Cancer treatment using a combination of antibodies that bind to LGR5 and EGFR and topoisomerase I inhibitors

Abstract

The present invention relates to means and methods for treating cancer. [Solution] The present invention describes an antibody or functional moiety, derivative and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, for use in the treatment of cancer, which is administered together with a topoisomerase I inhibitor.

Description

[Technical Field]

[0001] The present invention relates to means and methods relating to the treatment of cancer. In particular, the present invention relates to a method for treating a target cancer with a combination of antibodies that bind to LGR5 and EGFR and topoisomerase inhibitors. The present invention further relates to combinations for use in such a method, and combinations for use in manufacturing agents for the treatment of gastrointestinal cancer. [Background technology]

[0002] Colorectal cancer (CRC) is the third most common cancer in the world. While some new treatments for CRC have advanced, many have failed in clinical trials, and metastatic CRC remains largely incurable. The current standard treatment for advanced CRC in clinics involves chemotherapy regimens that disrupt the essential function of cancer cells and kill dividing cells.

[0003] Accumulated evidence suggests that cancer growth and regeneration after treatment-induced remission are caused by a population of cancer stem cells that evade chemotherapy. While not bound by theory, it is thought that the maintenance of these stem cells is regulated by the WNT signaling pathway.

[0004] While not constrained by theory, the EGFR (epidermal growth factor receptor) pathway is considered a second oncogenic pathway in CRCs that enhances cancer cell proliferation and apoptosis evasion. Several anti-EGFR drugs have demonstrated certain levels of efficacy as targeted therapies for metastatic CRCs (mCRCs). However, due to the heterogeneity of CRCs, oncogenic mutations in downstream KRAS genes lead to resistance to anti-EGFR therapy (approximately 40% of all mCRC patients), half of patients with wild-type KRAS have innate resistance to anti-EGFR therapy, and the majority of patients with cancers sensitive to anti-EGFR therapy later develop resistant cancer.

[0005] Merus' Biclonics® antibody program developed multispecific antibodies targeting EGFR and LGR5 (leucine-rich repeat-containing G protein-coupled receptors), which are stem cell markers in the WNT signaling pathway. The efficacy of these multispecific antibodies was evaluated in vitro and in vivo using patient-derived CRC organoid models and mouse PDX models. Multispecific antibodies targeting EGFR and LGR5 were shown to inhibit tumor growth. The efficacy of these inhibitory antibodies was shown to correlate with LGR5 RNA expression levels in cancer-derived cells.

[0006] This invention demonstrates that combination therapy, including the administration of multispecific antibodies targeting EGFR and LGR5 in combination with a topoisomerase I inhibitor, is remarkably effective when comparing the effects of the multispecific antibodies or topoisomerase antibodies separately. Such combination therapies can inhibit post-remission tumor metastasis and / or tumor regeneration induced by treatment in CRC patients, enabling longer-term remission. [Overview of the Initiative]

[0007] The present invention provides an antibody or functional moiety, derivative and / or analog thereof containing 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, wherein such antibody or functional moiety, derivative and / or analog is administered together with a topoisomerase I inhibitor. The cancer is preferably colorectal cancer, lung cancer, gastrointestinal cancer or ovarian cancer, and preferably colorectal cancer. The antibody or functional moiety, derivative and / or analog thereof and the topoisomerase I inhibitor are preferably administered simultaneously to the subject.

[0008] In one embodiment, an antibody or its functional portion, derivative, and / or analog is administered to the subject prior to a topoisomerase I inhibitor.

[0009] The variable domain that binds to the extracellular portion of EGFR may include the amino acid sequence of the VH chain MF3755 shown in Figure 8, or it may include the amino acid sequence of the VH chain MF3755 shown in Figure 8 having up to 15 amino acid modifications, preferably as many as 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, preferably as many as 5, 4, 3, 2, 1. These amino acid modifications include insertions, deletions, substitutions, or combinations thereof for the VH. The variable domain that binds to the extracellular portion of LGR5 may include the amino acid sequence of the VH chain MF5816 shown in Figure 8, or it may include the amino acid sequence of the VH chain MF5816 shown in Figure 8 having up to 15 amino acid modifications, preferably as many as 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, preferably as many as 5, 4, 3, 2, 1. This amino acid modification includes insertion, deletion, substitution, or a combination thereof for the VH. The antibody preferably contains both of the variable domains.

[0010] The LGR5-binding variable domain preferably binds to an epitope located within amino acid residues 21-118 of the human LGR5 sequence shown in Figure 1. In one embodiment, amino acid residues at positions 43, 44, 46, 67, 90, and 91 of human LGR5 are involved in the binding of the provided LGR5-binding variable domain to LGR5. The LGR5-binding variable domain preferably binds in small amounts to an LGR5 protein containing one or more amino acid residue mutations selected from 43A, 44A, 46A, 67A, 90A, and 91A.

[0011] The EGFR-binding variable domain preferably binds to an epitope located within amino acid residues 420-480 of the human EGFR sequence shown in Figure 2. In one embodiment, the amino acid residues at positions I462, G465, K489, I491, N493, and C499 of human EGFR are involved in the EGFR-binding variable domain provided to EGFR. The EGFR-binding variable domain preferably binds in small amounts to an EGFR protein containing one or more amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A, and C499A.

[0012] The antibodies or functional moieties, derivatives, and / or analogs provided herein preferably comprise both an LGR5-binding variable domain having epitope-binding properties as described above and an EGFR-binding variable domain having the epitope-binding properties described above.

[0013] In one embodiment, the topoisomerase I inhibitor is camptothecin or a derivative thereof. In another preferred embodiment, the topoisomerase I inhibitor is irinotecan or topotecan.

[0014] The antibody is preferably an ADCC-inducing antibody. In one embodiment, the antibody is an ADCC-enhancing antibody. In one embodiment, the antibody is afucosylated.

[0015] The present invention also provides a method for inhibiting the proliferation of cells expressing EGFR and LGR5 in a system that allows cell proliferation. This method includes providing a topoisomerase I inhibitor and a system having an antibody or a functional moiety, derivative and / or analog thereof comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5.

[0016] A method for treating cancer in a subject is further provided, which includes simultaneously or sequentially administering to the subject in need thereof a topoisomerase I inhibitor, and an antibody or a functional portion, derivative and / or analog thereof that includes a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5.

[0017] The cancer is preferably colorectal cancer, lung cancer, gastrointestinal cancer or ovarian cancer. In a preferred embodiment, the cancer is colorectal cancer.

[0018] The present invention also provides a pharmaceutical composition comprising an antibody or a functional portion, derivative and / or analog thereof that includes a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, and a topoisomerase I inhibitor. The antibody or a functional portion, derivative and / or analog thereof, and the topoisomerase I inhibitor can be provided as single agents. The antibody or a functional portion, derivative and / or analog thereof, and the topoisomerase I inhibitor can also be provided in separate formulations. When provided in separate formulations, both agents can be administered simultaneously or sequentially.

[0019] There is further provided a kit comprising an antibody or a functional portion, derivative and / or analog thereof that includes a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5; a topoisomerase I inhibitor, and instructions for using the antibody and the topoisomerase I inhibitor in the treatment described herein.

[0020] There is further provided an antibody or a functional portion, derivative and / or analog thereof that includes 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 gastrointestinal cancer in a subject. The antibody is administered simultaneously, separately or sequentially with a topoisomerase I inhibitor.

[0021] In one aspect, the present invention provides an antibody or a functional portion, derivative and / or analog thereof that includes 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 manufacture of a medicament for the treatment of cancer in a subject, wherein the antibody is administered with, simultaneously with, separately from or sequentially with a topoisomerase I inhibitor. The treatment is preferably for colorectal cancer, lung cancer, gastrointestinal cancer or ovarian cancer, and preferably for colorectal cancer.

[0022] In the treatment of gastrointestinal cancer in a subject, there is also provided herein a product comprising an antibody or a functional portion, derivative and / or analog thereof that includes a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, and a topoisomerase I inhibitor, as a combined formulation for simultaneous, separate or sequential use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] To facilitate a better understanding of the present specification, certain terms are first defined. Additional definitions are provided throughout the detailed description. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, and conventional methods in immunology, protein chemistry, biochemistry, recombinant DNA technology and pharmacology are employed.

[0024] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. The use of the term "including", as well as other forms such as "include", "includes", and "included", is not limiting.

[0025] As used herein, the term “antibody” means a protein molecule belonging to the immunoglobulin class of proteins containing one or more domains that bind to an epitope on an antigen, such domains originating from the variable region of the antibody or sharing sequence homology. Antibodies are typically composed of basic structural units, each having two heavy chains and two light chains. Antibodies for therapeutic purposes are preferably as close as possible to the natural antibody of the target being treated (e.g., a human antibody in the case of a human target). Antibodies according to the present invention are not limited to any particular format or method of preparation thereof.

[0026] A “bispecific antibody” is an antibody described herein in which one domain of the antibody binds to a first antigen while a second domain of the antibody binds to a second antigen, and the first and second antigens are not identical. 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 epitope on an antigen and a second VH / VL combination binds to a second epitope. This term further includes antibodies in which VH can specifically recognize the first antigen, and VL, paired with VH in the immunoglobulin variable region, can specifically recognize the second antigen. The resulting VH / VL pair binds to either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in International Publication Nos. 2008 / 027236, 2010 / 108127, and Schaefer et al (Cancer Cell 20, 472-486, October 2011). The bispecific antibodies according to the present invention are not limited to any particular bispecific format or method for generating it.

[0027] As used herein, the term “common light chain” refers to two light chains (or their VL portions) in a bispecific antibody. The binding specificity of the full-length antibody is unaffected, but the two light chains (or their VL portions) may be identical or have some differences in amino acid sequence. The terms “common light chain,” “common VL,” “single light chain,” and “single VL” are all used interchangeably herein, with or without the addition of the term “reconstituted.” “Common” refers to a functional equivalent of a light chain whose amino acid sequence is not identical. Many variants of the light chain exist, including mutations (deletions, substitutions, insertions, and / or additions) that do not affect the formation of the functional binding region. The light chain of the present invention may also be a light chain specified herein, having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof. For example, preparing or finding a light chain that is not identical but still functionally equivalent by introducing and testing conservative amino acid changes, or amino acid changes in regions that do not contribute or only partially contribute to binding specificity when paired with the heavy chain, is within the scope of the definition of a common light chain as used herein. The terms "full-length IgG" or "full-length antibody" according to the present invention are defined as containing essentially complete IgG, but not necessarily possessing all the functions of complete IgG. To avoid misunderstanding, full-length IgG contains two heavy chains and two light chains. Each chain contains a constant (C) region and a variable (V) region, which can be broken down into domains denoted as CH1, CH2, CH3, VH, and CL, VL. IgG antibodies bind to antigens via the variable region domain contained in the Fab portion, and after binding, can interact with molecules and cells of the immune system via the constant domain, mainly the Fc portion. Full-length antibodies according to the present invention contain IgG molecules in which mutations may exist to provide desired properties. Full-length IgG must not have any substantial deletions of any part of the region. However, IgG molecules with one or more amino acid residues deleted without essentially altering the binding properties of the resulting IgG molecule are included in the term "full-length IgG".For example, such an IgG molecule may have a deletion of 1 to 10 amino acid residues, preferably in the non-CDR region, and the deleted amino acids are not essential for the antigen-binding specificity of IgG.

[0028] In this specification, when referring to nucleic acids or amino acid sequences, "percent (%) identity" is defined as the percentage of residues in a candidate sequence that are identical to residues in a selected sequence after the sequences have been aligned for optimal comparison purposes. Percent sequence identity for comparing nucleic acid sequences is determined using the AlignX application of Vector NTI Advance® 11.5.2 software with default settings. These settings use a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, (1994) Nuc. Acid Res. 22(22):4673~4680), a swgapdnamt score matrix, a gap-open penalty of 15, and a gap-extension penalty of 6.66. Amino acid sequences are aligned by the AlignX application of Vector NTI Advance® 11.5.2 software with default settings. This configuration uses a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, (1994) Nuc. Acid Res. 22(22):4673~4680), a bloom62mt2 score matrix, a gap-open penalty of 10, and a gap-extension penalty of 0.1.

[0029] Antibodies typically recognize an epitope of an antigen, and since such epitopes may also be present in other compounds, they "specifically recognize" an antigen, such as EGFR or LGR5. Antibodies according to the present invention may similarly recognize other compounds if those compounds contain the same type of epitope. Therefore, the term "specifically recognizes" with respect to antigen-antibody interactions does not exclude the binding of antibodies to other compounds containing the same type of epitope.

[0030] The terms “epitope” or “antigenic determinant” refer to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from both continuous and discontinuous amino acids juxtaposed by the tertiary folding of proteins (so-called linear and conformational epitopes). Epitopes formed from continuous linear amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding typically lose their conformation upon treatment with denaturing solvents. Epitopes may typically contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in their unique spatial structure. Methods for determining the spatial conformation of an epitope are known to those skilled in the art, and include, depending on the properties of the epitope, X-ray crystallography, HDX-MS and two-dimensional nuclear magnetic resonance, PEP scan, and alanine scan (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)).

[0031] As used herein, the terms “subject” and “patient” are used synonymously and refer to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cattle, horses, and pigs (for example, human patients, including those suffering from cancer).

[0032] As used herein, the terms “treat,” “treating,” and “treatment” refer to any type of intervention or procedure performed, or administration of an activator or combination of activators, aimed at reversing, alleviating, restoring, inhibiting, slowing, or preventing the progression, development, severity, or relapse of any disease-related symptoms, complications, conditions, or biochemical signs.

[0033] As used herein, “effective treatment” or “positive treatment response” refers to a beneficial effect, such as a treatment that results in the recovery of at least one symptom of a disease or disorder, such as cancer. Beneficial effects can result in an improved state that exceeds a standard, such as an improvement beyond the measurement or observation made before the commencement of therapy by this method. Beneficial effects can result in a state that slows, stabilizes, stops, or reverses the progression of cancer in a subject at any clinical stage, for example, as demonstrated by a reduction or elimination of clinical or diagnostic symptoms of the disease, or a reduction or elimination of cancer markers. Effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, slow or inhibit tumor growth, and / or prevent or delay tumor recurrence or relapse.

[0034] The term “effective dose” or “therapeutic effective dose” refers to the amount of an agent or combination of agents that provides a desired biological, therapeutic, and / or prophylactic outcome. The outcome may be a reduction, recovery, remission, decrease, delay, and / or mitigation of one or more signs, symptoms, or causes of a disease, or other desired changes in the ecosystem. In some embodiments, the effective dose is sufficient to delay tumor development. In some embodiments, the effective dose is sufficient to prevent or delay tumor recurrence. The effective dose may be administered in one or more doses. An effective dose of the drug or composition may (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, block, slow, or halt, to some extent, cancer cell infiltration into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor development and / or recurrence; and / or (vii) alleviate, to some extent, one or more symptoms associated with cancer. In one example, the "effective dose" is the amount of a combination of an EGFR / LGR5 antibody and a topoisomerase I inhibitor that results in a reduction of cancer (e.g., a reduction in the number of cancer cells), slows the progression of cancer, or prevents cancer regeneration or recurrence, wherein the cancer is gastrointestinal cancer, preferably colorectal cancer.

[0035] The present invention further provides a method for inhibiting the growth of cells that express EGFR and LGR5 in a system that allows cell growth. The method comprises providing a system having the antibody and topoisomerase I inhibitor described herein. The system is preferably a culture system. The method preferably comprises culturing the cells in the system. Compared to the cell number or tumor volume / tumor size that would occur under the same conditions except in the absence of the antibody and / or topoisomerase I inhibitor of the present invention, the inhibition is preferably a reduction of at least 10% in cell number, tumor volume or tumor size. The inhibition is preferably a reduction of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% in cell number, tumor volume or tumor size and / or an increase in progression-free survival. Compared to the number of lumens occurring under the same conditions except in the absence of the antibody and / or topoisomerase I inhibitor of the present invention, inhibition may result in at least a 10% reduction in other parameters associated with tumor malignancy or malformation, such as the number of lumens per organoid. Preferably, inhibition is a reduction of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% in the number of lumens per organoid and / or an increase in progression-free survival.

[0036] To avoid misunderstanding, as used herein, reference to cell growth refers to a change in cell number. Inhibition of growth refers to a decrease in cell number that would have been obtained under the same conditions except in the absence of the antibody and / or topoisomerase I inhibitor of the present invention. Increased growth refers to an increase in cell number that would have otherwise been obtained. Cell growth typically refers to cell proliferation. This decrease is compared to the growth / proliferation of the same cells under the same conditions in the absence of the antibody and / or topoisomerase I inhibitor of the present invention.

[0037] The present invention also provides a method for treating individuals who have gastrointestinal cancer or are at risk of having such cancer. The method comprises administering the antibody of the present invention to an individual in need. The individual is preferably an individual with cancer. The cancer is preferably gastrointestinal cancer. In a preferred embodiment, the cancer is colorectal cancer.

[0038] The present invention also provides a method for preventing metastasis or tumor recurrence in individuals who have cancer, preferably gastrointestinal cancer, or who are at risk of having such cancer. The method comprises administering an antibody or functional portion thereof, derivatives and / or analogs, and a topoisomerase I inhibitor, to an individual in need, which include a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5. The individual is preferably an individual who has cancer, or, in the case of tumor recurrence, has a radiographic diagnosis of recurrence, or shows signs and symptoms of cancer recurrence after a period of improvement or response. The cancer is preferably gastrointestinal cancer. In a preferred embodiment, the cancer is colorectal cancer.

[0039] In a preferred embodiment, the prevention of metastasis involves preventing metastasis from gastrointestinal cancer to non-gastrointestinal cancers, such as metastasis to lung or liver tissue.

[0040] An effective dose of combination therapy is administered in accordance with the method described herein, in the form of an "effective regimen" referring to a combination of an EGFR / LGR5 antibody and a topoisomerase I inhibitor. In this case, the order of administration, the amount administered, and the number of doses are sufficient for effective treatment.

[0041] As described above, cancer types such as CRC may be associated with the presence of oncogenic mutations, such as those in the genes encoding phosphatidylinositol-4,5-bisphosphate-3-kinase catalytic subunit α (PIK3CA) or Kirsten rat sarcoma (KRAS). Mutations in both PIK3CA and KRAS are widely associated with cancer types such as colorectal cancer. The prevalence of KRAS and PIK3C mutations in metastatic CRC is 20-50% and up to 14.3%, respectively, across different ethnic populations. On the other hand, the PIK3CA C420R mutation has been detected in at least nine different types of cancer, including breast cancer, colorectal cancer, esophageal cancer, low-grade cerebral glioma, pulmonary squamous cell carcinoma, endometrial tumors, prostate cancer, gastric adenocarcinoma, and ovarian tumors (Prevalence of KRAS, BRAF, PI3K and EGFR mutations among Asian patients with metastatic colorectal cancer, Phua et al., Oncology Letters, 10:2519-2526 2014; the AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017;7(8):818-831.Dataset Version 4; https: / / www.cancer.gov / research / key-initiatives / ras / ras-central / blog / 2017 / pik3ca.pdf). By July 2019, the Catalogue of Somatic Mutations In Cancer (COSMIC), managed by the Sanger Institute (UK), had characterized 18 different types of tissue carrying the mutation PIK3C C420R (https: / / cancer.sanger.ac.uk / cosmic, mutation ID COSM757).In the mutation in PIK3CA C420R, cysteine ​​is replaced by arginine at position 420 of the protein's amino acid residues, while in KRAS G12D, the glycine residue at position 12(G) is mutated to aspartic acid (D) of KRAS.

[0042] One of the advantages of the present invention is that subjects with KRAS mutations and / or PIK3CA mutations who received combination therapy with antibodies binding to LGR5 and EGFR and a topoisomerase I inhibitor did not show significant weight loss throughout the entire treatment period. In particular, subjects with KRAS G12D and / or PIK3CA C420R mutations did not show statistically significant weight loss.

[0043] Therefore, in a preferred embodiment, the present invention relates to a method for treating cancer in subjects having mutations in the gene encoding KRAS, preferably mutations causing G12D, and / or mutations in the gene encoding PIK3CA, preferably mutations causing C420R.

[0044] The cancers treated with the methods or products for use in the treatment described herein are preferably breast cancer, colorectal cancer, esophageal cancer, cerebral glioma, preferably low-grade cerebral glioma, pulmonary squamous cell carcinoma, endometrial tumor, prostate cancer, gastric adenocarcinoma, or ovarian tumor. The cancers are preferably colorectal cancer, lung cancer, gastrointestinal cancer, or ovarian cancer, and preferably colorectal cancer. The cancers preferably have mutations in the gene encoding KRAS, the gene encoding PIK3CA, or a combination thereof. The KRAS mutation is preferably a mutation that causes a G12D amino acid substitution. The PIK3CA mutation is preferably a mutation that causes a C420R amino acid substitution.

[0045] A further advantage of the present invention is that, in subjects with KRAS and / or PIK3CA mutations, specifically those with KRAS G12D and / or PIK3CA C420R mutations, no apparent signs of toxicity were observed with combination therapy of antibodies binding to LGR5 and EGFR and topoisomerase I inhibitors.

[0046] As used herein, the terms “synergy,” “therapeutic synergy,” and “synergy” refer to the phenomenon in which treatment of a patient with a combination of therapeutic agents (e.g., an EGFR / LGR5 antibody combined with a topoisomerase I inhibitor) results in a therapeutically superior outcome than that achieved when each component of the combination is used alone (see, for example, THCorbet et al., 1982, Cancer Treatment Reports, 66, 1187). In this regard, therapeutically superior outcomes include one or more of the following: (a) a greater increase in the therapeutic response than either or both of the separate effects of each agent alone at the same dose as the combination; (b) a reduction in the dose of one or more agents in the combination without a decrease in therapeutic efficacy; (c) a reduction in the incidence of adverse events while achieving a therapeutic effect greater than or equal to that of each agent monotherapy at the same dose as the combination; (d) a reduction in dose-limiting toxicity while achieving a greater therapeutic effect than that of each agent monotherapy; and (e) a delay or minimization of the induction of drug resistance. In xenograft models, a combination of drugs used at their maximum tolerated dose, where each component generally does not exceed its individual maximum tolerated dose, demonstrates a therapeutic synergy if the reduction in tumor growth achieved by the administration of that combination is greater than the reduction in tumor growth achieved by the best component administered alone. The synergistic effect of drug combinations can be determined, for example, according to the Chou-Talalay combination index (CI) theorem (Chou et al., Adv. Enzyme Regul. 1984;22:27-55; Chou, Cancer Res. 2010;70(2):440-446).

[0047] "Relapse," "recurrence," or "resurgence" are used interchangeably herein and refer to the radiographic diagnosis of relapse, or to signs and symptoms of cancer recurrence after a period of improvement or response.

[0048] Topoisomerase inhibitors are compounds that block the action of topoisomerases (topoisomerase I and topoisomerase II). Topoisomerases are a type of enzyme that regulates changes in DNA structure during the normal cell cycle by catalyzing and rejoining the phosphodiester backbone of DNA strands.

[0049] Human topoisomerase is a target for cancer chemotherapy. While not theoretically bound, topoisomerase inhibitors are thought to induce single- and double-strand breaks in the cell genome, affecting the stability of the cell's genome. The introduction of such breaks can lead to apoptosis and cell death.

[0050] In the present invention, human topoisomerase inhibitors are preferably inhibitors of human topoisomerase I. A non-limiting example of such topoisomerase inhibitors is camptothecin (CPT). CPT has long been known to have anti-cancer properties. CPT has relatively low solubility. CPT derivatives possess better activity. CPT derivatives / analogs are approved and are used today in cancer chemotherapy. Examples of preferred topoisomerase I inhibitors in humans are camptothecin, topotecan, lamellarin D, and irinotecan. In one embodiment, as used herein, "topoisomerase I inhibitor" includes, but is not limited to, topotecan, irinotecan, gimatecan, camptothecin and its analogs, 9-nitrocamptothecin, and the high molecular weight camptothecin conjugate PNU-166148 (compound A1 in International Publication No. 99 / 17804).

[0051] Irinotecan (CPT-11) is a semi-synthetic derivative of camptothecin and is a topoisomerase I inhibitor active against various solid tumors, including colorectal cancer, lung cancer, gastric cancer, and ovarian cancer. Irinotecan is a prodrug and is hydrolyzed by hepatic carboxylesterases to produce the active metabolite SN-38. SN-38 is removed by glucuronidation dependent on the hepatic UDP-glucuronosyltransferase family 1 member A1 cluster (UGTA1) enzyme. Genotype UGT1A1 * 28 has been found to be associated with a decrease in SN-38 glucuronidation. Approximately 10% of Americans in North America have UGT1A1 * Two copies of 28 alleles (homozygous, UGT1A1) * 28 / * 28) Patients possessing UGT1A1 and undergoing irinotecan therapy after neutropenia (Dean L. Irinotecan Therapy and UGT1A1 Genotype. 2015 [Updated 2018 Apr 4]. In: Pratt V, McLeod H, Rubinstein W, et al., editors. Medical Genetics Summaries [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2012; available from https: / / www.ncbi.nlm.nih.gov / books / NBK294473 / ). The subjects are those with one or more UGT1A1 * The presence of 28 alleles can be screened. Preferably, subjects treated with irinotecan have one or more UGT1A1 alleles. * The subject does not possess 28 alleles, and more preferably, the subject is UGT1A1 * Homozygote is not present for all 28 alleles.

[0052] Irinotecan and other human topoisomerase inhibitors have been used in clinics for a very long time, and appropriate dosing information is readily available to those skilled in the art. For example, irinotecan is administered once a week, every other week, or every three weeks at doses of 70-350 mg / m². 2It can be administered. Other regimens are 120 - 150 mg / m on the first and eighth days every three weeks. 2 are provided. As yet another schedule, it includes 125 mg / m in four weeks 2 , followed by a two - week interval. 50 mg / m on the first to fifth days every three weeks 2 , and 20 mg / m on the first to fifth days of one, two, four, and five weeks 2 . The administration shown in this specification refers to the amount (mg) per unit body surface area (m 2 ) of the subject at the indicated time point.

[0053] The antibody or its functional part, derivative and / or analog described in this specification comprises a variable domain that binds to the extracellular part of the epidermal growth factor (EGF) receptor and a variable domain that binds to LGR5. EGFR is preferably human EGFR. LGR5 is preferably human LGR5. The antibody or its functional part, derivative and / or analog described in this specification comprises a variable domain that binds to the extracellular part of the human epidermal growth factor (EGF) receptor and a variable domain that binds to human LGR5.

[0054] The epidermal growth factor (EGF) receptor (EGFR, ErbB1, or HER1) is a member of a family of four receptor tyrosine kinases (RTKs), designated as Her-, or cErbB-1, cErbB-2, and cErbB-3. EGFR is known by various synonyms, the most common being EGFR. EGFR has an extracellular domain (ECD) consisting of four subdomains, two of which are involved in ligand binding, and two of which are involved in homodimerization and heterodimerization. EGFR integrates extracellular signals from various ligands to obtain diverse intracellular responses. The major signaling pathway activated by EGFR consists of the Ras mitogen-activated protein kinase (MAPK) mitotic signaling cascade. Activation of this pathway is initiated by the recruitment of Grb2 to tyrosine-phosphorylated EGFR. This leads to Ras activation via the Grb2-binding 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 more potent in the presence of ErbB-3 (HER3) co-expression. EGFR is involved in several human epithelial malignancies, particularly breast, bladder, non-small cell lung cancer, colon, ovarian, head and neck, and brain cancers. Activating mutations in this gene, as well as overexpression of the receptor and its ligand, have been found, resulting in an autocrine activation loop. Therefore, this RTK is widely used as a target in cancer therapy. Both RTKs directed at the extracellular ligand-binding domain and small molecule inhibitors targeting monoclonal antibodies (mAbs) have been developed, which have shown some clinical success, although so far, mostly with a select group of patients. The database registry number for the human EGFR protein and the gene encoding it is GenBank NM_005228.3.This registration number is primarily given to provide further methods for targeting and identifying the EGFR protein, and the actual sequence of the antibody-bound EGFR protein may be altered due to mutations in the coding gene, such as those occurring in some cancers. Unless otherwise specified, the terms cancer and tumor are used herein, and typically both refer to cancer.

[0055] In this specification, when EGFR is referred to, it refers to human EGFR unless otherwise specified. The variable domain antigen-binding site that binds to EGFR binds to various variants, including those expressed in EGFR and some EGFR-positive tumors.

[0056] 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, with LGR4, LGR5, and LGR6 being the most well-known.

[0057] LGR5 is a leucine-rich repeat-containing G protein-coupled receptor 5. Alternative names for this gene or protein include 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 of the present invention may also bind to such orthologues due to sequence and tertiary structure similarities between human and other mammalian orthologues, but this is not necessarily required. 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). These registration numbers are primarily provided to offer further methods for targeting and identifying LGR5, and the actual sequence of the bound LGR5 protein may vary due to mutations in the encoding gene, such as those occurring in some cancers. The LGR5 antigen-binding site binds to LGR5 and various variants of it, such as those expressed by LGR5 and some LGR5-positive tumor cells.

[0058] In relation to the present invention, cells are said to express LGR5 if they contain detectable RNA encoding LGR5. Expression can often be detected by incubating cells with an antibody that binds to LGR5. However, some cells do not express sufficiently high levels of the protein for such LGR5 antibody testing. In such cases, mRNA or other forms of nucleic acid sequence detection are preferred.

[0059] Where accession numbers or alternative names for proteins / genes are given herein, these are primarily provided to offer further methods for identifying the target proteins, and the actual sequence of the target protein bound to the antibody of the present invention may vary due to mutations and / or alternative splicing in the coding gene, such as those that occur in some cancers. The target protein is bound by the antibody insofar as the epitope is present in the protein and the epitope is accessible to the antibody.

[0060] The antibodies or their functional moieties, derivatives, and / or analogs described herein preferably interfere with the binding of EGFR ligands to EGFR. As used herein, the term “interferes with binding” means that the binding of the antibody or its functional moiety, derivative, and / or analog to EGFR competes with the ligand for binding to the EGF receptor. The antibody or its functional moiety, derivative, and / or analog may reduce ligand binding, replace the ligand if it is already bound to the EGF receptor, or at least partially prevent the ligand from binding to the EGF receptor, for example, by steric hindrance.

[0061] The EGFR antibody of the present invention preferably inhibits EGFR ligand-induced signaling, measured as ligand-induced proliferation of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058), or ligand-induced cell death of A431 cells (ATCC CRL-1555), respectively. The EGFR antibody mentioned can reduce ligand-induced signaling by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, preferably 40%, 45%, 50%, 55%, 60%, more preferably 70%, 80%, 85%, most preferably 90%, 95%, 99%, or 100%, compared to the ligand-induced effect in the presence of a neutral substance or negative control as measured by assays known in the art. EGFR can bind to numerous ligands and stimulate the proliferation of the BxPC3 or BxPC3-luc2 cells mentioned. The presence of an EGFR ligand stimulates the growth of BxPC3 or BxPC3-luc2 cells. EGFR ligand-induced growth of BxPC3 cells can be measured by comparing cell growth in the presence and absence of the ligand. A preferred EGFR ligand for measuring EGFR ligand-induced growth of BxPC3 or BxPC3-luc2 cells is EGF. Ligand-induced growth is preferably measured using a saturated amount of ligand. In a preferred embodiment, EGF is used in a culture medium at a volume of 100 ng / mL. The EGF is preferably EGF from R&D Systems catalog numbers 396-HB and 236-EG (see also International Publication No. 2017 / 069628, which is incorporated herein by reference).

[0062] The EGFR antibody of the present invention preferably inhibits EGFR ligand-induced proliferation of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058). The EGFR antibody mentioned may reduce ligand-induced proliferation signaling by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, preferably 40%, 45%, 50%, 55%, 60%, more preferably 70%, 80%, 85%, most preferably 90%, 95%, 99%, or 100%, compared to ligand-induced proliferation induced by a neutral substance or negative control as measured by assays known in the art. EGFR can bind to numerous ligands and stimulate the growth of the BxPC3 or BxPC3-luc2 cells mentioned. The growth of BxPC3 or BxPC3-luc2 cells is stimulated in the presence of ligands. EGFR ligand-induced proliferation of BxPC3 cells can be measured by comparing cell growth in the presence and absence of the ligand. A preferred EGFR ligand for measuring EGFR ligand-induced proliferation of BxPC3 or BxPC3-luc2 cells is EGF. Ligand-induced proliferation is preferably measured using a saturated amount of ligand. In a preferred embodiment, EGF is used in a culture medium at a volume of 100 ng / mL. The EGF is preferably R&D Systems' EGF (catalog numbers 396-HB and 236-EG) (see also International Publication 2017 / 069628, which is incorporated herein by reference).

[0063] Whether the antibody of the present invention inhibits signal transduction or amplification in a multispecific format is preferably determined by the method described herein using a monospecific monovalent or monospecific bivalent version of the antibody. Such antibodies preferably have a receptor binding site that determines the signal transduction. Monospecific monovalent antibodies may have a variable domain with irrelevant binding specificity, such as tetanus toxoid specificity. A preferred antibody is a bivalent monospecific antibody, in which the antigen-binding variable domain consists of a variable domain that binds to a member of the EGF receptor family.

[0064] The antibodies or functional portions thereof, derivatives, and / or analogs described herein include a variable domain that binds to the extracellular portion of LGR5.

[0065] 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 shown in Figure 1, in which amino acid residues D43, G44, M46, F67, R90, and F91 are involved in antibody binding to the epitope.

[0066] The LGR5 variable domain is preferably a variable domain in which one or more amino acid residue substitutions in LGR5, such as D43A, G44A, M46A, F67A, R90A, and F91A, reduce the binding of the variable domain to LGR5.

[0067] The epitopes on the extracellular portion of LGR5 are preferably located within amino acid residues 21-118 of the sequence shown in Figure 1. Preferably, these are epitopes in which one or more of the following amino acid residue substitutions in LGR5, D43A, G44A, M46A, F67A, R90A, and F91A, reduce the binding of the LGR5 variable domain to LGR5.

[0068] The present invention 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. The LGR5 variable domain binds to an epitope on LGR5 located within amino acid residues 21-118 of the sequence shown in Figure 1.

[0069] The epitope on LGR5 is preferably a conformational epitope. This epitope is preferably located within amino acid residues 40-95 of the sequence shown in Figure 1. Antibody binding to LGR5 is preferably reduced by using one or more of the following amino acid residue substitutions: D43A, G44A, M46A, F67A, R90A, and F91A.

[0070] While not bound by theory, M46, F67, R90, and F91 of LGR5 shown in Figure 1 are considered to be contact residues of the variable domain as shown herein, i.e., antigen-binding sites of the variable domain that bind to the LGR5 epitope. Amino acid residue substitutions D43A and G44A reduce antibody binding, which may be due to the fact that these substitutions are also contact residues. However, these amino acid residue substitutions may also induce (slight) conformational modifications of the portion of LGR5 having one or more other contact residues (i.e., at positions 46, 67, 90, or 91), and it is also possible that the conformational changes are large enough to reduce antibody binding. Epitopes are characterized by the amino acid substitutions mentioned. Whether an antibody binds to the same epitope can be determined in various ways. A preferred method is described in the examples. This method utilizes CHO cells. CHO cells express LGR5 on the cell membrane, or preferably on alanine substitution mutants containing one or more of the substitutions M46A, F67A, R90A, or F91A. Test antibodies are brought into contact with CHO cells, and the binding of the antibody to the cells is compared. The test antibody binds to LGR5, and if the degree of binding to LGR5 with the M46A, F67A, R90A, or F91A substitution is low, it binds to the epitope. It is preferable to compare binding using a panel of mutants, each containing one alanine residue substitution. Studies on these bindings are well-known in the art. In many cases, the panel contains single alanine substitution mutants that essentially cover all amino acid residues. For LGR5, the panel only needs to cover the extracellular portion of the protein and, naturally, the portion that ensures association with the cell membrane when cells are used. Expression of specific mutants may be impaired, which can be easily detected by one or more LGR5 antibodies that bind to one or more different regions. If the expression of these control antibodies is also reduced, then the protein level or its folding on the membrane is impaired for this particular mutant.The binding characteristics of the test antibody to the panel readily identify whether the test antibody exhibits reduced binding to variants having M46A, F67A, R90A, or F91A substitutions, and therefore whether the test antibody is the antibody of the present invention. Reduced binding to variants having M46A, F67A, R90A, or F91A substitutions also identifies an epitope for positioning within amino acid residues 21-118 of the sequence in Figure 1. In preferred embodiments, the panel includes both D43A and G44A substitution variants. Antibodies having the VH sequence of MF5816 exhibit reduced binding to these substitution variants.

[0071] While not bound by any particular theory, as shown in Figure 2, amino acid residues I462;G465;K489;I491;N493; and C499 are thought to be involved in epitope binding by antibodies containing the variable domain as indicated herein. Participation in binding is preferably determined by observing a reduction in the binding of the variable domain to EGFR having one or more amino acid residue substitutions selected from I462A;G465A;K489A;I491A;N493A; and C499A.

[0072] In one embodiment, the variable domain that binds to an epitope on the extracellular portion of human EGFR is a variable domain that binds to an epitope located within amino acid residues 420-480 of the sequence shown in Figure 2. Preferably, the binding of the variable domain to EGFR is reduced by one or more of the following amino acid residue substitutions in EGFR: I462A; G465A; K489A; I491A; N493A; and C499A. The binding of the antibody to human EGFR preferably interferes with the binding of EGF to the 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 Figure 2, preferably 430-480 of the sequence shown in Figure 2; preferably 438-469 of the sequence shown in Figure 2.

[0073] While not bound by theory, it is thought that the epitope contact residues, i.e., the sites where the variable domain contacts human EGFR, may be I462;K489;I491; and N493. Amino acid residues G465 and C499 are involved almost indirectly in antibody binding to EGFR. This is probably because mutations involving alanine substitution induce (slight) conformational changes in the epitope, leading to a reduction in epitope binding.

[0074] The variable domain that binds to human EGFR is preferably a variable domain having a heavy chain variable region that includes at least the CDR3 sequence of MF3755 VH shown in Figure 8, or a CDR3 sequence that differs from the CDR3 sequence of MF3755 VH shown in Figure 8 by a maximum of 3, preferably a maximum of 2, preferably 1 amino acid.

[0075] The variable domain that binds to human EGFR is preferably a variable domain having a heavy chain variable region containing the CDR1, CDR2, and CDR3 sequences of the VH of MF3755 shown in Figure 8, which has at least three, preferably two, and preferably one amino acid substitutions.

[0076] The variable domain that binds to human EGFR is preferably the VH chain sequence of MF3755 shown in Figure 8; or a variable domain having a heavy chain variable region containing the amino acid sequence of the VH chain MF3755 shown in Figure 8, with a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5, amino acid insertions, deletions, substitutions, or combinations thereof, relative to the VH chain of MF3755.

[0077] In one embodiment, the present invention provides an antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5. The heavy chain variable region of the variable domain includes at least a CDR3 sequence of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370;MF3755;MF4280 or MF4289 shown in Figure 8, or the heavy chain variable region of the variable domain includes a VH CDR3 sequence selected from the group consisting of MF3370;MF3755;MF4280 or MF4289 shown in Figure 8, and a heavy chain CDR3 sequence that differs by up to three, preferably up to two, preferably one amino acid. The variable domain preferably includes a heavy chain variable region that includes at least a CDR3 sequence of MF3370;MF3755;MF4280 or MF4289.

[0078] The variable domain preferably includes a heavy chain variable region containing at least the CDR1, CDR2, and CDR3 sequences of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370;MF3755;MF4280 or MF4289 shown in Figure 8, or a heavy chain variable region containing at least the CDR1, CDR2, and CDR3 sequences of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370;MF3755;MF4280 or MF4289, with up to three, preferably up to two, and preferably up to one amino acid difference. The variable domain preferably includes a heavy chain variable region containing at least the CDR1, CDR2, and CDR3 sequences of MF3370;MF3755;MF4280 or MF4289 shown in Figure 8. A preferred heavy chain variable region is MF3755. Another preferred heavy chain variable region is MF4280.

[0079] The antibody comprises a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, wherein the EGFR-binding variable domain has CDR3, and the CDR1, CDR2, and CDR3 and / or VH sequences as indicated herein preferably have a variable domain that binds to LGR5, which includes at least a CDR3 sequence of the LGR5-specific heavy chain variable region selected from the group consisting of MF5790;MF5803;MF5805;MF5808;MF5809;MF5814;MF5816;MF5817; or MF5818 shown in Figure 8, or a heavy chain CDR3 sequence that differs from a CDR3 sequence of VH selected from the group consisting of MF5790;MF5803;MF5805;MF5808;MF5809;MF5814;MF5816;MF5817; or MF5818 shown in Figure 8 by up to three, preferably up to two, preferably one amino acid. The variable domain preferably includes a heavy chain variable region containing at least the CDR3 sequences of MF5790;MF5803;MF5805;MF5808;MF5809;MF5814;MF5816;MF5817; or MF5818 as shown in Figure 8.

[0080] The LGR5 variable domain preferably includes a heavy chain variable region containing at least CDR1, CDR2, and CDR3 sequences of the LGR5 specific heavy chain variable region selected from the group consisting of MF5790;MF5803;MF5805;MF5808;MF5809;MF5814;MF5816;MF5817; or MF5818 as shown in Figure 8, or heavy chain CDR1, CDR2, and CDR3 sequences that differ from the CDR1, CDR2, and CDR3 sequences of the LGR5 specific heavy chain variable region selected from the group consisting of MF5790;MF5803;MF5805;MF5808;MF5809;MF5814;MF5816;MF5817; or MF5818 as shown in Figure 8 by up to three, preferably up to two, and preferably up to one amino acid. The variable domain preferably includes a heavy chain variable region containing at least the CDR1, CDR2, and CDR3 sequences of MF5790;MF5803;MF5805;MF5808;MF5809;MF5814;MF5816;MF5817; or MF5818 as shown in Figure 8. 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, with MF5814, MF5818, and MF5816 being particularly preferred. Another preferred heavy chain variable region is MF5818.

[0081] Antibodies containing the heavy chain variable region MF3755, or one or more variable domains having one or more CDRs thereof, have been shown to have good efficacy when used to inhibit the growth of cancer or cells responsive to EGFR ligands. In relation to bispecific or multispecific antibodies, the arms of antibodies containing the heavy chain variable region MF3755, or variable domains having one or more CDRs thereof, bind well to arms containing the heavy chain variable region MF5818, or variable domains having one or more CDRs thereof.

[0082] The VH chain of the variable domain that binds to EGFR or LGR5 may have one or more amino acid substitutions relative to the sequence shown in Figure 8. The VH chain preferably has up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and preferably has the amino acid sequence of EGFR VH or LGR5 VH in Figure 8, having 1, 2, 3, 4, or 5 of its amino acid insertions, deletions, substitutions, or combinations thereof relative to the VH chain sequence in Figure 8.

[0083] The CDR sequence may have one or more amino acid residue substitutions relative to the CDR sequence shown in the figure. Such one or more substitutions are made, for example, for optimization purposes, preferably to improve the binding strength or stability of the antibody. Optimization is carried out, for example, preferably by a mutagenesis procedure after the stability and / or binding affinity of the resulting antibody has been tested, and after an improved EGFR-specific or LGR5-specific CDR sequence has been preferably selected. Those skilled in the art will be able to generate antibody variants containing at least one altered CDR sequence of the present invention. For example, conservative amino acid substitutions may be applied. Examples of conservative amino acid substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another hydrophobic residue, and the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.

[0084] Preferably, the 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 of VH or VL as specified herein are preferably conservative amino acid substitutions. The amino acid insertions, deletions, and substitutions of VH or VL as specified herein are preferably not present in the CDR3 region. The amino acid insertions, deletions, and substitutions mentioned are preferably also not present in the CDR1 and CDR2 regions. The amino acid insertions, deletions, and substitutions mentioned are preferably also not present in the FR4 region.

[0085] The up to 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid substitutions mentioned are preferably conservative amino acid substitutions, and 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 region of the VH chain, or preferably not present in the FR4 region.

[0086] Antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 are preferably, -The amino acid sequence of VH chain MF3755 shown in Figure 8; or -The amino acid sequence of the VH chain MF3755 shown in Figure 8, having a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof for the VH; The VH chain of the variable domain that binds to LGR5 is -The amino acid sequence of VH chain MF5790 shown in Figure 8; or -The VH chain contains the amino acid sequence of MF5790 shown in Figure 8, which has a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0087] Antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 are preferably, -The amino acid sequence of VH chain MF3755 shown in Figure 8; or -The amino acid sequence of the VH chain MF3755 shown in Figure 8, having a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof for the VH; The VH chain of the variable domain that binds to LGR5 is -The amino acid sequence of VH chain MF5803 shown in Figure 8; or -The VH contains the amino acid sequence of the VH chain MF5803 shown in Figure 8, which has a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0088] Antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 are preferably, -The amino acid sequence of VH chain MF3755 shown in Figure 8; or -The amino acid sequence of the VH chain MF3755 shown in Figure 8, having a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof for the VH; The VH chain of the variable domain that binds to LGR5 is -The amino acid sequence of VH chain MF5814 shown in Figure 8; or -The VH contains the amino acid sequence of the VH chain MF5814 shown in Figure 8, which has a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0089] Antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 are preferably, -The amino acid sequence of VH chain MF3755 shown in Figure 8; or -The amino acid sequence of the VH chain MF3755 shown in Figure 8, having a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof for the VH; The VH chain of the variable domain that binds to LGR5 is -The amino acid sequence of VH chain MF5816 shown in Figure 8; or -The VH contains the amino acid sequence of the VH chain MF5816 shown in Figure 8, which has a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0090] Antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 are preferably, -The amino acid sequence of VH chain MF3755 shown in Figure 8; or -The amino acid sequence of the VH chain MF3755 shown in Figure 8, having a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof for the VH; The VH chain of the variable domain that binds to LGR5 is -The amino acid sequence of VH chain MF5817 shown in Figure 8; or -The VH contains the amino acid sequence of the VH chain MF5817 shown in Figure 8, which has a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0091] Antibodies containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 are preferably, -The amino acid sequence of VH chain MF3755 shown in Figure 8; or -The amino acid sequence of the VH chain MF3755 shown in Figure 8, having a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof for the VH; The VH chain of the variable domain that binds to LGR5 is -The amino acid sequence of VH chain MF5818 shown in Figure 8; or -The VH contains the amino acid sequence of the VH chain MF5818 shown in Figure 8, which has a maximum of 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or combinations thereof.

[0092] Further variants of the disclosed amino acid sequences that retain EGFR or LGR5 binding can be obtained, for example, from a phage display library containing a collection of VH regions incorporating amino acid substitutions into the amino acid sequences of the EGFR or LGR5 VH region, as disclosed herein as above (e.g., International Publication No. 2017 / 069628). By selecting phages encoding Fab regions that bind to EGFR or LGR5, analyzing them by flow cytometry, and sequencing them, variants that retain antigen binding with amino acid substitutions, insertions, deletions, or additions can be identified.

[0093] The light chain variable regions of the VH / VL EGFR and LGR5 variable domains of the EGFR / LGR5 antibody may be the same or different.

[0094] 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.

[0095] In certain embodiments, the light chain variable region of one or both VH / VL variable domains of the EGFR / LGR5 antibody includes a common light chain variable region. In some embodiments, the common light chain variable region of one or both VH / VL variable domains includes the germline IgVκ1-39 variable region V segment. In certain embodiments, the light chain variable region of one or both VH / VL variable domains includes the kappa light chain V segment IgVκ1-39 * Includes 01. IgVκ1-39 is a shortened form of the immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin variable kappa 1-39, IGKV139;IGKV1-39;external IDs for this gene are HGNC:5740, Entrez Gene:28930;Ensembl:ENSG00000242371. Preferred amino acid sequences of the V region are provided in Figure 9. The V region can be combined with one of five J regions. Preferred J regions are jk1 and jk5, and the conjugated sequences are shown as IGKV1-39 / jk1 and IGKV1-39 / jk5. Alternative name is IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * It is 01 (nomenclature according to the IMGT database on imgt.org on the World Wide Web). In certain embodiments, one or both light chain variable regions of the VH / VL variable domains are kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 or IgVκk1-39 * 01 / IGJκ1 * Includes 05 (shown in Figure 9).

[0096] In some embodiments, the light chain variable region of one or both VH / VL variable domains of an EGFR / LGR5 bispecific antibody includes LCDR1 containing the amino acid sequence QSISSY (shown in Figure 9), LCDR2 containing the amino acid sequence AAS (shown in Figure 9), and LCDR3 containing the amino acid sequence QQSYSTP (shown in Figure 9) (i.e., the CDR of IGKV1-39 by IMGT). In some embodiments, the light chain variable region of one or both VH / VL variable domains of an EGFR / LGR5 antibody includes LCDR1 containing the amino acid sequence QSISSY (shown in Figure 9), LCDR2 containing the amino acid sequence AASLQS (shown in Figure 9), and LCDR3 containing the amino acid sequence QQSYSTP (shown in Figure 9).

[0097] In some embodiments, one or both of the VH / VL variable domains of the EGFR / LGR5 antibody include a light chain variable region containing an amino acid sequence that is at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or 100% identical to the amino acid sequence described in Figure 9.

[0098] For example, in some embodiments, the variable light chain of one or both of the VH / VL variable domains of the EGFR / LGR5 antibody may have 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof relative to the sequence in Figure 9. In some embodiments, the light chain variable region of one or both of the VH / VL variable domains of the EGFR / LGR5 antibody may include 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1, or preferably 0, amino acid insertions, deletions, substitutions, additions, or combinations thereof relative to the indicated amino acid sequence.

[0099] In other embodiments, the light chain variable region of one or both VH / VL variable domains of the EGFR / LGR5 antibody contains the amino acid sequence shown in Figure 9. In specific embodiments, both VH / VL variable domains of the EGFR / LGR5 antibody contain the same VL region. In one embodiment, both VLs of the VH / VL variable domain of the EGFR / LGR5 bispecific antibody contain the amino acid sequence shown in Figure 9. In one embodiment, both VLs of the VH / VL variable domain of the EGFR / LGR5 bispecific antibody contain the amino acid sequence shown in Figure 9.

[0100] The EGFR / LGR5 antibodies described herein preferably have two variable domains and are bispecific antibodies, one of which binds to EGFR and the other to LGR5, as described herein.

[0101] EGFR / LGR5 bispecific antibodies for use in the methods disclosed herein can be provided in a number of formats. Many different formats of bispecific antibodies are known in the art and have been outlined by Kontermann (Drug Discov Today, 2015 Jul;20(7):838-47;MAb, 2012 Mar-Apr;4(2):182~97) and Spiess et al. (Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol.Immunol.(2015) http: / / dx.doi.org / 10.1016 / j.molimm.2015.01.003), which are incorporated herein by reference, respectively. For example, a bispecific antibody format other than a conventional antibody having two VH / VL combinations has a variable domain including at least a heavy chain variable region and a light chain variable region. This variable domain may be ligated to a single-chain Fv fragment, a monobody, a VH, and a Fab fragment that provides a second binding activity.

[0102] In some embodiments, the EGFR / LGR5 bispecific antibody used in the methods provided herein is generally of a human IgG subclass (e.g., IgG1, IgG2, IgG3, IgG4). In certain embodiments, the antibody is of a human IgG1 subclass. Full-length IgG antibodies are preferred due to their advantageous half-life and low immunogenicity. Therefore, in certain embodiments, the EGFR / LGR5 bispecific antibody is a full-length IgG molecule. In one embodiment, the EGFR / LGR5 bispecific antibody is a full-length IgG1 molecule.

[0103] Therefore, in certain embodiments, the EGFR / LGR5 bispecific antibody comprises a crystallizable fragment (Fc). The Fc of the EGFR / LGR5 bispecific antibody is preferably composed of a human constant region. The constant region or Fc of the EGFR / LGR5 bispecific antibody may contain one or more, preferably as many as 10, and preferably as many as 5, amino acids that are different 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 containing modifications that promote the formation of the bispecific antibody, modifications that promote stability, and / or other features described herein.

[0104] Bispecific antibodies are typically produced by cells expressing nucleic acids encoding the antibody. Therefore, in some embodiments, the bispecific EGFR / LGR5 antibodies disclosed herein are produced by providing cells containing one or more nucleic acids encoding the heavy and light chain variable and constant regions of the bispecific EGFR / LGR5 antibody. The cells are preferably animal cells, more preferably mammalian cells, more preferably primate cells, and most preferably human cells. Suitable cells are any cells that can contain, and preferably produce, the EGFR / LGR5 bispecific antibody.

[0105] Cells suitable for antibody production are known in the art and include hybridoma cells, Chinese hamster ovary (CHO) cells, NS0 cells, or PER-C6 cells. Various institutions and companies have developed cell lines for large-scale antibody production, 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, cells transformed with the adenovirus E1 region or its functional equivalent. A preferred example of such a cell line is the PER.C6 cell line or its equivalent. In a particularly preferred embodiment, the cells are CHO cells or their variants. Preferably, variants utilizing a glutamine synthetase (GS) vector system for antibody expression. In one preferred embodiment, the cells are CHO cells.

[0106] In some embodiments, cells express different light and heavy chains constituting an EGFR / LGR5 bispecific antibody. In certain embodiments, cells express two different heavy chains and at least one light chain. In one preferred embodiment, cells express a “common light chain” as described herein to reduce the number of different antibody species (combinations of different heavy and light chains). For example, each VH region is cloned into an expression vector together with a reconstituted human IGKV139 / IGKJ1(huVκ139) light chain using methods known in the art for the production of bispecific IgG (International Publication No. 2013 / 157954; incorporated herein by reference). It has been previously shown that huVκ139 pairs with multiple heavy chains, thereby generating antibodies with diverse specificities and promoting the creation of bispecific molecules (De Kruif et al. J.Mol.Biol.2009(387)548 58; International Publication No. 2009 / 157771).

[0107] Antibody-producing cells expressing a common light chain and two equal amounts of heavy chains typically produce 50% bispecific antibodies and 25% monospecific antibodies each (i.e., identical heavy / light chain combinations). Several methods have been described that favor the production of bispecific antibodies over the production of each monospecific antibody. Such methods are typically achieved by modifying the constant regions of the heavy chains so that they favor heterodimerization (i.e., dimerization with the heavy chain of another heavy / light chain combination) over homodimerization. In a preferred embodiment, the bispecific antibodies of the present invention comprise two different immunoglobulin heavy chains having compatible heterodimerization domains. Various compatible heterodimerization domains have been described in the art. This compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain. Various methods have been described in the art in which such heterodimerization of heavy chains can be achieved.

[0108] One preferred method for producing EGFR / LGR5 bispecific antibodies is disclosed in U.S. Patents 9,248,181 and 9,358,286. Specifically, preferred mutations for producing essentially bispecific full-length IgG molecules are amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain ("KK-mutant" heavy chain) and amino acid substitutions L351D and L368E in the second domain ("DE-mutant" heavy chain), or vice versa. As described above, the DE-mutant and KK-mutant preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of the DE-mutant heavy chain (DEDE homodimer) or the KK-mutant heavy chain (KKKK homodimer) rarely occurs due to the strong repulsive force between charged residues at the CH3-CH3 interface between identical heavy chains.

[0109] Therefore, in one embodiment, the heavy / light chain combination containing a variable domain that binds to EGFR includes a DE variant of the heavy chain. In this embodiment, the heavy / light chain combination containing a variable domain that binds to LGR5 includes a KK variant of the heavy chain.

[0110] Candidate EGFR / LGR5 IgG bispecific antibodies can be tested for binding using any suitable assay. For example, binding to membrane-expressed EGFR or LGR5 on CHO cells can be evaluated by flow cytometry (following the FACS procedure previously described in International Publication No. 2017 / 069628). In one embodiment, binding of a candidate EGFR / LGR5 bispecific antibody to LGR5 on CHO cells is demonstrated by flow cytometry performed according to a standard procedure known in the art. Binding to CHO cells is compared to CHO cells that have not been transfected with an EGFR and / or LGR5 expression cassette. Binding of candidate bispecific IgG1 to EGFR is determined using CHO cells transfected with an EGFR expression construct. LGR5 monospecific antibodies and EGFR monospecific antibodies, as well as unrelated IgG1 isotype control mAbs, are included in the assay as controls (e.g., antibodies that bind to LGR5 and another antigen such as tetanus toxin (TT)).

[0111] The affinity of candidate EGFR / LGR5 bispecific antibodies for LGR5 and EGFR against a target can be measured by surface plasmon resonance (SPR) technology using the BIAcore T100. Briefly, an anti-human IgG mouse monoclonal antibody (Becton and Dickinson, cat. Nr. 555784) is bound to the surface of a CM5 sensor chip using free amine chemistry (NHS / EDC). The bsAb is then captured on the sensor surface. Subsequently, recombinant purified antigens of human EGFR (Sino Biological Inc, cat. Nr. 11896-H07H) and human LGR5 protein are flowed over the sensor surface in a concentration range for measuring the on / off rate. After each cycle, the sensor surface is regenerated by a pulse of HCl to recapture the bsAb. From the obtained sensorgram, the on / off rate and affinity values ​​for binding to human LGR5 and EGFR are measured using BIAevaluation software, as previously described for CD3 in U.S. Patent No. 2016 / 0368988.

[0112] The antibodies of the present invention are typically bispecific full-length antibodies, preferably human IgG subclasses. Preferably, the antibodies of the present invention are human IgG1 subclasses. Such antibodies of the present invention have good ADCC properties, which can be enhanced by techniques known in the art, if desired, and have a favorable half-life when administered in vivo to humans. CH3 gene manipulation techniques can provide modified heavy chains that preferentially form heterodimers over homodimers when co-expressed in clonal cells.

[0113] The ADCC activity of an antibody can be improved by slightly modifying the constant region of the antibody if the antibody itself has low ADCC activity. Another way to improve the ADCC activity of an antibody is by enzymatically interfering with the glycosylation pathway, which results in a decrease in fucose. Several in vitro methods exist to determine the effectiveness of antibodies or effector cells in inducing ADCC. These include, among others, 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 specific surface exposure antigen is incubated with an antibody specific to that antigen. After washing, effector cells expressing the Fc receptor CD16 are incubated together with the antibody-labeled target cells. Lysis of the target cells is later measured by the release of the intracellular label by scintillation counter or spectrophotometric measurement.

[0114] In one embodiment, the bispecific antibody of the present invention may be ADCC-enhancing. In one embodiment, the bispecific antibody of the present invention may be afucosylated. The bispecific antibody of the present invention preferably includes a reduction in the amount of fucosylation of the N-linked carbohydrate structure in the Fc region compared to the same antibody produced in normal CHO cells.

[0115] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 may further comprise one or more additional variable domains capable of binding to one or more further targets. The further targets are preferably proteins, preferably membrane proteins including extracellular portions. Antibodies having three or more variable domains are known in the art. For example, it is possible to conjugate additional variable domains to the constant portion of the antibody. Antibodies having three or more variable domains are preferably polyvalent multimer antibodies as described in PCT / NL2019 / 050199, which is incorporated herein by reference.

[0116] In one embodiment, the antibody is a bispecific antibody comprising two variable domains. One variable domain binds to the extracellular portion of EGFR, and the other variable domain binds to the extracellular portion of LGR5. The variable domains are preferably the variable domains described herein.

[0117] To avoid misunderstanding, as used herein, reference to cell growth refers to a change in the number of cells. Inhibition of growth refers to a decrease in the number of cells that would otherwise have been achieved. Increase in growth refers to an increase in the number of cells that would otherwise have been achieved. Cell growth typically refers to cell proliferation.

[0118] As used herein, a membrane protein is a protein present in the outer membrane of a cell, and is a cell membrane protein, which is the membrane that separates the cell from the outside world. A membrane protein has an extracellular component. A membrane protein is present on the cell if it contains a transmembrane region that is present in the cell membrane of the cell.

[0119] Pharmaceutical compositions comprising an EGFR / LGR5 bispecific antibody, a topoisomerase I inhibitor, and a pharmaceutically acceptable carrier are also provided. As used herein, the term “pharmaceutically acceptable” means any and all physiologically compatible solvents, salts, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, etc., that are approved by a government regulatory body or listed in the United States Pharmacopeia or other universally recognized pharmacopoeias for use in animals, particularly humans. The term “carrier” refers to a diluent, adjuvant, excipient, or solvent on which the compound is administered. Such pharmaceutical carriers may be sterile liquids such as water and oil, and may be of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol, and ricinoleic acid. Water or saline, as well as aqueous solutions of dextrose and glycerol, can be used as carriers, particularly for injectable solutions. Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravesical, intratumoral, intravenous, intraperitoneal, intramuscular, intrathecal, and subcutaneous. Depending on the route of administration (e.g., intravenous, subcutaneous, intra-articular, etc.), the active compound may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate the compound.

[0120] Pharmaceutical compositions suitable for administration to human patients are typically formulated, for example, for parenteral administration in a liquid carrier, or are suitable for reconstitution into liquid solutions or suspensions for intravenous administration. The compositions can be formulated in dose unit form to facilitate administration and ensure uniformity of the dosage.

[0121] This also includes solid preparations intended to be converted into liquid preparations for oral or parenteral administration immediately before use. Examples of such liquid forms include liquid preparations, suspensions, and emulsions.

[0122] The compositions and methods provided herein are particularly useful for the treatment of cancer, especially gastrointestinal cancer, in patients. Therefore, the compositions and methods may be used for the treatment of various malignant tumors.

[0123] As used herein, concomitant administration (simultaneous administration) includes simultaneous, separate, or sequential administration of the EGFR / LGR5 bispecific antibody and the topoisomerase I inhibitor in the same or different dosage forms. Therefore, in some embodiments, the EGFR / LGR5 bispecific antibody can be used in a method for treating cancer in a subject, and the EGFR / LGR5 bispecific antibody may be administered simultaneously, separately, or sequentially with the topoisomerase I inhibitor. In other embodiments, the EGFR / LGR5 bispecific antibody can be used in the treatment of cancer in a subject, and the EGFR / LGR5 bispecific antibody may be administered simultaneously, separately, or sequentially with the topoisomerase I inhibitor.

[0124] In other embodiments, the EGFR / LGR5 bispecific antibody may be used for the manufacture of a drug for the treatment of cancer in a subject, and the EGFR / LGR5 bispecific antibody may be administered simultaneously, separately, or sequentially with the topoisomerase I inhibitor. In other embodiments, the EGFR / LGR5 bispecific antibody may be used for the manufacture of a drug for the treatment of cancer in a subject, and the EGFR / LGR5 bispecific antibody may be administered simultaneously, separately, or sequentially with the topoisomerase I inhibitor. The product comprising the EGFR / LGR5 bispecific antibody and the topoisomerase I inhibitor may be a combined formulation for simultaneous, separate, or sequential use when treating cancer in a subject.

[0125] EGFR / LGR5 bispecific antibodies and topoisomerase I inhibitors can be administered according to a suitable dosage and route (e.g., intravenous, intraperitoneal, intramuscular, intrathecal, or subcutaneous).

[0126] EGFR / LGR5 bispecific antibodies and topoisomerase I inhibitors can also be administered according to any preferred schedule. For example, EGFR / LGR5 bispecific antibodies and topoisomerase I inhibitors can be administered simultaneously as monotherapy or as separate agents. Alternatively, EGFR / LGR5 bispecific antibodies and topoisomerase I inhibitors can be formulated for separate administration and administered simultaneously or sequentially.

[0127] For example, in some embodiments, an EGFR / LGR5 bispecific antibody may be administered first, followed by a topoisomerase I inhibitor, or vice versa. The administration regimen in the above-described therapeutic method and method of use is adjusted to provide the optimal desired response (e.g., therapeutic response).

[0128] For example, a single bolus may be administered, or the dose may be administered in multiple doses over time. Alternatively, the dose may be proportionally reduced or increased as indicated by the urgent treatment situation. In one embodiment, the EGFR / LGR5 bispecific antibody is administered before the topoisomerase I inhibitor. For example, the EGFR / LGR5 bispecific antibody is administered to the patient first, followed by the administration of the topoisomerase I inhibitor. In another embodiment, the topoisomerase I inhibitor is administered before the EGFR / LGR5 bispecific antibody. For example, the topoisomerase I inhibitor is administered to the patient first, followed by the administration of the EGFR / LGR5 bispecific antibody (e.g., more than 1 minute, more than 1 hour, or more than 1 day after administration). Such simultaneous or sequential administration results in a state in which both the EGFR / LGR5 bispecific antibody and the topoisomerase I inhibitor are simultaneously present in the patient being treated. The simultaneous presence of both an EGFR / LGR5 bispecific antibody and a topoisomerase I inhibitor directs EGFR / LGR5 bispecific antibody-induced cancer treatment and EGFR / LGR5 bispecific antibody-mediated inhibition of EGFR / LGR5 signaling.

[0129] In another embodiment, a topoisomerase I inhibitor and an EGFR / LGR5 bispecific antibody are administered simultaneously.

[0130] In one embodiment, the subject is administered a single dose of a topoisomerase I inhibitor and a single dose of an EGFR / LGR5 bispecific antibody. In some embodiments, the EGFR / LGR5 bispecific antibody and the topoisomerase I inhibitor are administered repeatedly throughout the course of treatment. For example, in certain embodiments, multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the topoisomerase I inhibitor and multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the EGFR / LGR5 bispecific antibody are administered to the subject in need of treatment.

[0131] In some embodiments, the administration of the topoisomerase I inhibitor and the EGFR / LGR5 bispecific antibody may be once a week, every two weeks, or once a month, and in that regimen, they may be administered on the same day (e.g., simultaneously) or sequentially (e.g., more than one minute, several hours, or several days apart). When administered separately, the EGFR / LGR5 bispecific antibody and the topoisomerase I inhibitor may be administered according to the same administration (i.e., dose) protocol, but not necessarily. For example, one cycle of treatment may include one or more administrations of the EGFR / LGR5 bispecific antibody. On the other hand, a therapeutically effective dose of the topoisomerase I inhibitor may be administered with the EGFR / LGR5 bispecific antibody at either a high or low frequency. In certain embodiments, each dose of the topoisomerase I inhibitor and the EGFR / LGR5 bispecific antibody may be administered on the same day, or alternatively, the topoisomerase I inhibitor may be administered one or more days before or after the EGFR / LGR5 antibody.

[0132] In some embodiments, the dose of the EGFR / LGR5 bispecific antibody and / or topoisomerase I inhibitor changes over time. For example, the EGFR / LGR5 bispecific antibody and / or topoisomerase I inhibitor may be administered at a high dose initially and then decreased over time. In another embodiment, the EGFR / LGR5 bispecific antibody and / or topoisomerase I inhibitor may be administered at a low dose initially and then increased over time.

[0133] In another embodiment, the amount of EGFR / LGR5 bispecific antibody and / or topoisomerase I inhibitor administered is constant for each dose. In another embodiment, the amount of EGFR / LGR5 bispecific antibody and / or topoisomerase I inhibitor varies with each dose. For example, each maintenance (or subsequent) dose may be higher than or equal to the initial loading dose. In another embodiment, each maintenance dose may be lower than or equal to the loading dose. Clinicians may utilize preferred doses as a natural consequence of the patient's condition. Doses may vary depending on a number of factors, including the stage of the disease. The specific dose to be administered based on the presence of one or more of these factors is within the scope of the art of the art. Generally, treatment is initiated with a dose even lower than the optimal dose. Thereafter, the dose is increased in small increments until the optimal effect is achieved under those circumstances. For convenience, the total daily dose may be divided and administered in divided doses throughout the day, if necessary. Intermittent treatment (e.g., one week out of three or three weeks out of four) may also be used.

[0134] In certain embodiments, the EGFR / LGR5 bispecific antibody is administered in doses of 0.1, 0.3, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg body weight. In other embodiments, the EGFR / LGR5 bispecific antibody is administered in doses of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg body weight.

[0135] The treatment methods described herein are typically continued as long as the clinician supervising the patient's care considers the treatment to be effective, i.e., the patient is responding to the treatment. Non-limiting parameters indicating the effectiveness of a treatment method may include one or more of the following: reduction of tumor cells; inhibition of tumor cell proliferation; elimination of tumor cells; progression-free survival; and appropriate response to suitable tumor markers (if applicable).

[0136] Regarding the frequency of administration of EGFR / LGR5 bispecific antibodies, those skilled in the art will be able to determine an appropriate frequency. For example, a clinician may decide to administer EGFR / LGR5 bispecific antibodies at a relatively low frequency (e.g., once every two weeks) and gradually shorten the duration of administration as the patient tolerates. Regarding the frequency of administration of topoisomerase I inhibitors, the frequency of these drugs can be determined in a similar manner. Examples of lengths of time associated with the course of treatment by the claimed method include: approximately 1 week, 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, approximately 11 weeks, approximately 12 weeks, approximately 13 weeks, approximately 14 weeks, approximately 15 weeks, approximately 16 weeks, approximately 17 weeks, approximately 18 weeks, approximately 19 weeks, approximately 20 weeks, approximately 21 weeks, approximately 22 weeks, approximately 23 weeks, approximately 24 weeks, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 12 months, approximately 13 months, approximately 14 months, approximately 15 months, approximately 16 months, approximately 17 months, approximately 18 months, approximately 19 months, approximately 20 months, approximately 21 months, approximately 22 months, approximately 23 months, approximately 24 months, approximately 30 months, approximately 3 years, approximately 4 years, approximately 5 years, and indefinite (e.g., continuous maintenance therapy). The aforementioned period may correspond to one or more treatment rounds / cycles.

[0137] The efficacy of the therapeutic methods provided herein can be evaluated using any preferred means. In one embodiment, the clinical efficacy of combination therapy is analyzed using a reduction in the number of cancer cells as an objective response criterion. Patients, e.g., humans, treated according to the methods disclosed herein preferably experience improvement in at least one symptom of cancer. In some embodiments, one or more of the following may occur: the number of cancer cells can be reduced; cancer recurrence can be prevented or delayed; and one or more symptoms associated with cancer can be alleviated to some extent. Furthermore, an in vitro assay for determining T cell-mediated target cell lysis is also available.

[0138] In another embodiment, the treatment method produces a comparable clinical efficacy rate (CBR = CR (complete response), PR (partial response), or SD (stable disease) ≥ 6 months) which is better than that achieved by EGFR / LGR5 bispecific antibodies or topoisomerase I inhibitors (e.g., irinotecan) alone.

[0139] In some embodiments, tumor cells are no longer detectable after the treatment described herein. In some embodiments, the subject is in partial or complete remission. In certain embodiments, the subject has increased overall survival, median survival rate, and / or progression-free survival.

[0140] The combination of the present invention (for example, an EGFR / LGR5 bispecific antibody combined with a topoisomerase I inhibitor) may also be used in conjunction with other well-known therapies selected for their specific efficacy against the cancer being treated. Alternatively, the combination of the present invention may, where appropriate, be used sequentially with known pharmaceutically acceptable agents.

[0141] Methods for the safe and effective administration of chemotherapeutic agents are known to those skilled in the art. Furthermore, such administrations are described in standard literature. For example, many administrations of chemotherapeutic agents are described in the Physicians' Desk Reference (PDR), e.g., the 1996 edition (Medical Economics Company, Montvale, NJ07645-1742, USA), and its disclosure is incorporated herein by reference.

[0142] It will be apparent to those skilled in the art that the administration of chemotherapeutic agents and / or radiotherapy may vary depending on the disease being treated and the known effects of chemotherapeutic agents and / or radiotherapy on that disease. Furthermore, according to the knowledge of those skilled in the art, the treatment protocol (e.g., dosage and administration time) may be modified considering the observed effects of the administered therapeutic agent on the patient and the observed response of the disease to the administered therapeutic agent.

[0143] Kits or products comprising a pharmaceutical composition containing an EGFR / LGR5 bispecific antibody and a topoisomerase I inhibitor, and a pharmaceutically acceptable carrier in a therapeutically effective amount adapted for use in the manner described herein, are also provided herein. In some embodiments, the kit or product may optionally also include instructions, for example, a dosing schedule, for enabling a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein to a patient with cancer.

[0144] In some embodiments, the kit or product comprises multiple packages of single-dose pharmaceutical compositions, each containing an effective amount for a single dose of an EGFR / LGR5 bispecific antibody and a topoisomerase I inhibitor, according to the method provided above. Instruments or devices required to administer one or more pharmaceutical compositions may also be included in the kit or product. For example, the kit or product may provide one or more pre-filled syringes containing unit doses of an EGFR / LGR5 bispecific antibody and a topoisomerase I inhibitor, either in the same container or in separate containers to be administered as separate and distinct compositions.

[0145] In certain embodiments, one or both of the EGFR / LGR5 bispecific antibody and the topoisomerase I inhibitor are provided in a solid form suitable for reconstitution and subsequent administration according to the accompanying instructions.

[0146] The functional portion of the antibody described herein includes at least a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 as described herein. Therefore, it includes the antigen-binding portion of the antibody described herein and typically contains the variable domain of the antibody. The variable domain of the functional portion may be a single-chain Fv fragment or a so-called single-domain antibody fragment. A single-domain antibody fragment (sdAb) is an antibody fragment having a single monomeric variable antibody domain. Like the whole antibody, it can selectively bind to a particular antigen. With a molecular weight of only 12-15 kDa, a single-domain antibody fragment is much smaller than a typical antibody (150-160 kDa) composed of two heavy-chain proteins and two light chains, and even smaller than a Fab fragment (about 50 kDa, one light chain and half heavy chains) and a single-chain variable fragment (about 25 kDa, two variable domains, one from the light chain and one from the heavy chain). Single-domain antibodies themselves are not significantly smaller than normal antibodies (typically 90–100 kDa). Most single-domain antibody fragments are genetically engineered from heavy-chain antibodies found in camels, and these are called VHH fragments (nanobodies®). Some fish also possess heavy-chain-only antibodies (IgNAR, "immunoglobulin neoantigen receptor") from which single-domain antibody fragments called VNAR fragments can be obtained. An alternative approach is to split dimeric variable domains from common immunoglobulin G (IgG) from humans or mice into monomers. While most research on single-domain antibodies currently relies on heavy-chain variable domains, nanobodies derived from light chains have also been shown to specifically bind to target epitopes. Non-limiting examples of such variable domains in antibody moieties include VHH, human domain antibodies (dAbs), and unibodies. Preferred antibody moieties or derivatives have at least two variable domains in the antibody or its equivalent. Non-limiting examples of such variable domains or their equivalents include F(ab) fragments and single-chain Fv fragments. The functional portion of the bispecific antibody includes the antigen-binding portion of the bispecific antibody, or a derivative and / or analog of the binding portion.As described herein, the antibody binding portion is contained within the variable domain.

[0147] In further embodiments, the composition or combination or kit or product may include one or more additional activators.

[0148] All documents and references, including Genbank entries, patents and published patent applications, and websites, described herein are expressly incorporated herein by reference to the same extent as if they were described in whole or in part in this document.

[0149] For clarity and conciseness, features are described herein as part of the same or distinct embodiments, but it will be understood that the scope of the invention may include embodiments having all or some combinations of the described features.

[0150] Herein, the present invention will be described by reference to the following embodiments, which are illustrative only and not intended to limit the invention. Although the present invention has been described in detail and described by 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. [Brief explanation of the drawing]

[0151] [Figure 1] Human LGR5 sequence. [Figure 2] Human EGFR sequence. [Figure 3] Therapeutic effect on tumor volume in the M005 orthotopic PDX model of CRC. A: Injection frequency, injection administration, and injection site during the treatment period; B: Change in tumor volume multiplier over time; C: Dot plot showing the change in tumor volume multiplier per mouse at week 6. [Figure 4]A shows the tumor volume before and after treatment in mouse model M005. Treatment was stopped after 9 weeks, and tumor volume was monitored in the same mice for a further 3 weeks. The number below each group indicates why not all mice were included at week 12. B shows the body weight (model M005) in each group over time. [Figure 5] A is the number of mice with metastases at the time of sacrifice, detected macroscopically or evaluated by H&E staining; B is the residual disease at 12 weeks. [Figure 6] A shows the mouse model M001: injection frequency, dosage, and injection site during the treatment period; B shows the change in average tumor volume over time; C is a dot plot showing the tumor volume per mouse at week 6. [Figure 7] A shows the body weight of each group over time (model M001), and the combination therapy was not toxic; B shows that treatment with bispecific MF5816xMF3755 alone, or bispecific MF5816xMF3755 + irinotecan, prevented metastasis. Metastasis was evaluated histologically by macroscopic staining using H&E. [Figure 8-1] a) Along with common light chain variable regions such as the variable region of the human kappa light chain IgVκ1(39*01 / IGJκ1*01), the amino acid sequence of the heavy chain variable region of MF5816xMF3755 forms a variable domain that binds to LGR5 or EGFR. [Figure 8-2] b) refers to the CDR and framework areas. [Figure 8-3] c) shows the DNA sequence. Additional heavy chain variable regions that bind to EGFR and LGR5, which are suitable for combination with topoisomerase I inhibitors and for the production of bispecific antibodies, are further disclosed in this figure. [Figure 8-4] c) shows the DNA sequence. Additional heavy chain variable regions that bind to EGFR and LGR5, which are suitable for combination with topoisomerase I inhibitors and for the production of bispecific antibodies, are further disclosed in this figure. [Figure 8-5] c) shows the DNA sequence. Additional heavy chain variable regions that bind to EGFR and LGR5, which are suitable for combination with topoisomerase I inhibitors and for the production of bispecific antibodies, are further disclosed in this figure. [Figure 9-1] a) is the amino acid sequence of the common light chain amino acid sequence. b) is the DNA sequence and translation of the common light chain variable region (IGKV1-39 / jk1). c) is the DNA sequence and translation of the common light chain constant region. d) is the translation of the IGKV1-39 / jk5 common light chain variable region. e) is the V-region IGKV1-39A. [Figure 9-2] f) are the common light chains CDR1, CDR2, and CDR3. [Figure 10-1] IgG heavy chain for the generation of bispecific molecules. a) CH1 region. b) Hinge region. c) CH2 region. [Figure 10-2] IgG heavy chains for the generation of bispecific molecules. d) CH3 domains containing variations L351K and T366K(KK). e) CH3 domains containing variations L351D and L368E(DE). [Examples]

[0152] As used herein, “MFXXXX” (wherein X is independently a digit from 0 to 9) refers to a Fab containing a variable domain, where VH has the four-digit identified amino acid sequence shown in Figure 8. Unless otherwise specified, the light chain variable region of the variable domain typically has the sequence shown in Figure 9b). The light chain in the examples has the sequence shown in Figure 9a). “MFXXXX VH” refers to the four-digit identified amino acid sequence of VH. MF further comprises a constant region of the light chain and a constant region of the heavy chain that normally interacts with the constant region of the light chain. The VH / variable region of the heavy chain is different, typically a CH3 region, where one of the heavy chains has a KK mutation in its CH3 domain and the other has a complementary DE mutation in its CH3 domain, as shown in Figures 10d) and 10e) (see reference PCT / NL2013 / 050294 (published as International Publication No. 2013 / 157954)). The bispecific antibodies in the examples have a KK / DE CH3 heterodimer-forming domain, an Fc tail containing a CH2 domain and a CH1 domain as shown in Figure 10, a common light chain as shown in Figure 9a), and a VH specified by the MF number. For example, the bispecific antibody represented by MF3755xMF5816 has the above general sequence, as well as a variable domain having a VH with the sequence of MF3755 and a variable domain having a VH with the sequence of MF5816.

[0153] Example 1 Cell line: Freestyle 293F cells (catalog number p / n51-0029) were obtained from Invitrogen and routinely maintained in 293 Freestyle medium. HEK293T (ATCC-CRL-11268) and CHO-K1 (DSMZ ACC110) cell lines were purchased from ATCC and routinely maintained in DMEM / F12 (Gibco) supplemented with L-glutamine (Gibco) and FBS (Lonza).

[0154] The amino acid and nucleic acid sequences of various heavy chain variable regions (VHs) are shown in Figure 8. Among the other LGR5 and EGFR combinations shown in Figure 9a), the bispecific antibody EGFR / LGR5, MF3755xMF5814, which includes the heavy chain variable regions MF3755 and MF5816 and the common light chain, and includes ADCC modification enhanced by afucosylation, has been shown to be effective in International Publication No. 2017 / 069628 (page 138).

[0155] Production of bispecific antibodies Bispecific antibodies were generated by transient co-transfection of two plasmids encoding IgG having different VH domains, using a proprietary CH3 gene manipulation technique, to ensure efficient heterodimerization and formation of bispecific antibodies. The common light chain is also co-transfected into the same cell, either on the same plasmid or a different plasmid. The applicants' previous applications (e.g., International Publications 2013 / 157954 and 2013 / 157953, incorporated herein by reference) disclose methods and means for producing bispecific antibodies from a single cell, thereby providing means to favor the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can be suitably employed in the present invention. Specifically, preferred mutations for essentially generating only bispecific full-length IgG molecules are amino acid substitutions at positions 351 and 366 in the first CH3 domain, e.g., L351K and T366K (numbered according to EU numbering) ("KK-mutant" heavy chain), and amino acid substitutions at positions 351 and 368 in the second CH3 domain, e.g., L351D and L368E ("DE-mutant" heavy chain), or vice versa (see Figures 10d and 10e). It has been previously demonstrated in the cited 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 homodimer) or KK-mutant heavy chains (KK-KK homodimer) rarely occurs due to the strong repulsive force between charged residues at the CH3-CH3 interface between identical heavy chains.

[0156] The VH gene of the variable domain that binds to LGR5 was cloned into a vector encoding a positively charged CH3 domain. The VH gene of the variable domain that binds to EGFR, such as that disclosed in International Publication No. 2015 / 130172, was cloned into a vector encoding a negatively charged CH3 domain. A 293F freestyle cell suspension adapted for augmentation was cultured in a T125 flask on a shaking plateau until the density reached 3.0 × 10⁶ cells / mL. Cells were seeded into each well of a 24-deep-well plate at a density of 0.3–0.5 × 10⁶ viable cells / mL. Cells were transiently transfected with a mixture of two plasmids encoding different antibodies and cloned into proprietary vector systems. Seven days after transfection, the cell supernatant was collected and filtered through a 0.22 μM filter (Sartorius). The sterile supernatant was stored at 4°C until antibody purification.

[0157] IgG purification Purification was performed under sterile conditions in a filter plate using filtration. First, the pH of the culture medium was adjusted to pH 8.0, and then the IgG-containing supernatant was incubated with protein A Sepharose CL-4B beads (50% v / v) (Pierce) on a shaking platform at 600 rpm at 25°C for 2 hours. Next, the beads were collected by filtration. The beads were washed twice with PBS pH 7.4. Then, the bound IgG was eluted with 0.1 M citrate buffer at pH 3.0, and the eluate was immediately neutralized with Tris pH 8.0. Buffer exchange was performed by centrifugation using a Multiscreen Ultracel 10 multiplate (Millipore). Finally, the sample was collected in PBS pH 7.4. The IgG concentration was measured using Octet. The protein sample was stored at 4°C.

[0158] IgG quantification using Octet To determine the amount of purified IgG, whole human IgG (Sigma Aldrich, catalog number I4506) was used as a standard, and the antibody concentration was determined by Octet analysis using a protein A biosensor (Forte-Bio, as recommended by the supplier).

[0159] Preparation of mice and cells for engraftment Large tumor spheroid structures (tumoroids) were grown for 7 days before deaggregation into a single-cell suspension for injection. For all mouse studies, 6-8 week old female NOD.CB17 / AlhnRj-Prkdcscid / Rj mice (Janvier Labs) were used.

[0160] Method for preparing culture conditions and single cells Organoids derived from colorectal cancer samples were treated with 2 mM GlutaMax (Invitrogen), 10 mM HEPES (Invitrogen), 1x B27 retinoic acid-free supplement (Invitrogen), 50 ng / mL EGF (Peprotech), 0.1 μg / mL Noggin (Peprotech), Rock inhibitor Y-27632 (Sigma-Aldrich), 10 nM PGE2 (Sigma-Aldrich), and 3 μm SB20219. Organoids were cultured in 100% basement membrane extract (BME, Amsbio) at 37°C and 5% CO2 using a medium consisting of advanced DMEM / F12 (Invitrogen) supplemented with 0 (Sigma-Aldrich), 10 nM gastrin (Tocris), 1 μg / mL R-SPO1 (homemade), 10 mM nicotinoamide (Sigma-Aldrich), 1.25 mM N-acetylcysteine ​​(Sigma-Aldrich), and 0.5 μM A83-01 (Tocris). The organoids were deaggregated into single cells the day before analysis. For this purpose, the organoids were first released from the BME by removing the medium, the BME was resuspended in cell recovery solution (BD Biosciences), and incubated on ice for 1 hour. Subsequently, the organoids were centrifuged (all centrifugation steps were 5 minutes at 4°C and 200 g). The pellet was resuspended in 1 mL of 50% trypsin / EDTA solution (TE) and 50% PBS, and pipetted up and down until a single-cell suspension was obtained, with periodic visual evaluation. The TE was diluted in 10 mL of PBS and centrifuged. The cells were washed twice in 10 mL of PBS and then resuspended in BME, which was divided into 50 μL portions and dropped onto a preheated plate (37°C). After allowing the BME droplets to stand for 15 minutes, 500 μL of culture medium was added per drop. After 12 hours, the cells were isolated from the BME using the cell recovery solution. After 1 hour on ice, the cells were centrifuged and washed once in 10 mL of PBS containing 0.5% BSA and 0.5 mM EDTA (staining buffer). The pellet was then resuspended in staining buffer and counted.

[0161] Stem cells and cancer cells from VHIO generated CRCPDX model collections derived from surgically resected primary tumors (colon and rectum) and liver metastases. The PDX models are clinically and molecularly annotated and faithfully represent the clinical epidemiology of mCRC. These models can be injected subcutaneously or orthotopically into the cecal wall of immunodeficient mice. The orthotopic models generate local and distant metastases in lymph nodes, liver, lungs, and carcinomatosis, replicating the progressive disease in CRC patients. A set of PDX models with key molecular traits was selected to evaluate the efficacy of the anti-LGR5 / EGFR bispecific antibody of the present invention (see Table 1). The initial PDX set included multiple mutant and wild-type models. Other determinants, such as the relative expression of EGFR or LGR5, which can also determine the response to the developed EGFR / LGR5 antibody, were measured in these PDX models (Table 1). We selected PDX models (Table 1) derived from liver metastases in three patients with progressive CRC. Two of the models were KRAS variants (G13D and G12D for M005 and M001, respectively), and M005 was also an APC and PIK3CA112_112del variant.

[0162] Model M005:120 NOD-SCID mouse, derived from the M005 PDX model, with 1×10 6Individual tumor cells were injected orthotopically into the cecal wall. In this case, the model was essentially generated as described in Puig et al., A Personalized Preclinical Model to Evaluate the Metastatic Potential of Patient-Derived Colon Cancer Initiating Cells, Clin Cancer Res; 19(24), 6787-6801 (2013), which is incorporated in its entirety in this application. These human tumor cells were derived from CRC liver metastases and contained mutations in the KRAS gene (KRAS G13D) and the PIK3CA gene (PIK3CA 112_112del). See Sanger Institute (UK), which has 18 histological types possessing the mutated PIK3C C420R (https: / / cancer.sanger.ac.uk / cosmic, mutation ID COSM757). From day 15 post-injection, mice were monitored weekly using CT imaging to detect primary tumors in the cecum. Treatment was initiated after at least 80% of the animals had a primary tumor growing in the cecum. The following 18 mice were excluded: those that died after surgery (#5), those without a primary tumor (#7), those with tumors that were too small or too large (#2 and #1, respectively), those with low body weight (#2), and those exhibiting general signs of the disease (#1). Following Figure 3A, the remaining 102 animals were treated and imaged weekly using micro-CT. The frequency and size of metastatic lesions were also determined by histological evaluation of the liver and lungs (hematoxylin and eosin staining (H&E)). Peritoneal dissemination of colorectal cancer was detected macroscopically at necropsy and later confirmed histologically. Model M001: The M001 PDX model was essentially generated as described in Puig et al., A Personalized Preclinical Model to Evaluate the Metastatic Potential of Patient-Derived Colon Cancer Initiating Cells, Clin Cancer Res; 19(24), 6787-6801 (2013), which is incorporated in its entirety in this application. See Sanger Institute (UK), which has 18 histological types possessing the PIK3C C420R mutation (https: / / cancer.sanger.ac.uk / cosmic, mutation ID COSM757). In the second orthotopic model, the injected human tumor cells originally originated from CRC liver metastases with mutations such as KRAS G12D and PIK3CA-C420R. Injection of tumor cells was performed in the same manner as above. The following 18 mice were excluded. Specifically, these included mice that died after surgery (#11), mice without a primary tumor (#2), mice with an excessively large tumor (#2), mice with low body weight (#1), and mice exhibiting general signs of the disease (#2). The administration and treatment management system followed Figure 6A.

[0163] At week 6, all mice treated with the solvent or bispecific EGFR / LGR5 containing only MF3755 and MF5816 were sacrificed, and approximately half of the mice treated with irinotecan or bispecific EGFR / LGR5 containing MF3755 and MF5816 + irinotecan were also sacrificed.

[0164] result: Analysis Model M005 The mean tumor volume in mice treated with bispecific EGFR / LGR5 containing only MF3755 and MF5816 was lower than in mice given the solvent. However, it was not as low as the mean tumor volume in mice treated with irinotecan alone. Surprisingly, mice receiving bispecific EGFR / LGR5, including MF3755 and MF5816 plus irinotecan combination therapy, had lower tumor volumes compared to all other groups of mice (Figure 3B, Figure 3C). Interestingly, after the end of treatment, bispecific EGFR / LGR5 containing MF3755 and MF5816 extended the tumor growth blocking effect of irinotecan, as confirmed by a fold change in tumor volume (Figure 4A).

[0165] Primary tumors were collected from all mice at sacrifice, and the frequency and size of metastatic lesions were analyzed. Figure 5A shows the number of mice found to have metastatic lesions at sacrifice. This demonstrates that mice treated with bispecific EGFR / LGR5 containing MF3755 and MF5816 or irinotecan, either alone or in combination, had fewer metastases than untreated mice.

[0166] Tissue analysis was also performed on mice sacrificed after the end of treatment (9 weeks) and a 3-week untreated period. Smaller tumors were found to contain only necrotic cells or a small number of tumor cells, while the majority of larger tumors contained a large number of tumor cells (Figure 5B). This analysis showed that tumor volume and cecal weight were positively correlated with treated mice 3 weeks after the end of treatment (P<0.0001 for the Pearson correlation coefficient).

[0167] Analysis Model M001: The mean tumor volume in mice treated with bispecific EGFR / LGR5 containing only MF3755 and MF5816 was very similar to the mean tumor volume in mice treated with irinotecan alone. However, mice receiving bispecific EGFR / LGR5, including MF3755 and MF5816 plus irinotecan, had lower tumor volumes than any of the other groups of mice (Figure 6B, Figure 6C). No toxicity was observed in mice receiving the bispecific EGFR / LGR5 combination containing MF3755 and MF5816 plus irinotecan (Figure 7A). Histological analysis to determine metastatic lesions at sacrifice demonstrated that mice treated with bispecific EGFR / LGR5 containing either MF3755 and MF5816 or irinotecan alone or in combination had fewer metastases than untreated mice (Figure 7B).

[0168] To investigate their potential to inhibit tumor growth and metastatic potential, MF3755 and MF5816, along with the bispecific antibody EGFR / LGR5 containing the chemotherapy drug irinotecan, were tested individually and in combination in two orthotopic models, M005 and M001. In M005, MF3755 and MF5816, along with the bispecific antibody EGFR / LGR5 containing irinotecan alone, were able to delay primary tumor growth. However, the combination demonstrated that MF3755 and MF5816, along with the bispecific antibody EGFR / LGR5 containing irinotecan, promoted superior response. After treatment completion, the combination therapy completely eliminated primary tumors in 5 out of 5 surviving mice. This was the case for irinotecan monotherapy in only 1 out of 14 mice. Furthermore, this indicates that the bispecific antibody EGFR / LGR5, containing MF3755 and MF5816, enhances chemotherapy-induced complete tumor regression. Regarding metastatic potential, the bispecific antibody EGFR / LGR5, containing MF3755 and MF5816, inhibited the formation of distant metastases, similar to irinotecan. No metastases were observed in mice treated with a combination of irinotecan and the bispecific antibody EGFR / LGR5 containing MF3755 and MF5816.

[0169] The results from model M005 were confirmed in model M001. Bispecific antibodies containing gMF3755, MF5816, and irinotecan alone, including EGFR / LGR5, were equally effective in delaying primary tumor growth in M001. However, when administered together, combination therapy appears to be even more effective than either drug administered alone.

[0170] Of the data shown in Figure 6C, statistical analysis (ANCOVA) of tumor volume at week 6 showed that treatment significantly reduced tumor volume in all groups except between irinotecan and bispecific antibodies containing MF3755 and MF5816. (Solvent vs. MF3755 and MF5816, p<0.0001; solvent vs. irinotecan, p<0.0001; solvent vs. irinotecan + MF3755 and MF5816, p<0.0001; MF3755 and MF5816 vs. irinotecan, p<0.6429; MF3755 and MF5816 vs. irinotecan + MF3755 and MF5816, p<0.0001; irinotecan + MF3755 and MF5816 vs. irinotecan, p<0.0001.)

[0171] In M001, combination therapy was not more toxic than irinotecan alone. Regarding metastatic potential, EGFR / LGR5, MF3755, and MF5816 prevented the formation of distant metastases, similar to irinotecan. No metastases were observed in mice treated with irinotecan in combination with the bispecific antibody EGFR / LGR5, which includes MF3755 and MF5816.

[0172] In conclusion, using two orthotopic CRC tumor models, combination therapy with the bispecific antibody EGFR / LGR5, including MF3755 and MF5816 and irinotecan, resulted in a greater degree of tumor regression compared to when these drugs were administered alone. In addition, metastasis was found to be inhibited by bispecific antibody EGFR / LGR5, including monotherapy or combination therapy with MF3755 and MF5816 and irinotecan.

[0173] [Table 1] Table 1 | Characteristics of PDX models derived from liver metastases in CRC patients. LGR5, EGFR, and nuclear β-catenin were determined by immunofluorescence quantification. Mutation status of Wnt signaling proteins (APC, RSPO, RNF43, ZNRF3) and oncogenic proteins (KRAS, PIK3CA, TP53) was determined by genomic analysis. Sensitivity of PDX models (subcutaneous growth) to WNT inhibitors was shown for dark cells. PDX model T108 was not used in further experiments.

Claims

1. An antibody or functional moiety, derivative and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, for use in the treatment of cancer, which is administered together with a topoisomerase I inhibitor.

2. The antibody or functional portion, derivative and / or analog thereof according to claim 1, wherein the cancer is colorectal cancer, lung cancer, gastrointestinal cancer, or ovarian cancer.

3. The antibody or functional portion thereof, derivative and / or analog according to claim 1 or 2, wherein the cancer is colorectal cancer.

4. The antibody or its functional portion, derivative and / or analog according to any one of claims 1 to 3, wherein the antibody or its functional portion, derivative and / or analog, and the topoisomerase I inhibitor are administered simultaneously to a subject.

5. The antibody or its functional portion, derivative and / or analog according to any one of claims 1 to 4, wherein the antibody or its functional portion, derivative and / or analog is administered to the subject prior to the topoisomerase I inhibitor.

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

7. The antibody or functional moiety, derivative and / or analog thereof according to any one of claims 1 to 6, wherein the variable domain that binds to LGR5 binds to an epitope located within amino acid residues 21 to 118 of the human LGR5 sequence shown in Figure 1.

8. The antibody or functional moiety, derivative and / or analog thereof according to claim 7, 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.

9. The antibody or functional moiety, derivative and / or analog thereof according to claim 7 or 8, wherein the LGR5-binding variable domain binds in small amounts to an LGR5 protein containing one or more amino acid residue mutations selected from 43A, 44A, 46A, 67A, 90A and 91A.

10. The antibody or functional moiety, derivative and / or analog thereof according to any one of claims 1 to 9, wherein the variable domain that binds to EGFR binds to an epitope located within amino acid residues 420 to 480 of the human EGFR sequence shown in Figure 2.

11. The antibody or functional moiety, derivative and / or analog thereof according to claim 10, wherein the amino acid residues at positions I462, G465, K489, I491, N493 and C499 of human EGFR are involved in the EGFR-binding variable domain to EGFR.

12. The antibody or functional moiety, derivative and / or analog thereof according to claim 10 or 11, wherein the EGFR-binding variable domain binds in small amounts to an EGFR protein containing one or more amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A and C499A.

13. The antibody or functional portion, derivative and / or analog thereof according to any one of claims 1 to 12, wherein the topoisomerase I inhibitor is camptothecin or a derivative thereof.

14. The antibody or functional portion, derivative and / or analog thereof according to any one of claims 1 to 13, wherein the topoisomerase I inhibitor is irinotecan or topotecan.

15. The antibody according to any one of claims 1 to 14, or a functional portion thereof, derivative and / or analog thereof, wherein the antibody is ADCC-enhancing.

16. The antibody according to any one of claims 1 to 15, or a functional portion thereof, derivative and / or analog thereof, wherein the antibody is afucosylated.

17. A method for inhibiting the proliferation of cells expressing EGFR and LGR5 in a system that allows cell proliferation, the method comprising providing a topoisomerase I inhibitor and a system comprising an antibody or a functional portion thereof, a derivative and / or an analog, containing a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5.

18. A method for treating cancer in a target, comprising simultaneously or sequentially administering to a target requiring treatment an antibody containing a topoisomerase I inhibitor and 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 thereof, a derivative, and / or an analog.

19. The method for treating cancer according to claim 18, wherein the cancer is colorectal cancer, lung cancer, gastrointestinal cancer, or ovarian cancer.

20. The method for treating cancer according to claim 18, wherein the cancer is colorectal cancer.

21. A pharmaceutical composition comprising an antibody or a functional portion thereof, a derivative and / or an analog thereof, which includes a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, and a topoisomerase I inhibitor.

22. The pharmaceutical composition according to claim 21, wherein the antibody or a functional portion thereof, derivative and / or analog, comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, and a topoisomerase I inhibitor are provided as a monotherapy agent.

23. The pharmaceutical composition according to claim 22, wherein the antibody or a functional portion thereof, derivative and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, and a topoisomerase I inhibitor are provided in separate formulations.

24. A kit comprising an antibody or a functional portion thereof, a 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; a topoisomerase I inhibitor; and instructions for using the antibody and the topoisomerase I inhibitor in the treatment of any one of claims 1 to 16.

25. An antibody or a functional portion thereof, derivative and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, for use in the treatment of gastrointestinal cancer in a subject, wherein the antibody is administered simultaneously, separately, or sequentially with a topoisomerase I inhibitor.

26. An antibody or a functional portion thereof, derivative and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, for use in the manufacture of a drug for the treatment of cancer in a target, wherein the antibody is administered simultaneously, separately or sequentially, together with a topoisomerase I inhibitor.

27. The antibody or functional portion thereof, derivative and / or analog according to claim 26, for the treatment of colorectal cancer, lung cancer, gastrointestinal cancer, or ovarian cancer.

28. An antibody or functional portion thereof, derivative and / or analog according to claim 27, for the treatment of colorectal cancer.

29. A product comprising, for the treatment of gastrointestinal cancer in a target area, an antibody or functional portion thereof, derivatives and / or analogs, and a topoisomerase I inhibitor, as a combined formulation for simultaneous, separate, or continuous use, comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5.

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