Treatment of previously treated subjects having colorectal cancer using an Anti-EGFR rechallenge treatment with a bispecific antibody that binds EGFR and LGR5

Abstract

The disclosure relates to means and methods in the treatment of a subject having a colorectal cancer. The disclosure in particular relates to therapeutic uses and methods involving a bispecific antibody that binds EGFR and LGR5. Such bispecific antibody is particularly useful in the treatment of metastatic colorectal cancer or a locally advanced, unresectable colorectal cancer. The disclosure further relates to uses in such methods and to the use in the manufacture of a medicament for the treatment of such cancers.

Description

[0001] Title: Treatment of previously treated subjects having colorectal cancer using an anti-EGFR rechallenge treatment with a bispecific antibody that binds EGFR and LGR5 TECHNICAL FIELD

[0002] The disclosure relates to means and methods in the treatment of cancer. The disclosure in particular relates to a method of treating metastatic colorectal cancer (mCRC) or locally advanced, unresectable mCRC in an individual using a bispecific antibody that binds EGFR and LGR5. The disclosure further relates to the use in such methods and to use in the manufacture of a medicament for the treatment of mCRC. Such bispecific antibodies are particularly useful in the treatment of mCRC, such as in an individual which has received two to more prior anticancer treatments against colorectal cancer. BACKGROUND

[0003] Cancer is still a major cause of death in the world, in spite of the many advances that have been made in the treatment of the disease and the increased knowledge of the molecular events that lead to cancer. After lung cancer, the second leading cause of death from cancer is colorectal cancer with a mortality rate of 9.4% out of a total of 9.9 million deaths for 2020. In particular, the 5-year relative overall survival (OS) of patients with mCRC is lower than 15% (Sung, H. et al. CA Cancer J. Clin. 71, 209-249 (2021)).

[0004] Patients with initial benefit from EGFR blockade almost invariably develop resistance. Data suggest that after failure of an upfront anti-EGFR-based regimen, a subpopulation of patients can still benefit from further anti-EGFR blockade. In molecularly selected patients, an anti-EGFR rechallenge strategy achieved up to 30% response rate, with progression-free survival longer than 4 months and overall survival of more than 1 year (Sartore-Bianchi et al., Nature Medicine, Vol 28, pages 1612-1618, 2022), comparing favorably with other standard therapeutic options available for other pretreated patients. Thus, while various strategies have been attempted to address treatment of mCRC, treatment of patients who initially respond to therapeutic EGFR blockade and then progress, remains an unmet clinical need.

[0005] Therefore, a need exists for improved or alternative cancer treatments, in particular for treating mCRC in subjects who have received multiple lines of prior anticancer treatment against colorectal cancer.

[0006] SUMMARY

[0007] The disclosure provides the following preferred aspects. However, the disclosure is not limited thereto.

[0008] In certain aspects, the present disclosure relates to therapeutic methods or therapeutic uses in the treatment of metastatic colorectal cancer or locally advanced, unresectable colorectal cancer involving a bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF3755 heavy chain variable region, as depicted in Figure 8a and the second variable domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF5816 heavy chain variable region, as depicted in Figure 8a.

[0009] In certain aspects, the present disclosure relates to treatment of metastatic colorectal cancer (mCRC). In certain aspects, the present disclosure relates to treatment of locally advanced, unresectable CRC, such as locally advanced disease not amenable to standard therapy with curative intent. In certain aspects, the cancer has progressed after having received at least two prior anticancer therapies. In certain aspects, said at least two prior anticancer therapies relate to treatment of colorectal cancer, the origin, or primary tumor of which, is colorectal tissue and which subsequently progressed into said mCRC or into said locally advanced, unresectable CRC. In certain aspects, treatment of said subject according to the present disclosure thus relates to third line treatment where the prior first and second line treatments were targeted against colorectal cancer. In certain aspects, treatment with a bispecific antibody of the present disclosure comprises a fourth or fifth line treatment, where the prior first, second, third and optional fourth line treatments comprised anti-colorectal cancer.

[0010] In certain aspects, the present disclosure relates to therapeutic methods or therapeutic uses in the treatment of metastatic colorectal cancer or locally advanced, unresectable colorectal cancer involving a bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises the amino acid sequences of the MF3755 heavy chain variable region, as depicted in Figure 8a and the second variable domain comprises the MF5816 heavy chain variable region, as depicted in Figure 8a.

[0011] In certain aspects, the present disclosure relates to therapeutic methods or therapeutic uses in the treatment of metastatic colorectal cancer or locally advanced, unresectable colorectal cancer involving a bispecific antibody, or functional part thereof, of which both first and second variable domains comprise the amino acid sequences of an LCDR1, an LCDR2, and an LCDR3 light chain variable domain as depicted in Figure 9a, such as of IGKVl-39 / jkl.

[0012] In certain aspects, the present disclosure relates to therapeutic methods or therapeutic uses in the treatment of metastatic colorectal cancer or locally advanced, unresectable colorectal cancer involving a bispecific antibody, or functional part thereof, of which both first and second variable domains comprise a light chain variable domain which comprises an LCDR1 comprising the amino acid sequence QSISSY, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence QQSYSTP as depicted in Figure 9a.

[0013] In certain aspects, the present disclosure relates to therapeutic methods or therapeutic uses in the treatment of metastatic colorectal cancer or locally advanced, unresectable colorectal cancer using a bispecific antibody, or functional part thereof, of which both first and second variable domains comprise the amino acid sequences of the light chain variable domain as depicted in Figure 9a, such as of IGKVl-39 / jkl.

[0014] In certain aspects, the present disclosure relates to therapeutic methods or therapeutic uses in the treatment of metastatic colorectal cancer or locally advanced, unresectable colorectal cancer using a bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises a heavy chain variable domain that comprises a HCDR1 comprising the animo acid NYAMN, a HCDR2 comprising the amino acid sequence WINANTGDPTYAQGFTG, and a HCDR3 comprising the amino acid sequence ERFLEWLHFDY, wherein the second variable domain comprises a heavy chain variable domain that comprises a HCDR1 comprising the amino acid sequence SYTMN, HCDR2 comprising the amino acid sequence WINTDTGDPTYAQGFTG, and HCDR3 comprising the amino acid sequence GDCDSTSCYRYSYGYEDY, and wherein both variable domains comprise a light chain variable domain which comprises an LCDR1 comprising the amino acid sequence QSISSY, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence QQSYSTP.

[0015] In certain aspects, the present disclosure relates to therapeutic methods or therapeutic uses in the treatment of metastatic colorectal cancer or locally advanced, unresectable colorectal cancer using petosemtamab, or functional part thereof (cf. Recommended INN list 83, WHO Drug Information Vol. 34, No. 1, 2020).

[0016] In certain aspects, the present disclosure relates to a bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of a heavy chain variable region of MF3755 and the second variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the heavy chain variable region of MF5816, for use in the treatment of colorectal cancer in a subject, which cancer has progressed after having received at least two prior anticancer therapies. In certain aspects, said at least two prior anticancer therapies were for treatment of metastatic CRC. In certain aspects, said at least two prior anticancer therapies were for treatment of advanced CRC. In certain aspects, said subject is afflicted with metastatic CRC. In certain aspects, said subject is afflicted with advanced CRC, such as locally advanced, unresectable CRC.

[0017] In certain aspects, the present disclosure relates to a bispecific antibody as mentioned herein for use in the treatment of metastatic colorectal cancer or locally advanced, unresectable colorectal cancer in a subject, which cancer has progressed after having received at least two prior anticancer therapies. In certain aspects, the present disclosure relates to a method of treating a subject having metastatic colorectal cancer or locally advanced, unresectable colorectal cancer, wherein said subject has progressed after having received at least two prior anticancer therapies, the method comprising providing the subject with an effective amount of a bispecific antibody, or functional part thereof as mentioned herein. In certain aspects, the present disclosure relates to the use of a bispecific antibody, or functional part thereof, as mentioned herein, in the manufacture of a medicament for treating metastatic colorectal cancer or locally advanced, unresectable colorectal cancer in a subject, which cancer has progressed after having received at least two prior anticancer therapies.

[0018] In certain aspects, a cancer of the present disclosure is metastatic colorectal cancer (mCRC). In certain aspects, a cancer of the present disclosure is a locally advanced, unresectable colorectal cancer. In certain aspects, said cancer expresses EGFR and / or LGR5. In certain aspects, said metastatic CRC includes a cancer or tumor in bone tissue, lung tissue, a lymph node, the neck, liver or spleen. In certain aspects, the primary origin of the mCRC is a tumor located in colorectal tissue and the cancer is mCRC or locally advanced, unresectable CRC.

[0019] In certain aspects, a cancer of the present disclosure does not have an oncogenic mutation in KRAS, NRAS, BRAF and / or the EGFR ectodomain. In certain aspects, said cancer is negative for HER2 gene copy number amplification. In certain aspects, said cancer does not have or does not exhibit HER2 gene copy number amplification. In certain aspects, the HER2 gene copy number does not exceed 2.18. In certain aspects, the HER2 gene copy number is established using next- generation sequencing. In certain aspects, the HER2 gene copy number amplification is not associated with chromosomearm aneuploidy.

[0020] In certain aspects, said subject received two, three or four lines of said prior anticancer therapy. In certain aspects, said at least two, three or four prior anticancer therapies were for treatment of metastatic CRC. In certain aspects, said at least two, three or four prior anticancer therapies were for treatment of advanced CRC, such as locally advanced, unresectable CRC. In certain aspects, said subject received two lines of said prior anticancer therapy. In certain aspects, said subject received three lines of said prior anticancer therapy. In certain aspects, said subject received four lines of said prior anticancer therapy. In certain aspects, said prior anticancer therapy comprises chemotherapy, such as oxaliplatin, irinotecan and / or a fluoropyrimidine, and / or therapy with an anti-VEGF agent and / or an anti-EGFR inhibitor. In certain aspects, one of said prior anticancer therapies comprises chemotherapy, such as oxaliplatin, irinotecan and / or a fluoropyrimidine. In certain aspects, one of said prior anticancer therapies comprises therapy with an anti-VEGF agent. In certain aspects, one of said prior anticancer therapies comprises therapy with an anti-EGFR inhibitor. In certain aspects, said prior anticancer therapy was therapy for metastatic CRC.

[0021] In certain aspects, said subject is a mammal, such as a human. In certain aspects, said treatment comprises providing to the subject a flat dose of 1500 mg of a bispecific antibody of the present disclosure, such as petosemtamab. In certain aspects, petosemtamab is provided biweekly (q2w).

[0022] In certain aspects, the present disclosure relates to a method for classifying a subject having colorectal cancer for treatment with a bispecific antibody as mentioned herein, a

[0023] said method comprising establishing said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification in a cancer cell containing sample from said subject.

[0024] In certain aspects, the present disclosure relates to a method for selecting a subject having colorectal cancer for treatment with a bispecific antibody as mentioned herein, the method comprising establishing that said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for of HER2 gene copy number amplification in a cancer cell containing sample from said subject.

[0025] In certain aspects, the subject is selected for treatment or classified as being likely to respond to treatment with said antibody when said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and not in the EGFR ectodomain, and is negative for HER2 gene copy number amplification as determined in the cancer cell containing sample from said subject.

[0026] In certain aspects, the present disclosure relates to a method for selecting a subject having colorectal cancer for treatment with petosemtamab, the method comprising determining that said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and not in the EGFR ectodomain, and is negative for HER2 gene copy number amplification in a cancer cell containing sample from said subject.

[0027] In certain aspects, the present disclosure relates to petosemtamab for use in a method of treatment of a subject having colorectal cancer, wherein said subject is classified as being likely to respond to treatment with petosemtamab, or selected for treatment with petosemtamab, when said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and not in the EGFR ectodomain and is negative for HER2 gene copy number amplification as established in a cancer cell containing sample from the subject.

[0028] In certain aspects, the present disclosure relates to petosemtamab for use in a method of treatment of a subject having colorectal cancer, wherein said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and not in the EGFR ectodomain and is negative for HER2 gene copy number amplification.

[0029] In certain aspects, at least six months prior to being administered petosemtamab, said subject has not received any anticancer therapy against said colorectal cancer, such as an EGFR inhibitor, and / or which said colorectal cancer has progressed after having received at least two prior anticancer therapies.

[0030] In certain aspects, genotype status for KRAS, NRAS, BRAF and EGFR ectodomain is identified using a suitable assay designed to establish the respective genotype status or the HER2 gene copy number, in particular the assay is or comprises next generation sequencing performed on a ctDNA sample obtained from said subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Human LGR5 amino acid sequence.

[0032] Figure 2 Human EGFR amino acid sequence, with its ectodomain in underlining, which correspond to topological domain positions 25-645.

[0033] Figure 3 Human HER2 amino acid sequence

[0034] Figure 4 Human EGFR ectodomain amino acid sequence which corresponds to topological domain positions 25-645 of human EGFR.

[0035] Figure 5 Human KRAS amino acid sequence

[0036] Figure 6 Human NRAS amino acid sequence

[0037] Figure 7 Human BRAF amino acid sequence

[0038] Figure 8. Heavy chain amino acid sequences

[0039] Figure 8(a) Amino acid sequences of heavy chain variable regions that together with a common light chain variable region such as the variable region of the human kappa light chain IGKVl-39 / jkl, form a variable domain that binds LGR5 or EGFR. The heavy chain CDRs and framework regions are indicated in Figure 8b (KABAT numbering system).

[0040] Figure 9 (a) Common light chain variable domain (VL) and L-CDR amino acid sequences (according to IMGT numbering), b) Common light chain constant domain (CL) amino acid sequence.

[0041] Figure 10. IgG heavy chains for the generation of bispecific molecules, a) CHI region amino acid sequence, b) Hinge region amino acid sequence, c) CH2 region amino acid sequence, d) CH3 domain containing variations L351K and T366K (KK) amino acid sequence, e) CH3 domain containing variations L351D and L368E (DE) amino acid sequence. Residue positions are according to EU numbering.

[0042] DETAILED DESCRIPTION OF THE DISCLOSURE

[0043] In order that the present description may be more readily understood, certain terms are first defined. Additional definitions may be set forth throughout the detailed description where deemed required. Unless separately defined herein, 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 of immunology, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are employed.

[0044] As used herein, the singular forms “a”, “an” and “the” include plural referents. Use of the term “comprising”, “having”, “including”, as well as other forms, such as “comprise”, “comprises”, “comprised”, “has”, “have”, “had”, “include”, “includes”, and “included”, is not limiting. The term “antibody” as used herein means a proteinaceous molecule belonging to the immunoglobulin class of proteins, containing one or more domains that bind an epitope on an antigen, where such domains are or derived from or share sequence homology with the variable region of an antibody. Antibodies are typically made of basic structural units, each with two heavy chains and two light chains. A bispecific antibody according to the present disclosure is not limited to any particular format or method of producing it. In certain aspects, the term “binding arm” as used herein is or comprises a binding domain. In certain aspects, said binding domain comprises a variable heavy domain and a variable light domain. Said variable heavy domain (VH) in certain aspects binds EGFR, a membrane-associated member of the Wnt signaling pathway, such as LGR5, or cMET.

[0045] A “bispecific antibody” is an antibody as described herein wherein one domain of the antibody binds to a first antigen whereas a second domain of the antibody binds to a second antigen, wherein said first and second antigens are not. identical, or where one domain binds a first epitope on an antigen, wThereas a second domain binds to a second epitope on the antigen. The term “bispecific antibody” also encompasses antibodies wherein one heavy chain variable region / light chain variable region (VH / VL) combination binds a first antigen or epitope on an antigen and a second VH / VL combination binds a second antigen or epitope on the antigen. The term further includes antibodies wherein VH is capable of specifically recognizing a first antigen and the VL, paired with the VH in an immunoglobulin variable region, is capable of specifically recognizing a second antigen. The resulting VH / VL pair will bind either antigen 1 or antigen 2. Such so called “two-in-one antibodies”, are described in for instance WO 2008 / 027236, WO 2010 / 108127 and Schaefer et al (Cancer Cell 20, 472-486, October 2011). A bispecific antibody according to the present disclosure is not limited to any particular bispecific format or method of producing it.

[0046] The term ‘common light chain’ as used herein refers to the two light chains (or the VL part thereof) in the bispecific antibody. The two light chains (or the VL part thereof) may be identical or have some amino acid sequence differences while the binding specificity of the full-length antibody is not affected. The terms ‘common light chain’, ‘common VL’, ‘single light chain’, ‘single VL’, with or without the addition of the term ‘rearranged’ are all used herein interchangeably. “Common” also refers to functional equivalents of the light chain of which the amino acid sequence is not identical. Many variants of said light chain exist wherein mutations (deletions, substitutions, insertions and / or additions) are present that do not influence the formation of functional binding regions. In certain aspects, the light chain of the present disclosure can also be a light chain as specified herein, having from 0 to 10 amino acid insertions, deletions, substitutions, additions or a combination thereof. In certain aspects, the light chain of the present disclosure can also be a light chain as specified herein, having from 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof. It is for instance within the scope of the definition of common light chains as used herein, to prepare or find light chains that are not identical but still functionally equivalent, e.g., by introducing and testing conservative amino acid changes, changes of amino acids in regions that do not or only partly contribute to binding specificity when paired with the heavy chain, and the like. As used herein, “to comprise” and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb “to consist” may be replaced by “to consist essentially of’ meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the disclosure.

[0047] The term ‘full length IgG’ or ‘full length antibody’ according to the disclosure is defined as comprising an essentially complete IgG, which however does not necessarily have all functions of an intact IgG. For the avoidance of doubt, a full-length IgG contains two heavy and two light chains. Each chain contains constant (C) and variable (V) regions, which can be broken down into domains designated CHI, CH2, CHS, VH, and CL, VL. An IgG antibody binds to antigen via the variable region domains contained in the Fab portion, and after binding can interact with molecules and cells of the immune system through the constant domains, mostly through the Fc portion. Full length antibodies according to the disclosure encompass IgG molecules wherein variations may be present that provide desired characteristics. Full length IgG should not have deletions of substantial portions of any of the regions. However, IgG molecules wherein one or several amino acid residues are deleted, without essentially altering the binding characteristics of the resulting IgG molecule, are embraced within the term "full length IgG". For instance, such IgG molecules can have a deletion of between 1 and 10 amino acid residues, preferably in non-CDR regions, wherein the deleted amino acids are not essential for the antigen binding specificity of the IgG. In certain aspects, such IgG molecules can have a deletion of between 1 and 10 amino acid residues in non-CDR regions, wherein the deleted amino acids are not essential for the antigen binding specificity of the IgG.

[0048] In certain aspects, the present disclosure relates to a bispecific antibody or functional part thereof. A “functional part” as described herein comprises at least a variable domain that binds an extracellular part of the target of interest, meaning EGFR or LGR5 as described herein. It thus comprises the antigen binding parts of a bispecific antibody as described herein and typically contains the variable domains of the antibody. A variable domain of a functional part can be a single chain Fv-fragment or a so-called single domain antibody fragment. In certain aspects, the antibody or functional parts have at least two variable domains of an antibody or equivalents thereof. Non-limiting examples of such variable domains or equivalents thereof are F(ab) -fragments and Single chain Fv fragments. A functional part of a bispecific antibody comprises the antigen binding parts of the bispecific antibody. As mentioned herein above, the binding part of an antibody is encompassed in its variable domain (s).

[0049] As a bispecific antibody, functional part, or a binding domain thereof, typically recognizes an epitope of an antigen, and such an epitope may be present in other compounds as well, antibodies according to the present disclosure that “specifically recognize” an antigen, for example, EGFR or LGR5, may recognize other compounds as well, if such other compounds contain the same kind of epitope. Hence, the terms “specifically recognizes” with respect to an antigen and antibody interaction does not exclude binding of the antibodies to other compounds that contain the same kind of epitope.

[0050] The term “epitope” or “antigenic determinant” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein (so-called linear and conformational epitopes). Epitopes formed from contiguous, linear amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding conformation are typically lost on treatment with denaturing solvents. An epitope may typically include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation.

[0051] As used herein, the terms “subject” and “patient” are used interchangeably and refer to a mammal such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig and the like (e.g., a patient, such as a human patient, having cancer). In certain aspects, the term “subject” refers to a human.

[0052] The terms “treat,” “treating,” and “treatment,” as used herein, refer to any type of intervention or process performed on, or administering an active agent or combination of active agents to the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease. The terms “treatment” and “therapy” herein are used interchangeably.

[0053] As used herein, “effective treatment” or “positive therapeutic response” refers to a treatment producing a beneficial effect, e.g., amelioration of at least one symptom of a disease or disorder, e.g., cancer. A beneficial effect can take the form of an improvement over baseline, including an improvement over a measurement or observation made prior to initiation of therapy according to the method. For example, a beneficial effect can take the form of slowing, stabilizing, stopping or reversing the progression of a cancer in a subject at any clinical stage, as evidenced by a decrease or elimination of a clinical or diagnostic symptom of the disease, or of a marker of cancer. Effective treatment may, for example, decrease in tumor size, decrease the presence of circulating tumor cells, reduce or prevent metastases of a tumor, slow or arrest tumor growth and / or prevent or delay tumor recurrence or relapse.

[0054] The term “effective amount” or “therapeutically effective amount” refers to an amount of an agent or combination of agents that provides the desired biological, therapeutic, and / or prophylactic result. That result can be reduction, amelioration, palliation, lessening, delaying, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In terms of tumor development, an effective amount is an amount sufficient to delay tumor development. In terms of tumor recurrence, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations. The effective amount of the agent or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and may stop cancer cell infiltration into peripheral organs; (iv) inhibit tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer. In one aspect, an “effective amount” is the amount of a bispecific antibody as disclosed herein as the therapeutic agent to affect a decrease in a cancer (for example a decrease in the number of cancer cells); slowing of progression of a cancer or prevent regrowth or recurrence of the cancer. As mentioned before herein, the bispecific antibody or functional part thereof that binds EGFR and LGR5 of the present disclosure, is also referred herein to as a “therapeutic agent”. In certain aspects, the effective amount herein is a flat dose of 1500 mg administered on a biweekly basis (i.e., q2w) to a subject having a cancer of the present disclosure. In certain aspects, said therapeutic agent comprises or is petosemtamab.

[0055] The term “flat dose” herein refers to a dosing regimen wherein a subject is administered a fixed amount of a therapeutic substance over multiple administrations, independent of body weight of the subject. Flat dosing is typically abbreviated with qnw, wherein n is an integer indicating the interval and w is week. For instance, a q2w flat dose administration regimen of 1500 mg antibody means a fixed amount, independent of body weight, of 1500 mg bispecific antibody of the present disclosure that is administered once every 2 weeks. For instance, a q2w flat dose administration regimen of 1500 mg antibody means a fixed amount of 1500 mg bispecific antibody of the present disclosure is administered once every 2 weeks. Herein, in certain aspects, the therapeutic substance is a bispecific antibody binding EGFR and LGR5 that is administered using a q2w dosing regimen of 1500 mg to a human subject. In certain aspects, the subject has been administered at least 3 of such q2w flat dosages of 1500mg. In certain aspects, said administration is at least 4 dosages or more and may last until the patient shows sufficient clinical or radiological progression.

[0056] The flat dose may be premedicated, meaning medication is administered to the subject prior to being administered the antibody of the present disclosure. In certain aspects, said flat dose of antibody is premedicated with an antihistamine, pain reducing medication, fever reducing medication and / or anti-inflammatory medication.

[0057] The term “H-score”, sometimes referred to as “histo” score in the art, refers to a reproducible and standardized scoring methodology which can be used to semi-quantitatively calculate expression of a gene of interest in a tumor sample following a protocol based on methods of immunohistochemistry (IHC) or in situ hybridization techniques (ISH), all well-known with the skilled person and follow the ASCO April 10th, 2015 posting on how to calculate H-scores. See also Hirsch FR, Varella-Garcia M, Bunn PA Jr, et al: Epidermal growth factor receptor in non- small-cell lung carcinomas:

[0058] Correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 21:3798-3807, 2003; and John T, Liu G, Tsao M-S: Overview of molecular testing in non-small-cell lung cancer: Mutational analysis, gene copy number, protein expression and other biomarkers of EGFR for the prediction of response to tyrosine kinase inhibitors. Oncogene 28: S14-S23, 2009. The relevant teachings of these references are herein incorporated by reference. In the context of H scoring for EGFR, the term “determined using IHC” refers to a method that uses or comprises IHC as the basis for subsequently determining the H score, as opposed to methods alternative to IHC.

[0059] CDRs and framework regions of antibodies have been described and defined in the art using a number of different systems, including for instance Kabat (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991); Kabat et al., J. Biol. Chem.252:6609-6616 (1977)), IMGT (discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-21 1 1997), Chothia (Chothia and Lesk J. Mol. Biol. 196: 901 -917, 1987; Chothia et al., Nature 342: 877-883, 1989; Al-Lazikani et al., J. Mol. Biol. 273: 927-948, 1997), and the nomenclatures of Honnegher and Plukthun (Honneghei' and Plukthun, J. Mol. Biol. 309: 657-670, 2001), MacCallum (MacCallum et al., J. Mol. Biol.262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008)), and Lefranc (Lefranc M. P. et al., Dev. Comp. Immunol., 27: 55-77 (2003)). In general, it is irrelevant which numbering system is used, as an antibody exhibits its properties regardless of the numbering system used. When the amino acid sequence of a variable region is given, a skilled person can readily determine its CDRs based on different numbering systems. Thus, the present disclosure encompasses defining the CDRs in accordance with each numbering system available to a skilled person. In particular, the present disclosure encompasses defining the CDRs in accordance with the numbering systems of Kabat, IMGT, and Chothia. In certain aspects, the heavy chain (H)CDRs are as defined according to Kabat. In certain aspects, the light chain (L)CDRs are as defined according to IMGT. Amino acids in the constant regions are indicated according to the EU numbering system.

[0060] Epidermal growth factor (EGF) receptor (EGFR, ErbBl, or HER1) is a member of a family of four receptor tyrosine kinases (RTKs), named Her- or ErbB-1, -2, -3 and -4. EGFR is known under various synonyms, the most common of which is EGFR, EGFR has an extracellular domain (ECD) that is composed of four sub-domains, two of which are involved in ligand binding and two of which are involved in homo-dimerization and hetero-dimerization. EGFR integrates extracellular signals from a variety of ligands to yield diverse intracellular responses. A major signal transduction pathway activated by EGFR is composed of the Ras-mitogen-activated protein kinase (MAPK) mitogenic signaling cascade. Activation of this pathway is initiated by the recruitment of Grb2 to tyrosine phosphorylated EGFR. This leads to activation of Ras through the Grb2-bound Ras-guanine nucleotide exchange factor Son of Sevenless (SOS). In addition, the PI3-kinase-Akt signal transduction pathway is also activated by EGFR, although this activation is much stronger in case there is co-expression of ErbB-3 (HER3). EGFR is implicated in several human epithelial malignancies, notably cancers of the breast, bladder, non-small cell lung cancer, colon, ovarian head and neck and brain. Activating mutations in the gene have been found, as well as over-expression of the receptor and of its ligands, giving rise to autocrine activation loops. This RTK has therefore been extensively used as target for cancer therapy. Both small-molecule inhibitors targeting the RTK and monoclonal antibodies (mAbs) directed to the extracellular ligand-binding domains have been developed and have shown hitherto several clinical successes, albeit mostly for a select group of patients. The database accession number for the human EGFR protein and the gene encoding it is GenBank NM_005228.3 / NP_005219.2. This accession number is primarily given to provide a further method of identification of EGFR protein as a target, the actual sequence of the EGFR protein bound by an antibody may vary, for instance because of a mutation in the encoding gene such as those occurring in some cancers or the like. Human EGFR ectodomain sequence (UniProt P00533) is depicted in underlining in Figure 2 and 4, which correspond to topological domain positions 25-645.

[0061] Where reference herein is made to EGFR, the reference refers to human EGFR unless otherwise stated. The variable domain antigen-binding site that binds EGFR, binds EGFR and a variety of variants thereof such as those expressed on some EGFR positive tumors.

[0062] In certain aspects, EGFR is human EGFR. The EGFR that is bound by said antibody or functional part thereof of the present disclosure, includes EGFR without any mutation in its ectodomain, such as depicted in underlining in Figure 2 or depicted in Figure 4.

[0063] In its Biclonics® antibody program, Merus has developed multispecific antibodies that target EGFR and LGR5 (Leucine -rich repeat containing G protein-coupled receptor). The efficacy of such multispecific antibodies has been assessed in vitro and in vivo using patient-derived CRC organoids and mice PDX models, respectively (see, e.g., WO2017 / 069628; wdiich is incorporated by reference herein). Multispecific antibodies that target EGFR and LGR5 were shown to inhibit tumor growth. The potency of such inhibitory antibodies was shown to be correlated with the levels of LGR5 RNA expression by cells from the cancer. In certain aspects, said multispecific antibodies that target EGFR and LGR5 are as described in WO2017 / 069628.

[0064] The term “LGR” refers to the family of proteins known as Leucine-rich repeat-containing G-protein coupled receptors. Several members of the family are known to be involved in the WNT signaling pathway, of note LGR4, LGR5, and LGR6.

[0065] LGR5 is Leucine-Rich Repeat Containing G Protein-Coupled Receptor 5. Alternative names for the gene or protein are Leucine-Rich Repeat Containing G Protein-Coupled Receptor 5; Leucine-Rich Repeat- Containing G Protein-Coupled Receptor 5; G-Protein Coupled Receptor HG38; G-Protein Coupled Receptor 49; G-Protein Coupled Receptor 67; GPR67; GPR49; Orphan G Protein- Coupled Receptor HG38; G Protein-Coupled Receptor 49; GPR49; HG38 and FEX. A protein or antibody of the disclosure that binds LGR5, binds human LGR5. The LGR5 binding protein or antibody of the disclosure may, due to sequence and tertiary structure similarity between human and other mammalian orthologs, also bind such an ortholog but not necessarily so. Database accession numbers for the human LGR5 protein and the gene encoding it are (NC_000012.12;

[0066] NT_029419.13; NC_018923.2; NP_001264155.1; NP_001264156.1; in particular NP_003658.1). The accession numbers are primarily given to provide a further method of identification of LGR5 as a target, the actual sequence of the LGR5 protein bound may vary, for instance because of a mutation in the encoding gene such as those occurring in some cancers or the like. The LGR5 antigen binding site binds LGR5 and a variety of variants thereof, such as those expressed by some LGR5 positive tumor cells.

[0067] In certain aspects, KRAS herein is human KRAS, primarily referred to as uniprot P01116-1. Alternative names for the gene or protein are for instance Kirsten Rat Sarcoma Viral Oncogene Homolog and GTPase KRas. The GeneCards symbol given is KRAS and public database accession numbers for the human KRAS protein and the gene encoding it are HGNC: 6407; NCBI Gene: 3845; Ensembl: ENSG00000133703 or OMIM®: 190070. In certain aspects, the human KRAS wildtype amino acid sequence is depicted in Figure 5.

[0068] In certain aspects, NRAS herein is human NRAS, primarily referred to as uniprot P01111. Alternative names for the gene or protein are for instance NRAS 2, NRAS ProtoOncogene, GTPase. Its GeneCards symbol is KRAS, and public database accession numbers for the human NRAS protein and the gene encoding it are HGNC: 7989; NCBI Gene: 4893; Ensembl: ENSG00000213281 and OMIM®: 164790. In certain aspects, the human NRAS wildtype amino acid sequence is depicted in Figure 6.

[0069] In certain aspects, BRAF herein is human BRAF, primarily referred to as uniprot P15056. Alternative names for the gene or protein are for instance B-Raf Proto¬ Oncogene, Serine / Threonine Kinase. Its GeneCards symbol is BRAF, and public database accession numbers for the human BRAF protein and the gene encoding it are HGNC: 1097; NCBI Gene: 673; Ensembl: ENSG00000157764 and OMIM®: 164757. In certain aspects, the human BRAF wildtype amino acid sequence is depicted in Figure 7.

[0070] In certain aspects, HER2 herein is human HER2, primarily referred to as uniprot P04626-1. Alternative names for the gene or protein are for instance Erb-B2 Receptor Tyrosine Kinase and NEU. Its GeneCards symbol is ERBB2, and public database accession numbers for the human HER2 protein and the gene encoding it are HGNC: 3430; NCBI Gene: 2064; Ensembl: ENSG00000141736 and OMIM®: 164870. In certain aspects, the human HER2 wildtype amino acid sequence is depicted in Figure 3.

[0071] The present disclosure relates to a bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF3755 heavy chain variable region as depicted in Figure 8a, and the second variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF5816 heavy chain variable region as depicted in Figure 8a, for use in the treatment of colorectal cancer in a subject, which cancer has progressed after said subject has received at least two prior anticancer therapies.

[0072] The present disclosure relates to a use of a bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF3755 heavy chain variable region, as depicted in Figure 8a and the second variable domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF5816 heavy chain variable region, as depicted in Figure 8a, in the manufacture of a medicament for treating colorectal cancer in a subject, which cancer has progressed after said subject has received at least two prior anticancer therapies.

[0073] The present disclosure relates to a method of treating colorectal cancer in a subject, wherein said subject has progressed after having received at least two prior anticancer therapies, the method comprising providing the subject with an effective amount of a bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF3755 heavy chain variable region, as depicted in Figure 8a and the second variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF5816 heavy chain variable region, as depicted in Figure 8a.

[0074] In certain aspects, the present disclosure relates to a pharmaceutical or medicinal composition for (use in) the treatment of a subject having colorectal cancer, wherein said subject has progressed after having received at least two prior anticancer therapies, the method comprising providing the subject with an effective amount of a bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF3755 heavy chain variable region, as depicted in Figure 8a and the second variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF5816 heavy chain variable region, as depicted in Figure 8a.

[0075] In certain aspects, any prior anticancer treatment as mentioned herein has been administered to a subject suffering from cancer. In certain aspects, said subject has progressed after having received said prior anticancer treatment.

[0076] In certain aspects, said bispecific antibody is petosemtamab or a functional part thereof. In certain aspects, said bispecific antibody is petosemtamab.

[0077] In certain aspects, the EGFR binding arm comprises the heavy chain variable region that comprises the CDR1, CDR2 and CDR3 sequences of the VH of MF3755 as depicted in Figure 8b; or the CDR1, CDR2 and CDR3 sequences of the VH of MF3755 as depicted in Figure 8b with at most three, or at most two, or at most one amino acid substitutions.

[0078] In certain aspects, the variable domain that binds human EGFR, is a variable domain with a heavy chain variable region that comprises the sequence of the VH chain of MF3755 as depicted in Figure 8a; or the amino acid sequence of the VH chain of MF3755 depicted in Figure 8a having at most 15 (or in certain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in certain aspects 1, 2, 3, 4 or 5) amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain of MF3755, In certain aspects, the EGFR binding arm comprises the heavy chain variable region that comprises the sequence of MF3755 as depicted in Figure 8a.

[0079] In certain aspects, the second variable domain as described herein comprises a variable domain that binds an extracellular part of LGR5, in particular a variable domain that specifically binds an extracellular part of LGR5.

[0080] In certain aspects, said first variable domain as described herein comprises a variable domain that binds an extracellular part of EGFR and interferes with the binding of EGF to the receptor. In certain aspects, said second variable domain that binds LGR5 does not block the binding of an Rspondin (RSPO) to LGR5 an LGR5-expressing cell in the interaction of the LGR5 binding domain to LGR5. Methods for determining whether an antibody blocks or does not block the binding of an Rspondin to LGR5 are described in WO2017069528, which is hereby incorporated by reference.

[0081] In certain aspects, said binding moiety further comprises an LGR5 binding arm that comprises a heavy chain variable region comprising the CDR1, CDR2 and CDR3 sequences of a VH of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817 or MF5818 as depicted in Figure 8b with at most three, or at most two, or at most one amino acid substitution.

[0082] In certain aspects, said binding moiety further comprises an LGR5 binding arm that comprises a heavy chain variable region that comprises the CDR1, CDR2 and CDR3 sequences of a VH of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in Figure 8b.

[0083] In certain aspects, said binding moiety further comprises an LGR5 binding arm that comprises the heavy chain variable region that comprises the CDR1, CDR2 and CDR3 sequences of the VH of MF5816 as depicted in Figure 8.

[0084] In certain aspects, said binding moiety further comprises an LGR5 binding arm that comprises a heavy chain variable region comprising the amino acid sequence of a VH chain of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in Figure 8a having at most 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or having 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818, wherein the amino acid insertions, deletions, substitutions or a combination thereof are not present in the CDR sequences.

[0085] In certain aspects, said binding moiety further comprises an LGR5 binding arm that comprises a heavy7chain variable region comprising the sequence of a VH chain of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in Figure 8a.

[0086] In certain aspects, said binding arm which comprises a variable domain that binds LGR5 comprises CDR1, CDR2 and CDR3 amino acid sequences of the heavy chain variable region of MF5816. In certain aspects, said binding arm which comprises a variable domain that binds LGR5 comprises a heavy chain variable region with the amino acid sequence of MF5816 as depicted in Figure 8a having at most 10, or at most 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid insertions, deletions, substitutions or a combination thereof with respect to the indicated sequence. In certain aspects, the variable domain that binds LGR5 comprises a heavy chain variable region with the amino acid sequence of MF5816 having at most 0, 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof with respect to the indicated sequence. In certain aspects, said variable domain comprises the heavy chain variable region with the amino acid sequence of MF5816.

[0087] In certain aspects, said binding moiety further comprises an LGR5 binding arm that comprises the heavy chain variable region comprising the sequence of VH chain MF5816 as depicted in Figure 8a.

[0088] In certain aspects, said bispecific antibody is or comprises petosemtamab.

[0089] In certain aspects, the EGFR and LGR5 binding arms bind specifically to EGFR and LGR5, respectively. In certain aspects, the EGFR and LGR5 binding arms bind specifically to human EGFR and human LGR5, respectively.

[0090] VH chains of variable domains that bind EGFR or LGR5 can have one or more amino acid substitutions with respect to the sequences depicted in Figure 8a. In certain aspects, a VH chain has an amino acid sequence of an EGFR or LGR5 VH of Figure 8a, having at most 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and, in certain aspects, having 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof with respect to any of the VH chain sequences of Figure 8a.

[0091] Additional variants of the disclosed amino acid sequences which retain EGFR or LGR5 binding can be obtained, for example, from phage display libraries which contain the rearranged human IGKV1-39 / IGKJ1 VL region (De Kruif et al. Biotechnol Bioeng. 2010 (106)741-50), and a collection of VH regions incorporating amino acid substitutions into the amino acid sequence of an EGFR or LGR5 VH region mentioned herein, as previously described (e.g., WO2017 / 069628). Phages encoding Fab regions which bind EGFR or LGR5 may be selected and analyzed by flow cytometry, and sequenced to identify variants with amino acid substitutions, insertions, deletions or additions which retain antigen binding.

[0092] In certain aspects, the mentioned amino acid substitutions are conservative amino acid substitutions. In certain aspects, the insertions, deletions, substitutions or combination thereof are not in the CDR1, CDR2 or CDR3 region with respect to the indicated VH or VL sequence.

[0093] In certain aspects, the mentioned at most 15 (or in certain aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in certain aspects 1, 2, 3, 4 or 5) amino acid substitutions are conservative amino acid substitutions. In certain aspects, the insertions, deletions, substitutions or a combination thereof are not in the HCDR1, HCDR2 or HCDR3 region of the VH chain and in certain aspects, not in the FR4 region.

[0094] In certain aspects, the binding moiety of the present disclosure comprises a light chain variable region. In certain aspects, said binding moiety comprises a common light chain variable region. In certain aspects, said binding moiety comprises a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences of the VL as depicted in Figure 9a.

[0095] In certain aspects, said binding moiety comprises a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences, according to IMGT, of the VL as depicted in Figure 9a. In certain aspects, said binding moiety comprises the light chain variable region of the VL as depicted in Figure 9a.

[0096] Said light chain variable regions of the VH / VL EGFR and LGR5 variable domains of an EGFR / LGR5 antibody may be the same or different. In certain aspects, said VL region of the VH / VL EGFR variable domain of an EGFR / LGR5 bispecific antibody is similar to the VL region of the VH / VL LGR5 variable domain. In certain aspects, VL regions in the first and second VH / VL variable domains are identical.

[0097] In certain aspects, the light chain variable region of one or both VH / VL variable domains as mentioned herein comprises a common light chain variable region. In certain aspects, the common light chain variable region of one or both VH / VL variable domains comprises a germline IgVKl-39 variable region V- segment. In certain aspects, the light chain variable region of one or both VH / VL variable domains comprises the kappa light chain V-segment IgVKl-39*01. IgVKl-39 is short for Immunoglobulin Variable Kappa 1- 39 Gene. The gene is also known as Immunoglobulin Kappa Variable 1-39; IGKV139; IGKV1-39. External Ids for the gene are HGNC: 5740; Entrez Gene: 28930; Ensembl: ENSG00000242371. The V-region can be combined with one of five J-regions. In certain aspects, the J-regions are jkl and jko, and the joined sequences are indicated as IGKV1-39 / jkl and IGKVl-39 / jk5; alternative names are IgVKl-39*01 / IGJKl*01 or IgVul-39*01 / IGJK5*01 (nomenclature according to the IMGT database worldwide web at imgt.org). In certain aspects, the light chain variable region of one or both VH / VL variable domains comprises the kappa light chain IgVKl-39*01 / IGJKl*01 or IgVxl-39*01 / IGJK1*05 (described in Figure 9a).

[0098] In certain aspects, the light chain variable region of both VH / VL comprising variable domains of a bispecific antibody of the present disclosure comprises a LCDR1, LCDR2, and LCDR3 amino acid sequence. In certain aspects, said LCDR1, LCDR2 and LCDR3 sequences are according to Kabat or IMGT numbering. In certain aspects, said LCDR1, LCDR2 and LCDR3 sequences are according to IMGT numbering. In certain aspects, said LCDR1, LCDR2 and LCDR3 comprise the amino acid sequence QSISSY amino acid sequence AAS, and the amino acid sequence QQSYSTP. In certain aspects, said LCDR1, LCDR2 and LCDR3 comprise the amino acid sequence QSISSY (described in Figure 9a), the amino acid sequence AAS (described in Figure 9a), and the amino acid sequence QQSYSTPPT (described in Figure 9a). In certain aspects, said LCDR1, LCDR2 and LCDR3 comprise the amino acid sequence QSISSY (described in Figure 9a), the amino acid sequence AAS (described in Figure 9a), and the amino acid sequence QQSYSTPPIT (described in Figure 9a).

[0099] In certain aspects, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK or DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK with 0-10, or such as 0-5, amino acid insertions, deletions, substitutions, additions or a combination thereof. In certain aspects, the light chain variable region comprises 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, or 0-3, or 0-2, or 0-1 or 0 amino acid insertions, deletions, substitutions, additions with respect to the indicated amino acid sequence, or a combination thereof, wherein the insertions, deletions, substitutions, additions or a combination thereof are not in the LCDR1, LCDR2 or LCDR3. A combination of an insertion, deletion, addition or substitution is a combination as claimed if aligned sequences do not differ at more than 5 positions. In certain aspects, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PTFGQ GTKVE IK. In certain aspects, said light chain variable region comprises LCDR1, LCDR2, and LCDR3 amino acid sequences. In certain aspects, the light chain variable region comprises the amino acid sequence DIQMT QSPSS LSASV GDRVT ITCRA SQSIS SYLNW YQQKP GKAPK LLIYA ASSLQ SGVPS RFSGS GSGTD FTLTI SSLQP EDFAT YYCQQ SYSTP PITFG QGTRL EIK. In certain aspects, said light chain variable region comprises LCDR1, LCDR2, and LCDR3 amino acid sequences.

[0100] For example, the light chain variable region of both VH / VL comprising variable domains of said bispecific antibody can have from 0 to 10, or in certain aspects from 0 to 5 amino acid insertions, deletions, substitutions, additions or a combination thereof with respect to a sequence in Figure 9a. In certain aspects, the light chain variable region of one or both VH / VL variable domains of said bispecific antibody comprises from 0 to 9, from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, in certain aspects from 0 to 3, in certain aspects from 0 to 2, in certain aspects from 0 to 1 and in certain aspects 0 amino acid insertions, deletions, substitutions, additions with respect to the indicated amino acid sequence, or a combination thereof. In certain aspects, the mentioned at most 10 (or in certain aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in certain aspects 1, 2, 3, 4 oi' 5) amino acid substitutions are conservative amino acid substitutions. In certain aspects, the insertions, deletions, substitutions or a combination thereof are not in the LCDR1, LCDR2 or LCDR3 region of the VL chain and in certain aspects, not in the FR4 region.

[0101] Also, the light chain variable region of one or both VH / VL variable domains of said bispecific antibody may comprise the amino acid sequence of a sequence as depicted in Figure 9a. In certain aspects, both VH / VL variable domains of said bispecific antibody comprise identical VL regions. In certain aspects, the VL of both VH / VL variable domains of said bispecific antibody comprises the amino acid sequence set forth in Figure 9a. In certain aspects, the VL of both VH / VL variable domains of said EGFR / LGR5 bispecific antibody comprises the amino acid sequence set forth in Figure 9a.

[0102] In certain aspects, the mentioned at most 15 (or in certain aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or in certain aspects 1, 2, 3, 4 or 5) amino acid substitutions are conservative amino acid substitutions. In certain aspects, the insertions, deletions, substitutions or a combination thereof are not in the LCDR1, LCDR2 or LCDR3 region of the VL chain and in certain aspects, not in the FR4 region.

[0103] The term ‘bispecific’ (bs) in the context of the present disclosure means that a binding moiety, or antibody, is capable of binding two different targets or two epitopes on the same target, for example, where one variable domain of the antibody (as defined above) binds to an epitope on EGFR and a second variable domain binds to an epitope on LGR5. One arm of the bispecific antibody typically contains the variable domain of one antibody, and the other arm contains the variable domain of another antibody (i.e. one arm of the bispecific antibody is formed by one heavy chain paired with one light chain whereas the other arm is formed by a different heavy chain paired with a light chain). Thus, in certain aspects, the stoichiometry of a bispecific antibody of the present disclosure is 1:1 for EGFR: LGR5 binding.

[0104] A bispecific antibody of the present disclosure is typically a bispecific full-length antibody, in certain aspects of the human IgG subclass. In certain aspects, said antibody is of the human IgGl subclass. Such antibodies have good ADCC properties which can, if desired, be enhanced by techniques known in the art, have favorable half-life upon in vivo administration to humans and CH 3 engineering technology exists that can provide for modified heavy chains that preferentially form heterodimers over homodimers upon co-expression in clonal cells.

[0105] ADCC activity of an antibody can be improved when the antibody itself has a low ADCC activity, by modifying the constant region of the antibody. Another way to improve ADCC activity of an antibody is by enzymatically interfering with the glycosylation pathway resulting in a reduced fucose. Several in vitro methods exist for determining the efficacy of antibodies or effector cells in eliciting ADCC. Among these are chromium-51 [Cr51] release assays, europium [Eu] release assays, and sulfur-35 [S35] release assays. Usually, a labeled target cell line expressing a certain surface-exposed antigen is incubated with antibody specific for that antigen. After washing, effector cells expressing Fc receptor CD 16 are co-incubated with the antibody-labeled target cells. Target cell lysis is subsequently measured by release of intracellular label by a scintillation counter or spectrophotometry.

[0106] A bispecific antibody of the present disclosure typically is ADCC enhanced. In certain aspects, such a bispecific antibody is afucosylated. In certain aspects, said bispecific antibody comprises a reduced amount of fucosylation of the N-linked carbohydrate structure in the Fc region, when compared to the same antibody produced in a normal CHO cell. Low fucose levels are associated with increased CD 16 (FcyRIIIa) binding on NK effector cells, resulting in increased ADCC activity. In certain aspects, and in addition to its direct antitumor activity, said binding moiety can eliminate tumor cells following opsonization and subsequent natural killer (NK) cell-mediated ADCC activity and complement-dependent cytotoxic (CDC) activity.

[0107] In certain aspects, a bispecific antibody of the present disclosure is capable of activating the complement system, in particular of activating the Cl complex. In certain aspects, said bispecific antibody is capable of exhibiting Complement-Dependent Cytotoxicity (CDC). In certain aspects, activation of the complement system occurs under permissible conditions. In certain aspects, said bispecific antibody is an IgG having an Fc tail which allows recruitment and activation of Clq.

[0108] In certain aspects, said bispecific antibody capable of complement activation comprises an Fc tail (or CH2 and / or CH3 domain) which comprises contact residue (such as position 322 or an analogous position) which allows Clq binding to an IgG. Complement activation herein can be established by measuring deposition of one of the downstream activated components of the C DC pathway, such as C3b. For instance, CDC activity can be measured using the Incucyte® CDC assay using suitable target cells that express a target of interest for a bispecific antibody of the present disclosure to bind to and allow it to display CDC activity. In certain aspects, said assay will be performed using 10-step semi-log dilutions of IgG to establish a concentration range in the presence of complement-preserved serum (e.g. from Sigma (cat #S1764-5xlML). In certain aspects, measurements are performed every hour for 24 hours to establish CDC activity over said time points for the antibody of interest. In certain aspects, suitable target cells are A-431, CAL-27 or 293FF. In certain aspects, the CDC activity outcome is compared to a positive and / or a negative control sample. In certain aspects, the CDC activity outcome is compared to CDC activity of a well-investigated antibody such as cetuximab and / or Sym004.

[0109] In certain aspects, a bispecific antibody of the present disclosure, is generally of the human IgG subclass (e.g., for instance IgGl, IgG2, IgG3, IgG4). In certain aspects, said antibody is of the human IgGl subclass. In certain aspects, said bispecific antibody is a full-length IgG antibody because of their favorable half-life and for reasons of low immunogenicity. Accordingly, said bispecific antibody is in certain aspects, a full-length IgG molecule. In certain aspects, said bispecific antibody is a full length IgGl molecule.

[0110] In certain aspects, a bispecific antibody of the present disclosure comprises a fragment crystallizable (Fc). In certain aspects, the Fc of said binding moiety is comprised of a human constant region. A constant region or Fc of said binding moiety may contain one or more, or not more than 10, or not more than 5 amino- acid differences with a constant region of a naturally occurring human antibody. For example, each Fab-arm of said binding moiety may further include an Fc-region comprising modifications promoting the formation of a bispecific binding moiety, promoting stability and / or other features described herein.

[0111] When provided as bispecific antibody which comprises an EGFR binding arm and a binding arm that binds LGR5, such are typically produced by cells that express nucleic acid(s) encoding said binding moiety. Accordingly, in certain aspects, said bispecific antibodies disclosed herein are produced by providing a cell comprising one or more nucleic acids that encode the heavy and light chain variable regions and constant regions of said binding moieties. In certain aspects, the cell is an animal cell, such as a mammal cell, or a primate cell and in certain aspects a human cell, A suitable cell is any cell capable of comprising and preferably of producing the said antibodies.

[0112] Suitable cells for antibody production are known in the art and include a hybridoma cell, a Chinese hamster ovary (CHO) cell, an NSO cell or a PER-C6 cell. Various institutions and companies have developed cell lines for the large-scale production of antibodies, for instance for clinical use. Non-limiting examples of such cell lines are CHO cells, NSO cells or PER. C6 cells. In particular, said cell is a human cell. Preferably a cell is transformed by an adenovirus El region or a functional equivalent thereof. A preferred example of such a cell line is the PER. C6 cell line or equivalent thereof. In a particular, said cell is a CHO cell or a variant thereof. Preferably the variant makes use of a Glutamine synthetase (GS) vector system for expression of an antibody. In certain aspects, the cell is a CHO cell.

[0113] In certain aspects a bispecific antibody which comprises an EGFR binding arm and a binding arm that binds LGR5, is produced in a cell which expresses the different light and heavy chains that make up said bispecific antibody. In certain aspects, the cell expresses two different heavy chains and at least one light chain. In certain aspects, the cell expresses a “common light chain” as described herein to reduce the number of different antibody species (combinations of different heavy and light chains). For example, the respective VH regions are cloned into expression vectors using methods known in the art for production of bispecific IgG (WO2013 / 157954; incorporated herein by reference), in conjunction with the rearranged human IGKV1-39 / IGKJ1 (huVKl-39) light chain, previously shown to be able to pair with more than one heavy chain thereby giving rise to antibodies with diverse specificities, which facilitates the generation of bispecific molecules (De Kruif et al. J. Mol. Biol. 2009 (387) 54858; WO2009 / 157771).

[0114] In certain aspects, a method for producing an EGFR / LGR5 bispecific antibody is as disclosed in US 9,248,181 and US 9,358,286. In certain aspects, mutations to produce essentially only bispecific full length IgG molecules are the amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain (the ‘KK-variant’ heavy chain) and the amino acid substitutions L351D and L368E in the second domain (the ‘DE-variant’ heavy chain), or vice versa. As previously described, the DE-variant and KK-variant preferentially pair to form heterodimers (so-called ‘DEKK’ bispecific molecules). Homodimerization of DE-variant heavy chains (DEDE homodimers) or KK-variant heavy chains (KKKK homodimers) hardly occurs due to strong repulsion between the charged residues in the CH3-CH3 interface between identical heavy chains.

[0115] In certain aspects, the heavy chain / light chain combination that comprises the first variable domain that binds EGFR, comprises a DE variant of the heavy chain. In certain aspects, the second heavy chain / light chain combination that comprises the variable domain that binds LGR5 comprises a KK variant of the heavy chain. In certain aspects, a bispecific antibody of the present disclosure also comprises an Fc tail or a Constant Heavy (CH) domain. In certain aspects, said Fc domain or CH domain is an Fc domain or CH domain from a human IgG, such as from a human IgGl. In certain aspects, said Fc domain or CH domain is ADCC enhanced. In certain aspects, said Fc domain or CH domain is afucosylated. In certain aspects, ADCC functionality is enhanced by altering the Fc portion to increase binding affinity to FcyRIIIA. In certain aspects, ADCC functionality is enhanced by changing Fc domain glycosylation. In certain aspects, ADCC functionality is enhanced by removing or reducing Fc domain fucosylation. In certain aspects, said CH domain is a CH2 and / or CHS region, such as from human IgGl. In certain aspects, said Fc domain or CH domain has antibodydependent cell-mediated phagocytosis (ADCP) activity. In certain aspects, ADCP functionality is enhanced by altering the Fc portion. In certain aspects, ADCP functionality is enhanced by changing Fc domain glycosylation. In certain aspects, ADCP functionality is enhanced by removing or reducing Fc domain fucosylation. In certain aspects, said CH domain is a CH2 and / or CH3 region, such as from human IgGl.

[0116] In certain aspects, a bispecific antibody of the present disclosure comprises a heavy chain which comprises a constant region of an IgGl antibody, such as a human IgGl antibody. In certain aspects, the CH2 region of said IgGl constant region is engineered to alter ADCC activity of the antibody. In certain aspects, said alteration results in enhanced ADCC activity. In certain aspects, the CH3-region of said binding moiety is engineered to facilitate heterodimerization of heavy chains comprising a first heavy chain that binds EGFR and a second heavy chain that binds LGR5. In certain aspects, the CH3-region of said binding moiety is engineered to facilitate heterodimerization of heavy chains comprising a first heavy chain that binds EGFR and a second heavy chain binds cMET.

[0117] The Fc region mediates effector functions of a bispecific antibody such as mentioned herein, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). Depending on the therapeutic antibody or Fc fusion protein application, it may be desired to either reduce or increase the effector function. Reduced effector function can be desired when an immune response is to be activated, enhanced or stimulated as in some of the aspects of the present disclosure. Antibodies with reduced effector functions can be used to target cell-surface molecules of immune cells, among others.

[0118] In certain aspects, a bispecific antibody of the present disclosure, with reduced effector functions is an IgG antibody comprising a modified CH2 / lower hinge region, for instance to reduce Fc-receptor interaction or to reduce Clq binding. In certain aspects, said bispecific antibody is an IgG antibody with a mutant CH2 and / or lower hinge domain such that interaction of the bispecific IgG antibody to a Fc-gamma receptor is reduced. In certain aspects, a bispecific antibody of the present disclosure comprises a mutant CH2 region and is an IgGl antibody. In certain aspects, such a mutant IgGl CH2 and / or lower hinge domain comprise an amino substitution at position 235 and / or 236 (EU-numbering), such as an L235G and / or G236R substitution (Figure 10c). In certain aspects, said antibody is obtained from a producer cell line which stably co-expresses the RMD (GDP-6-deoxy-D-lyxo-4-hexulose reductase) enzyme to yield antibodies with a low level of fucose on their Fc region-associated glycan structures. The RMD enzyme redirects the de novo fucose synthesis pathway towards a sugar nucleotide that cannot be metabolized by the cell. Low fucose levels are associated with increased CD16 (FcyRIHa) binding on NK effector cells, resulting in increased ADCC activity. In certain aspects, said antibody is capable of inducing ADCP. In certain aspects, said antibody is capable of inducing ADCC. In certain aspects, said antibody is capable of inducing CDC.

[0119] In certain aspects, a bispecific antibody of the present disclosure is capable of activating the complement system, in particular of activating the Cl complex. In certain aspects, said antibody is capable of exhibiting Complement-Dependent Cytotoxicity (CDC). In certain aspects, said antibody comprises an Fc tail which allows recruitment and activation of Clq. Activation of the complement system occurs under permissible conditions, which conditions include a suitably functioning immune system of the subject to which said antibody is administered, or in particular a subject having a functional complement activation system which includes Clq and typically the downstream complement components to allow CDC to occur. In certain aspects, said suitably functioning immune system means the subject is capable of functional exhibiting CDC. In certain aspects, said antibody is an IgG, having an Fc tail which allows recruitment and activation of Clq.

[0120] Without being bound by any theory it is believed that amino acid residues 1462; G465; K489; 1491; N493; and C499 as depicted Figure 2 are involved in binding an epitope by a bispecific antibody of the present disclosure. In certain aspects, involvement in binding is determined by observing a reduced binding of the variable domain to an EGFR with one or more of the amino acid residue substitutions selected from I462A; G465A; K489A; 1491 A; N493A; and C499A.

[0121] In an exemplary method, CHO cells express EGFR on the cell membrane, or an alanine substitution mutant, such as a mutant comprising one or more of the substitutions selected from I462A; G465A; K489A; 1491 A; N493A; and C499A. A test antibody is contacted with the CHO cells and binding of the antibody to the cells compared. A test antibody binds the epitope if it binds to EGFR and to a lesser extent to an EGFR with a I462A; G465A; K489A; I491A; N493A; and C499A substitution. Comparing binding with a panel of mutants each comprising one alanine residue substitution is preferred. Such binding studies are well known in the art. Often the panel comprises single alanine substitution mutants covering essentially all amino acid residues. For EGFR the panel only needs to cover the extracellular part of the protein and a part that warrants association with the cell membrane of course, when cells are used. Expression of a particular mutant can be compromised but this is easily detected by one or more EGFR antibodies that bind to different region(s). If expression is also reduced for these control antibodies the level or folding of the protein on the membrane is compromised for this particular mutant. Binding characteristics of the test antibody to the panel readily identifies whether the test antibodies exhibit reduced binding to mutants with a I462A; G465A; K489A; I491A; N493A; and C499A substitution. In one aspect, the variable domain that binds an epitope on an extracellular part of human EGFR is a variable domain that binds an epitope that is located within amino acid residues 420-480 of the sequence depicted in Figure 2. In certain aspects, the binding of the variable domain to EGFR is reduced by one or more of the following amino acid residue substitutions I462A; G465A; K489A; I491A; N493A; and C499A in EGFR, In certain aspects, binding of the antibody to human EGFR interferes with the binding of EGF to the receptor. In certain aspects, the epitope on EGFR is a conformational epitope. In one aspect, the epitope is located within amino acid residues 420-480 of the sequence depicted in Figure 2, or within 430-480 of the sequence depicted in Figure 2. In certain aspects, said epitope is located within 438-469 of the sequence depicted in Figure 2.

[0122] Without being bound by theory it is believed that the contact residues of the epitope, i.e. where the variable domain contacts the human EGFR are likely 1462; K489; 1491; and N493. The amino acid residues G465 and C499 are likely indirectly involved in the binding of the antibody to EGFR.

[0123] Where herein accession numbers or alternative names of proteins / genes are given, they are primarily given to provide a further method of identification of the mentioned protein as a target, the actual sequence of the target protein bound by a bispecific antibody of the disclosure may vary, for instance because of a mutation and / or alternative splicing in the encoding gene such as those occurring in some cancers or the like. The target protein is bound by the antibody as long as the epitope is present in the protein and the epitope is accessible to the antibody.

[0124] For the avoidance of doubt the reference to the growth of a cell as used herein refers to a change in the number of cells. Inhibition of growth refers to a reduction in the number of cells that would otherwise have been obtained. Increase in growth refers to an increase in the number of cells that would otherwise have been obtained. The growth of a cell typically refers to the proliferation of the cell.

[0125] In certain aspects, the variable domain that binds an extracellular part of LGR5 binds an epitope that is located within amino acid residues 21-118 of the sequence of Figure 1 of which amino acid residues D43; G44, M46, F67, R90, and F91 are involved in binding of the antibody to the epitope.

[0126] In certain aspects, the LGR5 variable domain is a variable domain wherein one or more of the amino acid residue substitutions in LGR5 of D43A; G44A, M46A, F67A, R90A, and F91A reduces the binding of the variable domain to LGR5.

[0127] In certain aspects, the epitope on an extracellular part of LGR5 is located within amino acid residues 21-118 of the sequence of Figure 1. In certain aspects, it is an epitope wherein the binding of the LGR5 variable domain to LGR5 is reduced by one or more of the following amino acid residue substitutions D43A; G44A, M46A, F67A, R90A, and F91A in LGR5.

[0128] In certain aspects, the epitope on LGR5 is a conformational epitope. In certain aspects, the epitope is located within amino acid residues 40-95 of the sequence of Figure 1. In certain aspects, the binding of the antibody to LGR5 is reduced with one or more of the following amino acid residue substitutions D43A; G44A, M46A, F67A, R90A, and F91A.

[0129] Without being bound by theory it is believed that M46, F67, R90, and F91 of LGR5 as depicted in Figure 1, are contact residues for a variable domain as indicated herein above, i.e. the antigen-binding site of a variable domain that binds the LGR5 epitope. That amino acid residue substitution D43A and G44A reduces the binding of an antibody can be due to the fact that these are also contact residues, however, it is also possible that these amino acid residue substitutions induce a (slight) modification of the conformation of the part of LGR5 that has one or more of the other contact residues (i.e. at positions 46, 67, 90 or 91) and that conformation change is such that antibody binding is reduced. The epitope is characterized by the mentioned amino acid substitutions. Whether an antibody binds the same epitope can be determined in various ways. In an exemplary method, CHO cells express LGR5 on the cell membrane, or an alanine substitution mutant, such as a mutant comprising one or more of the substitutions M46A, F67A, R90A, or F91A. A test antibody is contacted with the CHO cells and binding of the antibody to the cells compared. A test antibody binds the epitope if it binds to LGR5 and to a lesser extent to an LGR5 with a M46A, F67A, R90A, or F91A substitution. Comparing binding with a panel of mutants each comprising one alanine residue substitution is preferred. Such binding studies are well known in the art. Often the panel comprises single alanine substitution mutants covering essentially all amino acid residues. For LGR5 the panel only needs to cover the extracellular part of the protein and a part that warrants association with the cell membrane of course, when cells are used. Expression of a particular mutant can be compromised but this is easily detected by one or more LGR5 antibodies that bind to different region(s). If expression is also reduced for these control antibodies the level or folding of the protein on the membrane is compromised for this particular mutant. Binding characteristics of the test antibody to the panel readily identifies whether the test antibodies exhibit reduced binding to mutants with a M46A, F67A, R90A, or F91A substitution and thus whether the test antibody is a bispecific antibody of the disclosure. Reduced binding to mutants with a M46A, F67A, R90A, or F91A substitution also identifies the epitope to be located within amino acid residues 21-118 of the sequence of Figure 1. In certain aspects, the panel includes a D43A substitution mutant; a G44A substitution mutant of both. The antibody with the VH sequence of the VH of MF5816 exhibits reduced binding to these substitution mutants.

[0130] In certain aspects, said cancer is metastatic ORC. In certain aspects, said cancer is locally advanced, unresectable CRC. In certain aspects, said subject received two, three or four lines of said prior anticancer therapy. In certain aspects, said prior anticancer therapy comprises chemotherapy, such as oxaliplatin, irinotecan and / or a fluoropyrimidine, and / or therapy with an anti-VEGF agent and / or an anti-EGFR inhibitor. In certain aspects, said subject received two, three or four lines of said prior anticancer therapy comprising therapy with an anti-VEGF agent and / or an anti-EGFR inhibitor.

[0131] In certain aspects, said subject has received multiple prior anticancer treatments against colorectal cancer, which multiple prior anticancer treatment are separate anticancer treatments. In certain aspects, said multiple prior anticancer treatments are not overlapping but separated over time. In certain aspects, a subject will have progressed from each said prior anticancer treatment before having started a subsequent anticancer therapy. In certain aspects, a subject has shown progressive disease from each said prior anticancer treatment before having started a subsequent anticancer therapy. Herein it is to be understood that where a subject is indicated to have received two prior anticancer treatments, said subject has received only two such prior treatments.

[0132] It is possible for a subject to have received a combination of several anticancer medications or agents, which together constitute a single prior anticancer treatment. Thus, in certain aspects, a prior anticancer treatment is a combination of several anticancer medications or agents. As is common practice in the art, several chemotherapeutic agents may be combined into a single anticancer treatment such as any one of the following combinations: FOLFOX, FOLFIRI or FOLFIRINOX. Also, such combinations of chemotherapeutic agents may be further combined with anticancer immune therapy or anticancer therapy involving small molecules thereby constituting a single anticancer therapy which comprises multiple different antineoplastic agents. For instance, FOLFOX may be combined with panitumumab, FOLFIRI may be combined with aflibercept, capecitabine may be combined with bevacizumab. In certain aspects, a prior anticancer therapy is a combination of different anticancer medications or agents but said prior anticancer treatment is non-overlapping in time with a subsequent anticancer treatment.

[0133] In certain aspects, a prior anticancer treatment is a particular line of prior anticancer treatment. In certain aspects, a prior anticancer treatment is a first, second, third, fourth line of prior anticancer treatment. Typically, a subject will have progressed after having received a particular second line of anticancer treatment before receiving the subsequent line of anticancer treatment. Similarly, a subject will have progressed after having received a particular third line of anticancer treatment before receiving the subsequent line of anticancer treatment. Similarly, a subject will have progressed after having received a particular fourth line of anticancer treatment before receiving the subsequent line of anticancer treatment. In certain aspects, said prior treatments comprise treatment for metastatic CRC. In certain aspects, said prior treatments are treatments for metastatic CRC. In certain aspects, said prior treatments comprise treatment for advanced CRC, such as locally advanced, unresectable CRC. In certain aspects, said prior treatments are treatments for advanced CRC, such as locally advanced, unresectable CRC.

[0134] Neoadjuvant therapy herein is used in its ordinary meaning in the art and refers to a therapy which has the purpose of reducing the size of a tumor, prior to removal of the tumor (such as via surgery or radiation) to facilitate subsequent anticancer treatment. Adjuvant therapy herein is used in its ordinary meaning in the art and refers to a therapy which has the purpose of lowering the risk that the cancer will return. Adjuvant therapy is given after a subject has received an anticancer treatment. If a subject has progressed within 6 months of completion of neoadjuvant or adjuvant therapy, said neoadjuvant or adjuvant therapy is considered the first prior anticancer treatment a subject has received. If a subject has progressed after 6 months of completion of neoadjuvant or adjuvant therapy, said neoadjuvant or adjuvant therapy is not considered the first prior anticancer treatment a subject has received.

[0135] In certain aspects, a subject of the present disclosure has not received neoadjuvant and / or adjuvant therapy. Said subject has received two, three or four prior anticancer treatments.

[0136] In certain aspects, a subject of the present disclosure has received n eoadjuvant and / or adjuvant therapy and has shown disease progression more than 6 months after completion of said neoadjuvant and / or adjuvant therapy. Said subject has received two, three or four prior anticancer treatments.

[0137] In certain aspects, where a subject has received neoadjuvant and / or adjuvant therapy and has shown disease progression within 6 months after completion of said neoadjuvant and / or adjuvant therapy, said subject is considered to have received said neoadjuvant and / or adjuvant therapy as a first prior anticancer treatment. Thus, in certain aspects, the at least two prior anticancer therapies of the present disclosure relate to treatment of colorectal cancer, wherein the first of said tw’O prior anticancer treatments comprise neoadjuvant and / or adjuvant therapy if said subject has shown progressive disease within 6 months from completion of said neoadjuvant and / or adjuvant therapy. In certain aspects, the at least three prior anticancer therapies of the present disclosure relate to treatment of colorectal cancer, wherein the first of said three prior anticancer treatments comprise neoadjuvant and / or adjuvant therapy if said subject has shown progressive disease within 6 months from completion of said neoadjuvant and / or adjuvant therapy. In certain aspects, the at least four prior anticancer therapies of the present disclosure relate to treatment of colorectal cancer, wherein the first of said four prior anticancer treatments comprise neoadjuvant and / or adjuvant therapy if said subject has shown progressive disease within 6 months from completion of said neoadjuvant and / or adjuvant therapy.

[0138] In certain aspects, said subject has received two prior anticancer treatments for metastatic CRC, the first of which treatments comprised neoadjuvant and / or adjuvant therapy and said subject showed progressive disease that started within 6 months from completion of said adjuvant and / or neoadjuvant therapy. In certain aspects, said subject has received three prior anticancer treatments for metastatic CRC, the first of which treatments comprised neoadjuvant and / or adjuvant therapy and said subject showed progressive disease that started within 6 months from completion of said adjuvant and / or neoadjuvant therapy. In certain aspects, said subject has received four prior anticancer treatments for metastatic CRC, the first of which treatments comprised neoadjuvant and / or adjuvant therapy and said subject showed progressive disease that started within 6 months from completion of said adjuvant and / or neoadjuvant therapy.

[0139] In certain aspects, said subject has received two prior anticancer treatments for advanced CRC, the first of which treatments comprised neoadjuvant and / or adjuvant therapy and said subject showed progressive disease that started within 6 months from completion of said adjuvant and / or neoadjuvant therapy. In certain aspects, said subject has received three prior anticancer treatments for advanced CRC, the first of which treatments comprised neoadjuvant and / or adjuvant therapy and said subject showed progressive disease that started within 6 months from completion of said adjuvant and / or neoadjuvant therapy. In certain aspects, said subject has received four prior anticancer treatments for advanced CRC, the first of which treatments comprised neoadjuvant and / or adjuvant therapy and said subject showed progressive disease that started within 6 months from completion of said adjuvant and / or neoadjuvant therapy. In certain aspects, said advanced CRC comprises locally advanced, unresectable CRC.

[0140] According to the clinical trial of Example 1, petosemtamab is considered first-line in the advanced setting if disease progression occurred within 6 months of completion of adjuvant and / or neoadjuvant therapy. When neoadjuvant and / or adjuvant therapy was completed and disease progression occurred within 6 months from the end of said therapy, then adjuvant or neoadjuvant treatment is to be considered as a treatment that was administered for advanced or metastatic CRC.

[0141] In certain aspects, at least six months prior to being administered a bispecific antibody or functional part thereof of the present disclosure, said subject has not shown progressive disease after having received neoadjuvant and / or adjuvant therapy. In certain aspects, at least six months prior to being administered said bispecific antibody or functional part thereof, said subject has not received any anticancer therapy against said cancer, such as a therapy comprising an EGFR inhibitor. In certain aspects, said subject has previously shown clinical efficacy to said prior anticancer therapy comprising a complete response (CR), a partial response (PR), or any tumor reduction to a prior EGFR inhibitor, such as received as monotherapy or in combination with chemotherapy. In certain aspects, said subject has received said prior anticancer therapy for a period of at least six months with an EGFR inhibitor without showing evidence of disease progression. Treatment of patients who initially respond to therapeutic EGFR blockade and then progress remains an unmet clinical need and are treated using a bispecific antibody of the present disclosure, such as after allowing regaining sensitivity to an EGFR blockade, e.g. using petosemtamab.

[0142] In certain aspects, the primary tumor sidedness of said colorectal cancer is left-sidedness CRC. In certain aspects, said primary CRC tumor sidedness of said colorectal cancer is the left-sided colon (or distal colon) as characterized by including the last one-third of the transverse colon, the descending colon, the sigmoid colon, and the rectum and / or originates from the hindgut and is supplied by the inferior mesenteric artery.

[0143] In certain aspects, the primary tumor sidedness of said colorectal cancer is right-sidedness CRC. In certain aspects, said primary CRC tumor sidedness of said colorectal cancer is the right- sided colon (or proximal colon) as characterized by including the cecum, the ascending colon, and the proximal two-thirds of the transverse colon and / or originates from the midgut and is supplied by the superior mesenteric artery. In certain aspects, the subject has been previously treated with two or more lines of standard approved therapy or standard of care. In certain aspects, a subject as mentioned herein has progressed after having received prior treatment into metastatic CRC or locally advanced, unresectable CRC. In certain aspects, a subject as mentioned herein has progressed after having received prior first and second line treatment. In certain aspects, a subject as mentioned herein is treated with a bispecific antibody of the present disclosure as third line, fourth, fifth line or more, after having progressed on prior anticancer treatment such as an-EGFR inhibitor or anti-VEGF inhibitor. In certain aspects, treatment of a subject as mentioned herein is for locally advanced unresectable or metastatic treatment of CRC or mCRC.

[0144] In certain aspects, a subject as mentioned herein has progressed after having received prior anticancer treatment which comprises or consists of FOLFIRINOX and panitumumab. In certain aspects, a subject as mentioned herein has progressed after having received prior anticancer treatment which comprises or consists of FOLFOX and panitumumab. In certain aspects, a subject as mentioned herein has progressed after having received prior anticancer treatment which comprises or consists of capecitabine and bevacizumab. In certain aspects, a subject as mentioned herein has progressed after having received prior anticancer treatment which comprises or consists of FOLFIRI and aflibercept. In certain aspects, a subject as mentioned herein has progressed after having received prior anticancer treatment which comprises or consists of FOLFIRINOX, panitumumab and FOLFOX. In certain aspects, a subject as mentioned herein has progressed after having received prior anticancer treatment which comprises or consists of FOLFOX, panitumumab, capecitabine, bevacizumab and aflibercept.

[0145] Although surgery or radiation therapy may be preferred for most patients with early or localized disease, and may be considered for locally advanced disease, it may not be possible to apply to all patients, for instance due to the anatomical location of the cancer. In certain aspects, the subject of the present disclosure has received prior treatment with standard approved therapy or standard of care which herein includes treatment of a chemotherapeutic agent, such as one or more of oxaliplatin, irinotecan, and a fluoropyrimidine or any combination thereof.

[0146] In certain aspects, the cancer expresses EGFR. In certain aspects, the cancer expresses LGR5. In certain aspects, the cancer expresses EGFR and LGR5.

[0147] As used herein, a cancer expresses EGFR if the cancer comprises cells that express EGFR. A cell which expresses EGFR comprises detectable levels of RNA that codes for EGFR. In certain aspects, EGFR expression is determined by ISH.

[0148] In certain aspects, EGFR protein expression is detected by IHC. In certain aspects, EGFR expression is determined by IHC using a commercially available EGFR detection kit, such as the EGFR pharmDx™ kit for a Dako autostainer (Agilent), using the manufacturer’s recommendations or the commercially available IHC EGFR detection kit based on EGFR clone 113 which binds the EGFR extracellular domain (Leica, https: / / shop.leicabiosystems.com / us / ihc-ish / ihc-primary-antibodies / pid-epidermal-growth- factor-receptor). Alternatively, EGFR expression is determined using the Novocastra™ Liquid Mouse Monoclonal Antibody Epidermal Growth Factor Receptor which is based on clone EGFR.113 (Product Code: NCL-L-EGFR, Epidermal Growth Factor Receptor -IHC Primary Antibodies by leicabiosystems.com).

[0149] Briefly, the commercially available EGFR pharmDx™ IHC kit system contains reagents required to complete an IHC staining procedure for routinely-fixed, paraffin-embedded specimens. Following incubation with the primary, non-Her2, Her3 and Her4 cross-reactive, monoclonal antibody (clone 2-18C9) to human EGFR protein, this kit employs a ready-to-use visualization reagent based on dextran technology. This reagent consists of both secondary goat anti-mouse antibody molecules and horseradish peroxidase molecules linked to a common dextran polymer backbone. The enzymatic conversion of the subsequently added chromogen results in formation of a visible reaction product at the antigen site. Results are routinely assessed using a light microscope. Control slides containing two formalin-fixed, paraffin-embedded human cell lines with staining intensity scores of 2+ and 0 are provided for quality control of the kit reagent performance.

[0150] Staining intensity is established as follows: 3+ (strong staining): visible at low levels of magnification, x5 objective lens which could be confirmed at higher levels as required; 2+ (moderate staining): visible at. intermediate levels of magnification, xlO or x20 objective lenses; 1+ (weak staining): only reliably confirmable at high magnification, x40 objective lens; 0 (no staining): no staining visible at high magnification.

[0151] In certain aspects, EGFR expression is determined using immunohistochemistry (IHC) and the cancer is IHC positive for EGFR. In certain aspects, the cancer is a cancer characterized by an EGFR IHC score of 1+, 2+ or 3+. In certain aspects, the cancer is a cancer characterized by an EGFR IHC score of 1+. In certain aspects, the cancer is a cancer characterized by an EGFR IHC score of 2+. In certain aspects, the cancer is a cancer characterized by an EGFR IHC score of 3+.

[0152] In certain aspects, EGFR expression is determined using immunohistochemistry (IHC) followed by assigning an H-score for EGFR using a range of 0-300. In certain aspects, the cancer of the present disclosure is gastric cancer, esophageal cancer, gastric-esophageal-junction cancer characterized by an H-score for EGFR of more than 200 on a scale of 0-300. In certain aspects, the EGFR H score is thus > 200 up to and including 300. In certain aspects, the cancer of the present disclosure is characterized by an H-score for EGFR of more than 50 on a scale of 0-300. In certain aspects, the cancer of the present disclosure is head and neck cancer characterized by an H-score for EGFR of more than 50 on a scale of 0-300. In certain aspects, the cancer of the present disclosure is characterized by an H-score for EGFR of more than 80 on a scale of 0-300. In certain aspects, the cancer of the present disclosure is head and neck cancer characterized by an H-score for EGFR of more than 80 on a scale of 0-300.

[0153] Herein, determining the H-score to assign the EGFR expression status involves a first step of establishing intensity of membrane staining (resulting in a scoring of 0, 1+, 2+, or 3+) which is determined for each cell in a predefined field as described herein. Subsequently, the percentage of cells at each staining intensity level is calculated, and finally, an H-score is assigned using the following formula: [1 x (% cells having 1+ staining) + 2 x (% cells having 2+ staining) + 3 x (% cells having 3+ staining)] resulting in an H-score for EGFR between 0-300. As a result, the H-score gives more relative weight to higher intensity or amount of staining in a given tumor sample.

[0154] Herein, the term “ctDNA” (circulating tumor DNA) is used interchangeably with “cfDNA” (cell-free tumor DNA).

[0155] In some disclosures, the cancer expresses LGR5 and EGFR. As used herein, a cancer expresses LGR5 if the cancer comprises cells that express LGR5. A cancer cell which expresses LGR5 comprises detectable levels of RNA that codes for LGR5.

[0156] Expression can often also be detected by incubating the cell with an antibody that binds to LGR5. However, some cells do not express the protein high enough for such an antibody test. In such cases mRNA or other forms of nucleic acid sequence detection is preferred. In certain aspects, LGR5 mRNA expression is detected. In certain aspects, LGR5 detection is by Tissue MicroArray (TMA) staining. In certain aspects, LGR5 expression is determined using In-Situ Hybridization (ISH). Thus in certain aspects, the cancer is ISH positive for LGR5. In certain aspects, ISH positive means that expression is characterized by an H-score of 1 or more. The techniques for detection and scoring on the basis thereof, by TMA, ISH and IHC are each well known in the art to the person of ordinary skill and typically commercially available as a standard kit. Quantifying mRNA levels using IHC and expressing such on the basis of an H-score, such as for LGR5, can be performed using commercially available kits, such as the RNAscope1' kit from Advanced Cell Diagnostics (Hayward, CA, USA) on a staining platform such as the BondRx platform (Leica, Wetzlar, Germany). Typically, the H-score ranges from 0-400. Alternatively, LGR5 expression is determined by RNA sequencing (RNAseq) or qPCR. Thus in certain aspects, the cancer is ISH positive for LGR5. In certain aspects, the cancer cell is negative for LGR5 expression. In certain aspects, a cancer cell does not comprise detectable levels of RNA that codes for LGR5. In certain aspects, a cancer cell of the present disclosure is characterized by an H-score of between 0 and 80. In certain aspects, a cancer cell of the present disclosure is characterized by an H-score of between 0 and 50. In certain aspects, a cancer cell of the present disclosure is characterized by an H-score of between 0 and 20. In certain aspects, the cancer cell is negative for LGR5 expression.

[0157] The words cancer and tumor are used herein and typically both refer to cancer, unless otherwise specifically stated.

[0158] In certain aspects, said cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and / or the EGFR ectodomain. In certain aspects, said cancer is negative for HER2 gene copy number amplification, in particular such a cancer has a HER2 copy number which does not exceed 2.18. In certain aspects, said cancer is a microsatellite stable tumor or wherein the MSI status of said cancer is not MSLH. Said genotypes can be established using a cancer cell containing sample from a subject having mCRC or locally advanced, unresectable CRC.

[0159] MSI is the manifestation of defective DNA mismatch repair (dMMR), which leads to dramatically increased mutation rates throughout the genome, including gain and / or loss of nucleotides within repeating motifs known as microsatellite tracts, from which the entity derives its name. In certain aspects, said cancer is a microsatellite stable tumor or wherein the MSI status of said cancer is not MSLH. In certain aspects, determination of MSI tumor status is performed using ctDNA-based genotyping, such as next-generation sequencing. As an example, such NGS can be performed and include BAT-25, BAT-26, and NR-21 microsatellite loci (e.g. such as described in Umar A et al., Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst 2004;96:261-8)).

[0160] In certain aspects, microsatellite stability to establish whether a cancer is MSS or not MSLH, is established using Guardant 360 (G360) technology, such as described in Willis et al., Clin Cancer Res 2019;25:7035-45. In certain aspects, a statistical probabilistic score is evaluated across 90 MSI loci for G360. Each locus is evaluated to determine whether it has a stable / unstable status and the number of unstable loci is referred to as the sample MSI score. If this score exceeds a threshold (in particular 6 loci), the sample is reported as MSLH. In certain aspects of G360 analytical validation, a 95% sensitivity level limit of detection is 0.1% MAF (mutant allele frequency) for samples with 30 ng input and 0.4% for samples with 5 ng input.

[0161] In certain aspects, said cancer cell containing sample comprises a blood sample, a sample taken from a solid tumor, a sample taken from said cancer, circulating tumor DNA (ctDNA) or cell-free DNA (cfDNA). Nucleic acids, including genomic DNA, can be readily isolated from such samples using any commercially available kit or other suitable method.

[0162] Samples which can be used in the present methods include samples containing genomic DNA, including tissues, cells, and / or biological fluids isolated from a subject. Examples of such samples include tissues, cells, biopsies, blood, lymph, serum, plasma, urine, saliva, mucus and tears. In certain aspects, said samples may be tissue samples (such as a biopsy taken from a solid tumor or cancer of the present disclosure). In certain aspects, said samples may be obtained directly from a subject (e.g., by blood or tissue sampling). In certain aspects, said nucleic acids may be isolated and / or purified prior to assaying. In certain aspects, said cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and / or the EGFR ectodomain and / or is negative for HER2 gene copy number amplification. In certain aspects, said cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification. In certain aspects, said cancer does not have an oncogenic mutation occurring at a position selected from G12, G13 or Q61 in KRAS or NRAS; position V600 in BRAF; a position selected from V441, S464, G465 or S492 in the EGFR ectodomain, and wherein said cancer has a HER2 gene copy number which does not exceed 2.18. In certain aspects, a subject does not have a KRAS mutation selected from G12A, G12D, G12R, G12C, G12S, G12V, G13D and Q61R. In certain aspects, a subject does not have an NRAS mutation selected from G12A, G12D, G12R, G12C, G12S, G12V, G13D and Q61R. In certain aspects, a subject does not have a BRAF mutation selected from V600E and V600K. In certain aspects, a subject does not have an oncogenic missense mutation in KRAS, NRAS, BRAF. In certain aspects, a subject does not have an oncogenic missense mutation in the EGFR ectodomain.

[0163] In certain aspects, a subject of the present disclosure is characterized as a subject having had a documented best response which is either a complete response (OR), a partial response (PR), or any tumor reduction from prior anticancer treatment with a EGFR inhibitor received as monotherapy anticancer treatment.

[0164] In certain aspects, a subject of the present disclosure is characterized as a subject having had a documented best response which is either a complete response (CR), a partial response (PR), or any tumor reduction from prior anticancer treatment with a EGFR inhibitor received in combination with chemotherapy.

[0165] In certain aspects, a subject of the present disclosure is characterized as having had at least 6 months of prior anticancer treatment with an EGFR inhibitor without disease progression.

[0166] In certain aspects, a subject of the present disclosure has not received an EGFR inhibitor as anticancer treatment for at least 6 months prior to administration of a bispecific antibody of the present disclosure.

[0167] In certain aspects, the present disclosure relates to a method for classifying a subject having colorectal cancer for treatment with a bispecific antibody as mentioned herein, said method comprising establishing said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification in a cancer cell containing sample from said subject.

[0168] In certain aspects, the present disclosure relates to a method for selecting a subject having colorectal cancer for treatment with a bispecific antibody as mentioned herein, the method comprising establishing that said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for of HER2 gene copy number amplification in a cancer cell containing sample from said subject.

[0169] In certain aspects, the subject is selected for treatment or classified as being likely to respond to treatment with said antibody when said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification as determined in the cancer cell containing sample from said subject.

[0170] In certain aspects, the subject is selected for treatment or classified as being likely to respond to treatment with said bispecific antibody when said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain, and is negative for HER2 gene copy number amplification as determined in the cancer cell containing sample from the subject.

[0171] In certain aspects, the present disclosure relates to a method for selecting a subject having colorectal cancer for treatment with a bispecific antibody as mentioned herein, such as petosemtamab, the method comprising determining that said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain, and is negative for HER2 gene copy number amplification in a cancer cell containing sample from said subject.

[0172] In certain aspects, the present disclosure relates to petosemtamab for use in a method of treatment of a subject having colorectal cancer, wherein said subject is classified as being likely to respond to treatment with petosemtamab, or selected for treatment with petosemtamab, when said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification as established in a cancer cell containing sample from the subject.

[0173] In certain aspects, the present disclosure relates to petosemtamab for use in a method of treatment of a subject having colorectal cancer, wherein said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification.

[0174] In certain aspects, at least six months prior to being administered a bispecific antibody as mentioned herein, such as petosemtamab, said subject has not received any anticancer therapy against said colorectal cancer, such as an EGFR inhibitor, and / or which said colorectal cancer has progressed after having received at least two prior anticancer therapies.

[0175] In certain aspects, genotype status for KRAS, NRAS, BRAF and EGFR ectodomain is identified using a suitable assay designed to establish the respective genotype status or the HER2 gene copy number, in particular next generation sequencing performed on a ctDNA sample obtained from said subject.

[0176] In certain aspects, a treatment of the present disclosure comprises or is preceded by a step of selecting said subject on the basis of having a cancer which does not have an oncogenic mutation in KRAS, NRAS, BRAF and / or the EGFR ectodomain and / or is negative for HER2 gene copy number amplification. In certain aspects, said treatment comprises or is preceded by a step of selecting said subject on the basis of said cancer not having one or more oncogenic mutations in KRAS, NRAS, BRAF and / or the EGFR ectodomain and for said cancer not having HER2 gene copy number amplification. In certain aspects, said cancer is screened by next- generation sequencing on a ctDNA plasma sample or tumor tissue sample designed to establish the KRAS, NRAS, BRAF and EGFR ectodomain genotype and the HER2 gene copy number.

[0177] Also provided is a bispecific antibody or functional part thereof, as disclosed herein (i.e., the therapeutic agent) and a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful in the treatment of cancer. As used herein, the term "pharmaceutically acceptable" means approved by a government regulatory agency or listed in the U. S. Pharmacopeia or another generally recognized pharmacopeia for use in animals, particularly in humans, and includes any and all solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol ricinoleate, and the like. Water or aqueous solution saline and aqueous dextrose and glycerol solutions may be employed 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., intravenously, subcutaneously, intra- articularly and the like) the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0178] Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, e.g., in a liquid carrier, or suitable for reconstitution into liquid solution or suspension for intravenous administration. The compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Also included are solid preparations which are intended for conversion, shortly before use, to liquid preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.

[0179] The therapeutic agent mentioned herein can be administered according to a suitable dosage, and suitable route (e.g., intravenous, intraperitoneal, intramuscular, intrathecal or subcutaneous). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In certain aspects, a subject is administered a single dose of the binding moiety or antibody as disclosed herein. In certain aspects, the therapeutic agent will be administered repeatedly, over a course of treatment. For example, in certain embodiments, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of the therapeutic agent are administered to a subject in need of treatment. In certain aspects, administrations of the therapeutic agent may be done weekly, biweekly or monthly.

[0180] A clinician may utilize preferred dosages as warranted by the condition of the patient being treated. The dose may depend upon a number of factors, including stage of disease, etc. Determining the specific dose that should be administered based upon the presence of one or more of such factors is within the skill of the artisan. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small amounts until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. Intermittent therapy (e.g., one week out of three weeks or three out of four weeks) may also be used.

[0181] In certain aspects, treatment with a bispecific antibody of the present disclosure comprises providing the subject with an effective amount thereof. In certain aspects, said treatment with said bispecific antibody comprises providing to the subject a flat dose of 1500 mg, in particular a flat dose of 1500 mg petosemtamab. A flat dosage offers several advantages over body-surface or weight dosing as it reduces preparation time and reduces potential dose calculation mistakes. As is understood by the skilled person, the dosage can be administered over time. For example, the dosage may be administered by IV, for example with a 1-6 hour infusion, preferably a 2-4 hour infusion. In certain aspects, the bispecific antibody administered once every 2 weeks. In particular, the flat dosages disclosed herein are suitable for use in adults and / or in subjects weighing at least 35 kg. In certain aspects, said bispecific antibody comprises or is petosemtamab. In certain aspects, said bispecific antibody, in particular petosemtamab, is provided biweekly (q2w). In certain aspects, said bispecific antibody, in particular petosemtamab, is provided intravenously to said subject.

[0182] In certain aspects, a premedication regimen may be used. Such a regimen may be useful to reduce the likelihood or severity of an infusion-related reaction. Generally, a steroid such as dexamethasone and / or an antihistamine such as dexchlorpheniramine, diphenhydramine, or chlorpheniramine is administered (e.g., orally, intravenously) prior to antibody treatment.

[0183] The treatment method described herein is typically continued for as long as the clinician overseeing the patient's care deems the treatment method to be effective, i.e., that the patient is responding to treatment. Non-limiting parameters that indicate the treatment method is effective may include one or more of the following: decrease in tumor cells; inhibition of tumor cell proliferation; tumor cell elimination; progression-free survival; appropriate response by a suitable tumor marker (if applicable).

[0184] The efficacy of the treatment methods provided herein can be assessed using any suitable means. In certain aspects, the clinical efficacy of the treatment is analyzed using cancer cell number reduction as an objective response criterion. Patients, e.g., humans, treated according to the methods disclosed herein preferably experience improvement in at least one sign of cancer. In certain aspects, one or more of the following can occur: the number of cancer cells can be reduced; cancer recurrence is prevented or delayed; one or more of the symptoms associated with cancer can be relieved to some extent. In addition, in vitro assays can be used to determine the T cell mediated target cell lysis. In certain aspects, tumor assessment is based on CT-scan and / or MRI scans, see, e.g., the RECIST 1.1 guidelines (Response Evaluation Criteria in Solid Tumours) (Eisenhauer et al., 2009 Eur J Cancer 45:228-247). Such assessments generally take place every 4-8 weeks after treatment.

[0185] In certain aspects, the tumor cells are no longer detectable following treatment as described herein. In certain aspects, a subject is in partial or full remission. In certain aspects, a subject has an increased overall survival, median survival rate, and / or progression free survival.

[0186] The compounds and compositions disclosed herein are useful as therapy and in therapeutic treatments and may thus be useful as medicaments and used in a method of preparing a medicament.

[0187] In certain aspects, at the start of treatment, at least one, more than one or all of the following inclusion factors IF1-IF16 are applicable to subjects for treatment. In certain aspects, the instructions for use of a bispecific antibody of the present disclosure, such as petosemtamab, comprise any one or all of the inclusion factors IF1-IF16. In certain aspects, the subject comprises or complies with all of IF1-IF16.

[0188] IF 1. Having an age of at least 18.

[0189] IF 2. Having a histologically or cytologically confirmed solid tumor with evidence of metastatic CRC or locally advanced CRC not amenable to standard therapy with curative intent, and disease progression on the last line of therapy.

[0190] IF 3. Not having an oncogenic mutation in KRAS, NRAS, BRAF, and the EGFR ectodomain

[0191] IF 4. Not having HER2 gene copy number amplification, for instance as detected in plasma by ctDNA NGS testing.

[0192] IF 5. Having a microsatellite stable (MSS) tumor.

[0193] IF 6. Having received two, three or four lines of prior therapy in the metastatic setting, which includes 1) chemotherapy with oxaliplatin, irinotecan, and a fluoropyrimidine, and 2) targeted therapy with an anti-VEGF and an anti-EGFR inhibitor. Petosemtamab treatment would be considered first-line in the advanced setting if disease progression had occurred within 6 months of completion of adjuvant or neoadjuvant therapy.

[0194] IF 7. Having a documented best response of complete response (CR), partial response (PR), or any tumor reduction on a prior EGFR inhibitor received in any line as monotherapy or in combination with chemotherapy or having had at least 6 months of treatment with an EGFR inhibitor without disease progression.

[0195] IF 8. Not having received any EGFR inhibitor for at least 6 months prior to administration of a bispecific antibody of the present disclosure.

[0196] IF 9. Availability of a formalin-fixed paraffin-embedded (FFPE) block with sufficient tumor material or a minimum of 20 unstained slides of archival tumor tissue (such as an FFPE block). For subjects who have progressed on prior treatments, biopsies may be collected after completion of such therapies. If archival tumor tissue is not available, a tumor biopsy may be performed prior to the start of treatment.

[0197] IF 10. Being amenable for biopsy, if safe and / or feasible.

[0198] IF 11. Having a measurable disease as defined by RECIST vl.l by radiologic methods. IF 12. Having an ECOG PS score of 0 or 1.

[0199] IF 13. Having a life expectancy of at least 12 weeks, typically determined as per investigator.

[0200] IF 14. Having a left ventricular ejection fraction (LVEF) of at least 50% by echocardiogram (ECHO) or multigated acquisition (MUGA) scan.

[0201] IF 15. Having adequate organ function, such as defined by IF15.1: An absolute neutrophil count (ANC) of at least 1.5 x 109 / L;

[0202] IF15.2: Having a hemoglobin level of at least 9 g / dL

[0203] IF15.3: Having a platelets count of at least 100 x 10-VL; having serum magnesium, sodium, corrected total calcium, phosphate, and potassium levels within normal ranges (or corrected with supplements or appropriate treatment);

[0204] IF15.4: Having an alanine aminotransferase (ALT) to aspartate aminotransferase (AST) ratio of not more than 2.5 times the upper limit of normal (ULN) and total bilirubin <1.5 times ULN, with the proviso that in case a subject has known Gilbert’s syndrome, said total bilirubin is not >3.0 times ULN or direct bilirubin is not >1.5 x ULN;

[0205] IF15.5: Having an ALT / AST ratio of < 5 times ULN and total bilirubin level of <1.5 times LTLN in cases of liver involvement, with the proviso that in case a subject has known Gilbert’s syndrome, said total bilirubin is 3.0 times ULN or less, or direct bilirubin is 1.5 times ULN or less.

[0206] IF15.6: Having a serum creatinine level of <1.5 times ULN or creatinine clearance (CrCl) of >60 mL / min calculated according to the Cockroft and Gault formula or Modification of Diet in Renal Disease (MDRD) formula for patients aged >65 years.

[0207] IF15.7: Having a serum albumin level of >3 g / dL.

[0208] IF15.8: Having an international normalized ratio (INR) or a prothrombin time (PT) of <1.5 times ULN, unless said subject is receiving anticoagulant therapy and in therapeutic range of intended used anticoagulant.

[0209] IF15.9: Having an activated partial thromboplastin time (APTT) or partial thromboplastin time (PTT) of <1.5 times ULN, unless said subject is receiving anticoagulant therapy and is in therapeutic range of intended used anticoagulant.

[0210] IF 16: Willing to undergo testing for human immunodeficiency virus (HIV) if not tested within the past 6 months, wherein known HIV-positive subjects are eligible provided the cluster of differentiation 4 (CD4+) count is >300 / pL, viral load is undetectable, and the subject is currently receiving highly active antiretroviral therapy (HAART).

[0211] In certain aspects, all values for organ function measurements according to IF15 have an upper limit observed with healthy subjects.

[0212] In certain aspects, the subject for treatment complies with factors IF2, IF3, IF4, IF5, IF6, IF7, IF8, IF11, IF12, IF13, IF14, IF15 and IF16. In certain aspects, the subject for treatment complies with factors IF2, IF3, IF4, IF5, IF6, IF7 and IF8.

[0213] In certain aspects, the instructions for use of a bispecific antibody of the present disclosure, such as petosemtamab, comprise any one or all of the exclusion factors EF1-EF15. In certain aspects, at the start of treatment (i.e. when administered the first dose of a bispecific antibody of the present disclosure), at least one of the following exclusion factors EF1-EF15 is applicable to subjects for treatment:

[0214] EF 1. Having a central nervous system metastasis which is untreated or symptomatic, or requires radiation, surgery, or continued steroid therapy to control symptoms within 14 days of first administration of a bispecific antibody of the present disclosure, such as petosemtamab.

[0215] EF 2. Having a leptomeningeal involvement. EF 3. Having participated in another clinical study or treatment with any investigational drug within 4 weeks prior to the first administration of a bispecific antibody of the present disclosure, such as petosemtamab.

[0216] EF 4. Having had any systemic anticancer therapy within 4 weeks or 5 halfdives, whichever is shorter, of the first administration of a bispecific antibody of the present disclosure (such as petosemtamab), wherein for cytotoxic agents that have major delayed toxicity (eg, mitomycin C, nitrosoureas), or anticancer immunotherapies, a washout period of 6 weeks is required.

[0217] EF 5. Having a requirement for immunosuppressive medication (eg, methotrexate, cyclophosphamide).

[0218] EF 6. Having had major surgery or radiotherapy within 3 weeks of the first administration of a bispecific antibody of the present disclosure, wherein subjects who received prior radiotherapy to at least 25% of their bone marrow are not eligible, irrespective of the timing when said prior radiotherapy was received.

[0219] EF 7. Having persistent Grade >1 clinically significant toxicities related to prior antineoplastic therapies (except for alopecia), with the proviso that stable sensory neuropathy Grade <2 (according to the National Cancer Institute— Common Terminology Criteria for Adverse Events (NCI-CTCAE) v4.03) is allowed.

[0220] EF 8. Having a history of hypersensitivity reaction to any of the excipients of petosemtamab or any human protein.

[0221] EF 9. Having uncontrolled hypertension (systolic blood pressure [BP] >150 mmHg and / or diastolic BP >100 mmHg) with appropriate treatment; unstable angina; history of congestive heart failure of Class II-IV New York Heart Association (NYHA) criteria, serious cardiac arrhythmia requiring treatment (except atrial fibrillation, paroxysmal supraventricular tachycardia); or history of myocardial infarction within 6 months of administration of a bispecific antibody of the present disclosure, such as petosemtamab. EF 10. History of prior malignancies with the exception of excised cervical intraepithelial neoplasia or nonmelanoma skin cancer, or curatively treated cancer deemed at low risk for recurrence with no evidence of disease for >3 years.

[0222] EF 11. Having current dyspnea at rest of any origin, or other diseases requiring continuous oxygen therapy, including subjects with a history of interstitial lung disease (ILD) (eg, pneumonitis or pulmonary fibrosis), or evidence of ILD on baseline chest computerized tomography (CT) scan.

[0223] EF 12. Having current serious illness or medical conditions including, but not limited to, uncontrolled active infection, clinically significant pulmonary, metabolic, or psychiatric disorders.

[0224] EF 13. Having any known infectious disease, such as

[0225] • Having an active hepatitis B infection (hepatitis B surface antigen [HbsAg] positive) without receiving antiviral treatment, with the proviso that if subjects who are HbsAg positive and receive antiviral treatment with lamivudine, tenofovir, entecavir, or other antiviral agents starting at least 7 days before administration of a bispecific antibody of the present disclosure or subjects with antecedents of hepatitis B (eg, anti¬ hepatitis B core (anti-HBc) positive, HbsAg, and hepatitis B virus [HBV] DNA negative) are not excluded from treatment.

[0226] • Having a positive test for hepatitis C virus (HCV) RNA, with the proviso that if subjects for whom said HCV infection resolved spontaneously (ie, positive HCV antibodies without detectable HCV RNA), or who achieved a sustained response after antiviral treatment and show absence of detectable HCV RNA >6 months (with the use of interferon [IFN]-free regimens) or >12 months (with the use of IFN-based regimens) after cessation of antiviral treatment, are not excluded from treatment.

[0227] EF 14, Being pregnant or breastfeeding,

[0228] EF 15. Having a documented oncogenic KRAS, NRAS, BRAF, or EGFR ectodomain mutation, a HER2 gene copy number amplification, such as identified by tumor test or ctDNA test in disease history, or during central ctDNA screening.

[0229] In certain aspects, the subject for treatment complies with any one or more factors selected from the group consisting of EF1-EF15.

[0230] ECOG Performance Status Scoring Grade Definition is as followed in the art, meaning: 0 Fully active, able to carry on all pre-disease performance without restriction. 1 Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, e.g., light housework, office work. 2 Ambulatory and capable of all self-care but unable to carry out any work activities. Up and about more than 50% of waking hours. 3 Capable of only limited self-care, confined to bed or chair more than 50% of waking hours. 4 Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair. 5 Dead.

[0231] The Child- Pugh scoring, also referred to as the Child-Pugh classification, the Child- Turcotte-Pugh (CTP) calculator or the Child Criteria, herein is applied in accordance with standard clinical practice. The Child-Pugh score is determined by scoring five clinical measures of liver disease and the possibility of eventual liver failure. A score of 1, 2, or 3 is given to each measure, with 3 being the most severe. The five clinical measures are total bilirubin, serum albumin level, prothrombin time (or prolongation or INR as a time for blood to clot), ascites, and hepatic encephalopathy. Class A means 5 to 6 points, least severe liver disease and a one- to five-year survival rate of 95 percent. Class B: 7 to 9 points, moderately severe liver disease and a one- to five-year survival rate of 75 percent. Class C: 10 to 15 points, most severe liver disease and a one- to five-year survival rate of 50 percent. Per clinical measure, the following points are given. Encephalopathy: None - 1 p, Grade 1 and 2 = 2 p, Grade 3 and 4 = 3 pts. Ascites: none = 1 pt, slight = 2 pts, moderate = 3 pts. Bilirubin: under 2 mg / ml = 1 pt, 2 to 3 mg / ml = 2 pts, over 3 mg / ml = 3 pts. Albumin: greater than 3.5 mg / ml = 1 pt, 2.8 to 3,5 mg / ml = 2 pts, less than 2.8 mg / ml = 3 pts. Prothrombin Time (PT, seconds prolonged): less than 4 sec = 1 pt, 4 to 6 sec = 2 pts, over 6 sec = 3 pts. Alternatively, the International Normalized Ratio (INR) will be used as a substitute for PT, with INR under 1.7 = 1 pt, INR 1.7 to 2.2 = 2 pts, INR above 2.2 = 3 pts.

[0232] Herein, calculation of renal clearance is according to Cockcroft & Gault formula for subjects aged <65 years: Male=1.25 x weight (kg) x (140-age) / serum creatinine (pmol / L). Female=1.04 x weight (kg) x (140-age) / serum creatinine (pmol / L).

[0233] Herein, calculation of MDRD (Modification of Diet in Renal Disease) is according to the following formula: Patients aged >65 years. For a male subject older than 65 years of age, calculation is as follows: Male=186 x (serum creatinine (pmol / L) x 0,0113)-l,154 x age- 0,203. For dark skinned subjects, the outcome is multiplied by 1.21. For female subjects, the outcome is multiplied by 0.742.

[0234] In certain aspects, said subject exhibits adequate organ function. In certain aspects, herein adequate organ function includes

[0235] an absolute neutrophil count (ANC) >1.5 x 109 / L;

[0236] a hemoglobin level of >9 g / dL;

[0237] platelet count of >100 x 109 / L;

[0238] serum magnesium, sodium, corrected total calcium, phosphate, and potassium within normal ranges (or corrected with supplements or appropriate treatment);

[0239] an alanine aminotransferase (ALT) to aspartate aminotransferase (AST) ratio of <2.5 x upper limit of normal (ULN) and total bilirubin <1.5 x ULN; serum creatinine <1.5 x ULN or creatinine clearance (CrCl) >60 mL / min calculated according to the Cockroft and Gault formula or Modification of Diet in Renal Disease (MDRD) formula for patients aged >65 years;

[0240] serum albumin >3 g / dL;

[0241] International normalized ratio (INR) or prothrombin time (PT) <1.5 x ULN unless patient is receiving anticoagulant therapy and in therapeutic range of intended used anticoagulant;

[0242] activated partial thromboplastin time (APTT) or partial thromboplastin time (PTT) <1.5 x ULN unless patient is receiving anticoagulant therapy and is in therapeutic range of intended used anticoagulant.

[0243] In certain aspects, adequate organ function does not include total bilirubin levels of >3.0 x ULN or direct bilirubin >1.5 x ULN if related to known Gilbert’s syndrome. In certain aspects, adequate organ functions does not include an ALT / AST ratio of <5 x ULN and total bilirubin <1.5 x ULN in cases of liver involvement or includes adequate organ function when due to known Gilbert’s syndrome when total bilirubin <3.0 x ULN or direct bilirubin <1.5 x ULN. In certain aspects, said adequate organ function is adequate human organ function.

[0244] All documents and references, including Genbank entries, patents and published patent applications, and websites, described herein are each expressly incorporated herein by reference to the same extent as if were written in this document in full or in part.

[0245] For the purpose of clarity and a concise description of features are described herein as part of the same or separate parts of the disclosure, however, it will be appreciated that the scope of the disclosure may include preferred aspects having combinations of all or some of the features described.

[0246] The disclosure is now described by reference to the following examples, which are illustrative only, and are not intended to limit the present disclosure. While the disclosure has been described in detail and with reference to specific aspects thereof, it will be apparent to one of skill in the art that various changes and modifications can be made thereto without departing from the spirit and scope thereof. List of clauses

[0247] 1. A bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises HCDR1, HCD R2 and HCDR3 amino acid sequences of the MF3755 heavy chain variable region as depicted in Figure 8a, and the second variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF5816 heavy chain variable region as depicted in Figure 8a, for use in the treatment of colorectal cancer in a subject, which cancer has progressed after having received at least two prior anticancer therapies.

[0248] 2. Use of a bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF3755 heavy chain variable region, as depicted in Figure 8a and the second variable domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF5816 heavy chain variable region, as depicted in Figure 8a, in the manufacture of a medicament for treating colorectal cancer in a subject, which cancer has progressed after having received at least two prior anticancer therapies.

[0249] 3. A method of treating a subject having colorectal cancer, wherein said subject has progressed after having received at least two prior anticancer therapies, the method comprising providing the subject with an effective amount of a bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF3755 heavy chain variable region, as depicted in Figure 8a and the second variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF5816 heavy chain variable region, as depicted in Figure 8a.

[0250] 4. The bispecific antibody, or the use or the method of any one of the preceding clauses, wherein said bispecific antibody is petosemtamab or a functional part thereof.

[0251] 5. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein at least six months prior to being administered said bispecific antibody or functional part thereof, said subject has not received any anticancer therapy against said cancer, such as a therapy comprising an EGFR inhibitor.

[0252] 6. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said cancer is metastatic CRC.

[0253] 7. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said cancer is locally advanced, unresectable CRC. 8. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and / or the EGFR ectodomain.

[0254] 9. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said cancer is negative for HER2 gene copy number amplification, in particular a cancer that has a HER2 copy number not exceeding 2.18.

[0255] 10. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said cancer is a microsatellite stable tumor or wherein the MSI status of said cancer is not MSI-H.

[0256] 11. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said subject received two, three or four lines of said prior anticancer therapy.

[0257] 12. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said prior anticancer therapy comprises chemotherapy, such as oxaliplatin, irinotecan and / or a fluoropyrimidine, and / or therapy with an anti-VEGF agent and / or an anti- EGFR inhibitor.

[0258] 13. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said subject received two, three or four lines of said prior anticancer therapy comprising therapy with an anti-VEGF agent and / or an anti-EGFR inhibitor.

[0259] 14. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said cancer expresses EGFR and / or LGR5.

[0260] 15. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said subject has previously shown clinical efficacy to said prior anticancer therapy comprising a complete response (OR), a partial response (PR), or any tumor reduction to a prior EGFR inhibitor, such as received as monotherapy or in combination with chemotherapy.

[0261] 16. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said subject has received said prior anticancer therapy for a period of at least six months with an EGFR inhibitor without showing evidence of disease progression.

[0262] 17. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said subject exhibits adequate organ function.

[0263] 18. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein the subject is a mammal, such as a human. 19. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said treatment with said bispecific antibody comprises providing the subject with an effective amount thereof.

[0264] 20. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said treatment with said bispecific antibody comprises providing to the subject a flat dose of 1500 mg, in particular a flat dose of 1500 mg petosemtamab.

[0265] 21. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said bispecific antibody, in particular petosemtamab, or a functional part thereof, is provided biweekly (q2w).

[0266] 22. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said bispecific antibody, in particular petosemtamab, or a functional part thereof, is provided intravenously to said subject.

[0267] 23. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and / or the EGFR ectodomain and / or is negative for HER2 gene copy number amplification.

[0268] 24. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said treatment comprises or is preceded by a step of selecting said subject on the basis of having a cancer which does not have an oncogenic mutation in KRAS, NRAS, BRAF and / or the EGFR ectodomain and / or is negative for HER2 gene copy number amplification.

[0269] 25. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said treatment comprises or is preceded by a step of selecting said subject on the basis of said cancer not having one or more oncogenic mutations in KRAS, NRAS, BRAF and / or the EGFR ectodomain and for said cancer not having HER2 gene copy number amplification.

[0270] 26. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said cancer does not have an oncogenic mutation occurring at a position selected from G12, G13 or Q61 in KRAS or NRAS; position V600 in BRAF; a position selected from V441, S464, G465 or S492 in the EGFR ectodomain, and wherein said cancer has a HER2 gene copy number which does not exceed 2.18.

[0271] 27. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein said cancer is screened by next-generation sequencing on a ctDNA plasma sample or tumor tissue sample designed to establish the KRAS, NRAS, BRAF and EGFR ectodomain genotype and the HER2 gene copy number. 28. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein the primary tumor sidedness of said colorectal cancer is left-sidedness CRC.

[0272] 29. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding clauses, wherein the primary CRC tumor sidedness of said colorectal cancer is the left-sided colon (or distal colon) as characterized by including the last one-third of the transverse colon, the descending colon, the sigmoid colon, and the rectum and / or originates from the hindgut and is supplied by the inferior mesenteric artery.

[0273] 30. The bispecific antibody or functional part thereof, or the use or the method of any one of clauses 1-27, wherein the primary tumor sidedness of said colorectal cancer is right-sidedness CRC.

[0274] 31. The bispecific antibody or functional part thereof, or the use or the method of any one of clauses 1-27, wherein the primary CRC tumor sidedness of said colorectal cancer is the right- sided colon (or proximal colon) as characterized by including the cecum, the ascending colon, and the proximal two-thirds of the transverse colon and / or originates from the midgut and is supplied by the superior mesenteric artery.

[0275] 32. A method for classifying a subject having colorectal cancer for treatment with the bispecific antibody of clause 1, said method comprising establishing said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification in a cancer cell containing sample from said subject.

[0276] 33. A method for selecting a subject having colorectal cancer for treatment with the bispecific antibody of clause 1, the method comprising establishing that said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for of HER2 gene copy number amplification in a cancer cell containing sample from said subject.

[0277] 34. The method of clause 32 or 33, wherein the subject is selected for treatment or classified as being likely to respond to treatment with said antibody when said colorectal cancer not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification as determined in the cancer cell containing sample from said subject.

[0278] 35. A method for selecting a subject having colorectal cancer for treatment with petosemtamab, the method comprising determining that said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain, and is negative for HER2 gene copy number amplification in a cancer cell containing sample from said subject. 36. The method of any one of clauses 29-33, wherein the subject is selected for treatment or classified as being likely to respond to treatment with said bispecific antibody when said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain, and is negative for HER2 gene copy number amplification as determined in the cancer cell containing sample from the subject.

[0279] 37. Petosemtamab for use in a method of treatment of a subject having colorectal cancer, wherein said subject is classified as being likely to respond to treatment with petosemtamab, or selected for treatment with petosemtamab, when said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification as established in a cancer cell containing sample from the subject.

[0280] 38. Petosemtamab for use in a method of treatment of a subject having colorectal cancer, wherein said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification.

[0281] 39. The method, use or petosemtamab according to any one of clauses 32-38, wherein at least six months prior to being administered petosemtamab, said subject has not received any anticancer therapy against said colorectal cancer, such as an EGFR inhibitor, and / or which said colorectal cancer has progressed after having received at least two prior anticancer therapies.

[0282] 40. The method, use or petosemtamab according to any one of clauses 32-39, wherein the genotype status for KRAS, NRAS, BRAF and EGFR ectodomain is identified using a suitable assay designed to establish the respective genotype status, or the HER2 gene copy number, in particular wherein said genotype status is identified using next generation sequencing performed on a ctDNA sample obtained from said subject.

[0283] 41. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the first variable domain comprises a heavy chain variable domain that comprises a HCDR1 comprising the animo acid NYAMN, a HCDR2 comprising the amino acid sequence WINANTGDPTYAQGFTG, and a HCDR3 comprising the amino acid sequence ERFLEWLHFDY, wherein the second variable domain comprises a heavy chain variable domain that comprises a HCDR1 comprising the amino acid sequence SYTMN, HCDR2 comprising the amino acid sequence WINTDTGDPTYAQGFTG, and HCDR3 comprising the amino acid sequence GDCDSTSCYRYSYGYEDY, and wherein both variable domains comprise a light chain variable domain which comprises an LCDR1 comprising the amino acid sequence QSISSY, an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence QQSYSTP or an LCDR3 comprising the amino acid sequence QQSYSTPPT.

[0284] 42. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the cancer is positive for EGFR expression. 43. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the cancer is characterized by an IHC score for EGFR of 2+ or 3+.

[0285] 44. The use or method according to any one of the preceding clauses, wherein the cancer is characterized by an H-score for EGFR of more than 50 on a scale of 0-300.

[0286] 45. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said antibody is an IgG antibody, such as an IgGl.

[0287] 46. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said antibody comprises an Fc region, in particular an ADCC enhanced Fc region.

[0288] 47. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said antibody comprises an Fc region, in particular an afucosylated Fc region.

[0289] 48. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said first variable domain comprises a heavy chain variable region that comprises the CDR1, CDR2 and CDR3 sequences of the VH of MF3370; MF3755;

[0290] MF4280 or MF4289 as depicted in Figure 8b, with at most three, or at most two, or at most one amino acid substitution.

[0291] 49. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said first variable domain comprises a heavy chain variable region that comprises the CDR1, CDR2 and CDR3 sequences of the VH of MF3370; MF3755;

[0292] MF4280 or MF4289 as depicted in Figure 8b.

[0293] 50. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said first variable domain comprises the heavy chain variable region that comprises the CDR1, CDR2 and CDR3 sequences of the VH of MF3755 as depicted in Figure 8b.

[0294] 51. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said first variable domain comprises a heavy chain variable region that comprises the amino acid sequence of MF3370; MF3755; MF4280; MF4289 as depicted in Figure 8a, having at most 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or having 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain of MF3370; MF3755; MF4280 or MF4289.

[0295] 52 The use, method or bispecific antibody according to any one of the preceding- clauses, wherein said first variable domain comprises a heavy chain variable region that comprises the sequence of MF3370; MF3755; MF4280 or MF4289 as depicted in Figure 8a. 53. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said first variable domain comprises the heavy chain variable region that comprises the sequence of MF3755 as depicted in Figure 8a.

[0296] 54. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said second variable domain does not block the binding of RSPO to LGR5.

[0297] 55. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said second variable domain specifically binds an extracellular part of LGR5.

[0298] 56. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said cancer is positive for LGR5 expression.

[0299] 57. The use, method or bispecific antibody according to clause 54, wherein positive LGR5 expression is established by detection through mRNA sequencing, cDNA sequencing, Tissue MicroArray (TMA) staining, In- Situ Hybridization (ISH) or by incubating a cancer cell with an antibody that binds to LGR5.

[0300] 58. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the cancer is ISH positive for LGR5 as characterized by an H-score of 1 or more, on a scale from 0-400.

[0301] 59. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said second variable domain comprises a heavy chain variable region comprising the CDR1, CDR2 and CDR3 sequences of a VH of MF5790; MF5803;

[0302] MF5805; MF5808; MF5809; MF5814; MF5816; MF5817 or MF5818 as depicted in Figure 8b with at most three, or at most two, or at most one amino acid substitution.

[0303] 60. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said second variable domain comprises a heavy chain variable region that comprises the CDR1, CDR2 and CDR3 sequences of a VH of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in Figure 8b.

[0304] 61. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said second variable domain comprises the heavy chain variable region that comprises the CDR1, CDR2 and CDR3 sequences of the VH of MF5816 as depicted in Figure 8b.

[0305] 62. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said second variable domain comprises a heavy chain variable region comprising the amino acid sequence of a VH chain of MF5790; MF5803; MF5805;

[0306] MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in Figure 8a having at most 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or having 1, 2, 3, 4 or 5 amino acid insertions, deletions, substitutions or a combination thereof with respect to the VH chain of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818.

[0307] 63. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said second variable domain comprises a heavy chain variable region comprising the sequence of a VH chain of MF5790; MF5803; MF5805; MF5808; MF5809; MF5814; MF5816; MF5817; or MF5818 as depicted in Figure 8a.

[0308] 64. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said second variable domain comprises the heavy chain variable region comprising the sequence of VH chain MF5816 as depicted in Figure 8a.

[0309] 65. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said bispecific antibody comprises petosemtamab.

[0310] 66. The use, method or bispecific antibody according to any one of the preceding- clauses, wherein said bispecific antibody comprises a light chain variable region.

[0311] 67. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said bispecific antibody comprises a common light chain variable region.

[0312] 68. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said bispecific antibody comprises a light chain variable region comprising the CDR1, CDR2 and CDR3 sequences of a VL as depicted in Figure 9a.

[0313] 69. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said bispecific antibody comprises a light chain variable region comprising the CDR1, CDR2 and CDR3 sequences, according to IMGT, of the IGKV1-39 / jkl VL as depicted in Figure 9a.

[0314] 70. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said bispecific antibody comprises the light chain variable domain of the IGKVl-39 / jklVL as depicted in Figure 9a.

[0315] 71 The use, method or bispecific antibody according to any one of the preceding clauses, wherein said bispecific antibody is capable of complement activation.

[0316] 72. The use, method or bispecific antibody according to any one of the preceding-clauses, wherein said bispecific antibody is capable of exhibiting Complement-Dependent Cytoxicity (CDC).

[0317] 73. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the amino acid insertions, deletions, substitutions or a combination thereof are not in the CDRl, CDR2 and CDR3 sequences. 74. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said subject is administered a biweekly infusion of petosemtamab of 1500 mg.

[0318] 75 The use, method or bispecific antibody according to any one of the preceding clauses, wherein said prior chemotherapy comprises oxaliplatin, irinotecan and 5-FU.

[0319] 76. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said prior 5-FU is administered orally or intravenously.

[0320] 77. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said prior anti-VEGF agent comprises ranibizumab, aflibercept, brolucizumab, bevacizumab, and faricimab.

[0321] 78. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said prior anti-VEGF agent comprises bevacizumab.

[0322] 79. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said prior anti-VEGF agent comprises aflibercept.

[0323] 80. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said prior anti-EGFR inhibitor comprises gefitnib, cetuximab, erlotinib, and trastuzumab.

[0324] 81. The use, method or bispecific antibody according to any one of the preceding clauses, wherein said prior anti-EGFR inhibitor comprises an antibody such as cetuximab, panitumumab or trastuzumab.

[0325] 82. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after prior anticancer treatment which comprises or consists of FOLFIRINOX and panitumumab.

[0326] 83. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after prior anticancer treatment which comprises or consists of FOLFOX and panitumumab.

[0327] 84 The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after prior anticancer treatment which comprises or consists of capecitabine and bevacizumab.

[0328] 85. The use, method or bispecific antibody according to any one of the preceding- clauses, wherein the subject has progressed after prior anticancer treatment which comprises or consists of FOLFIRI and aflibercept. 86. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after prior anticancer treatment which comprises or consists of FOLFIRINOX, panitumumab and FOLFOX.

[0329] 87. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after prior anticancer treatment which comprises or consists of FOLFOX, panitumumab, capecitabine, bevacizumab and aflibercept.

[0330] 88. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFOX and bevacizumab, and which is followed by a second prior anticancer treatment which comprises cetuximab and FOLFIRI.

[0331] 89. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first anticancer treatment comprises a combination of FOLFOX and bevacizumab, and which is followed by a second prior anticancer treatment which comprises panitumumab and FOLFIRI.

[0332] 90. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFOX and cetuximab, and which is followed by a second prior anticancer treatment which comprises bevacizumab and FOLFIRI.

[0333] 91. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFOX and panitumumab, and which is followed by a second prior anticancer treatment which comprises bevacizumab and FOLFIRI.

[0334] 92. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFIRI and cetuximab, and which is followed by a second prior anticancer treatment which comprises bevacizumab and FOLFOX.

[0335] 93. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFIRI and panitumumab, and which is followed by a second prior anticancer treatment which comprises bevacizumab and FOLFOX. 94. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFIRI and bevacizumab, and which is followed by a second prior anticancer treatment which comprises cetuximab and FOLFOX.

[0336] 95. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFIRI and bevacizumab, and which is followed by a second prior anticancer treatment which comprises panitumumab and FOLFOX.

[0337] 96. The use, method or bispecific antibody according to any one of the preceding clauses, wherein the subject received prior neoadjuvant and / or adjuvant therapy.

[0338] EXAMPLES

[0339] As used herein “MFXXXX” wherein X is independently a numeral 0-9, refers to a Fab comprising a variable domain wherein the VH has the amino acid sequence identified by the 4 digits depicted in Figure 8. Unless otherwise indicated the light chain variable region of the variable domain typically has a sequence of Figure 9. The light chain in the examples has a sequence as depicted in Figure 9a (VL IGKVl-39 / jkl) and Figure 9b (CL). “MFXXXX VH” refers to the amino acid sequence of the VH identified by the 4 digits. The MF further comprises a constant region of a light chain and a constant region of a heavy chain that normally interacts with a constant region of a light chain. The VH / variable region of the heavy chains differs and typically also the CH3 region, wherein one of the heavy chains has a KK mutation of its CH3 domain and the other has the complementing DE mutation of its CH3 domain (see for reference PCT / NL2013 / 050294 (published as WO2013 / 157954) and Figure lOd and lOe. Bispecific antibodies in the examples have an Fc tail with a KK / DE CH3 heterodimerization domain, a CH2 domain and a CHI domain as indicated in Figure 10, a common light chain as indicated in Figure 9a (i.e. IGKVl-39 / jkl) and 9b and a VH as specified by the MF number. For example, a bispecific antibody indicated by MF3755 xMF5816 has the above general sequences and a variable domain with a VH with the sequence of MF3755 and a variable domain with a VH with the sequence of MF5816.

[0340] The amino acid sequences of the various heavy chain variable regions (VH) are indicated in Figure 8a. Bispecific antibodies EGFR / LGR5, MF3755xMF5816; comprising heavy chain variable regions MF3755 and MF5816 and a common light chain and including modifications for enhanced ADCC from afucosylation, among other LGR5 and EGFR combinations as depicted in Figure 8a have been shown to be effective in WO2017 / 069628.

[0341] The following bispecific antibodies are suitable for use in Examples describing the use of a bispecific antibody that binds EGFR and LGR5 and for use in the methods of the disclosure: MF3370xMF5790, MF3370x5803, MF3370x5805, MF3370x5808, MF3370x5809, MF3370x5814, MF3370x5816, MF3370x5817, MF3370x5818, MF3755xMF5790, MF3755x5803, MF3755x5805, MF3755x5808, MF3755x5809, MF3755x5814, MF3755x5816, MF3755x5817, MF3755x5818, MF4280xMF5790, MF4280x5803, MF4280x5805, MF4280x5808, MF4280x5809, MF4280x5814, MF4280x5816, MF4280x5817, MF4280x5818, MF4289xMF5790, MF4289x5803, MF4289x5805, MF4289x5808, MF4289x5809, MF4289x5814, MF4289x5816, MF4289x5817, and MF4289x5818, as well as petosemtamab. Each bispecific antibody comprises two VH as specified by the MF numbers capable of binding EGFR and LGR5 respectively, further comprises an Fc tail with a KK / DE CH 3 heterodimerization domain (Figure lOd, lOe respectively), a CH2 domain (Figure 10c) and a CHI domain (Figure 10a), a common light chain (IGKV 1-39 / jk 1 of Figure 9a and the CL of Figure 9b).

[0342] Example 1. Dose expansion, and efficacy of single-agent petosemtamab for 3L+ mCRC patients.

[0343] STUDY DESIGN

[0344] This is a FIH Phase 1 / 2 open-label multicenter study combining an initial dose escalation part with a dose expansion part. The initial dose escalation part was completed, and the preliminary RP2D was established at 1500 mg Q2W. Single-agent cohorts of selected solid tumor indications for which there is evidence of EGFR dependency and potential sensitivity to EGFR inhibition will be evaluated in the dose expansion part of the study. The eligible solid tumor indications in this single-agent nonrandomized cohort include patients with solid tumors with evidence of metastatic or locally advanced disease not amenable to standard therapy with curative intent, and disease progression on the last line of therapy. Also eligible are patients with third-line and later (3L+) mCRC and no tumoral oncogenic mutations in Kirsten rat sarcoma (KRAS), NRAS, B-rapidly accelerated fibrosarcoma (BRAF), the EGFR ectodomain, or human epidermal growth factor receptor 2 (HER2) amplification, as determined by next generation sequencing (NGS)-based central assessment of plasma circulating tumor DNA (ctDNA).

[0345] Patients must sign a screening informed consent form (IGF). When the ICF is signed, a screening period of 28 days will start. All screening procedures are to take place within 28 days prior to the patient's first dose.

[0346] Petosemtamab will be administered IV as a flat dose over an infusion period of 2 to approximately 6 h, Q2W, with 4-week cycles (28 days); 1500 mg Q2W is the primary dose level under investigation in the dose expansion cohorts, with demonstration of clinical activity.

[0347] STUDY POPULATION

[0348] Inclusion criteria

[0349] Patients must fulfill the following requirements to enter the study: 1. Willing and able to provide signed ICF prior to initiation of any study procedures. Patients unable to provide their own consent will not be eligible to participate in the study.

[0350] 2. Age >18 years at signing of ICF.

[0351] 3. Histologically or cytologically confirmed solid tumors with evidence of metastatic or locally advanced disease not amenable to standard therapy with curative intent, and disease progression on the last line of therapy:

[0352] Third line + mCRC patients must have

[0353] • No oncogenic missense mutations in KRAS, NRAS, BRAF, or EGFR ectodomain, and no HER2 gene copy number amplification, as detected in plasma by ctDNA NGS central testing performed during screening.

[0354] • A microsatellite stable (MSS) tumor.

[0355] • Received >2 and no more than 4 lines of prior therapy in the metastatic setting including 1) chemotherapy with oxaliplatin, irinotecan, and a fluoropyrimidine, and 2) targeted therapy with an anti-VEGF and an anti-EGFR inhibitor. Petosemtamab treatment would be considered first-line in the advanced setting if disease progression had occurred within 6 months of completion of adjuvant or neoadjuvant therapy.

[0356] Patients with prior exposure to EGFR inhibitors should have shown the following criteria:* 1) a documented best response of complete response (CR), partial response (PR), or any tumor reduction on a prior EGFR inhibitor received in any line as monotherapy or in combination with chemotherapy; 2) >6 months of treatment with an EGFR inhibitor without progression; and 3) an interval of >6 months since the last administration of EGFR inhibitor at the time of study treatment.

[0357] 4. Able to provide a formalin-fixed paraffin-embedded (FFPE) block with sufficient tumor material or a minimum of 20 unstained slides of archival tumor tissue (FFPE blocks are preferred). For patients who have progressed on prior treatments, it is preferred that biopsies were collected after completion of such therapies. If archival tumor tissue is not available, a tumor biopsy may be performed prior to the start of treatment.

[0358] 5. Amenable for biopsy (if safe / feasible).

[0359] 6. Measurable disease as defined by RECIST vl.l by radiologic methods.

[0360] 7. ECOG PS of O or 1.

[0361] 8. Life expectancy >12 weeks, as per investigator.

[0362] 9. Left ventricular ejection fraction (LVEF) >50% by echocardiogram (ECHO) or multigated acquisition (MUGA) scan.

[0363] 10. Adequate organ function, as defined by:

[0364] • Absolute neutrophil count (ANC) >1.5 x 109 / L.

[0365] • Hemoglobin >9 g / dL.

[0366] • Platelets >100 x 109 / L.

[0367] • Serum magnesium, sodium, corrected total calcium, phosphate, and potassium within normal ranges (or corrected with supplements or appropriate treatment).

[0368] • Alanine aminotransferase (ALT), aspartate aminotransferase (AST) <2.5 x upper limit of normal (ULN) and total bilirubin <1.5 x ULN (unless due to known Gilbert’s syndrome who are excluded if total bilirubin >3.0 x ULN or direct bilirubin >1.5 x ULN); in cases of liver involvement, ALT / AST <5 x ULN and total bilirubin <1.5 x ULN will be allowed, unless due to known Gilbert’s syndrome when total bilirubin <3.0 x ULN or direct bilirubin <1.5 x ULN will be allowed. • Serum creatinine <1.5 x ULN or creatinine clearance (CrCl) >60 mL / min calculated according to the Cockroft and Gault formula or Modification of Diet in Renal Disease (MDRD) formula for patients aged >65 years.

[0369] • Serum albumin >3 g / dL.

[0370] • International normalized ratio (INR) or prothrombin time (PT) <1.5 x ULN unless patient is receiving anticoagulant therapy and in therapeutic range of intended used anticoagulant.

[0371] • Activated partial thromboplastin time (APTT) or partial thromboplastin time (PTT) <1.5 x ULN unless patient is receiving anticoagulant therapy and is in therapeutic range of intended used anticoagulant.

[0372] 11. Willing to undergo testing for human immunodeficiency virus (HIV) if not tested within the past 6 months. Known HIV-positive patients are eligible provided the cluster of differentiation 4 (CD4+) count is >300 / pL, viral load is undetectable, and the patient is currently receiving highly active antiretroviral therapy (HAART).

[0373] Exclusion Criteria

[0374] The presence of any of the following criteria excludes a patient from participating in the study:

[0375] 1. Central nervous system metastases that are untreated or symptomatic, or require radiation, surgery, or continued steroid therapy to control symptoms within 14 days of study entry.

[0376] 2. Known leptomeningeal involvement.

[0377] 3. Participation in another clinical study or treatment with any investigational drug within 4 weeks prior to study entry.

[0378] 4. Any systemic anticancer therapy within 4 weeks or 5 half-lives, whichever is shorter, of the first dose of study treatment. For cytotoxic agents that have major delayed toxicity (eg, mitomycin C, nitrosoureas), or anticancer immunotherapies, a washout period of 6 weeks is required.

[0379] 5. Requirement for immunosuppressive medication (eg, methotrexate, cyclophosphamide).

[0380] 6. Major surgery or radiotherapy within 3 weeks of the first dose of study treatment. Patients who received prior radiotherapy to >25% of bone marrow are not eligible, irrespective of when it was received.

[0381] 7. Persistent Grade >1 clinically significant toxicities related to prior antineoplastic therapies (except for alopecia); stable sensory neuropathy Grade <2 National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE) v4.03 is allowed.

[0382] 8. History of hypersensitivity reaction to any of the excipients of petosemtamab, human proteins, or any non-IMP treatment required for this study.

[0383] 9. Uncontrolled hypertension (systolic blood pressure [BP] >150 mmHg and / or diastolic BP >100 mmHg) with appropriate treatment; unstable angina; history of congestive heart failure of Class II-IV New York Heart Association (NYHA) criteria, or serious cardiac arrhythmia requiring treatment (except atrial fibrillation, paroxysmal supraventricular tachycardia); or history of myocardial infarction within 6 months of study entry. 10. History of prior malignancies with the exception of excised cervical intraepithelial neoplasia or nonmelanoma skin cancer, or curatively treated cancer deemed at low risk for recurrence with no evidence of disease for >3 years.

[0384] 11. Current dyspnea at rest of any origin, or other diseases requiring continuous oxygen therapy, including patients with a history of interstitial lung disease (ILD) (eg, pneumonitis or pulmonary fibrosis), or evidence of ILD on baseline chest computerized tomography (CT) scan.

[0385] 12. Current serious illness or medical conditions including, but not limited to, uncontrolled active infection, clinically significant pulmonary, metabolic, or psychiatric disorders.

[0386] 13. Patients with known infectious diseases:

[0387] • Active hepatitis B infection (hepatitis B surface antigen [HbsAg] positive) without receiving antiviral treatment. Note: Patients who are HbsAg positive must receive antiviral treatment with lamivudine, tenofovir, entecavir, or other antiviral agents, starting at least >7 days before the initiation of study treatment. Patients with antecedents of hepatitis B (eg, anti-hepatitis B core (anti-HBc) positive, HbsAg, and hepatitis B virus [HBV] DNA negative) are eligible.

[0388] • Positive test for hepatitis C virus (HCV) RNA. Note: Patients in whom HCV infection resolved spontaneously (ie, positive HCV antibodies without detectable HCV RNA), or who achieved a sustained response after antiviral treatment and show absence of detectable HCV RNA >6 months (with the use of interferon [IFN]-free regimens) or >12 months (with the use of IFN-based regimens) after cessation of antiviral treatment, are eligible.

[0389] 14. Pregnant or breastfeeding patients; patients of childbearing potential must use highly effective contraception methods prior to study entry, for the duration of study participation, and for 6 months after the last dose of petosemtamab.

[0390] 15. A documented oncogenic KRAS, NRAS, BRAF, or EGFR ectodomain mutation, or HER2 gene copy number amplification identified by tumor test or ctDNA test in disease history, or during central ctDNA screening.

[0391] Screening of 3L+ mCRC Patients

[0392] For 3L+ mCRC patients, central screening by plasma ctDNA NGS testing during the screening period is required to exclude somatic oncogenic mutations in KRAS / NRAS (G12, G13, Q61), BRAF (V600), and EGFR ectodomain (V441, S464, G465, S492), and to exclude HER2 gene copy number amplification. If any of these variants were detected historically in tumor tissue or ctDNA, the patient will not be considered for screening. Any other oncogenic mutations or copy number variations associated with resistance to antibody-based EGFR inhibition should also be reported, although these do not preclude eligibility. Results of ctDNA NGS central testing must be available prior to performing any other screening assessments, unless otherwise approved by Sponsor. For patients with confirmed molecular status available by local ctDNA NGS testing, central confirmation is required to confirm eligibility, however screening procedures may be performed in parallel with central ctDNA NGS testing.

[0393] Central screening by plasma ctDNA NGS testing during the screening period is required to exclude the presence of all of the following somatic mutations in oncogenic drivers associated with resistance to antibody-based EGFR inhibition: KRAS missense alterations: any KRAS alteration with protein effect in G12, G13, or Q61; NRAS missense alterations: any NRAS alteration with protein effect in G12, G13, or Q61;

[0394] BRAF missense alterations: any BRAF alteration with protein effect in V600; EGFR ectodomain missense alterations: any EGFR alteration with protein effect in V441, S464, G465, or S492 and HER2 gene copy number amplification. If any of these variants were detected historically in tumor tissue or ctDNA, the patient will not be considered for screening.

[0395] Petosemtamab Administration (IMP)

[0396] Petosemtamab is a bispecific human full-length immunoglobulin G1 (IgGl) antibody with enhanced antibody- dependent cell-mediated cytotoxicity (ADCC) activity targeting EGFR and LGR5. Petosemtamab is composed of 2 identical common light chains and 2 different heavy chains (HC1 and HC2), each containing a different antigen-binding fragment (Fab) (HC1, anti- EGFR and HC2, anti-LGR5). Petosemtamab is produced by recombinant expression in a suitable production cell line. Petosemtamab is administered by IV infusion Q2W. The dose level under investigation is 1500 mg Q2W, with demonstration of clinical activity. Infusions must be administered IV over approximately 6 h for the Cycle 1 Day 1 infusion. Subsequent infusions after Cycle 1 Day 1 can be reduced to 2 h (±15 min) at the investigator’s discretion and in the absence of infusion-related reactions. A cycle is considered 4 weeks. For each patient, a 1 h observation period will be implemented following the end of infusion (EOI).

[0397] Treatment Duration

[0398] Study treatment will be administered until disease progression (as per RECIST vl.l), unacceptable toxicity, withdrawal of patient consent, patient noncompliance, investigator decision (eg, clinical deterioration), or petosemtamab interruption >6 consecutive weeks. Upon discontinuation of study treatment, patients will have an End of Treatment (EOT) visit as close to the treatment discontinuation date as possible.

[0399] Patients will subsequently be followed up for safety data collection >35 ±5 days after treatment discontinuation at the Safety Follow Up (SFU) visit, or before the initiation of a new anticancer treatment, whichever comes first.

[0400] CONCOMITANT MEDICATIONS

[0401] Key permitted or prohibited medications are listed as follows.

[0402] Permitted: All medication necessary for the patient’s safety and wellbeing, and which is not expected to interfere with evaluation of the study drug, may be given at the investigator’s discretion. Concurrent radiation treatment during this study for symptom control without evidence of progression, only with authorization of the Sponsor.

[0403] Prohibited: Concomitant chronic oral corticosteroids (>10 mg / day prednisone equivalent), tumor necrosis factor (TNF)-alpha inhibitors, anti-T-cell antibodies, or other immunosuppressive medication. Any investigational drug or other anticancer therapy during the study. Major surgery or radiotherapy without Sponsor consent unless in emergency. Prior radiotherapy to >25% of bone marrow.

[0404] The primary objective is to determine the ORR per RECIST vl.l as assessed by investigator review and endpoint is the ORR. Secondary objectives include the objective response rate (ORR), duration of response (DOR), progression free survival (PFS). ORR: the proportion of patients with BOR of CR or PR in patients with measurable disease at baseline. ORR is defined as a CR or PR (RECIST vl.l) on 2 consecutive occasions >4 weeks apart. DOR: the time from the date of initial confirmed response (CR or PR) until the date of progression or death due to any cause. PFS: the time from treatment start until first documented radiographic progression as determined by the investigator using current RECIST vl.l or death due to any cause, whichever occurs first.

[0405] EFFICACY ASSESSMENTS

[0406] Tumor assessment will be based on CT / magnetic resonance imaging (MRI) with contrast and imaging required will be dependent on the tumor type of the patient. Imaging of the chest, abdomen, and pelvis is required and any other appropriate anatomy of tumor location per RECIST vl.l. Imaging assessments will be conducted at baseline and Q8W after treatment start for up to 12 months. After 12 months, tumor assessments can be conducted Q12W. Brain scans are optional at baseline and only required if clinically indicated and metastases are suspected to be present, and are repeated at the same frequency as the other imaging. Bone scans will be performed as clinically indicated for patients with suspected bone metastases at baseline outside of the CT scan regions, or suspected lesions on study. Imaging of all patients will undergo local investigator review: BICR of images will be performed in mCRC cohorts and may be performed in other cohorts at the discretion of the Sponsor.

[0407] Circulating blood tumor markers may be evaluated if appropriate for the tumor indication, at screening and on Day 1 of each cycle, EOT, and during the follow up period for patients with elevated levels at screening who discontinue for reasons other than progression.

[0408] BIOMARKERS

[0409] Tumor samples (archival or fresh FFPE) will be obtained at baseline from metastatic or primary tissue. Tumor samples (FFPE) will also be obtained during Cycle 2 Day 1, with an optional sample at any other time thereafter as per investigator’s judgment in agreement with the patient, and / or at EOT. In addition, an optional fresh tumor sample (living cells) may be collected at screening and EOT (within 35 ±5 days of last treatment), from patients who agree to have additional tissue taken for ex vivo analyses. EGFR and LGR5 expression in tumor tissue will be evaluated as exploratory endpoints.

[0410] Example 2. Patient FFPE samples

[0411] Specimen preparation

[0412] Biopsy specimens from Example 1 were handled to preserve the tissue for IHC staining. Standard methods of tissue processing should be used for all specimens. Specimens preserved in the following fixatives are suitable for testing with EGFR pharmDx: 10% (v / v) neutral buffered formalin, 10% (v / v) unbuffered formalin, 25% (v / v) unbuffered formalin, AFA (acetic formalin alcohol), Richard-Allen Scientific’s Pen-fix and Bouin’s fixative. Paraffin- embedded sections

[0413] Routinely processed and paraffin embedded tissues are suitable for use. Specimens from the biopsy should be blocked into a thickness of 3 or 4 mm and fixed for the time period appropriate to the fixative. The tissues are then dehydrated and cleared in a series of alcohols and xylene, followed by infiltration by melted paraffin. The paraffin temperature should not exceed 60°C. Properly fixed and embedded tissue blocks expressing the EGFR protein will keep indefinitely prior to sectioning and slide mounting if stored in a cool place (15-25°C).

[0414] Tissue specimens should be cut into sections of 3-5 pm. After sectioning, tissues should be mounted on slides and placed in drying racks. The following slides are recommended for use: Fisher’s SuperFrost Plus, Dako’s Silanized (code S3003), charged or poly-L-lysine coated slides. The slide racks should be pounded on an absorbent towel to remove water trapped under paraffin and on glass and then dried at room temperature for one hour. The rack of slides should then be placed in a 56-60°C incubator for one hour. Any excess water remaining on slides after removal from the incubator should be removed by pounding slides on towels and drying for one additional hour in the incubator. After removal from the incubator, slides should be held at room temperature until cool and paraffin has hardened. To preserve antigenicity, tissue sections, mounted on slides (Fisher’s SuperFrost Plus, poly-L-lysine, charged or Dako’s Silanized slides (code S3003), should be stained within 2 months of sectioning when held at room temperature (20- 25°C).

[0415] Example 3. EGFR scoring via IHC

[0416] EGFR expression on patient FFPE samples, such as obtained following Example 1 or 2, are evaluated by detection of Novocastra™ anti-EGFR mouse monoclonal antibody (clone EGFR.113, Leica Biosystems) using immunohistochemistry (IHC) on the Dako Autostainer Link 48 (Agilent). FFPE blocks are sectioned at 4pm, floated on a water bath, and picked up onto SuperFrost Plus positively charged microscope glass slides. Unstained slides are placed in a 58°C (±2°C) oven for one hour. For antigen retrieval, the slides are incubated at 97°C for 20 minutes in EnVision FLEX target retrieval High pH solution (Agilent Dako, # K800421-2), with pre-heat and cool down temperatures at 65°C. The EnVision FLEX+, Mouse, High pH kit (Agilent Dako, K800221-2) is used for the following steps. Slides are incubated with FLEX peroxidase block for 5 minutes, followed by CAS Block for 10 minutes. The slides are incubated with primary antibody (EGFR.113 mouse monoclonal antibody) for 1 hour, followed by FLEX+ Mouse Linker secondary antibody for 15 minutes. Then the slides are incubated with EnVision FLEX HRP polymer for 20 minutes, followed by FLEX DAB+ substrate for 2 x 5 minutes.

[0417] Finally, the slides are incubated with FLEX Hematoxylin (Agilent Dako, #K800821-2) for 5 minutes.

[0418] Interpretation of staining: Cells labeled by the EGFR.113 antibody display predominantly membranous staining.

[0419] EGFR-Negative Tumor: Absence of membrane staining above background in all tumor cells, or with a H-score of <3. EGFR-Positive Tumor: EGFR positive staining is defined as any IHC staining of tumor cell membranes of any staining intensity above background, whether it is complete or incomplete circumferential staining, with a H-score of >3 (Petersen I et al., EGFR immunohistochemistry as biomarker for antibody-based therapy of squamous NSCLC — Experience from the first ring trial of the German Quality Assurance Initiative for Pathology (QuIP®). Pathol Res Pract (2017) 213(12):1530- 1535.)

[0420] General scoring guidelines (see Table 1):

[0421] Histological evaluation is considered linear, either complete or incomplete membranous staining of tumor cells only.

[0422] Presence or absence of cytoplasmic expression will be captured.

[0423] Assessment of membranous staining in 4 staining intensity categories (0 to 3+). Intensity assessment is done applying the “magnification rule”.

[0424] For Histo-score calculation all membrane staining was considered independent of the completeness (complete and incomplete membrane staining).

[0425] Table 1. EGFR IHC staining intensity criteria

[0426] 3+ / Strong Staining Visible at low levels of magnification, x5 objective lens which could be confirmed at higher levels as required 2+ / Moderate Staining Visible at intermediate levels of magnification, xlO or x20 objective lenses

[0427] 1+ / Weak Staining Only reliably confirmable at high magnification, x40 objective lens

[0428]

[0429] 0 / No Staining No staining visible at high magnification

[0430] The final H- score is calculated as follows:

[0431] H-score = (% weak [1+] x 1) + (% moderate [2+] x 2) + (% strong [3+] x 3)

[0432] Example 4. LGR5 H-scoring

[0433] Patient samples, such as obtained following Example 1 or 2, are analyzed for LGR5 expression by using RNAscope® Technology in situ hybridization (ISH) assay (ACD), including the RNAscope™ 2.5 LS Reagent Kit — RED (ACD, #322150) and RNAscope™ LS 2.5 Probe- Hs-LGR5 (ACD, #311028). The samples are stained by using the Leica BondRx staining platform (Leica, #3219652 / #3219764) and BONDRx software v. 4.0. For control setup, the dapB (negative control, ACD, #312038) and PPIB (positive control, ACD, #313908) probes in patient tissue samples are prepared on extra control slides for each patient sample per run. For each stained patient slide with dapB, it shall result in no detectable signal or less than 1 signal in ten target cells (score 0). In case of a higher number of observed signals, the respective target probe slides shall not be evaluated. For each stained patient slide with PPIB, it shall result in at least 4-9 detectable signals per target cell (predominant category score 2). In case of a lower number of observed signals, the respective target probe slides shall not be evaluated. The percentage of tumor cells showing a pre- defined staining pattern / signal number will be determined per category, with categories defined as:

[0434] 0 = No staining or <1 dot per 10 cells (i.e.<10% positive cells)

[0435] 1 = 1-3 dots per cell

[0436] 2 = 4-9 dots per cell with no or very few signal clusters

[0437] 3 = 10-15 dots per cell and, if applicable, less than 10% of signals in clusters

[0438] 4 - >15 dots per cell and, if applicable, more than 10% of signals in clusters

[0439] The H-Score for all evaluation results is determined by using the following formula: H-Score = (0x% cat. 0) + (1 x % cat. 1) + (2 x % cat. 2) + (3 x % cat. 3) + (4 x % cat. 4)

[0440] Example 5. Patient ctDNA isolation

[0441] Blood from patients (e.g. following Example 1) is collected in 2x StreckCell-Free DNA BCT tubes (Streck, #230741) by venipuncture according to CLSI H3-A6 guidelines using a 21G or 22G needle. Streck Cell-Free DNA BCT should be drawn after the EDTA tube and before the fluoride oxalate (glycolytic inhibitor) tube. If a Cell-Free DNA BCT tube immediately follows a heparin tube in the draw order, collect 2 ml Red tube (discard tube) prior to collection in the Cell-Free DNA BCT. If Cell-Free DNA BCT is the first collection tube, draw the 2mL Red tube (discard tube) prior to the first specimen collection, to fill the blood collection set tubing's "dead space" with blood. The discard tube does not need to be filled completely. This step ensures maintenance of the proper blood-additive-ratio of the specimen. Fill the 2X10mL Cell-Free DNA BCT tubes completely using its vacuum. Overfilling or underfilling of tubes will result in an incorrect blood-to-additive ratio and may lead to incorrect analytic results or poor product performance. After the blood draw, remove tube from adapter and immediately mix by gentle inversion 8 to 10 times. Inadequate or delayed mixing may result in incorrect analytical results or poor product performance. One inversion is a complete turn of the wrist, 180 degrees, and back. Ensure the tube is labelled and kept at room temperature (6-37 Degrees Celsius). Do not refrigerate or freeze blood samples. The tubes are then centrifuged for 10 min, 10 °C at 1,600 x g followed by 10 min at 16000 x g. The plasma is collected into a conical screw cap tube. Isolated cfDNA (cell-free DNA) was subsequently quantified. A minimum of 5 ng of isolated cfDNA is used as a threshold for proceeding to the next steps. Next -generation sequencing liquid biopsy assay is done by Guardant Health (505 Penobscot Drive, Redwood City, CA 94063, USA) or using the Guardant360®CDx or Guardant Infinity platforms (Guardant Complete™).

[0442] Example 6. Next generation sequencing (NGS)

[0443] Sample preparation, NGS and data analysis is according to FDA approved PMA P200010 protocol which uses the NGS Oncology Panel for somatic or germline variant detection on a Guardant360®CDx by Guardant Health, Inc. Redwood City, CA 94063 USA (https: / / www.accessdata.fda.gov / cdrh_docs / pdf20 / P200010B.pdf). Briefly, Guardant360® CDx is a qualitative next generation sequencing-based in vitro diagnostic device that uses targeted high throughput hybridization-based capture technology for detection of single nucleotide variants (SNVs), insertions and deletions (indels) in 55 genes, copy number amplifications (CNAs) in two (2) genes, and fusions in four (4) genes, including KRAS, NRAS, BRAF and HER2 copy number variation.

[0444] Guardant360 CDx utilizes circulating cell-free DNA (cfDNA) from plasma of peripheral whole blood collected in Streck Cell-Free DNA Blood Collection Tubes (BCTs). The test includes reagents, software, and procedures for testing cfDNA from whole blood samples. The test uses 5-30 ng of cfDNA for library construction and next generation sequencing. Sequencing data is processed using a customized bioinformatics pipeline designed to detect several classes of genomic alterations, including nucleotide substitutions, indels, copy number amplifications, and genomic fusions / rearrangements. The device is designed to sequence 74 genes, but only report pre-defined and de novo alterations (Single Nucleotide Variants (SNVs), indels and copy number amplifications) within genes including KRAS, NRAS, BRAF and HER2.

[0445] In particular, the following mutations are screened for: KRAS or NRAS mutations involving amino acid positions G12 (e.g. G12A, G12D, G12R, G12C, G12S, G12V), G13 (e.g. G13D), Q61 (e.g. Q61R); BRAF mutation involving amino acid position V600 (e.g. V600E, V600K); EGFR ectodomain mutations at position V441, S464, G465, S492, and HER2 gene copy number amplification (CNA). The CNA calling property uses the HER2 copy number cut-off at > 2.18 as metric, following Table 5 of the referenced FDA document. No upper limit is provided.

[0446] Example 7.

[0447] A 46 year-old female patient was previously diagnosed with metastatic colorectal adenocarcinoma (mCRC). The primary tumor, located to the left side of the colon had not been removed after initial diagnosis. Metastases were present in the lung and in the liver at time of trial inclusion.

[0448] Over a period of about 5 years, the patient had received and progressed after prior treatment with the following three time-separated anticancer treatments: 1) FOLFIRINOX combined with panitumumab, 2) FOLFOX and 3) again FOLFOX. Best response as reported during treatment with FOLFIRINOX combined with panitumumab was a partial response. Following Example 1, the patient was treated with petosemtamab at 1500 mg Q2W for 6 cycles.

[0449] Absence of BRAF, KRAS and NRAS mutations was confirmed in a sample obtained from the patient. Also, the patient sample was identified to be without relevant EGFR ectodomain mutation, and was without reported HER2 amplification. All molecular genetic testing was performed on circulating tumor DNA using next-generation sequencing by Guardant360® / Guardant Health testing on a biological sample obtained at baseline. Tumor assessment was done after 8 weeks of treatment according to RECIST 1.1 criteria and revealed a partial response (PR) with a 54% shrinkage of target lesions. PR was confirmed at 16 weeks after having received the first dose of petosemtamab (i.e. Cycle 1, Day 1).

[0450] b

[0451] Example 8.

[0452] A 58 year-old male patient was previously diagnosed with metastatic colorectal adenocarcinoma. The primary tumor, located in the sigma (left side) of the colon was 0 previously removed. Liver metastases were previously removed by surgery. At time of trial inclusion, the patient had metastases in the bone, lung and lymph nodes.

[0453] Over a period of more than 4 years, the patient had received and progressed to prior treatment with the following five time-separated anticancer treatments: 1) FOLFOX 5 combined with panitumumab, 2) FOLFIRI combined with aflibercept, 3) LV5FU2, 4) capecitabine combined with bevacizumab and 5) fruquitinib. Best response as reported during treatment with FOLFOX combined with panitumumab was a partial response. Following Example 1, the patient has been treated with petosemtamab at 1500 mg Q2W for 11 cycles.

[0454] 0

[0455] Absence of BRAF, KRAS and NRAS mutations was confirmed in a sample obtained from the patient. Also, the patient sample was identified to be without relevant EGFR ectodomain mutation, and was without reported HER2 amplification. All molecular genetic testing was performed on circulating tumor DNA using next- generation

[0456] 5 sequencing by Guardant360® / Guardant Health testing on a biological sample obtained at baseline.

[0457] Tumor assessment was done after 8 weeks of treatment according to RECIST 1.1 criteria and revealed a partial response (PR) with a 43% shrinkage of target lesions. PR was 0 confirmed at 16 weeks after having received the first dose of petosemtamab (i.e. Cycle 1, Day 1).

Claims

Claims1. A bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises HCDR1, HCD R2 and HCDR3 amino acid sequences of the MF3755 heavy chain variable region as depicted in Figure 8a, and the second variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF5816 heavy chain variable region as depicted in Figure 8a, for use in the treatment of colorectal cancer in a subject, which cancer has progressed after having received at least two prior anticancer therapies.

2. Use of a bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR anda second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF3755 heavy chain variable region, as depicted in Figure 8a and the second variable domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF5816 heavy chain variable region, as depicted in Figure 8a, in the manufacture of a medicament for treating colorectal cancer in a subject, which cancer has progressed after having received at least two prior anticancer therapies.

3. A method of treating a subject having colorectal cancer, wherein said subject has progressed after having received at least two prior anticancer therapies, the method comprising providing the subject with an effective amount of a bispecific antibody, or functional part thereof, that comprises a first variable domain that binds an extracellular part of EGFR and a second variable domain that binds an extracellular part of LGR5, wherein the first variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF3755 heavy chain variable region, as depicted in Figure 8a and the second variable domain comprises HCDR1, HCDR2 and HCDR3 amino acid sequences of the MF5816 heavy chain variable region, as depicted in Figure 8a.

4. The bispecific antibody, or the use or the method of any one of the preceding claims, wherein said bispecific antibody is petosemtamab or a functional part thereof.

5. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein at least six months prior to being administered said bispecific antibody or functional part thereof, said subject has not received any anticancer therapy against said cancer, such as a therapy comprising an EGFR inhibitor.

6. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said cancer is metastatic CRC.

7. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said cancer is locally advanced, unresectable CRC.

8. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and / or the EGFR ectodomain.

9. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said cancer is negative for HER2 gene copy number amplification, in particular a cancer that has a HER2 copy number not exceeding 2.18.

10. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said cancer is a microsatellite stable tumor or wherein the MSI status of said cancer is not MSI-H.

11. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said subject received two, three or four lines of said prior anticancer therapy.

12. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said prior anticancer therapy comprises chemotherapy, such as oxaliplatin, irinotecan and / or a fluoropyrimidine, and / or therapy with an anti-VEGF agent and / or an anti- EGFR inhibitor.

13. The bi specific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said subject received two, three or four lines of said prior anticancer therapy comprising therapy with an anti-VEGF agent and / or an anti-EGFR inhibitor.

14. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said cancer expresses EGFR and / or LGR5.

15. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said subject has previously shown clinical efficacy to said prior anticancer therapy comprising a complete response (CR), a partial response (PR), or any tumor reduction to a prior EGFR inhibitor, such as received as monotherapy or in combination with chemotherapy.

16. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said subject has received said prior anticancer therapy for a period of at least six months with an EGFR inhibitor without showing evidence of disease progression.

17. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said subject exhibits adequate organ function.

18. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein the subject is a mammal, such as a human.

19. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said treatment with said bispecific antibody comprises providing the subject with an effective amount thereof.

20. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said treatment with said bispecific antibody comprises providing to the subject a flat dose of 1500 mg, in particular a flat dose of 1500 mg petosemtamab.

21. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said bispecific antibody, in particular petosemtamab, or a functional part thereof, is provided biweekly (q2w).

22. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said bispecific antibody, in particular petosemtamab, or a functional part thereof, is provided intravenously to said subject.

23. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and / or the EGFR ectodomain and / or is negative for HER2 gene copy number amplification.

24. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said treatment comprises or is preceded by a step of selecting said subject on the basis of having a cancer which does not have an oncogenic mutation in KRAS, NRAS, BRAF and / or the EGFR ectodomain and / or is negative for HER2 gene copy number amplification.

25. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said treatment comprises or is preceded by a step of selecting said subject on the basis of said cancer not having one or more oncogenic mutations in KRAS, NRAS, BRAF and / or the EGFR ectodomain and for said cancer not having HER2 gene copy number amplification.

26. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said cancer does not have an oncogenic mutation occurring at a position selected from G12, G13 or Q61 in KRAS or NRAS; position V600 in BRAF; a position selected from V441, S464, G465 or S492 in the EGFR ectodomain, and wherein said cancer has a HER2 gene copy number which does not exceed 2.18.

27. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein said cancer’ is screened by next- generation sequencing on a ctDNA plasma sample or tumor tissue sample designed to establish the KRAS, NRAS, BRAF and EGFR ectodomain genotype and the HER2 gene copy number.

28. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein the primary tumor sidedness of said colorectal cancer is left-sidedness CRC.

29. The bispecific antibody or functional part thereof, or the use or the method of any one of the preceding claims, wherein the primary CRC tumor sidedness of said colorectal cancer is the left-sided colon (or distal colon) as characterized by including the last one-third of the transverse colon, the descending colon, the sigmoid colon, and the rectum and / or originates from the hindgut and is supplied by the inferior mesenteric artery.

30. The bispecific antibody or functional part thereof, or the use or the method of any one of claims 1-27, wherein the primary tumor sidedness of said colorectal cancer is right-sidedness CRC.

31. The bispecific antibody or functional part thereof, or the use or the method of any one of claims 1-27, wherein the primary CRC tumor sidedness of said colorectal cancer is the right-sided colon (or proximal colon) as characterized by including the cecum, the ascending colon, and the proximal two-thirds of the transverse colon and / or originates from the midgut and is supplied by the superior mesenteric artery.

32. A method for classifying a subject having colorectal cancer for treatment with the bispecific antibody of claim 1, said method comprising establishing said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification in a cancer cell containing sample from said subject.

33. A method for selecting a subject having colorectal cancer for treatment with the bispecific antibody of claim 1, the method comprising establishing that said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for of HER2 gene copy number amplification in a cancer cell containing sample from said subject.

34. The method of claim 32 or 33, wherein the subject is selected for treatment or classified as being likely to respond to treatment with said antibody when said colorectal cancer not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification as determined in the cancer cell containing sample from said subject.

35. A method for selecting a subject having colorectal cancer for treatment with petosemtamab, the method comprising determining that said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain, and is negative for HER2 gene copy number amplification in a cancer cell containing sample from said subject.

36. The method of any one of claims 29-33, wherein the subject is selected for treatment or classified as being likely to respond to treatment with said bispecificantibody when said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain, and is negative for HER2 gene copy number amplification as determined in the cancer cell containing sample from the subject.

37. Petosemtamab for use in a method of treatment of a subject having colorectal cancer, wherein said subject is classified as being likely to respond to treatment with petosemtamab, or selected for treatment with petosemtamab, when said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification as established in a cancer cell containing sample from the subject,38. Petosemtamab for use in a method of treatment of a subject having colorectal cancer, wherein said colorectal cancer does not have an oncogenic mutation in KRAS, NRAS, BRAF and the EGFR ectodomain and is negative for HER2 gene copy number amplification.

39. The method, use or petosemtamab according to any one of claims 32-38, wherein at least six months prior to being administered petosemtamab, said subject has not received any anticancer therapy against said colorectal cancer, such as an EGFR inhibitor, and / or which said colorectal cancer has progressed after having received at least two prior anticancer therapies.

40. The use, method, bispecific antibody or petosemtamab according to any one of the preceding claims, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFOX and bevacizumab, and which is followed by a second prior anticancer treatment which comprises cetuximab and FOLFIRI.

41. The use, method, bispecific antibody or petosemtamab according to any one of the preceding claims, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first anticancer treatment comprises a combination of FOLFOX and bevacizumab, and which is followed by a second prior anticancer treatment which comprises panitumumab and FOLFIRI.

42. The use, method, bispecific antibody or petosemtamab according to any one of the preceding claims, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFOX and cetuximab, and which is followed by a second prior anticancer treatment which comprises bevacizumab and FOLFIRI.

43. The use, method, bispecific antibody or petosemtamab according to any one of the preceding claims, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFOX and panitumumab, and which is followed by a second prior anticancer treatment which comprises bevacizumab and FOLFIRI.

44. The use, method, bispecific antibody or petosemtamab according to any one of the preceding claims, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFIRI and cetuximab, and which is followed by a second prior anticancer treatment which comprises bevacizumab and FOLFOX.

45. The use, method, bispecific antibody or petosemtamab according to any one of the preceding claims, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFIRI and panitumumab, and which is followed by a second prior anticancer treatment which comprises bevacizumab and FOLFOX.

46. The use, method, bispecific antibody or petosemtamab according to any one of the preceding claims, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFIRI and bevacizumab, and which is followed by a second prior anticancer treatment which comprises cetuximab and FOLFOX.

47. The use, method, bispecific antibody or petosemtamab according to any one of the preceding claims, wherein the subject has progressed after having received two prior anticancer treatments, wherein the first prior anticancer treatment comprises a combination of FOLFIRI and bevacizumab, and which is followed by a second prior anticancer treatment which comprises panitumumab and FOLFOX.