Treatment of cancers with antibody that binds LGR5 and EGFR

Abstract

To provide, in one embodiment, cancer treatment, particularly treatment of gastric and esophageal cancer.SOLUTION: The disclosure relates to means and methods in the treatment of cancer. The disclosure in particular relates to a method of treating cancer in an individual with an antibody that binds LGR5 and EGFR. The invention further relates to the combination for use in such methods, and to the combination for use in the manufacture of a medicament for the treatment of gastrointestinal cancer. Such antibodies are particularly useful in the treatment of gastric, esophageal or gastro-esophageal-junction cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to means and methods in the treatment of cancer. In particular, the present disclosure relates to methods of treating cancer in individuals 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 a medicament for the treatment of gastrointestinal cancer. Such antibodies are particularly useful in the treatment of gastric cancer, esophageal cancer, or gastroesophageal junction cancer. [Background technology]

[0002] Traditionally, most cancer drug discovery has focused on agents that block essential cellular functions and kill dividing cells via chemotherapy. However, chemotherapy rarely results in a complete cure. In most cases, tumors in patients only stop growing or shrink temporarily (called remission), then start growing again, sometimes more rapidly (called relapse), and become increasingly difficult to treat. More recently, the focus of cancer drug development has shifted from broad-spectrum cytotoxic chemotherapy to targeted cell suppressive therapies with lower toxicity. Treatment of advanced cancers with targeted therapies that specifically inhibit signaling pathway components has been clinically validated in leukemia. However, in the majority of carcinomas, targeted approaches have still proven ineffective.

[0003] Cancer remains a leading cause of death worldwide, despite many advances made in the treatment of the disease and increasing knowledge of the molecular events that lead to cancer. Gastric cancer, for example, is the fifth most common cancer diagnosis and the third most lethal worldwide. In 2018, an estimated 783,000 deaths were attributed to gastric cancer. Esophageal cancer is the ninth most common cancer and the sixth most common cause of cancer death. Epidermal growth factor receptor (EGFR) has been reported to be overexpressed in more than 30% of gastric adenocarcinoma (GAC) and esophageal adenocarcinoma (EAC) cases. However, an analytical review of six different studies concluded that the addition of anti-EGFR agents to chemotherapy did not improve overall survival or progression-free survival in patients with advanced / metastatic EAC, GAC, or gastroesophageal junction adenocarcinoma (GEJAC) (Kim et al. 2017 Oncotarget. 2017 Nov 17;8(58):99033-99040). Thus, a need exists for cancer treatments, particularly for gastric and esophageal cancer. Summary of the Invention

[0004] The present disclosure provides the following preferred embodiments, however, the present invention is not limited to these embodiments.

[0005] In some embodiments, the disclosure provides an antibody, or a functional portion, derivative, and / or analog thereof, comprising a variable domain that binds to an extracellular portion of EGFR and a variable domain that binds to an extracellular portion of LGR5, for use in treating cancer in a subject, the use comprising providing to the subject a flat dose of 1500 mg of the antibody, or a functional portion, derivative, and / or analog thereof. The disclosure further provides a method of treating cancer in a subject, comprising providing to a subject in need thereof a flat dose of 1500 mg of the antibody, or a functional portion, derivative, and / or analog thereof.

[0006] In some embodiments, the present disclosure provides an antibody, or a functional portion, derivative, and / or analog thereof, comprising a variable domain that binds to an extracellular portion of EGFR and a variable domain that binds to an extracellular portion of LGR5, for use in treating gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a subject. The present disclosure further provides a method of treating gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a subject, comprising providing the antibody, or a functional portion, derivative, and / or analog thereof, to a subject in need thereof. Preferably, the use comprises providing a flat dose of 1500 mg of the antibody, or a functional portion, derivative, and / or analog thereof, to the subject.

[0007] In some embodiments, administration of the therapeutic compound may occur weekly, biweekly, or monthly, hi some embodiments, the therapeutic compound is administered once every two weeks.

[0008] In some embodiments, the present disclosure provides an antibody, or a functional part, derivative, and / or analog thereof, comprising a variable domain that binds to an extracellular portion of EGFR and a variable domain that binds to an extracellular portion of LGR5, for use in treating gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a Her2-negative subject. The present disclosure further provides a method of treating gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a Her2-negative subject, the method comprising providing the antibody, or a functional part, derivative, and / or analog thereof, to a subject in need thereof. Preferably, the use comprises providing a flat dose of 1500 mg of the antibody, or a functional part, derivative, and / or analog thereof, to the subject. In some embodiments, the administration of the therapeutic compound to the Her2-negative subject may be weekly, biweekly, or monthly. Preferably, the therapeutic compound is administered once every two weeks.

[0009] Preferably, the antibody, or functional part, derivative and / or analogue thereof, is provided intravenously.

[0010] Preferably, the cancer has a mutation in one or more genes selected from TP53, MLH1, PIK3CA, CDKN2A, UGT1A, UGT1A8, BRAF, PTEN, and KRAS, preferably, the cancer has a mutation in one or more genes selected from TP53, MLH1, CDKN2A, UGT1A, UGT1A8, BRAF, and PTEN. Preferably, the cancer has one or more mutations selected from TP53 R196T, TP53 R342T, TP53 R248Q, MLH1 V384D, PIK3CA H1047R, CDKN2A W110T, UGT1A1 G71R, UGT1A8 G71R, and KRAS G12C.

[0011] Preferably, the cancer has a mutation in the gene encoding TP53, preferably the mutation is R196T. Preferably, the cancer has a mutation in the gene encoding TP53, preferably the mutation is R342T, and the cancer has a mutation in the gene encoding MLH1, preferably the mutation is V384D.

[0012] Preferably, the cancer has a mutation in the gene encoding TP53, preferably the mutation is R248Q; the cancer has a mutation in the gene encoding PIK3CA, preferably the mutation is H1047R; the cancer has a mutation in the gene encoding CDKN2A, preferably the mutation is W110T; the cancer has a mutation in the gene encoding UGT1A1, preferably the mutation is G71R; the cancer has a mutation in the gene encoding UGT1A8, preferably the mutation is G71R.

[0013] Preferably, the cancer is esophageal cancer, preferably esophageal squamous cell carcinoma (ESCC).

[0014] Preferably, cancer has a mutation in the gene encoding BRAF.However, preferably, cancer does not have the mutation V600E in BRAF, and cancer has a mutation in the gene encoding PTEN.However, preferably, cancer also does not have the mutation R130Ter in PTEN.

[0015] Preferably, the cancer has a mutation in the gene encoding KRAS, preferably the mutation is G12C; the cancer has a mutation in the gene encoding UGT1A1, preferably the mutation is G71R; the cancer has a mutation in the gene encoding UGT1A8, preferably the mutation is G71R.

[0016] Preferably, the cancer has a mutation in the gene encoding UGT1A1, preferably the mutation is G71R, and the cancer has a mutation in the gene encoding UGT1A8, preferably the mutation is G71R. Preferably, the cancer further has a mutation in PIK3CA, preferably the mutation is E545K.

[0017] Preferably, the cancer is gastric cancer.

[0018] Preferably, the VH chain of the variable domain that binds EGFR comprises the amino acid sequence of VH chain MF3755 shown in Figure 3 as shown in Figure 3 with 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 insertions, deletions, substitutions, or a combination thereof, to the VH; and the VH chain of the variable domain that binds LGR5 comprises the amino acid sequence of VH chain MF5816 shown in Figure 3 as shown in Figure 3 as shown in Figure 3 with 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 insertions, deletions, substitutions, or a combination thereof to the VH.

[0019] Preferably, the LGR5-binding variable domain binds to an epitope located within amino acid residues 21 to 118 of the human LGR5 sequence shown in Figure 1. Preferably, 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 binds less to LGR5 proteins containing one or more of the amino acid residue mutations selected from 43A, 44A, 46A, 67A, 90A, and 91A.

[0020] Preferably, the EGFR-binding variable domain binds to an epitope located within amino acid residues 420 to 480 of the human EGFR sequence shown in Figure 2. Preferably, 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 binds less to EGFR proteins containing one or more of the amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A, and C499A.

[0021] Preferably, the antibody has enhanced ADCC. Preferably, the antibody is defucosylated. [Brief description of the drawings]

[0022] [Figure 1] Human LGR5 sequence, SEQ ID NO:1. [Diagram 2] Human EGFR sequence; SEQ ID NO:2. [Diagram 3] (a) Amino acid sequences of heavy chain variable regions (SEQ ID NOs: 3 to 15) that form variable domains that bind to LGR5 and EGFR together with a common light chain variable region such as the variable region of human kappa light chain IgVκ139*01 / IGJκ1*01. The CDR and framework regions are shown in FIG. 3b. The respective DNA sequences are shown in FIG. 3c. [Figure 4]a) Amino acid sequence of the common light chain amino acid sequence. b) Common light chain variable region DNA sequence and translation (IGKV1-39 / jk1). c) Light chain constant region DNA sequence and translation. d) V region IGKV1-39A. e) CDR1, CDR2, and CDR3 of the common light chain according to IMGT numbering. [Diagram 5] IgG heavy chain for the generation 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 mutations L351K and T366K (KK) DNA sequence and translation. e) CH3 domain containing mutations L351D and L368E (DE) DNA sequence and translation. Residue positions are according to EU numbering. [Figure 6] Data show the mean tumor size in a) gastric PDX model and b) esophageal PDX model, error bars are SEM. Two-way ANOVA test was used to calculate statistical significance at a given time point. ADC = adenocarcinoma. SCC = squamous cell carcinoma. Grey area represents treatment period. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] In order to make this description easier to understand, certain terms are defined first. Additional definitions are described throughout the detailed description. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, using conventional methods of immunology, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology.

[0024] As used herein, the singular forms "a," "an," and "the" include plural referents. Use of "comprising," "having," "including," and other forms of words such as "comprise," "comprises," "comprised," "has," "have," "had," "have," "include," "includes," and "included" is not limiting.

[0025] The term "antibody" as used herein means a protein molecule belonging to the immunoglobulin class of proteins that contains one or more domains that bind to an epitope on an antigen, such domains deriving from or sharing sequence homology with the variable regions of the antibody. Antibodies are typically composed of a basic structural unit having two heavy chains and two light chains each. The antibodies according to the present invention are not limited to any particular format or method of their production.

[0026] A "bispecific antibody" is an antibody as 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 a second VH / VL combination that binds to a second antigen or epitope on the antigen. The term further includes antibodies in which the VH can specifically recognize the first antigen and the VL can specifically recognize the second antigen in combination with the VH in the 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). Bispecific antibodies according to the invention are not limited to any particular bispecific format or method of their production.

[0027] The term "common light chain" as used herein refers to the two light chains (or VL portions thereof) in a bispecific antibody. The two light chains (or VL portions thereof) 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", "single VL", with or without the addition of the term "rearranged", are all used interchangeably herein. "Common" also refers to the functional equivalent of light chains that are not identical in amino acid sequence. There are many variants of the light chain in which there are mutations (deletions, substitutions, insertions and / or additions) that do not substantially affect the formation of a functional binding region. The light chain of the present invention may also be a light chain as described herein with 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof. It is within the scope of the definition of a common light chain as used herein, for example, to prepare or find variable light chains that are not identical but are still functionally equivalent, for example by introducing conservative amino acid changes, amino acid changes in regions that do not contribute, or only partially contribute, to binding specificity when paired with a heavy chain, and the like, and testing.

[0028] As used herein, "comprise" and its conjugations are used in their open-ended sense to mean that the items following the word are included, but not excluding items not specifically mentioned. Additionally, the verb "consisting of" may be replaced with "consisting essentially of," meaning that the compounds or sub-compounds defined herein may contain additional components other than those specifically identified, and that the additional components do not alter the inherent characteristics of the invention.

[0029] The term "full-length IgG" or "full-length antibody" according to the present invention is defined to include essentially a complete IgG, but not necessarily with all the functions of an intact IgG. For the avoidance of doubt, a full-length IgG comprises two heavy chains and two light chains. Each chain contains a constant (C) region and a variable (V) region, which may be classified into domains designated CH1, CH2, CH3, VH, and CL, VL. IgG antibodies bind to antigens via the variable region domains contained in the Fab portion, and after binding, can interact with molecules and cells of the immune system via the constant domains, primarily via the Fc portion. Full-length antibodies according to the present invention encompass IgG molecules in which mutations may be present that provide desired characteristics. Full-length IgG should not have a deletion of a substantial portion of any region. However, IgG molecules in which one or several amino acid residues have been deleted without essentially changing the binding properties of the resulting IgG molecule are encompassed by the term "full-length IgG". For example, such IgG molecules may have a deletion of 1-10 amino acid residues, preferably within the non-CDR regions, where the deleted amino acids are not essential for the antigen-binding specificity of the IgG.

[0030] An "antibody derivative" is a protein that deviates from the amino acid sequence of a native antibody in at most 20 amino acids, outside the CDR regions. The antibody derivatives disclosed herein are antibodies that deviate from the amino acid sequence in at most 20 amino acids.

[0031] "Percent identity" in the present specification with respect to nucleic acid or amino acid sequences is defined as the percentage of residues in a candidate sequence that are identical to the residues in a selected sequence after aligning the sequences for optimal comparison purposes. Percent sequence identity for comparing amino acid sequences is determined using the AlignX application of Vector NTI Advance® 11.5.2 software using the default settings with a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, (1994) Nuc. Acid Res. 22(22):4673-4680), swgapdnamt scoring matrix, gap opening penalty of 15, and gap extension penalty of 6.66. Amino acid sequences are aligned using the AlignX application of Vector NTI Program Advance® 11.5.2 software using default settings with a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, (1994) Nuc. Acid Res. 22(22):4673-4680), blosum62mt2 scoring matrix, a gap opening penalty of 10, and a gap extension penalty of 0.1.

[0032] Since an antibody usually recognizes an epitope of an antigen, and such an epitope may also exist in other compounds, an antibody according to the present invention that "specifically recognizes" an antigen, for example, EGFR or LGR5, may also recognize other compounds if such other compounds contain the same type of epitope. Thus, the term "specifically recognizes" with respect to the interaction between an antigen and an antibody does not exclude the binding of the antibody to other compounds that contain the same type of epitope.

[0033] "Epitope" or "antigenic 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 a protein (so-called linear and conformational epitopes). Epitopes formed from adjacent linear amino acids typically are retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding typically lose conformation upon treatment with denaturing solvents. Epitopes can typically include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

[0034] As used herein, the terms "subject" and "patient" are used interchangeably and refer to a mammal, such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, etc. (e.g., a patient, such as a human patient with cancer).

[0035] The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process of administering an active agent or combination of active agents to a subject for the purpose of reversing, alleviating, ameliorating, inhibiting, or slowing or preventing the progression, occurrence, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease.

[0036] As used herein, "effective treatment" or "positive therapeutic response" refers to a treatment that results in a beneficial effect, e.g., an improvement in at least one symptom of a disease or disorder, e.g., cancer. The beneficial effect can take the form of an improvement over a baseline, including an improvement over a measurement or observation made before starting treatment according to the method. For example, the beneficial effect can take the form of slowing, stabilizing, stopping, or reversing the progression of cancer in a subject at any clinical stage, as evidenced by a reduction or elimination of clinical or diagnostic symptoms of the disease or markers of cancer. An effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, slow or stop tumor growth, and / or prevent or delay tumor recurrence or relapse.

[0037] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent or combination of agents that provides a desired biological, therapeutic, and / or prophylactic result. The result can be reduction, amelioration, remission, lessening, delay, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. In some embodiments, an effective amount is an amount sufficient to delay tumor development. In some embodiments, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition can (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, delay, to some extent, and stop 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) relieve to some extent one or more of the symptoms associated with cancer. In one example, an "effective amount" is the amount of EGFR / LGR5 antibody that affects a reduction in cancer (eg, a reduction in the number of cancer cells), slows the progression of cancer, or prevents the regrowth or recurrence of cancer.

[0038] The present disclosure provides 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 cancer. The words cancer and tumor are used herein, unless otherwise specified, and generally both refer to cancer.

[0039] The epidermal growth factor "EGFR" 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 of which is EGFR. EGFR has an extracellular domain (ECD) composed of four subdomains, two of which are involved in ligand binding and two of which are involved in homodimerization and heterodimerization. EGFR integrates extracellular signals from various ligands, resulting in diverse intracellular responses. The major signaling pathway activated by EGFR consists of the Ras-mitogen-activated protein kinase (MAPK) mitogenic signaling cascade. Activation of this pathway is initiated by 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 much stronger in the presence of co-expression of ErbB-3 (HER3). EGFR is involved in several human epithelial malignancies, especially cancers of the breast, bladder, non-small cell lung cancer, colon, ovary, head and neck, and brain. Activating mutations in the gene and overexpression of the receptor and its ligands have been found, resulting in an autocrine activation loop. Therefore, this RTK is widely used as a target for cancer therapy. Both small molecule inhibitors targeting the RTK and monoclonal antibodies (mAbs) directed against the extracellular ligand-binding domain have been developed and have shown some clinical success so far, but mostly in selected patient groups. The database accession number for the human EGFR protein and its encoding gene is GenBank NM_005228.3. The accession numbers are provided primarily to provide an additional method of specification of the EGFR protein as a target, and the actual sequence of the EGFR protein bound by the antibody may vary due to mutations in the encoding gene, such as those that occur in some cancers.

[0040] When EGFR is mentioned herein, unless otherwise stated, reference is made to human EGFR. Variable domain antigen binding sites that bind EGFR bind to EGFR and its various mutants, including mutants expressed on some EGFR-positive tumors.

[0041] The term "LGR" refers to a family of proteins known as leucine-rich repeat-containing G protein-coupled receptors. Several members of this family are known to be involved in the WNT signaling pathway, notably LGR4, LGR5, and LGR6.

[0042] LGR5 is a leucine-rich repeat containing G protein-coupled receptor 5. Alternative names for the gene or protein are leucine-rich repeat containing G protein-coupled receptor 5, leucine-rich repeat containing G protein-coupled receptor 5, G protein-coupled receptor HG38, G protein-coupled receptor 49, G protein-coupled receptor 67, GPR67, GPR49, orphan G protein-coupled receptor HG38, G protein-coupled receptor 49, GPR49, HG38 and FEX. The protein or antibody of the present invention that binds to LGR5 binds to human LGR5. The LGR5 binding protein or antibody may also bind to other mammalian orthologues due to sequence and tertiary structure similarities between such orthologues, but this is not necessarily the case. The database accession 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). The accession numbers are provided primarily to provide a method of further specification of 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 that occur in some cancers. The LGR5 antigen binding site binds to LGR5 and various variants thereof, such as those expressed by some LGR5-positive tumor cells.

[0043] In some embodiments, the cancer is a gastrointestinal cancer, such as colon cancer. Preferably, the cancer is gastric cancer, esophageal cancer, or gastroesophageal junction cancer. Gastric cancer (also called stomach cancer) is a cancer that develops from the lining of the stomach, particularly the mucus-producing glandular cells found therein. Such cancers are also called gastric adenocarcinoma, or in this case gastric adenocarcinoma, because they arise from the stomach lining. In a preferred embodiment, the cancer is therefore a gastric adenocarcinoma or a cancer that develops from the stomach lining, which are used interchangeably herein. Esophageal cancer is a cancer that develops from the esophagus. The two main subtypes are ESCC (esophageal squamous cell carcinoma) and EAC (esophageal adenocarcinoma). Gastroesophageal junction cancer (also known as gastroesophageal junction adenocarcinoma) arises from the gastroesophageal junction.

[0044] In some embodiments, the cancer expresses LGR5 and / or expresses EGFR. As used herein, if the cancer contains cells that express LGR5, the cancer expresses LGR5. Cells that express LGR5 contain detectable levels of RNA that encodes LGR5. As used herein, if the cancer contains cells that express EGFR, the cancer expresses EGFR. Cells that express EGFR contain detectable levels of RNA that encodes LGR5. Expression can often also be detected by incubating cells with an antibody that binds to LGR5 or EGFR. However, some cells do not express high enough protein for such antibody testing. In such cases, mRNA or other forms of nucleic acid sequence detection are preferred.

[0045] In some embodiments, the present disclosure provides an antibody that binds to a variable domain that binds to an extracellular portion of EGFR and a variable domain that binds to an extracellular portion of LGR5, or a functional portion, derivative, and / or analog thereof, for use in treating gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a subject whose Her2 status is selected from Her2 positive, Her2 high, Her2 3+, Her2 2+, Her2 1+, Her2 0, or a Her2 negative subject. Preferably, the subject is Her2 negative. The present disclosure further provides a method of treating gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a Her2 negative subject, comprising providing the antibody, or a functional portion, derivative, and / or analog thereof, to a subject in need thereof. Preferably, the use comprises providing a flat dose of 1500 mg of the antibody, or a functional portion, derivative, and / or analog thereof, to the subject. In some embodiments, the administration of the therapeutic compound to the Her2 negative subject may be weekly, biweekly, or monthly. Preferably, the therapeutic compound is administered once every two weeks.

[0046] Methods for determining the expression of human epidermal growth factor receptor 2 (HER2) in a subject are well known in the art. For example, the expression level of Her2 can be established using immunohistochemistry (IHC) or (fluorescence) in-situ hybridization (ISH), which allows the identification of Her2 status, including the identification of Her2-negative subjects. IHC or ISH are both well-defined and standard procedures that are routinely used to establish Her2 status in human subjects. Reference is made herein, for example, to the ASCO / CAP guidelines by Bartley et al., (HER2 Testing and Clinical Decision Making in Gastroesophageal Adenocarcinoma. Arch Pathol Lab Med. 2016; 140: 1345-1363). For example, the use of an anti-Her-2 / neu antibody (clone 4B5) allows the semi-quantitative detection of HER-2 antigen in sections of FFPE gastric / esophageal adenocarcinoma, gastric cancer, esophageal cancer, or gastroesophageal junction cancer using IHC. Staining and scoring are performed according to consensus guidelines for this cancer type. Such IHC tests, which measure the amount of HER2 receptor protein on the surface of cells in a cancer tissue sample, usually give a score of 0 to 3+. Based on the IHC score, a patient can be classified as Her2 negative, such as when a score of 0 or 1+ is measured. When ISH tests are used to establish Her2 expression, such as using a HER2 probe (17q11.2-q12) and a centromere 17 probe (Cen17), the diagnosis is either "positive" or "negative," and is sometimes reported as "zero" for HER2. The therapeutic methods of the present disclosure are preferably for subjects who are Her2 negative as established by IHC and / or ISH.

[0047] A Her2 negative subject herein means a subject having cancer, cancer cells or a tumor, i.e., a subject that is Her2 negative. HER2 status may be determined according to IHC and / or ISH as described above.

[0048] Preferably, in some embodiments, a step of diagnosing the subject for Her2 status is performed prior to treatment with the antibody, or functional portion, derivative, and / or analog thereof. Preferably, in some embodiments, a subject with a Her2-negative status is selected for treatment. Preferably, in some embodiments, a step of diagnosing the subject with Her2-negative gastric cancer, esophageal cancer, or gastroesophageal junction cancer is performed prior to treatment of the subject. Such cancers treated by the methods of the present disclosure include gastric adenocarcinoma and esophageal cancer with squamous cell carcinoma histology diagnosis.

[0049] The Her2 negative diagnosis preferably involves ISH or IHC testing of Her2 status.

[0050] Preferably, in some embodiments, prior to treatment of a Her2-negative subject, a step of screening the subject for Her2-negative gastric, esophageal or gastroesophageal junction cancer is performed. Such cancers are, in particular, adenocarcinomas. The screening preferably involves ISH or IHC testing of Her2 status.

[0051] Cancers such as gastric, esophageal, or gastroesophageal junction cancers can be associated with the presence of mutations. Such mutations include mutations in known cancer genes such as PIK3CA, KRAS, and BRAF. Oncogenic mutations are generally described as activating mutations or mutations that result in new functions. Another type of cancer mutation involves tumor suppressor genes such as TP53, MLH1, CDKN2A, and PTEN. Mutations in tumor suppressor genes are generally inactivating.

[0052] TP53 encodes a transcription factor that regulates several activities, including stress response and cell proliferation. Mutations in TP53 are associated with various cancers and are estimated to occur in more than 50% of human cancers, including gastric and esophageal cancers. In particular, the TP53 R248Q mutation has been shown to be associated with cancers, including gastric and esophageal cancers (Pitolli et al. Int. J. Mol. Sci. 2019 20:6241). Nonsense mutations at positions R196 and R342 have been identified in several tumors, such as breast and esophageal, as well as ovarian, prostate, breast, pancreatic, gastric, colon / rectal, lung, esophageal, and bone, respectively (Priestly et al. Nature 2019 575:210-216). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers with TP53 mutations, particularly those that result in reduced TP53 expression or activity.

[0053] MLH1 (MutL homolog 1) encodes a protein involved in DNA mismatch repair and is a known tumor suppressor gene. Mutations in MLH1 are associated with various cancers, including gastrointestinal cancer. Low levels of MLH1 are also associated with esophageal cancer patients with a family history of esophageal cancer (Chang et al. Oncol Lett. 2015 9:430-436), and MLH1 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, e.g., 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 that have MLH1 mutations, particularly mutations that result in reduced MLH1 expression or activity.

[0054] PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) encodes the 110 kDa catalytic subunit of PI3 K (phosphatidylinositol 3-kinase). Mutations in PIK3CA are associated with various cancers, including gastrointestinal cancer. According to a report from the American Cancer Society, PIK3CA is mutated in 12.75% of patients with malignant solid tumors. Specifically, the PIK3CA H1047R mutation is present in 2.91% of all patients with malignant solid tumors, and the PIK3CA E545K mutation is present in 2.55% of all patients with malignant solid tumors (see The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831. Dataset Version 6.). In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers that have PIK3CA mutations, particularly oncogenic mutations in PIK2CA.

[0055] CDKN2A (cyclin-dependent kinase inhibitor 2A) encodes a protein that inhibits CDK4 and ARF. According to a report from the American Cancer Society, 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 with CDKN2A mutations, particularly those that result in reduced expression or activity of CDKN2A.

[0056] PTEN (phosphatase and tensin homolog) encodes phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase. According to a report by the American Cancer Society, 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 termination / stop codon) is present in 0.21% of all colorectal cancer patients (The AACR Prot Genie Consontium. 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 PTEN mutations, particularly those that result in reduced PTEN expression or activity.

[0057] BRAF encodes the serine / threonine-protein kinase B - Raf, which is involved in growth signaling. According to a report by the American Cancer Society, BRAF is mutated in 1.19% 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 that have a BRAF mutation, specifically, an oncogenic mutation in BRAF. However, in some embodiments, the therapeutic compounds disclosed herein are useful for treating gastric cancers that do not have the BRAF V600E mutation.

[0058] KRAS (Kirsten RAt sarcoma) encodes a protein that is a party of the RAS / MAPK pathway. According to a report by the American Cancer Society, KRAS is mutated in 14.7% of patients with malignant solid tumors, with KRAS G12C 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.

[0059] UGT1A1 (uridine diphosphateglucuronosyltransferase 1A1) and UGT1A8 (uridine diphosphateglucuronosyltransferase 1A8) encode enzymes in the glucuronidation pathway. Some polymorphisms that reduce enzyme activity are known to affect the metabolism and efficacy of irinotecan. For example, the UGT1A1*6 allele (G71R polymorphism) and the UGT1A1*28 allele (a dinucleotide repeat polymorphism in the TATA sequence in the promoter region), which have an allele frequency of about 0.13% in Chinese, Korean, and Japanese populations, are risk factors for irinotecan-induced neutropenia. In some embodiments, the therapeutic compounds disclosed herein are useful for treating cancers with mutations in UGT1A1 and / or UGT1A8, specifically mutations that result in reduced expression or activity of UGT1A1 and / or UGT1A8.

[0060] ATM (Ataxia Telangiectaisa Mutated) is a member of the serine-threonine kinase family that orchestrates the cellular response to DNA damage through activation of distinct DNA repair and signaling pathways. ATM germline mutations are associated with ataxia telangiectasia, and somatic ATM mutations are commonly observed in endometrial, colon, pancreatic, breast, and urothelial cancers.

[0061] In a preferred embodiment, the present disclosure provides a method for treating cancers with mutations in genes encoding TP53, MLH1, PIK3CA, CDKN2A, UGT1A, UGT1A8, BRAF, PTEN, and KRAS. Preferably, the cancer has one or more mutations selected from TP53 R196T, TP53 R342T, TP53 R248Q, MLH1 V384D, PIK3CA H1047R, PIK3CA E545K, CDKN2A W110T, UGT1A1 G71R, UGT1A8 G71R, and KRAS G12C. In some embodiments, the cancer is wild type for KRAS. Alternatively, the present disclosure provides a method for treating cancers with mutations in genes encoding ATM, in particular mutation W57T. Specifically, the present disclosure provides methods for treating esophageal cancer, particularly ESCC, having a mutation in the gene encoding ATM, particularly the mutation W57T.

[0062] In some embodiments, the cancer has a mutation in the gene encoding TP53, preferably the mutation is R342T, and the cancer has a mutation in the gene encoding MLH1, preferably the mutation is V384D.

[0063] In some embodiments, the cancer has a mutation in the gene encoding TP53, preferably the mutation is R248Q, the cancer has a mutation in the gene encoding PIK3CA, preferably the mutation is H1047R, the cancer has a mutation in the gene encoding CDKN2A, preferably the mutation is W110T, the cancer has a mutation in the gene encoding UGT1A1, preferably the mutation is G71R, the cancer has a mutation in the gene encoding UGT1A8, preferably the mutation is G71R. Preferably, the cancer is esophageal cancer, preferably esophageal squamous cell carcinoma (ESCC).

[0064] In some embodiments, the cancer has a mutation in the gene encoding BRAF. However, the cancer preferably does not have a mutation V600E in the gene encoding BRAF, and preferably does not have a mutation R130Ter in the gene encoding PTEN. In some embodiments, the cancer has a mutation in the gene encoding KRAS, preferably the mutation is G12C, the cancer has a mutation in the gene encoding UGT1A1, preferably the mutation is G71R, and the cancer has a mutation in the gene encoding UGT1A8, preferably the mutation is G71R. In some embodiments, the cancer has a mutation in the gene encoding UGT1A1, preferably the mutation is G71R, and the cancer has a mutation in the gene encoding UGT1A8, preferably the mutation is G71R. In some embodiments, the cancer has a mutation in PIK3CA, preferably the mutation is E545K. Preferably, the cancer is gastric cancer.

[0065] The antibodies described herein, or functional parts, derivatives, and / or analogs thereof, comprise a variable domain that binds to the extracellular portion of the epidermal growth factor (EGF) receptor and a variable domain that binds to LGR5. The EGFR is preferably human EGFR. The LGR5 is preferably human LGR5. The antibodies described herein, or functional parts, derivatives, and / or analogs thereof, comprise 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.

[0066] Preferably, the antibodies described herein, or functional parts, derivatives, and / or analogs thereof, 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, and the interaction of the antibody with LGR5 on an LGR5-expressing cell does not block Rspondin (RSPO) binding to LGR5. Methods for determining whether an antibody blocks or does not block Rspondin binding to LGR5 are described in WO2017 / 069528, which is incorporated herein by reference.

[0067] Protein / gene accession numbers or alternative names are given herein, which are given primarily to provide an additional method of identification of the above mentioned proteins as targets, and the actual sequence of the target protein bound by an antibody may vary due to, for example, mutations in the encoding gene and / or alternative splicing, such as those that occur in some cancers, etc. A target protein will be bound by an antibody as long as the epitope is present within the protein and is accessible to the antibody.

[0068] The antibody described herein, or its functional part, derivative, and / or analog, preferably prevents the binding of a ligand for EGFR to EGFR. As used herein, the term "prevents binding" means that the binding of the antibody, or its functional part, derivative, and / or analog, to EGFR competes with the ligand for binding to the EGF receptor. The antibody, or its functional part, derivative, and / or analog, may weaken the ligand binding, displace the ligand if it is already bound to the EGF receptor, or may at least partially prevent the ligand from binding to the EGF receptor, for example, through steric hindrance.

[0069] The EGFR antibodies disclosed herein preferably inhibit EGFR ligand-induced signaling, measured as ligand-induced growth of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058), or ligand-induced cell death of A431 cells (ATCC CRL-1555), respectively. EGFR can bind to several ligands and stimulate the proliferation of the above-mentioned BxPC3 cells or BxPC3-Luc2 cells. In the presence of an EGFR ligand, the proliferation of BxPC3 or BxPC3-Luc2 cells is stimulated. The EGFR ligand-induced proliferation of BxPC3 cells can be measured by comparing the proliferation of cells in the absence and presence of the ligand. A preferred EGFR ligand for measuring the EGFR ligand-induced proliferation of BxPC3 or BxPC3-Luc2 cells is EGF. The ligand-induced proliferation is preferably measured using a saturating amount of the ligand. In a preferred embodiment, EGF is used in the medium in an amount of 100 ng / ml. The EGF is preferably EGF R&D Systems, catalog numbers 396-HB and 236-EG (see also WO2017 / 069628, which is incorporated herein by reference).

[0070] The EGFR antibodies disclosed herein preferably inhibit EGFR ligand-induced proliferation of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058). EGFR can bind to several ligands and stimulate proliferation of the above-mentioned BxPC3 cells or BxPC3-Luc2 cells. In the presence of the ligand, proliferation of BxPC3 or BxPC 3-Luc2 cells is stimulated. EGFR ligand-induced proliferation of BxPC3 cells can be measured by comparing proliferation of cells in the absence and presence of 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 saturating amount of ligand. In a preferred embodiment, EGF is used in the medium in an amount of 100 ng / ml. The EGF is preferably EGF from R&D Systems, catalog numbers 396-HB and 236-EG (see also WO2017 / 069628, which is incorporated herein by reference).

[0071] For the avoidance of doubt, reference to cell growth as used herein refers to a change in the number of cells. Inhibition of growth refers to a reduction in the number of cells that would otherwise be obtained. Increased growth refers to an increase in the number of cells that would otherwise be obtained. Cell growth usually refers to cell proliferation.

[0072] Whether the antibodies described herein inhibit signaling or inhibit proliferation in a multispecific format is determined by the methods described herein above, preferably using a monospecific monovalent or monospecific bivalent version of the antibody. Such antibodies preferably have a binding site for the receptor for which signaling is determined. Monospecific monovalent antibodies may have variable domains with unrelated binding specificities, such as tetanus toxoid specificity. Preferred antibodies are bivalent monospecific antibodies, whose antigen-binding variable domains consist of variable domains that bind to EGF receptor family members.

[0073] In its Biclonics® antibody program, Merus has developed multispecific antibodies targeting EGFR and LGR5 (leucine-rich repeat containing G protein-coupled receptor). The efficacy of such multispecific antibodies has been evaluated in vitro and in vivo using patient-derived CRC organoids and mouse PDX models, respectively (see, e.g., WO2017 / 069628, which is incorporated herein by reference). Multispecific antibodies targeting EGFR and LGR5 have been shown to inhibit tumor growth. The efficacy of such inhibitory antibodies has been shown to correlate with the level of LGR5 RNA expression by cancer-derived cells. Particularly preferred are multispecific antibodies targeting EGFR and LGR5 described in WO2017 / 069628.

[0074] The antibodies, or functional portions, derivatives, and / or analogs thereof, described herein comprise 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 of Figure 1, where amino acid residues D43, G44, M46, F67, R90, and F91 are involved in binding of the antibody to the epitope.

[0075] The LGR5 variable domain is preferably one in which one or more of the following amino acid residue substitutions in LGR5: D43A, G44A, M46A, F67A, R90A, and F91A reduce binding of the variable domain to LGR5.

[0076] The epitope on the extracellular portion of LGR5 is preferably located within amino acid residues 21 to 118 of the sequence of Figure 1. Preferably, it is an epitope in which 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.

[0077] The 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 of Figure 1.

[0078] The epitope on LGR5 is preferably a conformational epitope. The epitope is preferably located within amino acid residues 40-95 of the sequence of Figure 1. Binding of the antibody to LGR5 is preferably reduced with one or more of the following amino acid residue substitutions: D43A, G44A, M46A, F67A, R90A, and F91A.

[0079] Without being bound by theory, M46, F67, R90, and F91 of LGR5 shown in Figure 1 are believed to be contact residues for the antigen binding site of the variable domain shown herein above, i.e., the variable domain that binds to the LGR5 epitope. The amino acid residue substitutions D43A and G44A reduce antibody binding, which may be due to the fact that they are also contact residues, but it may also be due to the fact that these amino acid residue substitutions induce (slight) modifications of the conformation of the part of LGR5 that has one or more of the other contact residues (i.e., at positions 46, 67, 90, or 91), and the conformational change is such that antibody binding is reduced. The epitope is characterized by the amino acid substitutions described above. 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 an alanine substitution mutant, preferably a mutant 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 an epitope if the epitope binds to LGR5 and binds to a lesser extent to LGR5 with the M46A, F67A, R90A, or F91A substitution. It is preferable to compare the binding to a panel of mutants each containing one alanine residue substitution. Such binding tests are well known in the art. In many cases, the panel will include single alanine substitution mutants covering substantially all amino acid residues. In the case of LGR5, the panel need of course only cover the extracellular portion of the protein and the portion that ensures association with the cell membrane when the cells are used. The expression of a particular mutant may be impaired, which is easily detected by one or more LGR5 antibodies that bind to different regions. If expression is also reduced for these control antibodies, then the level or folding of the protein on the membrane is impaired for this particular mutant. The binding characteristics of the test antibody to the panel will readily identify whether the test antibody shows reduced binding to mutants with the M46A, F67A, R90A, or F91A substitutions, and therefore whether the test antibody is an antibody of the invention.Reduced binding to mutants with M46A, F67A, R90A, or F91A substitutions also identifies an epitope located within amino acid residues 21-118 of the sequence in Figure 1. In a preferred embodiment, the panel includes a D43A substitution mutant, both G44A substitution mutants, and antibodies with the VH sequence of MF5816 show reduced binding to these substitution mutants.

[0080] Without being bound by any theory, it is believed that amino acid residues I462, G465, K489, I491, N493, and C499 as shown in Figure 2 are involved in binding to the epitope by an antibody comprising the variable domain as shown herein above. Participation in binding is preferably determined by observing reduced binding of the variable domain to EGFR having one or more of the amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A, and C499A.

[0081] 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 FIG. 2. Preferably, 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. Binding of the antibody to human EGFR preferably prevents binding of EGF to its receptor. The epitope on EGFR is preferably a conformational epitope. In one embodiment, the epitope is located within amino acid residues 420-480 of the sequence shown in FIG. 2, preferably within 430-480 of the sequence shown in FIG. 2, preferably within 438-469 of the sequence shown in FIG. 2.

[0082] Without being bound by theory, it is believed that the contact residues of the epitope, i.e., the regions where the variable domain contacts human EGFR, are likely to be 1462, K489, 1491, and N493. Amino acid residues G465 and C499 are likely indirectly involved in binding of the antibody to EGFR.

[0083] The variable domain that binds to human EGFR is preferably a variable domain having a heavy chain variable region comprising at least the CDR3 sequence of VH of MF3755 shown in Figure 3, or a CDR3 sequence that differs from the CDR3 sequence of VH of MF3755 shown in Figure 3 by up to 3 amino acids, preferably up to 2 amino acids, and preferably not more than 1 amino acid.

[0084] A variable domain that binds to human EGFR is preferably a variable domain having a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of VH of MF3755 shown in Figure 3, or the CDR1, CDR2, and CDR3 sequences of VH of MF3755 shown in Figure 3 with up to three, preferably up to two, preferably up to one amino acid substitutions.

[0085] The variable domain that binds to human EGFR is preferably a variable domain having a heavy chain variable region comprising the amino acid sequence of the VH chain of MF3755 shown in Figure 3, or the amino acid sequence of the VH chain of MF3755 shown 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 of MF3755.

[0086] In one embodiment, the disclosure provides an antibody comprising a variable domain that binds to an extracellular portion of EGFR and a variable domain that binds to an extracellular portion of LGR5, The heavy chain variable region of the variable domain comprises at least the 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 by up to 3 amino acids, preferably up to 2 amino acids, preferably no more than 1 amino acid, from the CDR3 sequence of a VH selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3. The variable domain preferably comprises a heavy chain variable region comprising at least the CDR3 sequence of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3.

[0087] The variable domain preferably comprises a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences that differ by up to 3, preferably up to 2, preferably up to 1 amino acid from the CDR1, CDR2, and CDR3 sequences of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3. The variable domain preferably comprises a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3. A preferred heavy chain variable region is MF3755. Another preferred heavy chain variable region is MF4280.

[0088] 5. An antibody comprising a variable domain which binds to the extracellular portion of EGFR and a variable domain which binds to the extracellular portion of LGR5, wherein the EGFR-binding variable domain having the CDR3, CDR1, CDR2, and CDR3, and / or VH sequences as shown herein above, preferably comprises a heavy chain CDR3 sequence which differs by up to 3, preferably up to 2, preferably not more than 1 amino acid from the CDR3 sequence of an LGR5-specific heavy chain variable region selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in FIG. 3, or from the CDR3 sequence of a VH selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in FIG. The variable domain preferably comprises a heavy chain variable region comprising at least the CDR3 sequence of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3.

[0089] The LGR5 variable domain preferably comprises a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of an LGR5-specific heavy chain variable region selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3, or a heavy chain CDR1, CDR2, and CDR3 sequence that differs by up to three, preferably up to two, preferably up to one amino acid from the CDR1, CDR2, and CDR3 sequence of an LGR5-specific heavy chain variable region selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3. The variable domain preferably comprises a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 as shown in Figure 3. 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 the heavy chain variable region MF5816 being particularly preferred. Another preferred heavy chain variable region is MF5818.

[0090] Antibodies comprising one or more variable domains having the heavy chain variable region MF3755 or one or more CDRs thereof have been shown to have better efficacy when used to inhibit the growth of EGFR ligand-responsive cancers or cells. In the context of bispecific or multispecific antibodies, an arm of an antibody comprising a variable domain having the heavy chain variable region MF3755 or one or more CDRs thereof binds well to an arm comprising a variable domain having the heavy chain variable region MF5818 or one or more CDRs thereof.

[0091] The VH chain of the variable domain that binds EGFR or LGR5 can have one or more amino acid substitutions relative to the sequence shown in Figure 3. The VH chain preferably has the amino acid sequence of the EGFR or LGR5 VH of Figure 3 with 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 of Figure 3.

[0092] The CDR sequences may have one or more amino acid residue substitutions with respect to the CDR sequences in the figures. 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, for example, by mutagenesis procedures, after which the stability and / or binding affinity of the resulting antibodies are preferably tested and improved EGFR-specific or LGR5-specific CDR sequences are preferably selected. The skilled person can generate antibody variants comprising at least one modified CDR sequence according to the invention. For example, conservative amino acid substitutions may be applied. Examples of conservative amino acid substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another hydrophobic residue, and the substitution of one polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.

[0093] 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 in the VH or VL specified herein are preferably conservative amino acid substitutions. The amino acid insertions, deletions and substitutions in the VH or VL specified herein are preferably not in the CDR3 region. The amino acid insertions, deletions and substitutions mentioned are preferably not in the CDR1 and CDR2 regions either. The amino acid insertions, deletions and substitutions mentioned are preferably not in the FR4 region either.

[0094] 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 are preferably conservative amino acid substitutions and the insertions, deletions, substitutions or combinations thereof are preferably not present in the CDR3 region of the VH chain, preferably not present in the CDR1, CDR2 or CDR3 regions of the VH chain, preferably not present in the FR4 region.

[0095] 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 is preferably the amino acid sequence of VH chain MF3755 shown in Figure 3, or - comprising the amino acid sequence of VH chain MF3755 as shown in Figure 3 with 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 for said VH, 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 - comprising the amino acid sequence of VH chain MF5790 as shown in Figure 3, with 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 for said VH.

[0096] 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 is preferably the amino acid sequence of VH chain MF3755 shown in Figure 3, or - comprising the amino acid sequence of VH chain MF3755 as shown in Figure 3 with 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 for said VH, 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 - comprising the amino acid sequence of VH chain MF5803 as shown in Figure 3, with 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 for said VH.

[0097] 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 is preferably the amino acid sequence of VH chain MF3755 shown in Figure 3, or - comprising the amino acid sequence of VH chain MF3755 as shown in Figure 3 with 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 for said VH, The VH chain of the variable domain that binds to LGR5 is the amino acid sequence of VH chain MF5814 as shown in FIG. 3, or - comprising the amino acid sequence of VH chain MF5814 as shown in Figure 3, with 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 for said VH.

[0098] 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 is preferably the amino acid sequence of VH chain MF3755 shown in Figure 3, or - comprising the amino acid sequence of VH chain MF3755 as shown in Figure 3 with 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 for said VH, The VH chain of the variable domain that binds to LGR5 is the amino acid sequence of VH chain MF5816 as shown in FIG. 3, or - comprising the amino acid sequence of VH chain MF5816 as shown in Figure 3, with 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 for said VH.

[0099] 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 is preferably the amino acid sequence of VH chain MF3755 shown in Figure 3, or - comprising the amino acid sequence of VH chain MF3755 as shown in Figure 3 with 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 for said VH, The VH chain of the variable domain that binds to LGR5 is the amino acid sequence of VH chain MF5817 as shown in FIG. 3, or - comprising the amino acid sequence of VH chain MF5817 as shown in Figure 3, with 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 for said VH.

[0100] 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 is preferably the amino acid sequence of VH chain MF3755 shown in Figure 3, or - comprising the amino acid sequence of VH chain MF3755 as shown in Figure 3 with 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 for said VH, The VH chain of the variable domain that binds to LGR5 is the amino acid sequence of VH chain MF5818 as shown in FIG. 3, or - comprising the amino acid sequence of VH chain MF5818 as shown in Figure 3, with 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 for said VH.

[0101] Additional variants of the disclosed amino acid sequences that retain EGFR or LGR5 binding can be obtained, for example, from phage display libraries containing rearranged human IGKV1-39 / IGKJ1 VL regions (De Kruif et al. Biotechnol Bioeng. 2010(106)741-50) and collections of VH regions that incorporate amino acid substitutions in the amino acid sequences of the EGFR or LGR5 VH regions disclosed herein, as previously described (e.g., WO2017 / 069628). Phages that encode Fab regions that bind EGFR or LGR5 can be selected, analyzed by flow cytometry, and sequenced to identify variants with amino acid substitutions, insertions, deletions, or additions that retain antigen binding.

[0102] The light chain variable regions of the VH / VL EGFR and LGR5 variable domains of an EGFR / LGR5 antibody may be the same or different. In some embodiments, the VL region of the VH / VL EGFR variable domain of an EGFR / LGR5 antibody is similar to 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.

[0103] In certain embodiments, the light chain variable regions of one or both of the VH / VL variable domains of the EGFR / LGR5 antibodies comprise 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 comprises a germline IgVκ1-39 variable region V-segment. In certain embodiments, the light chain variable region of one or both of the VH / VL variable domains comprises a kappa light chain V-segment IgVκ1-39*01. IgVκ1-39 is short for 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:ENSG00000242371. The amino acid sequences for suitable V regions are provided in FIG. 4. The V region can be combined with one of five J regions. Preferred J regions are jk1 and jk5, the linked sequences are designated IGKV1-39 / jk1 and IGKV1-39 / jk5, alternative names are IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (designation by the IMGT database world wide web at imgt.org). In certain embodiments, the light chain variable region of one or both VH / VL variable domains comprises the kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ1*05 (depicted in FIG. 4).

[0104] In some embodiments, the light chain variable region of one or both VH / VL variable domains of the EGFR / LGR5 bispecific antibody comprises an LCDR1 comprising the amino acid sequence QSISSY (as depicted in FIG. 4), an LCDR2 comprising the amino acid sequence AAS (as depicted in FIG. 4), and an LCDR3 comprising the amino acid sequence QQSYSTP (as depicted in FIG. 4) (i.e., the CDRs of IGKV1-39 by IMGT). In some embodiments, the light chain variable region of one or both VH / VL variable domains of the EGFR / LGR5 antibody comprises an LCDR1 comprising the amino acid sequence QSISSY (as depicted in FIG. 4), an LCDR2 comprising the amino acid sequence AASLQS (as depicted in FIG. 4), and an LCDR3 comprising the amino acid sequence QQSYSTP (as depicted in FIG. 4).

[0105] In some embodiments, one or both of the VH / VL variable domains of an EGFR / LGR5 antibody comprise a light chain variable region comprising 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% identical, or 100% identical to the amino acid sequence set forth in Figure 4. In some embodiments, one or both of the VH / VL variable domains of an EGFR / LGR5 antibody comprise a light chain variable region comprising 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% identical, or 100% identical to the amino acid sequence set forth in Figure 4.

[0106] For example, in some embodiments, one or both variable light chains of the VH / VL variable domains of the EGFR / LGR5 antibody can have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination 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 the EGFR / LGR5 antibody contains 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, preferably 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof with respect to the amino acid sequence shown.

[0107] In other embodiments, the light chain variable region of one or both VH / VL variable domains of the EGFR / LGR5 antibody comprises the amino acid sequence of the sequence shown in Figure 4. In certain embodiments, both VH / VL variable domains of the EGFR / LGR5 antibody comprise identical VL regions. In one embodiment, the VL of both VH / VL variable domains of the EGFR / LGR5 bispecific antibody comprises the amino acid sequence set forth in Figure 4. In one embodiment, the VL of both VH / VL variable domains of the EGFR / LGR5 bispecific antibody comprises the amino acid sequence set forth in Figure 4.

[0108] The EGFR / LGR5 antibodies described herein are preferably bispecific antibodies with two variable domains, one that binds EGFR and another that binds LGR5 as described herein. EGFR / LGR5 bispecific antibodies for use in the methods disclosed herein can be provided in several formats. Many different formats of bispecific antibodies are known in the art and are reviewed by Kontermann (Drug Discov Today, 2015 Jul;20(7):838-47, MAbs, 2012 Mar-Apr;4(2):182-97) and in Spiess et al., (Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol. Immunol. (2015) http: / / dx.doi.org / 10.1016 / j.molimm.2015.01.003), each of which is incorporated herein by reference. For example, bispecific antibody formats that are not classical antibodies with two VH / VL combinations have at least a variable domain comprising a heavy chain variable region and a light chain variable region, which may be linked to a single chain Fv fragment, a single VH and a Fab fragment providing a second binding activity.

[0109] In some embodiments, the EGFR / LGR5 bispecific antibodies used in the methods provided herein are generally of the human IgG subclass (e.g., IgG1, IgG2, IgG3, IgG4). In certain embodiments, the antibodies are of the human IgG1 subclass. Full-length IgG antibodies are preferred because of their favorable half-life and because of their low immunogenicity. Thus, in certain embodiments, the EGFR / LGR5 bispecific antibodies are full-length IgG molecules. In one embodiment, the EGFR / LGR5 bispecific antibodies are full-length IgG1 molecules.

[0110] Thus, 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 no more than 10, preferably no more than 5 amino acid differences from the constant region of a naturally occurring human antibody. For example, in certain embodiments, each Fab arm of the bispecific antibody may further comprise an Fc region that contains modifications that facilitate bispecific antibody formation, stability and / or other characteristics described herein.

[0111] Antibodies are typically produced by a cell expressing a nucleic acid encoding the antibody. Thus, in some embodiments, the bispecific EGFR / LGR5 antibodies disclosed herein are produced by providing a cell comprising one or more nucleic acids encoding the heavy and light chain variable and constant regions of the bispecific EGFR / LGR5 antibody. The cell is preferably an animal cell, more preferably a mammalian cell, more preferably a primate cell, and most preferably a human cell. A suitable cell is any cell that can comprise and preferably produce an EGFR / LGR5 bispecific antibody.

[0112] Suitable cells 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 have developed cell lines for large-scale production of antibodies, for example for clinical use. Non-limiting examples of such cell lines are CHO cells, NS0 cells or PER.C6 cells. In a particularly preferred embodiment, the cells are human cells. Preferably, the cells are transformed with an adenovirus E1 region or a functional equivalent thereof. A preferred example of such a cell line is the PER.C6 cell line or an equivalent thereof. In a particularly preferred embodiment, the cells are CHO cells or variants thereof. Preferably, the variants use the glutamine synthetase (GS) vector system for the expression of the antibody. In a preferred embodiment, the cells are CHO cells.

[0113] In some embodiments, the cells express different light and heavy chains that make up the EGFR / LGR5 bispecific antibody. In certain embodiments, the cells express two different heavy chains and at least one light chain. In a preferred embodiment, the cells express a "common light chain" as described herein to reduce the number of different antibody species (different heavy and light chain combinations). For example, each VH region is cloned into an expression vector using methods known in the art for the production of bispecific IgG (WO2013 / 157954, incorporated herein by reference) together with a rearranged human IGKV139 / IGKJ1 (huVκ139) light chain, which has previously been shown to be capable of pairing with two or more heavy chains, thereby resulting in antibodies with diverse specificities, facilitating the production of bispecific molecules (De Kruif et al. J. Mol. Biol. 2009(387)548 58; WO2009 / 157771).

[0114] 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., with the same heavy chain combination), respectively. Several methods have been published to favor the production of bispecific antibodies over the production of the respective monospecific antibodies. This is typically achieved by modifying the constant regions of the heavy chains such that they favor heterodimerization (i.e., dimerization with heavy chains of other heavy / light chain combinations) over homodimerization. In a preferred embodiment, the bispecific antibody of the present invention comprises two different immunoglobulin heavy chains with compatible heterodimerization domains. Various compatible heterodimerization domains have been described in the art. The compatible heterodimerization domains are preferably compatible immunoglobulin heavy chain CH3 heterodimerization domains. Various methods have been described in the art by which such heterodimerization of heavy chains can be achieved.

[0115] A preferred method for producing EGFR / LGR5 bispecific antibodies is disclosed in US 9,248,181 and US 9,358,286. In particular, preferred mutations that produce essentially only bispecific full-length IgG molecules are the amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain ("KK mutant" heavy chain) and L351D and L368E in the second domain ("DE mutant" heavy chain), or vice versa. As mentioned above, DE and KK mutants preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE mutant heavy chains (DEDE homodimers) or KK mutant heavy chains (KKKK homodimers) hardly occurs due to the strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0116] Thus, in one embodiment, the heavy / light chain combination comprising a variable domain that binds EGFR comprises a DE mutant of the heavy chain, in this embodiment, the heavy / light chain combination comprising a variable domain that binds LGR5 comprises a KK mutant of the heavy chain.

[0117] 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 assessed by flow cytometry (by a FACS procedure previously described in WO2017 / 069628). In one embodiment, binding of candidate EGFR / LGR5 bispecific antibodies to LGR5 on CHO cells is demonstrated by flow cytometry performed according to standard procedures known in the art. Binding to CHO cells is compared to CHO cells that are not transfected with expression cassettes for EGFR and / or LGR5. Binding of candidate bispecific IgG1 to EGFR is determined using CHO cells transfected with an EGFR expression construct, and LGR5 and EGFR monospecific antibodies as well as an irrelevant IgG1 isotype control mAb are included in the assay as controls (e.g., an antibody that binds to LGR5 and another antigen such as tetanus toxoid (TT)).

[0118] The affinity of the LGR5 and EGFR Fab of the candidate EGFR / LGR5 bispecific antibody to the target can be measured by surface plasmon resonance (SPR) technology using a BIAcore T100. Briefly, anti-human IgG mouse monoclonal antibody (Becton and Dickinson, Cat. No. 555784) is coupled onto the surface of a CM5 sensor chip using free amine chemistry (NHS / EDC). The bsAb is then captured onto the sensor surface. Recombinant purified antigen human EGFR (Sino Biological Inc, Cat. No. 11896-H07H) and human LGR5 proteins are then run over the sensor surface at a range of concentrations to measure the on-rates and off-rates. After each cycle, the sensor surface is regenerated by a pulse of HCl and the bsAb is captured again. From the resulting sensorgrams, on- and off-rates and affinity values ​​for binding to human LGR5 and EGFR are determined using BIAevaltion software previously described for CD3 in US2016 / 0368988.

[0119] The antibodies disclosed herein are typically bispecific full-length antibodies, preferably human IgG subclass, preferably human IgG1 subclass. Such antibodies can be enhanced, if desired, by techniques known in the art, and have good ADCC properties, with favorable half-life upon in vivo administration to humans, and CH3 engineering techniques exist that can provide modified heavy chains that form heterodimers preferentially over homodimers upon co-expression in clonal cells.

[0120] The ADCC activity of an antibody can be improved when the antibody itself has low ADCC activity by modifying the antibody's constant region. Another way to improve the ADCC activity of an antibody is by enzymatically interfering with the glycosylation pathway that results in reduced fucose. There are several in vitro methods to determine the effectiveness of an antibody or effector cell in inducing ADCC. Among them are the chromium-51 [Cr51] release assay, the europium [Eu] release assay, and the sulfur-35 [S35] release assay. Typically, a labeled target cell line expressing a particular surface-exposed antigen is incubated with an antibody specific for that antigen. After washing, effector cells expressing the Fc receptor CD16 are co-incubated with the antibody-labeled target cells. Target cell lysis is then measured by the release of intracellular label by scintillation counter or spectrophotometry.

[0121] The bispecific antibody disclosed herein can be enhanced in ADCC.The bispecific antibody can be afucosylated in one embodiment.The bispecific antibody preferably comprises a reduced amount of fucosylation of N-linked carbohydrate structures in the Fc region when compared with the same antibody produced in normal CHO cells.

[0122] 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 may further comprise one or more additional variable domains that can bind to one or more additional targets. The additional targets are preferably proteins, preferably membrane proteins that comprise an extracellular portion. A membrane protein as used herein is a cell membrane protein, such as a protein that is in the outer membrane of a cell, the membrane that separates the cell from the outside world. A membrane protein has an extracellular portion. A membrane protein is at least on a cell when it contains a transmembrane region that is in the cell membrane of the cell.

[0123] Antibodies having two or more variable domains are known in the art.For example, it is possible to combine additional variable domains with the constant part of the antibody.Antibodies having three or more variable domains are preferably multivalent multimeric antibodies as described in PCT / NL2019 / 050199, which is incorporated herein by reference.

[0124] In one embodiment, the antibody is a bispecific antibody comprising two variable domains, one variable domain that binds to the extracellular portion of EGFR and another variable domain that binds to the extracellular portion of LGR5. The variable domains are preferably variable domains as described herein.

[0125] The functional part of the antibody described herein comprises at least a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 described herein. It therefore comprises the antigen-binding portion of the antibody described herein, typically containing the variable domain of the antibody. The variable domain of the functional part can be a single chain Fv fragment or a so-called single domain antibody fragment. Single domain antibody fragments (sdAbs) are antibody fragments that have a single monomeric variable antibody domain. Like whole antibodies, these antibodies can selectively bind to a specific antigen. With a molecular weight of only 12-15 kDa, single domain antibody fragments are much smaller than a typical antibody (150-160 kDa) composed 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 very small compared to normal antibodies (typically 90-100 kDa). Single domain antibody fragments can be engineered primarily from heavy chain antibodies found in camelids, and these are called VHH fragments (Nanobody®). Some fish also have heavy chain only antibodies (IgNAR, "immunoglobulin novel antigen receptor"), from which single domain antibody fragments called VNAR fragments can be derived. An alternative approach is to split the dimeric variable domain from common immunoglobulin G (IgG) of human or mouse origin into monomers. Most of the research on single domain antibodies is currently based on heavy chain variable domains, but nanobodies derived from light chains have also been shown to specifically bind target epitopes. Non-limiting examples of such variable domains of antibody moieties are VHH, human domain antibodies (dAb), and unibodies. Preferred antibody moieties or derivatives have at least two variable domains of an antibody or its equivalent. Non-limiting examples of such variable domains or its equivalents are F(ab) fragments and single chain Fv fragments. The functional portion of a bispecific antibody comprises the antigen-binding portion of the bispecific antibody, or a derivative and / or analogue of the binding portion. As described herein above, the binding portion of an antibody is encompassed by the variable domain.

[0126] Also provided are antibodies disclosed herein, or functional portions, derivatives, and / or analogs thereof (i.e., therapeutic compounds), and pharma- ceutically acceptable carriers. Such pharmaceutical compositions are useful in the treatment of cancer, particularly for the treatment of gastric, esophageal, or gastroesophageal junction cancer. As used herein, the term "pharmaceutical acceptable" means approved by a government regulatory agency or listed in the United States Pharmacopeia or another generally recognized pharmacopoeia for use in animals, particularly humans, and includes any and all solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, glycerol ricinoleate polyethylene glycol, and the like. Water or saline and aqueous dextrose and glycerol solutions may be used as carriers, particularly for injectable solutions. Liquid compositions for parenteral administration may be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravesical, intratumoral, intravenous, intraperitoneal, intramuscular, intrathecal, and subcutaneous. Depending on the route of administration (e.g., intravenous, subcutaneous, intraarticular, etc.), the active compound may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0127] Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, for example, in liquid carriers, or for reconstitution into solutions or suspensions for intravenous administration.Compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage.Also included are solid preparations intended to be converted immediately before use into liquid preparations for either oral or parenteral administration.Such liquid forms include solutions, suspensions, and emulsions.

[0128] The disclosed therapeutic compounds may be administered at a suitable dose and according to a suitable route (e.g., intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously). 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 exigencies of the therapeutic situation. In one embodiment, the subject is administered a single dose of the antibody, or functional part, derivative, and / or analog thereof, disclosed herein. In some embodiments, the therapeutic compound is administered repeatedly over the course of treatment. For example, in certain embodiments, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) doses of the therapeutic compound are administered to the subject in need of treatment. In some embodiments, administration of the therapeutic compound may be weekly, biweekly, or monthly.

[0129] The clinician may utilize a preferred dose that is deemed appropriate by the condition of the patient being treated. The dose may depend on several factors, including the stage of the disease, etc. It is within the skill of the artisan to determine the specific dose to be administered based on the presence of one or more of such factors. Generally, treatment is initiated with a smaller dose that is less than the optimal dose of the compound. Thereafter, the dosage is increased by small increments until the optimal effect under the circumstances is reached. For convenience, the total daily dosage may be administered in divided doses throughout the day, as necessary. Intermittent therapy (e.g., one week out of three weeks or three weeks out of four weeks) may also be used.

[0130] In certain embodiments, the therapeutic compound is administered at a dose of 0.1, 0.3, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg body weight. In other embodiments, the therapeutic compound is administered at a dose of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg body weight.

[0131] In a preferred embodiment, the therapeutic compound (i.e., an antibody, or functional portion, derivative, and / or analog thereof, comprising a variable domain that binds to an extracellular portion of EGFR and a variable domain that binds to an extracellular portion of LGR5) is provided to the subject in a dose of 1500 mg. A flat dose offers several advantages over surface or body weight administration, as it reduces preparation time and reduces potential dose calculation errors. In some embodiments, the therapeutic compound is provided in a dose of at least 1100 mg, preferably a dose of 1100-2000 mg, more preferably a dose of 1100-1800 mg. As will be appreciated by those skilled in the art, this dose may be administered over time. For example, the dose may be administered by IV, e.g., in a 1-6 hour infusion, preferably a 2-4 hour infusion. In some embodiments, the therapeutic compound is administered once every two weeks. In some embodiments, the flat dose disclosed herein is suitable for use in adults and / or subjects weighing at least 35 kg. Preferably, the subject is suffering from gastric cancer, esophageal cancer, or gastroesophageal junction cancer.

[0132] In some embodiments, a premedication regimen may be used. Such a regimen may be useful to reduce the likelihood or severity of infusion-related reactions. Generally, a steroid such as dexamethasone and / or an antihistamine such as dexchlorpheniramine, diphenhydramine, or chlorpheniramine is administered (e.g., orally, intravenously) prior to antibody treatment.

[0133] The therapeutic method described herein is typically continued as long as the clinician supervising the care of the patient considers the therapeutic method to be effective, i.e., the patient responds to the treatment.The non-limiting parameters that indicate that the therapeutic method is effective 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 appropriate response (if applicable) by suitable tumor markers.

[0134] With regard to the frequency of administration of the therapeutic compound, a person skilled in the art would be able to determine the appropriate frequency. For example, a clinician may decide to administer the therapeutic compound relatively infrequently (e.g., once every two weeks), gradually shortening the period between doses tolerated by the patient. Examples of exemplary time periods associated with a course of therapy according to the claimed methods include about 1 week, 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 30 months, about 3 years, about 4 years, about 5 years, or permanently (e.g., continuing maintenance therapy). The aforementioned durations may relate to one or more rounds / cycles of treatment.

[0135] The efficacy of the therapeutic methods provided herein may be evaluated using any suitable means. In one embodiment, the clinical efficacy of the treatment is analyzed using cancer cell count reduction as an objective response criterion. Patients, e.g., humans, treated according to the methods disclosed herein preferably experience an improvement in at least one symptom of cancer. In some embodiments, one or more of the following may occur: the cancer cell count may be reduced, the recurrence of the cancer may be prevented or delayed, and one or more of the symptoms associated with the cancer may be alleviated to some extent. In addition, an in vitro assay to determine T cell-mediated target cell lysis. In some embodiments, tumor assessment is based on CT scans and / or MRI scans, see, e.g., RECIST 1.1 guidelines (Response Evaluation Criteria in Solid Tumors) (Eisenhauer et al., 2019 Eur J Cancer 45:228-247). Such assessments are generally performed every 4-8 weeks after treatment.

[0136] In some embodiments, tumor cells are not detectable following treatment as described herein. In some embodiments, the subject is in partial or complete remission. In certain embodiments, the subject has increased overall survival, median survival, and / or progression-free survival.

[0137] The therapeutic compound (i.e., 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, or a functional portion, derivative, and / or analog thereof) may be used in conjunction with other well-known therapies (e.g., chemotherapy or radiation therapy) that are selected for their particular usefulness against the cancer being treated.

[0138] Methods for safe and effective administration of chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in standard texts. For example, the administration of many chemotherapeutic agents is described in Physicians' Desk Reference (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, NJ07645-1742, USA), the disclosure of which is incorporated herein by reference.

[0139] It will be apparent to one skilled in the art that administration of chemotherapeutic agents and / or radiation therapy may vary depending on the disease being treated and the known effects of the chemotherapeutic agents and / or radiation therapy on that disease. Also, in accordance with the knowledge of the skilled artisan, treatment protocols (e.g., dosage and administration time) may be altered taking into account the observed effects of the administered therapeutic agent on the patient and the observed response of the disease to the administered therapeutic agent.

[0140] The compounds and compositions disclosed herein are useful as therapies and in therapeutic treatments, and therefore are useful as medicaments and can be used in methods of preparing medicaments.

[0141] All documents and references, including Genbank entries, patents, and published patent applications, and websites, mentioned herein are each expressly incorporated by reference in whole or in part to the same extent as if set forth herein.

[0142] Although for purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, it will be understood that the scope of the invention may include embodiments having all or any combination of the described features.

[0143] The present invention will now be described with reference to the following examples, which are illustrative only and are 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

[0144] As used herein, "MFXXXX", where X is independently a number 0-9, refers to a Fab comprising a variable domain, where the VH has the 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 of Figure 4b. The light chain in the examples has the sequence shown in Figure 4a. "MFXXXX VH" refers to the amino acid sequence of the VH identified by the four digits. The MF further comprises 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 regions of the heavy chains are different, and typically the CH3 regions are also different, with one of the heavy chains 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 with a KK / DE CH3 heterodimerization 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 designated by the MF number. For example, the bispecific antibody designated MF3755xMF5816 has a variable domain with the general sequence shown above, a VH with the sequence of MF3755, and a VH with the sequence of MF5816.

[0145] The amino acid and nucleic acid sequences of the various heavy chain variable regions (VH) are shown in Figure 3. Among other LGR5 and EGFR combinations shown in Figure 3, including the heavy chain variable regions MF3755 and MF5816 and a common light chain, the bispecific antibody EGFR / LGR5, MF3755 x MF5816, which includes modifications for enhanced ADCC from afucosylation, has been shown to be effective in WO2017 / 069628.

[0146] Generation of bispecific antibodies Bispecific antibodies were generated by transient co-transfection of two plasmids encoding IgG with different VH domains using a proprietary CH3 engineering technique that ensures efficient heterodimerization and bispecific antibody formation. A common light chain is also co-transfected in the same cell, either on the same plasmid or on a separate plasmid. In our applications (e.g. WO2013 / 157954 and WO2013 / 157953, incorporated herein by reference), methods and means for producing bispecific antibodies from a single cell are disclosed, thereby providing a 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 produce essentially 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 (numbering 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 negatively charged DE mutant heavy chains and positively charged KK mutant heavy chains preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE mutant heavy chains (DE-DE homodimers) or KK mutant heavy chains (KK-KK homodimers) hardly occurs due to the strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0147] The VH genes of the variable domains binding to LGR5 described above were cloned into a vector encoding a positively charged CH3 domain. The VH genes of the variable domains binding to EGFR, such as those disclosed in WO2015 / 130172 (incorporated herein by reference), were cloned into a vector encoding a negatively charged CH3 domain. 293F freestyle cells adapted to suspension growth were cultured in T125 flasks on a shaker plateau to a density of 3.0×10e6 cells / ml. Cells were seeded in each well of a 24 deep-well plate at a density of 0.3-0.5×10e6 viable cells / ml. Cells were transiently transfected with a mixture of two plasmids encoding different antibodies and cloned into a proprietary vector system. Seven days after transfection, cell supernatants were harvested and filtered through a 0.22 μM filter (Sartorius). Sterile supernatants were stored at 4°C until antibody purification.

[0148] IgG purification and quantification Purification was performed under sterile conditions on filter plates using Protein A affinity chromatography. First, the pH of the 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) for 2 hours at 25° C. at 600 rpm on a shaking platform. The beads were then collected by filtration. The beads were washed twice with PBS pH 7.4. Bound IgG was then eluted with 0.1 M citrate buffer at pH 3.0, and the eluate was immediately neutralized using Tris pH 8.0. Buffer exchange was performed by centrifugation using a multiscreen Ultracel 10 multiplate (Millipore). Samples were finally collected in PBS pH 7.4. IgG concentration was measured using an Octet. Protein samples were stored at 4° C.

[0149] To determine the amount of purified IgG, the concentration of the antibody was determined by means of Octet analysis using a Protein A biosensor (Forte-Bio, according to the supplier's recommendations) using total human IgG (Sigma Aldrich, catalogue no. I4506) as standard.

[0150] The following bispecific antibodies are suitable for use in this example and in the methods of the invention: MF3370xMF5790, MF3370x5803, MF3370x5805, MF3370x5808, MF3370x5809, MF3370x5814, MF3370x5816, MF3370x5817, MF3370x5818, MF3755xMF5790, MF3755x5803, MF3755x5805, MF3755x5808, MF3755x5809, MF3755x5814, MF3755x5816, MF3755x5817, MF3755x5818, 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 comprises two VHs designated by MF numbers capable of binding to EGFR and LGR5, respectively, and further comprises a KK / DE CH3 heterodimerization domain as shown in SEQ ID NO: 136 (FIG. 5d) and SEQ ID NO: 138 (FIG. 5e), respectively, a CH2 domain as shown in SEQ ID NO: 134 (FIG. 5c), and a CH1 domain as shown in SEQ ID NO: 131 (FIG. 5a), and an Fc tail with a common light chain as shown in SEQ ID NO: 121 (FIG. 4).

[0151] Example 1 Evaluation of anti-EGFR x anti-LGR5 candidates against various cancers: Mouse model selection Crown Biosciences Inc. has developed a collection of patient-derived xenograft (PDX) models derived from surgically resected human primary tumors (Crown Bioscience database, http: / / hubase.crownbio.com). The PDX models are clinically and molecularly annotated and faithfully represent the clinical epidemiology of the respective tumors. These models can be injected subcutaneously into the flank of immunodeficient mice. Different cancer models were tested for EGFR and LGR5 expression analyzed by RNA sequencing (RNAseq) (see Table 1). A series of esophageal and gastric cancer PDX models found to exhibit high EGFR and LGR5 expression levels were selected to test the efficacy of the MF3755 x MF5816 bispecific antibody. For esophageal cancer, four PDX models were selected and for gastric cancer, eight PDX models were selected. Detailed information of the PDX models, including cancer subtype, known driver mutations, EGFR / LGR5 expression, and time to randomization, are listed in (Table 1). [Table 1]

[0152] method Fresh tumor tissue for inoculation was harvested from mice bearing established primary human tumors. Tumor fragments (2-3 mm in diameter) were inoculated subcutaneously into the right upper dorsal flank of 6-8 week-old female BALB / cNude or NOD / SCID mice, depending on the model. When tumors were 100-150 mm 3 Mice were randomized when tumors reached a size of 100-200 μg / kg. A total of 6 mice per model were enrolled, 3 control mice and 3 MF3755×MF5816 bispecific antibody-treated mice. For 6 weeks, mice received 0.5 mg of bispecific antibody / week intraperitoneally in an injection volume of 200 μl (approximately 25 mg / kg / week), regardless of their body weight. Control mice were administered PBS (200 μL). After the 6-week treatment period, tumor growth was monitored for an additional 3 weeks. Mice were sacrificed before day 63 if they reached a humane point.

[0153] result: From the eight gastric PDX models tested, the treatment with the MF3755×MF5816 bispecific antibody dramatically reduced tumor growth in six models (FIG. 6a). From these eight models, three models (GA0429, GA6833, and GA6891) showed lower tumor volume at the end of the observation period than at the beginning of treatment, suggesting strong tumor inhibition of the bispecific antibody in gastric cancer. Model GA2434 showed a certain response. As for the esophageal PDX models, all four models tested responded to bispecific antibody treatment, with model ES2356 showing the strongest response (FIG. 6b). Results showing a statistically significant (p<0.0001) efficacy of treatment with the MF3755×MF5816 bispecific antibody of this example were obtained in model ES11065.

[0154] Example 2: Dose escalation and efficacy of anti-EGFR x anti-LGR5 antibodies for patients with EAC, GAC and GEJAC: Phase 1 dose escalation study in advanced solid tumors Study design A Phase 1 open-label, multicenter study was performed with an initial dose escalation portion to determine the recommended Phase 2 dose (RP2D) of the anti-EGFR x anti-LGR5 bispecific antibody for solid tumors in mCRC patients with a flat starting dose of 5 mg. Once the RP2D is established, the antibody will be further evaluated in the expansion portion of the study, including patients diagnosed with EAC, GAC, and GEJAC. 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.

[0155] Dose escalation In the dose escalation portion, patients with metastatic colorectal cancer (mCRC) adenocarcinoma previously treated in the metastatic setting with standard approved therapy including oxaliplatin, irinotecan and fluoropyrimidines (5-FU and / or capecitabine) with or without anti-angiogenesis and anti-EGFR for KRAS and NRAS wild-type RASwt were treated.

[0156] A PK model was generated based on available bispecific antibody serum concentration data from pilot studies and GLP cynomolgus monkey toxicity studies. Following allometric scaling, the model was used to predict antibody exposure in humans. The starting dose of the antibody is 5 mg (flat dose) IV every 2 weeks for 4-week cycles. Up to 11 dose levels will be investigated: 5, 20, 50, 90, 150, 225, 335, 500, 750, 1100, and 1500 mg (flat dose). The dose, 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.

[0157] Dose-limiting toxicity (DLT) Any of the following clinical toxicities and / or laboratory abnormalities occurring during the first cycle (28 days) and deemed by the investigator to be related to antibody treatment will be considered a DLT: ●Hematological toxicity: Grade 4 neutropenia (absolute neutrophil count [ANC] < 0.5 x 109 cells / L) for ≥ 7 days - Grade 3 or greater febrile neutropenia - Grade 4 thrombocytopenia - Grade 3 thrombocytopenia secondary to bleeding episodes - Other grade 4 hematologic toxicity Grade 3-4 non-hematological AEs and laboratory toxicities excluding: - Grade 3-4 infusion-related reaction - Grade 3 skin toxicity that resolves to grade 2 or less within 2 weeks with optimal treatment - Grade ≤1 or Grade 3 diarrhea, nausea, and / or vomiting that resolves to baseline within 3 days with optimal treatment Grade 3 electrolyte abnormality that resolves with optimal treatment within 48 hours Grade 3-4 liver abnormalities for 48 hours or less • Any liver function abnormality that meets the definition of Hy's law. Any drug-related toxicity lasting 15 days or more that prevents the next 2 doses.

[0158] Dose expansion In the expansion section, the bispecific antibody is administered at the RP2D for patients with EAC, GAC, or GEJAC. 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 preliminary evaluation of antitumor activity and biomarker evaluation. The malignancies treated are known to co-express both targets (i.e., LGR5 and EGFR) and may have a prior indication of sensitivity to EGFR inhibition.

[0159] Antibody treatment in patients with EAC, GAC, or GEJAC may be expanded to a maximum of 40 patients, with 10-20 patients for each indication, subject to signs of preliminary antitumor activity. Safety of RP2D will be continually evaluated during the expansion portion of the study by the Safety Monitoring Committee. If the incidence of DLT exceeds a predefined threshold of 33% for any cohort, enrollment for this cohort will be paused and a full safety, PK, and biomarker review will be performed by the SMC to determine whether it is safe to continue accrual in that cohort. At that point, the overall safety of the drug will also be interrogated.

[0160] Investigational Treatments and Regimens The anti-EGFR x anti-LGR5 bispecific antibody will be formulated as a clear liquid solution for IV infusion. IV infusions will be performed every 2 weeks using standard infusion procedures, with a starting dose of 5 mg (flat dose) and a recommended Phase 2 dose of 1500 mg (flat dose). Dose escalation was stopped after reaching the RP2D. Infusions should be administered over a minimum of 4 hours during Cycle 1. Subsequent infusions after Cycle 1 may be shortened to 2 hours at the investigator's discretion and in the absence of an IRR.

[0161] A cycle is considered to be 4 weeks. For each patient, an observation period of 6 hours after the start of infusion of the first antibody infusion, a 4 hour period for the second infusion, and a minimum of 2 hours for all subsequent administrations corresponding to at least the infusion duration was performed. The antibody was administered as a 2-4 hour IV infusion every 2 weeks in 4 week cycles. Day 1 of the subsequent cycle was day 29 or after recovery from any adverse effects related to the previous cycle.

[0162] Duration of treatment Study treatment will be administered until progressive disease (by RECIST 1.), unacceptable toxicity, withdrawal of consent, patient noncompliance, investigator decision (e.g., clinical deterioration), or antibody discontinuation for ≥6 consecutive weeks. Patients will be followed for safety for at least 30 days after the last antibody infusion and until resolution or stabilization of all associated toxicities, as well as disease progression and survival status for 12 months.

[0163] Efficacy evaluation Tumor assessments will be based on CT / MRI with contrast per RECIST 1.1 (Eisenhauer et al., 2009 Eur J Cancer 45:228-247) every 8 weeks after treatment initiation. Objective response must be confirmed at least 4 weeks after first observation. Bone scans will be performed as clinically indicated for patients with bone metastases at baseline or suspected study lesions. Circulating blood tumor markers, including carcinoembryonic antigen (CEA), will be assessed at screening and on day 1 of each cycle.

Claims

1. An antibody, or a functional portion, derivative, and / or analogue 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 treating cancer in a subject, said use comprising providing to the subject a flat dose of 1500 mg of the antibody, or a functional portion, derivative, and / or analogue thereof.

2. An antibody, or a functional part, derivative, and / or analogue 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 treating gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a subject.

3. An antibody, or a functional part, derivative, and / or analogue 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 treating gastric cancer, esophageal cancer, or gastroesophageal junction cancer in a Her2 negative subject.

4. 4. The antibody, or functional part, derivative and / or analogue thereof, for use according to claim 2 or 3, wherein said use comprises providing to said subject a flat dose of 1500 mg of said antibody, or functional part, derivative and / or analogue thereof.

5. 13. The antibody, or functional part, derivative and / or analogue thereof, for use according to any one of the preceding claims, wherein the antibody, or functional part, derivative and / or analogue thereof, is provided intravenously.

6. 5. The antibody or functional part, derivative and / or analogue thereof for use according to any one of the preceding claims, wherein the cancer has a mutation in one or more genes selected from TP53, MLH1, PIK3CA, CDKN2A, UGT1A, UGT1A8, BRAF, PTEN and KRAS, preferably has a mutation in one or more genes selected from TP53, MLH1, CDKN2A, UGT1A, UGT1A8, BRAF and PTEN.

7. 7. The antibody for use according to claim 6, or a functional part, derivative and / or analogue thereof, wherein the cancer has one or more mutations selected from TP53 R196T, TP53 R342T, TP53 R248Q, MLH1 V384D, PIK3CA H1047R, CDKN2A W110T, UGT1A1 G71R, UGT1A8 G71R, and KRAS G12C.

8. 10. The antibody, or functional part, derivative and / or analogue thereof, for use according to any one of the preceding claims, wherein administration of the antibody, or functional part, derivative and / or analogue thereof, is weekly, biweekly or monthly.

9. 10. The antibody, or functional part, derivative and / or analogue thereof, for use according to any one of the preceding claims, wherein administration of said antibody, or functional part, derivative and / or analogue thereof, is once every two weeks.

10. 7. The antibody, or a functional part, derivative and / or analogue thereof, for use according to claim 6, wherein the cancer has a mutation in the gene encoding TP53, preferably wherein the mutation is R196T.

11. 7. The antibody or a functional part, derivative and / or analogue thereof for use according to claim 6, wherein the cancer has a mutation in the gene encoding TP53, preferably the mutation is R342T, and the cancer has a mutation in the gene encoding MLH1, preferably the mutation is V384D.

12. 7. The antibody or a functional part, derivative and / or analogue thereof for use according to claim 6, wherein the cancer has a mutation in the gene encoding TP53, preferably wherein the mutation is R248Q, the cancer has a mutation in the gene encoding PIK3CA, preferably wherein the mutation is H1047R, the cancer has a mutation in the gene encoding CDKN2A, preferably wherein the mutation is W110T, the cancer has a mutation in the gene encoding UGT1A1, preferably wherein the mutation is G71R, the cancer has a mutation in the gene encoding UGT1A8, preferably wherein the mutation is G71R.

13. The antibody, or a functional part, derivative and / or analogue thereof, for use according to any one of claims 1 to 12, wherein said cancer is esophageal cancer, preferably esophageal squamous cell carcinoma (ESCC).

14. 7. The antibody, or a functional part, derivative and / or analogue thereof, for use according to claim 6, wherein the cancer is a gastric cancer having a mutation in a gene selected from UGT1A1, UGT1A8 and / or PIK3CA.

15. 7. The antibody or a functional part, derivative and / or analogue thereof for use according to claim 6, wherein the cancer has a mutation in the gene encoding KRAS, preferably the mutation is G12C, the cancer has a mutation in the gene encoding UGT1A1, preferably the mutation is G71R, and the cancer has a mutation in the gene encoding UGT1A8, preferably the mutation is G71R.

16. 3. The antibody or functional part, derivative and / or analogue thereof for use according to claim 2, wherein the cancer has a mutation in the gene encoding UGT1A1, preferably the mutation is G71R, and the cancer has a mutation in the gene encoding UGT1A8, preferably the mutation is G71R.

17. The antibody, or a functional part, derivative and / or analogue thereof, for use according to claim 10, wherein the cancer further has a mutation in PI3CA, preferably said mutation being E45K.

18. The antibody, or a functional part, derivative and / or analogue thereof, for use according to any one of claims 1 to 17, wherein the cancer is gastric cancer.

19. The VH chain of the variable domain that binds EGFR comprises the amino acid sequence of VH chain MF3755 shown in Figure 3 or the amino acid sequence of VH chain MF3755 shown in Figure 3 with 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 insertions, deletions, substitutions, or combinations thereof relative to said VH; and the VH chain of the variable domain that binds LGR5 comprises the amino acid sequence of VH chain MF3755 shown in Figure 3 3 or a functional part, derivative and / or analogue thereof, for use according to any one of the preceding claims, comprising the amino acid sequence of VH chain MF5816 as shown in Figure 3, or the amino acid sequence of VH chain MF5816 as shown in Figure 3 with 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 insertions, deletions, substitutions or combinations thereof relative to said VH.

20. 2. An antibody for use according to any one of the preceding claims, or a functional part, derivative and / or analogue thereof, wherein said variable domain which binds to LGR5 binds to an epitope located within amino acid residues 21 to 118 of the human LGR5 sequence shown in Figure 1.

21. 21. The antibody or functional part, derivative and / or analogue thereof for use according to claim 20, 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.

22. The antibody for use according to claim 20 or 21, or a functional part, derivative and / or analogue thereof, wherein the LGR5-binding variable domain binds less to an LGR5 protein containing one or more of the amino acid residue mutations selected from 43A, 44A, 46A, 67A, 90A and 91A.

23. 2. The antibody or functional part, derivative and / or analogue thereof for use according to any one of the preceding claims, wherein said variable domain which binds to EGFR binds to an epitope located within amino acid residues 420 to 480 of the human EGFR sequence shown in Figure 2.

24. 24. The antibody or functional part, derivative and / or analogue thereof for use according to claim 23, 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.

25. 25. The antibody for use according to claim 23 or 24, or a functional part, derivative and / or analogue thereof, wherein the EGFR-binding variable domain binds less to an EGFR protein comprising one or more of the amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A and C499A.

26. 10. An antibody, or a functional part, derivative and / or analogue thereof, for use according to any one of the preceding claims, wherein the antibody has enhanced ADCC.

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

28. A method of treating the stomach, esophagus, or gastroesophageal junction, comprising administering to a subject in need thereof 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.

29. 13. The antibody, or functional part, derivative and / or analogue thereof, for use according to any one of the preceding claims, wherein said treatment with said antibody, or functional part, derivative and / or analogue thereof, is carried out following a step of diagnosing said subject for Her2 status.

30. 30. The antibody, or functional part, derivative and / or analogue thereof, of claim 29, wherein the diagnosis is by ISH or IHC test for Her2 status.

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