Combination cancer therapy using at least an antibody that binds to EGFR and an immune checkpoint inhibitor

Abstract

The present disclosure relates to means and methods for treating cancer. In particular, the disclosure relates to methods of treating cancer in an individual with an antibody that binds at least EGFR and an immune checkpoint inhibitor (ICI). The invention further relates to the use in such methods and in the manufacture of a medicament for the treatment of cancer that has not previously been treated with a therapeutic agent having anti-cancer properties. Such combinations of the antibody and ICI are particularly useful in the treatment of cancers such as head and neck cancer.

Description

[Technical Field]

[0001] The present disclosure relates to means and methods for treating cancer. In particular, the disclosure relates to methods of treating cancer in an individual with antibodies that monovalently bind at least extracellular EGFR. The invention further relates to the use of such antibodies in such methods, and in the manufacture of a medicament for the treatment of head and neck cancer. Such antibodies are particularly useful in the treatment of cancers such as head and neck cancer. [Background technology]

[0002] Cancer remains a leading cause of death in the world, despite many advances made in the treatment of the disease and increasing knowledge about the molecular events that lead to cancer.

[0003] Traditionally, most cancer drug discovery has focused on agents that block essential cellular functions and kill dividing cells. However, in the case of advanced cancer, chemotherapy rarely results in a complete cure, no matter how aggressively it is administered, even to the point where patients experience life-threatening side effects from the treatment. In most cases, the patient's tumor only stops growing or shrinks temporarily (called remission), only to begin growing again, sometimes more rapidly (called relapse), becoming increasingly difficult to treat. More recently, the focus of cancer drug development has shifted from broad-spectrum cytotoxic chemotherapy to targeted, less toxic cell-suppressing therapies. Treatments for advanced cancers have been clinically tested in leukemia and several other cancers. However, in most carcinomas, targeted approaches have proven ineffective, with significant unmet medical needs remaining, and safer and more effective treatment options remain necessary.

[0004] Cancer targeting has been achieved using a variety of different methods, including, for example, small molecules directed against signaling proteins that cancers depend on for survival and / or growth, vaccines with tumor-specific proteins, cell therapy with antibodies that target immune cells and cytotoxic molecules to tumors that actively kill tumor cells, disrupting signal transduction and / or (re)directing the host's immune system against tumor cells.

[0005] In the United States, head and neck cancer, particularly in the oral cavity and pharynx, already accounts for 3% of all malignant tumors, with approximately 53,000 Americans developing this cancer each year and 10,800 dying from it (Siegel et al., CA Cancer J Clin. 2020;70(1):7. Epub 2020 Jan 8.). Furthermore, head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with the 5-year overall survival rate for patients with HNSCC reportedly being approximately 40-50% (Head and Neck Cancer, Union for International Cancer Control, 2014 Review of Cancer Medicines on the WHO List of Essential Medicines).

[0006] In the KEYNOTE-048 study (Burtness et al., Vol. 394, 10212, pages 1915-1928, November 23, 2019, The Lancet), pembrolizumab was administered as monotherapy at 200 mg IV q3w for up to 24 months. In patients with CPS ≥ 1, the mean OS was 12.3 months (95% confidence interval [CI]: 10.8, 14.9), the mean PFS was 3.2 months (95% CI: 2.2, 3.4), the best objective response (CR) or PR was confirmed in only 19% of patients (CI: 14.5, 24.4), and the mean duration of response (DOR) was 20.9 months (95% CI: 1.5, 34.8).

[0007] A recent phase 2 trial combining pembrolizumab and cetuximab in a population of HNSCC patients who had not previously received anti-PD1 or EGFR therapy showed that 15 of 33 participants achieved an objective response rate (ORR) by 6 months (45% [95% CI: 28, 62]), with a mean duration of response (DOR) of 13.1 months (95% CI: 6.5, not reached). One patient achieved a response after 6 months, thereby increasing the ORR to 48%. The mean progression-free survival (PFS) was 6.5 months (95% CI: 2.1, not reached), and the mean overall survival (OS) was 18.4 months (95% CI: 11.0, not reached) (Sacco et al., 2021, Lancet Oncol. Jun;22(6):883-892). In this phase 2 combination trial, observed adverse events (AEs) were as expected based on the individual toxicity profiles of each drug. Fatigue, skin events (rash and dry skin), hypomagnesemia, and hypertransaminasemia were among the most commonly reported TEAEs. Pembrolizumab-associated immune-related AEs were consistent with its toxicity profile. Potential overlapping toxicities were observed, including oral mucositis and skin toxicity.

[0008] A meta-analysis of locally advanced head and neck squamous cell carcinoma (LA-HNSCC) reported that adding anti-EGFR agents to radiotherapy or chemoradiotherapy did not improve clinical outcomes in patients with LA-HNSCC (Oncotarget.2017;8(60):102371-102380). The addition of anti-EGFR agents also increased the risk of skin toxicity and mucositis.

[0009] Thus, there is a need for improved cancer treatments, particularly for the treatment of head and neck cancer. Summary of the Invention

[0010] The present disclosure provides the following preferred embodiments, however, the present invention is not limited thereto.

[0011] The present disclosure relates to a combination therapy in which an antibody comprising an antigen-binding site that binds to the extracellular portion of EGFR, or a functional part, derivative, and / or analog thereof, is used together with an immune checkpoint inhibitor in a method of treating cancer in a subject in need thereof.

[0012] The present disclosure provides means and methods for the (re)direction of components of the immune system in the treatment of cancer.

[0013] The present disclosure also relates to an antibody, or a functional part, derivative, and / or analogue thereof, comprising a variable domain that binds to the extracellular portion of EGFR, for use in treating cancer in a subject, wherein the treatment further comprises administering an immune checkpoint inhibitor.

[0014] The present disclosure also relates to the use of an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative, and / or analog thereof, and an immune checkpoint inhibitor in the manufacture of one or more medicaments for treating cancer in a subject. In certain aspects, the antibody, or a functional portion, derivative, and / or analog thereof, and the immune checkpoint inhibitor are used to manufacture separate medicaments. In certain aspects, treating cancer comprises administering the antibody, or a functional portion, derivative, and / or analog thereof, and the immune checkpoint inhibitor.

[0015] The present disclosure also relates to a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative, and / or analog thereof, and an effective amount of an immune checkpoint inhibitor.

[0016] In certain embodiments, the immune checkpoint inhibitor comprises a PD-L1, PD-L2, or PD-1 inhibitor. In certain embodiments, the immune checkpoint inhibitor comprises or is a PD-L1, PD-L2, or PD-1 inhibitor. In certain embodiments, the immune checkpoint inhibitor comprises or is an antibody. In certain embodiments, the immune checkpoint inhibitor comprises or is an antibody that targets PD-L1, PD-L2, or PD-1. In certain embodiments, the immune checkpoint inhibitor comprises or is an antibody that inhibits PD-L1, PD-L2, or PD-1. In certain embodiments, the immune checkpoint inhibitor comprises or is an antibody that inhibits PD-L1. In certain embodiments, the immune checkpoint inhibitor comprises or is an antibody that inhibits PD-L2. In certain embodiments, the immune checkpoint inhibitor comprises or is an antibody that inhibits PD-1.

[0017] In certain aspects, the immune checkpoint inhibitor comprises or is nivolumab, pembrolizumab, cemiplimab, penprimimab, retifanlimab, sintilimab, tislelizumab, toripalimab, dostarimab, atezolizumab, avelumab, or durvalumab.

[0018] In certain aspects, the immune checkpoint inhibitor includes or is pembrolizumab, hi certain aspects, the immune checkpoint inhibitor includes or is nivolumab.

[0019] In certain embodiments, the cancer comprises or is adenocarcinoma, squamous cell carcinoma, or head and neck cancer, including squamous cell carcinoma of the head and neck (SCCHN).

[0020] In certain embodiments, the cancer comprises or is head and neck cancer, including squamous cell carcinoma of the head and neck (SCCHN).

[0021] In certain embodiments, the antibody, or functional part, derivative, and / or analog thereof, comprises or is a multispecific antibody, hi certain embodiments, the antibody, or functional part, derivative, and / or analog thereof, comprises or is a bispecific antibody.

[0022] In certain embodiments, the antibody, or functional portion, derivative, and / or analog thereof, of the present disclosure is a multispecific antibody that binds to at least EGFR. In certain embodiments, the antibody, or functional portion, derivative, and / or analog thereof, is a bispecific antibody that binds to at least EGFR. In certain embodiments, the antibody binds monovalently to EGFR. In certain embodiments, the antibody comprises a second variable domain that does not bind to EGFR. In certain embodiments, the antibody, or functional portion, derivative, and / or analog thereof, comprises a variable domain that binds to LGR5. In certain embodiments, the antibody, or functional portion, derivative, and / or analog thereof, is ADCC enhanced. Also, in certain embodiments, the antibody, or functional portion, derivative, and / or analog thereof, is afucosylated. In certain embodiments, the antibody that binds to at least EGFR comprises or is petosemutamab.

[0023] In certain aspects, the cancer expresses PD-L1, EGFR, and / or LGR5. In certain aspects, the cancer expresses PD-L1 and EGFR.

[0024] In certain embodiments, the treatment is a first line treatment. In certain embodiments, the subject receiving the treatment has not received prior or prior anti-cancer treatment for the cancer.

[0025] In certain embodiments, the antibody that binds to at least EGFR is petosemutamab and is administered at a dose of 1500 mg. In certain embodiments, petosemutamab is administered at a dose of 1500 mg every two weeks. In certain embodiments, the immune checkpoint inhibitor is pembrolizumab and is administered at a dose of 600 mg. In certain embodiments, the immune checkpoint inhibitor is pembrolizumab and is administered at a dose of 600 mg every six weeks.

[0026] In certain aspects, the subject of the present disclosure is a mammalian subject, such as a human subject.

[0027] The present disclosure further includes (pharmaceutical) combinations or kits of parts comprising an antibody that binds to at least EGFR of the present disclosure, or a functional portion, derivative, and / or analog thereof, in combination with an immune checkpoint inhibitor of the present disclosure. The combination, in certain embodiments, comprises a container containing the antibody that binds to at least EGFR, or a functional portion, derivative, and / or analog thereof, which are not physically linked, and a container containing the immune checkpoint inhibitor referred to herein. The combination, in certain embodiments, is accompanied by instructions for use. The instructions for use include clinically relevant information, such as instructions for intravenous administration, the dose to be administered, and the time interval between administrations. In certain embodiments, the antibody, or a functional portion, derivative, and / or analog thereof, particularly petosemutamab, and the immune checkpoint inhibitor, particularly pembrolizumab, are administered according to instructions for use after approval by the relevant authorities. [Brief explanation of the drawings]

[0028] [Figure 1] Human LGR5 sequence, SEQ ID NO:1. [Figure 2] Human EGFR sequence, SEQ ID NO:2. [Figure 3](a) Amino acid sequences of heavy chain variable regions (SEQ ID NOS: 3-15) that form variable domains that bind to LGR5 and EGFR together with common light chain variable regions, such as the variable region of human kappa light chain IgVκ139*01 / IGJκ1*01. The CDR and framework regions are shown in Figure 3b according to Kabat numbering. [Figure 4] a) Amino acid sequence of the common light chain amino acid sequence; b) Common light chain variable region (IGKV1-39 / jk1); c) Light chain constant region; d) V region IGKV1-39A; e) CDR1, CDR2, and CDR3 of the common light chain according to IMGT numbering. [Figure 5] IgG heavy chain for generating bispecific molecules. a) CH1 region. b) Hinge region. c) CH2 region. d) CH3 domain containing mutations L351K and T366K (KK). e) CH3 domain containing mutations L351D and L368E (DE). Residue positions are according to EU numbering. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present disclosure relates to a combination therapy in which an antibody comprising an antigen-binding site that binds to the extracellular portion of EGFR, or a functional part, derivative, and / or analog thereof, is used together with an immune checkpoint inhibitor in a method of treating cancer in a subject in need thereof.

[0030] The present disclosure relates to an antibody, or a functional part, derivative, and / or analogue thereof, comprising an antigen-binding site that binds to an extracellular portion of EGFR, for use in a method of treating cancer in a subject in need thereof, the method further comprising administering an immune checkpoint inhibitor for treating said cancer.

[0031] The present disclosure also relates to an antibody, or a functional part, derivative, and / or analogue thereof, comprising a variable domain that binds to the extracellular portion of EGFR, for use in a method of treating cancer in a subject, wherein the treatment further comprises the use of an immune checkpoint inhibitor.

[0032] The present disclosure also relates to a method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative, and / or analog thereof, and an effective amount of an immune checkpoint inhibitor.

[0033] The present disclosure also relates to the use of an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative, and / or analog thereof, and an immune checkpoint inhibitor in the manufacture of one or more medicaments for treating cancer in a subject. In certain embodiments, the antibody, or a functional portion, derivative, and / or analog thereof, and the immune checkpoint inhibitor are used to manufacture separate medicaments, e.g., two separate medicaments, one for the antibody, or a functional portion, derivative, and / or analog thereof, and one for the immune checkpoint inhibitor. The medicaments comprising the antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative, and / or analog thereof, may be contained in separate holders, meaning that they are not physically linked to the holder containing the immune checkpoint inhibitor as a medicament. In certain embodiments, treating cancer comprises administering at least an antibody that binds to EGFR, or a functional portion, derivative, and / or analog thereof, and an immune checkpoint inhibitor.

[0034] In certain aspects, the present disclosure provides the use of an antibody of the present disclosure, or a functional part, derivative, and / or analogue, comprising a variable domain capable of binding to the extracellular portion of EGFR, and an immune checkpoint inhibitor in the manufacture of a medicament for the treatment of cancer.

[0035] In certain aspects, the present disclosure provides use of an antibody of the present disclosure, or a functional part, derivative, and / or analogue, comprising a variable domain capable of binding to the extracellular portion of EGFR, in the manufacture of a medicament for increasing the effect of an immune checkpoint inhibitor for the treatment of cancer.

[0036] In certain aspects, the present disclosure provides for the use of an immune checkpoint inhibitor in the manufacture of a medicament for increasing the effect of an antibody, or functional part, derivative, and / or analog of the present disclosure, comprising a variable domain capable of binding to the extracellular portion of EGFR for the treatment of cancer.

[0037] The present disclosure also provides a kit of parts comprising an antibody, or a functional part, derivative, and / or analogue thereof, comprising a variable domain capable of binding to the extracellular portion of EGFR, an immune checkpoint inhibitor, and instructions for use of the antibody, or a functional part, derivative, and / or analogue thereof, and for use of the immune checkpoint inhibitor.

[0038] The present disclosure also provides a combination of an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR, or a functional part, derivative, and / or analogue thereof, and an immune checkpoint inhibitor as mentioned herein, for use in treating cancer in a subject in need thereof.

[0039] The present disclosure also provides a combination of an immune checkpoint inhibitor referred to herein, instructions for the use of said immune checkpoint inhibitor in the treatment of cancer in a subject, and instructions for the use of an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR referred to herein, or a functional part, derivative, and / or analogue thereof, in the treatment of said cancer in said subject.

[0040] The present disclosure also provides a combination of an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR referred to herein, or a functional part, derivative, and / or analogue thereof, instructions for the use of the antibody, or a functional part, derivative, and / or analogue thereof, in the treatment of cancer in a subject, and instructions for the use of an immune checkpoint inhibitor referred to herein in the treatment of cancer in a subject.

[0041] In certain aspects, the present disclosure provides pharmaceutical compositions comprising antibodies comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, or functional parts, derivatives, and / or analogs thereof, and instructions for their use with immune checkpoint inhibitors in the treatment of such cancers.

[0042] In certain aspects, the present disclosure provides pharmaceutical compositions for the treatment of cancer comprising an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, or a functional part, derivative, and / or analog thereof, as well as pharmaceutical compositions for the treatment of cancer comprising an immune checkpoint inhibitor of the present disclosure.

[0043] In certain aspects, the present disclosure provides a pharmaceutical composition for use in the treatment of cancer comprising an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, or a functional part, derivative, and / or analog thereof, wherein the pharmaceutical composition is administered in combination with an immune checkpoint inhibitor of the present disclosure.

[0044] In certain aspects, the present disclosure relates to a pharmaceutical composition for the treatment of cancer comprising an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, or a functional part, derivative, and / or analog thereof, wherein the subject to be treated is further administered prior to, concurrently with, or following administration of the bispecific antibody.

[0045] In certain aspects, the present disclosure relates to a pharmaceutical composition for the treatment of cancer in a subject comprising an immune checkpoint inhibitor, wherein the subject being treated is further administered an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR, or a functional part, derivative, and / or analog thereof, of the present disclosure, prior to, concurrently with, or following administration of the immune checkpoint inhibitor.

[0046] Accordingly, the present disclosure relates to a drug combination for the treatment of cancer in a subject, comprising administration to the subject of a plurality of different drugs for treating the cancer, wherein the treatment comprises simultaneous, sequential, or separate administration of the drugs. In certain aspects, the drugs comprise an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, or a functional part, derivative, and / or analog thereof, and the other different drugs comprise an immune checkpoint inhibitor.

[0047] In certain aspects, the antibodies comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, or functional portions, derivatives, and / or analogs thereof, can be administered simultaneously, sequentially, or separately with the immune checkpoint inhibitors of the present disclosure. Thus, such combinations of antibodies comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, or functional portions, derivatives, and / or analogs thereof, and immune checkpoint inhibitors encompass simultaneous, sequential, or separate administration.

[0048] Thus, in certain aspects, the present disclosure provides an antibody, or functional part, derivative, and / or analogue thereof, comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, for use in a method of treating cancer, wherein the treatment further comprises administering an immune checkpoint inhibitor, optionally wherein the antibody, or functional part, derivative, and / or analogue thereof is administered simultaneously, sequentially, or separately from the immune checkpoint inhibitor.

[0049] Accordingly, in certain aspects, the present disclosure provides methods of treating a subject having cancer, the method comprising administering to the subject an effective amount of an immune checkpoint inhibitor and an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure, or a functional part, derivative, and / or analogue thereof, optionally administered simultaneously, sequentially, or separately from the immune checkpoint inhibitor.

[0050] Thus, in certain aspects, the present disclosure provides use of an antibody, or functional portion, derivative, and / or analog thereof, comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure and an immune checkpoint inhibitor, in the manufacture of a medicament for the treatment of cancer, optionally wherein the antibody, or functional portion, derivative, and / or analog thereof is administered simultaneously, sequentially, or separately from the immune checkpoint inhibitor. In certain aspects, the antibody, or functional portion, derivative, and / or analog thereof is administered before, simultaneously, or after the administration of the immune checkpoint inhibitor.

[0051] In certain aspects, an antibody, or functional part, derivative, and / or analog thereof, comprising a variable domain capable of binding to the extracellular portion of EGFR of the present disclosure is for use in the manufacture of a medicament for the treatment of cancer, and an immune checkpoint inhibitor is for use in the manufacture of a medicament for the treatment of said cancer, optionally wherein the antibody, or functional part, derivative, and / or analog thereof is administered simultaneously, sequentially, or separately with said immune checkpoint inhibitor. Optionally, the antibody, or functional part, derivative, and / or analog thereof is administered before, simultaneously, or after administration of said immune checkpoint inhibitor.

[0052] In order to make this description more readily understandable, certain terms are defined herein. Additional definitions may be provided throughout the detailed description if deemed necessary. Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, employing conventional methods of immunology, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology.

[0053] As used herein, the singular forms "a," "an," and "the" include plural referents. Use of the terms "comprising," "having," "including," as well as other word forms such as "comprise," "comprises," "comprised," "has," "have," "had," "include," "includes," and "included" is not limiting.

[0054] As used herein, the term "antibody" refers to a protein molecule belonging to the immunoglobulin class of proteins that contains one or more domains that bind to an epitope on an antigen, such domains being derived from or sharing sequence homology with the variable regions of the antibody. Antibodies are typically composed of basic structural units, each of two heavy chains and two light chains. Antibodies according to the present invention are not limited to any particular format or method of production.

[0055] A "bispecific antibody" is an antibody described herein in which one domain of the antibody binds to one antigen while a further domain of the antibody binds to a further antigen, and the one and further antigens are not identical, or one domain binds to one epitope on the antigen while the further domain binds to a further epitope on the antigen. The term "bispecific antibody" also encompasses antibodies in which one heavy chain variable region / light chain variable region (VH / VL) combination binds to an antigen or epitope on the antigen and a further VH / VL combination that binds to a further antigen or epitope on the antigen. This term also includes antibodies in which the VH can specifically recognize one antigen and the VL paired with the VH in an immunoglobulin variable region can specifically recognize a further antigen. The resulting VH / VL pair binds to either antigen 1 or antigen 2. Such so-called "two-in-one antibodies" are described, for example, in WO2008 / 027236, WO2010 / 108127, and Schaefer et al (Cancer Cell 20, 472-486, October 2011). Bispecific antibodies according to the invention are not limited to any particular bispecific format or method of their production.

[0056] As used herein, the term "common light chain" refers to the two light chains (or their VL portions) in a bispecific antibody. The two light chains (or their VL portions) may be identical or may have some amino acid sequence differences, but the binding specificity of the full-length antibody is not affected. The terms "common light chain," "common VL," "single light chain," and "single VL" are all used interchangeably herein, regardless of whether the term "rearranged" is added. "Common" also refers to functional equivalents of light chains that do not have identical amino acid sequences. Many variants of the light chain exist in which mutations (deletions, substitutions, insertions, and / or additions) do not substantially affect the formation of a functional binding region. In certain embodiments, the light chain of the present invention may also be a light chain as defined herein, having 0 to 10 amino acid insertions, deletions, substitutions, additions, or a combination thereof. In certain embodiments, the 0 to 10 amino acid insertions, deletions, substitutions, additions, or a combination thereof are not within the CDR regions. In certain embodiments, the light chains of the present invention may also be light chains as defined herein that have 0 to 5 amino acid insertions, deletions, substitutions, additions, or combinations thereof. For example, it is within the scope of the definition of a consensus light chain as used herein to prepare or find variable light chains that are not identical but are still functionally equivalent by introducing, for example, conservative amino acid changes, amino acid changes in regions that do not contribute, or only partially contribute, to binding specificity when paired with a heavy chain, and the like, and testing such changes.

[0057] As used herein, "comprise" and its conjugations are used in their open-ended sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded. Additionally, the verb "consisting of" may be replaced with "consisting essentially of," meaning that a compound or sub-compound defined herein may contain additional components other than those specifically identified, and that such additional components do not alter the inherent characteristics of the invention.

[0058] An "antibody derivative" is a protein that deviates from the amino acid sequence of a native antibody in at most 20 amino acids, outside the CDR regions. Antibody derivatives disclosed herein are antibodies that deviate from the amino acid sequence in at most 20 amino acids. Functional parts, derivatives, and / or analogs maintain the binding specificity of (bispecific) antibodies. An "antibody analog" is a protein that may differ in structure, format, or origin, but maintains the binding specificity of the antibody it is an analog of.

[0059] "Percent identity" herein with respect to nucleic acid or amino acid sequences is defined as the percentage of residues in a candidate sequence that are identical to those in a selected sequence after aligning the sequences for optimal comparison purposes. Percent sequence identity for comparing nucleic acid sequences is determined using the AlignX application of Vector NTI Advance® 11.5.2 software using default settings with a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, (1994) Nuc. Acid Res. 22(22):4673-4680), a swgapdnamt scoring matrix, a gap opening penalty of 15, and a gap extension penalty of 6.66. Amino acid sequences were determined with the AlignX application of Vector NTI Advance® 11.5.2 software using default settings with a modified ClustalW algorithm (Thompson, JD, Higgins, DG, and Gibson TJ, (1994) Nuc. Acid Res. 22(22):4673-4680), blosum62mt2 scoring matrix, a gap opening penalty of 10, and a gap extension penalty of 0.1.

[0060] Because antibodies typically recognize an epitope on an antigen, and such epitopes may exist on other compounds as well, an antibody according to the invention that "specifically recognizes" an antigen, e.g., PD-L1, EGFR, or LGR5, may also recognize other compounds if such other compounds contain the same type of epitope. Thus, the term "specifically recognize" with respect to the interaction between an antigen and an antibody does not exclude the binding of the antibody to other compounds containing the same type of epitope.

[0061] "Epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein (so-called linear and conformational epitopes). Epitopes formed from contiguous linear amino acids typically are retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding typically lose their conformation upon treatment with denaturing solvents. Epitopes can typically include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

[0062] As used herein, the terms "subject" and "patient" are used interchangeably and refer to a mammal (e.g., a patient, such as a human patient with cancer), such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, etc.

[0063] The terms "treat," "treating," and "treatment," as used herein, refer to any type of intervention or process of administering an active agent or combination of active agents to a subject for the purpose of reversing, alleviating, ameliorating, inhibiting, or slowing or preventing the progression, occurrence, severity, or recurrence of symptoms, complications, conditions, or biochemical manifestations associated with a disease.

[0064] As used herein, "effective treatment" or "positive therapeutic response" refers to a treatment that results in a beneficial effect, e.g., an improvement in at least one symptom of a disease or disorder, e.g., cancer. A beneficial effect can take the form of an improvement over a baseline, including an improvement over measurements or observations made before initiating treatment according to the method. For example, a beneficial effect can take the form of slowing, stabilizing, halting, or reversing the progression of cancer in a subject at any clinical stage, as evidenced by a reduction or elimination of clinical or diagnostic symptoms of the disease or markers of cancer. An effective treatment can, for example, reduce tumor size, reduce the presence of circulating tumor cells, reduce or prevent tumor metastasis, slow or halt tumor growth, and / or prevent or delay tumor recurrence or relapse.

[0065] The term "effective amount" or "therapeutically effective amount" refers to the amount of an agent or combination of agents that provides a desired biological, therapeutic, and / or prophylactic result. That result can be reduction, amelioration, remission, alleviation, delay, 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. With respect to tumor development, an effective amount is an amount sufficient to delay tumor development. With respect to 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. An effective amount of an agent or composition can (i) reduce the number of cancer cells, (ii) reduce tumor size, (iii) inhibit, prevent, delay to some extent, and stop cancer cell invasion into peripheral organs, (iv) inhibit tumor metastasis, (v) inhibit tumor growth, (vi) prevent or delay tumor development and / or recurrence, and / or (vii) relieve to some extent one or more symptoms associated with cancer. In one aspect, an "effective amount" is the amount of an antibody disclosed herein as a therapeutic agent that affects a reduction in cancer (e.g., a reduction in the number of cancer cells), delays the progression of cancer, or prevents the regrowth or recurrence of cancer. The antibodies or functional portions, derivatives, and / or analogs thereof that bind to EGFR or that bind to EGFR and LGR5 disclosed herein are also referred to herein as "therapeutic agents." In a specific aspect, an effective amount of petosemutamab herein is a flat dose of 1500 mg administered every two weeks to a subject with cancer disclosed herein. Immune checkpoint inhibitors are also referred to herein as "therapeutic agents." In a specific aspect, an effective amount of an immune checkpoint inhibitor herein is a flat dose of 400 mg pembrolizumab administered once every six weeks to a subject with cancer disclosed herein. In a specific aspect, an effective amount of an immune checkpoint inhibitor herein is a flat dose of 200 mg pembrolizumab administered once every three weeks to a subject with cancer disclosed herein.

[0066] As used herein, the term "cancer" applies equally to the term "tumor," just as treating a tumor also applies to treating cancer.

[0067] The term "flat dose" herein refers to a dosing regimen in which a subject is administered a consistent amount of a therapeutic agent over multiple administrations, regardless of the subject's body weight. Flat doses are typically abbreviated as qnw, where n is an integer indicating the interval and w is weeks. For example, a q2w flat dose dosing regimen of 1500 mg of an antibody means that a consistent amount of 1500 mg of the antibody is administered every two weeks. In certain embodiments, the therapeutic agent is an antibody that binds to EGFR, or EGFR and LGR5, administered in a q2w dosing regimen of 1500 mg. In certain embodiments, administration to the subject comprises at least three q2w flat doses of 1500 mg. In certain embodiments, administration comprises at least four or more doses and may continue until the patient shows sufficient clinical or radiological progression. In certain embodiments, the therapeutic agent is pembrolizumab, administered in an effective amount of 400 mg q6w. In certain embodiments, the administration to the subject comprises at least three q6w flat doses of 400 mg, hi certain embodiments, the administration comprises at least four doses or more and can continue until the subject shows sufficient clinical or radiological progression.

[0068] The term "H-score" refers to a reproducible, standardized scoring methodology that can be used to semi-quantitatively calculate the expression of a gene of interest in a tumor sample according to protocols based on immunohistochemistry (IHC) or in situ hybridization (ISH) methods, sometimes referred to in the art as "Hist," all well known to those skilled in the art, and is incorporated herein by reference in the ASCO April 10, 2010, publication of the method for calculating the H-score. th, 2015. See also Hirsch FR, Varella-Garcia M, Bunn PA Jr, et al: Epidermal growth factor receptor in non-small-cell lung carcinomas: Correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 21:3798-3807, 2003, and John T, Liu G, Tsao MS: Overview of molecular testing in non-small-cell lung cancer: Mutational analysis, gene copy number, protein expression, and other biomarkers of EGFR for the prediction of response to tyrosine kinase inhibitors. Oncogene 28:S14-S23, 2009. The relevant teachings of these references are incorporated herein by reference. In the context of H-scoring for EGFR, the term "determined using IHC" refers to a method that uses or includes IHC as the basis for subsequently determining an H-score, as opposed to a method that replaces IHC.

[0069] Also provided in the present disclosure are antibodies, or functional parts, derivatives, and / or analogs thereof, comprising a variable domain that binds to the extracellular portion of EGFR, and immune checkpoint inhibitors, for use in treating cancer in a subject, wherein the cancer expresses EGFR characterized by an IHC score of 3+, and the variable domain comprises amino acids as further disclosed herein.

[0070] Also provided in the present disclosure are antibodies, or functional parts, derivatives, and / or analogs thereof, comprising a variable domain that binds to the extracellular portion of EGFR, and immune checkpoint inhibitors, for use in treating cancer in a subject, wherein the cancer expresses an EGFR characterized by an H-score for EGFR of greater than 200, and the variable domain comprises amino acids as further disclosed herein.

[0071] The present disclosure also provides a method of treating a subject having an EGFR-expressing cancer, wherein the subject has not received prior anti-cancer treatment, the method comprising providing to the subject an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative, and / or analog thereof, and an immune checkpoint inhibitor.

[0072] Also provided in the present disclosure are an antibody, or a functional part, derivative, and / or analogue thereof, comprising a variable domain that binds to the extracellular portion of EGFR, for use in treating head and neck cancer in a subject, wherein the cancer expresses EGFR characterized by an IHC score of 3+, and an immune checkpoint inhibitor.

[0073] EGFR, LGR5, PD-L1 / 2, and PD-1 as targets of the present disclosure The epidermal growth factor (EGF) receptor (EGFR, ErbB1, or HER1) is a member of a family of four receptor tyrosine kinases (RTKs), designated Her- or cErbB-1, -2, -3, and -4. EGFR is known by various synonyms, the most common of which is EGFR. EGFR has an extracellular domain (ECD) composed of four subdomains, two of which are involved in ligand binding and two of which are involved in homodimerization and heterodimerization. EGFR integrates extracellular signals from various ligands, resulting in diverse intracellular responses. The major signaling pathway activated by EGFR consists of the Ras-mitogen-activated protein kinase (MAPK) mitogenic signaling cascade. Activation of this pathway is initiated by recruitment of Grb2 to tyrosine-phosphorylated EGFR. This leads to activation of Ras by the Grb2-bound Ras-guanine nucleotide exchange factor Son of Sevenless (SOS). In addition, the PI3-kinase-Akt signaling pathway is also activated by EGFR, but this activation is much stronger when ErbB-3 (HER3) is coexpressed. EGFR is involved in several human epithelial malignancies, particularly cancers of the breast, bladder, non-small cell lung cancer, lung, colon, ovary, head and neck, and brain. Activating mutations in the gene and overexpression of the receptor and its ligands have been found, resulting in an autocrine activation loop. Therefore, this RTK is widely used as a target for cancer therapy. Both small molecule inhibitors targeting the RTK and monoclonal antibodies (mAbs) directed against the extracellular ligand-binding domain have been developed and have shown some clinical success, although mostly in select patient populations. The database accession number for the human EGFR protein and its encoding gene is GenBank NM_005228.3. The accession numbers are provided primarily to provide a further method of specification of the EGFR protein as a target, and the actual sequence of the EGFR protein bound by the antibody may vary due to mutations in the encoding gene, such as those that occur in some cancers.

[0074] When EGFR is mentioned herein, reference is made to human EGFR unless otherwise stated. Variable domain antigen-binding sites that bind to EGFR bind to EGFR and its various variants, such as those expressed on some EGFR-positive tumors.

[0075] The term "LGR" refers to a family of proteins known as leucine-rich repeat-containing G protein-coupled receptors. Several members of this family, notably LGR4, LGR5, and LGR6, are known to be involved in the WNT signaling pathway.

[0076] LGR5 is a leucine-rich repeat containing G protein-coupled receptor 5. Alternative names for the gene or protein are leucine-rich repeat containing G protein-coupled receptor 5, leucine-rich repeat-containing G protein-coupled receptor 5, G protein-coupled receptor HG38, G protein-coupled receptor 49, G protein-coupled receptor 67, GPR67, GPR49, orphan G protein-coupled receptor HG38, G protein-coupled receptor 49, GPR49, HG38, and FEX. Proteins or antibodies of the present invention that bind to LGR5 bind to human LGR5. Due to sequence and tertiary structure similarities between human and other mammalian orthologs, LGR5-binding proteins or antibodies may, but do not necessarily, also bind to such orthologs. The database accession numbers for the human LGR5 protein and its encoding gene are (NC_000012.12, NT_029419.13, NC_018923.2, NP_001264155.1, NP_001264156.1, NP_003658.1). The accession numbers are provided primarily to provide a means of further characterization of LGR5 as a target; the actual sequence of the bound LGR5 protein may vary due to mutations in the encoding gene, such as those that occur in some cancers. When LGR5 is mentioned herein, reference is made to human LGR5 unless otherwise specified. The LGR5 antigen-binding site binds to LGR5 and its various variants, such as those expressed by some LGR5-positive tumor cells.

[0077] Programmed death-ligand 1 (PD-L1, CD274, or B7 homolog 1 (B7-H1), HGNC:17635, NCBI, Entrez Gene:29126, UniProtKB / Swiss-Prot:Q9NZQ7) is a protein encoded by the CD274 gene in humans. This gene encodes an immunoinhibitory receptor ligand expressed by hematopoietic and nonhematopoietic cells, such as T cells and B cells, as well as various types of tumor cells. The encoded protein is a type I transmembrane protein with immunoglobulin V-like and C-like domains. Interaction of this ligand with its receptor inhibits T cell activation and cytokine production. During infection or inflammation of normal tissues, this interaction is important for preventing autoimmunity by maintaining homeostasis of the immune response. In the tumor microenvironment, this interaction provides immune escape for tumor cells via cytotoxic T cell inactivation.

[0078] PD-L1 is a type 1 transmembrane protein that plays a role in suppressing immune responses during certain events such as pregnancy, tissue allografts, autoimmune diseases, and other disease states such as hepatitis. PD-L1 is expressed in various types of cancer, especially NSCLC (Boland et al., 2013. Tumor B7-H1 and B7-H3 expression in squamous cell carcinoma of the lung. Clinical lung cancer Vol. 14, No. 2, 157-63; Velcheti et al., 2014. Programmed death ligand-1 expression in non-small cell lung cancer. Laboratory investigation. 94, 107-116), melanoma, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, as well as various leukemias and multiple myeloma (Bernstein et al., 2014. Radiation-induced modulation of costimulatory and coinhibitory T-cell signaling molecules on human prostate carcinoma cells promotes productive antitumor immune interactions. Cancer biotherapy and radiopharmaceuticals. Vol. 29, No. 4, 153-161; Thompson et al. (Dong et al., 2002. Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune invasion. Nature medicine. Vol 8 NO 8 793-800) PD-L1 is present in the cytoplasm and plasma membrane of cancer cells, although not all cancers or all cells within tumors express PD-L1 (Dong et al., 2002. Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune invasion. Nature medicine. Vol 8 NO 8 793-800).Multiple tumor microenvironment cells contribute to immune suppression by upregulating PD-L1 expression. This effect is called "adaptive immune resistance" because tumors protect themselves by inducing PD-L1 in response to IFN-γ produced by activated T cells (Sharma et al., 2017. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell. Vol. 168, 707-723). PD-L1 can also be regulated by oncogenes, a mechanism known as intrinsic immune resistance (Akbay et al., 2013. Activation of the PD-1 pathway contributes to immune excape in EGFR-driven lung tumors. Cancer discovery. 1355-1363). Within the tumor microenvironment, PD-L1 is also expressed on myeloid cells and activated T cells (Tumeh et al., 2014. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 515(7528):568-571). PD-L1 expression is induced by multiple proinflammatory molecules, including type I and type II IFN-γ, TNF-α, LPS, GM-CSF, and VEGF, as well as the cytokines IL-10 and IL-4, with IFN-γ being the most potent inducer (Sznol and Chen, 2013. Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer. Clin Cancer Res;19(5);1021-34).

[0079] The programmed cell death 1 protein (PD-1) is a cell surface receptor that belongs to the CD28 family of receptors and is expressed on T cells and pro-B cells. PD-1 is currently known to bind to two ligands, PD-L1 and PD-L2. Functioning as an immune checkpoint, PD-1 plays an important role in downregulating the immune system by inhibiting T cell activation, which in turn reduces autoimmunity and promotes self-tolerance. The inhibitory effect of PD-1 is thought to be achieved through a dual mechanism: promoting apoptosis (programmed cell death) in antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis in regulatory T cells (suppressor T cells). PD-1 is also known by a number of different names, including PDCD1, programmed cell death 1, systemic lupus erythematosus susceptibility 2, protein PD-1, HPD-1, PD1, programmed cell death 1 protein, CD279 antigen, CD279, HPD-L, HSLE1, SLEB2, and PD-1. The external IDs for PD-1 are HGNC:8760, Entrez Gene:5133, Ensembl:ENSG00000188389, OMIM:600244, and UniProtKB:Q15116. PD-1 inhibitors, a new class of drugs that block the activity of PD-1, activate the immune system to attack tumors and thus have been used successfully to treat several types of cancer. When PD-1 is mentioned herein, reference is made to human PD-1 unless otherwise specified. The PD-1 antigen-binding site binds to PD-1 and its various variants, for example, variants expressed by some PD-1-positive tumor cells.

[0080] Binding of PD-L1 to PD-1 or B7.1 (CD80) transmits an inhibitory signal that reduces the proliferation of PD-1-expressing T cells. PD-1 is thought to be able to control the accumulation of foreign antigen-specific T cells through apoptosis. PD-L1 is expressed by various cancer cells, and its expression is thought to be at least partially involved in the attenuation of immune responses to cancer cells. PD-L1 is a member of the B7 family of proteins and is known by various other names, including CD274 molecule, CD274 antigen, B7 homolog 1, PDCD1 ligand 1, PDCD1LG1, PDCD1L1, B7H1, PDL1, programmed cell death 1 ligand 1, programmed death ligand 1, B7-H1, and B7-H. The external Ids for CD274 are HGNC:17635, Entrez Gene:29126, Ensembl:ENSG00000120217, OMIM:605402, UniProtKB:Q9NZQ7. When PD-L1 is referred to herein, reference is made to human PD-L1 unless otherwise stated. The PD-L1 antigen-binding site binds to PD-L1 and its various variants, for example, variants expressed by some PD-L1-positive tumor cells.

[0081] PD-L2 is the second ligand for PD-1. Engagement of PD-1 by PD-L2 inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2 / PD-1 binding inhibits B7-CD28 signaling. At high antigen concentrations, PD-L2 / PD-1 binding reduces cytokine production. PD-L expression is upregulated on antigen-presenting cells by interferon-gamma treatment. It is expressed in several normal tissues and various tumors. PD-L1 and PD-L2 are thought to have overlapping functions and regulate T cell responses. The protein is known by several other names, including programmed cell death 1 ligand 2, B7 dendritic cell molecule, programmed death ligand 2, butyrophilin B7-DC, PDCD1 ligand 2, PD-1 ligand 2, PDCD1L2, B7-DC, CD273, B7DC, PDL2, PD-1-ligand 2, CD273 antigen, BA574F11.2, and Btdc. The external identifiers for PD-L2 are HGNC:18731, Entrez Gene:80380, Ensembl:ENSG00000197646, OMIM:605723, and UniProtKB:Q9BQ51. When PD-L2 is referred to herein, reference is made to human PD-L2 unless otherwise specified. The PD-L2 antigen-binding site binds to PD-L2 and its various variants, for example, variants expressed by some PD-L2-positive tumor cells.

[0082] Immune checkpoint inhibitors In certain embodiments, the immune checkpoint inhibitor comprises a PD-L1, PD-L2, or PD-1 inhibitor. In certain embodiments, the immune checkpoint inhibitor comprises or is an antibody. In certain embodiments, the immune checkpoint inhibitor comprises or is an antibody that targets PD-L1, PD-L2, or PD-1. In certain embodiments, the immune checkpoint inhibitor comprises or is an antibody that inhibits PD-L1, PD-L2, or PD-1. In certain embodiments, the PD-1, PD-L2, or PD-L1 inhibitor is an antibody or other binding molecule. In certain embodiments, the immune checkpoint inhibitor comprises or is a PD-L1 inhibitor. In certain embodiments, the immune checkpoint inhibitor comprises or is a PD-1 inhibitor. In certain embodiments, the immune checkpoint inhibitor comprises or is an anti-PD-L1 antibody. In certain embodiments, the immune checkpoint inhibitor comprises or is an anti-PD-1 antibody, hi certain embodiments, the immune checkpoint inhibitor is an antibody that binds to or is capable of binding to PD-L1 or PD-1.

[0083] In certain aspects, the immune checkpoint inhibitor comprises or is nivolumab, pembrolizumab, cemiplimab, penprimimab, retifanlimab, sintilimab, tislelizumab, toripalimab, dostarimab, atezolizumab, avelumab, or durvalumab.

[0084] Immune checkpoint inhibitors, such as PD-1 inhibitors, are known in the art. They include antibodies that bind to and block PD-1. For example, pembrolizumab is a humanized antibody used in cancer immunotherapy to treat melanoma, lung cancer, head and neck cancer, Hodgkin's lymphoma, gastric cancer, and cervical cancer. It is administered by slow intravenous injection. Pembrolizumab is a therapeutic antibody that binds to and blocks PD-1, located on lymphocytes. This receptor is a so-called "immune checkpoint" and is therefore generally involved in preventing the immune system from attacking the body's own tissues. Normally, the PD-1 receptor on activated T cells binds to PD-L1 or PD-L2 ligands present on normal cells in the body, inactivating any potential cell-mediated immune response against these cells. Many cancers produce proteins, such as PD-L1, that also bind to the PD-1 receptor, thus blocking the body's ability to kill the cancer. Pembrolizumab works by inhibiting lymphocyte PD-1 receptors, inactivating them and blocking ligands that can prevent an immune response. This allows the immune system to target and destroy cancer cells, but it also blocks an important mechanism that prevents the immune system from attacking the body itself.

[0085] Pembrolizumab was approved for medical use in the United States in 2014. In 2017, the U.S. Food and Drug Administration (FDA) approved it for any unresectable or metastatic solid tumor with certain genetic abnormalities (mismatch repair deficiency or microsatellite instability).

[0086] In certain embodiments, the immune checkpoint inhibitor is nivolumab, pembrolizumab, cemiplimab, penprimimab, retifanlimab, sintilimab, tislelizumab, toripalimab, or dostarimab. In certain embodiments, the immune checkpoint inhibitor is pembrolizumab. In certain embodiments, the immune checkpoint inhibitor is nivolumab. In certain embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In certain embodiments, the PD-L1 inhibitor is atezolizumab, avelumab, or durvalumab. The amino acid sequences of all such immune checkpoint inhibitors are well known in the art.

[0087] Durvalumab, sold under the brand name Imfinzi™, is an FDA-approved immune checkpoint inhibitor for the treatment of cancers such as bladder and lung cancer. Durvalumab is a human immunoglobulin G1 kappa (IgG1κ) monoclonal antibody that binds to PD-L1 and blocks its interaction with PD-1 (CD279). Durvalumab is an immune checkpoint inhibitor, or sometimes referred to as an immune checkpoint inhibitor drug.

[0088] Pembrolizumab (sold under the brand name Keytruda™) is a humanized antibody used in cancer immunotherapy to treat various cancers, including melanoma, lung cancer, and Hodgkin lymphoma, and functions as an immune checkpoint inhibitor. It is an IgG4 isotype antibody that targets the programmed cell death protein 1 (PD-1) receptor on lymphocytes. Pembrolizumab was approved for medical use in the United States in 2014. In 2017, the U.S. Food and Drug Administration (FDA) approved it for any unresectable or metastatic solid tumor with certain genetic abnormalities. It is listed on the World Health Organization's Essential Medicines List.

[0089] Nivolumab (sold under the brand name Opdivo™) is an immune checkpoint inhibitor used to treat many cancers, including melanoma, lung cancer, malignant pleural mesothelioma, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, urothelial carcinoma, colon cancer, esophageal squamous cell carcinoma, liver cancer, gastric cancer, and esophageal or gastroesophageal junction (GEJ) cancer. Nivolumab is a human IgG4 monoclonal antibody that blocks PD-1. Nivolumab was approved for medical use in the United States in 2014. It is listed on the World Health Organization's list of essential medicines. Nivolumab is the second FDA-approved systemic therapy for mesothelioma and the first FDA-approved immunotherapy for the first-line treatment of gastric cancer.

[0090] Atezolizumab (sold under the brand name Tecentriq™) is a monoclonal antibody drug used to treat urothelial carcinoma, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), small cell lung cancer (SCLC), and hepatocellular carcinoma (HCC). It is a humanized monoclonal antibody of the IgG1 isotype that targets programmed death-ligand 1 (PD-L1). Atezolizumab was the first PD-L1 inhibitor approved by the U.S. Food and Drug Administration.

[0091] Retifanlimab (formerly known as MGA012) is a humanized anti-PD-1 monoclonal antibody being developed for use as monotherapy and in combination with other cancer therapeutics. Retifanlimab is undergoing clinical trials (NCT04472429 and NCT04205812) as monotherapy for patients with microsatellite instability-high endometrial cancer, Merkel cell carcinoma, and squamous cell carcinoma of the anal canal (SCAC), and in combination with platinum-based chemotherapy for patients with non-small cell lung cancer and SCAC. Retifanlimab has received orphan drug designation by the FDA for the treatment of anal cancer and may be administered intravenously at 500 mg every four weeks (500 mg q4w).

[0092] Cemiplimab (sold under the brand name Libtayo®) is a monoclonal antibody drug for the treatment of squamous cell skin cancer. Cemiplimab belongs to a class of drugs that bind to programmed death receptor 1 (PD-1) and block the PD-1 / PD-L1 pathway. In September 2018, it was approved by the FDA for the treatment of patients with metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC who are not candidates for curative surgery or curative radiation therapy. In June 2019, it was approved for medical use in the European Union.

[0093] Dostarimab (Jemperli) is a PD-1 blocking antibody indicated for the treatment of adult patients with mismatch repair deficient (dMMR) recurrent or advanced endometrial cancer that has progressed on or after prior treatment with a platinum-containing regimen, as determined by an FDA-approved test. Dosage and administration include 500 mg every 3 weeks (q3w) for doses 1 to 4, followed by 1,000 mg every 6 weeks thereafter starting 3 weeks after dose 4 (dose 5 onwards). The dosage form and strength is an injection of a 500 mg / 10 mL (50 mg / mL) solution in a single-dose vial.

[0094] Avelumab (or Bavencio®) is a PD-L1 blocking antibody indicated for Merkel cell carcinoma, urothelial carcinoma, and renal cell carcinoma. Its dosage and indication include premedication for the first four infusions, followed by as-needed infusions. Avelumab is used for Merkel cell carcinoma, urothelial carcinoma, and herein as 800 mg every two weeks (q2w), 800 mg every two weeks. Optionally, avelumab is administered orally twice daily at 800 mg every two weeks in combination with axitinib 5 mg, and avelumab is administered as an intravenous infusion over 60 minutes. The dosage form and strength is an injection of a 200 mg / 10 mL (20 mg / mL) solution in a single-dose vial.

[0095] Penprimab is a humanized anti-PD-1 monoclonal antibody developed by Akeso Biopharma in collaboration with Chia Tai Tianqing for the treatment of various cancers, including Hodgkin lymphoma, nasopharyngeal carcinoma, non-small cell lung cancer (NSCLC), and solid tumors. Penprimab is an immunoglobulin G1 monoclonal antibody engineered to completely eliminate Fcγ receptor binding and Fc-mediated effector functions, which may compromise antitumor activity. In August 2021, penprimab received its first approval in China for the treatment of adult patients with relapsed or refractory classical Hodgkin lymphoma who have received at least second-line chemotherapy. The recommended dose of penprimab is 200 mg administered intravenously once every two weeks (200 mg q2w) until disease progression or unacceptable toxicity occurs.

[0096] Sintilimab (Tyvyt®) is an anti-PD-1, fully human IgG4 monoclonal antibody that binds to PD-1 on the surface of T cells, blocking the PD-1 / PD-ligand 1 (PD-L1) pathway and reactivating T cells to kill cancer cells. Sintilimab was developed by Innovent Biologics and Eli Lilly and Company and is approved by the State Food and Drug Administration of the People's Republic of China to treat relapsed or refractory classical Hodgkin lymphoma in patients who have received two or more lines of systemic chemotherapy. Instructions for its use include an intravenous injection of 200 mg once every three weeks (q3w).

[0097] Tislelizumab is a humanized IgG4 anti-PD-1 monoclonal antibody developed both as monotherapy and in combination with other therapies. The BLA submission was based on data from the phase III RATIONALE 302 trial, which demonstrated a 30% reduction in the risk of death (HR=0.70, 95% CI: 0.57-0.85, p=0.0001) and a 2.3-month extension in median overall survival compared with chemotherapy in people with unresectable, recurrent, locally advanced, or metastatic esophageal squamous cell carcinoma who had received prior systemic therapy. Pharmacokinetics, safety, and antitumor activity data from both phase IA and phase IB studies determined the recommended tislelizumab dose of 200 mg intravenously every 3 weeks (200 mg q3w), and therefore, this was the dose and schedule recommended for incorporation into subsequent clinical trials.

[0098] In certain aspects, the immune checkpoint inhibitor is a full-length antibody or a fragment of an antibody, such as a Fab fragment or a single-chain variable fragment (scFv). In certain aspects, the immune checkpoint inhibitor is a full-length antibody or a fragment thereof according to the uses or methods of the present disclosure. In certain aspects, the immune checkpoint inhibitor is or comprises an antibody selected from nivolumab, pembrolizumab, cemiplimab, dostarimab, atezolizumab, avelumab, and durvalumab, or a functional fragment or variant thereof. In certain aspects, the immune checkpoint inhibitor is or comprises pembrolizumab, or a functional fragment or variant thereof.

[0099] In certain aspects, the immune checkpoint inhibitor comprises or consists of the amino acid sequence of, or having substantial sequence identity to, the amino acid sequence of the antigen-binding site of one of nivolumab, pembrolizumab, cemiplimab, dostarimab, atezolizumab, avelumab, and durvalumab. In certain aspects, the immune checkpoint inhibitor comprises or consists of the amino acid sequence of, or having substantial sequence identity to, the amino acid sequence of the antigen-binding site of pembrolizumab.

[0100] Toripalimab (or Tuoyi™) is a selective, recombinant, humanized monoclonal antibody against PD-1 developed by Shanghai Junshi Bioscience Co., Ltd. Toripalimab can bind to PD-1 and block its interaction with its ligands. Toripalimab received conditional approval in China in December 2018 for the treatment of melanoma. It was also approved for the treatment of nasopharyngeal carcinoma and urothelial carcinoma in 2021. Furthermore, the U.S. Food and Drug Administration has granted several orphan drug designations to toripalimab. In a first-in-human phase I study, NCT02836795, the recommended phase II dose was determined to be 3 mg / kg Q2W. In the study, patients in the dose-escalation cohorts received intravenous infusions at 1 mg / kg, 3 mg / kg, and 10 mg / kg Q2W, and 28 days after the first dose, subjects continued to receive toripalimab at their intended dose level Q2W.

[0101] In certain aspects, the immune checkpoint inhibitor is or comprises at least one, more preferably at least two, and even more preferably three heavy chain CDRs that are identical or substantially identical to the CDRs of one of nivolumab, pembrolizumab, cemiplimab, dostarimab, atezolizumab, avelumab, and durvalumab, particularly pembrolizumab. In certain embodiments, the PD-1 or PD-L1 inhibitor comprises at least one, more preferably at least two, and even more preferably three CDRs that are identical to the CDRs found in pembrolizumab. In certain aspects, the immune checkpoint inhibitor is or comprises a heavy chain CDR1 identical to CDR1 from one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, and durvalumab, a heavy chain CDR2 identical to CDR2 from one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, and durvalumab, or a heavy chain CDR3 identical to CDR3 from one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, and durvalumab. In certain aspects, the immune checkpoint inhibitor is or comprises a heavy chain CDR1 and a heavy chain CDR2 that are identical to CDR1 and CDR2 from one of nivolumab, pembrolizumab, cemiplimab, dostarimab, atezolizumab, avelumab, and durvalumab; a heavy chain CDR1 and a heavy chain CDR3 that are identical to CDR1 and CDR3 from one of nivolumab, pembrolizumab, cemiplimab, dostarimab, atezolizumab, avelumab, and durvalumab; or a heavy chain CDR2 and a heavy chain CDR3 that are identical to CDR2 and CDR3 from one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, and durvalumab. In certain aspects, the immune checkpoint inhibitor is or comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 that are identical to the CDR1, CDR2, and CDR3 from one of nivolumab, pembrolizumab, cemiplimab, dostarimab, atezolizumab, avelumab, and durvalumab.In certain aspects, the immune checkpoint inhibitor comprises a heavy chain CDR1, a light chain CDR2, and a CDR3 that are identical to the CDR1, CDR2, and CDR3 from pembrolizumab.

[0102] Methods for determining or annotating the CDR sequences of a given antibody are known to those skilled in the art, for example using annotation systems such as IMGT, Chothia, Kabat, or other suitable annotation systems.

[0103] In certain aspects, the immune checkpoint inhibitor is or comprises at least one, more preferably at least two, and even more preferably three light chain CDRs that are identical or substantially identical to the CDRs of one of nivolumab, pembrolizumab, cemiplimab, dostarimab, atezolizumab, avelumab, and durvalumab, particularly pembrolizumab. In certain embodiments, the PD-1 or PD-L1 inhibitor comprises at least one, more preferably at least two, and even more preferably three CDRs that are identical to the CDRs found in pembrolizumab. In certain aspects, the immune checkpoint inhibitor is or comprises a light chain CDR1 identical to CDR1 from one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, and durvalumab, a light chain CDR2 identical to CDR2 from one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, and durvalumab, or a light chain CDR3 identical to CDR3 from one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, and durvalumab. In certain aspects, the immune checkpoint inhibitor is or comprises a light chain CDR1 and a light chain CDR2 that are identical to CDR1 and CDR2 from one of nivolumab, pembrolizumab, cemiplimab, dostarimab, atezolizumab, avelumab, and durvalumab; a light chain CDR1 and a light chain CDR3 that are identical to CDR1 and CDR3 from one of nivolumab, pembrolizumab, cemiplimab, dostarimab, atezolizumab, avelumab, and durvalumab; or a CDR2 and CDR3 that are identical to CDR2 and CDR3 from one of nivolumab, pembrolizumab, cemiplimab, dostarlimab, atezolizumab, avelumab, and durvalumab. In certain aspects, the immune checkpoint inhibitor is or comprises a light chain CDR1, light chain CDR2, and light chain CDR3 that are identical to the CDR1, CDR2, and CDR3 from one of nivolumab, pembrolizumab, cemiplimab, dostarimab, atezolizumab, avelumab, and durvalumab.In certain aspects, the immune checkpoint inhibitor comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3 that are identical to the CDR1, CDR2, and CDR3 from pembrolizumab.

[0104] In certain embodiments, immune checkpoint inhibitors are administered according to current recommendations established by FDA approval. Such recommendations typically consist of instructions for use in treating cancer in a subject. Thus, in certain embodiments, the present disclosure relates to instructions for use of such immune checkpoint inhibitors.

[0105] Cancer of the present disclosure The words cancer and tumor are used herein, unless otherwise specified, and generally both refer to cancer.

[0106] Cancers collectively known as head and neck cancers typically arise from squamous cells lining the moist mucosal surfaces inside the head and neck, such as the inside of the mouth, nose, and throat. These squamous cell cancers are often referred to as squamous cell carcinoma of the head and neck, and are treated in certain aspects of the present disclosure. Although rare, head and neck cancers can also arise in the salivary glands. In certain aspects, head and neck cancers can arise within the oral cavity, including the lips, the front two-thirds of the tongue, the gums, the inner lining of the cheeks and lips, the floor of the mouth under the tongue, the hard palate, and a small area of ​​gum behind the wisdom teeth.

[0107] Thus, in certain aspects, the head and neck cancer is squamous cell carcinoma, including laryngeal cancer, hypopharyngeal cancer, nasal cancer, paranasal cancer, oral cancer, oropharynx cancer, or salivary gland cancer. In certain aspects, the present disclosure relates to the treatment of cancers, including squamous cell head and neck cancer, such as those located in the oropharynx, hypopharynx, larynx, oral cavity, or tongue.

[0108] In certain embodiments, the cancer comprises or is adenocarcinoma, squamous cell carcinoma, or head and neck cancer, including squamous cell carcinoma of the head and neck (SCCHN).

[0109] In certain embodiments, the cancer is squamous cell carcinoma of the head and neck. In certain embodiments, the cancer is squamous cell carcinoma of the head and neck that expresses EGFR and PD-L1. In certain embodiments, the cancer is squamous cell carcinoma that expresses EGFR and PD-L1.

[0110] In certain aspects, the cancer is a cancer of the pharynx, including the oropharynx and hypopharynx, oral cavity, larynx, paranasal sinuses, nasal cavity, or salivary glands.

[0111] In certain aspects, the cancer is a cancer of the oropharynx, oral cavity, hypopharynx, or larynx.

[0112] In certain aspects, the primary location of the cancer is in the oropharynx, oral cavity, hypopharynx, or larynx. In certain aspects, the head and neck cancer is squamous cell carcinoma of unknown primary (also referred to in the art as carcinoma of unknown primary or CUP).

[0113] In certain aspects, the cancer is a locally advanced, unresectable, or metastatic cancer, such as head and neck squamous cell carcinoma.

[0114] Expression of targets of the present disclosure by cancer In certain aspects, the cancer expresses EGFR, LGR5, and / or PD-L1. In certain aspects, the cancer expresses EGFR. In certain aspects, the cancer expresses LGR5. In certain aspects, the cancer expresses PD-L1. In certain aspects, the cancer expresses EGFR and PD-L1. In certain aspects, the cancer expresses EGFR, LGR5, and PD-L1.

[0115] As used herein, a cancer expresses EGFR if it contains cells that express EGFR. Cells that express EGFR contain detectable levels of RNA encoding EGFR. In certain embodiments, EGFR expression is determined by ISH. As used herein, a cancer expresses LGR5 if it contains cells that express LGR5. Cells that express LGR5 contain detectable levels of RNA encoding LGR5.

[0116] In certain embodiments, EGFR protein expression is detected by IHC. In certain embodiments, EGFR expression is determined by IHC using a commercially available EGFR detection kit, such as the EGFR pharmDx™ kit for a Dako automated stainer (Agilent) using the manufacturer's recommendations, or a commercially available IHC EGFR detection kit based on EGFR clone 113, which binds to the EGFR extracellular domain (Leica, https: / / shop.leicabiosystems.com / us / ihc-ish / ihc-primary-antibodies / pid-epidermal-growth-factor-receptor). Alternatively, EGFR expression is determined using Novocastra™ Liquid Mouse Monoclonal Antibody Epidermal Growth Factor Receptor (product code: NCL-L-EGFR, epidermal growth factor receptor-IHC primary antibody from leicabiosystems.com), based on clone EGFR.113.

[0117] Briefly, the commercially available EGFR pharmDx™ IHC kit system contains the necessary reagents to complete the IHC staining procedure on routinely fixed, paraffin-embedded specimens. After incubation with a primary, non-Her2, Her3, and Her4 cross-reactive monoclonal antibody (clone 2-18C9) against the human EGFR protein, the kit employs a ready-to-use visualization reagent based on dextran technology. This reagent consists of both a secondary goat anti-mouse antibody molecule and a horseradish peroxidase molecule linked to a common dextran polymer backbone. Subsequent enzymatic conversion of the added chromogen results in the formation of a visible reaction product at the antigen site. Results are routinely evaluated 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 kit reagent performance.

[0118] The staining intensity is established as follows: 3+ (strong staining): visible at high levels if necessary, at low levels of magnification, visible with a 5x objective; 2+ (moderate staining): visible at medium levels of magnification, at a 10x or 20x objective; 1+ (weak staining): visible at high magnification, only reliably with a 40x objective; 0 (no staining): no visible staining at high magnification.

[0119] In certain embodiments, EGFR expression is determined using immunohistochemistry (IHC), and the cancer is IHC-positive for EGFR. In certain embodiments, the cancer is characterized by an EGFR IHC score of 2+ or 3+. In certain embodiments, the cancer is characterized by an H-score for EGFR greater than 50, greater than 80, or greater than 200 but less than or equal to 300.

[0120] In certain embodiments, EGFR expression is determined using immunohistochemistry (IHC), followed by assigning an H-score for EGFR using a range of 0 to 300. In certain embodiments, the cancer of the present disclosure is a cancer characterized by an H-score for EGFR greater than 200 on a scale of 0 to 300. Thus, in certain embodiments, the EGFR H-score is greater than 200 and less than or equal to 300. In certain embodiments, the cancer of the present disclosure is characterized by an H-score for EGFR greater than 50 on a scale of 0 to 300. In certain embodiments, the cancer of the present disclosure is a head and neck cancer characterized by an H-score for EGFR greater than 50 on a scale of 0 to 300. In certain embodiments, the cancer of the present disclosure is characterized by an H-score for EGFR greater than 80 on a scale of 0 to 300. In certain embodiments, the cancer of the present disclosure is a head and neck cancer characterized by an H-score for EGFR greater than 80 on a scale of 0 to 300.

[0121] In another embodiment, the cancer is head and neck cancer characterized by an EGFR IHC score of 2+ or 3+.

[0122] As used herein, determining an H-score to assign an EGFR expression status involves a first step of establishing the intensity of membrane staining (resulting in a score of 0, 1+, 2+, or 3+) determined for each cell within a given field, as described herein. The percentage of cells at each staining intensity level is then calculated, and finally, an H-score is assigned using the following formula: [1 × (% of cells with 1+ staining) + 2 × (% of cells with 2+ staining) + 3 × (% of cells with 3+ staining)], resulting in an H-score for EGFR between 0 and 300. As a result, the H-score gives greater relative weight to higher intensity or amount of staining in a given tumor sample.

[0123] Optionally, treatment with the antibody, or functional part, derivative, and / or analog thereof, includes (or in certain embodiments is followed by) diagnosing the subject for EGFR status. In certain embodiments, subjects with an IHC score of 3+, or whose cancer is characterized by an H-score for EGFR greater than 200 on a scale of 0 to 300, are selected for treatment. In certain embodiments, treatment of the subject is followed by diagnosing the subject with an H-score for EGFR greater than 200 on a scale of 0 to 300.

[0124] In certain embodiments, the cancer expresses LGR5. As used herein, a cancer expresses LGR5 if the cancer contains cells that express LGR5. Cells that express LGR5 contain detectable levels of RNA encoding LGR5.

[0125] Expression can often also be detected by incubating cells with an antibody that binds to LGR5. However, some cells do not express the protein at a high enough level for such antibody testing. In such cases, mRNA or other forms of nucleic acid sequence detection are preferred. In certain embodiments, LGR5 is detected via mRNA expression. In certain embodiments, LGR5 detection is by RNA sequencing. In certain embodiments, LGR5 detection is by tissue microarray (TMA) staining. In certain embodiments, LGR5 expression is determined using in situ hybridization (ISH). Thus, preferably, the cancer is ISH-positive for LGR5. ISH-positive preferably means that expression is characterized by an H-score of 1 or greater.

[0126] Techniques for detection and scoring based on TMA, ISH, and IHC are each well known to those skilled in the art and are usually commercially available as standard kits. For example, for LGR5, quantifying mRNA levels using ISH and expressing such expression based on an H score can be performed using commercially available kits such as the RNAscope® kit from Advanced Cell Diagnostics (Hayward, CA, USA) on a staining platform such as the BondRx platform (Leica, Wetzlar, Germany). Typically, the ISH H score for LGR5 quantification ranges from 0 to 400. Alternatively, LGR5 expression can be determined by RNA sequencing (RNAseq).

[0127] In certain embodiments, the cancer expresses LGR5 at a level sufficient for an antibody that binds to the LGR5 protein, e.g., an antibody comprising an LGR5-binding variable domain comprising the amino acid sequence of the VH chain of MF5816 shown in Figure 3, or a VH chain of an surrogate variable domain that binds to LGR5 described herein. In certain embodiments, the cancer expresses EGFR at a level sufficient for an antibody that binds to the EGFR protein, e.g., an antibody comprising an EGFR-binding variable domain comprising the amino acid sequence of the VH chain of MF3755 shown in Figure 3, or a VH chain of an surrogate variable domain that binds to EGFR described herein.

[0128] In certain embodiments, the cancer expresses PD-L1. In certain embodiments, PD-L1 protein expression is detected by IHC. In certain embodiments, combined positive score (CPS) scoring is performed on a sample obtained from the subject. In certain embodiments, CPS is determined by an FDA-approved test. In certain embodiments, CPS is determined using IHC, particularly using clone 22C3 (Agilent). In certain embodiments, CPS is determined using the pharmDx PD-L1 IHC kit using clone 22C3. In certain embodiments, the combined positive score is determined according to Example 4. In certain embodiments, PD-L1 expression is determined by IHC using a commercially available PD-L1 detection kit, such as the PD-L1 IHC22C3 pharmDx™ assay for a Dako automated stainer (Agilent Technologies, Carpinteria, CA, USA), using the manufacturer's recommendations.

[0129] In certain embodiments, the cancer has a CPS of 1 or greater but 100 or less for PD-L1 expression. As used herein, CPS is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells multiplied by 100. While the result of the calculation may exceed 100, the maximum score is defined as a CPS of 100. In certain embodiments, the CPS is determined using IHC. In certain embodiments, the CPS is determined by IHC using clone 22C3. In certain embodiments, the cancer has a CPS score of ≥1 to <20. In certain embodiments, the cancer has a CPS score of ≥20 to 100. In certain embodiments, the subject is positive for p16 status as determined in the subject or cancer, particularly oropharyngeal cancer, as described herein below. In certain embodiments, the subject is negative for p16 status as determined in a sample obtained from the subject or, particularly in the case of head and neck cancer other than oropharyngeal cancer, the subject.

[0130] In certain embodiments, CPS is determined by IHC using clone 22C3. In certain embodiments, PD-L1 protein expression is detected by IHC using PD-L1 IHC22C3, a qualitative immunohistochemistry assay using a monoclonal mouse anti-PD-L1, clone 22C3, intended for use in detecting PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) cancer tissues, using the EnVision FLEX visualization system on the automated stainer link 48.

[0131] In summary, the PD-L1 IHC22C3 pharmDx Kit contains all the necessary reagents for immunohistochemical staining (excluding wash buffer), control slides representing different expression levels of PD-L1 protein, and detailed instructions. The kit is specifically tailored for use with the Link 48 Autostainer. The PD-L1 IHC22C3 pharmDx kit contains the optimized reagents and protocols required to complete the IHC staining procedure for FFPE specimens using the Link 48 Autostainer and the PT Link Pretreatment Module. After incubation with a primary monoclonal antibody against PD-L1 or a negative control reagent, the specimen is incubated with a linker antibody specific to the host species of the primary antibody, followed by incubation with a ready-to-use visualization reagent consisting of a secondary antibody molecule and a horseradish peroxidase molecule linked to a dextran polymer backbone. Subsequent enzymatic conversion of the added chromogen results in the precipitation of a visible reaction product at the antigen site. The color of the chromogen reaction is modified by a chromogen enhancement reagent. The specimen can then be counterstained and coverslipped. Results are interpreted using a light microscope. Control slides containing two FFPE human cell lines are provided to verify staining performance.

[0132] In certain embodiments, CPS is determined by IHC using clone 28-8. In certain embodiments, PD-L1 protein expression is detected by IHC using PD-L1 IHC28-8, which is a qualitative immunohistochemistry assay using monoclonal rabbit anti-PD-L1, clone 28-8, intended for use in detecting PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) squamous cell carcinoma of the head and neck (SCCHN) tissue using the EnVision FLEX visualization system on the automated stainer Link 48.

[0133] In certain embodiments, samples obtained from subjects with cancer of the present disclosure are tested for their PD-L1 CPS score and tested for p16 status. Relevant CPS scores herein are CPS scores of ≧1 to 100, or ≧1 to <20, or ≧20 to 100. Relevant p16 status outcomes are p16 positive or negative.

[0134] Thus, in certain embodiments, subjects with cancer treated according to the methods or uses of the present disclosure have a CPS score of ≧1-100 and are positive for p16 status.

[0135] Alternatively, in certain embodiments, subjects with cancer treated according to the methods or uses of the present disclosure have a CPS score of ≧1-100 and are negative for p16 status.

[0136] Alternatively, in certain embodiments, subjects with cancer treated according to the methods or uses of the present disclosure have a CPS score of ≧1-20 and are positive for p16 status.

[0137] Alternatively, in certain embodiments, subjects with cancer treated according to the methods or uses of the present disclosure have a CPS score of ≧1-20 and are negative for p16 status.

[0138] Alternatively, in certain embodiments, subjects with cancer treated according to the methods or uses of the present disclosure have a CPS score of ≧20-100 and are positive for p16 status.

[0139] Alternatively, in certain embodiments, subjects with cancer treated according to the methods or uses of the present disclosure have a CPS score of ≧20-100 and are negative for p16 status.

[0140] Optionally, treatment with the antibody, or functional part, derivative, and / or analog thereof, and the immune checkpoint inhibitor optionally includes a step of diagnosing the subject for PD-L1 status. In certain embodiments, treatment is after the step of diagnosing the subject for PD-L1 status. In certain embodiments, subjects with head and neck cancer having a CPS score of at least 1 and less than or equal to 100 are selected for treatment. In certain embodiments, treatment of the subject is after a step of diagnosing the subject for a CPS score for PD-L1 of 1 to 100. In certain embodiments, the subject is diagnosed for PD-L1 status by having a CPS score of ≧1 to <20, or a CPS score of ≧20 to 100. Furthermore, treatment with the antibody, or functional part, derivative, and / or analog thereof, and the immune checkpoint inhibitor, optionally, is after a step of diagnosing the subject for p16 status. The subject may be determined to be p16 positive or negative, or may be diagnosed.

[0141] In certain aspects, the present disclosure provides methods of diagnosing a subject with head and neck cancer and selecting the subject for treatment with an antibody, or a functional portion, derivative, and / or analog thereof, disclosed herein, and an immune checkpoint inhibitor disclosed herein, comprising determining a CPS score for PD-L1 (of the subject or of a sample obtained from the cancer), selecting the subject for the treatment if the CPS score is greater than or equal to 1, and optionally less than or equal to 100, and treating the subject for the cancer. In certain aspects, the subject is selected for treatment if a CPS score of ≧1 to <20 is established. In certain aspects, the subject is selected for treatment if a CPS score of ≧20 to 100 is established. Furthermore, diagnosing and selecting the subject optionally comprises determining the p16 status of the subject or of a sample obtained from the cancer, and selecting the subject for treatment if the p16 status is positive, particularly in the case of oropharyngeal cancer. Furthermore, diagnosing and selecting a subject optionally includes determining the p16 status of a sample obtained from the subject or cancer, and selecting the subject for treatment if the p16 status is negative, particularly in the case of head and neck cancer other than oropharyngeal cancer.

[0142] Also provided are methods for selecting a subject having head and neck cancer for treatment with an antibody, or a functional portion, derivative, and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR as disclosed herein, and an immune checkpoint inhibitor as disclosed herein, the methods comprising: a) determining a combined positive score (CPS) for PD-L1 expression in a sample obtained from the subject; and b) selecting the subject for treatment if the sample has a CPS for PD-L1 expression of 1 or greater, but optionally less than or equal to 100. In certain aspects, the subject has a CPS score of ≧1 to <20 and is selected for treatment, or a CPS score of ≧20 to 100. Furthermore, the methods may optionally comprise the further step of determining the p16 status of a sample obtained from the subject or cancer, and selecting the subject for treatment if the p16 status is positive, particularly in the case of oropharyngeal cancer. Furthermore, the method may optionally include the further step of determining the p16 status of a sample obtained from the subject or cancer, and selecting the subject for treatment if the p16 status is negative, particularly in the case of head and neck cancer other than oropharyngeal cancer.

[0143] Also disclosed is a method for establishing whether a subject with head and neck cancer is likely to respond to treatment with an antibody, or a functional portion, derivative, and / or analog thereof, disclosed herein, comprising a variable domain that binds to the extracellular portion of EGFR and an immune checkpoint inhibitor disclosed herein, the method comprising: a) determining a combined positive score (CPS) for PD-L1 expression in a sample obtained from the subject; and b) selecting samples that exhibit a CPS expression of PD-L1 of 1 or greater, but optionally 100 or less, thereby establishing that the subject from whom the sample was derived is likely to respond to the treatment. In certain aspects, the subject has a CPS score of ≥ 1 to < 20, thereby establishing that the subject is likely to respond to the treatment. In certain aspects, the subject has a CPS score of ≥ 20 to 100, thereby establishing that the subject is likely to respond to the treatment. Furthermore, the method may optionally comprise the further step of determining the p16 status of a sample obtained from the subject or cancer. In certain embodiments, the subject has p16 positive status, particularly in the case of oropharyngeal cancer, thereby establishing that the subject is likely to respond to the treatment. In certain embodiments, the subject has p16 negative status, particularly in the case of head and neck cancer other than oropharyngeal cancer, thereby establishing that the subject is likely to respond to the treatment.

[0144] Also provided is a method for classifying a subject having head and neck cancer based on a combined positive score for PD-L1 expression before treatment with an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional portion, derivative, and / or analog thereof, and an immune checkpoint inhibitor disclosed herein, the method comprising: a) determining a combined positive score for PD-L1 expression in a sample obtained from the subject; and b) classifying the subject from whom the sample was obtained as eligible for the treatment if the sample shows a CPS expression for PD-L1 of 1 or greater, but optionally 100 or less. In certain aspects, if in step b) the sample shows a CPS expression for PD-L1 of ≧1-20, the subject is classified as eligible for the treatment. In certain aspects, if in step b) the sample shows a CPS expression for PD-L1 of ≧20-100, the subject is classified as eligible for the treatment. Furthermore, the method may optionally comprise the further step of determining the p16 status of the sample obtained from the subject or the cancer. In certain embodiments, the subject has p16 positive status, particularly in the case of oropharyngeal cancer, thereby classifying the subject as eligible for the treatment. In certain embodiments, the subject has p16 negative status, particularly in the case of head and neck cancer other than oropharyngeal cancer, thereby classifying the subject as eligible for the treatment.

[0145] In certain embodiments, the combined positive score is determined by an FDA-approved test. In certain embodiments, the combined positive score is determined using IHC, particularly clone 22C3.

[0146] In certain aspects, the CPS is a histologically confirmed CPS having a value of 1 or greater, and optionally 100 or less, where the CPS is defined as the number of PD-L1 positive tumor cells, lymphocytes, and macrophages divided by the total number of tumor cells multiplied by 100. In certain embodiments, a subject of the present disclosure has a PD-L1 CPS score of ≧1 and < 20. In certain embodiments, a subject of the present disclosure has a PD-L1 CPS score of CPS ≧20, but e.g., a CPS score of 100 or less.

[0147] p16 (INK4a, cyclin-dependent kinase inhibitor 2A (CDKN2A)) is typically used as a marker for HPV status. In certain embodiments, p16 status is determined by immunohistochemistry (IHC). In certain embodiments, testing for p16 IHC status is based on p16 / HPV testing for head and neck cancer according to CAP guidelines 2018. IHC testing for p16 status can be performed using any commercially available test according to the manufacturer's instructions. Examples include commercially available test kits such as IHC tests based on clone E6H4 (CINtec® Histology, Roche Diagnostics) or anti-p16 primary antibody 6H12 (e.g., catalog number: PA0016, Leica Biosystems).

[0148] In certain embodiments, testing for p16 status follows the CAP guideline 2018 (Lewis et al., Human Papillomavirus Testing in Head and Neck Carcinomas; Guideline From the College of American Pathologists, Arch Pathol Lab Med, Vol 142, May 2018). In certain embodiments, statement 8 of the guideline is followed, which states that pathologists should report p16 IHC positivity as a surrogate for high-risk HPV in tissue specimens if there is at least 70% nuclear and cytoplasmic expression with at least moderate to strong intensity. In certain embodiments, p16 testing is performed on a sample obtained from a subject with oropharyngeal cancer or an oropharyngeal cancer / tumor sample. In certain embodiments, testing for p16 status is according to Example 6.

[0149] Previous treatment In certain embodiments, the subject has not undergone prior anti-cancer therapy for the treatment of the cancer.

[0150] In certain embodiments, the subject has not received prior treatment with anti-PD-L1, anti-PD-1, or anti-EGFR therapy. The treatments of the present disclosure are particularly effective in subjects who have not yet received any prior anti-cancer treatment. Also, exposing treatment-naive cancers to the treatments of the present disclosure is particularly effective. Treatments involving subjects or cancers who have not previously received anti-cancer treatment are also referred to as first-line treatments. Thus, in certain embodiments, the antibodies of the present disclosure, or functional portions thereof, derivatives and / or analogs thereof, and immune checkpoint inhibitors are used as first-line treatments.

[0151] In certain embodiments, the subject has not received prior anti-cancer therapy for the treatment of the cancer at least six months prior to receiving the antibody, or functional part, derivative, and / or analog thereof, and the immune checkpoint inhibitor.

[0152] In certain embodiments, the prior anti-cancer treatment comprises chemotherapy, immunotherapy, an anti-EGFR agent, an antibody targeting EGFR, cetuximab, a PD-1 inhibitor, or a PD-L1 inhibitor.

[0153] In certain embodiments, the subject has not been previously treated with an anti-EGFR agent. In certain embodiments, the subject has not been treated with an antibody targeting EGFR. In certain embodiments, the subject has not been treated with cetuximab. Such subjects are also referred to as cetuximab-naive subjects or anti-EGFR-naive subjects. In other words, the subject's cancer has not been previously treated with any anti-cancer agent, such as an anti-EGFR agent. In certain embodiments, the subject's cancer has not been treated with an antibody targeting EGFR. In certain embodiments, the subject's cancer has not been treated with cetuximab. Such subjects are also referred to as cetuximab-naive subjects or anti-EGFR-naive subjects.

[0154] In certain embodiments, the subject of the present disclosure has not received prior treatment with an anti-cancer immunotherapy. In certain embodiments, the immunotherapy includes an anti-PD-L1 or anti-PD-1 therapy, including pembrolizumab, nivolumab, atezolizumab, retifanlimab, cemiplimab, or other anti-PD1, anti-PD-L1 antibodies approved or in development. In certain embodiments, the anti-cancer immunotherapy includes an immune checkpoint inhibitor, including prior pembrolizumab treatment.

[0155] In certain embodiments, the subject of the present disclosure has not received prior anti-cancer therapy for the treatment of the cancer at least six months prior to receiving the antibody, or functional portion, derivative, and / or analog thereof, and the immune checkpoint inhibitor. In certain embodiments, the subject has received multimodal therapy, including surgery, radiation therapy, and / or platinum-containing chemotherapy, such as cisplatin, six or more months prior to receiving the antibody, or functional portion, derivative, and / or analog thereof, and the immune checkpoint inhibitor. In certain embodiments, the subject has received the multimodal therapy to treat a curable localized disease.

[0156] An antibody of the present disclosure, or a functional part, derivative, and / or analogue thereof, that binds to EGFR, and optionally LGR5. In certain aspects, the antibody, or functional part, derivative, and / or analog thereof, comprises a variable domain that binds to the extracellular portion of EGFR. Such variable domains are further described herein. In certain aspects, the antibody, or functional part, derivative, and / or analog thereof, comprises a variable domain that binds to the extracellular portion of LGR5. Such variable domains are further described herein. In certain aspects, the antibody, or functional part, derivative, and / or analog thereof, comprises a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5.

[0157] In certain embodiments, the antibody, or functional part, derivative, and / or analog thereof, comprises or is a multispecific antibody, hi certain embodiments, the antibody, or functional part, derivative, and / or analog thereof, comprises or is a bispecific antibody.

[0158] In certain embodiments, an antibody disclosed herein, or a functional portion, derivative, and / or analog thereof, is a multispecific antibody. In certain embodiments, the antibody is a bispecific antibody. The multispecific or bispecific antibody, or a functional portion, derivative, and / or analog thereof, in certain embodiments, comprises a variable domain that binds to the extracellular portion of the epidermal growth factor (EGF) receptor and, in certain embodiments, a variable domain that does not bind to EGFR. In certain embodiments, the antibody, or a functional portion, derivative, and / or analog thereof, binds monovalently to EGFR. Also, in certain embodiments, the multispecific or bispecific antibody, or a functional portion, derivative, and / or analog thereof, comprises a variable domain that binds to LGR5.

[0159] In certain embodiments, the EGFR is human EGFR. EGFRs bound by the antibodies, or functional portions, derivatives, and / or analogs thereof, of the present disclosure include wild-type EGFR and EGFRs with oncogenic driver mutations. In certain embodiments, the oncogenic driver mutations are activating EGFR mutations. In certain embodiments, such mutations do not structurally alter the epitope bound by the antibodies of the present disclosure. In certain embodiments, EGFR mutations of the present disclosure include exon 18 mutations including G719A, G719C, 2E709_T710D, E709A, and G719S; exon 19 deletion mutations including deletions of LREA or VAIKEL; exon 19 point mutations G735S, P753L, L747S, and D761Y; exon 20 mutations including 1-7 amino acid in-frame exon 20 insertion mutations V765A, T783A, V774A, S784P, V769M, and T790M; and exon 21 mutations including L858R, T854A, A871E, L861A, L861C, L861S, V843I, or P848L. Antibodies of the present disclosure bind to epitopes that are not located in close proximity to the mutations. In particular, the EGFR mutation is S492R, which results in the loss of cetuximab binding to EGFR. The antibody of the present disclosure binds to an epitope different from the epitope recognized by cetuximab. Without being bound by any theory, it is believed that the amino acid residues I462, G465, K489, I491, N493, and C499 shown in FIG. 2 are involved in epitope binding by the antibody of the present disclosure. In certain embodiments, involvement in binding is determined by observing reduced binding of a variable domain to EGFR having one or more amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A, and C499A. In certain embodiments, epitope binding for EGFR and LGR5 is established using shotgun mutagenesis analysis.

[0160] In one embodiment, the variable domain that binds to an epitope on the extracellular portion of human EGFR is a variable domain that binds to an epitope located within amino acid residues 420-480 of the sequence shown in Figure 2. In certain embodiments, binding of the variable domain to EGFR is reduced by one or more of the following amino acid residue substitutions in EGFR: I462A, G465A, K489A, I491A, N493A, and C499A. In certain embodiments, binding of the antibody to human EGFR prevents EGF from binding to its receptor. In certain embodiments, the epitope on EGFR is a conformational epitope. In one embodiment, the epitope is located within amino acid residues 420-480 of the sequence shown in Figure 2 or within residues 430-480 of the sequence shown in Figure 2. In certain embodiments, the epitope is located within residues 438-469 of the sequence shown in Figure 2.

[0161] Without being bound by theory, it is believed that the contact residues of the epitope, i.e., where the variable domain contacts human EGFR, are likely to be 1462, K489, 1491, and N493. Amino acid residues G465 and C499 are likely indirectly involved in binding of the antibody to EGFR.

[0162] In certain embodiments, the variable domain binds to LGR5. In certain embodiments, the LGR5 is human LGR5. The multispecific or bispecific antibodies, or functional parts, derivatives, and / or analogs thereof described herein comprise a variable domain that binds to the extracellular portion of the human epidermal growth factor (EGF) receptor, and in certain embodiments, a variable domain that binds to human LGR5.

[0163] In certain aspects, the antibodies described herein, or functional portions, derivatives, and / or analogs thereof, comprise a variable domain that binds to the extracellular portion of the epidermal growth factor (EGF) receptor and prevents EGF from binding to the receptor, and a variable domain that binds to LGR5, wherein interaction of the antibody with LGR5 on an LGR5-expressing cell does not block Rspondin (RSPO) binding to LGR5. Methods for determining whether an antibody blocks Rspondin binding to LGR5 are described in WO2017069528, which is incorporated herein by reference.

[0164] Protein / gene accession numbers or alternative names are given herein primarily to provide an additional method of identification of the proteins described as targets; the actual sequence of the target protein bound by an antibody may vary, for example, due to mutations and / or alternative splicing in the encoding gene, such as those that occur in some cancers. A target protein will be bound by an antibody as long as the epitope is present within the protein and is accessible to the antibody.

[0165] In certain aspects, the antibodies described herein, or functional portions, derivatives, and / or analogs thereof, interfere with the binding of an EGFR ligand to EGFR. As used herein, the term "interfere with binding" means that the binding of the antibody, or functional portion, derivative, and / or analog thereof, to EGFR competes with the ligand for binding to the EGF receptor. The antibody, or functional portion, derivative, and / or analog thereof, may weaken ligand binding, displace the ligand if it is already bound to the EGF receptor, or at least partially prevent the ligand from binding to the EGF receptor, e.g., through steric hindrance.

[0166] In certain embodiments, the EGFR antibodies disclosed herein inhibit EGFR ligand-induced signaling, as measured by ligand-induced proliferation of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058), or ligand-induced cell death of A431 cells (ATCC CRL-1555), respectively. EGFR binds to several ligands and can stimulate proliferation of the described BxPC3 or BxPC3-luc2 cells. In the presence of an EGFR ligand, proliferation of BxPC3 or BxPC3-luc2 cells is stimulated. EGFR ligand-induced proliferation of BxPC3 cells can be measured by comparing cell proliferation in the absence and presence of a ligand. A preferred EGFR ligand for measuring EGFR ligand-induced proliferation of BxPC3 or BxPC3-luc2 cells is EGF. In certain embodiments, ligand-induced proliferation is measured using a saturating amount of ligand. In certain embodiments, EGF is used in an amount of 100 ng / ml of medium, hi certain embodiments, the EGF is EGF R&D Systems, catalog numbers 396-HB and 236-EG (see also WO2017 / 069628, which is incorporated herein by reference).

[0167] In certain embodiments, the EGFR antibodies disclosed herein inhibit EGFR ligand-induced proliferation of BxPC3 cells (ATCC CRL-1687) or BxPC3-luc2 cells (Perkin Elmer 125058). EGFR binds to several ligands and can stimulate proliferation of the described BxPC3 or BxPC3-luc2 cells. In the presence of the ligand, proliferation of BxPC3 or BxPC3-luc2 cells is stimulated. EGFR ligand-induced proliferation of BxPC3 cells can be measured by comparing cell proliferation in the absence and presence of a ligand. In certain embodiments, the EGFR ligand used to measure EGFR ligand-induced proliferation of BxPC3 or BxPC3-luc2 cells is EGF. In certain embodiments, ligand-induced proliferation is measured using a saturating amount of ligand. In certain embodiments, EGF is used in an amount of 100 ng / ml of medium. In certain embodiments, the EGF is R&D Systems EGF, catalog numbers 396-HB and 236-EG (see also WO2017 / 069628, which is incorporated herein by reference).

[0168] For the avoidance of doubt, references to cell growth as used herein refer to a change in cell number. Inhibition of growth refers to a reduction in the number of cells that would otherwise be obtained. Increased growth refers to an increase in the number of cells that would otherwise be obtained. Cell growth usually refers to cell proliferation.

[0169] In certain embodiments, whether an antibody described herein inhibits signaling in a multispecific format or inhibits proliferation is determined by the methods described herein above using a monospecific monovalent or monospecific bivalent version of the antibody. In certain embodiments, such an antibody has a binding site for the receptor for which signaling is determined. A monospecific monovalent antibody may have a variable domain with an unrelated binding specificity, such as tetanus toxoid specificity. In certain embodiments, the antibody is a bivalent monospecific antibody in which the antigen-binding variable domain consists of a variable domain that binds to an EGF receptor family member.

[0170] In its Biclonics® antibody program, Merus has developed multispecific antibodies targeting EGFR and LGR5 (leucine-rich repeat containing G protein-coupled receptors). The efficacy of such multispecific antibodies has been evaluated in vitro and in vivo using patient-derived CRC organoids and mouse PDX models, respectively (see, e.g., WO2017 / 069628, which is incorporated herein by reference). Multispecific antibodies targeting EGFR and LGR5 have been shown to inhibit tumor growth. The efficacy of such inhibitory antibodies has been shown to correlate with the level of LGR5 RNA expression by cancer-derived cells. In certain embodiments, the multispecific antibodies targeting EGFR and LGR5 are those described in WO2017 / 069628.

[0171] The antibodies, or functional portions, derivatives, and / or analogs thereof, described herein comprise a variable domain that binds to the extracellular portion of LGR5. In certain embodiments, the variable domain that binds to the extracellular portion of LGR5 binds to an epitope located within amino acid residues 21-118 of the sequence in Figure 1, in which amino acid residues D43, G44, M46, F67, R90, and F91 are involved in binding of the antibody to the epitope.

[0172] In certain aspects, the LGR5 variable domain is a variable domain in which one or more of the following amino acid residue substitutions in LGR5 reduce binding of the variable domain to LGR5: D43A, G44A, M46A, F67A, R90A, and F91A.

[0173] In certain embodiments, the epitope on the extracellular portion of LGR5 is located within amino acid residues 21-118 of the sequence of Figure 1. In certain embodiments, it is an epitope in which binding of the LGR5 variable domain to LGR5 is reduced by one or more of the following amino acid residue substitutions in LGR5: D43A, G44A, M46A, F67A, R90A, and F91A.

[0174] The present disclosure further provides an antibody having a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, wherein the LGR5 variable domain binds to an epitope on LGR5 located within amino acid residues 21 to 118 of the sequence of Figure 1.

[0175] In certain embodiments, the epitope on LGR5 is a conformational epitope. In certain embodiments, the epitope is located within amino acid residues 40-95 of the sequence of Figure 1. In certain embodiments, binding of the antibody to LGR5 is reduced by one or more of the following amino acid residue substitutions: D43A, G44A, M46A, F67A, R90A, and F91A.

[0176] Without being bound by theory, M46, F67, R90, and F91 of LGR5 shown in Figure 1 are thought to be contact residues for the antigen-binding site of the variable domain shown hereinabove, i.e., the variable domain that binds to the LGR5 epitope. The reduced antibody binding of the amino acid residue substitutions D43A and G44A may 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 portion of LGR5 that has one or more of the other contact residues (i.e., at positions 46, 67, 90, or 91), and the conformational change is such that antibody binding is reduced. An epitope is characterized by the amino acid substitutions mentioned. Whether antibodies bind to the same epitope can be determined in various ways. In an exemplary method, CHO cells express LGR5 or an alanine substitution mutant, such as a mutant containing one or more of the substitutions M46A, F67A, R90A, or F91A, on the cell membrane. A test antibody is contacted with the CHO cells, and the binding of the antibody to the cells is compared. A test antibody binds to an epitope if it binds to LGR5 and, to a lesser extent, to LGR5 with the M46A, F67A, R90A, or F91A substitution. Preferably, binding is compared with a panel of mutants each containing a single alanine residue substitution. Such binding tests are well known in the art. Often, the panel includes single alanine substitution mutants covering substantially all amino acid residues. In the case of LGR5, the panel need, of course, cover only the extracellular portion of the protein and the portion that ensures association with the cell membrane, when cells are used. Expression of a particular mutant may be impaired, but this is easily detected by one or more LGR5 antibodies that bind to different regions. If expression is also reduced for these control antibodies, then the level or folding of the protein on the membrane is impaired for this particular mutant. The binding characteristics of the test antibody to the panel will readily identify whether the test antibody shows reduced binding to mutants with the M46A, F67A, R90A, or F91A substitutions, and therefore whether the test antibody is an antibody of the invention.Reduced binding to mutants with M46A, F67A, R90A, or F91A substitutions also identifies an epitope located within amino acid residues 21-118 of the sequence of Figure 1. In certain embodiments, the panel includes both the D43A and G44A substitution mutants. Antibodies with the VH sequence of MF5816 show reduced binding to these substitution mutants.

[0177] Without being bound by any theory, it is believed that amino acid residues I462, G465, K489, I491, N493, and C499 shown in Figure 2 are involved in binding to the epitope by antibodies comprising the variable domains shown hereinabove. In certain embodiments, involvement in binding is determined by observing reduced binding of the variable domain to EGFR having one or more amino acid residue substitutions selected from I462A, G465A, K489A, I491A, N493A, and C499A. In an exemplary method, CHO cells express EGFR or an alanine substitution mutant, such as a mutant comprising one or more of the substitutions I462A, G465A, K489A, I491A, N493A, and C499A, on the cell membrane. A test antibody is contacted with the CHO cells, and binding of the antibody to the cells is compared. A test antibody binds to an epitope if it binds to EGFR, and to a lesser extent to EGFR with the I462A, G465A, K489A, I491A, N493A, and C499A substitutions. It is preferable to compare binding with a panel of mutants, each containing a single alanine residue substitution. Such binding tests are well known in the art. Often, the panel includes single alanine substitution mutants covering substantially all amino acid residues. In the case of EGFR, the panel need only cover the extracellular portion of the protein and the portion that ensures association with the cell membrane, if cells are used. Expression of a specific mutant may be impaired, which is easily detected by one or more EGFR antibodies that bind to different regions. If expression is also reduced with these control antibodies, then the level or folding of the protein on the membrane is impaired for this particular mutant. The binding characteristics of a test antibody to the panel readily identify whether the test antibody exhibits reduced binding to variants with the I462A, G465A, K489A, I491A, N493A, and C499A substitutions.

[0178] In one embodiment, the variable domain that binds to an epitope on the extracellular portion of human EGFR is a variable domain that binds to an epitope located within amino acid residues 420-480 of the sequence shown in Figure 2. In certain embodiments, binding of the variable domain to EGFR is reduced by one or more of the following amino acid residue substitutions in EGFR: I462A, G465A, K489A, I491A, N493A, and C499A. In certain embodiments, binding of the antibody to human EGFR prevents EGF from binding to its receptor. In certain embodiments, the epitope on EGFR is a conformational epitope. In one embodiment, the epitope is located within amino acid residues 420-480 of the sequence shown in Figure 2, such as within residues 430-480 of the sequence shown in Figure 2. In certain embodiments, the epitope is located within residues 438-469 of the sequence shown in Figure 2.

[0179] Without being bound by theory, the contact residues of the epitope, i.e., where the variable domain contacts human EGFR, are thought to be 1462, K489, 1491, and N493. Amino acid residues G465 and C499 are likely indirectly involved in the binding of the antibody to EGFR.

[0180] In certain embodiments, the variable domain that binds to human EGFR is a variable domain having a heavy chain variable region comprising at least the CDR3 sequence of the VH of MF3755 shown in Figure 3, or a CDR3 sequence that differs from the CDR3 sequence of the VH of MF3755 shown in Figure 3 by at most three, at most two, or no more than one amino acid.

[0181] In certain embodiments, the variable domain that binds to human EGFR is a variable domain having a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of the VH of MF3755 shown in Figure 3, or the CDR1, CDR2, and CDR3 sequences of the VH of MF3755 shown in Figure 3 with at most three, or at most two, or at most one amino acid substitution.

[0182] In certain aspects, the variable domain that binds to human EGFR is a variable domain having a heavy chain variable region comprising the sequence of the VH chain of MF3755 shown in Figure 3, or the amino acid sequence of the VH chain of MF3755 shown in Figure 3 with 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 disclosure provides an antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, The present invention provides an antibody wherein the heavy chain variable region of the variable domain comprises at least the CDR3 sequence of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or the heavy chain variable region of the variable domain comprises a heavy chain CDR3 sequence that differs by at most three, at most two, or no more than one amino acid from the CDR3 sequence of a VH selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3. In certain aspects, the variable domain comprises a heavy chain variable region comprising at least the CDR3 sequence of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3.

[0183] In certain embodiments, the variable domain comprises a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences that differ by at most three, at most two, or at most one amino acid from the CDR1, CDR2, and CDR3 sequences of an EGFR-specific heavy chain variable region selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3. In certain embodiments, the variable domain comprises a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3. In certain embodiments, the heavy chain variable region is MF3755. In one particular embodiment, the heavy chain variable region is MF4280.

[0184] In certain embodiments, an antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, wherein the EGFR-binding variable domain and the LGR5-binding variable domain have the CDR3, CDR1, CDR2, and CDR3, and / or VH sequences shown hereinabove, are selected from the group consisting of at least MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5815, MF5816, MF5817, MF5818, MF5819, MF5820, MF5821, MF5822, MF5823, MF5824, MF5825, MF5826, MF5827, MF5828, MF5829, MF5830, MF5831, MF5832, MF5833, MF5834, MF5835, MF5836, MF5837, MF5838, MF5839, MF5840, MF5841, MF5842, MF5843, MF5844, MF5845, MF5846, MF5847, MF5848, MF5849, MF5850, MF5851, MF5852, MF5853, MF5854, MF5855, MF5856, MF5857, MF5858, MF5859, MF5860, MF5861, MF5862, MF5863, MF5864, MF5865, MF5866, MF5867, The variable domain comprises a heavy chain CDR3 sequence that differs by at most three, at most two, or no more than one amino acid from the CDR3 sequence of an LGR5-specific heavy chain variable region selected from the group consisting of MF5816, MF5817, or MF5818, or the CDR3 sequence of a VH selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3. In certain embodiments, the variable domain comprises a heavy chain variable region comprising at least the CDR3 sequence of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3.

[0185] In certain embodiments, the LGR5 variable domain comprises at least the CDR1, CDR2, and CDR3 sequences of an LGR5-specific heavy chain variable region selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3, or a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 sequences that differ by at most three, or at most two, or at most one amino acid from the CDR1, CDR2, and CDR3 sequences of an LGR5-specific heavy chain variable region selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3. In certain aspects, the variable domain comprises a heavy chain variable region comprising at least the CDR1, CDR2, and CDR3 sequences of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 as shown in Figure 3. In certain aspects, the heavy chain variable region is MF5790, MF5803, MF5814, MF5816, MF5817, or MF5818. In certain aspects, the heavy chain variable region is MF5790, MF5814, MF5816, and MF5818. In certain aspects, the heavy chain variable region is MF5814, MF5818, or MF5816. In certain aspects, the heavy chain variable region is MF5816. In certain aspects, the heavy chain variable region is MF5818.

[0186] Antibodies comprising the heavy chain variable region MF3755 or one or more variable domains having one or more CDRs thereof have been shown to have better efficacy when used to inhibit the growth of EGFR ligand-responsive cancers or cells. In the context of bispecific or multispecific antibodies, an antibody arm comprising the heavy chain variable region MF3755 or a variable domain having one or more CDRs thereof pairs well with an arm comprising the heavy chain variable region MF5818 or a variable domain having one or more CDRs thereof. The VH chain of the variable domain binding to EGFR or LGR5 can have one or more amino acid substitutions with respect to the sequence shown in Figure 3. In certain embodiments, the VH chain has the amino acid sequence of the EGFR or LGR5 VH of Figure 3 with at most 15, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and in certain embodiments, 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, with respect to the VH chain sequence of Figure 3.

[0187] The CDR sequences may have one or more amino acid residue substitutions with respect to the CDR sequences shown in the figures. Such one or more substitutions are made for optimization purposes, such as to improve the binding strength or stability of the antibody. Optimization is preferably carried out by mutagenesis procedures, for example, after testing the stability and / or binding affinity of the resulting antibody and selecting an improved EGFR-specific or LGR5-specific CDR sequence. Those skilled in the art can generate antibody variants comprising at least one modified CDR sequence according to the present invention. For example, conservative amino acid substitutions can be applied. Examples of conservative amino acid substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another hydrophobic residue, and the substitution of one polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.

[0188] In certain aspects, the 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 referred to in a VH or VL designated herein are conservative amino acid substitutions. In certain aspects, the amino acid insertions, deletions, and substitutions referred to in a VH or VL designated herein are not in the CDR3 region. In certain aspects, the amino acid insertions, deletions, and substitutions referred to are also not in the CDR1 and CDR2 regions. In certain aspects, the amino acid insertions, deletions, and substitutions referred to are also not in the FR4 region.

[0189] In certain embodiments, the at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid substitutions referred to are conservative amino acid substitutions. In certain embodiments, the insertions, deletions, substitutions, or a combination thereof, are not in the CDR3 region of the VH chain, and in certain embodiments, are not in the CDR1, CDR2, or CDR3 regions of the VH chain, and in certain embodiments, are not in the FR4 region.

[0190] In certain embodiments, a treatment of the present disclosure comprises an antibody comprising a variable domain that binds to the extracellular portion of EGFR, and in certain embodiments, a variable domain that binds to the extracellular portion of LGR5; the amino acid sequence of VH chain MF3755 shown in Figure 3, or - comprising the amino acid sequence of VH chain MF3755 as shown in Figure 3 with at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or a combination thereof for said VH; The variable domain VH chain that binds to LGR5 is the amino acid sequence of VH chain MF5790 shown in Figure 3, or - With respect to the VH, it comprises the amino acid sequence of VH chain MF5790 as shown in Figure 3, with at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or a combination thereof.

[0191] In certain embodiments, a therapy of the present disclosure comprises a variable domain that binds to the extracellular portion of EGFR, and in certain embodiments, a variable domain that binds to the extracellular portion of LGR5; the amino acid sequence of VH chain MF3755 shown in Figure 3, or - comprising the amino acid sequence of VH chain MF3755 as shown in Figure 3 with at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or a combination thereof for said VH; The variable domain VH chain that binds to LGR5 is the amino acid sequence of VH chain MF5803 shown in Figure 3, or - With respect to the VH, it comprises the amino acid sequence of VH chain MF5803 as shown in Figure 3, with at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or a combination thereof.

[0192] In certain embodiments, a therapy of the present disclosure comprises a variable domain that binds to the extracellular portion of EGFR, and in certain embodiments, a variable domain that binds to the extracellular portion of LGR5; the amino acid sequence of VH chain MF3755 shown in Figure 3, or - comprising the amino acid sequence of VH chain MF3755 as shown in Figure 3 with at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or a combination thereof for said VH; The variable domain VH chain that binds to LGR5 is the amino acid sequence of VH chain MF5814 shown in Figure 3, or - Use an antibody comprising the amino acid sequence of VH chain MF5814 shown in Figure 3, with at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or a combination thereof, for the VH.

[0193] In certain embodiments, a therapy of the present disclosure comprises a variable domain that binds to the extracellular portion of EGFR, and in certain embodiments, a variable domain that binds to the extracellular portion of LGR5; the amino acid sequence of VH chain MF3755 shown in Figure 3, or - comprising the amino acid sequence of VH chain MF3755 as shown in Figure 3 with at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or a combination thereof for said VH; The variable domain VH chain that binds to LGR5 is the amino acid sequence of VH chain MF5816 shown in Figure 3, or - Use an antibody comprising the amino acid sequence of VH chain MF5816 shown in Figure 3, with at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or a combination thereof, for the VH.

[0194] In certain embodiments, a therapy of the present disclosure comprises a variable domain that binds to the extracellular portion of EGFR, and in certain embodiments, a variable domain that binds to the extracellular portion of LGR5; the amino acid sequence of VH chain MF3755 shown in Figure 3, or - comprising the amino acid sequence of VH chain MF3755 as shown in Figure 3 with at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or a combination thereof for said VH; The variable domain VH chain that binds to LGR5 is the amino acid sequence of VH chain MF5817 shown in Figure 3, or - Use an antibody comprising the amino acid sequence of VH chain MF5817 shown in Figure 3, with at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or a combination thereof, for the VH.

[0195] In certain embodiments, a therapy of the present disclosure comprises a variable domain that binds to the extracellular portion of EGFR, and in certain embodiments, a variable domain that binds to the extracellular portion of LGR5; the amino acid sequence of VH chain MF3755 shown in Figure 3, or - comprising the amino acid sequence of VH chain MF3755 as shown in Figure 3 with at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or a combination thereof for said VH; The variable domain VH chain that binds to LGR5 is the amino acid sequence of VH chain MF5818 shown in Figure 3, or - Use an antibody that comprises the amino acid sequence of VH chain MF5818 shown in Figure 3, with at most 15 (or in certain embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or in certain embodiments 1, 2, 3, 4, or 5) amino acid insertions, deletions, substitutions, or a combination thereof, for its VH.

[0196] In certain embodiments, the variable domain that binds to the extracellular portion of EGFR comprises a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences of a variable region selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, and the variable domain that binds to the extracellular portion of LGR5 comprises a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences of a variable region selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3.

[0197] In certain embodiments, the variable domain that binds to the extracellular portion of EGFR comprises a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences of the variable region of MF3755 shown in Figure 3, and the variable domain that binds to the extracellular portion of LGR5 comprises a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences of the variable region of MF5816 shown in Figure 3.

[0198] In certain embodiments, the VH chain of the variable domain that binds EGFR comprises the amino acid sequence of VH chain MF3370, MF3755, MF4280, or MF4289 shown in FIG. 3 with up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less, amino acid modifications including the amino acid sequence of VH chain MF3370, MF3755, MF4280, or MF4289 shown in FIG. 3, or insertions, deletions, substitutions, or combinations thereof relative to said VH; and the VH chain of the variable domain that binds LGR5 comprises the amino acid sequence of VH chain MF3370, MF3755, MF4280, or MF4289 shown in FIG. 3 with up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, amino acid modifications including insertions, deletions, substitutions, or combinations thereof relative to said VH. The amino acid sequence of VH chain MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3, or the amino acid sequence of VH chain MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3, with up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, and preferably 5, 4, 3, 2, or 1 or less amino acid modifications, including insertions, deletions, substitutions, or combinations thereof, in said VH.

[0199] In certain embodiments, the VH chain of the variable domain that binds to EGFR has the amino acid sequence of VH chain MF3755 shown in FIG. 3 or the amino acid sequence of VH chain MF3755 shown in FIG. 3 with up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less, amino acid modifications, including insertions, deletions, substitutions, or a combination thereof, relative to the VH. The VH chain of the variable domain comprising the sequence and binding to LGR5 comprises the amino acid sequence of VH chain MF5816 shown in Figure 3 or the amino acid sequence of VH chain MF5816 shown in Figure 3 with up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less, amino acid modifications including insertions, deletions, substitutions, or combinations thereof, relative to the VH.

[0200] In certain aspects, the VH chain of the variable domain that binds to EGFR comprises the amino acid sequence of VH chain MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, and the VH chain of the variable domain that binds to LGR5 comprises the amino acid sequence of VH chain MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3.

[0201] In certain embodiments, the VH chain of the variable domain that binds to EGFR comprises the amino acid sequence of VH chain MF3755 shown in Figure 3, and the VH chain of the variable domain that binds to LGR5 comprises the amino acid sequence of VH chain MF5816 shown in Figure 3.

[0202] In certain embodiments, both the variable domain that binds EGFR and the variable domain that binds LGR5 comprise the CDR1, CDR2, and CDR3 regions of a light chain variable region as shown in Figure 4b.

[0203] In certain embodiments, the variable domains that bind EGFR and that bind LGR5 both comprise a light chain variable region as shown in Figure 4b, wherein the variable light chain region comprises 0 to 10 amino acid insertions, deletions, substitutions, additions, or a combination thereof, and the amino acid insertions, deletions, and substitutions are not present in the CDR1, CDR2, and CDR3 light chain variable regions.

[0204] Additional variants of the disclosed amino acid sequences that retain EGFR or LGR5 binding can be obtained, for example, from phage display libraries containing rearranged human IGKV1-39 / IGKJ1 VL regions (De Kruif et al. Biotechnol Bioeng. 2010(106)741-50) and collections of VH regions that incorporate amino acid substitutions in the amino acid sequences of the EGFR or LGR5 VH regions disclosed herein, as previously described (e.g., WO2017 / 069628). Phage encoding Fab regions that bind to EGFR or LGR5 can be selected, analyzed by flow cytometry, and sequenced to identify variants with amino acid substitutions, insertions, deletions, or additions that retain antigen binding.

[0205] The light chain variable regions of the VH / VL EGFR and LGR5 variable domains of an EGFR / LGR5 antibody can be the same or different. In certain embodiments, the VL region of the VH / VL EGFR variable domain of an EGFR / LGR5 antibody is the same as the VL region of the VH / VL LGR5 variable domain. In certain embodiments, the VL regions of the VH variable domain / VL variable domain that bind to EGFR and LGR5 are identical.

[0206] In certain embodiments, the light chain variable regions of one or both of the VH variable domains / VL variable domains of the EGFR / LGR5 antibodies comprise a common light chain variable region. In certain embodiments, the common light chain variable region of one or both of the VH variable domains / VL variable domains comprises a germline IgVκ1-39 variable region V-segment. In certain embodiments, the light chain variable region of one or both of the VH variable domains / VL variable domains comprises a kappa light chain V-segment, IgVκ1-39*01. IgVκ1-39 stands for immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39, IGKV139, or IGKV1-39. The external Ids for this gene are HGNC:5740, Entrez Gene:28930, and Ensembl:ENSG00000242371. The amino acid sequence for a suitable V-region is provided in FIG. 4. The V region can be combined with one of five J regions. In certain aspects, the J regions are jk1 and jk5, and the combined sequences are designated IGKV1-39 / jk1 and IGKV1-39 / jk5, alternatively designated IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ5*01 (designation according to the IMGT database world wide web at imgt.org). In certain aspects, the light chain variable region of one or both of the VH / VL variable domains comprises the kappa light chain IgVκ1-39*01 / IGJκ1*01 or IgVκ1-39*01 / IGJκ1*05 (depicted in Figure 4).

[0207] In certain embodiments, the light chain variable region of Figure 4d) comprises LCDR1, LCDR2, and LCDR sequences. Such sequences may be determined or annotated by one of skill in the art using, for example, an annotation system such as, for example, IMGT, Chothia, Kabat, or other suitable annotation systems. In certain embodiments, the light chain variable region is comprised of one or both VH variable domains / VL variable domains, present in an EGFR-binding antibody of the disclosure, and comprises LCDR1, LCDR2, and LCDR sequences. Such sequences may be determined or annotated by one of skill in the art using, for example, an annotation system such as, for example, IMGT, Chothia, Kabat, or other suitable annotation systems.

[0208] In certain embodiments, the light chain variable region of one or both of the VH variable domains / VL variable domains of the EGFR / LGR5 bispecific antibody comprises an LCDR1 comprising the amino acid sequence QSISSY (as depicted in Figure 4), an LCDR2 comprising the amino acid sequence AAS (as depicted in Figure 4), and an LCDR3 comprising the amino acid sequence QQSYSTP (as depicted in Figure 4). In certain embodiments, these CDRs are according to IMGT. In certain embodiments, the light chain variable region of one or both of the VH variable domains / VL variable domains of the EGFR / LGR5 antibody comprises an LCDR1 comprising the amino acid sequence QSISSY, an LCDR2 comprising the amino acid sequence AASSLQS, and an LCDR3 comprising the amino acid sequence QQSYSTP.

[0209] In certain embodiments, the light chain variable region of one or both of the VH variable domains / VL variable domains of an EGFR / LGR5 bispecific antibody comprises an LCDR1 comprising the amino acid sequence QSISSY (as depicted in Figure 4), an LCDR2 comprising the amino acid sequence AAS (as depicted in Figure 4), and an LCDR3 comprising the amino acid sequence QQSYSTPPT (i.e., a CDR according to IMGT) (as depicted in Figure 4). In certain embodiments, the light chain variable region of one or both of the VH variable domains / VL variable domains of an EGFR / LGR5 antibody comprises an LCDR1 comprising the amino acid sequence QSISSY, an LCDR2 comprising the amino acid sequence AASSLQS, and an LCDR3 comprising the amino acid sequence QQSYSTPPT.

[0210] In certain embodiments, one or both VH variable domains / VL variable domains of an EGFR / LGR5 antibody comprise a light chain variable region comprising an amino acid sequence that is at least 90%, and in certain embodiments, at least 95%, and in certain embodiments, at least 97%, and in certain embodiments, at least 98%, and in certain embodiments, at least 99%, or in certain embodiments, 100% identical to the amino acid sequence set forth in Figure 4. In certain embodiments, one or both VH variable domains / VL variable domains of an EGFR / LGR5 antibody comprise a light chain variable region comprising an amino acid sequence that is at least 90%, and in certain embodiments, at least 95%, and in certain embodiments, at least 97%, and in certain embodiments, at least 98%, and in certain embodiments, at least 99% identical, or in certain embodiments, 100% identical to the amino acid sequence set forth in Figure 4.

[0211] For example, the variable light chain of one or both of the VH variable domain / VL variable domain of the EGFR / LGR5 antibody may have 0 to 10, and in certain embodiments, 0 to 5, amino acid insertions, deletions, substitutions, additions, or a combination thereof with respect to the sequence in Figure 4. In certain embodiments, the light chain variable region of one or both of the VH variable domain / VL variable domain of the EGFR / LGR5 antibody contains 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, in certain embodiments, 0 to 3, in certain embodiments, 0 to 2, in certain embodiments, 0 to 1, and in certain embodiments, 0 amino acid insertions, deletions, substitutions, additions, or a combination thereof with respect to the amino acid sequence shown.

[0212] Alternatively, the light chain variable region of one or both of the VH variable domains / VL variable domains of an EGFR / LGR5 antibody may comprise the amino acid sequence shown in Figure 4. In certain embodiments, both the VH variable domains / VL variable domains of an EGFR / LGR5 antibody comprise the same VL region. In certain embodiments, the VL of both the VH variable domains / VL variable domains of an EGFR / LGR5 bispecific antibody comprises the amino acid sequence set forth in Figure 4.

[0213] In certain embodiments, the EGFR / LGR5 antibodies described herein are bispecific antibodies having two variable domains, one that binds to EGFR and another that binds to LGR5 as described herein. EGFR / LGR5 bispecific antibodies for use in the methods disclosed herein can be provided in several formats. Many different formats of bispecific antibodies are known in the art and are reviewed by Kontermann (Drug Discov Today, 2015 Jul;20(7):838-47; MAbs, 2012 Mar-Apr;4(2):182-97) and Spiess et al., (Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol. Immunol. (2015) http: / / dx.doi.org / 10.1016 / j.molimm.2015.01.003), each of which is incorporated herein by reference. For example, bispecific antibody formats that are not classical antibodies with two VH / VL combinations have at least a variable domain comprising a heavy chain variable region and a light chain variable region, which may be linked to a single chain Fv fragment, a single VH, and a Fab fragment that provides a second binding activity.

[0214] In certain embodiments, the EGFR / LGR5 bispecific antibodies used in the methods provided herein are generally of the human IgG subclass (e.g., IgG1, IgG2, IgG3, IgG4). In certain embodiments, the antibodies are of the human IgG1 subclass. Full-length IgG antibodies are preferred because of their favorable half-life and low immunogenicity. Thus, in certain embodiments, the EGFR / LGR5 bispecific antibodies are full-length IgG molecules. In certain embodiments, the EGFR / LGR5 bispecific antibodies are full-length IgG1 molecules.

[0215] Thus, in certain embodiments, the EGFR / LGR5 bispecific antibody comprises a crystallizable fragment (Fc). In certain embodiments, the Fc of the EGFR / LGR5 bispecific antibody consists of a human constant region. The constant region or Fc of the EGFR / LGR5 bispecific antibody may contain one or more, or no more than 10, or no more than 5 amino acid differences from the constant region of a naturally occurring human antibody. For example, each Fab arm of the bispecific antibody may further comprise an Fc region containing modifications that facilitate bispecific antibody formation, stability, and / or other characteristics described herein.

[0216] Antibodies are typically produced by cells expressing nucleic acids encoding the antibodies. Thus, in certain embodiments, the bispecific EGFR / LGR5 antibodies disclosed herein are produced by providing a cell containing one or more nucleic acids encoding the heavy and light chain variable and constant regions of the bispecific EGFR / LGR5 antibody. In certain embodiments, the cell is an animal cell, such as a mammalian cell or a primate cell, and in certain embodiments, a human cell. Suitable cells are any cells capable of containing, and preferably producing, an EGFR / LGR5 bispecific antibody.

[0217] Cells suitable for antibody production are known in the art and include hybridoma cells, Chinese hamster ovary (CHO) cells, NSO cells, or PER-C6 cells. Various institutions and companies are developing cell lines for large-scale production of antibodies, for example, for clinical use. Non-limiting examples of such cell lines are CHO cells, NSO cells, or PER.C6 cells. In particular, the cells are human cells. Preferably, the cells are transformed with an adenovirus E1 region or a functional equivalent thereof. A preferred example of such a cell line is the PER.C6 cell line or its equivalent. In particular, the cells are CHO cells or variants thereof. Preferably, the variants use a glutamine synthetase (GS) vector system for antibody expression. In a specific embodiment, the cells are CHO cells.

[0218] In certain embodiments, the cells express different light and heavy chains that make up the EGFR / LGR5 bispecific antibody. In certain embodiments, the cells express two different heavy chains and at least one light chain. In certain embodiments, the cells express a "common light chain" as described herein to reduce the number of different antibody species (combinations of different heavy and light chains). For example, each VH region, along with a rearranged human IGKV1-39 / IGKJ1 (huVκ1 39) light chain, is cloned into an expression vector using methods known in the art for producing bispecific IgG (WO 2013 / 157954, incorporated herein by reference), and has previously been shown to be capable of pairing with two or more heavy chains, thereby generating antibodies with diverse specificities, facilitating the production of bispecific molecules (De Kruif et al. J. Mol. Biol. 2009(387)548 58; WO 2009 / 157771).

[0219] Antibody-producing cells expressing a common light chain and equal amounts of two heavy chains typically produce 50% bispecific antibodies and 25% monospecific antibodies (i.e., with identical heavy chain combinations). Several methods have been published to favor the production of bispecific antibodies over the production of individual monospecific antibodies. This is typically achieved by modifying the constant regions of the heavy chains so that they favor heterodimerization (i.e., dimerization with heavy chains of other heavy chain / light chain combinations) over homodimerization. In certain embodiments, the bispecific antibodies of the present invention comprise two different immunoglobulin heavy chains with compatible heterodimerization domains. Various compatible heterodimerization domains have been described in the art. In certain embodiments, the compatible heterodimerization domain is a compatible immunoglobulin heavy chain CH3 heterodimerization domain. Various methods by which such heavy chain heterodimerization can be achieved have been described in the art.

[0220] Preferred methods for producing EGFR / LGR5 bispecific antibodies are disclosed in US Pat. Nos. 9,248,181 and 9,358,286. Specifically, preferred mutations that produce essentially only bispecific full-length IgG molecules are amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain ("KK variant" heavy chain) and L351D and L368E in the second domain ("DE variant" heavy chain), or vice versa. As mentioned above, DE and KK variants 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) rarely occurs due to strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0221] Thus, in certain embodiments, a heavy / light chain combination comprising a variable domain that binds EGFR comprises a DE variant of the heavy chain, hi certain embodiments, a heavy / light chain combination comprising a variable domain that binds LGR5 comprises a KK variant of the heavy chain.

[0222] Candidate EGFR / LGR5 IgG bispecific antibodies can be tested for binding using any suitable assay. For example, binding to membrane-expressed EGFR or LGR5 on CHO cells can be assessed by flow cytometry (using the FACS procedure previously described in WO 2017 / 069628). In certain embodiments, binding of candidate EGFR / LGR5 bispecific antibodies to LGR5 on CHO cells is demonstrated by flow cytometry performed according to standard procedures known in the art. Binding to CHO cells is compared to CHO cells not transfected with an EGFR and / or LGR5 expression cassette. Binding of candidate bispecific IgG1 to EGFR is determined using CHO cells transfected with an EGFR expression construct, and LGR5 and EGFR monospecific antibodies, as well as an irrelevant IgG1 isotype control mAb, are included in the assay as controls (e.g., an antibody that binds to LGR5 and another antigen, such as tetanus toxoid (TT)).

[0223] The affinity of the LGR5 and EGFR Fabs of candidate EGFR / LGR5 bispecific antibodies to their targets can be measured by surface plasmon resonance (SPR) technology using a BIAcore T100. Briefly, an anti-human IgG mouse monoclonal antibody (Becton and Dickinson, catalog number 555784) is coupled to the surface of a CM5 sensor chip using free amine chemistry (NHS / EDC). The bsAb is then captured on the sensor surface. Subsequently, recombinant purified antigen human EGFR (Sino Biological Inc., catalog number 11896-H07H) and human LGR5 protein are run over the sensor surface at a range of concentrations to measure on- and off-rates. After each cycle, the sensor surface is regenerated with a pulse of HCl, and the bsAb is recaptured. From the resulting sensorgrams, on- and off-rates and affinity values ​​for binding to human LGR5 and EGFR are determined using BIAevaluation software previously described for CD3 in US2016 / 0368988.

[0224] The antibodies disclosed herein are typically bispecific full-length antibodies, in certain embodiments of the human IgG subclass. In certain embodiments, the antibodies are of the human IgG1 subclass. Such antibodies can be enhanced, if desired, by techniques known in the art, and have good ADCC properties, with favorable half-lives upon in vivo administration to humans, and CH3 engineering techniques exist that can provide modified heavy chains that form heterodimers preferentially over homodimers upon co-expression in clonal cells.

[0225] The ADCC activity of an antibody can be improved by modifying the antibody's constant region if the antibody itself has low ADCC activity. Another method for improving antibody ADCC activity is by enzymatically interfering with the glycosylation pathway that results in reduced fucose. Several in vitro methods exist for determining the effectiveness of an antibody or effector cell in inducing ADCC. These include the chromium-51 [Cr51] release assay, europium [Eu] release assay, and sulfur-35 [S35] release assay. Typically, a labeled target cell line expressing a specific surface-exposed antigen is incubated with an antibody specific for that antigen. After washing, effector cells expressing the Fc receptor CD16 are co-incubated with the antibody-labeled target cells. Target cell lysis is then measured by the release of intracellular label using a scintillation counter or spectrophotometrically.

[0226] The bispecific antibodies disclosed herein may have enhanced ADCC. In certain embodiments, such bispecific antibodies are afucosylated. In certain embodiments, the bispecific antibodies comprise a reduced amount of fucosylation of N-linked carbohydrate structures in the Fc region compared to the same antibody produced in normal CHO cells. Low fucose levels are associated with increased CD16 (FcγRIIIa) binding on NK effector cells, resulting in increased ADCC activity. In certain embodiments, in addition to their direct antitumor activity, the bispecific antibodies of the present disclosure can eliminate tumor cells following opsonization and subsequent natural killer (NK) cell-mediated ADCC and complement-dependent cytotoxicity (CDC) activity.

[0227] An antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 may further comprise one or more additional variable domains that can bind to one or more additional targets. In certain embodiments, the additional targets are proteins, such as membrane proteins, that have an extracellular portion. As used herein, a membrane protein refers to a cell membrane protein, such as a protein located in the outer membrane of a cell, which is the membrane that separates the cell from the outside world. A membrane protein has an extracellular portion. A membrane protein is at least present on a cell when it contains a transmembrane region that is located within the cell membrane of the cell.

[0228] Antibodies having two or more variable domains are known in the art. For example, it is possible to combine additional variable domains. In certain embodiments, the antibody having three or more variable domains is a multivalent multimeric antibody as described in PCT / NL2019 / 050199, which is incorporated herein by reference.

[0229] In certain embodiments, the antibody is a bispecific antibody comprising two variable domains, one variable domain that binds to the extracellular portion of EGFR and another variable domain that binds to the extracellular portion of LGR5, hi certain embodiments, the variable domains are variable domains described herein.

[0230] The functional portion of an antibody described herein comprises at least a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5 described herein. It therefore comprises the antigen-binding portion of an antibody described herein, typically containing the variable domain of the antibody. The variable domain of the functional portion can be a single-chain Fv fragment or a so-called single-domain antibody fragment. In certain embodiments, the antibody portion or derivative comprises at least two variable domains of an antibody or its equivalent. Non-limiting examples of such variable domains or their equivalents are F(ab) fragments and single-chain Fv fragments. The functional portion of a bispecific antibody comprises the antigen-binding portion of the bispecific antibody, or a derivative and / or analog of the binding portion. As described herein above, the binding portion of an antibody is encompassed by the variable domain.

[0231] In certain embodiments, the antibody comprising one variable domain that binds to the extracellular portion of EGFR is selected from amivantamab (Janssen Biotech), bafizontamab (EpimAb Biotherapeutics), REGN-7075 (Regeneron Pharmaceuticals Inc.), BCA-101 (Bicara Therapeutics), PM-1080 (Biotheus Inc.), durigotuzumab (Genentech, Inc.), AFM-24 (Affimed GmbH).

[0232] Formulations and Pharmaceutical Compositions In certain aspects, the treatments disclosed herein utilize an antibody, or a functional portion, derivative, and / or analog thereof (i.e., as a therapeutic agent), and a pharmaceutically acceptable carrier and an immune checkpoint inhibitor (i.e., as an additional therapeutic agent), as disclosed herein, and a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful in the treatment of cancer, particularly head and neck cancer. As used herein, the term "pharmaceutically acceptable" means approved by a government regulatory agency or listed in the United States Pharmacopoeia or another generally recognized pharmacopoeia for use in animals, especially humans, and includes any and all physiologically compatible solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, glycerol ricinoleate, polyethylene glycol distearate, and the like. Water or saline and aqueous dextrose and glycerol solutions may be used as carriers, particularly for injectable solutions. Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravesical, intratumoral, intravenous, intraperitoneal, intramuscular, intrathecal, and subcutaneous. Depending on the route of administration (e.g., intravenous, subcutaneous, intraarticular, etc.), the active compound may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0233] The pharmaceutical composition comprising an antibody, functional part, derivative, and / or analogue thereof comprising a variable domain that binds to the extracellular portion of EGFR may be contained in a separate holder, meaning that it is not physically linked to the holder containing the pharmaceutical composition comprising the immune checkpoint inhibitor.

[0234] In certain embodiments, the pharmaceutical composition comprising an antibody, functional portion, derivative, and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR and LGR5 may be contained in a separate holder, meaning that it is not physically linked to the holder containing the pharmaceutical composition comprising the immune checkpoint inhibitor, such as pembrolizumab.

[0235] In certain aspects, the pharmaceutical composition comprises petosemutamab and is contained within a separate holder, meaning that it is not physically linked to the holder containing the pharmaceutical composition comprising the immune checkpoint inhibitor, such as pembrolizumab.

[0236] Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, for example, in a liquid carrier, or suitable for reconstitution into a solution or suspension for intravenous administration.The composition can be formulated in dosage unit form for ease of administration and uniformity of dosage.Also included are solid preparations intended to be converted into liquid preparations for either oral or parenteral administration immediately before use.Such liquid forms include solutions, suspensions, and emulsions.

[0237] Instructions for use of the EGFR binding antibodies and immune checkpoint inhibitors of the present disclosure. In certain embodiments, nivolumab is administered at 240 mg every 2 weeks, or 480 mg every 4 weeks, or 1 mg / kg on the same day every 3 weeks, followed by four doses of 3 mg / kg ipilimumab, then 240 mg every 2 weeks, or 480 mg every 4 weeks. In certain embodiments, nivolumab is administered at 3 mg / kg every 2 weeks with 1 mg / kg ipilimumab every 6 weeks, or 360 mg every 3 weeks with 1 mg / kg ipilimumab every 6 weeks, and two cycles of platinum-doublet chemotherapy. In certain embodiments, nivolumab is administered at 360 mg every 3 weeks with 1 mg / kg ipilimumab every 6 weeks. In certain embodiments, nivolumab is administered at 240 mg every 2 weeks, or 480 mg every 4 weeks, or 3 mg / kg every 2 weeks, or 3 mg / kg on the same day every 3 weeks, followed by 4 doses of 1 mg / kg ipilimumab, then 240 mg every 2 weeks, or 480 mg every 4 weeks.

[0238] Ipilimumab (sold under the brand name Yervoy™) is a monoclonal antibody and immune checkpoint inhibitor that works to activate the immune system by targeting CTLA-4, a protein receptor that downregulates the immune system. Ipilimumab was approved by the U.S. Food and Drug Administration (FDA) in March 2011 for the treatment of melanoma.

[0239] In one particular embodiment, the immune checkpoint inhibitor is nivolumab, administered at 3 mg / kg as an intravenous infusion (e.g., over 60 minutes) every two weeks until disease progression or unacceptable toxicity.

[0240] In certain embodiments, nivolumab is used for head and neck cancer, such as recurrent or metastatic squamous cell carcinoma, and is administered at 240 mg every two weeks or 480 mg every four weeks.

[0241] In certain embodiments, the dosage form and strength of nivolumab is an injection of a 40 mg / 4 mL (i.e., 10 mg / mL), 100 mg / 10 mL (i.e., 10 mg / mL), 120 mg / 12 mL (i.e., 10 mg / mL), or 240 mg / 24 mL (i.e., 10 mg / mL) solution in a single-dose vial. In certain embodiments, nivolumab administration is by 30-minute intravenous infusion.

[0242] In certain embodiments, the immune checkpoint inhibitor is cemiplimab, typically administered in an amount of 350 mg as an intravenous infusion (e.g., over 30 minutes) every three weeks until disease progression or unacceptable toxicity. In certain embodiments, administration is by intravenous infusion over 30 minutes via an intravenous line containing a sterile, in-line, or add-on 0.2 micron to 5 micron filter. In certain embodiments, the dosage form and strength of cemiplimab is an injection of a 350 mg / 7 mL (i.e., 50 mg / mL) solution in a single-dose vial.

[0243] In certain embodiments, the immune checkpoint inhibitor is dostarimab (or Jemperli) administered as an intravenous infusion (e.g., over 30 minutes) at doses 1 through 4 of 500 mg every 3 weeks, followed by the following dosing starting 3 weeks after dose 4 (dose 5 onwards): 1,000 mg every 6 weeks until disease progression or unacceptable toxicity. In certain embodiments, the dosage form and strength of dostarimab is an injection of a 500 mg / 10 mL (i.e., 50 mg / mL) solution in a single-dose vial. In certain embodiments, administration is by intravenous infusion over 30 minutes through an intravenous line using tubing made of polyvinyl chloride or platinum-cured silicone, fittings made of polyvinyl chloride or polycarbonate, and a sterile, non-pyrogenic, low protein-binding, 0.2 micron, in-line, or add-on filter.

[0244] In certain embodiments, the immune checkpoint inhibitor is atezolizumab, typically administered as an intravenous infusion (e.g., over 60 minutes) at 840 mg every 2 weeks, 1200 mg every 3 weeks, or 1680 mg every 4 weeks until disease progression or unacceptable toxicity. If the first infusion is tolerated, all subsequent infusions are delivered over 30 minutes. In certain embodiments, the injection is an 840 mg / 14 mL (i.e., 60 mg / mL) or 1200 mg / 20 mL (i.e., 60 mg / mL) solution in a single-dose vial.

[0245] In certain embodiments, the immune checkpoint inhibitor is avelumab, typically administered in an amount of 10 mg / kg as an intravenous infusion (e.g., over 60 minutes) every two weeks until disease progression or unacceptable toxicity. In certain embodiments, administration is typically in an amount of 800 mg as an intravenous infusion (e.g., over 60 minutes) every two weeks until disease progression or unacceptable toxicity. In certain embodiments, the injection is a 200 mg / 10 mL (i.e., 20 mg / mL) solution in a single-dose vial.

[0246] In certain embodiments, the immune checkpoint inhibitor is avelumab, administered orally at 800 mg every two weeks (800 mg q2w), optionally in combination with axitinib 5 mg twice daily. Avelumab is administered as an intravenous infusion over 60 minutes. The dosage form and strength is an injection of a 200 mg / 10 mL (20 mg / mL) solution in a single-dose vial.

[0247] In certain embodiments, the immune checkpoint inhibitor is durvalumab and is administered at 10 mg / kg administered as an intravenous infusion (e.g., over 60 minutes) every two weeks until disease progression or unacceptable toxicity. In certain embodiments, the injection is a 500 mg / 10 mL (i.e., 50 mg / mL) or 120 mg / 2.4 mL (i.e., 50 mg / mL) solution in a single-dose vial.

[0248] In certain embodiments, the immune checkpoint inhibitor is pembrolizumab and is administered in an amount of 200 mg as an intravenous infusion every three weeks, hi certain embodiments, the administration is a flat dose of 200 mg every three weeks until disease progression or unacceptable toxicity.

[0249] In certain embodiments, the immune checkpoint inhibitor is pembrolizumab and is administered as an intravenous infusion in an amount of 400 mg every six weeks, hi certain embodiments, the administration is a flat dose of 400 mg every six weeks until disease progression or unacceptable toxicity.

[0250] In certain embodiments, the immune checkpoint inhibitor is pembrolizumab, administered at 2 mg / kg (maximum 200 mg) as an intravenous infusion every three weeks for pediatrics.

[0251] In certain embodiments, the dosage form and strength of pembrolizumab injection is a 100 mg / 4 mL (or 25 mg / mL) solution in a single-dose vial. In certain embodiments, pembrolizumab is used as a single agent and is indicated for the first-line treatment of subjects with metastatic or unresectable, recurrent HNSCC whose tumors express PD-L1 [combined positive score (CPS) > 1] as determined by an FDA-approved test. In certain embodiments, pembrolizumab administration is by 30-minute intravenous infusion.

[0252] In certain embodiments, the immune checkpoint inhibitor is penprimab, and the instructions for its use include administering 200 mg once every two weeks (200 mg q2w), typically by intravenous injection.

[0253] In certain embodiments, the immune checkpoint inhibitor is sintilimab and the instructions for its use include administering 200 mg once every three weeks (q3w), typically by intravenous injection.

[0254] In certain embodiments, the immune checkpoint inhibitor is tislelizumab and the instructions for its use include administering 200 mg once every three weeks (200 mg q3w), typically by intravenous injection.

[0255] In certain embodiments, the immune checkpoint inhibitor is retifanlimab and the instructions for its use include administering 500 mg once every four weeks (500 mg q4w), typically by intravenous injection.

[0256] In certain embodiments, the immune checkpoint inhibitor is toripalimab, whose recommended Phase II dose is determined to be 3 mg / kg Q2W. Instructions for use include administering 3 mg / kg every two weeks. Administration is typically by intravenous injection.

[0257] Also, in certain aspects, the present disclosure includes instructions for use of such antibodies, or functional portions, derivatives, and / or analogs thereof, that bind to EGFR, and optionally LGR5, e.g., petosemutamab. These instructions for use are provided in more detail in the present disclosure and relate to use (e.g., treatment of head and neck cancer), route of administration (e.g., intravenous injection), dosage (e.g., 1500 mg amount), dosage regardless of subject weight (i.e., flat dose), and dosing interval (e.g., once every two weeks).

[0258] In certain embodiments, the antibody, or functional part, derivative, and / or analog thereof, comprises petosemutamab (see Recommended INN list 83, WHO Drug Information Vol. 34, No. 1, 2020), and the instructions for use comprise administering 1500 mg as an intravenous infusion once every two weeks. In certain embodiments, the dose is a flat dose of 1500 mg.

[0259] In certain embodiments, the antibody, or functional portion, derivative, and / or analog thereof, comprises petosemutamab, and the instructions for use comprise administering an amount of 1100 mg as an intravenous infusion once every two weeks. In certain embodiments, the dose is a flat dose of 1100 mg.

[0260] In certain embodiments, petosemutamab and pembrolizumab are administered sequentially. In certain embodiments, when pembrolizumab and petosemutamab are administered on the same day, pembrolizumab is administered after petosemutamab.

[0261] In certain embodiments, petosemutamab is administered in an amount of 1500 mg once every two weeks and pembrolizumab is administered in an amount of 400 mg once every six weeks, hi certain embodiments, both dosages are flat doses.

[0262] In certain embodiments, petosemutamab is administered in an amount of 1100 mg once every two weeks and pembrolizumab is administered in an amount of 400 mg once every six weeks, hi certain embodiments, both such doses are flat doses.

[0263] Administration, Dosage Regimen, and Treatment Disclosed therapeutic agents (e.g., petrolatum) may be administered at a suitable dosage and according to a suitable route (e.g., intravenously, intraperitoneally, intramuscularly, intrathecally, or subcutaneously). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In certain aspects, a subject is administered a single dose of an antibody, or functional part, derivative, and / or analog thereof, disclosed herein. In certain aspects, a therapeutic agent is administered repeatedly over the course of treatment. For example, in certain aspects, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of a therapeutic agent are administered to a subject in need of treatment. In certain aspects, administration of a therapeutic agent may be weekly, biweekly, or monthly.

[0264] Clinicians may utilize a preferred dose that is deemed appropriate depending on the condition of the patient being treated. The dose may depend on several factors, including the stage of the disease. It is within the skill of those skilled in the art to determine the specific dose to be administered based on the presence of one or more of these factors. Generally, treatment is initiated with a smaller dose that is less than the optimal dose of the compound. Thereafter, the dosage is increased by small increments until the optimal effect under the circumstances is reached. For convenience, the total daily dosage may be administered in divided doses throughout the day as needed. Intermittent therapy (e.g., one week out of three weeks or three weeks out of four weeks) may also be used.

[0265] In certain embodiments, the therapeutic agent is administered at a dose of 0.1, 0.3, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg body weight. Alternatively, the therapeutic agent is administered at a dose of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg body weight. In certain embodiments, a therapeutic agent (e.g., petosemutamab) is provided to a subject using a flat dose of 1500 mg. Flat doses offer several advantages over surface or body weight administration, such as reduced preparation time and reduced potential dose calculation errors. In certain embodiments, the therapeutic agent is provided in a dose of at least 500 mg. In certain embodiments, the dose is 1100-2000 mg. In certain embodiments, the dose is 1100-1800 mg. As will be appreciated by those skilled in the art, this dosage can be administered over time. For example, the dosage can be administered via IV, e.g., as a 1-6 hour infusion, preferably as a 2-4 hour infusion. In certain embodiments, the therapeutic agent is administered once every two weeks. In particular, the flat doses disclosed herein are suitable for use in adults and / or subjects weighing at least 35 kg. In certain embodiments, the subject is afflicted with head and neck cancer.

[0266] In certain embodiments, the antibody, or functional part, derivative, and / or analog thereof, comprises petosemutamab and is administered in an amount of 1100 mg as an intravenous infusion once every two weeks, hi certain embodiments, the dose is a flat dose of 1100 mg.

[0267] In certain embodiments, the antibody, or functional portion, derivative, and / or analog thereof, comprises petosemutamab and is administered in an amount that achieves at least 90%, at least 95%, or at least 99% human receptor target engagement for both EGFR and LGR5 across a relevant body weight range in a statistically significant number of subjects. The 90% amount can be achieved using a flat dose of about 1000 mg Q2W. The 95% amount can be achieved using a flat dose of about 1100 to about 1200 mg Q2W.

[0268] In certain embodiments, a premedication regimen may be used. Such a regimen may be useful for reducing the likelihood or severity of infusion-related reactions. Generally, a steroid such as dexamethasone and / or an antihistamine, such as dexchlorpheniramine, diphenhydramine, or chlorpheniramine, is administered (e.g., orally, intravenously) prior to treatment with the therapeutic agents referred to herein.

[0269] The therapeutic methods described herein are typically continued as long as the clinician supervising the patient's care deems the therapeutic method effective, i.e., the patient is responding to the treatment. Non-limiting parameters that indicate that the therapeutic method is effective may include one or more of the following: reduction of tumor cells, inhibition of tumor cell proliferation, elimination of tumor cells, progression-free survival, and appropriate response (if applicable) by suitable tumor markers.

[0270] With regard to the frequency of administration of the therapeutic agent, one skilled in the art would be able to determine an appropriate frequency. For example, a clinician may decide to administer the therapeutic agent relatively less frequently (e.g., once every two weeks) and gradually shorten the period between doses as tolerated by the patient. Examples of exemplary time periods associated with a course of therapy according to the claimed methods include about 1 week, 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, about 24 months, about 30 months, about 3 years, about 4 years, about 5 years, or permanently (e.g., continuing maintenance therapy). The aforementioned time periods may relate to one or more rounds / cycles of treatment.

[0271] The efficacy of the therapeutic methods provided herein can be assessed using any suitable means. In certain embodiments, the clinical efficacy of the treatment is analyzed using a reduction in cancer cell count as an objective response criterion. Patients, e.g., humans, treated according to the methods disclosed herein preferably experience an improvement in at least one symptom of cancer. In certain embodiments, one or more of the following may occur: a reduction in cancer cell count; prevention or delay of cancer recurrence; and partial alleviation of one or more symptoms associated with cancer. In addition, an in vitro assay for determining T cell-mediated target cell lysis may be used. In certain embodiments, tumor assessment is based on CT scans and / or MRI scans; see, e.g., RECIST 1.1 guidelines (Response Evaluation Criteria in Solid Tumors) (Eisenhauer et al., 2009 Eur J Cancer 45:228-247). Such assessments are generally performed every 4 to 8 weeks after treatment.

[0272] In certain embodiments, tumor cells are no longer detectable after treatment as described herein. In certain embodiments, the subject is in partial or complete remission. In certain embodiments, the subject has an increase in overall survival, median survival, and / or progression-free survival.

[0273] The therapeutic agent (i.e., an antibody comprising a variable domain that binds to the extracellular portion of EGFR and a variable domain that binds to the extracellular portion of LGR5, or a functional portion, derivative, and / or analog thereof) may also be used in conjunction with immune checkpoint inhibitors that are selected as additional therapeutic agents for their particular usefulness against the cancer being treated.

[0274] In certain embodiments, at the start of treatment, at least one, more than one, or all of the following inclusion factors IF1-IF16 apply to the subject of treatment. In certain embodiments, the subject includes or complies with all of IF1-IF16: IF 1. You are at least 18 years of age. IF 2. Have histologically or cytologically confirmed solid tumors with evidence of metastatic or locally advanced disease that is not amenable to standard therapy for subjects with locally advanced unresectable or metastatic disease for HNSCC with tumors expressing PD-L1, CPS ≥ 1 (as determined by an FDA-approved test in the US or an approved equivalent test in other countries). IF 3. No prior systemic therapy administered in the recurrent or metastatic setting, but prior systemic therapy as part of multimodal treatment for locally advanced disease is acceptable if completed at least 6 months prior to administration of petosemutamab and / or pembrolizumab. IF 4. HNSCC primary tumor location in the oropharynx, oral cavity, hypopharynx, or larynx. IF 5. No prior anti-cancer treatment with anti-PD-L1, anti-PD-1, or anti-EGFR therapy. IF 6. Have a baseline tumor sample (e.g., formalin-fixed, paraffin-embedded block, [FFPE]) from a metastatic or primary site. If the subject has such available tumor samples (at least 20 slides with >20% tumor content) from a sample collection with sufficient material and has not received further anti-cancer treatment since the sample collection, a new tumor biopsy at baseline is not required. Archival FFPE slides are not acceptable. Primary tumor material is acceptable only if the patient has not been treated with anti-EGFR. IF 7. Suitable for biopsy. IF 8. Have measurable disease as defined by RECIST version 1.1 by radiological methods. Have an Eastern Cooperative Oncology Group (ECOG) performance status of IF 9.0 or 1. IF 10. Life expectancy ≥ 12 weeks (typically assessed by investigator). IF 11. Left ventricular ejection fraction (LVEF) by echocardiogram (ECHO) or multiple gated acquisition scan (MUGA) of at least 50%. IF 12. Adequate organ function: IF 12.1 at least 1.5 x 10 9 and an absolute neutrophil count (ANC) of 1 / L. ·IF 12.2 Have a hemoglobin level of at least 9g / dL. IF 12.3 at least 100 x 10 9 Having a platelet level of / L. ·IF 12.4 Have corrected total serum calcium within normal range. ·IF 12.5 Have serum magnesium levels within normal range or corrected with supplements. IF 12.6 Alanine aminotransferase (ALT), aspartate aminotransferase (AST) ≤ 2.5 times the upper limit of normal (ULN), and total bilirubin ≤ 1.5 times the ULN, except that if the subject has Gilbert syndrome, total bilirubin ≤ 3.0 times the ULN or direct bilirubin ≤ 1.5 times the ULN; in the case of hepatic involvement, ALT / AST ≤ 5 times the ULN and total bilirubin ≤ 2 times the ULN; or in the case of hepatocellular carcinoma (HCC) with a Child-Pugh class A score, total bilirubin < 3 mg / dL. IF 12.7: Serum creatinine level less than 1.5 × ULN or creatinine clearance of at least 60 mL / min calculated according to the Cockroft and Gault formula or the Modification of Diet in Renal Disease (MDRD) formula for patients 65 years of age or older. IF 12.8: Serum albumin level of at least 3 g / dL. IF 12.9 Having an international normalized ratio (INR) or prothrombin time (PT) level less than 1.5 × ULN, unless the patient is receiving anticoagulation therapy and is within the therapeutic range of the anticoagulant used as intended. IF 12.10 Having an activated partial thromboplastin time (APTT) or PTT that is less than 1.5 x ULN unless the patient is receiving anticoagulant therapy and is within the therapeutic range of the anticoagulant used as intended. · IF 13.1. Have a negative test result for human immunodeficiency virus infection on a test date not more than six months prior to treatment with this disclosure. · IF 13.2 If there is a positive test result for human immunodeficiency virus infection, the CD4+ count is at least 300 / μL, the viral load is undetectable, and the subject is being treated with highly active antiretroviral therapy (HAART). IF 14. Have an active hepatitis B (HBsAg positive) infection but are receiving antiviral treatment with lamivudine, tenofovir, entecavir, or other antiviral agents starting at least 7 days prior to initiating treatment with the present disclosure. ·IF 15. Presence of prodromal symptoms of hepatitis B (anti-HBc positive, HBsAg and hepatitis B virus [HBV]-DNA negative). IF 16. Subjects who have tested positive for hepatitis C virus (HCV) ribonucleic acid (RNA) and whose HCV infection has spontaneously cleared (i.e., have positive HCV antibodies without detectable HCV-RNA) or who have achieved a sustained response after antiviral treatment and have demonstrated the absence of detectable HCV RNA for at least 6 months with the use of an IFN-free regimen or at least 12 months with the use of an IFN-based regimen.

[0275] In certain embodiments, all values ​​for IF12 organ function measurements have upper limits observed in healthy subjects.

[0276] In certain embodiments, the subject of treatment complies with one or more factors selected from the group consisting of IF1-IF16. In certain embodiments, the subject of treatment complies with factors IF2, IF3, IF4, IF5, IF8, IF9, IF10, IF11, IF12, IF13, IF14, IF15, and IF16. In certain embodiments, the subject of treatment complies with factors IF2, IF3, IF4, and IF5. In certain embodiments, the subject of treatment complies with factor IF3.

[0277] In certain embodiments, at the start of treatment, at least one, two or more, or all of the following exclusion factors EF1 to EF apply to a person who is a candidate for treatment. EF 1. Have central nervous system metastases that are untreated or symptomatic, or require radiation, surgery, or continuous steroid therapy to control symptoms within 14 days of initiating treatment according to the present disclosure. EF 2. Having leptomeningeal involvement. EF 3. Participation in further clinical trials or treatment with any investigational drug within 4 weeks prior to initiation of treatment according to this disclosure. EF 4. Have any systemic anti-cancer therapy within 4 weeks or 5 half-lives, whichever is longer, of the first dose according to the present disclosure. For cytotoxic agents with significant delayed toxicity (e.g., mitomycin C, nitrosoureas) or anti-cancer immunotherapies, a 6-week washout period is required before initiation of treatment according to the present disclosure. EF 5. Have requirement for immunosuppressive medications (e.g., methotrexate, cyclophosphamide). EF 6. Major surgery or radiation therapy within 3 weeks of initiating treatment according to the present disclosure. Patients who have received prior radiation therapy to at least 25% of their bone marrow, regardless of when it was received, are excluded. EF 7. Have persistent Grade >1 clinically significant toxicity (excluding alopecia) related to prior antineoplastic therapy, provided that Grade 2 (or less) National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v4.03 or v5.0 stable sensory neuropathy is tolerated at the time of administration or is current. EF 8. History of hypersensitivity reaction or any toxicity due to human proteins or excipients that justified permanent discontinuation of these agents. EF 9. Demonstrate uncontrolled hypertension (systolic BP > 150mmHg and / or diastolic BP > 100mmHg) with appropriate treatment or unstable angina. EF 10. History of Class II-IV New York Heart Association (NYHA) congestive heart failure or severe cardiac arrhythmias requiring treatment (excluding atrial fibrillation and paroxysmal supraventricular tachycardia). EF 11. Have had a myocardial infarction within 6 months of starting treatment according to the present disclosure. EF 12. Have a history of prior malignancy, excluding excised cervical intraepithelial neoplasia or non-melanoma skin cancer, or curatively treated cancer that has had no evidence of disease for at least three years prior to initiation of treatment according to this disclosure and is considered at low risk of recurrence. EF 13. Current dyspnea at rest of any origin or other illness requiring continuous oxygen therapy. EF 14. Have a history of interstitial lung disease (ILD) (e.g., pneumonia or pulmonary fibrosis) or evidence of ILD on a baseline chest computed tomography (CT) scan. EF 15. Having a serious illness or psychiatric disorder, including but not limited to, uncontrolled active infection, clinically significant pulmonary, metabolic or psychiatric disease, at the time of initiation of treatment according to the present disclosure. EF 16. Subject has active hepatitis B surface antigen infection (HBsAg positive) not receiving antiviral treatment. EF 17. Subject has Child-Pugh class B or C cirrhosis status, subject has fibrolamellar HCC, subject has sarcomatoid HCC, or subject has mixed cholangiocarcinoma and HCC. EF 18. Pregnant or nursing subjects, and subjects of childbearing potential, should use highly effective methods of contraception before initiating treatment according to the present disclosure, during said treatment, and for 6 months after the last dose of petosemutamab. EF 19. Have a diagnosis of immunodeficiency or have received systemic steroid therapy or any form of immunosuppressive therapy within 7 days prior to initiation of treatment according to the present disclosure. Of note, the use of corticosteroids as premedication for allergic or infusion-related reactions as specified herein is permitted. EF 20. Have an active autoimmune disease that has required systemic immunosuppressive treatment within two years prior to initiation of treatment according to this disclosure, provided that receiving replacement therapy (e.g., thyroxine, insulin, or physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency) is not considered immunosuppressive treatment. EF 21. Having had an allogeneic tissue / solid organ transplant prior to initiation of treatment according to the present disclosure. EF 22. Have a primary tumor site in the nasopharynx of any histology.

[0278] In certain embodiments, the subject of treatment complies with one or more factors selected from the group consisting of EF1-EF22. In certain embodiments, the subject of treatment complies with all of factors EF1-EF22. In certain embodiments, the subject of treatment complies with factors IF13, IF16, IF19, IF20, and IF21.

[0279] ECOG performance status scoring grade definitions are known in the art as follows: 0 Fully active, able to perform all pre-disease functions without limitation; 1 Limited physically strenuous activity but ambulatory and able to perform light or sedentary tasks, e.g., light housework, office work; 2 Ambulatory and able to perform all self-care tasks but unable to work; more than 50% of waking hours out of bed; 3 Only limited self-care; more than 50% of waking hours confined to bed or a chair; 4 Totally immobile; unable to perform any self-care tasks; entirely confined to bed or a chair; 5 Death.

[0280] Child-Pugh scoring, also referred to as the Child-Pugh classification, Child-Turcotte-Pugh (CTP) calculator, or Child criteria, is applied herein according to standard clinical practice. The Child-Pugh score is determined by scoring five clinical measures of liver disease and the likelihood of eventual liver failure. Each measure is given a score of 1, 2, or 3, with 3 being the most severe. The five clinical measures are total bilirubin, serum albumin level, prothrombin time (or prolonged INR, or the time it takes for blood to clot), ascites, and hepatic encephalopathy. Class A: 5-6 points, representing mild liver disease and a 95% 1-5 year survival rate; Class B: 7-9 points, representing moderate liver disease and a 75% 1-5 year survival rate; and Class C: 10-15 points, representing severe liver disease and a 50% 1-5 year survival rate. The following points are assigned for each clinical measure: Encephalopathy: None = 1 pt, Grade 1 and 2 = 2 pts, Grade 3 and 4 = 3 pts. Ascites: None = 1 pt, Mild = 2 pts, Moderate = 3 pts. Bilirubin: Less than 2 mg / ml = 1 pt, 2-3 mg / ml = 2 pts, Greater than 3 mg / ml = 3 pts. Albumin: Greater than 3.5 mg / ml = 1 pt, 2.8-3.5 mg / ml = 2 pts, Less than 2.8 mg / ml = 3 pts. Prothrombin time (PT, prolonged): Less than 4 seconds = 1 pt, 4-6 seconds = 2 pts, Greater than 6 seconds = 3 pts. Alternatively, the international normalized ratio (INR) is used as a surrogate for PT, with an INR of less than 1.7 = 1 pt, an INR of 1.7-2.2 = 2 pts, and an INR of greater than 2.2 = 3 pts.

[0281] Administration of the therapeutic agents of the present disclosure may be premedicated, meaning that the drug is administered to the subject prior to administration of the antibody or immune checkpoint of the present invention. In certain embodiments, a 1500 mg dose of petosemutamab is premedicated with an antihistamine, an analgesic, an antipyretic, and / or an anti-inflammatory.

[0282] The main approved medications are: In certain embodiments, the treatments of the present disclosure include pre-medication with paracetamol / acetaminophen, antihistamines, or corticosteroids. In certain embodiments, such pre-medication is administered in the event of infusion-related reactions, hypersensitivity, and / or allergic reactions, according to standard local clinical practice.

[0283] In certain embodiments, the treatment of the present disclosure further includes all medications necessary for patient safety and well-being, which are not expected to interfere with the evaluation of the study drug but may be administered at the investigator's discretion. Also permitted are supportive care for symptoms and adverse events, or standard treatment for comorbid conditions, including aspirin, transfusion support, granulocyte colony-stimulating factor, antibiotics, inhaled steroids (for asthma), antiemetics, antidiarrheals (such as loperamide), and bisphosphonates (depending on their product licensing and routine clinical practice). Also permitted are adjuvant hormonal therapy agents for curatively treated cancers with no evidence of disease and deemed low risk of recurrence. This may include adjuvant luteinizing hormone-releasing hormone (LHRH) agonists (for 5 years) for early-stage breast cancer in combination with an antiestrogen, and LHRH agonists + / - antiandrogens for localized prostate cancer. Concurrent radiation therapy during the treatment for symptom control without evidence of progression is also permitted.

[0284] The major prohibited medications are as follows: In certain embodiments, the treatments of the present disclosure do not include concomitant medications due to the risk of immunosuppression. Such medications typically include chronic oral corticosteroids (>10 mg / day prednisone equivalent, excluding inhaled and topical steroids), tumor necrosis factor (TNF) alpha inhibitors, anti-T cell antibodies, and other immunosuppressants. In certain embodiments, the treatments of the present disclosure do not include any investigational drugs or other anti-cancer therapies during the treatment or, if known, within four weeks or five half-lives, whichever is longer, prior to administration of the first dose of the treatment. In certain embodiments, the treatments of the present disclosure do not include cytotoxic agents with significant delayed toxicity requiring a six-week washout period (e.g., mitomycin C, nitrosoureas, or anti-cancer immunotherapy). In certain embodiments, the treatments of the present disclosure do not include herbal therapies for cancer treatment that were not initiated before the first administration of the therapeutic agents of the present disclosure. Continuing herbal therapies for cancer treatment during the treatment is permitted.

[0285] In certain aspects, the treatments of the present disclosure do not include major surgery or radiation therapy within three weeks prior to administration of the first dose of the treatment, or prior radiation therapy to at least 25% of the total bone marrow of the subject being treated.

[0286] Concomitant use of chronic oral corticosteroids (e.g., >10 mg / day prednisone equivalent, excluding inhaled and topical steroids), tumor necrosis factor (TNF)-alpha inhibitors, anti-T cell antibodies, or other immunosuppressive medications is not permitted during such treatment. Thus, in certain embodiments, there is no concomitant use of chronic oral corticosteroids (e.g., >10 mg / day prednisone equivalent, excluding inhaled and topical steroids), tumor necrosis factor (TNF)-alpha inhibitors, anti-T cell antibodies, or other immunosuppressive medications during treatment according to the present disclosure.

[0287] The compounds and compositions disclosed herein are useful as therapies and in therapeutic treatments, and therefore are useful as medicaments and can be used in methods for preparing medicaments.

[0288] Kits of parts and therapeutic combinations In certain aspects, the present disclosure also provides a kit of parts comprising an antibody as defined herein, or a functional part, derivative, and / or analog thereof, an immune checkpoint inhibitor as defined herein, and instructions for using the antibody, or a functional part, derivative, and / or analog thereof, and / or for using the immune checkpoint inhibitor.

[0289] In certain aspects, the present disclosure also provides a kit of parts comprising an antibody as defined herein, or a functional part, derivative, and / or analog thereof, instructions for using the antibody, or a functional part, derivative, and / or analog thereof, and instructions for using an immune checkpoint inhibitor as defined herein.

[0290] The present disclosure also provides a kit of parts comprising an antibody, or a functional part, derivative, and / or analogue thereof, comprising the extracellular portion of EGFR and optionally a variable domain capable of binding to LGR5, an immune checkpoint inhibitor, and instructions for use of the antibody, or a functional part, derivative, and / or analogue thereof, and for use of the immune checkpoint inhibitor.

[0291] The present disclosure also provides a combination of an antibody, or a functional part, derivative, and / or analogue thereof, comprising a variable domain capable of binding to the extracellular portion of EGFR and optionally LGR5, and an immune checkpoint inhibitor as mentioned herein, for use in treating cancer in a subject in need thereof.

[0292] The present disclosure also provides a combination of an immune checkpoint inhibitor referred to herein, instructions for the use of said immune checkpoint inhibitor in the treatment of cancer in a subject, and instructions for the use of an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR and optionally LGR5 referred to herein, or a functional part, derivative, and / or analogue thereof, in the treatment of said cancer in said subject.

[0293] The present disclosure also provides a combination of an antibody, or a functional part, derivative, and / or analogue thereof, comprising a variable domain capable of binding to the extracellular portion of EGFR and optionally LGR5 as referred to herein, instructions for the use of the antibody, or a functional part, derivative, and / or analogue thereof in the treatment of cancer in a subject, and instructions for the use of an immune checkpoint inhibitor as referred to herein in the treatment of cancer in a subject.

[0294] In certain aspects, the present disclosure provides pharmaceutical compositions comprising antibodies comprising variable domains capable of binding to the extracellular portion of EGFR and optionally LGR5 of the present disclosure, or functional parts, derivatives, and / or analogs thereof, and instructions for their use with immune checkpoint inhibitors in the treatment of such cancers.

[0295] In certain aspects, the present disclosure provides pharmaceutical compositions for the treatment of cancer, comprising an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR and optionally LGR5 of the present disclosure, or a functional part, derivative, and / or analog thereof, and a pharmaceutical composition for the treatment of said cancer, comprising an immune checkpoint inhibitor of the present disclosure.

[0296] In certain aspects, the present disclosure provides a pharmaceutical composition for use in the treatment of cancer comprising an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR and optionally LGR5 of the present disclosure, or a functional part, derivative, and / or analog thereof, administered in combination with an immune checkpoint inhibitor of the present disclosure.

[0297] In certain aspects, the present disclosure relates to a pharmaceutical composition for the treatment of cancer comprising an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR and optionally LGR5 of the present disclosure, or a functional part, derivative, and / or analog thereof, wherein the subject to be treated is administered the treatment prior to, concurrently with, or after administration of the bispecific antibody.

[0298] In certain aspects, the present disclosure relates to pharmaceutical compositions for the treatment of cancer in a subject comprising an immune checkpoint inhibitor, wherein the subject to be treated is administered an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR and optionally LGR5, or a functional part, derivative, and / or analog thereof, of the present disclosure, prior to, concurrently with, or following administration of the immune checkpoint inhibitor.

[0299] Thus, the present disclosure relates to a drug combination for the treatment of cancer in a subject, comprising administration to the subject of a plurality of different drugs for treating the cancer, wherein the treatment comprises simultaneous, sequential, or separate administration of the drugs. In certain aspects, the drugs comprise an antibody comprising a variable domain capable of binding to the extracellular portion of EGFR and optionally LGR5 of the present disclosure, or a functional part, derivative, and / or analog thereof, and the other different drugs comprise an immune checkpoint inhibitor.

[0300] In certain embodiments, the kit or combination of parts includes instructions for dosing petosemutamab at 1500 mg. In certain embodiments, the kit includes instructions for using petosemutamab for dosing at 1500 mg once every two weeks. In certain embodiments, the kit includes instructions for using an immune checkpoint inhibitor, such as petosemutamab and pembrolizumab, in the treatment of head and neck cancer. In certain embodiments, the kit includes instructions for using petosemutamab and the immune checkpoint inhibitor in the treatment of head and neck squamous cell carcinoma. In certain embodiments, the kit includes instructions for using petosemutamab and pembrolizumab. In certain embodiments, the kit includes instructions for using petosemutamab and pembrolizumab as referred to herein, e.g., pembrolizumab 400 mg q6w and petosemutamab 1500 mg q2w. In certain embodiments, the kit includes instructions for uniform dosing of petosemutamab and uniform dosing of pembrolizumab.

[0301] In certain aspects, the present disclosure also provides a combination of an antibody, or a functional part, derivative, and / or analog thereof, as defined herein, and an immune checkpoint inhibitor, as defined herein, for use in treating cancer in a subject in need thereof.

[0302] In certain aspects, the present disclosure also provides an immune checkpoint inhibitor, as defined herein, in a subject in need of treatment for cancer, wherein the immune checkpoint inhibitor is for simultaneous or sequential administration with an antibody, or functional part, derivative, and / or analogue thereof, as defined herein.

[0303] In certain embodiments, the antibody, or functional portion, derivative, and / or analog thereof, is administered to a subject with cancer who has been or will be administered the immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is administered to a subject with cancer who has been or will be administered the antibody, or functional portion, derivative, and / or analog thereof.

[0304] In certain aspects, the present disclosure also provides a combination of an antibody as defined herein, or a functional part, derivative, and / or analog thereof, instructions for the use of the antibody, or a functional part, derivative, and / or analog thereof in the treatment of cancer in a subject, and instructions for the use of an immune checkpoint inhibitor as defined herein in the treatment of cancer in a subject.

[0305] In certain aspects, the present disclosure also provides a combination of an immune checkpoint inhibitor as defined herein, instructions for the use of said inhibitor in the treatment of cancer in a subject, and instructions for the use of an antibody as defined herein, or a functional part, derivative, and / or analogue thereof, in the treatment of said cancer.

[0306] In certain embodiments, the instructions for use include the amount of immune checkpoint inhibitor to be used and the amount of the antibody, or functional portion, derivative, and / or analog thereof, the dosage interval, and the cancer to be treated.

[0307] All documents and references, including Genbank entries, patents and published patent applications, and websites, mentioned herein are each expressly incorporated by reference to the same extent as if fully or partially set forth herein.

[0308] For purposes of clarity and conciseness of description, features are described herein as part of the same or separate portions of this disclosure, but it will be understood that the scope of the present invention may include preferred embodiments having all or any combination of the described features.

[0309] The present invention will now be described with reference to the following examples, which are illustrative only and are not intended to limit the invention. Although the present invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope thereof.

[0310] List of clauses Terms 1. An antibody, or a functional part, derivative, and / or analogue thereof, comprising a variable domain that binds to the extracellular portion of EGFR, for use in treating cancer in a subject, wherein the treatment further comprises administering an immune checkpoint inhibitor. 2. Use of an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative, and / or analogue thereof, and an immune checkpoint inhibitor in the manufacture of one or more medicaments for treating cancer in a subject. 3. The use according to clause 2, wherein the antibody, or functional part, derivative and / or analogue thereof, and the immune checkpoint inhibitor are used to manufacture separate medicaments or medicament formulations. 4. The use according to clause 2 or 3, wherein the treatment of cancer comprises the administration of an antibody, or a functional part, derivative, and / or analogue thereof, and an immune checkpoint inhibitor. 5. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an antibody, or a functional part, derivative, and / or analog thereof, comprising a variable domain that binds to the extracellular portion of EGFR and an effective amount of an immune checkpoint inhibitor. 6. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the immune checkpoint inhibitor comprises a PD-L1 inhibitor, a PD-L2, or a PD-1 inhibitor. 7. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the immune checkpoint inhibitor comprises a PD-L2 inhibitor. 8. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the immune checkpoint inhibitor comprises a PD-1 inhibitor. 9. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the immune checkpoint inhibitor comprises nivolumab, pembrolizumab, cemiplimab, penprimimab, retifanlimab, sintilimab, tislelizumab, toripalimab, dostarimab, atezolizumab, avelumab, or durvalumab, in particular pembrolizumab. 10. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the immune checkpoint inhibitor comprises or is pembrolizumab. 11. The antibody, or functional part, derivative and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the cancer is adenocarcinoma or squamous cell carcinoma, or in particular head and neck cancer. 12. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the cancer is head and neck cancer. 13. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the cancer is squamous cell carcinoma of the head and neck. 14. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the cancer is cancer of the pharynx (including the nasopharynx, oropharynx, and hypopharynx), oral cavity, larynx, paranasal sinuses, nasal cavity, or salivary glands. 15. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the cancer is a cancer of the oropharynx, oral cavity, hypopharynx, or larynx. 16. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the primary location of the cancer is in the oropharynx, oral cavity, hypopharynx, or larynx. 17. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the cancer expresses EGFR and optionally LGR5. 18. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the cancer expresses EGFR characterized by an IHC score of 2+ or 3+, or an H-score for EGFR of more than 50, such as 80, 100, or 200 but not more than 300. 19. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the cancer expresses PD-L1. 20. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the cancer has a combined positive score (CPS) for PD-L1 expression of 1 or greater but 100 or less. 21. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the cancer has a combined positive score (CPS) for PD-L1 expression of from 1 (inclusive) to 20 (exclusive). 22. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the cancer has a combined positive score (CPS) for PD-L1 expression of from 20 (inclusive) to 100 (inclusive). 23. The antibody, or functional part, derivative and / or analogue thereof, or the use or method according to any one of clauses 20 to 22, wherein CPS is determined using IHC. 24. The antibody, or functional part, derivative and / or analogue thereof, or use or method according to any one of clauses 20 to 23, wherein CPS is determined by IHC using clone 22C3. 25. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the cancer is positive for p16 status. 26. The antibody or functional part, derivative and / or analogue thereof, or the use or method according to clause 25, wherein the cancer or the subject is suffering from (primary) oropharyngeal cancer. 27. The antibody, or functional part, derivative and / or analogue thereof, or use or method according to any one of clauses 1 to 24, wherein the subject is negative for p16 status. 28. The antibody, or functional part, derivative and / or analogue thereof, or use or method according to clause 27, wherein the cancer is head and neck cancer other than (primary) oropharyngeal cancer. 29. The antibody, or functional part, derivative and / or analogue thereof, or use or method according to any one of clauses 25 to 28, wherein p16 status is determined using IHC. 30. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the cancer expresses LGR5. 31. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the cancer is ISH-positive for LGR5. 32. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to clause 31, wherein LGR5 expression is determined using in situ hybridization (ISH), RNA sequencing, tissue microarray (TMA) staining, or by calculating an H-score for LGR5. 33. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the subject has not received any prior anti-cancer therapeutic agent for the treatment of said cancer. 34. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the subject has not received any prior anti-cancer therapy for the treatment of said cancer at least 6 months prior to receiving said antibody, or functional part, derivative, and / or analogue thereof, and / or said immune checkpoint inhibitor. 35. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to clause 34, wherein the subject has undergone multimodal treatment including surgery, radiotherapy, and / or platinum-containing chemotherapy, such as cisplatin, more than 6 months prior to receiving the antibody, or functional part, derivative, and / or analogue thereof, and the immune checkpoint inhibitor. 36. The antibody, or functional part, derivative and / or analogue thereof, or use or method according to clause 35, wherein said subject has undergone said multimodal treatment to treat a curable localized disease. 37. The antibody, or functional part, derivative and / or analogue thereof, or use or method according to clause 34, wherein the prior anti-cancer treatment comprises treatment with anti-PD-L1, anti-PD-1, or anti-EGFR therapy. 38. An antibody, or functional part, derivative, and / or analogue thereof, or use or method as defined in clause 34, wherein the prior anti-cancer treatment comprises treatment with chemotherapy, immunotherapy, an anti-EGFR agent, an antibody targeting EGFR, cetuximab, a PD-1 inhibitor, or a PD-L1 inhibitor. 39. An antibody, or a functional part, derivative and / or analogue thereof, or a use or a method according to any one of the preceding clauses, wherein the antibody is mammalian. 40. The antibody, or functional part, derivative and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the subject is a human subject. 41. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the treatment comprises administration of the antibody, or functional part, derivative, and / or analogue thereof weekly, biweekly, or every three weeks, preferably biweekly, and more preferably the subject is administered at least three or more biweekly dosages of the antibody, or functional part, derivative, and / or analogue thereof. 42. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the treatment comprises administering to the subject petosemutamab as the antibody every two weeks. 43. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the treatment comprises the administration of 1100 or 1500 mg to the subject every two weeks. 44. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the treatment comprises administering to the subject a dose of 1100 or 1500 mg of petosemutamab. 45. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the antibody, or functional part, derivative, and / or analogue thereof, is administered intravenously to the subject. 46. ​​The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the treatment comprises administering to the subject pembrolizumab as an immune checkpoint inhibitor at a dose of 400 mg once every six weeks. 47. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the administration of petosemutamab and pembrolizumab is administered sequentially. 48. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein petosemutamab and pembrolizumab are administered on the same day, with pembrolizumab being administered after petosemutamab. 49. An antibody or functional part, derivative, and / or analogue thereof, or a use or method according to any one of the preceding clauses, wherein concomitant administration of chronic oral corticosteroids (excluding inhaled and topical steroids, such as prednisone equivalents >10 mg / day), tumor necrosis factor (TNF) alpha inhibitors, anti-T cell antibodies, or other immunosuppressive drugs is not permitted during the treatment. 50. An antibody, or a functional part, derivative, and / or analogue thereof, or a use or a method according to any one of the preceding clauses, wherein the antibody, or functional part, derivative, and / or analogue thereof, has enhanced ADCC. 51. An antibody, or a functional part, derivative, and / or analogue thereof, or a use or a method according to any one of the preceding clauses, wherein the antibody, or functional part, derivative, and / or analogue thereof, is afucosylated. 52. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the cancer is recurrent, unresectable, locally advanced, and / or metastatic cancer. 53. An antibody, or a functional part, derivative, and / or analogue thereof, or a use or a method according to any one of the preceding clauses, wherein the antibody, or functional part, derivative, and / or analogue thereof, is multispecific. 54. An antibody, or a functional part, derivative, and / or analogue thereof, or a use or a method according to any one of the preceding clauses, wherein the antibody, or functional part, derivative, and / or analogue thereof, is bispecific. 55. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the antibody comprises a variable domain that does not bind to EGFR. 56. An antibody, or a functional part, derivative, and / or analogue thereof, or a use, or a method according to any one of the preceding clauses, wherein the antibody comprises a variable domain that binds to LGR5. 57. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of clauses 1 to 55, wherein the antibody is a monovalent antibody or wherein the antibody comprises the EGFR-binding variable domain as the only variable domain. 58. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein treatment comprises or is followed by a step of diagnosing or testing the subject for EGFR, LGR5, and / or PD-L1 expression. 59. The antibody, or functional part, derivative, and / or analogue thereof, or method according to any one of the preceding clauses, wherein treatment comprises or is followed by a step of diagnosing or testing the subject for PD-L1 expression. 60. An antibody, or a functional part, derivative and / or analogue thereof, or a method according to clause 58 or 59, wherein the diagnosis or testing is by ISH or IHC. 61. The antibody, or functional part, derivative, and / or analogue thereof, or method according to any one of clauses 58 to 60, wherein the diagnosis or testing for PD-L1 expression is by IHC. 62. The antibody, or functional part, derivative, and / or analogue thereof, or method according to any one of clauses 58 to 60, wherein diagnosing or testing for PD-L1 expression is by establishing a CPS for PD-L1. 63. The antibody, or functional part, derivative, and / or analogue thereof, or method according to clause 63, wherein the diagnosis or testing for PD-L1 expression is by establishing a CPS for PD-L1 for expression determined by IHC. 64. The antibody, or functional part, derivative, and / or analogue thereof, or method according to clause 63, wherein the CPS for PD-L1 expression is by IHC using clone 22C3. 65. The variable domain that binds to the extracellular portion of EGFR is - at least the CDR3 sequence of the VH of MF3370, MF3755, MF4280 or MF4289 shown in Figure 3 or a CDR3 sequence which differs from the CDR3 sequence of the VH of MF3370, MF3755, MF4280 or MF4289 shown in Figure 3 by at most three, preferably at most two, preferably no more than one amino acid; at least the CDR1, CDR2 and CDR3 sequences of the VH of MF3370, MF3755, MF4280 or MF4289 shown in Figure 3, or the CDR1, CDR2 and CDR3 sequences of the VH of MF3370, MF3755, MF4280 or MF4289 shown in Figure 3 with at most three, preferably at most two, preferably at most one amino acid substitution; or 3. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the heavy chain variable region comprises the sequence of the VH chain of MF3370, MF3755, MF4280, or MF4289 as shown in Figure 3, or the amino acid sequence of the VH chain of MF3370, MF3755, MF4280, or MF4289 as shown in Figure 3 with at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof, with respect to the VH chain of MF3370, MF3755, MF4280, or MF4289. 66. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the EGFR-binding variable domain binds to an epitope located within amino acid residues 420 to 480 of the human EGFR sequence shown in Figure 2. 67. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein binding of the variable domain to EGFR is reduced by one or more of the following amino acid residue substitutions in EGFR: I462A, G465A, K489A, I491A, N493A, and C499A, compared to an EGFR protein not comprising such substitutions. 68. The variable domain that binds to LGR5 is - at least the CDR3 sequence of the VH of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3, or a CDR3 sequence that differs from the CDR3 sequence of the VH of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3 by at most three, preferably at most two, preferably no more than one amino acid; - at least the CDR1, CDR2 and CDR3 sequence of the VH of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817 or MF5818 shown in Figure 3, or the CDR1, CDR2 and CDR3 sequence of the VH of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817 or MF5818 shown in Figure 3 with at most three, preferably at most two, preferably at most one amino acid substitution; or For the sequence of the VH chain of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3, or the VH chain of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818, at most 15, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 5. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the heavy chain variable region comprises the amino acid sequence of the VH chain of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 as shown in Figure 3, having 1, 2, 3, 4, or 5 amino acid insertions, deletions, substitutions, or a combination thereof. 69. The antibody, functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the variable domain binding to the extracellular portion of EGFR comprises a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences of a variable region selected from the group consisting of MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, and the variable domain binding to the extracellular portion of LGR5 comprises a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences of a variable region selected from the group consisting of MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3. 70. An antibody, or a functional part, derivative, and / or analogue thereof, or a use or method according to any one of the preceding clauses, wherein the variable domain that binds to the extracellular portion of EGFR comprises a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences of the variable region of MF3755 shown in Figure 3, and the variable domain that binds to the extracellular portion of LGR5 comprises a heavy chain variable region comprising the CDR1, CDR2, and CDR3 sequences of the variable region of MF5816 shown in Figure 3. 71. The VH chain of the variable domain that binds to EGFR comprises the amino acid sequence of VH chain MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, or the amino acid sequence of VH chain MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, with up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less, amino acid modifications including insertions, deletions, substitutions, or combinations thereof relative to the VH; and the VH chain of the variable domain that binds to LGR5 comprises the amino acid sequence of VH chain MF5790, MF5803, MF5805, MF5808, 3. The antibody, or functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, comprising the amino acid sequence of VH chain MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 as shown in Figure 3, having up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less amino acid modifications including insertions, deletions, substitutions, or combinations thereof in said VH. 72. The VH chain of the variable domain that binds to EGFR comprises the amino acid sequence of VH chain MF3755 shown in Figure 3 or the amino acid sequence of VH chain MF3755 shown in Figure 3 with up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less, amino acid modifications including insertions, deletions, substitutions, or a combination thereof relative to the VH; and the VH chain of the variable domain that binds to LGR5 comprises the amino acid sequence of VH chain MF3755 shown in Figure 3 with up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, amino acid modifications including insertions, deletions, substitutions, or a combination thereof relative to the VH. 3 , or the amino acid sequence of VH chain MF5816 as shown in Figure 3 with up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less amino acid modifications, including insertions, deletions, substitutions, or combinations thereof, relative to the VH, or a functional part, derivative, and / or analogue thereof, or a use or method thereof. 73. The antibody, or a functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the VH chain of the variable domain that binds to EGFR comprises the amino acid sequence of VH chain MF3370, MF3755, MF4280, or MF4289 shown in Figure 3, and the VH chain of the variable domain that binds to LGR5 comprises the amino acid sequence of VH chain MF5790, MF5803, MF5805, MF5808, MF5809, MF5814, MF5816, MF5817, or MF5818 shown in Figure 3. 74. The antibody, or a functional part, derivative, and / or analogue thereof, or use or method according to any one of the preceding clauses, wherein the VH chain of the variable domain that binds to EGFR comprises the amino acid sequence of VH chain MF3755 shown in Figure 3, and the VH chain of the variable domain that binds to LGR5 comprises the amino acid sequence of VH chain MF5816 shown in Figure 3. 75. An antibody, or a functional part, derivative, and / or analogue thereof, or a use or method according to any one of the preceding clauses, wherein the variable domains that bind to EGFR and that bind to LGR5 both comprise the CDR1, CDR2, and CDR3 regions of the light chain variable region shown in Figure 4b. 76. The antibody, or functional part, derivative, and / or analogue thereof, or use or method, according to any one of the preceding clauses, wherein the variable domains that bind EGFR and that bind LGR5 both comprise a light chain variable region as shown in Figure 4b, and wherein the variable light chain region comprises 0 to 10 amino acid insertions, deletions, substitutions, additions, or a combination thereof, and wherein the amino acid insertions, deletions, and substitutions are not present in the CDR1, CDR2, and CDR3 light chain variable regions. 77 An antibody, or a functional part, derivative, and / or analogue thereof, or a use or method thereof, according to any one of the preceding clauses, wherein the variable domain that binds to LGR5 binds to an epitope located within amino acid residues 21 to 118 of the human LGR5 sequence shown in Figure 1. 78. The antibody, or functional part, derivative, and / or analogue thereof, or use, or method according to any one of the preceding clauses, wherein the antibody that binds to the extracellular portion of EGFR is petosemutamab. 79. A kit of parts comprising an antibody as defined in any one of the preceding clauses, or a functional part, derivative, and / or analogue thereof, an immune checkpoint inhibitor as defined in any one of the preceding clauses, and instructions for use of the antibody, or a functional part, derivative, and / or analogue thereof, and for use of the immune checkpoint inhibitor. 80. The kit of clause 79, wherein the instructions for use of the antibody, or functional part, derivative, and / or analogue thereof, include instructions for dosing at 1500 mg. 81. The kit of clause 79 or 80, wherein the instructions for use of the antibody, or functional part, derivative, and / or analogue thereof, include instructions for dosing at 1500 mg once every two weeks. 82. A kit of parts according to any one of clauses 79 to 81, wherein the kit comprises instructions for use of the antibody, or functional part, derivative, and / or analogue thereof, and an immune checkpoint inhibitor in the treatment of head and neck cancer. 83. A kit of parts according to any one of clauses 79 to 82, wherein the kit comprises instructions for use of the antibody, or functional part, derivative, and / or analogue thereof, and an immune checkpoint inhibitor in the treatment of head and neck cancer. 84. A combination of an antibody as defined in any one of clauses 1 to 78, or a functional part, derivative and / or analogue thereof, and an immune checkpoint inhibitor as defined in any one of clauses 1 to 78, for use in the treatment of cancer in a subject in need thereof. 85. An immune checkpoint inhibitor as defined in any one of clauses 1 to 78 for the treatment of cancer in a subject in need thereof, wherein the immune checkpoint inhibitor is for simultaneous or sequential administration with an antibody, or functional part, derivative, and / or analogue thereof, as defined in any one of clauses 1 to 78. 86. An antibody, or a functional part, derivative and / or analogue thereof, as defined in any one of clauses 1 to 78, for use in the treatment of cancer in a subject who has been administered or will be administered an immune checkpoint inhibitor as defined in any one of clauses 1 to 78. 87. An immune checkpoint inhibitor as defined in any one of clauses 1 to 78 for use in the treatment of cancer in a subject to whom an antibody as defined in any one of clauses 1 to 78, or a functional part, derivative and / or analogue thereof, has been or will be administered. 88. A combination of an antibody as defined in any one of claims 1 to 78, or a functional part, derivative, and / or analogue thereof, instructions for use of the antibody, or a functional part, derivative, and / or analogue thereof in treating cancer in a subject, and instructions for use of an immune checkpoint inhibitor as defined in any one of claims 1 to 78 in treating cancer in a subject. 89. A combination of an immune checkpoint inhibitor as defined in any one of clauses 1 to 78, instructions for the use of said inhibitor in the treatment of cancer in a subject, and instructions for the use of an antibody as defined in any one of claims 1 to 78, or a functional part, derivative, and / or analogue thereof, in the treatment of cancer in a subject. 90. The combination according to clause 88 or 89, wherein the instructions for use include the amount of immune checkpoint inhibitor to be used and the amount of antibody, or functional part, derivative, and / or analogue thereof, the dosage interval, and the cancer to be treated. 91. A method for selecting a subject having head and neck cancer for treatment with an antibody comprising a variable domain that binds to the extracellular portion of EGFR according to any one of clauses 1 to 78, or a functional part, derivative, and / or analogue thereof, and an immune checkpoint inhibitor according to any one of clauses 1 to 78, the method comprising: a) determining a combined positive score (CPS) for PD-L1 expression in a sample obtained from the subject; and b) selecting the subject for said treatment if the sample has a CPS for PD-L1 expression of 1 or more, but optionally 100 or less. 92. A method for selecting a subject having head and neck cancer for treatment with an antibody comprising a variable domain that binds to the extracellular portion of EGFR as defined in any one of clauses 1 to 78, or a functional part, derivative, and / or analogue thereof, and an immune checkpoint inhibitor as defined in any one of clauses 1 to 78, the method comprising: a) determining a combined positive score (CPS) for PD-L1 expression in a sample obtained from the subject; and b) selecting the subject for said treatment if the sample has a CPS for PD-L1 expression of ≧1 and <20. 93. A method for selecting a subject having head and neck cancer for treatment with an antibody comprising a variable domain that binds to the extracellular portion of EGFR according to any one of clauses 1 to 78, or a functional part, derivative, and / or analogue thereof, and an immune checkpoint inhibitor according to any one of clauses 1 to 78, the method comprising: a) determining a combined positive score (CPS) for PD-L1 expression in a sample obtained from the subject; and b) selecting the subject for said treatment if the sample has a CPS for PD-L1 expression of ≧20 to 100. 94. The method of any one of clauses 91 to 93, wherein the method further comprises determining the p16 status in a sample from the subject. 95. The method of clause 94, wherein said sample from said subject is positive for p16 status. 96. The method of clause 94, wherein said sample from said subject is negative for p16 status. 97. A method for establishing whether a subject having head and neck cancer is likely to respond to treatment for head and neck cancer with an antibody comprising a variable domain that binds to the extracellular portion of EGFR as defined in any one of clauses 1 to 88, or a functional part, derivative, and / or analogue thereof, and an immune checkpoint inhibitor as defined in any one of clauses 1 to 78, the method comprising: a) determining a combined positive score (CPS) for PD-L1 expression in samples obtained from the subject; and b) selecting samples that show a CPS expression of PD-L1 of 1 or greater, optionally 100 or less, thereby establishing that the subject from whom the samples were derived is likely to respond to the treatment. 98. A method of establishing whether a subject having head and neck cancer is likely to respond to treatment with an antibody, or a functional part, derivative, and / or analogue thereof, as disclosed in any one of clauses 1 to 78, comprising a variable domain that binds to the extracellular portion of EGFR, and an immune checkpoint inhibitor as defined in any one of clauses 1 to 78, the method comprising: a) determining a combined positive score (CPS) for PD-L1 expression in samples obtained from the subject; and b) selecting samples that show a CPS expression of PD-L1 of ≧1 and <20, thereby establishing that the subject from whom the sample was derived is likely to respond to said treatment. 99. A method of establishing whether a subject having head and neck cancer is likely to respond to treatment with an antibody of any one of clauses 1 to 78, or a functional part, derivative and / or analogue thereof, comprising a variable domain that binds to the extracellular portion of EGFR and an immune checkpoint inhibitor that binds to any one of clauses 1 to 78, said method comprising: a) determining the combined positive score (CPS) for PD-L1 expression in samples obtained from the subject; and b) selecting samples that show a CPS expression of PD-L1 of ≧20 to 100, thereby establishing that the subject from whom said samples were derived is likely to respond to said treatment. 100. The method of any one of clauses 97 to 99, wherein the method further comprises determining the p16 status in a sample from the subject. 101. The method of clause 100, wherein said sample from said subject is positive for p16 status. 102. The method of clause 100, wherein said sample from said subject is negative for p16 status. 103. A method of classifying a subject having head and neck cancer based on a combined positive score for PD-L1 expression prior to treatment with an antibody comprising a variable domain that binds to the extracellular portion of EGFR as defined in any one of clauses 1 to 78, or a functional part, derivative, and / or analogue thereof, and an immune checkpoint inhibitor as defined in any one of clauses 1 to 78, the method comprising: a) determining a combined positive score for PD-L1 expression in a sample obtained from the subject; and b) classifying the subject from whom the sample was obtained as eligible for the treatment if the sample shows a CPS expression for PD-L1 of 1 or more, but optionally 100 or less. 104. A method of classifying a subject having head and neck cancer based on a combined positive score for PD-L1 expression prior to treatment with an antibody comprising a variable domain that binds to the extracellular portion of EGFR as defined in any one of clauses 1 to 78, or a functional part, derivative, and / or analogue thereof, and an immune checkpoint inhibitor as defined in any one of clauses 1 to 78, the method comprising: a) determining a combined positive score for PD-L1 expression in a sample obtained from the subject; and b) classifying the subject from whom the sample was obtained as eligible for the treatment if the sample shows a PD-L1 CPS expression of ≧1 to <20. 105. A method of classifying a subject having head and neck cancer based on a combined positive score for PD-L1 expression prior to treatment with an antibody comprising a variable domain that binds to the extracellular portion of EGFR according to any one of clauses 1 to 78, or a functional part, derivative, and / or analogue thereof, and an immune checkpoint inhibitor according to any one of clauses 1 to 78, the method comprising: a) determining a combined positive score for PD-L1 expression in a sample obtained from the subject; and b) classifying the subject from whom the sample was obtained as eligible for the treatment if the sample has a CPS for PD-L1 expression of ≧20-100. 106. The method of any one of clauses 103 to 105, wherein the method further comprises determining the p16 status in a sample from the subject. 107. The method of clause 106, wherein said sample from said subject is positive for p16 status. 108. The method of clause 106, wherein said sample from said subject is negative for p16 status. 109. The method of any one of clauses 91 to 108, wherein the CPS for PD-L1 expression is determined using IHC. 110. The method of any one of clauses 91 to 109, wherein the CPS for PD-L1 expression is determined by IHC using clone 22C3. 111. The method of any one of clauses 91 to 110, wherein the sample comprises or is a tumor sample or a cancer sample. 112. The method of any one of clauses 91 to 111, wherein the cancer comprises or is head and neck cancer. 113. The method of any one of clauses 91 to 112, wherein the cancer comprises or is head and neck squamous cell carcinoma (HNSCC). 114. The method of any one of clauses 91 to 113, further comprising administering to said subject selected or eligible for treatment, or said subject likely to respond to treatment, an effective amount of an antibody comprising a variable domain that binds to the extracellular portion of EGFR, or a functional part, derivative, and / or analogue thereof, and an effective amount of an immune checkpoint inhibitor. 115. The method of any one of clauses 91-114, wherein the subject or a sample thereof has a CPS score of ≧1-100 and is positive for p16 status. 116. The method of any one of clauses 91-114, wherein the subject or a sample thereof has a CPS score of ≧1-100 and is negative for p16 status. 117. The method of any one of clauses 91 to 114, wherein the subject or a sample thereof has a CPS score of ≧1 to <20 and is positive for p16 status. 118. The method of any one of clauses 91 to 114, wherein the subject or a sample thereof has a CPS score of ≧1 and <20 and is negative for p16 status. 119. The method of any one of clauses 91 to 114, wherein the subject or a sample thereof has a CPS score of ≧20 to 100 and is positive for p16 status. 120. The method of any one of clauses 91 to 114, wherein the subject or a sample thereof has a CPS score of ≧20 to 100 and is negative for p16 status. [Example]

[0311] As used herein, "MFXXXX," where X is independently a number from 0 to 9, refers to a Fab comprising a variable domain, and the VH has the amino acid sequence identified by the four digits shown in Figure 3. Unless otherwise indicated, the light chain variable region of the variable domain typically has the sequence shown in Figure 4b. In the examples, the light chain has the sequence shown in Figure 4a. "MFXXXX VH" refers to the amino acid sequence of the VH identified by the four digits. The MF further comprises a light chain constant region and a heavy chain constant region that typically interacts with the light chain constant region. The heavy chain VH / variable regions differ, and typically the CH3 regions also differ, with one heavy chain having a KK mutation in its CH3 domain and the other having a complementary DE mutation in its CH3 domain (see reference PCT / NL2013 / 050294 (published as WO2013 / 157954) and Figures 5d and 5e). In the examples, the bispecific antibody has an Fc tail with a KK / DE CH3 heterodimerization domain, a CH2 domain, and a CH1 domain as shown in Figure 5, a common light chain as shown in Figure 4a, and a VH designated by the MF number. For example, the bispecific antibody designated MF3755xMF5816 has a variable domain with the general sequence shown above, a VH with the sequence of MF3755, and a VH with the sequence of MF5816.

[0312] The amino acid sequences of various heavy chain variable regions (VH) are shown in Figure 3. Among other LGR5 and EGFR combinations shown in Figure 3, the bispecific antibody EGFR / LGR5, MF3755 x MF5816, which contains the heavy chain variable regions MF3755 and MF5816 and a common light chain and includes modifications for enhanced ADCC from afucosylation, has been shown to be effective in WO2017 / 069628.

[0313] Example 1. Generation of bispecific antibodies Bispecific antibodies have been generated by transient co-transfection of two plasmids encoding IgGs with different VH domains using a proprietary CH3 engineering technique that ensures efficient heterodimerization and bispecific antibody formation. A common light chain is also co-transfected into the same cell, either on the same plasmid or on a separate plasmid. Our applications (e.g., WO2013 / 157954 and WO2013 / 157953, incorporated herein by reference) disclose methods and means for producing bispecific antibodies from a single cell, thereby providing a means for favoring the formation of bispecific antibodies over the formation of monospecific antibodies. These methods can also be advantageously used in the present invention. Specifically, preferred mutations that produce essentially only bispecific full-length IgG molecules are amino acid substitutions at positions 351 and 366 in the first CH3 domain, e.g., L351K and T366K (EU numbering) ("KK variant" heavy chain), and amino acid substitutions at positions 351 and 368 in the second CH3 domain, e.g., L351D and L368E ("DE variant" heavy chain), or vice versa (see Figures 5d and 5e). It has been demonstrated that, in the indicated indications, negatively charged DE variant heavy chains and positively charged KK variant heavy chains preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE variant heavy chains (DE-DE homodimers) or KK variant heavy chains (KK-KK homodimers) rarely occurs due to strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0314] The VH gene of the variable domain binding to LGR5 described above was cloned into a vector encoding a positively charged CH3 domain. The VH gene of the variable domain binding to EGFR, such as that disclosed in WO2015 / 130172 (incorporated herein by reference), was cloned into a vector encoding a negatively charged CH3 domain. 293F Freestyle cells adapted for suspension growth were cultured in T125 flasks on a shaker plateau to a density of 3.0 x 10e6 cells / ml. Cells were seeded into each well of a 24-deep-well plate at a density of 0.3-0.5 x 10e6 viable cells / ml. Cells were transiently transfected with a mixture of two plasmids encoding different antibodies and cloned into a proprietary vector system. Seven days after transfection, the cell supernatant was collected and filtered through a 0.22 μM filter (Sartorius). The sterilized supernatant was stored at 4°C until antibody purification.

[0315] Example 2: IgG purification and quantification Purification was performed under sterile conditions on filter plates using Protein A affinity chromatography. First, the pH of the medium was adjusted to pH 8.0. Subsequently, the IgG-containing supernatant was incubated with Protein A Sepharose CL-4B beads (50% v / v) (Pierce) for 2 hours at 25°C on a shaking platform at 600 rpm. The beads were then collected by filtration. The beads were washed twice with PBS pH 7.4. Bound IgG was then eluted with 0.1 M citrate buffer at pH 3.0, and the eluate was immediately neutralized with Tris pH 8.0. Buffer exchange was performed by centrifugation using a Multiscreen Ultracel 10 multiplate (Millipore). Samples were finally collected in PBS pH 7.4. IgG concentration was measured using an Octet. Protein samples were stored at 4°C. To determine the amount of purified IgG, the concentration of the antibody was determined by Octet analysis using a Protein A biosensor (Forte-Bio, according to the supplier's recommendations) using total human IgG (Sigma Aldrich, catalog no. I4506) as a standard.

[0316] The following bispecific antibodies are suitable for use in this example and in the methods of the invention: MF3370xMF5790, MF3370x5803, MF3370x5805, MF3370x5808, MF3370x5809, MF3370x5814, MF3370x5816, MF3370x5817, MF3370x5818, MF3755xMF5790, MF3755x5803, MF3755x5805, MF3755x5808, MF3755x5809, MF3755x5814, MF3755x5816, MF3755x5817, MF3755x5818, F4280×MF5790, MF4280×5803, MF4280×5805, MF4280×5808, MF4280×5809, MF4280×5814, MF4280×5816, MF4280×5817, MF4280×5818, MF4289×MF5790, MF4289×5803, MF4289×5805, MF4289×5808, MF4289×5809, MF4289×5814, MF4289×5816, MF4289×5817, and MF4289×5818. Each bispecific antibody comprises two VHs designated by MF numbers capable of binding to EGFR and LGR5, respectively, and further comprises a KK / DE CH3 heterodimerization domain shown in SEQ ID NO: 117 (FIG. 5d) and SEQ ID NO: 118 (FIG. 5e), respectively, a CH2 domain shown in SEQ ID NO: 116 (FIG. 5c), a hinge region shown in SEQ ID NO: 115, and a CH1 domain shown in SEQ ID NO: 114 (FIG. 5a), and an Fc tail with a common light chain shown in SEQ ID NO: 107 (FIG. 4).

[0317] Example 3: Dose expansion with petosemutamab and pembrolizumab in patients with HNSCC The combination of petosemutamab and pembrolizumab will be explored first in patients with HNSCC. Patients with HNSCC who have received first-line systemic treatment for recurrent / metastatic disease are eligible. The combination will begin with full doses of both agents.

[0318] Study design A Phase 1, open-label, multicenter study was conducted with an initial dose-escalation portion to determine the recommended Phase 2 dose (RP2D) of petosemutamab, an anti-EGFRxanti-LGR5 bispecific antibody, for solid tumors in patients with mCRC, starting at a flat dose of 5 mg. With the RP2D established at 1500 mg Q2W, the antibody will be further evaluated in an expansion portion of the study, including patients diagnosed with head and neck cancer, including squamous cell carcinoma of the head and neck (SCCHN). The antibody's safety, PK, immunogenicity, and preliminary antitumor activity will be characterized in all patients, and biomarker analyses, including EGFR and LGR5 status, will be performed.

[0319] Inclusion criteria. 1. Signed an Informed Consent Form (ICF) before the start of any study procedures. 2. Age ≥ 18 years at the time of signing the informed consent. 3. Histologically or cytologically confirmed solid HNSCC tumors with evidence of metastatic or locally advanced disease not amenable to standard therapy with curative intent: Expansion cohort: Patients with locally advanced unresectable or metastatic disease for the following indications: First-line HNSCC: Patients are eligible to receive pembrolizumab as first-line monotherapy in tumors expressing PD-L1, CPS ≥ 1, as determined by an FDA-approved test in the United States or an equivalent test approved in other countries. Patients should not have received prior systemic therapy in the recurrent or metastatic setting, although prior systemic therapy as part of multimodal treatment for locally advanced disease is permitted if it was completed ≥ 6 months before signing the ICF or if progressive disease was completed ≥ 6 months after the last platinum-containing therapy dose. Eligible HNSCC primary tumor locations are the oropharynx, oral cavity, hypopharynx, and larynx. Prior treatment with anti-PD-L1 or anti-EGFR therapy is not permitted. 4. Documentation of p16 status (positive or negative) by local laboratory IHC for patients with primary oropharyngeal cancer. 5. A baseline new tumor specimen (formalin-fixed, paraffin-embedded [FFPE]) from a metastatic or primary site. If the patient has a tumor specimen as an FFPE block with sufficient material (at least 20 slides with >20% tumor content) and has not received further anti-cancer treatment since specimen collection, a new tumor biopsy at baseline is not necessary. Archival FFPE slides are not acceptable. Primary tumor material from HNSCC is acceptable only if the patient has not been treated with anti-EGFR or anti-human epidermal growth factor receptor (HER)-2 therapy. 6. Suitable for biopsy 7. Measurable disease as defined by RECIST version 1.1 by radiological methods Eastern Cooperative Oncology Group (ECOG) performance status of 8.0 or 1 9. Researchers say life expectancy is ≥ 12 weeks 10. Left ventricular ejection fraction (LVEF) ≥ 50% by echocardiogram (ECHO) or multiple gated acquisition scan (MUGA) 11. Proper organ function: ANC≧1.5×10 9 / L Hemoglobin ≥ 9g / dL Platelets ≥ 100 × 109 / L Corrected total serum calcium within normal range Serum magnesium within normal range (or corrected with supplements) Alanine aminotransferase (ALT), aspartate aminotransferase (AST) ≤ 2.5 x upper limit of normal (ULN), total bilirubin ≤ 1.5 x ULN (except in cases due to known Gilbert syndrome, which is excluded if total bilirubin > 3.0 x ULN or direct bilirubin > 1.5 x ULN). In the case of liver disease, total bilirubin ≤ 3.0 x ULN or direct bilirubin ≤ 1.5 x ULN is acceptable, except in cases due to known Gilbert syndrome, or total bilirubin < 3 mg / dL is acceptable. ALT / AST ≤ 5 x ULN and total bilirubin ≤ 2 x ULN are acceptable, except in cases due to hepatocellular carcinoma (Child-Pugh class A). Serum creatinine ≤ 1.5 × ULN or creatinine clearance ≥ 60 mL / min was calculated according to the Cockroft and Gault formula or the Modification of Diet in Renal Disease (MDRD) formula for patients aged > 65 years. Serum albumin ≥ 3g / dL International normalized ratio (INR) or prothrombin time (PT) ≤ 1.5 x ULN (within the intended therapeutic range of the anticoagulant used, unless the patient is receiving anticoagulant therapy) Activated partial thromboplastin time (APTT) or PTT ≤ 1.5 x ULN (within the therapeutic range of the intended anticoagulant used, unless the patient is receiving anticoagulant therapy) 12. Willingness to undergo human immunodeficiency virus (HIV) testing if not tested within 6 months (at the time of study enrollment). HIV-positive patients are eligible if they have a CD4+ count ≥ 300 / μL, an undetectable viral load, and are currently receiving highly active antiretroviral therapy (HAART).

[0320] Exclusion criteria 1. Central nervous system metastases that are untreated or symptomatic, or require radiation, surgery, or continuous steroid therapy to control symptoms within 14 days of study enrollment 2. Known leptomeningeal involvement 3. Participation in another clinical trial or treatment with any investigational drug within 4 weeks prior to study enrollment 4. Any systemic anti-cancer therapy within 4 weeks or 5 half-lives of the first dose of study treatment, whichever is longer. For cytotoxic agents with significant delayed toxicity (e.g., mitomycin C, nitrosoureas) or anti-cancer immunotherapies, a 6-week washout period is required. 5. Immunosuppressant medication (e.g., methotrexate, cyclophosphamide) requirements 6. Major surgery or radiation therapy within 3 weeks of the first dose of study treatment. Patients who have received prior radiation therapy to ≥ 25% of the bone marrow, regardless of when it was received, are not eligible. 7. Persistent grade > 1 clinically significant toxicity (excluding alopecia) related to prior antineoplastic therapy; stable sensory neuropathy ≤ grade 2 according to the National Cancer Institute's Common Terminology Criteria for Adverse Events (NCI-CTCAE) v4.03 or v5.0 is acceptable. 8. History of hypersensitivity reaction or any toxicity due to any human protein or excipient, such as from pembrolizumab or petosemutamab, that justified permanent discontinuation of these agents. 9. Uncontrolled hypertension (systolic BP > 150mmHg and / or diastolic BP > 100mmHg) with appropriate treatment or unstable angina. 10. History of Class II-IV New York Heart Association (NYHA) congestive heart failure or severe cardiac arrhythmia requiring treatment (excluding atrial fibrillation and paroxysmal supraventricular tachycardia) 11. History of myocardial infarction within 6 months of study enrollment 12. History of previous malignancies, except for cervical intraepithelial neoplasia or non-melanoma skin cancer, or curatively treated cancers that have been free of evidence of disease for at least 3 years and are considered at low risk of recurrence 13. Current respiratory distress of any origin at rest or other illness requiring continuous oxygen therapy 14. Patients with a history of interstitial lung disease (ILD) (e.g., pneumonia or pulmonary fibrosis) or evidence of ILD on baseline chest computed tomography (CT) scan 15. Current serious illness or psychiatric disorder, including but not limited to uncontrolled active infection, clinically significant pulmonary, metabolic, or psychiatric disease. 16.Patients with the following infections: Active hepatitis B surface antigen infection (HBsAg positive) not receiving antiviral treatment. Patients with active hepatitis B (HBsAg positive) must be receiving antiviral treatment with lamivudine, tenofovir, entecavir, or other antiviral agents starting ≥7 days before initiation of study treatment. Patients with prodromal symptoms of hepatitis B (anti-HBc positive, HBsAg and hepatitis B virus [HBV]-DNA negative) are eligible. Positive test for hepatitis C virus (HCV) ribonucleic acid (RNA). Note: Patients whose HCV infection has spontaneously cleared (i.e., positive HCV antibodies without detectable HCV-RNA) or who have achieved a sustained response after antiviral treatment and demonstrate the absence of detectable HCV RNA for ≥ 6 months (using IFN-free regimens) or ≥ 12 months (using IFN-based regimens) after discontinuation of antiviral treatment are eligible. 17. Patients with current cirrhosis status of Child-Pugh class B or C, known fibrolamellar HCC, sarcomatoid HCC, or mixed cholangiocarcinoma and HCC 18. Pregnant or lactating women and patients of childbearing potential must use highly effective methods of contraception before study enrollment, during study participation, and for 6 months after the last dose of petosemutamab. 19. Have a diagnosis of immunodeficiency or have received systemic steroid therapy or any form of immunosuppressive therapy within 7 days prior to dose 1. Use of corticosteroids as premedication for protocol-specified allergic reactions or IRR is permitted. 20. Have an active autoimmune disease that has required systemic immunosuppressive treatment within the past 2 years; replacement therapy (e.g., thyroxine, insulin, or physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency) is not considered immunosuppressive treatment. 21. Have received an allogeneic tissue / solid organ transplant 22. Patient has a primary tumor site (any histology) in the nasopharynx.

[0321] Dose expansion In the expansion portion, petosemutamab will be administered at the RP2D to patients with head and neck cancer, specifically SCCHN. The RP2D is defined as 1500 mg Q2W, and additional patients will be treated at this dose and schedule to further characterize the antibody's safety, tolerability, PK, and immunogenicity, and to conduct preliminary evaluations of antitumor activity and biomarker assessments. Alternatively, petosemutamab will be used at a flat dose of 1100 mg Q2W or dosing that achieves at least 95% or at least 99% human receptor occupancy for both EGFR and LGR5.

[0322] Antibody therapy in patients with head and neck cancer, particularly SCCHN, is explored for antitumor activity. The overall safety of the drug is also questioned.

[0323] Investigational Therapies and Regimen Petosemutamab, an anti-EGFR x anti-LGR5 bispecific antibody, is formulated as a clear liquid solution for IV infusion. IV infusions are administered every 2 weeks using standard infusion procedures, with a starting dose of 5 mg (flat dose) and a recommended phase 2 dose of 1500 mg (flat dose). Dose escalation was stopped after reaching the RP2D. Infusions must be administered over a minimum of 4 hours during Cycle 1. Subsequent infusions after Cycle 1 may be shortened to 2 hours at the investigator's discretion and in the absence of an IRR. A cycle is considered 4 weeks long.

[0324] Pembrolizumab administration Pembrolizumab dosing is 400 mg Q6W, but can also be 200 mg Q3W. Administration is by 30-minute intravenous infusion and / or according to label instructions in the country of administration. Injections are a 100 mg / 4 mL (25 mg / mL) solution in a single-dose vial.

[0325] Premedication During Cycle 1, all infusions will be administered over a period of at least 4 hours according to the following premedication regimen: 24 hours before the start of the infusion, 8 mg of dexamethasone PO will be administered 1 hour before the start of the infusion, and each patient will receive dexamethasone 20 mg IV, dexchlorpheniramine 5 mg IV or diphenhydramine 50 mg PO or chlorpheniramine 10 mg IV, ranitidine 50 mg IV or 150 mg PO, and paracetamol 1 g IV or 650 mg PO.

[0326] Treatment duration Study treatment will be administered until progressive disease (per RECIST 1.1), unacceptable toxicity, consent withdrawal, patient noncompliance, investigator decision (e.g., clinical deterioration), or antibody discontinuation for more than 6 consecutive weeks. Patients will be followed for safety for at least 30 days after the last antibody infusion and until resolution or stabilization of all associated toxicities, as well as for disease progression and survival status for 12 months.

[0327] Efficacy evaluation Tumor assessment will be based on CT / MRI with contrast according to RECIST 1.1 (Eisenhauer et al., 2009 Eur J Cancer 45:228-247) every 8 weeks after treatment initiation. Objective response must be confirmed at least 4 weeks after initial observation. Bone scans will be performed as clinically indicated for patients with bone metastases at baseline or suspected study lesions. Circulating blood tumor markers, including carcinoembryonic antigen (CEA), will be assessed at screening and on day 1 of each cycle.

[0328] Concomitant medications Key permitted medications are as follows: Paracetamol / acetaminophen, antihistamines, and corticosteroids are permitted as premedication treatment regimens but may also be administered in case of IRR, hypersensitivity, or allergic reaction, according to standard local clinical practice. All medications necessary for patient safety and well-being and not expected to interfere with the evaluation of study medication may be given at the investigator's discretion. Concurrent radiation therapy during this study for symptom control without evidence of progression. Major prohibited medications include: concomitant chronic oral corticosteroids (>10 mg / day prednisone equivalent), tumor necrosis factor (TNF) alpha inhibitors, anti-T cell antibodies, or other immunosuppressants; any investigational drug or other anticancer therapy within 4 weeks (or 5 half-lives, if known), whichever is longer, prior to administration of the first dose of study treatment; a 6-week washout period is required for cytotoxic agents with significant delayed toxicity (e.g., mitomycin C, nitrosoureas) or anticancer immunotherapies; major surgery or radiation therapy within 3 weeks prior to the first dose of study treatment or prior radiation therapy to ≥ 25% of the bone marrow.

[0329] Example 4. CPS Scoring in HNSCC Cancer Patients Prior to initiating combination therapy, under the clinical trial protocol, patients with HNSCC are required to undergo prescreening to detect tumors expressing PD-L1. PD-L1 testing must be performed in a CLIA-certified laboratory (or equivalent) using an FDA-approved test in the United States or an equivalent approved test in other countries. To be eligible for inclusion, patients with HNSCC must have a histological CPS of ≥ 1, defined as the number of PDL1-positive tumor cells, lymphocytes, and macrophages divided by the total number of tumor cells multiplied by 100. If local testing is not available, testing will be performed at a central laboratory qualified to perform such testing.

[0330] Reagent preparation The following reagents must be prepared prior to staining: EnVision FLEX Target Retrieval Solution, Low pH (50x). Prepare Low pH (50x) 1:50 by diluting a sufficient amount of 1x target retrieval solution with distilled or deionized water (reagent-quality water). The pH of the 1x target retrieval solution should be 6.1 ± 0.2. A 1x target retrieval solution pH below 5.9 may produce erroneous results. One 30 mL bottle of Low pH (50x) target retrieval solution diluted 1:50 provides 1.5 L of 1x reagent, enough to fill one PT Link tank for processing up to 24 slides per use. Discard the 1x target retrieval solution after three uses and do not use it for five days after dilution.

[0331] EnVision FLEX Wash Buffer (20x). Prepare a sufficient amount of wash buffer for the wash step by diluting the wash buffer (20x) 1:20 using distilled or deionized water (reagent-quality water). Store any unused 1x solution at 2-8°C for no more than one month. Discard the buffer if it appears cloudy.

[0332] DAB+ Substrate-Chromogen Solution. This solution must be mixed thoroughly before use. Any precipitate that forms in the solution will not affect staining quality. To prepare the DAB+ Substrate-Chromogen solution, add 1 drop of liquid DAB+ Chromogen per mL of DAB+ Substrate Buffer and mix. The prepared substrate-chromogen is stable for 5 days when stored in the dark at 2-8°C.

[0333] Specimen preparation Tissue specimens must be processed to preserve the tissue for IHC staining. Standard tissue processing methods should be used for all specimens. Paraffin-embedded (FFPE) specimens are preferred. Alternative fixatives have not been validated and may give erroneous results. Fixation in 10% neutral buffered formalin (NBF) for 12 to 72 hours is recommended. Fixation times of ≤3 hours should be avoided. Specimens should be blocked at 3 or 4 mm thickness, fixed in formalin, dehydrated, cleared through a series of alcohols and xylene, and subsequently infiltrated with molten paraffin. Paraffin temperatures should not exceed 60°C. NSCLC FFPE tissue blocks older than 5 years may result in loss of PD-L1 immunoreactivity.

[0334] The tissue specimens should be cut into 4-5 μm sections. After sectioning, the tissues should be mounted on Dako FLEX IHC microscope slides (code K8020) or Superfrost Plus slides and then placed in an oven at 58±2°C for 1 hour.

[0335] Recommendations for storage of cut sections. To maintain antigenicity, tissue sections, once mounted on slides, should be kept in the dark at 2-8°C (preferred), or at room temperature up to 25°C. Slide storage and handling conditions should not exceed 25°C at any time after mounting to ensure tissue integrity and antigenicity. HNSCC sections should be stained within 6 months if stored at 2-8°C (preferred), and within 4 months if stored at 25°C.

[0336] Staining procedure for the Link 48 solution automatic stainer. All reagents must be equilibrated to room temperature (20-25°C) prior to immunostaining. Similarly, all incubations must be performed at room temperature. Tissue sections must not be allowed to dry out during the staining procedure; dried tissue sections may exhibit increased nonspecific staining. All steps and incubation times required for staining are pre-programmed into the Dako Link software.

[0337] Staining protocol The PD-L1 IHC 22C3 pharmDx staining protocol is selected from the Dako Link drop-down menu options. All steps and incubation times required for staining are pre-programmed into the Link 48 automated stainer. Step 1: Deparaffinization, rehydration, and target retrieval (3-in-1) procedure Preheat and cool the PT Link (code PT100 / PT101 / PT200) to 65°C. Set the heat to 97°C for 20 minutes. Fill the PT Link tank with 1.5 L of target retrieval solution, low pH, 1x working solution, to cover the tissue sections. Preheat the target retrieval solution to 65°C. Immerse the autostainer racks containing the mounted FFPE tissue sections in the preheated low pH target retrieval solution (1x working solution) in the PT Link tank. Incubate at 97°C for 20 minutes. Once the target retrieval incubation is complete and the temperature has cooled to 65°C, remove each autostainer slide rack with slides from the PT Link tank and immediately place the autostainer rack with slides in a tank (e.g., PT Link Rinse Station, code PT109) containing diluted room temperature wash buffer (code K8007). Incubate the slides in the diluted room temperature wash buffer for 5 minutes. Step 2: Staining Procedure. After the deparaffinization, rehydration, and target retrieval (3-in-1) procedure, the automated stainer rack with slides is placed on the automated stainer link 48. The instrument executes the staining process by applying the appropriate reagents, monitoring incubation times, and rinsing the slides between reagents. Reagent times are pre-programmed into the Dako Link software. Step 3: Counterstaining. Slides must be counterstained with hematoxylin (code K8008) for 5 minutes. The hematoxylin incubation time is pre-programmed into the protocol. Step 4: Mounting. A non-aqueous permanent mounting medium is required.

[0338] quality control The reagents in PD-L1 IHC22C3 pharmDx are quality controlled by immunohistochemistry using targeted retrieval and staining procedures. Quality controls should be included with each staining run. These quality controls include: H&E-stained patient tissue specimens, laboratory-provided positive and negative control tissues, and Dako-provided control cell line slides.

[0339] Assay Validation. Prior to the first use of a staining system in a diagnostic procedure, the user should validate the performance of the assay by testing it on a set of laboratory-provided tissues with known IHC performance characteristics representing known positive and negative tissues. These quality control procedures should be repeated with each new antibody lot or whenever there are changes in the assay parameters.

[0340] Scoring interpretation. All viable tumor cells throughout the tissue section must be evaluated and included in the PD-L1 expression assessment. PD-L1 expression is determined by CPS, which is the number of PD-L1-stained cells (tumor cells, lymphocytes, and macrophages) divided by the total number of viable tumor cells and multiplied by 100. Distinguishing between viable tumor cells, lymphocytes, and macrophages is essential for accurate denominator estimation. While the calculation may result in a score greater than 100, the maximum score is defined as a CPS of 100. Slide evaluation should be performed by a pathologist using a light microscope. For evaluation of immunohistochemical staining, a 10-20x magnification objective is appropriate. For determination of PD-L1 expression, a 20x magnification objective is required. By definition, PD-L1-stained cells are: Tumor cells with confirmatory partial or complete linear membrane staining (at any intensity) that is recognized as distinct from cytoplasmic staining, and Lymphocytes and macrophages (mononuclear inflammatory cells, MIC) within the tumor nest and / or adjacent supportive stroma with confirmatory membrane and / or cytoplasmic staining (of any intensity). MIC should be directly related to the response to the tumor. For each staining run, slides should be examined in the order presented in Table 2 to determine the adequacy of the staining run and to allow for evaluation of the staining of the sample tissue. When evaluating PD-L1 expression, examine patient specimens stained with PD-L1 and the negative control reagent from PD-L1 IHC22C3 pharmDx. Specimens stained with the negative control reagent should have 0 specific staining and ≦1+ nonspecific staining.

[0341] HNSCC-CPS Interpretation. At least 100 viable tumor cells must be present on the PD-L1 stained slide for the specimen to be considered sufficient for PD-L1 assessment. The CPS denominator includes all viable invasive tumor cells (PD-L1 stained and unstained). All immune cells, benign cells, necrotic or nonviable tumor cells, carcinoma in situ, stromal cells (including fibroblasts), and necrotic cells and / or cellular debris are excluded. Table 1 provides details of the tissue elements included and excluded from the CPS numerator in HNSCC. [Table 1] [Table 2-1] [Table 2-2] [Table 2-3]

[0342] Example 5. EGFR Scoring via IHC The EGFR pharmDx™ assay is a qualitative immunohistochemistry (IHC) kit system for identifying epidermal growth factor receptor (EGFR) expression in routinely fixed normal and tumor tissues for histological evaluation. EGFR pharmDx specifically detects EGFR (HER1) protein in EGFR-expressing cells.

[0343] The EGFR pharmDx™ assay uses an EGFR antibody, clone 2-18C9 (2-18C9), to detect EGFR protein. Clone 2-18C9 has been tested for reactivity with cell lines expressing EGFR, HER2, HER3, and HER4. In Western blots of SKBR3 and A431 cell lysates, 2-18C9 recognized a 170 kD band consistent with the known molecular weight of EGFR. Clone 2-18C9 has also been found to recognize the EGFRvIII (145 kD) form of the receptor in immunohistochemistry, flow cytometry, and Western blotting of EGFRvIII-transfected cell lines. In Western blotting experiments, 2-18C9 was unreactive with HER2-positive CAMA-1 cell lysates, HER3-transformed E. coli BL-21 protein extracts, and CHO-HER4-transfected cell lysates. Furthermore, Chinese hamster ovary (CHO) transfectants expressing myc (vector tag), alone or coexpressing one of the HER family members, were grown in formalin-fixed, paraffin-embedded chamber slides and stained with anti-myc and 2-18C9. The myc antibody stained all five CHO transfectants, whereas 2-18C9 stained only the HER1-transfected CHO cells.

[0344] EGFR scoring is performed using the Dako EGFR pharmDx™ user protocol according to the manufacturer's instructions and recommendations, see the World Wide Web at agilent.com / cs / library / usermanuals / public / 08052_egfr_pharmdx_interpretation_manual.pdf.

[0345] Specimen preparation Biopsy specimens were processed to preserve 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 (acetate formalin alcohol), Richard-Allen Scientific Pen-fix, and Bouin's fixative.

[0346] Paraffin-embedded sections Routinely processed, paraffin-embedded tissue is suitable for use. Specimens from biopsies should be cut into blocks 3 or 4 mm thick and fixed in a fixative for the appropriate period. The tissue is then dehydrated and cleared through a series of alcohols and xylene, followed by infiltration with molten paraffin. The paraffin temperature should not exceed 60°C. When stored in a cool place (15–25°C), properly fixed and embedded tissue blocks expressing EGFR protein can be kept indefinitely before sectioning and slide mounting. Tissue specimens must be cut into 3-5 μm sections. After sectioning, the tissue must be mounted on slides and placed on a drying rack. The following slides are recommended: Fisher's SuperFrost Plus, Dako's Silanized (code S3003), and charged or poly-L-lysine-coated slides. The slide rack must be tapped on an absorbent towel to remove any moisture trapped under the paraffin and on the glass, and then allowed to dry at room temperature for 1 hour. The slide rack must then be placed in an incubator at 56-60°C for 1 hour. After removal from the incubator, any excess water remaining on the slides must be removed by tapping the slides on a towel and drying in the incubator for an additional hour. After removal from the incubator, the slides must be kept at room temperature until they have cooled and the paraffin has hardened. To maintain 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 kept at room temperature (20-25°C).

[0347] The slides required for EGFR assessment and verification of tumor presence must be prepared simultaneously.

[0348] At least five slides are recommended: one slide for tumor presence, two slides for EGFR protein assessment (one slide for the primary antibody and one slide for the negative control reagent), and two slides for backup.

[0349] Reagent preparation Prepare the following reagents prior to staining: Wash Buffer: For the wash steps, prepare a sufficient amount of wash buffer by diluting it 1:10 using distilled or deionized water (reagent-quality water). Discard the buffer if it appears cloudy. Substrate-Chromogen Solution (DAB+): This solution must be mixed thoroughly before use. Any precipitate that occurs in the solution will not affect staining quality. To prepare the DAB+ Substrate-Chromogen Solution, add 11 drops of liquid DAB+ Chromogen to one vial of DAB+ Substrate Buffer and mix. Discard any unused solution. Dilute according to the guidelines above. Adding excess liquid DAB+ Chromogen to DAB+ Substrate Buffer will result in a deterioration of the positive signal. Counterstain. Prepare aqueous ammonia for counterstain bluing, if needed. Ammonia water (0.037 mol / L) is prepared by mixing 2.5 (±0.5) mL of 15 mol / L (concentrated) ammonium hydroxide with 1 liter of reagent-quality water. Unused 0.037 mol / L aqueous ammonia may be stored in a tightly capped bottle at room temperature (20-25°C) for up to 12 months. Mounting media. For aqueous mounting, mounting media such as Dako's Faramount Aqueous Mounting Medium, Ready-to-use (code S3025) or Dako's Glycergel Mounting Medium (code C0563) are recommended. Liquefy the glycergel by warming to approximately 40 (±5)°C before use. Non-aqueous permanent mounting media such as Dako's Ultramount (code S1964) are also suitable.

[0350] Dako Autostainer Staining Procedure All reagents must be equilibrated to room temperature (20-25°C) prior to immunostaining. Similarly, all incubations must be performed at room temperature. Tissue sections must not be allowed to dry out during the staining procedure; dried tissue sections may exhibit increased nonspecific staining. Deparaffinization and rehydration. Prior to staining, tissue slides must be deparaffinized and rehydrated to remove the embedding medium. Avoid incomplete removal of paraffin; residual embedding medium can result in increased nonspecific staining. Step 1. Place slides in a xylene bath and incubate for 5 (±1) minutes. Change the bath and repeat once. Step 2. Tap off excess liquid and place slides in absolute ethanol for 3 (±1) minutes. Repeat once, changing the bath. Step 3. Tap off excess liquid and place slides in 95% ethanol for 3 (±1) minutes. Repeat once, changing the bath. Step 4. Tap off excess liquid and place slides in reagent-grade water for 5 (± 1) minutes. Step 5. Tap off excess liquid and place slides in wash buffer. Begin the staining procedure as outlined in the staining protocol.

[0351] The xylene and alcohol solutions should be changed after 40 slides. Toluene or xylene substitutes, such as Histoclear, can be used instead of xylene. EGFR pharmDx includes a pretreatment with a proteolytic enzyme digestion step. Tissue sections can sometimes be over-digested, causing destruction of cell membranes and overall tissue structure. The duration of the proteolysis step should be taken into consideration when performing the assay.

[0352] Post-fixation procedures 1. Deparaffinize sections and immerse in reagent-quality water. 2. Immerse slides in 10% neutral buffered formalin for 10 minutes. 3. Rinse the slides twice with deionized or distilled water. 4. Proceed with the EGFR pharmDx staining procedure.

[0353] Automated Staining Protocol Step 1. Select the desired protocol and program the staining run. Step 2. Set up the program using the automatic program and start the EGFR pharmDx program. Step 3. Place reagent vials into the DAKO automated stainer reagent rack according to the computer-generated reagent map. Step 4. Load the slides into the DAKO automated stainer according to the computer-generated slide map. Step 5. Start running. Step 6. Remove slides from the DAKO autostainer. Proceed to counterstaining and mounting. After the DAB + substrate-chromogen solution step, rinse the slides with reagent-quality water. (DAKO autostainer hardware versions 02 and 03 rinse the slides with reagent-quality water after the substrate-chromogen step. The 01 hardware version of the DAKO autostainer rinses the slides with buffer. Therefore, slides stained with the 01 hardware need to be rinsed with reagent-quality water after removal from the autostainer.)

[0354] Interpretation of staining procedures Slide evaluation should be performed by a pathologist using a light microscope. All evaluation should be performed in the tumor region of the specimen. For evaluation of immunocytochemical staining and scoring, a 10x or 20x objective is appropriate. Use intact cells to interpret staining results. Necrotic or degenerated cells often stain nonspecifically. Positive and negative cell lines are included with each EGFR pharmDx kit to validate the staining run each time the assay is performed. Appropriate staining of the control cell lines provides evidence that the EGFR pharmDx assay is functioning properly. Absence of membrane staining in the CAMA-1 control cell line (0) and moderate brown, complete or incomplete membrane staining in the HT-29 control cell line (2+) indicate a valid staining run. If the staining intensity of the positive control cell line is too weak or too strong, false-negative or false-positive results may be obtained and the test should be repeated. Reference images are available in the EGFR pharmDx Interpretation Guide. EGFR pharmDx primarily stains the cell membrane, demonstrating both complete and incomplete circumferential staining. The immunostaining pattern is often heterogeneous, showing variable staining intensity within a single neoplasm. Staining has also been observed in the cytoplasm and extracellular space. Cytoplasmic staining is common, but if significant cytoplasmic staining makes it difficult to distinguish from membrane staining and interpret the results, the test should be repeated. Tumors should be reported as EGFR-positive or EGFR-negative, using membrane staining as the assessable structure. Tumor cells are EGFR-positive if they have any membrane staining above background, regardless of whether it is completely circumferential. Tumors without membrane staining above background in any tumor cells are reported as EGFR-negative tumors. Depending on the length of incubation and the potency of the hematoxylin used, counterstaining will result in a light to dark blue coloration of the cell nuclei. Excessive or incomplete counterstaining can impair the interpretation of the results.

[0355] The staining intensity is established as follows: 3+ (strong staining): visible at high levels if necessary, at low levels of magnification, visible with a 5x objective; 2+ (moderate staining): visible at medium levels of magnification, at a 10x or 20x objective; 1+ (weak staining): visible at high magnification, only reliably with a 40x objective; 0 (no staining): no visible staining at high magnification. [Table 3]

[0356] EGFR H scoring The evaluation of membranous staining using IHC classifies samples into four staining intensity categories (0-3+). Note that only linear intercellular staining of tumor cells is considered positive, while complete and incomplete membranous staining is considered and recorded. Also, for the Histo-score calculation, all membranous staining is considered independent of completeness (complete and incomplete membranous staining).

[0357] H-scores are assigned using the following formula: [1 x (% cells with 1+ staining) + 2 x (% cells with 2+ staining) + 3 x (% cells with 3+ staining)], resulting in an H-score for EGFR between 0 and 300.

[0358] Example 6 The p16 status of the samples is determined using a commercially available test kit based on clone E6H4 (CINtec® Histology, Roche Diagnostics) and, depending on the required throughput and demand, using an automated slide preparation system such as BenchMark reader technology, such as BenchMark XT, BenchMark ULTRA, or BenchMark GX.

[0359] Samples are scored according to the CAP guidelines 2018 by reporting p16 IHC positivity if there is at least 70% nuclear and cytoplasmic expression with at least moderate to strong intensity. If results cannot be assessed due to external factors that interfere with interpretation, the test result is considered indeterminate. p16 testing can be performed on samples obtained from subjects with oropharyngeal cancer or on oropharyngeal cancer / tumor samples.

[0360] Example 7 A 66-year-old male patient diagnosed with incurable, progressive disease of head and neck cancer, specifically a form of squamous cell carcinoma with a primary tumor location in the larynx, was treated with petosemtamab 1500 mg Q2W and pembrolizumab 400 mg Q6W. The patient tested PD-L1+ with a CPS score of 1, as established using the pharmDx PD-L1 IHC kit, using clone 22C3 (Agilent).

[0361] Tumor assessment less than 8 weeks later using RECIST 1.1 criteria revealed a partial response (PR) with a 76% reduction in target lesions. The PR was confirmed after a second tumor assessment showing an 81% reduction in target lesions.

[0362] Example 8 A 66-year-old female patient diagnosed with untreatable, progressive disease of head and neck cancer, specifically a form of squamous cell carcinoma with a primary tumor location in the oral cavity, was treated with petosemutamab 1500 mg Q2W and pembrolizumab 400 mg Q6W. The patient tested positive for PD-L1 with a CPS score of >1 using the pharmDx PD-L1 IHC kit using clone 22C3 (Agilent).

[0363] Tumor assessment after 7 weeks using RECIST 1.1 criteria revealed a complete response (CR) with 100% shrinkage of the target lesion. The CR was confirmed after a second tumor assessment.

[0364] Example 9 A 63-year-old male patient diagnosed with head and neck cancer, specifically squamous cell carcinoma with a primary tumor of unknown location, was treated with petosemtamab 1500 mg Q2W and pembrolizumab 400 mg Q6W for progressive disease in the neck, lymph nodes, lung, and liver. The patient tested PD-L1+ with a CPS score of 43, as established using the pharmDx PD-L1 IHC kit, using clone 22C3 (Agilent).

[0365] Tumor assessment less than 8 weeks later using RECIST 1.1 criteria revealed a partial response (PR) with a 51% reduction in target lesions. The PR was confirmed after a second tumor assessment showing a 62% reduction in target lesions.

Claims

1. It includes a variable domain that binds to the extracellular portion of EGFR, which includes at least the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region as defined by Sequence ID No. 4, and A variable domain that binds to LGR5, comprising at least the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region as defined by Sequence ID No. 13, A bispecific antibody, or one containing its functional portion, A pharmaceutical composition for use in the treatment of head and neck cancer in human subjects, Both the variable domain that binds to EGFR and the variable domain that binds to LGR5 include the CDR1, CDR2, and CDR3 regions of the light chain variable region as defined by Sequence ID No.

108. A pharmaceutical composition wherein the treatment further comprises administering pembrolizumab.

2. A pharmaceutical composition comprising pembrolizumab for use in the treatment of head and neck cancer in human subjects, The aforementioned treatment, It includes a variable domain that binds to the extracellular portion of EGFR, which includes at least the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region as defined by Sequence ID No. 4, and A variable domain that binds to LGR5, comprising at least the CDR1, CDR2, and CDR3 sequences of the heavy chain variable region as defined by Sequence ID No. 13, The method further includes administering a bispecific antibody or a functional portion thereof. A pharmaceutical composition in which both the variable domain that binds to EGFR and the variable domain that binds to LGR5 include the CDR1, CDR2, and CDR3 regions of the light chain variable region defined by Sequence ID No.

108.

3. The pharmaceutical composition according to claim 1 or 2, wherein the cancer is squamous cell carcinoma (SCCHN) of the head and neck.

4. The pharmaceutical composition according to claim 1 or 2, wherein the cancer is a cancer of the pharynx, oral cavity, larynx, paranasal sinuses, nasal cavity, or salivary gland.

5. The pharmaceutical composition according to claim 4, wherein the pharyngeal cancer includes nasopharyngeal, oropharyngeal, and hypopharyngeal cancers.

6. The pharmaceutical composition according to claim 1 or 2, wherein the subject has not received prior anti-cancer treatment for the treatment of the cancer.

7. The heavy chain variable region defined by SEQ ID NO: 4 includes CDR1 containing the amino acid sequence of SEQ ID NO: 24, CDR2 containing the amino acid sequence of SEQ ID NO: 26, and CDR3 containing the amino acid sequence of SEQ ID NO: 28; The heavy chain variable region defined by SEQ ID NO: 13 includes CDR1 containing the amino acid sequence of SEQ ID NO: 87, CDR2 containing the amino acid sequence of SEQ ID NO: 89, and CDR3 containing the amino acid sequence of SEQ ID NO: 91; and The light chain variable region defined by SEQ ID NO: 108 includes CDR1 containing the amino acid sequence of SEQ ID NO: 111, CDR2 containing the amino acid sequence of SEQ ID NO: 112, and CDR3 containing the amino acid sequence of SEQ ID NO:

113. The pharmaceutical composition according to claim 1 or 2.

8. The heavy chain variable region contained in the variable domain that binds to the extracellular portion of EGFR contains the amino acid sequence defined by SEQ ID NO: 4, or the VH, having up to 15 amino acid modifications, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less, including insertions, deletions, substitutions, or combinations thereof, and The heavy chain variable region contained in the variable domain that binds to the extracellular portion of LGR5 contains an amino acid sequence defined by SEQ ID NO: 13, or an amino acid sequence containing up to 15, preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less, preferably 5, 4, 3, 2, or 1 or less amino acid modifications to the VH, including insertions, deletions, substitutions, or combinations thereof, and includes the amino acid sequence defined by SEQ ID NO:

13. The pharmaceutical composition according to claim 1 or 2.

9. The heavy chain variable region contained in the variable domain that binds to the extracellular portion of EGFR includes the amino acid sequence defined by Sequence ID No. 4, and The heavy chain variable region contained in the variable domain that binds to the extracellular portion of LGR5 includes the amino acid sequence defined by Sequence ID No.

13. The pharmaceutical composition according to claim 1 or 2.

10. Both the variable domain that binds to the extracellular portion of EGFR and the variable domain that binds to the extracellular portion of LGR5 include a light chain variable region as defined by Sequence ID No. 108, wherein the light chain variable region includes 0 to 10 amino acid insertions, deletions, substitutions, additions, or combinations thereof, and the amino acid insertions, deletions, and substitutions are not present in CDR1, CDR2, and CDR3 of the light chain variable region. The pharmaceutical composition according to claim 1 or 2.

11. The pharmaceutical composition according to claim 1 or 2, wherein the variable domain that binds to the extracellular portion of EGFR comprises an amino acid sequence defined by SEQ ID NO: 4, the variable domain that binds to the extracellular portion of LGR5 comprises an amino acid sequence defined by SEQ ID NO: 13, and the light chain variable region is defined by SEQ ID NO:

108.

12. The pharmaceutical composition according to claim 1 or 2, wherein the bispecific antibody is petosemtamab.

13. The pharmaceutical composition according to claim 12, wherein the treatment comprises administering petosemtamab to the subject at a dose of 1500 mg once every two weeks.

14. The pharmaceutical composition according to claim 1 or 2, wherein the treatment comprises administering to the subject a dose of 400 mg of pembrolizumab once every six weeks.

15. The pharmaceutical composition according to claim 12, wherein petosemtamab and pembrolizumab are administered sequentially.

16. The pharmaceutical composition according to claim 1 or 2, wherein the antibody, or a functional portion thereof, or pembrolizumab is provided intravenously to the subject.

17. The pharmaceutical composition according to claim 1 or 2, wherein the antibody is afucosylated.

18. The pharmaceutical composition according to claim 1 or 2, wherein the cancer is recurrent, unresectable, locally advanced, and / or metastatic cancer.

19. The pharmaceutical composition according to claim 1 or 2, wherein the dual-characteristic antibody is petosemtamab, and the treatment comprises i) administering petosemtamab to a subject at a dose of 1,500 mg once every two weeks, and ii) administering pembrolizumab to a subject at a dose of 400 mg once every six weeks, wherein the cancer is squamous cell carcinoma of the head and neck (SCCHN), and the subject has not received prior anti-cancer treatment for the treatment of the cancer.