and / or HN5125 (see Table 1), more preferably HN2167, HN2590, HN2579, HN5124, HN3642, HN3411 and / or HN5125. The mutations are preferably somatic mutations, missense mutations, frameshift mutations, deletions, or any combination thereof.

[0073] Preferably, head and neck cancer, specifically laryngeal cancer, or one or more mutations in model HN2167 are CDKN2A (HGNC:1787), CREBBP (HGNC:2348), CUL1 (HGNC:2551), EPHA3 (HGNC:3387), EXT1 (HGNC:3512), FAT2 (HGNC:3596), FOXP1 (HGNC:3823), HIST1H3B (HGNC:4776), HSP90AB1 (HGNC:5258), IKZF The mutations are selected from the group consisting of 3 (HGNC:13178), IL6ST (HGNC:6021), INHBA (HGNC:6066), LMO1 (HGNC:6641), LPP (HGNC:6679), MSR1 (HGNC:7376), NBN (HGNC:7652), RAD54B (HGNC:17228), RGS3 (HGNC:9999), TAOK1 (HGNC:29259), TP53 (HGNC:11998), and WNK1 (HGNC:14540). More preferably, one or more mutations in head and neck cancer, specifically squamous cell laryngeal cancer, include CDKN2A, CREPPB, CUL1, and / or TP53. The mutations are preferably somatic mutations, missense mutations, frameshift mutations, deletions, or any combination thereof. CDKN2A preferably includes deletions and / or frameshift mutations, specifically deletions of the amino acid RAGAR at positions 99-103 of the CDKN2A protein, and more specifically, deletions of the nucleic acid GGGCCGGGGCGCGG at positions 296-309 of the CDKN2A coding sequence. CREPPB preferably includes the mutation R1446C, or a C>T missense mutation leading to an R>C amino acid change, or the missense mutation C4336T in the coding sequence (CDS) of the codon CGC of the CREPPB gene. CUL1 preferably includes the mutation D483N, a G>A missense mutation leading to a D>N amino acid change, or the missense mutation G1447A in the coding sequence (CDS) of the codon GAT of the CUL1 gene. TP53 preferably contains the mutation R273C, or a C>T missense mutation leading to an R>C amino acid change, or a missense mutation C817T in the coding sequence (CDS) of the codon CGT of the TP53 gene.

[0074] Preferably, head and neck cancer, specifically squamous cell carcinoma of the tongue, or one or more mutations in model HN2590 are AHR (HGNC:348), ALK (HGNC:427), ATP6AP2 (HGNC:18305), CDKN2A (HGNC:1787), EP300 (HGNC:3373), FGFR1 (HGNC:3688), FLT4 (HGNC:3767), FN1 (HGNC:3778), H The mutations are selected from the group consisting of LA-B (HGNC:4932), IREB2 (HGNC:6115), MCM8 (HGNC:16147), PLCG2 (HGNC:9066), RB1 (HGNC:9884), THRAP3 (HGNC:22964), TP53 (HGNC:11998), WNK1 (HGNC:14540), YBX1 (HGNC:8014), and ZNF638 (HGNC:17894). More preferably, one or more mutations in head and neck cancer, specifically tongue cancer, include EP300, PLCG2, and / or TP53. The mutations are preferably somatic mutations, missense mutations, frameshift mutations, deletions, or any combination thereof. EP300 preferably contains the mutation S1730C, or a C>G missense mutation leading to an S>C amino acid change, or a missense mutation C5189G in the coding sequence (CDS) of the codon TCT of the EP300 gene. PLCG2 preferably contains the mutation R956H, or a G>A missense mutation leading to an R>H amino acid change, or a missense mutation G2867A in the coding sequence (CDS) of the codon CGC of the PLCG2 gene. TP53 preferably contains the mutation G245S, or a G>A missense mutation leading to a G>S amino acid change, or a missense mutation G733A in the coding sequence (CDS) of the codon GGC of the TP53 gene.

[0075] Preferably, one or more mutations in head and neck cancer, specifically squamous cell carcinoma of the buccal mucosa, or in model HN2579, are selected from the group consisting of DCC (HGNC:2701), DLC1 (HGNC:2897), HRAS (HGNC:5173), LZTS1 (HGNC:13861), SMARCA4 (HGNC:11100), and WRN (HGNC:12791). More preferably, one or more mutations in head and neck cancer, specifically squamous cell carcinoma of the buccal mucosa, include HRAS. The mutations are preferably somatic mutations, missense mutations, frameshift mutations, deletions, or any combination thereof. HRAS preferably includes the mutation G12S in its protein sequence, or a G>A missense mutation leading to a G>S amino acid change, more preferably the missense mutation G34A in the coding sequence (CDS) of the codon GGC of the HRAS gene.

[0076] Preferably, one or more mutations in head and neck cancer, specifically squamous cell carcinoma of the head and neck, or in model HN5124, are selected from the group consisting of APC (HGNC:583), ERCC6 (HGNC:3438), MAD1L1 (HGNC:6762), and ROS1 (HGNC:10261). The mutations are preferably somatic mutations, missense mutations, frameshift mutations, deletions, or any combination thereof. APC preferably includes the mutation R2505Q in its protein sequence, or a G>A missense mutation leading to an R>Q change, more preferably the missense mutation G7514A in the coding sequence (CDS) of codon CGA of the APC gene.

[0077] Preferably, head and neck cancer, specifically adenocarcinoma or parotid gland cancer, or one or more mutations in model HN3164 are selected from the group consisting of DLC1 (HGNC:2897), EPHA4 (HGNC:3388), KIAA1549 (HGNC:22219), MAP2K1 (HGNC:6840), MSH3 (HGNC:7326), and TP53 (HGNC:11998). The mutations are preferably somatic mutations, missense mutations, frameshift mutations, deletions, or any combination thereof. MAP2K1 preferably includes the mutation L375R in its protein sequence, or a T>G missense mutation leading to an L>R amino acid change, more preferably the missense mutation T1124G in the coding sequence (CDS) of the codon CTC of the MAP2K1 gene. TP53 preferably contains the mutation Y234C in its protein sequence, or the A>G missense mutation that leads to a Y>C amino acid change, and more preferably the missense mutation A701G in the coding sequence (CDS) of codon TAC of the TP53 gene.

[0078] Preferably, one or more mutations in head and neck cancer, specifically squamous cell carcinoma of the neck, or model HN5125, are selected from the group consisting of ATM (HGNC:795), ECT2L (HGNC:21118), HLA-B (HGNC:4932), ITGA9 (HGNC:6145), RB1 (HGNC:9884), RGS3 (HGNC:9999), SOX17 (HGNC:18122), and TP53 (HGNC:11998). The mutations are preferably somatic mutations, missense mutations, frameshift mutations, deletions, or any combination thereof. SOX17 preferably includes the mutation L156P in its protein sequence, or the T>C missense mutation leading to an L>P amino acid change, more preferably the missense mutation T467C in the coding sequence (CDS) of codon CTG of the SOX17 gene. TP53 preferably contains the mutation R337C in its protein sequence, or the C>T missense mutation that leads to an R>C amino acid change, and more preferably the missense mutation C1009T in the coding sequence (CDS) of the codon CGC of the TP53 gene.

[0079] The antibodies described herein, or their functional portions, derivatives, and / or analogs, include a variable domain that binds to the extracellular portion of the epidermal growth factor (EGF) receptor and a variable domain that binds to LGR5. EGFR is preferably human EGFR. LGR5 is preferably human LGR5. The antibodies described herein, or their functional portions, derivatives, and / or analogs, include a variable domain that binds to the extracellular portion of the human epidermal growth factor (EGF) receptor and a variable domain that binds to human LGR5.

[0080] Preferably, the antibodies described herein, or their functional portions, derivatives, and / or analogs, comprise a variable domain that binds to the extracellular portion of the epidermal growth factor (EGF) receptor and prevents EGF from binding to the receptor, and a variable domain that binds to LGR5, such that the interaction between the antibody and LGR5 on LGR5-expressing cells does not block the binding of Rspondin (RSPO) to LGR5. A method for determining whether or not an antibody blocks the binding of Rspondin to LGR5 is described in WO2017 / 069528, which is incorporated herein by reference.

[0081] In this specification, protein / gene acceptance numbers or alternative names are given, primarily to provide specific further methods for targeting the aforementioned proteins, and the actual sequence of the antibody-bound target protein may be altered due to mutations and / or alternative splicing in the coding gene, such as mutations occurring in some cancers. The target protein is bound by the antibody insofar as the epitope is present within the protein and the epitope is accessible to the antibody.

[0082] The antibodies, or their functional moieties, derivatives, and / or analogs described herein, preferably interfere with the binding of ligands for EGFR to EGFR. As used herein, the term “interferes with binding” means that the binding of the antibody, or its functional moiety, derivative, and / or analog to EGFR competes with the ligand for binding to the EGF receptor. The antibody, or its functional moiety, derivative, and / or analog may weaken ligand binding, or, if it is already bound to the EGF receptor, may move the ligand, or this may at least partially prevent the ligand from binding to the EGF receptor, for example, via steric hindrance.

[0083] The EGFR antibodies described herein preferably inhibit EGFR ligand-induced signaling, which is measured as ligand-induced proliferation of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058), or ligand-induced cell death of A431 cells (ATCC CRL-1555). EGFR can bind to several ligands and stimulate the proliferation of the aforementioned BxPC3 or BxPC3-luc2 cells. In the presence of an EGFR ligand, the proliferation of BxPC3 or BxPC3-luc2 cells is stimulated. EGFR ligand-induced proliferation of BxPC3 cells can be measured by comparing cell proliferation in the absence and presence of the ligand. A preferred EGFR ligand for measuring EGFR ligand-induced proliferation of BxPC3 or BxPC3-luc2 cells is EGF. Ligand-induced proliferation is preferably measured using a saturated amount of ligand. In a preferred embodiment, EGF is used in a culture medium at a volume of 100 ng / ml. The EGF is preferably the EGF R&D system, catalog numbers 396-HB and 236-EG (see also WO2017 / 069628, which is incorporated herein by reference).

[0084] The EGFR antibodies described herein preferably inhibit EGFR ligand-induced proliferation of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer125058). EGFR can bind to several ligands and stimulate the proliferation of the aforementioned BxPC3 or BxPC3-luc2 cells. In the presence of a ligand, the proliferation of BxPC3 or BxPC3-luc2 cells is stimulated. EGFR ligand-induced proliferation of BxPC3 cells can be measured by comparing cell proliferation in the absence and presence of the ligand. A preferred EGFR ligand for measuring EGFR ligand-induced proliferation of BxPC3 or BxPC3-luc2 cells is EGF. Ligand-induced proliferation is preferably measured using a saturated amount of ligand. In a preferred embodiment, EGF is used in a culture medium at a volume of 100 ng / ml. The EGF is preferably the EGF of the R&D system, catalog numbers 396-HB and 236-EG (see also WO2017 / 069628, which is incorporated herein by reference).

[0085] To avoid misunderstanding, as used herein, references to cell proliferation refer to a change in the number of cells. Inhibition of proliferation refers to a reduction in the number of cells that could have otherwise been obtained. Increased proliferation refers to an increase in the number of cells that could have otherwise been obtained. Cell growth usually refers to cell proliferation.

[0086] Whether the antibodies described herein inhibit signal transduction or proliferation in a multispecific format is preferably determined by the method described above herein, using monospecific monovalent or monospecific bivalent versions of the antibody. Such antibodies preferably have a binding site for the receptor to which signal transduction is determined. Monospecific monovalent antibodies may have a variable domain having irrelevant binding specificity, such as tetanus toxoid specificity. A preferred antibody is a bivalent monospecific antibody in which the antigen-binding variable domain consists of a variable domain that binds to members of the EGF receptor family.

[0087] In its Biclonics® antibody program, Merus developed multispecific antibodies targeting EGFR and LGR5 (leucine-rich repeats containing G protein-coupled receptors). The efficacy of these multispecific antibodies has been evaluated in vitro and in vivo using patient-derived CRC organoids and mouse PDX models, respectively (see, for example, WO2017 / 069628, which is incorporated herein by reference). Multispecific antibodies targeting EGFR and LGR5 have been shown to inhibit tumor growth. The efficacy of these inhibitory antibodies has been shown to correlate with the level of LGR5 RNA expression in cancer-derived cells. The multispecific antibodies targeting EGFR and LGR5 described in WO2017 / 069628 are particularly preferred.

[0088] The antibodies described herein, or their functional portions, derivatives, and / or analogs, include a variable domain that binds to the extracellular portion of LGR5. The variable domain that binds to the extracellular portion of LGR5 preferably binds to an epitope located within amino acid residues 21-118 of the sequence in Figure 1, in which amino acid residues D43, G44, M46, F67, R90, and F91 are involved in antibody binding to the epitope.

[0089] The LGR5 variable domain is preferably a variable domain in which one or more amino acid residue substitutions in LGR5, such as D43A, G44A, M46A, F67A, R90A, and F91A, reduce the binding of the variable domain to LGR5.

[0090] The extracellular epitope of LGR5 is preferably located within amino acid residues 21-118 of the sequence shown in Figure 1. Preferably, it is an epitope in which the binding of the LGR5 variable domain to LGR5 is reduced by one or more of the following amino acid residue substitutions in LGR5: D43A, G44A, M46A, F67A, R90A, and F91A.

[0091] This disclosure further provides an antibody having a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, wherein the LGR5 variable domain binds to an epitope on LGR5 located within amino acid residues 21-118 of the sequence shown in Figure 1.

[0092] The epitopes on LGR5 are preferably structural epitopes. The epitopes are preferably located within amino acid residues 40-95 of the sequence in Figure 1. Antibody binding to LGR5 is preferably reduced by one or more of the following amino acid residue substitutions: D43A, G44A, M46A, F67A, R90A, and F91A.

[0093] While not bound by theory, M46, F67, R90, and F91 of LGR5 shown in Figure 1 are considered in this specification to be contact residues for the antigen-binding site of the variable domain, i.e., the variable domain that binds to the LGR5 epitope. The reduction in antibody binding by amino acid substitutions D43A and G44A may be due to them also being contact residues, but it is also possible that these amino acid substitutions induce a (slight) conformational modification of a portion of LGR5 having one or more other contact residues (i.e., at positions 46, 67, 90, or 91), and that the conformational change is such that antibody binding is reduced. Epitopes are characterized by the aforementioned amino acid substitutions. Whether an antibody binds to the same epitope can be determined in various ways. In an exemplary method, CHO cells express LGR5 on the cell membrane or on alanine substitution mutants, preferably mutants containing one or more of the substitutions M46A, F67A, R90A, or F91A. A test antibody is contacted with the CHO cells, and the binding of the antibody to the cells is compared. The test antibody binds to the epitope if the epitope binds to LGR5, and if the degree of binding to LGR5 is reduced due to the M46A, F67A, R90A, or F91A substitution. It is preferable to compare binding to a panel of mutants, each containing a single alanine residue substitution. Such binding tests are well known in the art. Often, the panel includes single alanine substitution mutants covering substantially all amino acid residues. In the case of LGR5, the panel should, of course, cover only the portion that ensures the extracellular portion of the protein and its relevance to the cell membrane when the cells are used. Expression of certain mutants may be impaired, which can be readily detected by one or more LGR5 antibodies binding to different regions. If the expression of these control antibodies is also reduced, the level or folding of the protein on the membrane will be impaired for this particular variant. The binding characteristics of the test antibody to the panel allow for easy identification of whether the test antibody shows reduced binding to variants having the M46A, F67A, R90A, or F91A substitutions, and therefore whether the test antibody is the antibody of the present invention.By reducing binding to mutants with M46A, F67A, R90A, or F91A substitutions, epitopes located within amino acid residues 21-118 of the sequence in Figure 1 are also identified. In preferred embodiments, the panel includes the D43A substitution mutant and both G44A substitution mutants. Antibodies with the VH sequence of MF5816 show reduced binding to these substitution mutants.

[0094] While not bound by any theory, the amino acid residues I462, G465, K489, I491, N493, and C499 shown in Figure 2 are thought to be involved in the binding of antibodies containing the variable domains described above to the epitope. Involvement in binding is preferably determined by observing a reduction in the binding of the variable domain to EGFR having one or more amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A, and C499A.

[0095] In one embodiment, the variable domain that binds to an epitope on the extracellular portion of human EGFR is a variable domain that binds to an epitope located within amino acid residues 420-480 of the sequence shown in Figure 2. Preferably, the binding of the variable domain to EGFR is reduced by one or more of the following amino acid residue substitutions in EGFR: I462A, G465A, K489A, I491A, N493A, and C499A. The binding of the antibody to human EGFR preferably prevents the binding of EGF to its receptor. The epitope on EGFR is preferably a structural epitope. In one embodiment, the epitope is located within amino acid residues 420-480 of the sequence shown in Figure 2, preferably within 430-480 of the sequence shown in Figure 2, and preferably within 438-469 of the sequence shown in Figure 2.

[0096] While not bound by theory, it is considered highly probable that the epitope contact residues, i.e., the regions where the variable domain contacts human EGFR, are I462, K489, I491, and N493. Amino acid residues G465 and C499 are likely to be indirectly involved in antibody binding to EGFR.

[0097] The variable domain that binds to human EGFR is preferably a variable domain having a heavy chain variable region including at least the CDR3 sequence of MF3755 VH shown in Figure 3, or a CDR3 sequence that differs from the CDR3 sequence of MF3755 VH shown in Figure 3 by a maximum of 3 amino acids, preferably a maximum of 2 amino acids, and preferably 1 amino acid or less.

[0098] The variable domain that binds to human EGFR is preferably a variable domain having a heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences of the VH of MF3755 shown in Figure 3, or up to three, preferably up to two, and preferably up to one amino acid substitution.

[0099] The variable domain that binds to human EGFR is preferably a variable domain having a heavy chain variable region containing the amino acid sequence of the VH chain of MF3755 shown in Figure 3, or up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, relative to the VH chain of MF3755.

[0100] In one embodiment, 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 at least a CDR3 sequence of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or the heavy chain variable region of the variable domain comprises a heavy chain CDR3 sequence that differs from a VH CDR3 sequence selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3 by up to three amino acids, preferably up to two, preferably up to one amino acid. The variable domain preferably comprises a heavy chain variable region comprising at least a CDR3 sequence of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3.

[0101] The variable domain preferably includes a heavy chain variable region containing 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 shown in Figure 3, or a heavy chain variable region containing 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 shown in Figure 3, differing by up to three amino acids, preferably up to two, preferably up to one. The variable domain preferably includes a heavy chain variable region containing at least the CDR1, CDR2, and CDR3 sequences of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3. The preferred heavy chain variable region is MF3755. Another preferred heavy chain variable region is MF4280.

[0102] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, wherein the EGFR-binding variable domain having the CDR3, CDR1, CDR2, and CDR3 and / or VH sequences shown above in this specification preferably has a variable domain that binds to LGR5 containing at least a CDR3 sequence of the LGR5-specific heavy chain variable region selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3, and a VH CDR3 sequence selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818, which contains a heavy chain CDR3 sequence that differs from the heavy chain CDR3 sequence by up to 3, preferably up to 2, preferably 1 or less amino acids. The variable domain preferably includes a heavy chain variable region containing at least one CDR3 sequence of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 as shown in Figure 3.

[0103] The LGR5 variable domain preferably includes a heavy chain variable region containing at least CDR1, CDR2, and CDR3 sequences of an LGR5-specific heavy chain variable region selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3, or a heavy chain variable region containing heavy chain CDR1, CDR2, and CDR3 sequences that differ by a maximum of three, preferably a maximum of two, preferably a maximum of one amino acid. The variable domain preferably includes a heavy chain variable region containing at least the CDR1, CDR2, and CDR3 sequences of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 as shown in Figure 3. Preferred heavy chain variable regions are MF5790, MF5803, MF5814, MF5816, MF5817, or MF5818. Particularly preferred heavy chain variable regions are MF5790, MF5814, MF5816, and MF5818, preferably MF5814, MF5818, and MF5816, with heavy chain variable region MF5816 being particularly preferred. Another preferred heavy chain variable region is MF5818.

[0104] Antibodies containing one or more variable domains having the heavy chain variable region MF3755, or one or more CDRs thereof, have been shown to exhibit better efficacy when used to inhibit the proliferation of EGFR ligand-responsive cancers or cells. In the context of bispecific or multispecific antibodies, an arm of an antibody containing a variable domain having the heavy chain variable region MF3755, or one or more CDRs thereof, binds well to an arm containing a variable domain having the heavy chain variable region MF5818, or one or more CDRs thereof.

[0105] The VH chain of the variable domain that binds to EGFR or LGR5 may have one or more amino acid substitutions relative to the sequence shown in Figure 3. Preferably, the VH chain has the amino acid sequence of EGFR or LGR5 VH in Figure 3, having up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or combinations thereof relative to the VH chain sequence in Figure 3.

[0106] A CDR sequence may have one or more amino acid residue substitutions with respect to the CDR sequence shown in the figure. Such one or more substitutions are preferably made for optimization purposes, for example, to improve the binding strength or stability of the antibody. Optimization is performed by a mutagenesis procedure, for example, after the resulting antibody stability and / or binding affinity is preferably tested and an improved EGFR-specific or LGR5-specific CDR sequence is preferably selected. Those skilled in the art can generate antibody mutants containing at least one modified CDR sequence according to the present invention. For example, conservative amino acid substitutions may be applied. Examples of conservative amino acid substitutions include the substitution of one hydrophobic residue, e.g., isoleucine, valine, leucine, or methionine, with another hydrophobic residue, and the substitution of one polar residue with another polar residue, e.g., arginine to lysine, glutamic acid to aspartic acid, or glutamine to asparagine.

[0107] Preferably, up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid substitutions mentioned in the VH or VL specified herein are preferably conservative amino acid substitutions. Amino acid insertions, deletions, and substitutions in the VH or VL specified herein are preferably not located in the CDR3 region. Amino acid insertions, deletions, and substitutions mentioned are preferably not located in the CDR1 and CDR2 regions either. Amino acid insertions, deletions, and substitutions mentioned are preferably not located in the FR4 region either.

[0108] A maximum of 15 amino acid substitutions, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5, are preferably conservative amino acid substitutions, and insertions, deletions, substitutions, or combinations thereof are preferably not located within the CDR3 region of the VH chain, preferably not within the CDR1, CDR2, or CDR3 regions of the VH chain, and preferably not within the FR4 region.

[0109] Antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 are preferably, - The amino acid sequence of VH chain MF3755 shown in Figure 3, or -The VH chain MF3755 has an amino acid sequence as shown in Figure 3, which includes up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, The VH chain of the variable domain that binds to LGR5 is - The amino acid sequence of VH chain MF5790 shown in Figure 3, or - The VH chain contains the amino acid sequence of MF5790 shown in Figure 3, which has up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0110] Antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 are preferably, - The amino acid sequence of VH chain MF3755 shown in Figure 3, or -The VH chain MF3755 has an amino acid sequence as shown in Figure 3, which includes up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, The VH chain of the variable domain that binds to LGR5 is - The amino acid sequence of VH chain MF5803 shown in Figure 3, or -The VH contains the amino acid sequence of the VH chain MF5803 shown in Figure 3, which has up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0111] Antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 are preferably, - The amino acid sequence of VH chain MF3755 shown in Figure 3, or -The VH chain MF3755 has an amino acid sequence as shown in Figure 3, which includes up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, The VH chain of the variable domain that binds to LGR5 is - The amino acid sequence of VH chain MF5814 shown in Figure 3, or -The VH contains the amino acid sequence of the VH chain MF5814 shown in Figure 3, which has up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0112] Antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 are preferably, - The amino acid sequence of VH chain MF3755 shown in Figure 3, or -The VH chain MF3755 has an amino acid sequence as shown in Figure 3, which includes up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, The VH chain of the variable domain that binds to LGR5 is - The amino acid sequence of VH chain MF5816 shown in Figure 3, or -The VH contains the amino acid sequence of the VH chain MF5816 shown in Figure 3, which has up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0113] Antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 are preferably, - The amino acid sequence of VH chain MF3755 shown in Figure 3, or -The VH chain MF3755 has an amino acid sequence as shown in Figure 3, which includes up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, The VH chain of the variable domain that binds to LGR5 is - The amino acid sequence of VH chain MF5817 shown in Figure 3, or - The VH chain contains the amino acid sequence of the VH chain MF5817 shown in Figure 3, which has up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0114] Antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 are preferably, - The amino acid sequence of VH chain MF3755 shown in Figure 3, or -The VH chain MF3755 has an amino acid sequence as shown in Figure 3, which includes up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof, The VH chain of the variable domain that binds to LGR5 is - The amino acid sequence of VH chain MF5818 shown in Figure 3, or -The VH contains the amino acid sequence of the VH chain MF5818 shown in Figure 3, which has a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0115] Variants of the amino acid sequences disclosed herein that retain EGFR or LGR5 binding can be obtained, for example, from a phage display library containing rearranged human IGKVl-39 / IGKJl VL region (De Kruif et al. Biotechnol Bioeng. 2010(106)741-50) and from a previously described (e.g., WO2017 / 069628) collection of VH regions incorporating amino acid substitutions into the amino acid sequences of the EGFR or LGR5 VH regions disclosed herein. Phages encoding Fab regions that bind to EGFR or LGR5 can be selected, analyzed by flow cytometry, and sequenced to identify variants having amino acid substitutions, insertions, deletions, or additions that retain antigen binding.

[0116] The light chain variable regions of the VH / VL EGFR and LGR5 of the EGFR / LGR5 antibody may be the same or different. In some embodiments, the VL region of the VH / VL EGFR variable domain of the EGFR / LGR5 antibody is the same as the VL region of the VH / VL LGR5 variable domain. In certain embodiments, the VL regions in the first and second VH / VL variable domains are identical.

[0117] In certain embodiments, one or both of the light chain variable regions of the VH / VL variable domains of the EGFR / LGR5 antibody contain a common light chain variable region. In some embodiments, the common light chain variable region of one or both of the VH / VL variable domains contains a germline IgVκ1-39 variable region V segment. In certain embodiments, one or both of the light chain variable regions of the VH / VL variable domains are the kappa light chain V segment IgVκ1-39 * including 01. IgVκ1-39 is an abbreviation of the immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39, IGKV139, IGKV1-39. The external Ids for this gene are HGNC:5740, Entrez Gene:28930, Ensembl:ENSG(00000242371). The amino acid sequences for suitable V regions are provided in Figure 4. The V region can be combined with one of five J regions. The preferred J regions are jk1 and jk5, and the ligated sequences are shown as IGKV1-39 / jk1 and IGKV1-39 / jk5, and alternative names are IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * 01 (named by the IMGT database world wide web at imgt.org). In certain embodiments, the light chain variable regions of one or both of the VH / VL variable domains are the kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ1 * including 05 (described in Figure 4).

[0118] In some embodiments, the light chain variable region of one or both VH / VL variable domains of an EGFR / LGR5 bispecific antibody includes LCDR1 containing the amino acid sequence QSISSY (shown in Figure 4), LCDR2 containing the amino acid sequence AAS (shown in Figure 4), and LCDR3 containing the amino acid sequence QQSYSTP (shown in Figure 4) (i.e., CDR of IGKV1-39 by IMGT). In some embodiments, the light chain variable region of one or both VH / VL variable domains of an EGFR / LGR5 antibody includes LCDR1 containing the amino acid sequence QSISSY (shown in Figure 4), LCDR2 containing the amino acid sequence AASLQS (shown in Figure 4), and LCDR3 containing the amino acid sequence QQSYSTP (shown in Figure 4).

[0119] In some embodiments, one or both VH / VL variable domains of an EGFR / LGR5 antibody include a light chain variable region containing an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or 100% identical to the amino acid sequence shown in Figure 4.

[0120] For example, in some embodiments, the variable light chain of one or both VH / VL variable domains of an EGFR / LGR5 antibody may have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof with respect to the sequence in Figure 4. In some embodiments, the light chain variable region of one or both VH / VL variable domains of an EGFR / LGR5 antibody may include 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, and preferably 0, amino acid insertions, deletions, substitutions, additions, or combinations thereof with respect to the indicated amino acid sequence.

[0121] In other embodiments, the light chain variable region of one or both VH / VL variable domains of the EGFR / LGR5 antibody contains the amino acid sequence shown in Figure 4. In certain embodiments, both VH / VL variable domains of the EGFR / LGR5 antibody contain the same VL region. In one embodiment, the VL of both VH / VL variable domains of the EGFR / LGR5 bispecific antibody contains the amino acid sequence shown in Figure 4. In one embodiment, the VL of both VH / VL variable domains of the EGFR / LGR5 bispecific antibody contains the amino acid sequence shown in Figure 4.

[0122] The EGFR / LGR5 antibodies described herein are preferably bispecific antibodies having two variable domains: one that binds to EGFR and another that binds to LGR5 as described herein. EGFR / LGR5 bispecific antibodies for use in the methods disclosed herein may be provided in several formats. Many different 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), which are incorporated herein by reference, respectively. For example, a bispecific antibody format that is not a classical antibody having two VH / VL combinations has at least a variable domain including a heavy chain variable region and a light chain variable region. This variable domain may be linked to a single-chain Fv fragment, a monolayer, a VH, and a Fab fragment that provides a second binding activity.

[0123] In some embodiments, the EGFR / LGR5 bispecific antibody used in the methods provided herein is generally of a human IgG subclass (e.g., IgG1, IgG2, IgG3, IgG4). In certain embodiments, the antibody is of a human IgG1 subclass. Full-length IgG antibodies are preferred due to their preferred half-life and lower immunogenicity. Therefore, in certain embodiments, the EGFR / LGR5 bispecific antibody is a full-length IgG molecule. In one embodiment, the EGFR / LGR5 bispecific antibody is a full-length IgG1 molecule.

[0124] Therefore, in certain embodiments, the EGFR / LGR5 bispecific antibody comprises a crystallizable fragment (Fc). The Fc of the EGFR / LGR5 bispecific antibody preferably consists of a human constant region. The constant region or Fc of the EGFR / LGR5 bispecific antibody may contain one or more, preferably 10 or fewer, and preferably 5 or fewer amino acids from the constant region of a naturally occurring human antibody. For example, in certain embodiments, each Fab arm of the bispecific antibody may further contain an Fc region containing modifications that promote the formation of the bispecific antibody and enhance stability and / or other features described herein.

[0125] Antibodies are typically produced by cells that express the nucleic acid encoding the antibody. Therefore, in some embodiments, the bispecific EGFR / LGR5 antibodies disclosed herein are produced by providing cells containing one or more nucleic acids encoding the heavy and light chain variable regions and constant region of the bispecific EGFR / LGR5 antibody. These cells are preferably animal cells, more preferably mammalian cells, more preferably primate cells, and most preferably human cells. Suitable cells may contain, and preferably produce, the EGFR / LGR5 bispecific antibody.

[0126] Cells suitable for antibody production are known in the art and include hybridoma cells, Chinese hamster ovary (CHO) cells, NS0 cells, or PER-C6 cells. Various institutions and companies are developing cell lines for large-scale production of antibodies for clinical use, for example. Non-limiting examples of such cell lines are CHO cells, NS0 cells, or PER.C6 cells. In a particularly preferred embodiment, the cells are human cells. Preferably, the cells are transformed with the adenovirus E1 region or its functional equivalent. A preferred example of such a cell line is the PER.C6 cell line or its equivalent. In a particularly preferred embodiment, the cells are CHO cells or their variants. Preferably, the variants use a glutamine synthase (GS) vector system for antibody expression. In one preferred embodiment, the cells are CHO cells.

[0127] In some embodiments, cells express different light and heavy chains constituting an EGFR / LGR5 bispecific antibody. In certain embodiments, cells express two different heavy chains and at least one light chain. In one preferred embodiment, cells express a “common light chain” as described herein to reduce the number of different antibody species (combinations of different heavy and light chains). For example, each VH region, in combination with a rearranged human IGKV1 39 / IGKJ1 (huVκ1 39) light chain, is cloned into an expression vector using methods known in the art for the production of bispecific IgG (WO2013 / 157954, incorporated herein by reference), and has been previously shown to pair with two or more heavy chains, thereby yielding antibodies with diverse specificities, thereby promoting the production of bispecific molecules (De Kruif et al. J.Mol.Biol.2009(387)548 58;WO2009 / 157771).

[0128] Antibody-producing cells expressing a common light chain and equal amounts of two heavy chains typically produce 50% bispecific antibodies and 25% monospecific antibodies (i.e., those with the same heavy chain combination). Several methods have been described to favor the production of bispecific antibodies over the production of each monospecific antibody. This is typically achieved by modifying the constant regions of the heavy chains so that they favor heterodimerization (i.e., dimerization with heavy chains of other heavy / light chain combinations) over homodimerization. In a preferred embodiment, the bispecific antibodies of the present invention comprise two distinct immunoglobulin heavy chains having compatible heterodimerization domains. Various compatible heterodimerization domains have been described in the art. The compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain. Various methods have been described in the art that can achieve heterodimerization of such heavy chains.

[0129] A preferred method for producing EGFR / LGR5 bispecific antibodies is disclosed in US9,248,181 and US9,358,286. Specifically, preferred mutations that essentially produce only bispecific full-length IgG molecules are amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain ("KK mutant" heavy chain) and amino acid substitutions L351D and L368E in the second domain ("DE mutant" heavy chain), or vice versa. As described above, the DE mutant and KK mutant preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of the DE mutant heavy chain (DEDE homodimer) or the KK mutant heavy chain (KKKK homodimer) hardly occurs due to strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0130] Therefore, in one embodiment, the heavy / light chain combination containing a variable domain that binds to EGFR includes a DE variant of the heavy chain. In this embodiment, the heavy / light chain combination containing a variable domain that binds to LGR5 includes a KK variant of the heavy chain.

[0131] Candidate EGFR / LGR5 IgG bispecific antibodies can be tested for binding using any suitable assay. For example, binding to membrane-expressed EGFR or LGR5 on CHO cells can be evaluated by flow cytometry (by the FACS procedure previously described in WO2017 / 069628). In one embodiment, the binding of a candidate EGFR / LGR5 bispecific antibody to LGR5 on CHO cells is demonstrated by flow cytometry performed according to a standard procedure well known in the art. Binding to CHO cells is compared to CHO cells that have not been transfected with an EGFR and / or LGR5 expression cassette. Binding of a candidate bispecific IgG1 to EGFR is determined using CHO cells transfected with an EGFR expression construct, and LGR5 monospecific antibodies and EGFR monospecific antibodies, as well as unrelated IgG1 isotype control mAbs, are included in the assay as controls (e.g., antibodies that bind to LGR5 and another antigen such as tetanus toxin (TT)).

[0132] The affinity of candidate EGFR / LGR5 bispecific antibodies for the target to LGR5 and EGFR Fab can be measured by surface plasmon resonance (SPR) technology using the BIAcore T100. Briefly, an anti-human IgG mouse monoclonal antibody (Becton and Dickinson, catalog number 555784) is conjugated to the surface of a CM5 sensor chip using free amine chemistry (NHS / EDC). The bsAb is then captured on the sensor surface. Subsequently, recombinant purified antigens of human EGFR (Sino Biological Inc, catalog number 11896-H07H) and human LGR5 protein are run across the sensor surface in a certain concentration range, and the on and off speeds are measured. After each cycle, the sensor surface is regenerated by a pulse of HCl, and the bsAb is captured again. From the obtained sensorgrams, the on-rate and off-rate and affinity values ​​for binding to human LGR5 and EGFR are determined using the BIAevaluation software previously described for CD3 in US2016 / 0368988.

[0133] The antibodies disclosed herein are typically bispecific full-length antibodies, preferably of the human IgG subclass, and preferably of the human IgG1 subclass. Such antibodies may be enhanced by techniques known in the art as desired, and possess excellent ADCC properties, for which there are CH3 engineering techniques that can provide modified heavy chains that have a favorable half-life upon in vivo administration to humans and preferentially form heterodimers over homodimers upon co-expression in clonal cells.

[0134] The ADCC activity of an antibody can be improved by modifying the constant region of the antibody, if the antibody itself has low ADCC activity. Another way to improve the ADCC activity of an antibody is by enzymatically interfering with the glycosylation pathway that results in reduced fucose. Several in vitro methods exist to determine the effectiveness of an antibody or effector cell in inducing ADCC. These include chromium-51 [Cr51] release assays, europium [Eu] release assays, and sulfur-35 [S35] release assays. Typically, labeled target cell lines expressing an antigen exposed to a particular surface are incubated with an antibody specific to that antigen. After washing, effector cells expressing the Fc receptor CD16 are co-incubated with antibody-labeled target cells. Subsequently, target cell lysis is measured by the release of intracellular labels using a scintillation counter or spectrophotometric method.

[0135] The bispecific antibodies disclosed herein are preferably ADCC-enhanced. In one embodiment, the bispecific antibodies may be defucosylated. The bispecific antibodies preferably involve a reduction in the amount of fucosylation of the N-linked carbohydrate structure within the Fc region when compared to the same antibody produced in normal CHO cells.

[0136] Antibodies comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 may further comprise one or more additional variable domains capable of binding to one or more further targets. These further targets are preferably proteins, preferably membrane proteins including an extracellular portion. As used herein, membrane proteins are cell membrane proteins, such as proteins located in the outer membrane of a cell, which separates the cell from the outside world. Membrane proteins have an extracellular portion. Membrane proteins are at least on the cell if they contain a transmembrane region within the cell membrane.

[0137] Antibodies having two or more variable domains are known in the art. For example, it is possible to conjugate additional variable domains to the constant portion of the antibody. Antibodies having three or more variable domains are preferably polyvalent multimer antibodies as described in PCT / NL2019 / 050199, which is incorporated herein by reference.

[0138] In one embodiment, the antibody is a bispecific antibody comprising two variable domains, one of which binds to the extracellular portion of EGFR and the other variable domain binds to the extracellular portion of LGR5. The variable domains are preferably the variable domains described herein.

[0139] The functional portion of the antibodies described herein includes at least a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 as described herein. Therefore, it includes the antigen-binding portion of the antibodies described herein and typically contains the variable domain of the antibody. The variable domain of the functional portion may be a single-chain Fv fragment or a so-called single-domain antibody fragment. A single-domain antibody fragment (sdAb) is an antibody fragment having a single monomeric variable antibody domain. Like the whole antibody, this antibody can selectively bind to a specific antigen. With a molecular weight of only 12-15 kDa, single-domain antibody fragments are much smaller than typical antibodies (150-160 kDa) which consist of two heavy protein chains and two light chains, and even smaller than Fab fragments (about 50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (about 25 kDa, one variable domain from one light chain and one variable domain from one heavy chain). Single-domain antibodies themselves are not significantly smaller than typical antibodies (typically 90-100 kDa). Single-domain antibody fragments can be manipulated primarily from heavy-chain antibodies found in camelids, which are called VHH fragments (Nanobodies®). Some fish also possess heavy-chain-only antibodies (IgNAR, "novel immunoglobulin antigen receptors"), from which single-domain antibody fragments called VNAR fragments can be obtained. An alternative approach is to split dimeric variable domains derived from common immunoglobulin G (IgG) of human or mouse origin into monomers. While most single-domain antibody research currently relies on heavy-chain variable domains, it has also been shown that nanobodies derived from light chains can specifically bind to target epitopes. Non-limiting examples of such variable domains in antibody moieties include VHH, human domain antibodies (dAb), and unibodies. Preferred antibody moieties or derivatives have at least two variable domains in the antibody or its equivalent. Non-limiting examples of such variable domains or its equivalents include F(ab) fragments and single-chain Fv fragments. The functional portion of a bispecific antibody includes the antigen-binding portion of the bispecific antibody, or derivatives and / or analogs of the binding portion. As described herein, the antibody-binding portion is contained within a variable domain.

[0140] Antibodies, or functional portions thereof, derivatives, and / or analogues thereof (i.e., therapeutic compounds), as well as pharmaceutically acceptable carriers, are disclosed herein. Such pharmaceutical compositions are useful in the treatment of cancer, particularly for the treatment of head and neck cancers. As used herein, the term “pharmaceutically acceptable” means approved by a government regulatory body or listed in the United States Pharmacopeia or another generally accepted pharmacopoeia for use in animals, particularly humans, and includes any and all physiologically compatible solvents, salts, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, etc. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle on which the compound is administered. Such pharmaceutically acceptable carriers may be sterile liquids such as water and oil, including petroleum, animal, plant, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol ricinoleate, polyethylene glycol, etc. Water or saline and aqueous dextrose and aqueous glycerol solutions may be used as carriers, in particular for injectable solutions. Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravesical, intratumoral, intravenous, intraperitoneal, intramuscular, subarachnoid, and subcutaneous. Depending on the route of administration (e.g., intravenous, subcutaneous, intra-articular, etc.), the active compound may be coated with a material to protect it from the action of acids and other natural conditions that can inactivate the compound.

[0141] Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, for example, in a liquid carrier, or for reconstitution into a solution or suspension for intravenous administration. Compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Solid preparations intended to be converted into liquid preparations for either oral or parenteral administration immediately before use are also included. Such liquid forms include solutions, suspensions, and emulsions.

[0142] The disclosed therapeutic compounds may be administered according to a preferred dose and preferred route (e.g., intravenous, intraperitoneal, intramuscular, subarachnoid, or subcutaneous). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. In one embodiment, a subject is administered a single dose of the antibody disclosed herein, or its functional portion, derivative, and / or analog. In some embodiments, the therapeutic compound is administered repeatedly over the course of treatment. For example, in a particular embodiment, multiple doses (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) of the therapeutic compound are administered to a subject in need of treatment. In some embodiments, the therapeutic compound may be administered weekly, bi-weekly, or monthly. Preferably, the antibody of the present invention is administered bi-weekly.

[0143] Clinicians may use a preferred dose deemed appropriate based on the patient's condition. The dose may depend on several factors, including the stage of the disease. Determining a specific dose to be administered based on the presence of one or more such factors is within the scope of the skills of those skilled in the art. Generally, treatment is initiated with a dose lower than the optimal dose of the compound. The dosage is then increased in small increments until the optimal effect is achieved under these circumstances. For convenience, the total daily dose may be divided and administered throughout the day, if necessary. Intermittent therapy (e.g., one week out of three weeks or three weeks out of four weeks) may also be used.

[0144] In one particular embodiment, the therapeutic compound is administered in doses of 0.1, 0.3, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg body weight. In another embodiment, the therapeutic compound is administered in doses of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg body weight.

[0145] In preferred embodiments, the therapeutic compound (i.e., an antibody, or a functional portion thereof, derivative, and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5) is provided to the subject in a dose of 1500 mg. Uniform dosing offers several advantages over surface or body weight administration to reduce preparation time and potential dose calculation errors. In some embodiments, the therapeutic compound is provided in a dose of at least 1100 mg, preferably 1100-2000 mg, more preferably 1100-1800 mg. As will be understood by those skilled in the art, this dose may be administered over time. For example, the dose may be administered IV, for example, by infusion over 1-6 hours, preferably 2-4 hours. In some embodiments, the therapeutic compound is administered once every two weeks. In some embodiments, the uniform doses disclosed herein are suitable for use in adult subjects and / or subjects with a body weight of at least 35 kg. Preferably, the subjects have head and neck cancer.

[0146] This disclosure provides that premedication regimens may be used. Such regimens may be applied to reduce the likelihood or severity of infusion-related reactions. Preferably, steroids or corticosteroids (such as dexamethasone) and / or antihistamines or H1 antagonists (such as dexchlorpheniramine, diphenhydramine, or chlorpheniramine), or drugs to reduce gastric acid production (such as ranitidine) are administered before antibody therapy (e.g., orally or intravenously). Drugs to reduce, treat, or alleviate pain or fever may also be premedicated by administering paracetamol, etc.

[0147] A preferred premedication regimen includes dexamethasone 20 mg (IV), dexchlorpheniramine 5 mg (IV) or diphenhydramine 50 mg (PO) or chlorpheniramine 10 mg (IV) and ranitidine 50 mg (IV) or 150 mg (PO), and paracetamol 1 g (IV) or 650 mg (PO).

[0148] The treatments described herein are typically continued as long as the clinician supervising the patient's care considers the treatment to be effective, i.e., the patient is responding to the treatment. Non-limiting parameters indicating the effectiveness of a treatment may include one or more of the following: reduction of tumor cells, inhibition of tumor cell proliferation, elimination of tumor cells, progression-free survival, and an appropriate response to a suitable tumor marker (if applicable).

[0149] Those skilled in the art will be able to determine an appropriate frequency for administering therapeutic compounds. For example, a clinician may decide to administer the therapeutic compound at a relatively low frequency (e.g., once every two weeks) and gradually shorten the interval between doses that is tolerable for the patient. Examples of exemplary periods relating to the course of therapy as described in the claims include approximately 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 30 months, 3 years, 4 years, 5 years, and permanent (e.g., continuing maintenance therapy). The aforementioned duration may be associated with one or more rounds / cycles of treatment.

[0150] The effectiveness of the treatment methods provided herein may be evaluated using any preferred means. In one embodiment, the clinical effectiveness of the treatment is analyzed using a reduction in the number of cancer cells as the objective response criterion. Patients treated according to the methods disclosed herein, e.g., humans, preferably experience improvement in at least one sign of cancer. In some embodiments, one or more of the following may occur: a reduction in the number of cancer cells, prevention or delay of cancer recurrence, and some degree of relief of one or more of the symptoms associated with cancer. In addition, an in vitro assay for determining T cell-mediated target cell lysis. In some embodiments, tumor evaluation is based on CT scans and / or MRI scans; see, for example, the RECIST 1.1 guidelines (response evaluation criteria in solid tumors) (Eisenhauer et al., 2009 Eur J Cancer 45:228-247). Such evaluations are generally performed every 4 to 8 weeks after treatment.

[0151] In some embodiments, tumor cells are no longer detectable after the treatment described herein. In some embodiments, the subjects achieve partial remission or complete remission. In certain embodiments, the subjects have an increase in overall survival, median survival, and / or progression-free survival.

[0152] Therapeutic compounds (i.e., antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, or functional portions thereof, derivatives, and / or analogs) may be used in conjunction with other well-known therapies (e.g., chemotherapy or radiotherapy) selected for their specific utility against the cancer being treated.

[0153] 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 Physicians' Desk Reference (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, NJ 07645-1742, USA), and its disclosure is incorporated herein by reference.

[0154] It will be apparent to those skilled in the art that the administration of chemotherapeutic agents and / or radiotherapy may vary depending on the disease being treated and the known effects of chemotherapeutic agents and / or radiotherapy on that disease. Furthermore, according to the knowledge of those skilled in the art, treatment protocols (e.g., dosage and administration timing) may be modified in consideration of the observed effects of the administered therapeutic agent on the patient and the observed response of the disease to the administered therapeutic agent.

[0155] Preferably, the human subject meets one or all of the following requirements. 1. I signed informed consent before commencing any of the test procedures. 2. At the time of signing the informed consent form, the person must be 18 years of age or older. 3. Histologically or cytologically confirmed solid tumors with evidence of metastatic or locally advanced disease that are not suitable for standard curative therapy: Expanded cohort non-CRC tumor type: Patients with advanced or metastatic head and neck squamous cell carcinoma may be sought, regardless of whether they have previously been treated with at least two standard approved therapies. 4. Fresh baseline tumor samples from metastatic or primary sites (FFPE, provided sufficient material has been frozen). 5. Suitable for biopsy. 6. Measurable diseases as defined in RECIST version 1.1 by radiological methods. Performance status of Eastern Cooperative Oncology Group (ECOG) 7.0 or 1. 8. According to researchers, the average life expectancy is ≥12 weeks. 9. According to echocardiography (ECHO) or multi-gated acquisition scan (MUGA), the left ventricular ejection fraction (LVEF) is ≥50%. 10. Appropriate organ function: The absolute neutrophil count (ANC) is ≥ 1.5 × 10⁹ / L. • Hemoglobin level is ≥9 g / dL. • The platelet count is ≥100 x 10⁹ / L. • Corrected total serum calcium within the normal range • Serum magnesium levels within the normal range (or corrected with supplements) Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are ≤2.5 × upper limit of normal (ULN), and total bilirubin is ≤1.5 × ULN (except in cases of known Gilbert's syndrome where ALT / AST ≤5 × ULN and total bilirubin ≤2 × ULN are acceptable, except in cases of known Gilbert's syndrome where total bilirubin ≤3.0 × ULN or direct bilirubin ≤1.5 × ULN is acceptable, or in cases of hepatocellular carcinoma [Child-Pugh class A] where total bilirubin <3 mg / dL is acceptable). • Serum creatinine ≤ 1.5 × ULN or creatinine clearance ≥ 60 mL / min was calculated according to the Cockroft and Gault formula or MDRD formula for patients aged >65 years or older. • Serum albumin level is >3.3 g / dL.

[0156] The compounds and compositions disclosed herein are useful as therapies and in therapeutic treatments, and therefore are useful as drugs and can be used in methods for preparing drugs.

[0157] All documents and references described herein, including Genbank entries, patents, and published patent applications, as well as websites, are expressly incorporated herein by reference, in whole or in part, to the same extent as those described herein.

[0158] For the purposes of clarity and concise description, features are described herein as part of the same or distinct embodiments, but it will be understood that the scope of the invention may include embodiments or models having all or some combinations of the described features.

[0159] Herein, the present invention will be described with reference to the following embodiments, which are illustrative and not intended to limit the invention. Although the present invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope thereof. [Examples]

[0160] As used herein, “MFXXXX,” where X is independently a digit from 0 to 9, refers to a Fab containing a variable domain, where VH has an amino acid sequence identified by the four digits shown in Figure 3. Unless otherwise indicated, the light chain variable region of the variable domain typically has the sequence shown in Figure 4b. The light chain in the examples has the sequence shown in Figure 4a. “MFXXXX VH” refers to the amino acid sequence of VH identified by the four digits. MF further includes a constant region of the light chain and a constant region of the heavy chain that typically interacts with the constant region of the light chain. The VH / variable region of the heavy chain is different, and typically the CH3 region is also different, with one heavy chain having a KK mutation in its CH3 domain and the other having a complementary DE mutation in its CH3 domain (see reference PCT / NL2013 / 050294 (published as WO2013 / 157954), and Figures 5d and 5e). The bispecific antibodies in the examples have an Fc tail containing a KK / DE CH3 heterodimerized domain, a CH2 domain, and a CH1 domain as shown in Figure 5, a common light chain as shown in Figure 4a, and a VH specified by the MF number. For example, the bispecific antibody represented by MF3755×MF5816 has the above general sequence, a variable domain having a VH with the sequence of MF3755, and a variable domain having a VH with the sequence of MF5816.

[0161] The amino acid and nucleic acid sequences of various heavy chain variable regions (VHs) are shown in Figure 3. Among the other LGR5 and EGFR combinations shown in Figure 3, the bispecific antibody EGFR / LGR5, MF3755×MF5816, which includes the heavy chain variable regions MF3755 and MF5816 and a common light chain, and which includes modifications for enhancement of ADCC from afucosylation, has been shown to be effective in WO2017 / 069628.

[0162] Production of bispecific antibodies Bispecific antibodies were generated by transient cotransfection of two plasmids encoding IgG having different VH domains, using proprietary CH3 engineering techniques to ensure efficient heterodimerization and bispecific antibody formation. The common light chain is also cotransfected within the same cell, either on the same plasmid or on a different plasmid. Our applications (e.g., WO2013 / 157954 and WO2013 / 157953, incorporated herein by reference) disclose methods and means for producing bispecific antibodies from a single cell, thereby providing means to favor the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can also be advantageously used in the present invention. Specifically, preferred mutations that essentially produce only bispecific full-length IgG molecules are amino acid substitutions at positions 351 and 366 in the first CH3 domain ("KK mutant" heavy chain), e.g., L351K and T366K (numbered according to EU numbering), and amino acid substitutions at positions 351 and 368 in the second CH3 domain ("DE mutant" heavy chain), e.g., L351D and L368E, or vice versa (see Figures 5d and 5e). It was demonstrated in the aforementioned application that the negatively charged DE mutant heavy chain and the positively charged KK mutant heavy chain preferentially pair to form a heterodimer (the so-called "DEKK" bispecific molecule). Homodimerization of the DE mutant heavy chain (DE-DE homodimer) or the KK mutant heavy chain (KK-KK homodimer) hardly occurs due to strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0163] The VH gene of the variable domain that binds to LGR5, as described above, was cloned into a vector encoding a positively charged CH3 domain. The VH gene of the variable domain that binds to EGFR, such as that disclosed in WO2015 / 130172 (incorporated herein by reference), was cloned into a vector encoding a negatively charged CH3 domain. 293F freestyle cells adapted for suspension growth were cultured in T125 flasks on a shaker plateau until a density of 3.0 × 10⁶ cells / ml. Cells were seeded in each well of a 24-deep-well plate at a density of 0.3–0.5 × 10⁶ live cells / ml. Cells were transiently transfected with a mixture of two plasmids encoding different antibodies and cloned into proprietary vector systems. Seven days after transfection, the cell supernatant was collected and filtered through a 0.22 μM filter (Sartorius). The sterile supernatant was stored at 4°C until the antibodies were purified.

[0164] IgG purification and quantification Purification was performed using Protein A affinity chromatography under sterile conditions on a filter plate. First, the pH of the culture medium was adjusted to pH 8.0, and then the IgG-containing supernatant was incubated with Protein A Sepharose CL-4B beads (50% v / v) (Pierce) on a shaking platform at 600 rpm for 2 hours at 25°C. Next, the beads were collected by filtration. The beads were washed twice with PBS pH 7.4. Then, the bound IgG was eluted with 0.1 M citrate buffer at pH 3.0, and the eluate was immediately neutralized with Tris pH 8.0. Buffer exchange was performed by centrifugation using a Multiscreen Ultracel 10 multiplate (Millipore). The sample was finally collected in PBS pH 7.4. The IgG concentration was measured using Octet. The protein sample was stored at 4°C.

[0165] To determine the amount of purified IgG, the antibody concentration was determined by Octet analysis using a Protein A biosensor (Forte-Bio, as recommended by the supplier) with total human IgG (Sigma Aldrich, catalog number I4506) as the standard.

[0166] The following bispecific antibodies are suitable for use in this embodiment and in the method of the present invention: MF3370×MF5790, MF3370×5803, MF3370×5805, MF3370×5808, MF3370×5809, MF3370×5814, MF3370×5816, MF3370×5817, MF3370×5818, MF3755×MF5790, MF3755×5803, MF3755×5805, MF3755×5808, MF3755×5809, MF3755×5814, MF3755×5816, MF3755×5817, MF3755×5818, M F4280×MF5790, MF4280×5803, MF4280×5805, MF4280×5808, MF4280×5809, MF4280×5814, MF4280×5816, MF4280×5817, MF4280×5818, MF4289×MF5790, MF4289×5803, MF4289×5805, MF4289×5808, MF4289×5809, MF4289×5814, MF4289×5816, MF4289×5817, and MF4289×5818. Each bispecific antibody contains two VHs, each designated by an MF number capable of binding to EGFR and LGR5, and further includes a KK / DE CH3 heterodimerized domain as shown in SEQ ID NO: 136 (Figure 5d) and SEQ ID NO: 138 (Figure 5e), a CH2 domain as shown in SEQ ID NO: 134 (Figure 5c), a CH1 domain as shown in SEQ ID NO: 131 (Figure 5a), and a common light chain-containing Fc tail as shown in SEQ ID NO: 121 (Figure 4).

[0167] Example 1: In vivo evaluation of anti-EGFRx anti-LGR5 antibody against head and neck cancer using a patient-derived xenograft (PDX) mouse model. Mouse PDX model Crown Biosciences Inc. has developed a collection of patient-derived xenograft (PDX) models derived from surgically resected human primary tumors. The PDX models used herein are clinically and molecularly annotated and faithfully reproduce the clinical epidemiology of each tumor. These models can be subcutaneously injected into the flanks of immunodeficient mice. Using different head and neck PDX models, the therapeutic efficacy of full-length IgG1 bispecific antibodies containing MF3755×MF5816 and further related domains as shown (i.e., CH1, CH2, KK / DE modified CH3 heterodimerization domain and common light chain) was evaluated. Detailed information on these models, including cancer subtype, presence of genomic mutations, and EGFR / LGR5 expression levels, is listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0168] Tumor inoculation and randomization Fresh tumor tissue for inoculation was collected from mice with established primary human tumors. The fresh tumor was cut into small pieces (approximately 2-3 mm in diameter) and subcutaneously transplanted into the right dorsal flank of the mice. The tumor pieces were inoculated into 6-8 week old female BALB / c Nude or NOD / SCID mice with an average body weight of approximately 16-20 g. Mice were randomized when the average tumor size reached 100-150 mm³. A total of 16 mice were enrolled in the study per model (4 control mice and 12 antibody-treated mice). Randomization was performed using the "matched distribution" method (StudyDirector™ software, version 3.1.399.19). Control mice were administered PBS.

[0169] Treatment and sampling schedule The first treatment was administered on the day of randomization, which was considered day 0 of the experiment. All mice were intraperitoneally (ip) injected once a week for 6 weeks using a 200 μl injection volume with a freshly prepared dose from 20 mg / mL stock solution antibody prior to administration. Control mice were administered PBS, and antibody-treated mice were treated with a dose adjusted relative to body weight (dose = 10 μL / g). As detailed in Table 2, each mouse received 0.5 mg of antibody (approximately 25 mg / kg), regardless of their body weight. After the end of the treatment period, all mice had to undergo a 3-week observation period. The observation period was extended if the tumor did not grow to the maximum ethically acceptable tumor size. Control mice were injected with PBS using the same injection volume. [Table 2]

[0170] Observation, sample collection, and data collection After tumor inoculation, animals were checked daily for morbidity and mortality. During routine monitoring, animals were checked for any effects of tumor growth, behavioral changes, changes in mobility, food and water consumption, weight gain / loss (weight was measured twice a week after randomization), eye / coat matting, and other abnormalities. Mortality and observed clinical signs were recorded for each individual animal.

[0171] Tumor volume was measured in two dimensions twice one week after randomization using calipers, and the volume was expressed in mm using the formula: V=(L×W×W) / 2 3 The formula is expressed as follows: V = tumor volume, L = tumor length (longest tumor dimension), and W = tumor width (longest tumor dimension perpendicular to L). Body weight and tumor volume were recorded using StudyDirector™ software (version 3.1.399.19). Mice were monitored at the end of week 9, or when the animals reached a humane endpoint (e.g., when the tumor volume reached 2000 mm²). 3 They were slaughtered when either their weight exceeded 15% of their starting weight, or when their weight loss exceeded 15% of their starting weight, whichever came first.

[0172] result Treatment with bispecific antibodies demonstrated therapeutic efficacy in 7 out of 7 head and neck squamous cell carcinomas tested during the study period among the head and neck cancer PDX models tested (Figure 6). Significant reductions in tumor amplification were also observed in 3 models. Models HN2167 and HN2590 showed lower tumor volumes at the end of the observation period compared to the start of treatment, suggesting tumor inhibition mediated by bispecific antibodies in head and neck cancer. Mice HN2579, HN5124, HN3642, HN3411, and HN5125 also responded well to antibody treatment and showed reductions in tumor volume compared to vehicle-treated mice.

[0173] statistical analysis To compare tumor volumes in different groups on pre-specified days, Bartlett's test was performed first to confirm the assumption of homogeneity of variance across all groups. If the p-value of Bartlett's test was >0.05, a one-way ANOVA was performed to test the overall equivalence of the means across all groups. If the p-value of the one-way ANOVA was <0.05, Tukey's HSD (honest significant difference) test was performed for all pairwise comparisons, and Dunnett's test was performed to compare each treatment group to the vehicle group. If the p-value of Bartlett's test was <0.05, the Kruskal-Wallis test was performed to test the overall equivalence of the medians across all groups. If the p-value of the Kruskal-Wallis test was <0.05, further post-hoc tests were performed by performing Conover's non-parametric test, either for all pairwise comparisons or to compare each treatment group to the vehicle group, both using single-step p-adjustment. All statistical analyses were performed using the Ra language and environment for statistical calculations and graphics (version 3.3.1). All tests are two-sided unless otherwise specified, and a p-value < 0.05 is considered statistically significant.

[0174] Example 2: Dose expansion and efficacy of anti-EGFRx anti-LGR5 antibody in patients with head and neck cancer: Phase 1 dose escalation study in advanced solid tumors Test design A Phase 1 open-label, multicenter trial was conducted using the initial dose escalation portion to determine the recommended Phase 2 dose (RP2D) of the anti-EGFR × anti-LGR5 bispecific antibody of this disclosure for solid tumors in mCRC patients, with a uniform starting dose of 5 mg. Once the RP2D is established, the antibody will be further evaluated in an expanded portion of the trial, including patients diagnosed with cervical cancer. The safety, PK, immunogenicity, and preliminary antitumor activity of the antibody will be characterized in all patients, and biomarker analyses, including EGFR and LGR5 status, will be performed.

[0175] Selection Criteria Patients must meet all of the following requirements to participate in the trial. 1. I signed informed consent before commencing any of the test procedures. 2. At the time of signing the informed consent form, the person must be 18 years of age or older. 3. Histologically or cytologically confirmed solid tumors with evidence of metastatic or locally advanced disease that are not suitable for standard curative therapy: Expanded cohort non-CRC tumor type: Patients with advanced or metastatic head and neck squamous cell carcinoma may be sought, regardless of whether they have previously been treated with at least two standard approved therapies. 4. Fresh baseline tumor samples from metastatic or primary sites (FFPE, provided sufficient material has been frozen). 5. Suitable for biopsy. 6. Measurable diseases as defined in RECIST version 1.1 by radiological methods. Performance status of Eastern Cooperative Oncology Group (ECOG) 7.0 or 1. 8. According to researchers, the average life expectancy is ≥12 weeks. 9. According to echocardiography (ECHO) or multi-gated acquisition scan (MUGA), the left ventricular ejection fraction (LVEF) is ≥50%. 10. Appropriate organ function: The absolute neutrophil count (ANC) is ≥ 1.5 × 10⁹ / L. • Hemoglobin level is ≥9 g / dL. • The platelet count is ≥100 x 10⁹ / L. • Corrected total serum calcium within the normal range • Serum magnesium levels within the normal range (or corrected with supplements) Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are ≤2.5 × upper limit of normal (ULN), and total bilirubin is ≤1.5 × ULN (except in cases of known Gilbert's syndrome where ALT / AST ≤5 × ULN and total bilirubin ≤2 × ULN are acceptable, except in cases of known Gilbert's syndrome where total bilirubin ≤3.0 × ULN or direct bilirubin ≤1.5 × ULN is acceptable, or in cases of hepatocellular carcinoma [Child-Pugh class A] where total bilirubin <3 mg / dL is acceptable). • Serum creatinine ≤ 1.5 × ULN or creatinine clearance ≥ 60 mL / min was calculated according to the Cockroft and Gault formula or MDRD formula for patients aged >65 years or older. • Serum albumin level is >3.3 g / dL.

[0176] Exclusion criteria A patient will be excluded from the trial if any of the following criteria are met. 1. Central nervous system metastases that are untreated, symptomatic, or require radiation, surgery, or continuous steroid therapy to control symptoms within 14 days of trial enrollment. 2. Known involvement of the leptomeninges. 3. Participation in any other clinical trial or treatment involving the investigational drug within four weeks prior to enrollment in the clinical trial. 4. Any systemic anticancer therapy within 4 weeks of the first dose of the investigational drug or within 5 half-lives, whichever is longer. A 6-week washout period is required for cytotoxic agents with significant delayed toxicity (e.g., mitomycin C, nitrosourea) or for anticancer immunotherapy. 5. Requirements for immunosuppressants (e.g., methotrexate, cyclophosphamide) 6. Major surgery or radiation therapy within three weeks of the first dose of the investigational treatment. Patients who have previously received radiation therapy to ≥25% of the bone marrow are ineligible, regardless of when they received it. 7. Clinically significant toxicity of sustained grade > 1 associated with existing anti-cancer therapy (excluding alopecia); stable sensory neuropathy ≤ grade 2 NCI-CTCAE v4.03 is acceptable. 8. A history of hypersensitivity reactions or toxicity of either human protein or excipients that justifies the permanent discontinuation of these drugs. 9. Uncontrolled hypertension with appropriate treatment or unstable angina (systolic > 150 mmHg and / or diastolic > 100 mmHg). 10. A history of congestive heart failure according to Class II-IV New York Heart Association (NYHA) criteria, or severe cardiac arrhythmias requiring treatment (excluding atrial fibrillation and paroxysmal supraventricular tachycardia). 11. A history of myocardial infarction within 6 months of clinical trial registration. 12. A history of previous malignancies, excluding cervical intraepithelial neoplasia or non-melanoma skin cancer, or cancers that have been curatively treated and for which there has been no evidence of disease for at least three years and the risk of recurrence is considered low. 13. Current dyspnea or other condition requiring continuous oxygen therapy in the remainder of either origin. 14. Patients with a history of interstitial lung disease (e.g., pneumonia or pulmonary fibrosis) or evidence of interstitial lung disease (ILD) on baseline chest CT scan. 15. An uncontrolled active infection, a clinically significant pulmonary, metabolic or psychiatric disorder, or any other current serious illness or mental disorder. 16. Active HIV, HBV, or HCV infection requiring treatment. 17. Patients with current cirrhosis of Child-Pugh class B or C, known fibrous lamellar HCC, sarcomatoid HCC, or mixed cholangiocarcinoma and HCC 18. Pregnant or breastfeeding women, and patients who may become pregnant, must use a highly effective method of contraception before enrollment in the clinical trial, during participation in the clinical trial, and for six months after the last dose of the antibody.

[0177] Dosage gradual increase In the dose-escalation portion, patients with metastatic colorectal cancer (mCRC) adenocarcinoma who had previously been treated in a metastatic setting with standard approved therapy including oxaliplatin, irinotecan, and fluoropyrimidine (5-FU and / or capecitabine) were treated with or without anti-angiogenic and anti-EGFR KRAS and NRAS wild-type RASwt.

[0178] A PK model was generated based on available bispecific antibody serum concentration data from preliminary studies and GLP cynomolgus monkey toxicity studies. Following allometric scaling, this model was used to predict antibody exposure in humans. The starting antibody dose is IV at 5 mg (uniform dose) every two weeks in a four-week cycle. Up to 11 dose levels, 5, 20, 50, 90, 150, 225, 335, 500, 750, 1100, and 1500 mg (uniform doses), will be investigated. Dosage, dose escalation, and dosing frequency for each patient and each cohort may vary based on patient safety, PK, and PD data, but the dose will not exceed 4500 mg per cycle.

[0179] Dose-limiting toxicity (DLT) Any of the following clinical toxicities and / or laboratory abnormalities that occur during the first cycle (28 days) and that researchers consider to be related to antibody therapy are considered DLTs. ●Hematological toxicity: - Grade 4 neutropenia lasting 7 days or more (absolute neutrophil count [ANC] < 0.5 × 10⁹ cells / L) - Grade 3-4 febrile neutropenia - Grade 4 thrombocytopenia - Grade 3 thrombocytopenia associated with a bleeding episode - Other Grade 4 hematological toxicity ●Grade 3-4 non-hematological AEs and experimental toxicity, excluding the following: - Grade 3-4 infusion-related reactions - Grade 3 skin toxicity that resolves to Grade 2 or lower within 2 weeks with optimal treatment. - Grade 3 diarrhea, nausea, and / or vomiting that resolve to Grade 1 or below or to baseline within 3 days with optimal treatment. - Grade 3 electrolyte abnormalities that resolve within 48 hours with optimal treatment - Grade 3-4 liver abnormalities within 48 hours ● Any liver function abnormality that satisfies the definition of Hy's Law. ● Prevent any drug-related toxicity lasting ≥ 15 days from the next two doses.

[0180] Dosage expansion In the expanded section, the bispecific antibody of this disclosure is administered in a RP2D (Rehabilitation Program) to patients with head and neck cancer. Once the RP2D is defined, additional patients will be treated at this dose and schedule to further characterize the safety, tolerability, PK, and immunogenicity of the antibody, and to perform a preliminary evaluation of its antitumor activity and biomarker capabilities. The treated malignancies are known to co-express both targets (i.e., LGR5 and EGFR) and may have prior indicators of sensitivity to EGFR inhibition.

[0181] Antibody therapy in patients with head and neck cancer will explore 10-20 patients for each indication, potentially expanding to up to 40 patients, for example, conditional on preliminary signs of antitumor activity. The safety of RP2D will be continuously assessed by the Safety Monitoring Committee during the expanded portion of the trial. If the incidence of DLT exceeds a predetermined threshold of 33% for any cohort, enrollment for that cohort will be paused, and a full review of safety, PK, and biomarkers will be conducted by the SMC to determine whether it is safe to continue the occurrence in that cohort. At that point, the overall safety of the drug will also be questioned.

[0182] Investigational Therapies and Regimens The anti-EGFR × anti-LGR5 bispecific antibody is formulated as a clear liquid solution for intravenous infusion. Intravenous infusions are administered every two weeks using a standard infusion procedure, with an initial dose of 5 mg (uniform dose) and a recommended phase 2 dose of 1500 mg (uniform dose). Dose escalation was stopped after reaching RP2D. Infusions must be administered over a minimum of 4 hours during cycle 1. Subsequent infusions after cycle 1 may be shortened to 2 hours at the discretion of the investigator and in the absence of IRR.

[0183] Premedication During Cycle 1, all infusions are administered over at least 4 hours using the following premedication regimen: 8 mg of dexamethasone PO is administered 24 hours prior to the start of the infusion, and 1 hour prior to the start of the infusion, each patient is given dexamethasone 20 mg IV, dexchlorpheniramine 5 mg IV or diphenhydramine 50 mg PO or chlorpheniramine 10 mg IV, ranitidine 50 mg IV or 150 mg PO, and paracetamol 1 g IV or 650 mg PO.

[0184] If the patient tolerates all cycle 1 infusions without IRR and the investigator deems it appropriate, the patient may continue to receive further antibody infusions without dexamethasone premedication, and the duration of infusions may be reduced to 2 hours. In such cases, the duration of infusions may be extended up to approximately 4 hours if deemed appropriate to avoid or reduce the incidence or severity of IRR. For the first antibody infusion (cycle 1 on day 1), each patient is observed for 6 hours from the start of the infusion and for 4 hours from the start of the second infusion. Thereafter, the patient is observed for all subsequent administration periods (minimum 2 hours).

[0185] A cycle is considered to be 4 weeks. For each patient, a 6-hour observation period was observed after the start of the first antibody infusion, a 4-hour period after the second infusion, and a minimum 2-hour period after all subsequent administrations corresponding to at least one infusion period. Antibodies were administered as 2-4 hour IV infusions every 2 weeks in a 4-week cycle. Day 1 of the subsequent cycle was either day 29 or after recovery from any adverse effects related to the previous cycle.

[0186] Treatment period The investigational treatment will be administered until progressive disease (according to RECIST 1.1), unacceptable toxicity, withdrawal of consent, patient non-compliance, investigator's decision (e.g., clinical exacerbation), or antibody discontinuation for more than 6 consecutive weeks is confirmed. Patients will be followed for safety for at least 30 days after the last antibody infusion and until all related toxicities are resolved or stabilized, as well as for disease progression and survival for 12 months.

[0187] Effectiveness evaluation Tumor evaluation is based on contrast-enhanced CT / MRI according to RECIST 1.1 (Eisenhauer et al., 2009 Eur J Cancer 45:228-247) every 8 weeks after the start of treatment. Objective response must be confirmed at least 4 weeks after the first observation. Bone scans are performed as clinically directed for patients with bone metastases at baseline or suspected study lesions. Circulating blood tumor markers, including cancer embryo antigen (CEA), are evaluated at screening and on day 1 of each cycle.

Claims

1. An antibody, or a functional portion, derivative, and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, for use in the treatment of head and neck cancer in a subject, wherein the use comprises providing the subject with a uniform dose of 1500 mg of the antibody, or a functional portion, derivative, and / or analog thereof.

2. The antibody for use according to claim 1, or a functional portion, derivative, and / or analog thereof, wherein the subject is a human subject.

3. The antibody, or a functional portion thereof, derivative, and / or analog thereof, for use according to any one of the prior claims, wherein the antibody, or a functional portion thereof, derivative, and / or analog thereof, is provided intravenously.

4. An antibody for use according to any one of the prior claims, or a functional portion, derivative, and / or analog thereof, wherein the head and neck cancer is squamous cell carcinoma or adenocarcinoma.

5. The antibody for use according to any one of the prior claims, or a functional portion, derivative, and / or analog thereof, wherein the head and neck cancer is squamous cell carcinoma.

6. The antibody for use according to any one of the prior claims, or a functional portion, derivative, and / or analog thereof, wherein the head and neck cancer is nasopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, oral cancer, and oropharyngeal cancer.

7. The antibody for use according to any one of the prior claims, or a functional portion, derivative, and / or analog thereof, wherein the head and neck cancer is oropharyngeal squamous cell carcinoma.

8. An antibody for use according to any one of the prior claims, or a functional portion, derivative, and / or analog thereof, wherein the head and neck cancer has one or more mutations in the LGR5 and / or EGFR pathway present in a model selected from HN5124, HN5125, HN2579, HN2590, and HN2167.

9. The antibody for use according to any one of the prior claims, or a functional portion, derivative, and / or analog thereof, wherein the cancer is characterized by the expression of LGR5 and / or EGFR.

10. The VH chain of the variable domain that binds to EGFR contains the amino acid sequence of the VH chain MF3755 shown in Figure 3, or an amino acid modification of up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less, including insertions, deletions, substitutions, or combinations thereof to the VH, and the VH chain of the variable domain that binds to LGR5 is as shown in Figure 3 An antibody for use according to any one of the prior claims, or a functional moiety, derivative, and / or analogue thereof, comprising the amino acid sequence of the VH chain MF5816 shown in Figure 3, or having up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less amino acid modifications, including the amino acid sequence of the VH chain MF5816 shown in Figure 3, or insertions, deletions, substitutions, or combinations thereof to the VH.

11. An antibody for use according to any one of the prior claims, or a functional portion, derivative, and / or analog thereof, 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.

12. The antibody for use according to claim 11, or a functional portion, derivative, and / or analog thereof, wherein the amino acid residues at positions 43, 44, 46, 67, 90, and 91 of human LGR5 are involved in the binding of the LGR5-binding variable domain to LGR5.

13. The antibody for use according to claim 11 or 12, or a functional portion, derivative, and / or analog thereof, wherein the LGR5-binding variable domain binds less to an LGR5 protein containing one or more amino acid residue mutations selected from 43A, 44A, 46A, 67A, 90A, and 91A.

14. An antibody for use according to any one of the prior claims, or a functional portion, derivative, and / or analog thereof, wherein the variable domain that binds to EGFR binds to an epitope located within amino acid residues 420-480 of the human EGFR sequence shown in Figure 2.

15. The antibody for use according to claim 14, or a functional portion, derivative, and / or analog thereof, wherein the amino acid residues at positions I462, G465, K489, I491, N493, and C499 of human EGFR are involved in the binding of the EGFR-binding variable domain to EGFR.

16. The antibody for use according to claim 14 or 15, or a functional portion, derivative, and / or analog thereof, wherein the EGFR-binding variable domain binds less to an EGFR protein containing one or more amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A, and C499A.

17. The antibody for use according to any one of the prior claims, or a functional portion, derivative, and / or analog thereof, wherein the antibody is ADCC-enhancing.

18. The antibody for use according to any one of the prior claims, or a functional portion, derivative, and / or analog thereof, wherein the antibody is defucosylated.

19. A method for treating head and neck cancer, comprising administering an antibody, or a functional portion thereof, a derivative, and / or an analog thereof, to a subject in need of treatment for head and neck cancer, the 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.

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