Multispecific antibodies against CD40 and CD137 in combination with anti-PD1AB and chemotherapy

Abstract

The present invention relates to combination therapies using binding agents that bind to human CD40 and human CD137 in combination with checkpoint inhibitors that are inhibitors of the PD-1 / PD-L1 axis (particularly pembrolizumab) and chemotherapy to reduce or prevent the progression of head and neck squamous cell carcinoma (HNSCC) or to treat HNSCC.

Description

[Technical Field]

[0001] The present invention relates to combination therapies using binding agents that bind to human CD40 and human CD137 in combination with checkpoint inhibitors that are inhibitors of the PD-1 / PD-L1 axis (particularly pembrolizumab) and chemotherapy to reduce or prevent the progression of head and neck squamous cell carcinoma (HNSCC) or to treat HNSCC. [Background technology]

[0002] CD40 is a member of the tumor necrosis factor (TNF) receptor (TNFR) family and is known as a costimulatory protein found on various cell types. CD40 is constitutively expressed by antigen-presenting cells (APCs) such as dendritic cells (DCs), B cells, and macrophages. It can also be expressed by endothelial cells, platelets, smooth muscle cells, fibroblasts, and epithelial cells. Consistent with its widespread expression on normal cells, CD40 is also expressed on a variety of tumor cells.

[0003] antigen-specific CD4 + Presentation of peptide antigens in association with MHC class II molecules to T cells is mediated by CD4, together with costimulatory signals (from CD80 and / or CD86). + This results in T cell activation and upregulation of the DC licensing factors CD40 ligand (CD40L) and lymphotoxin-α1β2 (LTα1β2). Expression of CD40L and LTα1β2 on activated antigen-specific CD4+ T cells induces signaling through CD40 and the LTβ receptor (LTβR), which in turn induces DCs to express CD8 +It provides a license to induce T cell responses. CD40 signaling leads to the production of interleukin-12 (IL-12) and upregulation of CD70, CD86, 4-1BB ligand (4-1BBL), OX40 ligand (OX40L), and GITR ligand (GITRL), whereas LTβR signaling leads to the production of type I interferon (IFN). The signaling system that controls the activity of nuclear factor kappa B (NF-κB) responds to virtually all TNFR superfamily members. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) also contribute to these events. MHC class I-restricted peptide-mediated CD8 + T cell priming results in upregulation of CD27, 4-1BB, OX40, and glucocorticoid-inducible TNFR-related protein (GITR). + Stimulation of these receptors in T cells with their cognate TNF superfamily ligands, in combination with IL-12 and type I IFN, results in robust CD8 + T cell activation, proliferation and effector function, as well as CD8 +CD40 antibodies can exert various effects, including the death of CD40-expressing tumor cells by inducing antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or antibody-dependent cell-mediated phagocytosis (ADCP), as well as direct apoptosis or cell signaling to induce growth arrest, independent of CD40 expression on tumor cells via APC licensing to stimulate anti-cancer immune responses. Antibodies that bind to CD40 can initiate the priming of effector cytotoxic T lymphocytes (CTLs) via CD40 on APCs, induce the release of IL-2 by these cells, and indirectly activate NK cells. Antibodies that stimulate CD40 have been disclosed in the prior art, including the human IgG2 antibody CP-870,893 (WO03 / 040170), the humanized IgG1 antibody dacetuzumab (WO00 / 075348), and the chimeric IgG1 antibody Chi Lob7 / 4 (US2009 / 0074711). Additionally, a CD40 antagonist antibody, the human IgG1 antibody lucatumumab (WO02 / 028481), has been disclosed.

[0004] CD137 (4-1BB) is also a member of the TNFR family. +CD137 is a costimulatory molecule on CD4+ T cells, regulatory T cells (Tregs), natural killer T cells (NK(T) cells), B cells, and neutrophils. In T cells, CD137 is not constitutively expressed but is induced upon T cell receptor (TCR) activation, e.g., on tumor-infiltrating lymphocytes (TILs) (Gros et al., J. Clin Invest 2014;124(5):2246-59). Stimulation via its natural ligand 4-1BBL or agonist antibodies triggers signal transduction using TRAF-2 and TRAF-1 as adaptors. Early signaling by CD137 involves K-63 polyubiquitination, which ultimately leads to activation of the nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase pathways. Signaling leads to increased T cell costimulation, proliferation, cytokine production, maturation, and long-term CD8+ T cell survival. Agonistic antibodies against CD137 have been shown to promote anti-tumor control by T cells in various preclinical models (Murillo et al., Clin Cancer Res 2008;14(21):6895-906). Antibodies that stimulate CD137 can induce T cell survival and proliferation, thereby enhancing anti-tumor immune responses. Antibodies that stimulate CD137 have been disclosed in the prior art, including the human IgG4 antibody urelumab (AU2004279877) and the human IgG2 antibody utomilumab (Fisher et al., 2012, Cancer Immunol. Immunother. 61:1721-1733).

[0005] Westwood JA, et al., Leukemia Research 38 (2014), 948-954, discloses "Combination anti-CD137 and anti-CD40 antibody therapy in murine myc-driven hematological cancers." WO2018 / 011421 provides binding agents, such as bispecific antibodies, that bind to human CD40 and human CD137. Such bispecific antibodies crosslink CD40 on antigen-presenting cells (APCs) with 4-1BB on activated T cells, thereby inducing conditional stimulatory and costimulatory activity in both cell types, useful for the treatment of solid tumors.

[0006] PD-1, CTLA4, PD-L1, TIM-3, KIR, and LAG-3 are inhibitory checkpoint molecules that regulate the immune system and enable self-tolerance, making them ideal targets for cancer immunotherapy.

[0007] In tumor-draining lymph nodes and within the tumor microenvironment, 4-1BB is expressed by subsets of CD4+ and CD8+ T cells characterized by coexpression of multiple TCR-inducible molecules, including high levels of programmed cell death 1 (PD-1) (Gros et al., J. Clin Invest 2014;124(5):2246-59; Seifert et al., Cancers (Basel) 12; Simoni et al., Nature 557: 575-579). Upregulation of PD-1 on T cells may contribute to T cell exhaustion and reduce T cell activation upon binding to its ligand, programmed cell death 1 ligand 1 (PD-L1) (Yu et al., Eur J Pharmacol 881: 173240). PD-L1 expression is often upregulated by tumor cells, especially in inflamed tumors (Teng, et al., Cancer Res 75: 2139-2145). This allows tumor cells to provide inhibitory signals to activated T cells, allowing them to escape T cell-mediated cytotoxicity. Antibodies that block the PD-1 / PD-L1 inhibitory axis can restore T cell function (Boussiotis et al., N Engl J Med 375: 1767-1778; Chen et al., Nature 541: 321-330).

[0008] Head and neck squamous cell carcinoma (HNSCC) is a diagnosis made more than 600,000 times annually worldwide. In 2020, approximately 65,630 new cases of oral cavity, pharyngeal, and laryngeal cancer and an estimated 14,500 deaths are expected to occur in the United States over the same period (Clinical Practice Guidelines in Oncology, version 2, 2021). Tobacco use, alcohol use, and human papillomavirus (HPV) infection increase the risk of developing HNSCC. Patients with locally HPV-positive HNSCC have improved treatment outcomes compared with patients with HPV-negative disease. For patients with recurrent or metastatic HNSCC, pembrolizumab / platinum (cisplatin or carboplatin) / 5-FU and pembrolizumab monotherapy are recommended 1L regimens, but the median overall survival (mOS) is less than 15 months ( Clinical Practice Guidelines in Oncology, version 2, 2021 ). Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, HNSCC remains an area of ​​high unmet medical need, and further opportunities exist to improve outcomes with novel treatment approaches. [Means for solving the problem]

[0010] The inventors have surprisingly found that a combination of (i) stimulation with a binding agent that binds to human CD40 and human CD137; (ii) inhibition of the checkpoint PD-1 / PD-L1 axis (particularly using pembrolizumab); and (iii) chemotherapy using a combination comprising a platinum-based chemotherapy agent (particularly cisplatin or carboplatin) and 5-fluorouracil amplifies the immune response against HNSCC.

[0011] Accordingly, in a first aspect, the present disclosure provides a binding agent for use in a method for reducing or preventing the progression of, or treating, HNSCC in a subject, said method comprising administering to said subject (i) a binding agent, (ii) an inhibitor of the checkpoint PD-1 / PD-L1 axis (hereinafter also referred to as a PD-1 / PD-L1 checkpoint inhibitor), in particular pembrolizumab, and (iii) a chemotherapy combination comprising a platinum-based chemotherapy agent (in particular cisplatin or carboplatin) and 5-fluorouracil, wherein the binding agent comprises a first binding region that binds CD40 and a second binding region that binds CD137.

[0012] In a second aspect, the present disclosure provides a method for reducing or preventing the progression of or treating HNSCC in a subject, comprising administering to the subject (i) a binding agent, (ii) a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab), and (iii) a chemotherapy combination comprising a platinum-based chemotherapy agent (particularly cisplatin or carboplatin) and 5-fluorouracil, wherein the binding agent comprises a first binding region that binds CD40 and a second binding region that binds CD137.

[0013] In further aspects, the present disclosure provides kits comprising (i) a binding agent comprising a first binding region that binds to CD40 and a second binding region that binds to CD137, (ii) a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab), (iii) a platinum-based chemotherapeutic agent (particularly cisplatin and / or carboplatin), and (iv) 5-fluorouracil, as well as kits for use, e.g., in methods for reducing or preventing the progression of HNSCC or for treating HNSCC in a subject. [Brief explanation of the drawings]

[0014] [Figure 1]Figure 1 shows a schematic diagram of the expected mechanism of action of the CD40x4-1BB bispecific antibody. CD40 is expressed on antigen-presenting cells (APCs) as well as tumor cells. 4-1BB (CD137) is expressed on activated T cells. DuoBody-CD40x4-1BB (GEN1042 / BNT312) is a bispecific antibody that crosslinks CD40 on antigen-presenting cells (APCs) with 4-1BB on activated T cells, thereby conditionally stimulating both cell types. The CD40x4-1BB bispecific antibody can thereby enhance DC licensing, T cell clonal expansion, cytokine production, T cell survival, and T cell- and NK cell-mediated cytotoxicity. [Figure 2] Figure 2 shows an MC38 syngeneic tumor model established by subcutaneous implantation of 1 x 10 MC38 cells into hCD40xh4-1BB dKI C57BL / 6 mice. Once tumors reached a mean volume of 37 mm, mice were randomized and treated with a chemotherapy regimen consisting of GEN1042-mIgG2a (1 mg / kg, BIW x 3), anti-mouse PD-1 antibody (anti-mPD-1; 10 mg / kg, BIW x 3), carboplatin (20 mg / kg, BIW x 3), and 5-fluorouracil (5-FU, 25 mg / kg, Q3D x 5), either alone or in combination. A control group received both PBS and 0.9% saline (BIW x 3). (A) Data shown are median tumor volumes per treatment group (n = 10); data for animals that met termination criteria were carried forward. Arrows indicate days of treatment. (B) Data shown are tumor volumes for the different treatment groups on day 20, including the number of mice showing complete tumor regression (CR), with all groups still intact on the last day (vertical dotted line in A). Mann-Whitney analysis was used to compare tumor volumes between treatment groups (p<0.05 = *). [Figure 3A] Figure 3A shows the best change in target lesions in subjects with previously untreated recurrent or metastatic HNSCC treated with the chemotherapy agents pembrolizumab and GEN1042. Figure 3B shows the change in target lesions over time in the subjects. The data cutoff date was October 3, 2022. [Figure 3B] Same as above. [Figure 4A] Figure 4A shows the best change in target lesions in subjects with previously untreated recurrent or metastatic HNSCC treated with pembrolizumab and GEN1042. Figure 4B shows the change in target lesions over time in the subjects. The data cutoff date was October 7, 2022. [Figure 4B] Same as above. [Figure 5] Figure 5 shows an increase in pro-inflammatory IFNg in patient serum following administration of GEN1042+SoC. Circulating IFNg (interferon gamma) levels were measured in serum samples at baseline and at multiple time points after administration of GEN1042+SoC during cycles 1 and 2 (days 1, 3, 8, and 15), as well as before administration in cycle 3. Nine patients were analyzed and grouped by treatment. Because interim data limit maturation data at all time points, the maximum n available per regimen at any given time point was GEN1042+pembro [5] and GEN1042+chemo+pembro [4]. IFNg levels in serum samples were determined by Meso Scale Discovery (MSD) multiplex immunoassay. Abbreviations: IFN = interferon, 1042 = GEN1042, Chemo = 5FU + carbo / cisplatin, pembro = pembrolizumab, pg = picograms, mL = milliliters, SEM = standard error of mean (SEM), pre = pre-dose, SoC = standard of care. Study reference range: IFNg (pg / mL) < 11.81; clinical data cutoff = September 26, 2022. [Figure 6]Figure 6 shows the increase in TARC in the serum of patients receiving GEN1042+SoC. Circulating TARC levels were measured in serum samples at baseline and at multiple time points after administration of GEN1042+SoC during cycles 1 and 2 (days 1, 3, 8, and 15), as well as before administration in cycle 3. Seven patients were analyzed and grouped by treatment. Because interim data limit maturation data at all time points, the maximum n available per regimen at any given time point was GEN1042+pembro [3] and GEN1042+chemo+pembro [4]. TARC levels in serum samples were determined by Meso Scale Discovery (MSD) multiplex immunoassay. Abbreviations: TARC = thymus and activation-regulated chemokine, 1042 = GEN1042, Chemo = 5FU + carbo / cisplatin, pembro = pembrolizumab, pg = picograms, mL = milliliters, SEM = standard error of mean (SEM), pre = pre-dose, DC = dendritic cells, SoC = standard of care. Study reference range: TARC (pg / mL) < 513; clinical data cutoff = September 26, 2022. [Figure 7A]Figure 7 shows immune cell trafficking / marginalization after GEN1042+SoC administration. Peripheral blood immunophenotyping was performed on whole blood collected at baseline and multiple time points after GEN1042+SoC administration during cycles 1 and 2 (days 1, 3, 8, and 15), as well as before administration in cycle 3. Six patients were analyzed and grouped by treatment. Because interim data limit maturation data at all time points, the maximum n available per regimen at any given time point was GEN1042+pembro[3] and GEN1042+chemo+pembro[3]. Immune cell frequencies were assessed in whole blood samples by flow cytometry. Figure 7A shows the results for CD8+ T cells. Figure 7B shows the results for B cells. Abbreviations: 1042 = GEN1042, Chemo = 5FU+carbo / cisplatin, pembro = pembrolizumab, uL = microliter, SEM = standard error of mean, pre = pre-dose, abs = absolute count, SoC = standard of care. Clinical data cutoff = September 26, 2022. [Figure 7B] Same as above. [Figure 8A]Figure 8 shows T cell proliferation after administration of GEN1042+SoC. Peripheral blood immunophenotyping was performed at baseline and at multiple time points after administration of GEN1042+SoC during cycles 1 and 2 (days 1, 3, 8, and 15), as well as on whole blood collected prior to administration in cycle 3. Seven patients were analyzed and grouped by treatment. Because interim data limit maturation data at all time points, the maximum n available per regimen at any given time point was GEN1042+pembro [3] and GEN1042+chemo+pembro [4]. The frequency of proliferating (%Ki67) T cells was assessed in whole blood samples by flow cytometry. Figure 8A shows the results for proliferating (%Ki67) CD8+ T cells. Figure 8B shows the results for proliferating (%Ki67) effector memory CD8+ T cells (CD45RA-, CCR7-). Abbreviations: 1042 = GEN1042, Chemo = 5FU + carbo / cisplatin, pembro = pembrolizumab, uL = microliter, SEM = standard error of mean, pre = pre-dose, abs = absolute count, SoC = standard of care, Tem = T effector memory. Clinical data cutoff = September 26, 2022. [Figure 8B] Same as above. [Figure 9A]Figure 9 shows T cell activation after administration of GEN1042 + SoC. Peripheral blood immunophenotyping was performed at baseline and at multiple time points after administration of GEN1042 + SoC during cycles 1 and 2 (days 1, 3, 8, and 15), as well as on whole blood collected prior to administration in cycle 3. Seven patients were analyzed and grouped by treatment. Because interim data limit maturation data at all time points, the maximum n available per regimen at any given time point was GEN1042 + pembro [3] and GEN1042 + chemo + pembro [4]. The frequency of activated (%4-1BB) CD8+ T cells was assessed in whole blood samples by flow cytometry. Figure 9A shows the results for CD8+ T cells. Figure 9B shows the results specifically for effector memory CD8+ T cells (CD45RA-, CCR7-). Abbreviations: 1042 = GEN1042, Chemo = 5FU + carbo / cisplatin, pembro = pembrolizumab, uL = microliter, SEM = standard error of mean, pre = pre-dose, abs = absolute count, SoC = standard of care, Tem = T effector memory. Clinical data cutoff = September 26, 2022. [Figure 9B] Same as above. [Figure 10]Figure 10 shows B cell activation after GEN1042+SoC administration. Peripheral blood immunophenotyping was performed at baseline and at multiple time points after GEN1042+SoC administration during cycles 1 and 2 (days 1, 3, 8, and 15), as well as on whole blood collected prior to administration in cycle 3. Six patients were analyzed and grouped by treatment. Because interim data limit maturation data at all time points, the maximum n available per regimen at any given time point was 1042+pembro[3] and 1042+chemo+pembro[3]. The frequency of activated (%4-1BB+) B cells was assessed in whole blood samples by flow cytometry. Abbreviations: 1042 = GEN1042, Chemo = 5FU+carbo / cisplatin, pembro = pembrolizumab, uL = microliter, SEM = standard error of mean, pre = pre-dose, abs = absolute count, SoC = standard of care. Clinical data cutoff = September 26, 2022. DETAILED DESCRIPTION OF THE INVENTION

[0015] Table 1 - Sequences [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0016] SEQ ID NO: 63 HCDR1 (MAB-19-0618) Kabat and IMGT consensus SEQ ID NO: 64HCDR1 (MAB-19-0618) Kabat SEQ ID NO: 45HCDR1 (MAB-19-0618) IMGT SEQ ID NO: 65 HCDR2 (MAB-19-0618) Kabat and IMGT consensus (=IMGT) SEQ ID NO: 66HCDR2 (MAB-19-0618) Kabat SEQ ID NO: 47 HCDR3 (MAB-19-0618) Kabat and IMGT consensus (=Kabat) SEQ ID NO: 67HCDR3 (MAB-19-0618) IMGT SEQ ID NO: 48 LCDR1 (MAB-19-0618) Kabat and IMGT consensus (=IMGT) SEQ ID NO: 68LCDR1 (MAB-19-0618) Kabat SEQ ID NO: 49 LCDR2 (MAB-19-0618) Kabat and IMGT consensus (=IMGT) SEQ ID NO: 69LCDR2 (MAB-19-0618) Kabat SEQ ID NO: 50LCDR3 (MAB-19-0618) Intersection = Kabat = IMGT

[0017] The present disclosure is further described in more detail below, but it is understood that this disclosure is not limited to the particular methodologies, protocols, and reagents described herein, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0018] The elements of the present disclosure are described in more detail below. While these elements are listed with specific embodiments, it is understood that they can be combined in any manner and in any order to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the present disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments that combine the explicitly described embodiments with various disclosed and / or preferred elements. Furthermore, all permutations and combinations of all elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise. For example, if in a preferred embodiment of the binding agent used herein the first heavy chain comprises, or consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 26 or 34 [IgG1-Fc_FEAR] and in another preferred embodiment of the binding agent used herein the second heavy chain comprises, or consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 25 or 33 [IgG1-Fc_FEAL], then in a further preferred embodiment of the binding agent used herein the first heavy chain comprises, or consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 26 or 34 [IgG1-Fc_FEAR] and the second heavy chain comprises, or consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 25 or 33 [IgG1-Fc_FEAL].

[0019] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0020] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemical, biochemical, cell biological, immunological, and recombinant DNA techniques described in the art (e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Beyer / Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Römpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. See Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0021] All methods described herein may be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "for example," etc.), are intended merely to better illustrate the present disclosure and do not otherwise impose limitations on the scope of the claimed present disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0022] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise stated herein, each separate value is incorporated into the specification as if it were individually listed herein.

[0023] Numerous documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0024] definition The following definitions are provided and apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise specified. All terms not defined have their art-recognized meanings.

[0025] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to mean the inclusion of a stated element, integer, or step or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step or group of elements, integers, or steps. The term "consisting essentially of" means the exclusion of other elements, integers, or steps of some essential importance. The term "comprising" encompasses the term "essentially consisting of," which in turn encompasses the term "consisting of." Thus, at each occurrence in this application, the term "comprising" can be replaced with the term "essentially consisting of" or "consisting of." Similarly, at each occurrence in this application, the term "essentially consisting of" can be replaced with the term "consisting of."

[0026] As used in the context of describing the present disclosure (particularly in the context of the claims), the terms "a," "an," and "the" and similar references shall be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context.

[0027] "And / or," as used herein, shall be construed as a specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" shall be construed as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were individually set forth herein.

[0028] In the context of the present disclosure, the term "about" refers to a range of accuracy that a person skilled in the art would understand to still ensure the technical function of the characteristic in question. This term typically refers to a deviation from the specified numerical value of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, for example, a deviation of ±0.01%. As would be understood by a person skilled in the art, the specific deviation from the numerical value for such a given technical function will depend on the nature of the technical function. For example, a natural or biological technical function may generally have a greater deviation than an artificial or engineered technical function.

[0029] The term "binding agent," in the context of the present disclosure, refers to any substance capable of binding to a desired antigen. In certain embodiments of the present disclosure, the binding agent is an antibody, an antibody fragment, or a construct thereof. The binding agent may also include synthetic, modified, or non-naturally occurring moieties, particularly non-peptide moieties. Such moieties can, for example, link a desired antigen-binding functional group or region, such as an antibody or antibody fragment. In one embodiment, the binding agent is a synthetic construct comprising an antigen-binding CDR or variable region.

[0030] "Immune checkpoint," as used herein, refers to regulators of the immune system, particularly costimulatory and inhibitory signals that modulate the extent and quality of T cell receptor recognition of antigens. In certain embodiments, the immune checkpoint is an inhibitory signal. In certain embodiments, the inhibitory signal is the interaction of PD-1 with PD-L1 and / or PD-L2. In certain embodiments, the inhibitory signal is the interaction of CTLA-4 with CD80 or CD86 to displace CD28 binding. In certain embodiments, the inhibitory signal is the interaction of LAG-3 with an MHC class II molecule. In certain embodiments, the inhibitory signal is the interaction of TIM-3 with one or more of its ligands, such as galectin-9, PtdSer, HMGB1, and CEACAM1. In certain embodiments, the inhibitory signal is the interaction of one or more KIR with their ligands. In certain embodiments, the inhibitory signal is the interaction of TIGIT with one or more of its ligands, PVR, PVRL2, and PVRL3. In certain embodiments, the inhibitory signal is the interaction of CD94 / NKG2A with HLA-E. In certain embodiments, the inhibitory signal is the interaction of VISTA with its binding partner. In certain embodiments, the inhibitory signal is the interaction of one or more Siglecs with their ligands. In certain embodiments, the inhibitory signal is the interaction of GARP with one or more of its ligands. In certain embodiments, the inhibitory signal is the interaction of CD47 with SIRPα. In certain embodiments, the inhibitory signal is the interaction of PVRIG with PVRL2. In certain embodiments, the inhibitory signal is the interaction of CSF1R with CSF1. In certain embodiments, the inhibitory signal is the interaction of BTLA with HVEM. In certain embodiments, the inhibitory signal is part of the adenosinergic pathway, for example, the interaction of A2AR and / or A2BR produced by CD39 and CD73 with adenosine. In certain embodiments, the inhibitory signal is the interaction of B7-H3 with its receptor and / or B7-H4 with its receptor.In certain embodiments, the inhibitory signal is mediated by IDO, CD20, NOX, or TDO.

[0031] The terms "checkpoint inhibitor" (CPI) and "immune checkpoint (ICP) inhibitor" are used synonymously herein. The terms refer to molecules, such as binding agents, that completely or partially reduce, inhibit, interfere with, or negatively modulate one or more checkpoint proteins, or to molecules, such as binding agents, that completely or partially reduce, inhibit, interfere with, or negatively modulate the expression of one or more checkpoint proteins, such as molecules that inhibit immune checkpoints, particularly binding agents that inhibit the inhibitory signals of immune checkpoints. In one embodiment, an immune checkpoint inhibitor binds to one or more checkpoint proteins. In one embodiment, an immune checkpoint inhibitor binds to one or more molecules that regulate checkpoint proteins. In one embodiment, an immune checkpoint inhibitor binds to precursors of one or more checkpoint proteins, e.g., at the DNA or RNA level. Any agent that functions as a checkpoint inhibitor can be used in accordance with the present disclosure. The term "partial" as used herein means at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% at a level, e.g., at a level of inhibition of a checkpoint protein.

[0032] In one embodiment, a checkpoint inhibitor may be any compound, e.g., any binding agent, that inhibits the inhibitory signal of an immune checkpoint, where the inhibitory signal is the interaction of PD-1 with PD-L1 and / or PD-L2 (checkpoint inhibitors that inhibit such interactions between PD-1 and PD-L1 and / or PD-L2 are also referred to herein as PD-1 / PD-L1 checkpoint inhibitors); the interaction of CTLA-4 with CD80 or CD86 to displace CD28 binding; the interaction of LAG-3 with MHC class II molecules; the interaction of TIM-3 with one or more of its ligands, e.g., galectin-9, PtdSer, HMGB1, and CEACAM1; the interaction of one or more KIR with its ligand; TIGIT with its ligand; interaction between one or more of: VISTA and its binding partner; interaction between one or more Siglecs and their ligands; interaction between GARP and one or more of its ligands; interaction between CD47 and SIRPα; interaction between PVRIG and PVRL2; interaction between CSF1R and CSF1; interaction between BTLA and HVEM; interaction between a part of the adenosinergic pathway, for example, interaction between A2AR and / or A2BR and adenosine produced by CD39 and CD73; interaction between B7-H3 and its receptor and / or B7-H4 and its receptor; inhibitory signals mediated by IDO, CD20, NOX or TDO. In one embodiment, the checkpoint inhibitor is at least one selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a KIR inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a VISTA inhibitor, and a GARP inhibitor. In one embodiment, the checkpoint inhibitor may be a blocking antibody, such as a PD-1 blocking antibody, a CTLA4 blocking antibody, a PD-L1 blocking antibody, a PD-L2 blocking antibody, a TIM-3 blocking antibody, a KIR blocking antibody, a LAG-3 blocking antibody, a TIGIT blocking antibody, a VISTA blocking antibody, or a GARP blocking antibody.Examples of PD-1 blocking antibodies include pembrolizumab, nivolumab, cemiplimab, and spartalizumab. Examples of CTLA4 blocking antibodies include ipilimumab and tremelimumab. Examples of PD-L1 blocking antibodies include atezolizumab, durvalumab, and avelumab.

[0033] In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 43, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 44.

[0034] In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 45; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 47 Includes; The light chain variable region is (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 49; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 50 Includes:

[0035] In one embodiment of the anti-PD-1 antibodies described herein, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:43 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:44.

[0036] In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 81; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 82; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 83 and the light chain variable region comprises: (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 84; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 85; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 86 Includes:

[0037] In one embodiment of the anti-PD-1 antibodies described herein, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 87, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 88. In one embodiment of the anti-PD-1 antibodies described herein, the heavy chain comprises the amino acid sequence of SEQ ID NO: 89, and the light chain comprises the amino acid sequence of SEQ ID NO: 90.

[0038] In one embodiment, an immune checkpoint inhibitor suitable for use in the methods disclosed herein is an antibody that targets PD-1 or PD-L1. In a preferred embodiment, an immune checkpoint inhibitor suitable for use in the methods disclosed herein is pembrolizumab.

[0039] The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably antigen receptors, such as antibodies or B-cell receptors (BCRs). Immunoglobulins are characterized by structural domains, i.e., immunoglobulin domains with a characteristic immunoglobulin (Ig) fold. The term encompasses soluble immunoglobulins as well as membrane-bound immunoglobulins. Membrane-bound immunoglobulins are also called surface or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally called antibodies.

[0040] The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2 nded. Raven Press, NY (1989). Briefly, immunoglobulins generally comprise a number of chains, typically two identical heavy chains and two identical light chains linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains or regions, e.g., V L or VL (variable light chain) domain / region, C L or CL (constant light chain) domain / region, V H or a VH (variable heavy chain) domain / region, and C H or CH (constant heavy chain) domain / region C H 1(CH1), C H 2(CH2), C H 3(CH3), and C HThe heavy chain constant region is typically composed of three domains, CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interaction between the two heavy chains in an IgG molecule. Each light chain is typically composed of a VL and a CL. The light chain constant region is typically composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (or hypervariable regions that may be highly variable in sequence and / or in the form of structurally defined loops), also called complementarity-determining regions (CDRs), interspersed with more highly conserved regions called framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise specified or contradicted by context, CDR sequences herein are identified according to the rules of IMGT using DomainGapAlign (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); see also the internet http address www.imgt.org. Unless otherwise specified or contradicted by context, references to amino acid positions in constant regions in the present disclosure are according to EU numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).

[0041] There are five types of mammalian immunoglobulin heavy chains: α, δ, ε, γ, and μ, which constitute the different classes of antibodies: IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at their carboxy termini. In mammals, there are two types of light chains: lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is substantially conserved among different immunoglobulin isotypes, while the variable region is highly diverse and is responsible for antigen recognition.

[0042] The terms "amino acid" and "amino acid residue" may be used interchangeably herein and are not to be understood as limiting. Amino acids are organic compounds containing an amine (-NH2) and a carboxyl (-COOH) functional group, along with a side chain (R group) specific to each amino acid. In the context of the present disclosure, amino acids can be classified based on their structure and chemical characteristics. Thus, the classes of amino acids may be reflected in one or both of the following tables:

[0043] Table 2: Major classifications based on the structure and general chemical characteristics of the R group [Table 2]

[0044] Table 3: Physical and functional classification of amino acid residue alternatives [Table 3]

[0045] For purposes of the present disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those occurring in nature. The term "variant" particularly includes fragments of an amino acid sequence.

[0046] Amino acid insertion variants include the insertion of a single, two or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, but random insertions with appropriate screening of the resulting product are also possible.

[0047] Amino acid addition variants include amino and / or carboxy terminal fusions of one or more amino acids, for example 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

[0048] Amino acid deletion mutants are characterized by the removal of one or more amino acids from the sequence, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions can be at any position in the protein. Amino acid deletion mutants containing deletions at the N-terminus and / or C-terminus of the protein are also referred to as N-terminal and / or C-terminal truncation mutants.

[0049] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Substitution of one amino acid for another may be classified as conservative or non-conservative substitution. Preferably, the amino acid sequence is modified at a position that is not conserved between homologous proteins or peptides, and / or the amino acid is replaced with another amino acid with similar properties. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitution of an amino acid with an amino acid with a similar charge or no charge. Conservative amino acid changes involve the substitution of a member of a family of amino acids that are related in their side chains. In the context of the present disclosure, a "conservative substitution" refers to the substitution of one amino acid with another amino acid with similar structural and / or chemical characteristics, e.g., the substitution of one amino acid residue with another amino acid residue of the same class as defined in either of the two tables above, e.g., leucine may be substituted with isoleucine, since both are aliphatic, branched, hydrophobic substances. Similarly, aspartic acid may be substituted with glutamic acid because both are residues with small negative charges. Naturally occurring amino acids can also generally be divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: - glycine, alanine; - valine, isoleucine, leucine; - Aspartic acid, glutamic acid; - Asparagine, glutamine; - serine, threonine; - lysine, arginine; and - Phenylalanine, tyrosine.

[0050] The term "amino acid corresponding to position" and similar expressions, as used herein, refers to the number of an amino acid position in a human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence "corresponds to" an amino acid or segment in another sequence means that the amino acid or segment in one sequence, when aligned with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW, or similar, typically with default settings, has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. It is considered well known in the art how to align sequences or segments in sequences to determine positions in sequences that correspond to amino acid positions according to the present disclosure.

[0051] The term "antibody" (Ab), in the context of the present disclosure, refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, that has the ability to specifically bind to an antigen (particularly an epitope on an antigen) under typical physiological conditions, preferably with a half-life of a significant duration, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, etc., or any other relevant, functionally defined period of time (e.g., a period of time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or a period of time sufficient for the antibody to recruit effector activity). In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination of the foregoing. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to herein as variable and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more highly conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of the VH are referred to as HCDR1, HCDR2, and HCDR3 (or CDR-H1, CDR-H2, and CDR-H3), and the CDRs of the VL are referred to as LCDR1, LCDR2, and LCDR3 (or CDR-L1, CDR-L2, and CDR-L3). The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), and the CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged from the amino terminus to the carboxy terminus in the following order: CH1, CH2, CH3). The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or an immunologically active portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies can exist in various forms, such as polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0052] The variable regions of the heavy and light chains of immunoglobulin molecules contain binding domains that interact with antigens. The terms "binding region" and "antigen-binding region" are used interchangeably herein and refer to the region that interacts with antigens and includes both VH and VL regions. Antibodies as used herein include not only monospecific antibodies, but also multispecific antibodies that contain multiple, for example, two or more, for example, three or more different antigen-binding regions.

[0053] As indicated above, the term antibody as used herein, unless otherwise specified or clearly contradicted by the context, includes fragments of antibodies that are antigen-binding fragments, i.e., fragments of antibodies that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include: (i) Fab' or Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains, or monovalent antibodies as described in WO2007 / 059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody; (v) dAb fragments (Ward et al., Nature 341, 544-546 (1989)), also called domain antibodies (Holt et al.; Trends Biotechnol. 2003 Nov;21(11):484-90); (vi) camelid or nanobody molecules (Revets et al.; Expert Opin Biol Ther. 2005 Jan;5(1):111-24); and (vii) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be combined using recombinant methods with a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain antibodies or single-chain Fvs (scFvs), see, for example, Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed by the term antibody unless otherwise specified or clearly indicated by the context.Although such fragments are generally included within the meaning of "antibody," they are collectively and independently a unique feature of the present disclosure, exhibiting various biological properties and usefulness. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific versions of such fragments, are discussed further herein. Unless otherwise specified, the term "antibody" should also be understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant technology, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen.

[0054] Antibodies as produced may have a certain isotype. The term "isotype," as used herein, refers to the class of immunoglobulin (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgD, IgA (e.g., IgA1, IgA2), IgE, IgM, or IgY) encoded by heavy chain constant region genes. When a particular isotype, e.g., IgG1, is mentioned herein, the term is not limited to a specific isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g., IgG1, than to other isotypes. Thus, for example, the IgG1 antibodies disclosed herein may be sequence variants of naturally occurring IgG1 antibodies that contain changes in the constant region.

[0055] IgG1 antibodies can exist in multiple polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7), any of which are suitable for use in some of the embodiments described herein. Common allotypic variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In any of the embodiments described herein, the antibody may comprise a heavy chain Fc region comprising a human IgG Fc region. In a further embodiment, the human IgG Fc region comprises human IgG1.

[0056] The term "multispecific antibody," in the context of the present disclosure, refers to an antibody having at least two different antigen-binding regions defined by different antibody sequences. In some embodiments, the different antigen-binding regions bind to different epitopes on the same antigen. However, in preferred embodiments, the different antigen-binding regions bind to different target antigens. In one embodiment, the multispecific antibody is a "bispecific antibody" or "bs." Multispecific antibodies, e.g., bispecific antibodies, may have any format, including any of the bispecific or multispecific antibody formats described herein below.

[0057] The term "full length" when used in the context of an antibody indicates that the antibody is not a fragment, but contains all of the domains of a particular isotype that are normally found in that isotype in nature, e.g., in the case of an IgG1 antibody, the VH, CH1, CH2, CH3, hinge, VL and CL domains.

[0058] The term "human antibody," as used herein, is intended to include antibodies having variable and framework regions derived from human germline immunoglobulin sequences and human immunoglobulin constant domains. The human antibodies disclosed herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations, insertions, or deletions introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another non-human species, such as a mouse, have been grafted onto human framework sequences.

[0059] The term "chimeric antibody," as used herein, refers to an antibody whose variable region is derived from a non-human species (e.g., rodent) and whose constant region is derived from a different species, e.g., human. Chimeric antibodies may be produced by antibody engineering. "Antibody engineering" is a term commonly used to describe various types of antibody modifications, and processes for antibody engineering are well known to those skilled in the art. In particular, chimeric antibodies can be produced using standard DNA techniques, such as those described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, chimeric antibodies can be genetically or enzymatically engineered recombinant antibodies. Producing chimeric antibodies is within the knowledge of those skilled in the art, and therefore, chimeric antibody production can be performed by methods other than those described herein. Chimeric monoclonal antibodies for therapeutic use in humans have been developed to reduce the expected antibody immunogenicity of non-human antibodies, e.g., rodent antibodies. These typically contain a non-human (e.g., mouse or rabbit) variable region specific for an antigen of interest, and may contain human constant antibody heavy and light chain domains. The terms "variable region" or "variable domain," when used in the context of a chimeric antibody, refer to the region comprising the CDRs and framework regions of both the heavy and light chains of an immunoglobulin, as described below.

[0060] The term "humanized antibody," as used herein, refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that have been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity-determining regions (CDRs) that together form the antigen-binding site onto homologous human acceptor framework regions (FRs) (see WO 92 / 22653 and EP 0629240). Substitution (backmutation) of framework residues from the parent antibody (i.e., non-human antibody) with human framework regions may be necessary to fully reconstitute the binding affinity and specificity of the parent antibody. Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may contain non-human CDR sequences, primarily human framework regions that may contain one or more amino acid backmutations relative to the non-human amino acid sequences, and a fully human constant region. To obtain humanized antibodies with favorable characteristics, such as affinity and biochemical properties, additional amino acid modifications may be applied, and such modifications may not necessarily be back mutations.

[0061] As used herein, a protein "derived from" another protein, e.g., a parent protein, means that one or more amino acid sequences of the protein are identical to or similar to one or more amino acid sequences in the other or parent protein. For example, in an antibody, binding arm, antigen-binding region, constant region, etc. derived from another or parent antibody, binding arm, antigen-binding region, or constant region, one or more amino acid sequences are identical to or similar to the amino acid sequence of the other or parent antibody, binding arm, antigen-binding region, or constant region. Examples of such one or more amino acid sequences include, but are not limited to, the amino acid sequences of one or more or all of the VH and VL CDRs and / or framework regions, VH, VL, CL, hinge, or CH regions. For example, a humanized antibody may be described herein as "derived from" a non-human parent antibody, meaning that at least the VL and VH CDR sequences are identical to or similar to the VH and VL CDR sequences of said non-human parent antibody. Chimeric antibodies may be described herein as "derived from" a non-human parent antibody, which typically means that the VH and VL sequences may be identical or similar to those of the non-human parent antibody. Another example is a binding arm or antigen-binding region that may be described herein as "derived from" a particular parent antibody, which typically means that the binding arm or antigen-binding region comprises VH and / or VL CDRs or VH and / or VL sequences that are identical to or similar to those of the binding arm or antigen-binding region of the parent antibody. However, as described elsewhere herein, amino acid modifications, e.g., mutations, may be made in the CDRs, constant regions, or elsewhere in the antibody, binding arm, antigen-binding region, etc., to introduce desired characteristics. When used in the context of one or more sequences derived from a first or parent protein, a "similar" amino acid sequence preferably has at least about 50%, e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 97%, 98% or 99% sequence identity.

[0062] Non-human antibodies can be produced in a variety of different species, for example, mice, rabbits, chickens, guinea pigs, llamas, and goats.

[0063] Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody techniques, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Other techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using libraries of antibody genes, may also be employed; such methods are well known to those skilled in the art.

[0064] Hybridoma production in such non-human species is a very well-established procedure. Immunization protocols and techniques for isolation of splenocytes from immunized animals / non-human species for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0065] As used herein, unless contradictory in context, the term "Fab arm" or "arm" refers to one heavy chain-light chain pair and is used synonymously herein with "half molecule."

[0066] The term "binding arm comprising an antigen-binding region" refers to an antibody molecule or fragment that comprises an antigen-binding region. Thus, a binding arm may comprise, for example, six VH and VL CDR sequences, a VH and VL sequence, a Fab or Fab' fragment, or a Fab arm.

[0067] As used herein, unless contradicted by context, the term "Fc region" refers to the region of an antibody consisting of two Fc sequences of an immunoglobulin heavy chain, wherein the Fc sequences include at least a hinge region, a CH2 domain, and a CH3 domain. In one embodiment, the term "Fc region" as used herein refers to the region of an antibody that includes, from the N-terminus to the C-terminus, at least the hinge region, the CH2 domain, and the CH3 domain. The Fc region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system.

[0068] In the context of the present disclosure, the term "induce Fc-mediated effector functions to a lesser extent" as used in relation to antibodies, including multispecific antibodies, means that the antibody induces Fc-mediated effector functions, such as functions selected from the list of IgG Fc receptor (Fc gamma R, FcγR) binding, C1q binding, ADCC or CDC, to a lesser extent compared to a human IgG1 antibody that (i) has the same CDR sequences as said antibody, in particular CDR sequences comprising the same first and second antigen-binding regions, and (ii) two heavy chains comprising human IgG1 hinge, CH2 and CH3 regions.

[0069] Fc-mediated effector function can be measured by binding to FcγR, binding to C1q, or induction of Fc-mediated cross-linking through FcγR.

[0070] The term "hinge region" as used herein refers to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to the EU numbering system set forth in Kabat (Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91-3242, pp. 662, 680, 689 (1991)). However, the hinge region may also be of any of the other subtypes described herein.

[0071] The term "CH1 region" or "CH1 domain," as used herein, refers to the CH1 region of an immunoglobulin heavy chain. Thus, for example, the CH1 region of a human IgG1 antibody corresponds to amino acids 118-215 according to the EU numbering system set forth in Kabat (ibid.). However, the CH1 region may also be of any of the other subtypes described herein.

[0072] The term "CH2 region" or "CH2 domain," as used herein, refers to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to the EU numbering system set forth in Kabat (ibid.). However, the CH2 region may also be of any of the other subtypes described herein.

[0073] The term "CH3 region" or "CH3 domain," as used herein, refers to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to the EU numbering system set forth in Kabat (ibid.). However, the CH3 region may also be of any of the other subtypes described herein.

[0074] The term "monovalent antibody", in the context of the present disclosure, means that the antibody molecule is capable of binding to a single molecule of antigen and is therefore not capable of cross-linking antigens.

[0075] A "CD40 antibody" or "anti-CD40 antibody" is an antibody that specifically binds to the antigen CD40, as described above.

[0076] A "CD137 antibody" or "anti-CD137 antibody" is an antibody that specifically binds to the antigen CD137 as described above.

[0077] A "CD40xCD137 antibody" or "anti-CD40xCD137 antibody" is a bispecific antibody that contains two different antigen-binding regions, one of which specifically binds to the antigen CD40 and the other of which specifically binds to the antigen CD137.

[0078] As used herein, the terms "binding" or "capable of binding" refer to the binding of an antibody to a predetermined antigen or epitope, typically as determined using biolayer interferometry (BLI) or, for example, as determined using surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument using an antigen as the ligand and an antibody as the analyte, typically in the range of about 10 to about 100. -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 K of M D , or even lower K D An antibody will bind with an affinity equivalent to its K for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). D at least 10 times lower, such as at least 100 times lower, for example at least 1,000 times lower, such as at least 10,000 times lower, for example at least 100,000 times lower DThe antibody binds to a predetermined antigen with an affinity equivalent to the K D It is therefore dependent on the K D is very low (i.e., the antibody is highly specific), the affinity for the antigen can be at least 10,000 times lower than the affinity for a non-specific antigen.

[0079] The term "k" d ”(seconds -1 ) as used herein refers to the dissociation rate constant of a particular antibody-antigen interaction. The value is also referred to as k off Also called the value.

[0080] The term “K D " (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0081] Two antibodies have the "same specificity" if they bind to the same antigen or the same epitope. Whether an antibody being tested recognizes the same epitope as a particular antigen-binding antibody, i.e., whether the antibodies bind to the same epitope, can be tested by various methods well known to those skilled in the art.

[0082] Competition between antibodies can be detected by cross-blocking assay. For example, competitive ELISA assay can be used as a cross-blocking assay. For example, a target antigen can be coated on the well of a microtiter plate, and an antibody that binds to the antigen and a candidate competing test antibody can be added. The amount of antigen-bound antibody in the well is indirectly correlated with the binding ability of the candidate competing test antibody that competes with it for binding to the same epitope. Specifically, the greater the affinity of the candidate competing test antibody for the same epitope, the less antibody will bind to the antigen bound to the well coated with the antigen. The amount of antibody bound to the antigen bound to the well can be measured by labeling the antibody with a detectable or measurable labeling substance.

[0083] An antibody that competes for binding to an antigen with another antibody, e.g., an antibody comprising the heavy and light chain variable regions described herein, or with an antibody having specificity for the antigen of another antibody, e.g., an antibody comprising the heavy and light chain variable regions described herein, may be an antibody comprising a variant of the heavy and / or light chain variable regions described herein, e.g., an antibody comprising modifications in the CDRs and / or a particular degree of identity as described herein.

[0084] An "isolated multispecific antibody," as used herein, is intended to refer to a multispecific antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated bispecific antibody that specifically binds to CD40 and CD137 is substantially free of monospecific antibodies that specifically bind to CD40 or CD137).

[0085] The term "monoclonal antibody," as used herein, refers to a preparation of antibody molecules of single molecular composition, displaying a single binding specificity and affinity for a particular epitope.

[0086] The term "heterodimeric interaction between the first and second CH3 regions" as used herein refers to the interaction between the first CH3 region and the second CH3 region in a first CH3 / second CH3 heterodimeric antibody.

[0087] The term "homodimeric interaction of the first and second CH3 regions," as used herein, refers to the interaction of a first CH3 region with another first CH3 region in a first CH3 / first CH3 homodimeric antibody, and the interaction of a second CH3 region with another second CH3 region in a second CH3 / second CH3 homodimeric antibody.

[0088] The term "homodimeric antibody," as used herein, refers to an antibody comprising two first Fab arms or half molecules, wherein the amino acid sequences of said Fab arms or half molecules are the same.

[0089] The term "heterodimeric antibody," as used herein, refers to an antibody comprising a first and a second Fab arm or half molecule, wherein the amino acid sequences of said first and second Fab arms or half molecules are different. In particular, the CH3 region or the antigen-binding region, or the CH3 region and the antigen-binding region, of said first and second Fab arms / half molecules are different.

[0090] The term "reducing conditions" or "reducing environment" refers to conditions or circumstances in which a substrate, such as a cysteine ​​residue in the hinge region of an antibody, is more likely to become reduced than oxidized.

[0091] The present disclosure also describes multispecific antibodies, e.g., bispecific antibodies, that comprise functional variants of one or more of the VL regions, VH regions, or CDRs of the bispecific antibodies of the examples. Functional variants of VL, VH, or CDRs used in the context of bispecific antibodies still enable each antigen-binding region of the bispecific antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or more) of the affinity and / or specificity / selectivity of the parent bispecific antibody, and in some cases, such bispecific antibodies may be associated with greater affinity, selectivity, and / or specificity than the parent bispecific antibody.

[0092] Such functional variants typically retain significant sequence identity with the parent bispecific antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., % homology = number of identical positions / total number of positions × 100). The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4, 11-17 (1988), as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0093] In the context of the present disclosure, unless otherwise specified, the following notation is used to describe mutations: i) substitution of an amino acid at a given position is designated, for example, as K409R, which means substitution of lysine with arginine at position 409 of the protein; and ii) for specific variants, specific three-letter or one-letter codes are used, including the codes Xaa and X, to indicate any amino acid residue. Thus, substitution of lysine with arginine at position 409 is designated as K409R, and substitution of lysine at position 409 with any amino acid residue is designated as K409X. In the case of deletion of lysine at position 409, this is designated by K409*.

[0094] Exemplary variants include those that differ from the VH and / or VL and / or CDRs of a parent sequence primarily by conservative substitutions; for example, 12, for example 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements.

[0095] In the context of the present disclosure, conservative substitutions can be defined by substitutions within the classes of amino acids defined in Tables 2 and 3.

[0096] The term "CD40," as used herein, refers to CD40, also known as tumor necrosis factor receptor superfamily member 5 (TNFRSF5), which is a receptor for the ligand TNFSF5 / CD40L. CD40 is known to activate ERK in macrophages and B cells and transduce TRAF6- and MAP3K8-mediated signals, leading to the induction of immunoglobulin secretion by B cells. Other synonyms used for CD40 include, but are not limited to, B-cell surface antigen CD40, Bp50, CD40L receptor, and CDw40. In one embodiment, CD40 is human CD40, having UniProt accession number P25942. The sequence of human CD40 is also set forth in SEQ ID NO: 35. Amino acids 1-20 of SEQ ID NO:35 correspond to the signal peptide of human CD40; while amino acids 21-193 of SEQ ID NO:35 correspond to the extracellular domain of human CD40; the remainder of the protein, i.e., amino acids 194-215 and 216-277 of SEQ ID NO:35, are the transmembrane and cytoplasmic domains, respectively.

[0097] The term "CD137," as used herein, refers to CD137(4-1BB), also known as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is a receptor for the ligand TNFSF9 / 4-1BBL. CD137(4-1BB) is thought to be involved in T cell activation. Other synonyms for CD137 include, but are not limited to, 4-1BB ligand receptor, CDw137, T cell antigen 4-1BB homolog, and T cell antigen ILA. In one embodiment, CD137(4-1BB) is human CD137(4-1BB) having UniProt accession number Q07011. The sequence of human CD137 is also set forth in SEQ ID NO: 37. Amino acids 1 to 23 of SEQ ID NO: 37 correspond to the signal peptide of human CD137; while amino acids 24 to 186 of SEQ ID NO: 37 correspond to the extracellular domain of human CD137; the remainder of the protein, i.e., amino acids 187 to 213 and 214 to 255 of SEQ ID NO: 37, are the transmembrane and cytoplasmic domains, respectively.

[0098] The "programmed death-1 (PD-1)" receptor refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 (also known as CD279) is predominantly expressed on preactivated T cells in vivo and binds to two ligands, PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273). The term "PD-1," as used herein, includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs that share at least one epitope with hPD-1. The sequence of human PD-1 is also set forth in SEQ ID NO: 39. "Programmed death-ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1," as used herein, includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, such as macaque (cynomolgus), African elephant, wild boar, and mouse PD-L1 (see, for example, Genbank accession numbers NP_054862.1, XP_005581836, XP_003413533, XP_005665023, and NP_068693, respectively), as well as analogs that share at least one epitope in common with hPD-L1. The sequence of human PD-L1 is also set forth in SEQ ID NO: 40, in which amino acids 1-18 are predicted to be the signal peptide. The sequence of macaque (cynomolgus) PD-L1 is also set forth in SEQ ID NO: 41, in which amino acids 1-18 are predicted to be the signal peptide. The term "PD-L2," as used herein, includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, as well as analogs that share at least one epitope with hPD-L2. The ligands for PD-1 (PD-L1 and PD-L2) are expressed on the surface of antigen-presenting cells, such as dendritic cells or macrophages, and other immune cells. Binding of PD-1 to PD-L1 or PD-L2 results in downregulation of T cell activation.Cancer cells expressing PD-L1 and / or PD-L2 can switch off PD-1-expressing T cells, resulting in suppression of anti-cancer immune responses. The interaction of PD-1 with its ligands results in a reduction in tumor-infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and immune evasion by cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and this effect is additive when the interaction between PD-1 and PD-L2 is similarly blocked.

[0099] Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) (also known as CD152) is a T-cell surface molecule and a member of the immunoglobulin superfamily. This protein downregulates the immune system by binding to CD80 (B7-1) and CD86 (B7-2). The term "CTLA-4," as used herein, includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, as well as analogs that share at least one epitope with hCTLA-4. CTLA-4 is a homolog of the stimulatory checkpoint protein CD28, which has higher binding affinity to CD80 and CD86. CTLA4 is expressed on the surface of activated T cells, and its ligand is expressed on the surface of professional antigen-presenting cells. Binding of CTLA4 to its ligand prevents the costimulatory signal of CD28 and produces an inhibitory signal. Thus, CTLA-4 downregulates T-cell activation. The sequence of human CTLA-4 is also shown in SEQ ID NO:42.

[0100] "T cell immunoreceptor with Ig and ITIM domains" (TIGIT, also known as WUCAM or Vstm3) is an immunoreceptor on T cells and natural killer (NK) cells that binds to PVR (CD155) on DCs and macrophages, as well as PVRL2 (CD112; nectin-2) and PVRL3 (CD113; nectin-3), and regulates T cell-mediated immunity. The term "TIGIT," as used herein, includes human TIGIT (hTIGIT), variants, isoforms, and species homologs of hTIGIT, and analogs that share at least one common epitope with hTIGIT. The term "PVR," as used herein, includes human PVR (hPVR), variants, isoforms, and species homologs of hPVR, and analogs that share at least one common epitope with hPVR. The term "PVRL2," as used herein, includes human PVRL2 (hPVRL2), variants, isoforms, and species homologs of hPVRL2, and analogs that share at least one epitope in common with hPVRL2. The term "PVRL3," as used herein, includes human PVRL3 (hPVRL3), variants, isoforms, and species homologs of hPVRL3, and analogs that share at least one epitope in common with hPVRL3.

[0101] "B and T lymphocyte attenuator" (BTLA, also known as CD272) is a TNFR family member expressed in Th1 but not Th2 cells. BTLA expression is induced during T cell activation, particularly on the surface of CD8+ T cells. As used herein, the term "BTLA" includes human BTLA (hBTLA), variants, isoforms, and species homologs of hBTLA, as well as analogs that share at least one epitope with hBTLA. BTLA expression is gradually downregulated during differentiation of human CD8+ T cells into an effector cell phenotype. Tumor-specific human CD8+ T cells express high levels of BTLA. BTLA binds to "herpesvirus entry mediator" (HVEM, also known as TNFRSF14 or CD270) and is involved in T cell inhibition. The term "HVEM" as used herein includes human HVEM (hHVEM), variants, isoforms, and species homologs of hHVEM, as well as analogs that share at least one epitope with hHVEM. The BTLA-HVEM complex negatively regulates T cell immune responses.

[0102] "Killer cell immunoglobulin-like receptors" (KIRs) are receptors for MHC class I molecules on NK T cells and NK cells that are involved in the differentiation between healthy and diseased cells. KIRs bind to human leukocyte antigens (HLA) A, B, and C, which suppress normal immune cell activation. The term "KIR," as used herein, includes human KIR (hKIR), variants, isoforms, and species homologs of hKIR, as well as analogs that share at least one epitope with hKIR. The term "HLA," as used herein, includes variants, isoforms, and species homologs of HLA, as well as analogs that share at least one epitope with HLA. KIR, as used herein, specifically refers to KIR2DL1, KIR2DL2, and / or KIR2DL3.

[0103] "Lymphocyte activation gene-3 (LAG-3)" (also known as CD223) is an inhibitory receptor involved in the inhibition of lymphocyte activity by binding to MHC class II molecules. This receptor enhances the function of Treg cells and inhibits the function of CD8+ effector T cells, which leads to suppression of the immune response. LAG-3 is expressed on activated T cells, NK cells, B cells, and DCs. The term "LAG-3," as used herein, includes human LAG-3 (hLAG-3), variants, isoforms, and species homologs of hLAG-3, as well as analogs that share at least one common epitope.

[0104] "T-cell membrane protein-3 (TIM-3)" (also known as HAVcr-2) is an inhibitory receptor involved in inhibiting lymphocyte activity by inhibiting Th1 cell responses. Its ligand is galectin 9 (GAL9), which is upregulated in various types of cancer. Other TIM-3 ligands include phosphatidylserine (PtdSer), high-mobility group protein 1 (HMGB1), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). The term "TIM-3," as used herein, includes human TIM3 (hTIM-3), variants, isoforms, and species homologs of hTIM-3, as well as analogs that share at least one common epitope. The term "GAL9," as used herein, includes human GAL9 (hGAL9), variants, isoforms, and species homologs of hGAL9, as well as analogs that share at least one common epitope. The term "PdtSer" as used herein includes mutants and analogs that share at least one common epitope. The term "HMGB1" as used herein includes human HMGB1 (hHMGB1), mutants, isoforms, and species homologs of hHMGB1, and analogs that share at least one common epitope. The term "CEACAM1" as used herein includes human CEACAM1 (hCEACAM1), mutants, isoforms, and species homologs of hCEACAM1, and analogs that share at least one common epitope.

[0105] "CD94 / NKG2A" is an inhibitory receptor predominantly expressed on the surface of natural killer cells and CD8+ T cells. As used herein, the term "CD94 / NKG2A" includes human CD94 / NKG2A (hCD94 / NKG2A), hCD94 / NKG2A variants, isoforms, and species homologs, as well as analogs sharing at least one common epitope. The CD94 / NKG2A receptor is a heterodimer containing CD94 and NKG2A. It inhibits NK cell activation and CD8+ T cell function, possibly through binding to ligands such as HLA-E. CD94 / NKG2A limits cytokine release and cytotoxic responses of natural killer cells (NK cells), natural killer T cells (NK-T cells), and T cells (α / β and γ / δ). NKG2A is frequently expressed on tumor-infiltrating cells, while HLA-E is overexpressed in numerous cancers.

[0106] "Indoleamine 2,3-dioxygenase" (IDO) is a tryptophan catabolic enzyme with immunosuppressive properties. As used herein, the term "IDO" includes human IDO (hIDO), hIDO variants, isoforms, and species homologs, as well as analogs sharing at least one common epitope. IDO is the rate-limiting enzyme in tryptophan degradation, catalyzing its conversion to kynurenine. Therefore, IDO is involved in the depletion of essential amino acids. It is known to be involved in the suppression of T and NK cells, the generation and activation of Tregs and myeloid-derived suppressor cells, and the promotion of tumor angiogenesis. IDO is overexpressed in many cancers and has been shown to promote immune system evasion by tumor cells and, when induced by local inflammation, facilitate chronic tumor progression.

[0107] As used herein, the "adenosinergic pathway" or "adenosine signaling pathway" involves the conversion of ATP to adenosine by the ectonucleotidases CD39 and CD73, resulting in inhibitory signaling via adenosine binding by one or more of the inhibitory adenosine receptors, the "adenosine A2A receptor" (A2AR, also known as ADORA2A) and the "adenosine A2B receptor" (A2BR, also known as ADORA2B). Adenosine is a nucleoside with immunosuppressive properties, present at high concentrations in the tumor microenvironment, limiting immune cell infiltration, cytotoxicity, and cytokine production. Thus, adenosine signaling is a strategy used by cancer cells to evade clearance from the host immune system. Adenosine signaling via A2AR and A2BR is a key checkpoint in cancer therapy, typically activated by the high adenosine concentrations present in the tumor microenvironment. CD39, CD73, A2AR, and A2BR are expressed by most immune cells, including T cells, invariant natural killer cells, B cells, platelets, mast cells, and eosinophils. Adenosine signaling via A2AR and A2BR counteracts immune cell activation mediated by the T cell receptor, resulting in an increase in the number of Tregs and a decrease in the activation of DCs and effector T cells. The term "CD39," as used herein, includes human CD39 (hCD39), hCD39 variants, isoforms, and species homologs, as well as analogs that share at least one common epitope. The term "CD73," as used herein, includes human CD73 (hCD73), hCD73 variants, isoforms, and species homologs, as well as analogs that share at least one common epitope. The term "A2AR," as used herein, includes human A2AR (hA2AR), hA2AR variants, isoforms, and species homologs, as well as analogs that share at least one common epitope. The term "A2BR," as used herein, includes human A2BR (hA2BR), variants, isoforms, and species homologs of hA2BR, and analogs that share at least one common epitope.

[0108] "V-domain Ig suppressor of T-cell activation" (VISTA, also known as C10orf54) shares homology with PD-L1 but displays a unique expression pattern restricted to the hematopoietic compartment. The term "VISTA," as used herein, includes human VISTA (hVISTA), variants, isoforms, and species homologs of hVISTA, and analogs that share at least one common epitope. VISTA induces T-cell suppression and is expressed by leukocytes within tumors.

[0109] Members of the "sialic acid-binding immunoglobulin-type lectin" (Siglec) family recognize sialic acid and are involved in the discrimination between "self" and "non-self." The term "Siglec," as used herein, includes human Siglecs (hSiglecs), variants, isoforms, and species homologs of hSiglecs, as well as analogs that share at least one epitope with one or more hSiglecs. The human genome contains 14 Siglecs, some of which are involved in immunosuppression, including, but not limited to, Siglec-2, Siglec-3, Siglec-7, and Siglec-9. Siglec receptors bind to sialic acid-containing glycans but differ in their recognition of the linkage regiochemistry and spatial distribution of sialic acid residues. Family members also have distinct expression patterns. A wide range of malignant tumors overexpress one or more Siglecs.

[0110] "CD20" is an antigen expressed on the surface of B and T cells. High expression of CD20 can be found in cancers such as B cell lymphoma, hairy cell leukemia, B cell chronic lymphocytic leukemia, and melanoma cancer stem cells. The term "CD20," as used herein, includes human CD20 (hCD20), hCD20 variants, isoforms, and species homologs, as well as analogs that share at least one common epitope.

[0111] Glycoprotein A repeats predominant (GARP) play a role in immune tolerance and the tumor's ability to evade the patient's immune system. The term "GARP," as used herein, includes human GARP (hGARP), hGARP variants, isoforms, and species homologs, as well as analogs that share at least one common epitope. GARP is expressed on lymphocytes, such as Tregs in peripheral blood and tumor-infiltrating T cells at tumor sites. It can bind to latent transforming growth factor-β (TGF-β). Disruption of GARP signaling in Treg cells leads to a decrease in tolerance and inhibits Treg migration to the gastrointestinal tract and increased proliferation of cytotoxic T cells.

[0112] "CD47" is a transmembrane protein that binds to the ligand "signal regulatory protein alpha" (SIRPα). As used herein, the term "CD47" includes human CD47 (hCD47), variants, isoforms, and species homologs of hCD47, as well as analogs that share at least one epitope with hCD47. As used herein, the term "SIRPα" includes human SIRPα (hSIRPα), variants, isoforms, and species homologs of hSIRPα, as well as analogs that share at least one epitope with hSIRPα. CD47 signaling is involved in various cellular processes, including apoptosis, proliferation, adhesion, and migration. CD47 is overexpressed in many cancers and functions as a "don't eat me" signal to macrophages. Blocking CD47 signaling via inhibitory anti-CD47 or anti-SIRPα antibodies allows macrophage phagocytosis of cancer cells and promotes the activation of cancer-specific T lymphocytes.

[0113] "Poliovirus receptor-related immunoglobulin domain containing" (PVRIG, also known as CD112R) binds to "poliovirus receptor-related 2" (PVRL2). PVRIG and PVRL2 are overexpressed in many cancers. PVRIG expression also induces TIGIT and PD-1 expression, and PVRL2 and PVR (TIGIT ligand) are co-overexpressed in a number of cancers. Blockade of the PVRIG signaling pathway results in increased T cell function and CD8+ T cell responses, thus reducing immunosuppression and increasing interferon responses. The term "PVRIG," as used herein, includes human PVRIG (hPVRIG), variants, isoforms, and species homologs of hPVRIG, as well as analogs that share at least one epitope with hPVRIG. "PVRL2," as used herein, includes hPVRL2, as defined above.

[0114] The "colony-stimulating factor 1" (CSF1) pathway is another checkpoint that can be targeted according to the present disclosure. CSF1R is a myeloid growth factor receptor that binds to CSF1. Blocking CSF1R signaling can functionally reprogram macrophage responses, thereby enhancing antigen presentation and anti-tumor T cell responses. The term "CSF1R," as used herein, includes human CSF1R (hCSF1R), variants, isoforms, and species homologs of hCSF1R, as well as analogs that share at least one common epitope with hCSF1R. The term "CSF1," as used herein, includes human CSF1 (hCSF1), variants, isoforms, and species homologs of hCSF1, as well as analogs that share at least one common epitope with hCSF1.

[0115] "Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase" refers to an enzyme in the NOX family of enzymes in myeloid cells that generates immunosuppressive reactive oxygen species (ROS). Five NOX enzymes (NOX1-NOX5) have been found to be involved in cancer development and immunosuppression. Elevated ROS levels have been detected in almost all cancers and promote many aspects of tumor development and progression. ROS produced by NOX impairs NK and T cell function, and inhibition of NOX in myeloid cells improves the antitumor function of neighboring NK and T cells. As used herein, the term "NOX" includes human NOX (hNOX), hNOX mutants, isoforms, and species homologs, as well as analogs that share at least one epitope with hNOX.

[0116] Another immune checkpoint that can be targeted according to the present disclosure is signaling mediated by "tryptophan-2,3-dioxygenase" (TDO). TDO represents an alternative pathway to IDO in tryptophan degradation and is involved in immunosuppression. Because tumor cells can catabolize tryptophan via TDO instead of IDO, TDO may represent an additional target for checkpoint blockade. Indeed, numerous cancer cell lines have been found to upregulate TDO, and TDO can complement IDO inhibition. As used herein, the term "TDO" includes human TDO (hTDO), mutants, isoforms, and species homologs of hTDO, as well as analogs that share at least one epitope with hTDO.

[0117] Many immune checkpoints are regulated by the interaction of specific receptors with ligand pairs, such as those described above. Thus, immune checkpoint proteins mediate immune checkpoint signaling. For example, checkpoint proteins directly or indirectly regulate T cell activation, T cell proliferation, and / or T cell function. Cancer cells often utilize these checkpoint pathways to protect themselves from immune system attack. Therefore, the function of checkpoint proteins is typically to regulate T cell activation, T cell proliferation, and / or T cell function. Immune checkpoint proteins thus regulate and maintain self-tolerance and the duration and scope of physiological immune responses. Many immune checkpoint proteins belong to the B7:CD28 family or the tumor necrosis factor receptor (TNFR) superfamily and activate signaling molecules recruited to their cytoplasmic domains upon binding to specific ligands (Suzuki et al., 2016, Jap J Clin Onc, 46:191-203).

[0118] The term "dysfunctional," as used herein, refers to immune cells in a state of reduced immune responsiveness to antigenic stimulation, including unresponsiveness to antigen recognition and impaired ability to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2), and / or target cell killing.

[0119] The term "anergy," as used herein, refers to a state of unresponsiveness to antigenic stimulation resulting from defective or insufficient signals delivered via the T cell receptor (TCR). T cell anergy can also occur upon stimulation with an antigen in the absence of costimulation, resulting in cells becoming resistant to subsequent activation by the antigen, even in the presence of costimulation. The state of unresponsiveness can often be negated by the presence of IL-2. Anergic T cells do not undergo clonal expansion and / or acquire effector functions.

[0120] The term "exhaustion," as used herein, refers to immune cell exhaustion, such as the T cell failure caused by persistent TCR signaling that occurs during many chronic infections and cancers. It is distinct from anergy in that it results from persistent signaling, rather than from defective or deficient signaling. Exhaustion is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional landscape that differs from that of functional effector or memory T cells. Exhaustion prevents optimal control of disease (e.g., infection or tumor). Exhaustion can result from both extrinsic negative regulatory pathways (e.g., immunomodulatory cytokines) as well as cell-intrinsic negative regulatory pathways (inhibitory immune checkpoint pathways, such as those described herein).

[0121] "Enhancing T cell function" means inducing, causing, or stimulating T cells to have sustained or amplified biological function, or reviving or reactivating exhausted or inactive T cells. Examples of enhancing T cell function include increased gamma interferon secretion from CD8+ T cells, increased proliferation, and increased antigen responsiveness (e.g., tumor clearance) compared to their levels before the intervention. In one embodiment, the level of enhancement is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 200%, or more. Methods for measuring this enhancement are known to those skilled in the art.

[0122] The term "inhibitory nucleic acid" or "inhibitory nucleic acid molecule," as used herein, refers to a nucleic acid molecule, e.g., DNA or RNA, that reduces, inhibits, interferes with, or negatively modulates, fully or partially, one or more checkpoint proteins. Inhibitory nucleic acid molecules include, but are not limited to, oligonucleotides, siRNAs, shRNAs, antisense DNA or RNA molecules, and aptamers (e.g., DNA or RNA aptamers).

[0123] The term "oligonucleotide," as used herein, refers to a nucleic acid molecule capable of reducing protein expression, particularly the expression of a checkpoint protein, such as a checkpoint protein described herein. Oligonucleotides are short DNA or RNA molecules, typically containing 2 to 50 nucleotides. Oligonucleotides may be single-stranded or double-stranded. Checkpoint inhibitor oligonucleotides may be antisense oligonucleotides.

[0124] Antisense oligonucleotides are single-stranded DNA or RNA molecules that are complementary to a given sequence, particularly the nucleic acid sequence (or fragments thereof) of a checkpoint protein. Antisense RNA is typically used to prevent the protein translation of mRNA, for example, mRNA encoding a checkpoint protein, by binding to the mRNA. Antisense DNA is typically used to target specific, complementary (coding or non-coding) RNA. Upon binding, such DNA / RNA hybrids can be degraded by the enzyme RNase H. Furthermore, morpholino antisense oligonucleotides can be used for gene knockdown in vertebrates. For example, Kryczek et al., 2006 (J Exp Med, 203:871-81) designed a B7-H4-specific morpholino that specifically blocked B7-H4 expression in macrophages, resulting in increased T cell proliferation and reduced tumor volume in mice bearing tumor-associated antigen (TAA)-specific T cells.

[0125] The terms "siRNA," "short interfering RNA," or "small inhibitory RNA" are used interchangeably herein to refer to double-stranded RNA molecules, typically 20-25 base pairs in length, that interfere with the expression of specific genes, such as genes encoding checkpoint proteins with complementary nucleotide sequences. In one embodiment, siRNA interferes with mRNA, thereby blocking translation, for example, of immune checkpoint proteins. Transfection of exogenous siRNA can be used for gene knockdown, but this effect may be only transient, especially in rapidly dividing cells. Stable transfection can be achieved, for example, by RNA modification or by using an expression vector. Useful modifications and vectors for stable transfection of cells with siRNA are known in the art. siRNA sequences can also be modified to introduce a short loop between the two strands, resulting in "small hairpin RNAs" or "shRNAs." shRNAs can be processed into functional siRNAs by Dicer. shRNAs have relatively slow rates of degradation and turnover. Thus, the immune checkpoint inhibitor can be an shRNA.

[0126] The term "aptamer," as used herein, refers to a single-stranded nucleic acid molecule, typically 25-70 nucleotides in length, e.g., DNA or RNA, capable of binding to a target molecule, such as a polypeptide. In one embodiment, an aptamer binds to an immune checkpoint protein, e.g., an immune checkpoint protein described herein. For example, an aptamer according to the present disclosure can specifically bind to an immune checkpoint protein or polypeptide, or to a molecule in a signaling pathway that modulates the expression of an immune checkpoint protein or polypeptide. The generation and therapeutic use of aptamers is well known in the art (see, e.g., U.S. Pat. No. 5,475,096).

[0127] The term " small molecule inhibitor " or " small molecule " is used interchangeably herein and refers to a low molecular weight organic compound, usually up to 1000 daltons, that can completely or partially reduce, inhibit, interfere with or negatively modulate one or more checkpoint proteins as mentioned above.Such small molecule inhibitors are usually synthesized by organic chemistry, but can also be isolated from natural sources such as plants, fungi and microorganisms.Small molecular weight allows small molecule inhibitors to penetrate cell membranes and diffuse quickly.For example, various A2AR antagonists known in the art are organic compounds with molecular weights of less than 500 daltons.

[0128] The term "cell-based therapy" refers to the transplantation of cells (e.g., T lymphocytes, dendritic cells, or stem cells) that express an immune checkpoint inhibitor into a subject for the purpose of treating a disease or disorder (e.g., a cancer disease).

[0129] The term "oncolytic virus" as used herein refers to a virus that selectively replicates in cancerous or hyperproliferative cells, either in vitro or in vivo, with no or minimal effect on normal cells, and can slow the growth of or induce the death of such cells. Oncolytic viruses for the delivery of immune checkpoint inhibitors include expression cassettes that can encode immune checkpoint inhibitors, such as inhibitory nucleic acid molecules such as siRNA, shRNA, oligonucleotides, antisense DNA or RNA, aptamers, antibodies or fragments thereof, or soluble immune checkpoint proteins or fusions. The oncolytic virus is preferably replication-competent, and the expression cassette is under the control of a viral promoter, such as a synthetic early / late poxvirus promoter. Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), rhabdoviruses (e.g., picornaviruses, such as Seneca Valley virus; SVV-001), coxsackieviruses, parvoviruses, Newcastle disease virus (NDV), herpes simplex virus (HSV; OncoVEX GMCSF), retroviruses (e.g., influenza virus), measles virus, reovirus, Sindbis virus, vaccinia virus, such as those representatively described in WO2017 / 209053 (including the Copenhagen, Western Reserve, and Wyeth strains), and adenoviruses (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAd1, H101, AD5 / 3-D24-GMCSF). Methods for producing and using recombinant oncolytic viruses containing soluble forms of immune checkpoint inhibitors are disclosed in WO2018 / 022831, which is incorporated herein by reference in its entirety. The oncolytic virus can be used as an attenuated virus.

[0130] A "treatment cycle" is defined herein as the period within which separate dosages of a binding agent are added due to their pharmacodynamics, or in other words, the period after the administered binding agent has been substantially eliminated from the subject's body. Multiple smaller doses over a short time frame, e.g., within 2-24 hours, e.g., within 2-12 hours, or on the same day, may be equivalent to a larger single dose.

[0131] In the context of the present invention, the terms "treatment," "treating," or "therapeutic intervention" refer to the management and care of a subject for the purpose of combating a condition, such as a disease or disorder. This term is intended to include all areas of treatment for a given condition from which a subject is afflicted, such as the administration of therapeutically effective compounds to alleviate symptoms or complications, delay the progression of the disease, disorder, or condition, reduce or alleviate symptoms and complications, and / or cure or eliminate the disease, disorder, or condition, as well as to prevent the condition, where prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition, or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications. In one embodiment, "treatment" refers to the administration of an effective amount of a therapeutically active binding agent, such as the administration of a therapeutically active antibody of the present disclosure, for the purpose of alleviating, alleviating, arresting, or eradicating (curing) the symptoms or pathology.

[0132] Resistance to, failure to respond to, and / or recurrence from treatment with the binding agents of the present disclosure can be determined according to the Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST criteria v1.1). The RECIST criteria are listed in the table below (LD: longest dimension).

[0133] Table 4: Response definitions (RECIST criteria v1.1) [Table 4]

[0134] "Best overall response" is the best response recorded from the start of treatment until disease progression / recurrence (the minimum measurement recorded from the start of treatment will be used as the reference for PD). Subjects with CR or PR are considered to have an objective response. Subjects with CR, PR, or SD are considered to have controlled disease. Subjects with NE are counted as non-responders. "Best overall response" is the best response recorded from the start of treatment until disease progression / recurrence (the minimum measurement recorded from the start of treatment will be used as the reference for PD). Subjects with CR, PR, or SD are considered to have controlled disease. Subjects with NE are counted as non-responders.

[0135] Objective response rate (ORR) is the percentage of all subjects in a study or treatment group who have either a partial or complete response to treatment. ORR can be calculated by adding the number of subjects with a CR and the number of subjects with a PR and dividing the resulting total by the total number of subjects in the treatment group. ORR eval , i.e., ORR of all evaluable subjects in a study or treatment group is the percentage of all evaluable subjects in a study or treatment group who have either a partial or complete response to treatment.

[0136] Disease control rate (DCR) is the percentage of all subjects in a study or treatment group who have a complete response, partial response, or stable disease (CR, PR, or SD) to treatment.DCR can be calculated by adding the number of subjects with CR, the number of subjects with PR, and the number of subjects with SD, and dividing the resulting total by the total number of subjects in the treatment group.DCR eval , i.e., the DCR of all evaluable subjects in a study or treatment group is the percentage of all evaluable subjects in a study or treatment group who have either a complete response, partial response, or stable disease (CR, PR, or SD) to treatment.

[0137] "Duration of response (DOR)" applies only to subjects whose confirmed best overall response is CR or PR and is defined as the time from first evidence of objective tumor response (CR or PR) to the date of first PD or death from the underlying cancer.

[0138] "Progression-free survival (PFS)" is defined as the number of days from Day 1 of Cycle 1 to first documented progression or death from any cause.

[0139] "Overall survival (OS)" is defined as the number of days from day 1 of cycle 1 to death from any cause. If it is not known whether the subject died, OS will be adjusted to the latest date the subject is known to be alive (on or before the date of censoring).

[0140] In the context of the present disclosure, the term "treatment regimen" refers to a structured treatment plan designed to improve and maintain health.

[0141] The term "effective amount" or "therapeutically effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount of a binding agent, e.g., an antibody such as a multispecific antibody or a monoclonal antibody, may vary according to factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the binding agent to elicit a desired response in the individual. A therapeutically effective amount is also an amount such that the therapeutically beneficial effects outweigh any toxic or harmful effects of the binding agent or fragment thereof. If the patient does not respond adequately to the initial dose, a higher dose (or a higher dose, effectively achieved by a different, more localized route of administration) may be used. If unwanted side effects occur in a patient using a given dose, a lower dose (or a lower dose, effectively achieved by a different, more localized route of administration) may be used.

[0142] As used herein, the term "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. "Cancer cells" refer to abnormal cells that develop by rapid and uncontrolled cell proliferation and continue to grow after the stimulus that initiated the new growth has ceased.

[0143] The term "cancer" according to the present disclosure includes leukemia, seminoma, melanoma, sarcoma, myeloma, teratoma, lymphoma, mesothelioma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, renal cancer, urothelial cancer, adrenal cancer, adrenocortical cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastric cancer, digestive cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, penile cancer, uterine cancer, ovarian cancer and lung cancer, and metastases thereof. Examples thereof are lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer or metastases of the above mentioned cancer types or tumors.

[0144] The term "cancer," according to the present disclosure, also includes cancer metastasis. "Metastasis" refers to the spread of cancer cells from their original site to another part of the body. The formation of metastasis is a highly complex process that relies on the detachment of malignant cells from the primary tumor, infiltration of the extracellular matrix, penetration of the endothelial basement membrane into body cavities and blood vessels, and subsequent infiltration of the target organ after transport by blood. Ultimately, the growth of new tumors at the target site, i.e., secondary or metastatic tumors, depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor because tumor cells or elements remain and may develop metastatic potential. In one embodiment, the term "metastasis," according to the present disclosure, refers to "distant metastasis," which refers to metastasis far from the primary tumor and regional lymph node system.

[0145] Terms such as "reduce," "inhibit," "interfere," and "negatively modulate," as used herein, refer to the ability to cause an overall decrease in levels, for example, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete or substantially complete inhibition, i.e., a reduction to zero or substantially to zero.

[0146] Terms such as "increase" or "enhance" in one embodiment relate to an increase or enhancement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[0147] "Physiological pH," as used herein, refers to a pH of about 7.5.

[0148] "Wt. %," as used in disclosing the present invention, refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g), expressed as a percentage of the total weight of the total composition in grams (g).

[0149] The term "freeze" refers to the solidification of a liquid, usually by the removal of heat.

[0150] The term "lyophilize" or "freeze-drying" refers to freeze-drying a substance by freezing the substance and then reducing the surrounding pressure (e.g., to less than 15 Pa, e.g., less than 10 Pa, less than 5 Pa, or 1 Pa or less) to cause the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilize" and "freeze-dry" are used interchangeably herein.

[0151] The term "recombinant," in the context of the present disclosure, means "made by genetic engineering." In one embodiment, "recombinant," in the context of the present disclosure, is non-naturally occurring.

[0152] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a naturally occurring peptide or nucleic acid is a peptide or nucleic acid that exists in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory. The term "naturally occurring" means "naturally occurring", and includes not only known objects, but also objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0153] The term "peptide," according to the present disclosure, includes oligopeptides and polypeptides, and refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together via peptide bonds. The term "protein" refers to larger peptides, particularly peptides having at least about 151 amino acids, although the terms "peptide" and "protein" are generally used interchangeably herein.

[0154] A "therapeutic protein," when provided to a subject in a therapeutically effective amount, has a positive or beneficial effect on the subject's condition or pathology. In one embodiment, a therapeutic protein has curative or palliative properties and can be administered to ameliorate, alleviate, relieve, reverse, delay the onset, or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic protein may also have prophylactic properties and can be used to delay the onset of a disease or lessen the severity of such a disease or pathological condition. The term "therapeutic protein" includes whole proteins or peptides and may also refer to therapeutically active fragments thereof. It may also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, antigens for vaccination and immunostimulants, such as cytokines.

[0155] The term "portion" refers to a given portion. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" may refer to a contiguous or discontinuous portion of said structure.

[0156] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure such as an amino acid sequence or a protein refers to a continuous element of the structure. When used in the context of a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0157] "Fragment", when referring to an amino acid sequence (peptide or protein), refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. C-terminally truncated fragments (N-terminal fragments) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. N-terminally truncated fragments (C-terminal fragments) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence.

[0158] According to the present disclosure, a portion or fragment of a peptide or protein preferably possesses at least one functional property of the peptide or protein from which it is derived. Such functional properties include pharmacological activity, interaction with other peptides or proteins, enzymatic activity, interaction with antibodies, and selective binding of nucleic acids. For example, a pharmacologically active fragment of a peptide or protein possesses at least one pharmacological activity of the peptide or protein from which it is derived. A portion or fragment of a peptide or protein preferably comprises a sequence of at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, or at least 50 consecutive amino acids of the peptide or protein. A portion or fragment of a peptide or protein preferably comprises a sequence of up to 8, particularly up to 10, at most 12, at most 15, at most 20, at most 30, or at most 55 consecutive amino acids of the peptide or protein.

[0159] "Mutant" as used herein refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence, or may be a modified version of the wild-type amino acid sequence. Preferably, the mutant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid modifications, preferably 1 to about 10 or 1 to about 5 amino acid modifications, compared to the parent.

[0160] "Wild-type" or "WT" or "native," as used herein, refers to an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0161] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is expected to be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably expressed over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably expressed for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, and in some embodiments for consecutive such amino acids. In some embodiments, the degree of similarity or identity is expressed for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0162] "Sequence similarity" refers to the percentage of either identical amino acids or amino acids that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of identical amino acids between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of identical nucleotides between the sequences.

[0163] The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of identical nucleotides or amino acids between the sequences to be compared in an optimally aligned state. The percentage is purely statistical, although the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the sequences to be compared. Comparison of two sequences is usually carried out by comparing the sequences after optimal alignment over a segment or "window of comparison" to identify local regions of corresponding sequences. Optimal alignment for comparison may be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithms, available at the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi).In some embodiments, the algorithm parameters used for the BLASTN algorithm at the NCBI website include: (i) an expected threshold set to 10; (ii) a character size set to 28; (iii) maximum match in the query range set to 0; (iv) match / mismatch scores set to 1, -2; (v) a gap cost set to linear; and (vi) a filter if low complexity regions are used. In some embodiments, the algorithm parameters used for the BLASTP algorithm at the NCBI website include: (i) an expected threshold set to 10; (ii) a character size set to 3; (iii) maximum match in the query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap costs set to presence:11, extension:1; and (vi) an adjustment of the conditional compositional score matrix.

[0164] Percentage identity is obtained by determining the number of identical positions where the sequences being compared match, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0165] In some embodiments, the degree of similarity or identity is expressed over a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference amino acid sequence consists of 200 amino acid residues, the degree of identity is expressed over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acid residues, in some embodiments, for consecutive such amino acid residues. In some embodiments, the degree of similarity or identity is expressed over the entire length of the reference sequence.

[0166] Homologous amino acid sequences exhibit, according to the present disclosure, at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0167] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily prepared with the aid of known peptide synthesis techniques, for example, by solid-phase synthesis and similar methods.

[0168] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical to or similar to those of the amino acid sequence from which it is derived, i.e., functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant," as used herein, particularly refers to a variant molecule or sequence that contains an amino acid sequence in which one or more amino acids have been altered compared to the amino acid sequence of a parent molecule or sequence, yet is still capable of performing one or more of the functions of the parent molecule or sequence, e.g., capable of inducing an immune response. In one embodiment, the alterations in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced, but still significantly present, for example, the immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence, however, in other embodiments, the immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0169] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a designated amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, substantially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence can be a variant of the particular sequence or a fragment thereof. For example, one of skill in the art will understand that antigens suitable for use herein may be altered to have a sequence that differs from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0170] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-native environment, such as a host cell. In a preferred embodiment, the binding agent used in the present disclosure is in a substantially purified form.

[0171] The term "genetic modification" or simply "modification" includes the transfection of a cell with a nucleic acid. The term "transfection" refers to the introduction of a nucleic acid, particularly RNA, into a cell. For purposes of the present disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such a cell, where the cell may be present in a subject, e.g., a patient. Thus, according to the present disclosure, cells for transfection of nucleic acids described herein may be present in vitro or in vivo; for example, the cells may form part of an organ, tissue, and / or organism of a patient. According to the present disclosure, transfection may be transient or stable. For some applications of transfection, transient expression of the transfected genetic material is sufficient. RNA may be transiently transfected into cells to express its encoded protein. Typically, nucleic acids introduced during the transfection process are not integrated into the nuclear genome, resulting in the exogenous nucleic acid being diluted or degraded through mitosis. Cells that allow episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must be performed.Such stable transfection can be achieved by using a virus-based system or a transposon-based system for transfection.Generally, the nucleic acid encoding the antigen is transiently transfected into the cell.RNA can also be transfected into the cell to transiently express its encoded protein.

[0172] According to the present disclosure, a peptide or protein analog is a modified form of the peptide or protein from which it is derived, retaining at least one functional property of the peptide or protein. For example, a pharmacologically active analog of a peptide or protein retains at least one pharmacological activity of the peptide or protein from which it is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of any molecule associated with the protein or peptide, such as carbohydrates, lipids, and / or proteins or peptides. In one embodiment, a protein or peptide "analog" includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to an antibody or another cellular ligand. The term "analog" also covers all functional chemical equivalents of the proteins and peptides.

[0173] "Activation" or "stimulation" as used herein refers to the state of immune effector cells, such as T cells, that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector function. The term "activated immune effector cells" refers, inter alia, to immune effector cells that have undergone cell division.

[0174] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, causing it to differentiate into an effector cell, such as an effector T cell.

[0175] The term "clonal expansion" or "expansion" refers to the process by which a specific entity multiplies. In the context of the present disclosure, the term is preferably used in the context of an immune response, in which immune effector cells are stimulated by an antigen and proliferate, resulting in the amplification of specific immune effector cells that recognize said antigen. Preferably, clonal expansion leads to the differentiation of immune effector cells.

[0176] "Antigen" according to the present disclosure encompasses any substance expected to elicit an immune response and / or any substance against which an immune response or immune mechanism, e.g., a cellular response, is directed. This also includes situations in which an antigen is processed into antigenic peptides, particularly when presented in the context of MHC molecules, and an immune response or immune mechanism is directed against one or more antigenic peptides. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with antibodies or T lymphocytes (T cells). The term "antigen," according to the present disclosure, includes any molecule that contains at least one epitope, e.g., a T cell epitope. Preferably, an antigen in the context of the present disclosure is a molecule that, after appropriate processing, preferably induces an immune response specific to the antigen (including cells expressing the antigen). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0177] According to the present disclosure, any suitable antigen that is a candidate for an immune response can be used, and in this case, the immune response may be both a humoral and a cellular immune response. In the context of some embodiments of the present disclosure, the antigen is preferably presented by cells, preferably by antigen-presenting cells in the context of MHC molecules, thereby generating an immune response against the antigen. The antigen is preferably a product corresponding to or derived from a naturally occurring antigen. Such naturally occurring antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents, and the pathogen or antigen may also be a tumor antigen. According to the present disclosure, the antigen may be a naturally occurring product, such as a viral protein, or a portion thereof.

[0178] The term "disease-associated antigen" is used in its broadest sense and refers to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that is expected to stimulate the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by microorganisms, typically microbial antigens (e.g., bacterial or viral antigens), or antigens associated with cancer, typically tumors, such as tumor antigens.

[0179] In a preferred embodiment, the antigen is a tumor antigen, i.e., a part of a tumor cell, particularly one that is present primarily intracellularly or as a surface antigen of the tumor cell. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, for example, an antigen derived from a virus, bacterium, unicellular organism, or parasite, for example, a viral antigen such as a viral ribonucleoprotein or capsid protein. In particular, the antigen is capable of inducing the activation of cells of the immune system, preferably CD4, in particular through modulation of the activity of T cell receptors. + and CD8 + It must be presented by MHC molecules leading to lymphocyte modulation, particularly activation.

[0180] The term "tumor antigen" refers to a component of a cancer cell, which may be derived from the cytoplasm, cell surface, or cell nucleus. This particularly refers to an antigen produced intracellularly or as a surface antigen on tumor cells. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin, and fetal sulfoglycoprotein, α2-H-iron protein, and γ-fetoprotein, as well as various viral tumor antigens. According to the present disclosure, tumor antigens preferably include any antigen characteristic of tumors or cancers in terms of type and / or expression level, as well as tumor or cancer cells.

[0181] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0182] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A bacterial antigen may be derived from the bacterial cell wall or cytoplasmic membrane.

[0183] The term "epitope" refers to an antigenic determinant in a molecule, such as an antigen; that is, a portion or fragment of a molecule that is recognized by the immune system, e.g., by antibodies, T cells, or B cells, particularly when presented in the context of an MHC molecule. In one embodiment, "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes typically consist of surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that binding to the former is lost in the presence of denaturing solvents, but not to the latter. Epitopes may include amino acid residues directly involved in binding as well as other amino acid residues not directly involved in binding, e.g., amino acid residues that are effectively blocked or covered by the specifically antigen-binding peptide (in other words, amino acid residues within the footprint of the specifically antigen-binding peptide).

[0184] An epitope of a protein preferably comprises a contiguous or discontinuous portion of said protein and is preferably about 5 to about 100, preferably about 5 to about 50, more preferably about 8 to about 0, and most preferably about 10 to about 25 amino acids in length; for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. It is particularly preferred that the epitope in the context of the present disclosure is a T cell epitope.

[0185] Terms such as "epitope," "antigen fragment," "immunogenic peptide," and "antigenic peptide" are used interchangeably herein and preferably refer to an incomplete representation of an antigen that is preferably capable of eliciting an immune response against an antigen or a cell that expresses or contains the antigen, preferably a cell that presents the antigen. Preferably, these terms refer to the immunogenic site of an antigen. Preferably, the immunogenic site is a part of the antigen that is recognized by (i.e., specifically binds to) a T cell receptor, especially when presented in the context of an MHC molecule. Certain preferred immunogenic sites bind to MHC class I or class II molecules. The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. An epitope of an antigen may include a contiguous or discontinuous portion of the antigen and may be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 8 to about 25 amino acids in length. For example, an epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, the epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0186] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of MHC molecules. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or diseased cells in immune responses; in this case, MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, the binding peptides are typically about 8 to about 10 amino acids in length, although longer or shorter peptides can also be effective. For class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer and shorter peptides may also be effective.

[0187] Peptide and protein antigens may be 2 to 100 amino acids in length, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In some embodiments, peptides may be greater than 50 amino acids. In some embodiments, peptides may be greater than 100 amino acids.

[0188] The peptide or protein antigen can be any peptide or protein that is capable of inducing or increasing the ability of the immune system to generate antibody and T cell responses against the peptide or protein.

[0189] In one embodiment, the vaccine antigen, i.e., the antigen against which an immune response is induced upon inoculation into a subject, is recognized by immune effector cells. Preferably, when recognized by immune effector cells, the vaccine antigen can, in the presence of an appropriate costimulatory signal, induce the stimulation, priming, and / or expansion of immune effector cells bearing antigen receptors that recognize the vaccine antigen. In the context of the disclosed embodiments of the present invention, the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen-presenting cell. In one embodiment, the antigen is presented by a diseased cell (e.g., a tumor cell or an infected cell). In one embodiment, the antigen receptor is a TCR that binds to an epitope of an antigen presented in the context of an MHC. In one embodiment, when the TCR is expressed by and / or present on a T cell, binding to an antigen presented by a cell, such as an antigen-presenting cell, results in the stimulation, priming, and / or expansion of the T cell. In one embodiment, when a TCR is expressed by and / or present on a T cell, binding to an antigen presented on the diseased cell results in cytolysis and / or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors such as perforin and granzymes.

[0190] In one embodiment, the antigen receptor is an antibody or B cell receptor that binds to an epitope in the antigen. In one embodiment, the antibody or B cell receptor binds to a native epitope of the antigen.

[0191] The terms "expressed on the cell surface" or "associated with the cell surface" mean that a molecule, such as an antigen, is associated with and positioned on the plasma membrane of a cell, where at least a portion of the molecule faces the extracellular space of the cell and is accessible from outside the cell, e.g., by an antibody located outside the cell. In this context, the portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association may be direct or indirect. For example, the association may be via one or more transmembrane domains, one or more lipid anchors, or via interaction with any other protein, lipid, sugar, or other structure that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion, or may be a protein that associates with the surface of a cell through interaction with another protein that is a transmembrane protein.

[0192] "Cell surface" or "surface of a cell" is used according to its ordinary meaning in the art and thus includes the outside of a cell that is available for binding by proteins and other molecules. An antigen is expressed on the surface of a cell when it is on the surface of said cell and is available for binding, for example, by an antigen-specific antibody added to the cell.

[0193] The term "extracellular portion" or "exodomain" in the context of the present disclosure refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and that is preferably available from the outside of said cell for binding to a molecule, such as an antibody, that is on the outside of said cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0194] The terms "T cell" and "T lymphocyte" are used interchangeably herein, examples of which include T helper cells (CD4 + T cells), and cytotoxic T cells (CTL, CD8 +T cells). The term "antigen-specific T cells" or similar terms particularly relates to T cells that recognize the antigen that targets the T cell when presented on the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, in the context of an MHC molecule, and preferably exert T cell effector function. A T cell is considered antigen-specific if it kills a target cell expressing the antigen. T cell specificity can be assessed using any of a variety of standard techniques, for example, a chromium release assay or a proliferation assay. Alternatively, the synthesis of lymphokines (e.g., interferon-γ) may be measured. In certain embodiments of the present disclosure, RNA (particularly mRNA) encodes at least one epitope.

[0195] The term "target" is intended to mean a substance, such as a cell or tissue, that is the target for an immune response, such as a cellular immune response. Targets include cells that present an antigen or an antigenic epitope, i.e., a peptide fragment derived from an antigen. In one embodiment, the target cell is a cell that expresses the antigen, preferably a cell that presents said antigen in association with class I MHC.

[0196] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of said antigen (e.g., degradation of a protein into peptides), and the association (e.g., via binding) of one or more of these fragments with an MHC molecule for presentation to specific T cells by a cell, preferably an antigen-presenting cell.

[0197] "Antigen-responsive CTL" refers to a CD8 CTL that is responsive to an antigen or a peptide derived from the antigen presented together with class I MHC on the surface of an antigen-presenting cell. + It means T cells.

[0198] According to the present disclosure, CTL responsiveness can include sustained calcium flux, cell division, production of cytokines such as IFNγ and TNFα, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of tumor antigen expressing target cells. CTL responsiveness can also be determined using artificial reporters that accurately represent CTL responsiveness.

[0199] The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense and refer to an integrated body response to an antigen, preferably a cellular immune response, a humoral immune response, or both. According to the present disclosure, the terms "immune response to" or "immune response against" a substance, such as an antigen, cell, or tissue, relates to an immune response, such as a cellular response, directed against such substance. An immune response involves the development of antibodies against one or more antigens, as well as the activation of antigen-specific T lymphocytes, preferably CD4 + and CD8 + T-lymphocytes, more preferably CD8 + The expansion of T-lymphocytes may include one or more responses selected from the group consisting of: expansion of T-lymphocytes, which can be detected by various in vitro proliferation or cytokine production tests.

[0200] The terms "inducing an immune response" and "eliciting an immune response" and similar terms in the context of the present disclosure refer to the induction of an immune response, preferably a cellular immune response, a humoral immune response, or both. The immune response may be protective / preventative / prophylactic and / or therapeutic. The immune response may be directed against any immunogen or antigen or antigenic peptide, preferably a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (e.g., influenza virus (A, B, or C), CMV, or RSV)). "Inducing" in this context can mean that prior to induction, there was no immune response against a particular antigen or pathogen, but it can also mean that prior to induction, there was a certain level of immune response against a particular antigen or pathogen, and that after induction, said immune response is enhanced. Thus, "inducing an immune response" in this context also includes "enhancing an immune response." Preferably, after inducing an immune response in an individual, said individual is protected from developing a disease, such as an infectious disease or a cancerous disease, or the disease state is alleviated by inducing an immune response.

[0201] The terms "cellular immune response," "cellular response," "cell-mediated immunity," or similar terms are meant to include cellular responses directed against cells characterized by antigen expression and / or antigen presentation with class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as either "helpers" or "killers." Helper T cells (CD4 + T cells (also called T cells) play a central role by regulating the immune response and are responsible for the production of killer cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs) kill cells such as diseased cells.

[0202] The term "humoral immune response" refers to the process in an organism in which antibodies are produced in response to substances or organisms, ultimately neutralizing and / or eliminating them. The specificity of the antibody response is mediated by T and / or B cells through membrane-bound receptors that bind to a single antigen. After binding the appropriate antigen and receiving various other activation signals, B lymphocytes divide, thereby producing antibody-secreting plasma cell clones in addition to memory B cells, each of which produces antibodies that recognize the same antigen epitope as that recognized by its antigen receptor. Memory B lymphocytes remain quiescent until later activated by their specific antigen. These lymphocytes provide the cellular basis of memory, resulting in the recruitment of antibody responses upon re-exposure to the specific antigen.

[0203] The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and relate to the procedure of administering to an individual one or more immunogens or antigens or derivatives thereof, particularly in the form of RNA (particularly mRNA) encoding them as described herein, to stimulate an immune response against said one or more immunogens or antigens or cells characterized by the presentation of said one or more immunogens or antigens.

[0204] "Cells characterized by antigen presentation" or "cells presenting antigens" or "MHC molecules presenting antigens on the surface of antigen-presenting cells" or similar expressions refer to cells such as diseased cells, in particular tumor or infected cells, or antigen-presenting cells that present antigens or antigenic peptides, either directly or after processing, in the environment of MHC molecules, preferably MHC class I and / or MHC class II molecules, most preferably MHC class I molecules.

[0205] The term "transcription", in the context of the present disclosure, relates to the process by which the genetic code in a DNA sequence is transcribed into RNA (especially mRNA), which can then be translated into peptides or proteins.

[0206] The term "expression," as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence. With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes where a strand of mRNA directs the assembly of a sequence of amino acids to produce a peptide or protein.

[0207] The terms "optionally" or "optionally," as used herein, mean that the subsequently described event, circumstance, or condition may or may not occur, and the description includes cases where said event, circumstance, or condition occurs and cases where it does not occur.

[0208] "Endogenous," as used herein, refers to any substance that is from or produced within an organism, cell, tissue, or system.

[0209] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining together of two or more elements or components or domains.

[0210] The term "disease" (also referred to herein as "disorder") refers to an abnormal condition affecting an individual's body. Disease is often understood as a medical condition associated with specific symptoms and signs. Diseases may be caused by factors originally from external sources, such as infectious diseases, or by internal malfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems, or death in the affected individual, or similar problems in those who come into contact with that individual. In this broader sense, "disease" sometimes includes injury, disability, disorder, syndrome, infection, isolated symptoms, deviant behavior, and abnormal changes in structure or function, while in other contexts and for other purposes, these may be considered distinct categories. Diseases typically affect individuals not only physically but also emotionally, as suffering from and living with many illnesses can alter a person's outlook on life and personality.

[0211] The term "therapeutic treatment" refers to any treatment that improves the health and / or prolongs (increases) the lifespan of an individual. The treatment may eliminate the disease in an individual, stop or slow the onset of the disease in an individual, inhibit or slow the onset of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual who currently has or previously had the disease.

[0212] The term "prophylactic treatment" or "preventive treatment" relates to any treatment intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein. Similarly, the term "method for preventing" in the context of disease progression, such as tumor or cancer progression, relates to any method intended to prevent a disease from progressing in an individual.

[0213] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate), or any other non-mammal, such as a bird (chicken), fish, or any other animal species, that may or may not have a disease or disorder, or that may be in need of preventative intervention such as vaccination, or in need of intervention such as protein replacement, that may be susceptible to or susceptible to a disease or disorder (e.g., cancer, infectious disease). In many embodiments, an individual is a human being. Unless otherwise specified, the terms "individual" and "subject" do not designate a particular age, and thus encompass adults, elderly people, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient."

[0214] The term "patient" refers to an individual or subject for treatment, in particular an affected individual or subject.

[0215] Aspects and embodiments of the present disclosure In a first aspect, the present disclosure provides a binding agent for use in a method for reducing or preventing the progression of, or treating, head and neck squamous cell carcinoma (HNSCC) in a subject, the method comprising administering to the subject: (i) the binding agent; (ii) a checkpoint inhibitor that is an inhibitor of the PD-1 / PD-L1 axis (i.e., a PD-1 / PD-L1 checkpoint inhibitor, particularly pembrolizumab); and (iii) a chemotherapy combination comprising (a) a platinum-based chemotherapy agent and (b) 5-fluorouracil, wherein the binding agent comprises a first binding region that binds CD40 and a second binding region that binds CD137.

[0216] As demonstrated in the present disclosure, a chemotherapy combination based on (i) stimulation with a binding agent that binds to human CD40 and human CD137, (ii) inhibition of the PD-1 / PD-L1 axis, and (iii) a combination of platinum-based chemotherapy and 5-fluorouracil amplifies the immune response. Without being bound by any theory, the rationale behind this surprising discovery may be as follows: CD137 binds to PD-1 + PD-L1 / PD-1 signaling is co-expressed on T cells. Therefore, blockade of PD-L1 / PD-1 signaling and costimulation via CD137 can synergize to enhance T cell effector function and improve response duration. Through conditional activation of CD40 and CD137, binding agents targeting CD40 and CD137 induce potent antitumor activity through enhanced T cell priming, cytokine and chemokine production, and expansion and survival of antigen-experienced T cells. The PD-(L)1 pathway is expected to be activated during priming as well as during continuous antigen exposure, which may reduce the magnitude of the immune response induced by binding agents targeting CD40 and CD137.

[0217] Pembrolizumab, or pembrolizumab combined with platinum and 5-fluorouracil (5-FU), has become the global standard of care (SOC) for patients with previously untreated recurrent or metastatic HNSCC based on the results of the KEYNOTE-048 (KN-048) trial. In the KN-048 trial, patients were randomized to receive pembrolizumab, pembrolizumab plus platinum (cisplatin or carboplatin) and 5-FU, or the EXTREME regimen (cisplatin or carboplatin + 5-FU + cetuximab).

[0218] In KN-048, an overall survival (OS) benefit was observed in 1L HNSCC patients with PD-L1 CPS ≥ 1 in the pembrolizumab + platinum + 5-FU arm vs. the EXTREME regimen (median OS was 13.6 vs. 10.4 months, respectively); however, there was no improvement in ORR (36.4% vs. 35.7%) or PFS (5.1 vs. 5.0 months) between treatment groups (EMA Assessment Report, 2019). Based on available data from the GCT1042-01 study at the time of data cutoff, the addition of a binding agent that binds to CD40 and CD137 as disclosed herein (specifically GEN1042) to the pembrolizumab + platinum + 5-FU regimen combination appears to improve ORR (36.4% vs. 66.7%) compared with platinum + 5-FU + pembrolizumab alone.

[0219] Thus, in one embodiment, the binder for use according to the first aspect of the present disclosure provides an increased ORR compared to SOC, for example, compared to a dosing regimen of the combination of pembrolizumab, a platinum-based chemotherapy agent, and 5-fluorouracil alone, or compared to a dosing regimen of the combination of the binder and pembrolizumab alone. For example, each of the combination of the binder, pembrolizumab, and chemotherapy may be administered at a dose that increases the ORR compared to standard of care, for example, compared to a dosing regimen of the combination of pembrolizumab, a platinum-based chemotherapy agent, and 5-fluorouracil alone, or compared to a dosing regimen of the combination of the binder and pembrolizumab alone. In some of the above embodiments, the ORR is at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, %, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%.

[0220] Alternatively, or in addition, in one embodiment, the binder for use according to the first aspect of the present disclosure exhibits an increased ORR compared to SOC, for example compared to a dosing regimen of a combination of pembrolizumab, a platinum-based chemotherapy agent, and 5-fluorouracil alone, or compared to a dosing regimen of a combination of the binder and pembrolizumab alone. eval For example, each of the combinations of the binder, pembrolizumab, and chemotherapy has an ORR compared to standard of care, e.g., compared to a dosing regimen of the combination of pembrolizumab, a platinum-based chemotherapy agent, and 5-fluorouracil alone, or compared to a dosing regimen of the combination of the binder and pembrolizumab alone. eval In some of the above embodiments, the ORR eval is at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, may be increased by at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

[0221] Alternatively, or in addition, in one embodiment, the binder for use according to the first aspect of the present disclosure provides an increased DCR compared to SOC, for example, compared to the administration regimen of a combination of pembrolizumab, a platinum-based chemotherapy agent, and 5-fluorouracil alone, or compared to the administration regimen of a combination of the binder and pembrolizumab alone. For example, each of the binder, pembrolizumab, and chemotherapy combination may be administered at a dose that increases the DCR compared to standard of care, for example, compared to the administration regimen of a combination of pembrolizumab, a platinum-based chemotherapy agent, and 5-fluorouracil alone, or compared to the administration regimen of a combination of the binder and pembrolizumab alone. In some of the above embodiments, the DCR is at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, %, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%.

[0222] Alternatively, or in addition, in one embodiment, the binder for use according to the first aspect of the present disclosure exhibits an increased DCR compared to SOC, for example compared to a dosing regimen of a combination of pembrolizumab, a platinum-based chemotherapy agent and 5-fluorouracil alone, or compared to a dosing regimen of a combination of the binder and pembrolizumab alone. eval For example, each of the combinations of the binder, pembrolizumab, and chemotherapy has a DCR greater than or equal to a standard of care, e.g., a dosing regimen of the combination of pembrolizumab, a platinum-based chemotherapy agent, and 5-fluorouracil alone, or a dosing regimen of the combination of the binder and pembrolizumab alone. eval In some of the above embodiments, the DCR eval is at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, may be increased by at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

[0223] Binding agents that bind to CD40 and CD137 In one embodiment, the CD40 is human CD40, particularly human CD40 comprising the sequence set forth in SEQ ID NO: 36. In one embodiment, the CD137 is human CD137, particularly human CD137 comprising the sequence set forth in SEQ ID NO: 38. In one embodiment, the CD40 is human CD40 and CD137 is human CD137. In one embodiment, the CD40 is human CD40 comprising the sequence set forth in SEQ ID NO: 36 and the CD137 is human CD137 comprising the sequence set forth in SEQ ID NO: 38.

[0224] In one embodiment of the binder according to the first aspect, a) a first binding region that binds to human CD40 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 7 or 9, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 8 or 10; b) The second antigen-binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 17 or 19, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18 or 20.

[0225] In one embodiment of the binder according to the first aspect, a) a first binding region that binds to human CD40 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; b) The second antigen-binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 11, 12, and 13, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively.

[0226] In one embodiment of the binder according to the first aspect, a) a first binding region that binds to human CD40 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 7 or 9, and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or 10; b) The second binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 17 or 19, and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 18 or 20.

[0227] In one embodiment of the binder according to the first aspect, a) the first binding region that binds to human CD40 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 7 or 9, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; b) The second binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 18 or 20.

[0228] In one embodiment of the binder according to the first aspect, a) the first binding region that binds to human CD40 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 10; b) The second binding region that binds to human CD137 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 19, and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0229] The binding agent may in particular be an antibody, such as a multispecific antibody, e.g., a bispecific antibody. The binding agent may also be in the form of a full-length antibody or an antibody fragment.

[0230] It is further preferred that the binding agent is a human or humanized antibody.

[0231] Each variable region may comprise three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).

[0232] The complementarity determining regions (CDRs) and framework regions (FRs) may be arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0233] In one embodiment of the first aspect, the binder is i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and ii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH). Includes:

[0234] In one embodiment of the first aspect, the binder is i) a polypeptide comprising the first light chain variable region (VL) and further comprising a first light chain constant region (CL); and ii) a polypeptide comprising the second light chain variable region (VL) and further comprising a second light chain constant region (CL). Includes:

[0235] In one embodiment of the first aspect, the binding agent is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises: i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH), and ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL). Includes; The second binding arm is iii) a polypeptide comprising the second heavy chain variable region (VH) and the second heavy chain constant region (CH); and iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL). Includes:

[0236] In one embodiment of the first aspect, the binding agent comprises i) a first heavy chain and a first light chain comprising the antigen-binding region capable of binding to CD40, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; and ii) a second heavy chain and a second light chain comprising the antigen-binding region capable of binding to CD137, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region.

[0237] Each of the first and second heavy chain constant regions (CH) may comprise one or more of the constant heavy chain 1 (CH1) region, the hinge region, the constant heavy chain 2 (CH2) region and the constant heavy chain 3 (CH3) region, preferably at least the hinge region, the CH2 region and the CH3 region.

[0238] Each of the first and second heavy chain constant regions (CHs) may comprise a CH3 region, and the two CH3 regions may comprise asymmetric mutations. Asymmetric mutations mean that the sequences of the first and second CH3 regions contain amino acid substitutions at positions that are not identical. For example, one of the first and second CH3 regions contains a mutation at position 405 in the human IgG1 heavy chain according to EU numbering, and the other of the first and second CH3 regions contains a mutation at position 409 in the human IgG1 heavy chain according to EU numbering.

[0239] In the first heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering may be substituted, and in the second heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering may be substituted. In certain embodiments, the first and second heavy chains are not substituted at the same positions (i.e., the first and second heavy chains contain asymmetric mutations).

[0240] In one embodiment of the binding agent according to the first aspect, (i) the amino acid at the position corresponding to F405 in human IgG1 heavy chain according to EU numbering is L in said first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 in human IgG1 heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in human IgG1 heavy chain according to EU numbering is R in said first heavy chain and the amino acid at the position corresponding to F405 in human IgG1 heavy chain according to EU numbering is L in said second heavy chain.

[0241] In one embodiment of the first aspect, the binding agent induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CHs) comprising human IgG1 hinge, CH2 and CH3 regions.

[0242] In one particular embodiment of the binding agent according to the first aspect, the first and second heavy chain constant regions (CH) are modified such that the antibody induces Fc-mediated effector function to a lesser extent compared to an otherwise identical antibody comprising the unmodified first and second heavy chain constant regions (CH). In particular, each or both of the unmodified first and second heavy chain constant regions (CH) may comprise, consist of, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 21 or 29.

[0243] Fc-mediated effector function can be determined by measuring the binding of a binding agent to Fcγ receptors, binding to C1q, or induction of Fc-mediated cross-linking of Fcγ receptors. In particular, Fc-mediated effector function can be determined by measuring the binding of a binding agent to C1q.

[0244] The first and second heavy chain constant regions of the binding agent may be modified such that binding of C1q to said antibody is reduced compared to the wild-type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, where C1q binding is preferably determined by ELISA.

[0245] In one embodiment of the binding agent according to the first aspect, in at least one of said first and second heavy chain constant regions (CH), one or more amino acids at positions corresponding to positions L234, L235, D265, N297 and P331 in a human IgG1 heavy chain according to EU numbering are not L, L, D, N and P, respectively.

[0246] In one embodiment of the binding agent according to the first aspect, the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering may be F and E in said first and second heavy chains, respectively.

[0247] In particular, the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering may be F, E, and A in the first and second heavy chain constant regions (HC), respectively.

[0248] In one embodiment of the binding agent according to the first aspect, the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F and E, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is L.

[0249] In one embodiment of the binding agent according to the first aspect, the positions corresponding to L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F, E, and A, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is L.

[0250] In one embodiment of the binding agent according to the first aspect, the constant region of said first and / or second heavy chain comprises: a) the sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 29 [IgG1-FC]; b) a subsequence of the sequence of a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions, e.g. at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2 or at most 1 substitution compared to the amino acid sequence defined in a) or b). The amino acid sequence comprises an amino acid sequence selected from the group consisting of:

[0251] In one embodiment of the binding agent according to the first aspect, the constant region of the first or second heavy chain, e.g. of the second heavy chain, comprises: a) the sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 30 [IgG1-F405L]; b) a subsequence of the sequence of a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 9 substitutions, e.g. at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2 or at most 1 substitution compared to the amino acid sequence defined in a) or b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0252] In one embodiment of the binding agent according to the first aspect, the constant region of the first or second heavy chain, e.g. the first heavy chain, comprises: a) the sequence set forth in SEQ ID NO: 23 or 31 [IgG1-F409R]; b) a subsequence of the sequence of a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions, e.g. at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2 or at most 1 substitution compared to the amino acid sequence defined in a) or b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0253] In one embodiment of the binding agent according to the first aspect, the constant region of said first and / or second heavy chain comprises: a) the sequence set forth in SEQ ID NO: 24 or SEQ ID NO: 32 [IgG1-Fc_FEA]; b) a subsequence of the sequence of a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 7 substitutions, e.g. at most 6 substitutions, at most 5, at most 4, at most 3, at most 2 or at most 1 substitution compared to the amino acid sequence defined in a) or b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0254] In one embodiment of the binding agent according to the first aspect, the constant region of said first and / or second heavy chain, e.g. of the second heavy chain, comprises: a) the sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 33 [IgG1-Fc_FEAL]; b) a subsequence of the sequence of a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 6 substitutions, e.g. at most 5 substitutions, at most 4 substitutions, at most 3, at most 2 or at most 1 substitution compared to the amino acid sequence defined in a) or b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0255] In one embodiment of the binding agent according to the first aspect, the constant region of said first and / or second heavy chain, e.g. of the first heavy chain, comprises: a) the sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 34 [IgG1-Fc_FEAR]; b) a subsequence of the sequence of a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 6 substitutions, e.g. at most 5 substitutions, at most 4, at most 3, at most 2 or at most 1 substitution compared to the amino acid sequence defined in a) or b). The amino acid sequence of the present invention comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of:

[0256] In one embodiment of the first aspect, the binding agent comprises a kappa (κ) light chain constant region.

[0257] In one embodiment of the first aspect, the binding agent comprises a lambda (λ) light chain constant region.

[0258] In one embodiment of the binding agent according to the first aspect, the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

[0259] In one embodiment of the binding agent according to the first aspect, the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

[0260] In one embodiment of the binding agent according to the first aspect, the first light chain constant region is a kappa (κ) light chain constant region and the second light chain constant region is a lambda (λ) light chain constant region, or the first light chain constant region is a lambda (λ) light chain constant region and the second light chain constant region is a kappa (κ) light chain constant region.

[0261] In one embodiment of the binding agent according to the first aspect, the kappa (κ) light chain is a) the sequence set forth in SEQ ID NO: 27; b) a subsequence of the sequence of a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions, e.g. at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2 or at most 1 substitution compared to the amino acid sequence defined in a) or b). The amino acid sequence comprises an amino acid sequence selected from the group consisting of:

[0262] In one embodiment of the binding agent according to the first aspect, the lambda (λ) light chain is a) the sequence set forth in SEQ ID NO: 28; b) a subsequence of the sequence of a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions, e.g. at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2 or at most 1 substitution compared to the amino acid sequence defined in a) or b). The amino acid sequence comprises an amino acid sequence selected from the group consisting of:

[0263] The binding agent (particularly the antibody) according to the first aspect is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In particular, the binding agent may be a full-length IgG1 antibody. In a preferred embodiment of the first aspect, the binding agent (particularly the antibody) is of the IgG1m(f) allotype.

[0264] In one embodiment, the binding agent is the antibody GEN1042 disclosed herein (i.e., this antibody contains the humanized VH and VL sequences set forth in Table 1, a human kappa light chain, and a human IgG1 heavy chain; the CD40-binding arm was produced using a human IgG1 heavy chain containing the following amino acid mutations: L234F, L235E, D265A, and F405L (FEAL), where the amino acid positions are numbered according to EU numbering (corresponding to SEQ ID NO: 33); and the CD137-binding arm was produced using a human IgG1 heavy chain containing the following amino acid mutations: L234F, L235E, D265A, and K409R (FEAR), where the amino acid positions are numbered according to EU numbering (corresponding to SEQ ID NO: 34)).

[0265] Preferably, the binding agent is administered in a suitable amount, i.e., the amount of binding agent administered, e.g., in each dose and / or treatment cycle, is an amount capable of inducing intracellular signaling upon binding to CD137 expressed on another cell. Thus, a suitable amount of a binding agent according to the present disclosure can transactivate two different cell types. In humans, CD40 is expressed on numerous cells, such as antigen-presenting cells (APCs), e.g., dendritic cells, whereas CD137 is expressed on T cells and other cells. Therefore, a suitable amount of a binding agent that binds to CD40 and CD137 according to the present disclosure can simultaneously bind to APCs and T cells expressing these receptors. Therefore, without being bound by theory, the binding agent can (i) mediate cell-to-cell interactions between APCs and T cells through receptor binding and (ii) activate both CD40 and CD137 simultaneously, which is primarily induced by cross-linking and receptor clustering upon cell-to-cell interaction and does not necessarily depend on the agonistic activity of the parent monospecific bivalent antibody. Thus, these transactivating binding agents exert costimulatory activity in the context of APC:T cell interactions, enabling T cell responses against tumor cells. Therefore, this mechanism of action may mirror natural T cell activation through antigen presentation by activated APCs, allowing APCs to present various tumor-specific antigens to T cells. Without being limited by theory, costimulatory activity may result in one or more of the following: (i) activation of specific T cells only (i.e., those in contact with APCs), as opposed to any T cells; (ii) reactivation of exhausted T cells through strong costimulation via activated APCs and triggering of CD137; and (iii) priming of T cells by inducing antigen presentation by activated APCs and simultaneously triggering CD137.

[0266] The amount of binding agent administered in each dose and / or treatment cycle may be within a range in which, inter alia, more than 5%, preferably more than 10%, more preferably more than 15%, even more preferably more than 20%, even more preferably more than 25%, even more preferably more than 30%, even more preferably more than 35%, even more preferably more than 40%, even more preferably more than 45%, and most preferably more than 50% of the binding agent binds to both CD40 and CD137.

[0267] In a preferred embodiment, the amount of binding agent administered is about 50-150 mg / day (e.g., about 60-140 mg / day, about 70-130 mg / day, about 80-120 mg / day, about 90-110 mg / day, or about 95-105 mg / day, e.g., about 100 mg / day), or about 0.62-1.88 mg / kg body weight / day (e.g., about 0.75-1.75 mg / kg body weight / day, about 0.87-1.63 mg / kg body weight / day, 1.00-1.50 mg / kg body weight / day, 1.12-1.38 mg / kg body weight / day, or 1.18-1.31 mg / kg body weight / day, e.g., about 1.25 mg / kg body weight / day), for each dose and / or each treatment cycle.

[0268] In a preferred embodiment, the amount of binding agent administered is, for example, about 335×10 -9 ~1020×10 -9 mol / day (e.g., approximately 400 × 10 -9 ~950×10 -9 mol / day, approximately 470×10 -9 ~880×10 -9 mol / day, approximately 540×10 -9 ~810×10 -9 mol / day, approximately 600×10 -9 ~750×10 -9 mol / day, or approximately 640 × 10 -9 ~710×10 -9 mol / day, e.g., about 675 × 10 -9 mol / day), or approximately 4.1 × 10 -9 ~12.7×10 -9mol / kg body weight / day (e.g., 5.0 × 10 -9 ~11.9×10 -9 mol / kg body weight / day, 5.8 × 10 -9 ~11.0×10 -9 mol / kg body weight / day, 6.7 × 10 -9 ~10.1×10 -9 mol / kg body weight / day, 7.5 × 10 -9 ~9.4×10 -9 mol / kg body weight / day, or 8.0 × 10 -9 ~8.9×10 -9 mol / kg body weight / day, e.g., about 8.4 mol / kg body weight / day).

[0269] According to these embodiments, the dose defined in mg / kg may be converted to a flat dose based on the median body weight of subjects to whom the binding agent is administered being 80 kg, or vice versa.

[0270] The binding agent can be administered by any method and by any route known in the art, hi a preferred embodiment, the binding agent is administered systemically, e.g., parenterally, particularly intravenously.

[0271] The binding agent can be administered in the form of any suitable pharmaceutical composition described herein. In a preferred embodiment, the binding agent is administered in the form of an infusion.

[0272] The binding agent can be administered before, simultaneously with, or after administration of an inhibitor of the PD-1 / PD-L1 axis (a PD-1 / PD-L1 checkpoint inhibitor).

[0273] In one embodiment, the binding agent is administered prior to administration of the PD-1 / PD-L1 checkpoint inhibitor. For example, the gap between the end of administration of the binding agent and the beginning of administration of the PD-1 / PD-L1 checkpoint inhibitor can be at least about 10 minutes, e.g., at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes, or up to about 12 hours, e.g., up to about 6 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2.5 hours, or up to about 2 hours.

[0274] In one embodiment, the binding agent is administered after administration of the PD-1 / PD-L1 checkpoint inhibitor. For example, the gap from the end of administration of the PD-1 / PD-L1 checkpoint inhibitor to the beginning of administration of the binding agent can be at least about 10 minutes, e.g., at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 120 minutes, or up to about 12 hours, e.g., up to about 6 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2.5 hours, or up to about 2 hours.

[0275] In one embodiment, the binding agent is administered simultaneously with the PD-1 / PD-L1 checkpoint inhibitor. For example, the binding agent and the PD-1 / PD-L1 checkpoint inhibitor may be administered using a composition containing both drugs. Alternatively, the binding agent may be administered to one limb of the subject, and the PD-1 / PD-L1 checkpoint inhibitor may be administered to another limb of the subject.

[0276] PD-1 / PD-L1 checkpoint inhibitors In one embodiment, immune checkpoint inhibitors suitable for use in the methods disclosed herein are antagonists of the inhibitory signals of the PD-1 / PD-L1 axis, such as antibodies that target PD-1 or PD-L1. These ligands and receptors, as well as other checkpoint proteins, are reviewed in Pardoll, D., Nature. 12: 252-264, 2012. Additional immune checkpoint proteins that can be targeted in accordance with the present disclosure are described herein.

[0277] In one embodiment, the immune checkpoint inhibitor prevents inhibitory signals associated with an immune checkpoint. In one embodiment, the immune checkpoint inhibitor is an antibody or fragment thereof that disrupts or inhibits inhibitory signaling associated with an immune checkpoint. In one embodiment, the immune checkpoint inhibitor is a small molecule inhibitor that disrupts or inhibits inhibitory signaling. In one embodiment, the immune checkpoint inhibitor is a peptide-based inhibitor that disrupts or inhibits inhibitory signaling. In one embodiment, the immune checkpoint inhibitor is an inhibitory nucleic acid molecule that disrupts or inhibits inhibitory signaling.

[0278] Inhibiting or blocking inhibitory immune checkpoint signaling results in the prevention or reversal of immune suppression and the establishment or enhancement of T cell immunity against cancer cells, as described herein. In one embodiment, inhibition of immune checkpoint signaling reduces or inhibits immune system dysfunction, as described herein. In one embodiment, inhibition of immune checkpoint signaling results in less dysfunction of impaired immune cells, as described herein. In one embodiment, inhibition of immune checkpoint signaling results in less dysfunction of impaired T cells, as described herein.

[0279] In one embodiment, the PD-1 / PD-L1 immune checkpoint inhibitor prevents the interaction between the checkpoint blocker proteins PD-1 and PD-L1 or PD-L2.

[0280] The PD-1 / PD-L1 immune checkpoint inhibitor may be an antibody, an antigen-binding fragment thereof, or a construct thereof comprising a portion of an antibody together with an antigen-binding fragment having the required specificity. The antibody or antigen-binding fragment thereof is as described herein. Antibodies or antigen-binding fragments that are immune checkpoint inhibitors include, in particular, antibodies or antigen-binding fragments thereof that bind to immune checkpoint proteins, such as immune checkpoint receptors or immune checkpoint receptor ligands. The antibody or antigen-binding fragment may also be conjugated to a further moiety as described herein. In particular, the antibody or antigen-binding fragment thereof is a chimerized, humanized, or human antibody. Preferably, the immune checkpoint inhibitor antibody or antigen-binding fragment thereof is an immune checkpoint receptor antagonist or an immune checkpoint receptor ligand antagonist.

[0281] In a preferred embodiment, the antibody that is an immune checkpoint inhibitor is an isolated antibody.

[0282] In one embodiment, the PD-1 / PD-L1 immune checkpoint inhibitor is an antibody, fragment thereof, or construct that prevents the interaction between the checkpoint blocker protein PD-1 and PD-L1 or PD-L2. In one embodiment, such an antibody, fragment thereof, or construct comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 81, 82, and 83, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 84, 85, and 86, respectively. In one embodiment, such an antibody, fragment thereof, or construct comprises a heavy chain variable region comprising an amino acid sequence having at least 85% (e.g., at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87, and a light chain variable region comprising an amino acid sequence having at least 85% (e.g., at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 88. In one embodiment, such an antibody, fragment or construct comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 88. In one embodiment, such an antibody, fragment or construct comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 89, and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0283] PD-1 / PD-L1 immune checkpoint inhibitors may be inhibitory nucleic acid molecules, such as oligonucleotides, siRNAs, shRNAs, antisense DNA or RNA molecules, and aptamers (e.g., DNA or RNA aptamers), particularly antisense oligonucleotides. In one embodiment, PD-1 / PD-L1 immune checkpoint inhibitors that are siRNAs interfere with mRNA and thus block translation, for example, of immune checkpoint proteins.

[0284] The PD-1 / PD-L1 checkpoint inhibitor may also be a soluble form of the molecule (or a variant thereof) itself, such as a soluble PD-L1 or a PD-L1 fusion.

[0285] In the context of the present disclosure, more than one checkpoint inhibitor can be used, and in this case, more than one checkpoint inhibitor targets different checkpoint pathways or targets the same checkpoint pathway.Preferably, more than one checkpoint inhibitor is a different checkpoint inhibitor.Preferably, when more than one different checkpoint inhibitor is used, particularly at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 different checkpoint inhibitors are used, preferably 2, 3, 4 or 5 different checkpoint inhibitors are used, more preferably 2, 3 or 4 different checkpoint inhibitors are used, even more preferably 2 or 3 different checkpoint inhibitors are used, and most preferably 2 different checkpoint inhibitors are used.

[0286] In one embodiment, the PD-1 / PD-L1 checkpoint inhibitor (or inhibitory immunomodulator or immune checkpoint blocker) is a component of the PD-1 / PD-L1 or PD-1 / PD-L2 signaling pathway. Thus, in one embodiment of the present disclosure, the PD-1 / PD-L1 checkpoint inhibitor is an inhibitor of the PD-1 signaling pathway. In a specific embodiment, the checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 inhibitor. In a specific embodiment, the checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 ligand inhibitor, for example, a PD-L1 inhibitor or a PD-L2 inhibitor. In a preferred embodiment, the checkpoint inhibitor of the PD-1 signaling pathway is an antibody, antigen-binding portion thereof, or construct thereof that disrupts or inhibits the interaction of the PD-1 receptor with one or more of its ligands, PD-L1 and / or PD-L2. Antibodies that bind to PD-1 and disrupt or inhibit the interaction of PD-1 with one or more of its ligands are known in the art. In certain embodiments, the antibody, antigen-binding portion thereof, or construct thereof specifically binds to PD-1. In certain embodiments, the antibody, antigen-binding portion thereof, or construct thereof specifically binds to PD-L1 and disrupts or inhibits its interaction with PD-1, thereby increasing immune activity. In certain embodiments, the antibody, antigen-binding portion thereof, or construct thereof specifically binds to PD-L2 and disrupts or inhibits its interaction with PD-1, thereby increasing immune activity.

[0287] Exemplary PD-1 inhibitors include, but are not limited to, BGB-A317 (BeiGene; see US 8,735,553, WO 2015 / 35606 and US 2015 / 0079109), lambrolizumab (e.g., as disclosed in WO 2008 / 156712 as hPD109A and its humanized derivatives h409A1, h409A16 and h409A17), AB137132 (Abcam), EH12.2H7 and RMP1-14 (#BE0146; Bioxcell Lifesciences Pvt. LTD.), MIH4 (Affymetrix eBioscience), nivolumab (Opdivo, BMS-936558; Bristol Myers Squibb), and ribozyme inhibitors (Rx100, Rx101, Rx102, Rx103, Rx104, Rx105, Rx106, Rx107, Rx108, Rx110, Rx119, Rx120, Rx121, Rx122, Rx123, Rx124, Rx125, Rx126, Rx127, Rx128, Rx129, Rx130, Rx131, Rx132, Rx133, Rx134, Rx135, Rx136, Rx137, Rx138, Rx139, Rx140, Rx141, Rx142, Rx143, Rx144, Rx145, Rx146, Rx147, Rx148, Rx149, Rx149, Rx149, Rx149, Rx14 Squibb; see U.S. Patent No. 8,008,449; WO2013 / 173223; WO2006 / 121168), pembrolizumab (Keytruda; MK-3475; Merck; see WO2008 / 156712), pidilizumab (CT-011; CureTech; Hardy et al., 1994, Cancer Res., 54(22):5793-6 and WO2009 / 101611), PDR001 (Novartis; see WO2015 / 112900), MEDI0680 (AMP-514; AstraZeneca; see WO2012 / 145493), TSR-042 (see WO2014 / 179664), cemiplimab (REGN-2810; Regeneron; H4H7798N; see US2015 / 0203579 and WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., 2007, J. Hematol. Oncol. 70: 136), AMP-224 (GSK-2661380; Li et al., 2016, Int J Mol Sci 17(7):1151 and WO2010 / 027827 and WO2011 / 066342), PF-06801591 (Pfizer), tislelizumab (BGB-A317; BeiGene; see WO2015 / 35606, U.S. Patent No. 9,834,606, and US2015 / 0079109), BI754091, SHR-1210 (see WO2015 / 085847), and antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, INCSHR1210 (Jiangsu Hengrui), as described in WO2006 / 121168. Medicine; also known as SHR-1210; see WO2015 / 085847), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang et al., 2017, J. Hematol. Oncol. 70: 136), STI-1110 (Sorrento Therapeutics; see WO2014 / 194302), AGEN2034 (Agenus; see WO2017 / 040790), MGA012 (Macrogenics; see WO2017 / 19846), IBI308 (Innovent; see WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540), cetrelimab (JNJ-63723283; JNJ-3283; see Calvo et al., J. Clin. Oncol. 36, no. 5_suppl (2018) 58), genolizumab (CBT-501; Patel et al., J. ImmunoTher. Cancer, 2017, 5(Suppl 2):P242), sasanlimab (PF-06801591; Youssef et al., Proc. Am. Assoc. Cancer Res. Ann.Meeting 2017; Cancer Res 2017;77(13 Suppl):see Abstract), toripalimab (JS-001; see US2016 / 0272708), camrelizumab (SHR-1210; INCSHR-1210; see US2016 / 376367; Huang et al., Clin. Cancer Res. 2018;24(6):1296-1304), spartalizumab (PDR001; see WO2017 / 106656; see Naing et al., J. Clin. Oncol. 34, no. 15_suppl (2016) 3060-3060), BCD-100 (JSC BIOCAD, Russia; see WO2018 / 103017), balstilimab (AGEN2034; see WO2017 / 040790), sintilimab (IBI-308; see WO2017 / 024465 and WO2017 / 133540), and ezabenlimab (BI-754091; US2017 / 334995; Johnson et al., J. Clin. Oncol. 36, no.5_suppl (2018) 212-212), zimberelimab (GLS-010; see WO2017 / 025051), LZM-009 (see US2017 / 210806), AK-103 (see WO2017 / 071625, WO2017 / 166804, and WO2018 / 036472), retifanlimab (MGA-012; see WO2017 / 019846), Sym-021 (see WO2017 / 055547 anti-PD-1 antibodies such as CS1003 (see CN107840887), anti-PD-1 antibodies such as the IgG1-PD1 disclosed herein (i.e., comprising a VH sequence as defined in SEQ ID NO: 43, a VL sequence as defined in SEQ ID NO: 44, an Fc sequence as defined in SEQ ID NO: 61, and a kappa sequence as defined in SEQ ID NO: 27), e.g., those disclosed in U.S. Pat. No. 7,488,802, U.S. Pat. No. 8,008,449, U.S. Pat. No. 8,168,757, , WO03 / 042402, WO2010 / 089411 (which further disclose anti-PD-L1 antibodies), WO2010 / 036959, WO2011 / 159877 (which further disclose antibodies against TIM-3), WO2011 / 082400, WO2011 / 161699, WO2009 / 014708, WO03 / 099196, WO2009 / 114335, WO2012 / 145493 (which further disclose antibodies against PD-L1 and anti-PD-1 antibodies described in WO2015 / 035606, WO2014 / 055648 (which further disclose anti-KIR antibodies), US2018 / 0185482 (which further discloses anti-PD-L1 and anti-TIGIT antibodies), US8,008,449, US8,779,105, US6,808,710, US8,168,757, US2016 / 0272708, and US8,354,509, e.g., Shaabani et al., 2018, Expert Op Ther Pat., 28(9):665-678 and Sasikumar and Ramachandra, 2018, BioDrugs, 32(5):481-497, small molecule antagonists against the PD-1 signaling pathway, e.g., siRNAs directed against PD-1 disclosed in WO2019 / 000146 and WO2018 / 103501, soluble PD-1 proteins disclosed in WO2018 / 222711, and oncolytic viruses containing soluble forms of PD-1, e.g., described in WO2018 / 022831.

[0288] In certain embodiments, the PD-1 inhibitor is nivolumab (Opdivo; BMS-936558), pembrolizumab (Keytruda; MK-3475), pidilizumab (CT-011), PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF-06801591, BGB-A317, BI754091, or SHR-1210. In one embodiment, the PD-1 inhibitor is an IgG1-PD1 as disclosed herein.

[0289] In certain embodiments, the PD-1 / PD-L1 inhibitory immunomodulatory agent is an anti-PD-1 antibody or antigen-binding fragment thereof comprising the complementarity-determining regions (CDRs) of one of the anti-PD-1 antibodies or antigen-binding fragments described above, for example, an anti-PD-1 antibody or antigen-binding fragment thereof comprising the CDRs of one anti-PD-1 antibody or antigen-binding fragment selected from the group consisting of pembrolizumab, nivolumab, Amp-514, tislelizumab, cemiplimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, CS1003, and IgG1-PD1.

[0290] In some embodiments, the CDRs of an anti-PD-1 antibody are delineated using the Kabat numbering scheme (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NTH Publication No. 91-3242).

[0291] In certain embodiments, the PD-1 / PD-L1 inhibitory immunomodulatory agent is an anti-PD-1 antibody or antigen-binding fragment thereof comprising the heavy chain variable region and light chain variable region of one of the anti-PD-1 antibodies or antigen-binding fragments described above, for example, the heavy chain variable region and light chain variable region of one anti-PD-1 antibody or antigen-binding fragment selected from the group consisting of pembrolizumab, nivolumab, Amp-514, tislelizumab, cemiplimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, CS1003, and IgG1-PD1.

[0292] In certain embodiments, the PD-1 / PD-L1 inhibitory immunomodulatory agent is an anti-PD-1 antibody or antigen-binding fragment thereof selected from the group consisting of pembrolizumab, nivolumab, Amp-514, tislelizumab, cemiplimab, TSR-042, JNJ-63723283, CBT-501, PF-06801591, JS-001, camrelizumab, PDR001, BCD-100, AGEN2034, IBI-308, BI-754091, GLS-010, LZM-009, AK-103, MGA-012, Sym-021, CS1003, IgG1-PD1.

[0293] In certain embodiments, the PD-1 / PD-L1 inhibitory immunomodulator is pembrolizumab or an antigen-binding fragment thereof.

[0294] The anti-PD-1 antibodies of the present disclosure are preferably monoclonal, and may be multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fragments produced by an Fab expression library, and PD-1-binding fragments of any of the above. In some embodiments, the anti-PD-1 antibodies described herein specifically bind to PD-1 (e.g., human PD-1). The immunoglobulin molecules of the present disclosure may belong to any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

[0295] In certain embodiments of the present disclosure, the anti-PD-1 antibody is an antigen-binding fragment (e.g., a human antigen-binding fragment) described herein, including, but not limited to, Fab, Fab' and F(ab'), Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and V L or V H Antigen-binding fragments include fragments comprising any of the variable domains. Antigen-binding fragments, including single-chain antibodies, may comprise the variable region alone or in combination with all or part of the following: hinge region, CH1, CH2, CH3, and CL domains. Also included within the present disclosure are antigen-binding fragments comprising any combination of the variable region with the hinge region, CH1, CH2, CH3, and CL domains. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.

[0296] The anti-PD-1 antibodies disclosed herein may be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of PD-1, or may be specific for both PD-1 and a heterologous protein. See, e.g., PCT Publications WO93 / 17715; WO92 / 08802; WO91 / 00360; WO92 / 05793; Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547-1553.

[0297] The anti-PD-1 antibodies disclosed herein may be described or identified in terms of the particular CDRs they contain. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including, but not limited to, Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745." ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plueckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001;309(3):657-70 ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272 ("AbM" numbering scheme). The boundaries of a given CDR may vary depending on the scheme used for identification. In some embodiments, the CDRs or individual identified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (e.g., its variable region) shall be understood to encompass (or be specific for) the CDRs defined in any of the foregoing schemes. For example, a particular CDR (e.g., CDR-H3) may be a CDR of a given V. H or V L When a variable region is described as containing the amino acid sequence of a corresponding CDR in the amino acid sequence of the variable region, it is understood that such CDR has the sequence of a corresponding CDR (e.g., CDR-H3) within the variable region as defined by any of the aforementioned schemes. A particular CDR or scheme for identifying a CDR may be specified, for example, a CDR defined by the Kabat, Chothia, AbM, or IMGT method.

[0298] In some embodiments, the numbering of amino acid residues in the CDR sequences of the anti-PD-1 antibodies or antigen-binding fragments thereof provided herein is according to the IMGT numbering scheme described in Lefranc, MP et al., Dev. Comp. Immunol., 2003, 27, 55-77.

[0299] In some embodiments, the anti-PD-1 antibodies disclosed herein comprise the CDRs of the antibody nivolumab. See WO2006 / 121168. In some embodiments, the CDRs of the antibody nivolumab are detailed using the Kabat numbering scheme (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NTH Publication No. 91-3242). The present disclosure encompasses anti-PD-1 antibodies or derivatives thereof comprising a heavy or light chain variable domain, the variable domain comprising (a) a set of three CDRs, the set of CDRs being from the monoclonal antibody nivolumab, and (b) a set of four framework regions, the set of framework regions being different from the set of framework regions in the monoclonal antibody nivolumab, and the anti-PD-1 antibody or derivative thereof binds to PD-1. In a specific embodiment, the anti-PD-1 antibody is nivolumab.

[0300] In some embodiments, the anti-PD-1 antibodies disclosed herein comprise the CDRs of the antibody pembrolizumab. See WO2008 / 156712. In some embodiments, the CDRs of the antibody pembrolizumab are detailed using the Kabat numbering scheme (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NTH Publication No. 91-3242). The present disclosure encompasses anti-PD-1 antibodies or derivatives thereof comprising a heavy or light chain variable domain, the variable domain comprising (a) a set of three CDRs from the monoclonal antibody pembrolizumab, and (b) a set of four framework regions, the set of framework regions differing from the set of framework regions in the monoclonal antibody pembrolizumab, and the anti-PD-1 antibody or derivative thereof binds to PD-1. In a specific embodiment, the anti-PD-1 antibody is pembrolizumab.

[0301] The anti-PD-1 antibodies disclosed herein may also be described or specified in terms of their binding affinity to PD-1 (e.g., human PD-1). Preferred binding affinities include those with a binding affinity of 5×10 -2 Under M, 10 -2 Less than M, 5 x 10 -3 Under M, 10 -3 Less than M, 5 x 10 -4 Under M, 10 -4 Less than M, 5 x 10 -5 Under M, 10 -5 Less than M, 5 x 10 -6 Under M, 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Under M, 10 -9 Less than M, 5 x 10-10 Under M, 10 -10 Less than M, 5 x 10 -11 Under M, 10 -11 Less than M, 5 x 10 -12 Under M, 10 -12 Less than M, 5 x 10 -13 Under M, 10 -13 Less than M, 5 x 10 -14 Under M, 10 -14 Less than M, 5 x 10 -15 Less than M or 10 -15 These include those with a dissociation constant or Kd less than M.

[0302] Anti-PD-1 antibodies also include modified derivatives and constructs, i.e., derivatives and constructs modified by the covalent attachment of any type of molecule to the antibody such that the covalent attachment does not interfere with the antibody's binding to PD-1. For example, without limitation, anti-PD-1 antibody derivatives include modified antibodies, such as those modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Any of a number of chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivatives or constructs may contain one or more non-classical amino acids.

[0303] Exemplary PD-1 ligand inhibitors are PD-L1 inhibitors and PD-L2 inhibitors, including, but not limited to, MEDI4736 (durvalumab; AstraZeneca; see WO2011 / 066389), MSB-0010718C (see US2014 / 0341917), YW243.55.S70 (see SEQ ID NO: 20 in WO2010 / 077634 and US8,217,149), MIH1 (Affymetrix eBioscience; see EP3230319), MDX-1105 (Roche / Genentech; see WO2013019906 and US8,217,149), STI-1014 (Sorrento; see WO2013 / 181634), CK-301 (checkpoint therapy), KN035 (3D Med / Alphamab; see Zhang et al., 2017, Cell Discov. 3:17004), atezolizumab (Tecentriq; RG7446; MPDL3280A; R05541267; see US9,724,413), BMS-936559 (Bristol Myers Squibb; US 7,943,743, see WO2013 / 173223), avelumab (Bavencio; see US 2014 / 0341917), LY3300054 (Eli Lilly Co.), CX-072 (Proclaim-CX-072; also known as CytomX; see WO2016 / 149201), FAZ053, KN035 (see WO2017020801 and WO2017020802), MDX-1105 (see US2015 / 0320859), anti-PD-L1 antibodies such as those disclosed in US 7,943,743, e.g., 3G10, 12A4 (BMS -936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, etc., WO2010 / 077634, US8,217,149, WO2010 / 036959, WO2010 / 077634, WO2011 / 066342, US8,217,149, US7,943,743, WO2010 / 089411, US7,635,757, US8,2 17,149, US2009 / 0317368, WO2011 / 066389, WO2017 / 034916, WO2017 / 020291, WO2017 / 020858, WO2017 / 020801 , WO2016 / 111645, WO2016 / 197367, WO2016 / 061142, WO2016 / 149201, WO2016 / 000619, WO2016 / 160792, WO2016 Examples of such antibodies include the anti-PD-L1 antibodies described in WO2015 / 022630, WO2016 / 007235, WO2015 / 179654, WO2015 / 173267, WO2015 / 181342, WO2015 / 109124, WO2018 / 222711, WO2015 / 112805, WO2015 / 061668, WO2014 / 159562, WO2014 / 165082, and WO2014 / 100079.

[0304] In certain embodiments, the PD-L1 inhibitor is atezolizumab (Tecentriq; RG7446; MPDL3280A; R05541267; see US Pat. No. 9,724,413).

[0305] In certain embodiments, the PD-1 / PD-L1 inhibitory immunomodulator is an anti-PD-L1 antibody or antigen-binding fragment thereof that comprises the complementarity-determining region (CDR) of one of the anti-PD-L1 antibodies or antigen-binding fragments described above, such as the CDRs of atezolizumab or its antigen-binding fragment.

[0306] In some embodiments, the CDRs of an anti-PD-L1 antibody are delineated using the Kabat numbering scheme (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NTH Publication No. 91-3242).

[0307] In a specific embodiment, the PD-1 / PD-L1 inhibitory immunomodulatory agent is an anti-PD-L1 antibody or antigen-binding fragment thereof that comprises the heavy chain variable region and light chain variable region of one of the anti-PD-L1 antibodies or antigen-binding fragments described above, for example, the heavy chain variable region and light chain variable region of atezolizumab or its antigen-binding fragment.

[0308] The anti-PD-L1 antibodies of the present disclosure are preferably monoclonal, and may be multispecific, human, humanized, or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab') fragments, fragments produced by an Fab expression library, and PD-L1-binding fragments of any of the above. In some embodiments, the anti-PD-L1 antibodies described herein specifically bind to PD-L1 (e.g., human PD-L1). The immunoglobulin molecules of the present disclosure may belong to any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

[0309] In certain embodiments of the present disclosure, the anti-PD-L1 antibody is an antigen-binding fragment (e.g., a human antigen-binding fragment) described herein, including, but not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and V L or V H Antigen-binding fragments include fragments comprising any of the variable domains. Antigen-binding fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with all or part of the following: hinge region, CH1, CH2, CH3, and CL domains. Also included within the disclosure are antigen-binding fragments comprising any combination of the variable region(s) with the hinge region, CH1, CH2, CH3, and CL domains. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof is human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.

[0310] The anti-PD-L1 antibodies disclosed herein may be monospecific, bispecific, trispecific, or may have greater multispecificity. Multispecific antibodies may be specific for different epitopes of PD-L1, or may be specific for both PD-L1 and a heterologous protein. See, e.g., PCT Publications WO93 / 17715; WO92 / 08802; WO91 / 00360; WO92 / 05793; Tutt, et al., 1991, J. Immunol. 147:60 69; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547 1553.

[0311] The anti-PD-L1 antibodies disclosed herein may be described or identified in terms of the particular CDRs that they contain. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including, but not limited to, Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745." ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plueckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001;309(3):657-70 ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272 ("AbM" numbering scheme). The boundaries of a given CDR may vary depending on the scheme used for identification. In some embodiments, the CDRs or individual identified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (e.g., its variable region) shall be understood to encompass (or be specific for) the CDRs defined in any of the foregoing schemes. For example, a particular CDR (e.g., CDR-H3) may be a CDR of a given V. H or V L When a variable region is described as containing the amino acid sequence of a corresponding CDR in the amino acid sequence of the variable region, it is understood that such CDR has the sequence of a corresponding CDR (e.g., CDR-H3) within the variable region as defined by any of the aforementioned schemes. A particular CDR or scheme for identifying a CDR may be specified, for example, a CDR defined by the Kabat, Chothia, AbM, or IMGT method.

[0312] In some embodiments, the numbering of the amino acid residues in the CDR sequences of the anti-PD-L1 antibodies or antigen-binding fragments thereof provided herein is according to the IMGT numbering scheme described in Lefranc, MP et al., Dev. Comp. Immunol., 2003, 27, 55-77.

[0313] In some embodiments, the anti-PD-L1 antibodies disclosed herein comprise the CDRs of the antibody atezolizumab. See US 9,724,413. In some embodiments, the CDRs of the antibody atezolizumab are detailed using the Kabat numbering scheme (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NTH Publication No. 91-3242). The present disclosure encompasses anti-PD-L1 antibodies or derivatives thereof comprising a heavy or light chain variable domain comprising (a) a set of three CDRs, wherein the set of CDRs is from the monoclonal antibody atezolizumab, and (b) a set of four framework regions, wherein the set of framework regions differs from the set of framework regions in the monoclonal antibody atezolizumab, and wherein the anti-PD-L1 antibody or derivative thereof binds to PD-L1. In a specific embodiment, the anti-PD-L1 antibody is atezolizumab.

[0314] The anti-PD-L1 antibodies disclosed herein may also be described or specified in terms of their binding affinity to PD-L1 (e.g., human PD-L1). Preferred binding affinities include those with a binding affinity of 5×10 -2 Under M, 10 -2 Less than M, 5 x 10 -3 Under M, 10 -3 Less than M, 5 x 10 -4 Under M, 10 -4 Less than M, 5 x 10 -5 Under M, 10 -5 Less than M, 5 x 10 -6 Under M, 10 -6 Less than M, 5 x 10 -7 Under M, 10 -7 Less than M, 5 x 10 -8 Under M, 10 -8 Less than M, 5 x 10 -9 Under M, 10-9 Less than M, 5 x 10 -10 Under M, 10 -10 Less than M, 5 x 10 -11 Under M, 10 -11 Less than M, 5 x 10 -12 Under M, 10 -12 Less than M, 5 x 10 -13 Under M, 10 -13 Less than M, 5 x 10 -14 Under M, 10 -14 Less than M, 5 x 10 -15 Less than M or 10 -15 These include those with a dissociation constant or Kd less than M.

[0315] Anti-PD-L1 antibodies also include modified derivatives and constructs, i.e., derivatives and constructs that have been modified by the covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not interfere with the binding of the antibody to PD-L1. For example, without limitation, anti-PD-L1 antibody derivatives include modified antibodies, such as those modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Any of a number of chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivatives or constructs may contain one or more non-classical amino acids.

[0316] According to the present disclosure, PD-1 / PD-L1 immune checkpoint inhibitors are inhibitors of inhibitory checkpoint proteins, but preferably not inhibitors of stimulatory checkpoint proteins.

[0317] In a preferred embodiment, the inhibitory PD-1 / PD-L1 immune checkpoint inhibitor is an antibody, particularly an antagonist or blocking antibody, that disrupts or inhibits the inhibitory immune checkpoint signaling pathway (the interaction of PD-1 with one or more of its ligands (e.g., PD-L1 and / or PD-L2)). In a preferred embodiment, the immune checkpoint inhibitor is an antibody, particularly an antagonist or blocking antibody, that disrupts or inhibits the interaction between PD-1 and PD-L1.

[0318] Checkpoint inhibitors, e.g., PD-1 / PD-L1 checkpoint inhibitors, can be administered in the form of an immune checkpoint inhibitor, e.g., an inhibitory nucleic acid molecule or nucleic acid, e.g., a DNA or RNA molecule, encoding an antibody or fragment thereof. For example, an antibody encoded by an expression vector can be delivered as described herein. Thus, the nucleic acid molecule can be delivered, for example, in the form of a plasmid or mRNA molecule, or complexed with a delivery vehicle, e.g., a liposome, lipoplex, or nucleic acid-lipid particle. Checkpoint inhibitors can also be administered via oncolytic viruses containing an expression cassette encoding the checkpoint inhibitor. Checkpoint inhibitors can also be administered by administering endogeneic or allogeneic cells capable of expressing the checkpoint inhibitor, e.g., in the form of cell-based therapy.

[0319] In one embodiment, the cell-based therapy comprises genetically engineered cells. In one embodiment, the genetically engineered cells express an immune checkpoint inhibitor, such as an immune checkpoint inhibitor described herein. In one embodiment, the genetically engineered cells express an immune checkpoint inhibitor that is an inhibitory nucleic acid molecule, such as an siRNA, shRNA, an oligonucleotide, an antisense DNA or RNA, an aptamer, an antibody or fragment thereof, or a soluble immune checkpoint protein or fusion. The genetically engineered cells can also express additional agents that enhance T cell function. Such agents are known in the art. Cell-based therapies for use in inhibiting immune checkpoint signaling are disclosed, for example, in WO2018 / 222711, the entire contents of which are incorporated herein by reference.

[0320] Preferably, the checkpoint inhibitor, e.g., a PD-1 / PD-L1 checkpoint inhibitor, is administered in a suitable amount; i.e., the amount of checkpoint inhibitor administered, e.g., in each dose and / or treatment cycle, may be an amount that completely or partially reduces, inhibits, interferes with, or negatively modulates one or more checkpoint proteins, or may be an amount that completely or partially reduces, inhibits, interferes with, or negatively modulates the expression of one or more checkpoint proteins. Thus, a suitable amount of a checkpoint inhibitor according to the present disclosure can completely or partially reduce, inhibit, interfere with, or negatively modulate one or more checkpoint proteins, or completely or partially reduces, inhibits, interferes with, or negatively modulates the expression of one or more checkpoint proteins. Therefore, the checkpoint inhibitor preferably prevents inhibitory signals associated with immune checkpoints, resulting in the prevention or reversal of immunosuppression and the establishment or enhancement of T cell immunity against cancer cells.

[0321] The amount of checkpoint inhibitor administered in each dose and / or treatment cycle may, inter alia, be within a range in which more than 5%, preferably more than 10%, more preferably more than 15%, even more preferably more than 20%, even more preferably more than 25%, even more preferably more than 30%, even more preferably more than 35%, even more preferably more than 40%, even more preferably more than 45%, and most preferably more than 50% of said checkpoint inhibitor is bound to the checkpoint protein.

[0322] In some embodiments, the amount of PD-1 / PD-L1 checkpoint inhibitor administered is about 100-300 mg / day (e.g., about 120-280 mg / day, about 140-260 mg / day, about 160-240 mg / day, about 180-220 mg / day, or about 190-210 mg / day, e.g., about 200 mg / day), or about 1.25-3.75 mg / kg body weight / day (e.g., about 1.50-3.50 mg / kg body weight / day, about 1.75-3.25 mg / kg body weight / day, about 2.0-3.0 mg / kg body weight / day, about 2.25-2.75 mg / kg body weight / day, or about 2.37-2.63 mg / kg body weight / day, e.g., about 2.50 mg / kg body weight / day), e.g., for each dose and / or each treatment cycle.

[0323] In a preferred embodiment, the amount of pembrolizumab administered is about 150-250 mg / day (e.g., about 160-240 mg / day, about 170-230 mg / day, about 180-220 mg / day, about 190-210 mg / day, or about 195-205 mg / day, e.g., about 200 mg / day), or about 1.87-3.13 mg / kg body weight / day (e.g., about 1.75-3.00 mg / kg body weight / day, about 2.12-2.88 mg / kg body weight / day, about 2.25-2.75 mg / kg body weight / day, about 2.37-2.63 mg / kg body weight / day, or about 2.43-2.56 mg / kg body weight / day, e.g., about 2.50 mg / kg body weight / day), e.g., for each dose and / or each treatment cycle.

[0324] In a preferred embodiment, the amount of pembrolizumab administered is about 10×10, e.g., at each dose and / or each treatment cycle. -9 ~1710×10 -9 mol / day (e.g., approximately 1090 × 10 -9 ~1640×10 -9 mol / day, approximately 1160×10 -9 ~1570×10 -9 mol / day, approximately 1230×10 -9 ~1500×10 -9 mol / day, approximately 1295×10 -9 ~1435×10 -9 mol / day, or approximately 1330 × 10 -9 ~1400×10 -9 mol / day, e.g., about 1365 × 10 -9 mol / day), or approximately 12.7 × 10 -9 ~21.4×10 -9 mol / kg body weight / day (e.g., 13.6 × 10 -9 ~20.5×10 -9 mol / kg body weight / day, 14.5 × 10 -9 ~19.6×10 -9 mol / kg body weight / day, 15.3 × 10 -9 ~18.8×10 -9 mol / kg body weight / day, 16.1 × 10 -9 ~18.0×10 -9 mol / kg body weight / day, or 16.6 × 10 -9 ~17.5×10 -9 mol / kg body weight / day, e.g., about 17.1 mol / kg body weight / day).

[0325] Checkpoint inhibitors, such as PD-1 / PD-L1 checkpoint inhibitors, may be administered in any manner and by any route known in the art. The mode and route of administration will depend on the type of checkpoint inhibitor to be used. In a preferred embodiment, the checkpoint inhibitor is administered systemically, for example parenterally, and particularly intravenously.

[0326] The checkpoint inhibitor, e.g., the PD-1 / PD-L1 checkpoint inhibitor, may be administered in the form of any suitable pharmaceutical composition described herein. In a preferred embodiment, the checkpoint inhibitor is administered in the form of an infusion.

[0327] Additional therapeutic agents In addition to the binding agent and the PD-1 / PD-L1 checkpoint inhibitor, the treatment regimen according to the first aspect of the present disclosure further comprises administering to the subject a chemotherapy combination comprising: (a) a platinum-based chemotherapy agent; and (b) 5-fluorouracil.

[0328] The platinum-based chemotherapeutic agent and 5-fluorouracil can be administered by any method and route known in the art. The mode and route of administration will depend on the type of platinum-based chemotherapeutic agent to be used. In a preferred embodiment, the platinum-based chemotherapeutic agent and 5-fluorouracil are administered systemically, for example, parenterally, particularly intravenously.

[0329] In one embodiment, the platinum-based compound is selected from platinum-based compounds commonly used in the treatment of tumors or cancer, particularly HNSCC, such as cisplatin, oxaliplatin, and carboplatin.

[0330] In one embodiment, the platinum-based chemotherapy agent is carboplatin or cisplatin. In one embodiment, the chemotherapy combination is cisplatin and 5-fluorouracil. In another embodiment, the chemotherapy combination is carboplatin and 5-fluorouracil.

[0331] In a preferred embodiment, the amount of cisplatin administered is, for example, about 50-150 mg / m for each dose and / or each treatment cycle. 2 / day, e.g., about 60-140 mg / m 2 / day, about 70~130mg / m 2 / day, about 80~120mg / m2 / day, about 90~110mg / m 2 / day, or approximately 95-105 mg / m 2 / day, e.g., about 100 mg / m 2 / day.

[0332] In a preferred embodiment, the amount of carboplatin administered is, for example, AUC=about 4 to AUC=about 6, preferably AUC=about 5, for each dose and / or each treatment cycle.

[0333] Carboplatin dosing can be calculated by using the following equation (Calvert's equation; see also Calvert AH, et al, J Clin Oncol. (1989); 7:1748-1756): Carboplatin dose (mg) = target area under the curve (AUC mg / mL / min) × (GFR + 25) where GFR is the glomerular filtration rate, which can be estimated by creatinine clearance (CrCl), calculated using the following equation (the Cockcroft-Gault equation; see also Cockcroft DW, et al., Nephron. (1976); 16:31-41):

[0334]

number

[0335] In one embodiment, a minimum serum creatine of 0.7 mg / dL is used (particularly if the subject has an abnormally low serum creatinine level, such as an elderly subject or a subject in poor health).

[0336] In one embodiment, adjusted body weight is used (particularly if the subject is overweight or obese), in which case adjusted body weight can be calculated as follows: Adjusted weight (kg) = ideal body weight (IBW) + 0.4 × (total body weight [TBW] - IBW). Further information regarding Calvert's equation is known to those of skill in the art (e.g., US Food & Drug Administration. Carboplatin dosing., available at https: / / wayback.archive-it.org / 7993 / 20170113081146 / http: / / www.fda.gov / AboutFDA / CentersOffices / OfficeofMedicalProductsandTobacco / CDER / ucm228974.htm., updated November 27, 2015; "Updated FAQ's for dosing of carboplatin" [Newsletter], Philadelphia, PA: Gynecologic Oncology Group Newsletter; Spring 2011 issue, available at https: / / www.gog.org; Marina NM, et al., J Clin Oncol. (1993); 11:554-560; Newell DR, et al., J Clin Oncol. (1993); 11(12):2314-2323; Pinkerton CR, et al., Br J Cancer. (1990); 62(2):257-262; Mann JR, et al., Med Pediatr Oncol. (1998); 20(4):217-227; Schwartz GJ, et al., J Am Soc Nephrol. (2009); 20(3):629-637; all of which are incorporated by reference).

[0337] In one embodiment, the maximum carboplatin dose is capped according to the desired AUC to avoid potential toxicity due to overdosing. The maximum dose may be based on an estimated GFR of up to 125 mL / min (especially for subjects with normal renal function). Thus, in one embodiment, the maximum carboplatin dose is (i) 600 mg (=(125+25)×4) if AUC=4; (ii) 750 mg (=(125+25)×5) if AUC=5; or (iii) 900 mg (=(125+25)×6) if AUC=6.

[0338] In some embodiments, the amount of carboplatin administered is, for example, about 300-600 mg / day (if AUC=4), about 350-750 mg / day (if AUC=5), or about 400-900 mg / day (if AUC=6) for each dose and / or treatment cycle. In some embodiments, the amount of carboplatin administered is, for example, about 3.7-7.5 mg / kg body weight / day (if AUC=4), about 4.3-9.4 mg / kg body weight / day (if AUC=5), or about 5.0-11.3 mg / kg body weight / day (if AUC=6) for each dose and / or treatment cycle.

[0339] In a preferred embodiment, the amount of 5-fluorouracil administered is about 500-1500 mg / m, e.g., at each dose and / or each treatment cycle. 2 / day, e.g., about 600-1400 mg / m 2 / day, about 700~1300mg / m 2 / day, about 800~1200mg / m 2 / day, about 900~1100mg / m 2 / day, or approximately 950-1050 mg / m 2 / day, e.g., about 1000 mg / m 2 / day.

[0340] Subject and tumor or cancer to be treated The subjects to be treated according to the present disclosure are preferably human subjects.

[0341] The tumor or cancer to be treated is head and neck squamous cell carcinoma (HNSCC). More than 600,000 cases of HNSCC are diagnosed worldwide each year. In the United States, approximately 65,630 new cases of oral cavity, pharynx, and larynx cancer and an estimated 14,500 deaths are expected to occur in 2020 over the same period (NCCN, 2021b). Tobacco use, alcohol use, and human papillomavirus (HPV) infection increase the risk of developing HNSCC. Patients with locally HPV-positive HNSCC have improved treatment outcomes compared with patients with HPV-negative disease. For patients with recurrent or metastatic HNSCC, pembrolizumab / platinum (cisplatin or carboplatin) / 5-FU and pembrolizumab monotherapy (for patients with a PD-L1 combined positive score [CPS] ≥ 20 or ≥ 1) are recommended 1L regimens; however, median overall survival (mOS) is less than 15 months (NCCN, 2021b). Therefore, HNSCC remains an area of ​​significant unmet medical need, and further opportunities exist to improve outcomes using novel treatment approaches.

[0342] In one embodiment, histologically or cytologically confirmed recurrent or metastatic HNSCC is considered untreatable by local therapy.

[0343] In one embodiment, the subject has not received prior anticancer therapy for recurrent or metastatic disease (e.g., systemic anticancer therapy administered in the recurrent or metastatic setting). Anticancer therapy completed more than six months prior to signing the consent is permitted if given as part of multimodal treatment for locally advanced disease.

[0344] In one embodiment, eligible primary tumor locations are the oropharynx, oral cavity, hypopharynx, and larynx.

[0345] In one embodiment, the subject does not have a primary tumor site (any tissue structure) in the nasopharynx.

[0346] In one embodiment, the subject has a tumor PD-L1 combined positive score (CPS) (e.g., tumor PD-L1 IHC CPS) of ≧1, preferably ≧1 and ≦19 (which can be determined by local (preferably FDA-approved testing) or central laboratory testing (central testing is required in expansion phases)). In one embodiment, the subject has a tumor PD-L1 CPS (e.g., tumor PD-L1 IHC CPS) of ≧20.

[0347] In one embodiment, the subject has not received prior treatment with an immune checkpoint (ICP) inhibitor, i.e., prior to treatment according to the first aspect, the subject has not received treatment with an ICP inhibitor.

[0348] In one embodiment, the subject, e.g., a human subject (e.g., a human subject with a PD-L1 CPS >= 1 and <= 19 or a PD-L1 CPS >= 20), has not received prior treatment with a checkpoint inhibitor and / or an anti-cancer therapy for recurrent or metastatic disease. In one embodiment, the subject, e.g., a human subject (e.g., a human subject with a PD-L1 CPS >= 1 and <= 19 or a PD-L1 CPS >= 20), has not received prior treatment with any anti-cancer therapy, or with a checkpoint inhibitor and any anti-cancer therapy.

[0349] Treatment regimen The binding agent, the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab), and the chemotherapeutic agent (i.e., a platinum-based chemotherapeutic agent (e.g., cisplatin or carboplatin) and 5-fluorouracil) may be administered in any suitable manner, for example, intravenously, intraarterially, subcutaneously, intradermally, intramuscularly, intranodally, or intratumorally.

[0350] In one embodiment of the first aspect, the binding agent is administered to the subject, particularly by systemic administration.Preferably, the binding agent is administered to the subject by intravenous injection or infusion.In one embodiment, the binding agent is administered in at least one treatment cycle.

[0351] In one embodiment, the binding agent is administered for at least one treatment cycle (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, or at least 35 treatment cycles). In one embodiment, the binding agent is administered for at least 6 treatment cycles, e.g., at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or until the end of treatment.

[0352] In one embodiment, the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered to a subject by systemic administration. Preferably, the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered to a subject by intravenous injection or infusion.

[0353] In one embodiment, the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered for at least one treatment cycle (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, or at least 35 treatment cycles). In one embodiment, the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered for at least 6 treatment cycles, e.g., at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or until the end of treatment.

[0354] In one embodiment, the chemotherapeutic agents (i.e., platinum-based chemotherapeutic agents (e.g., cisplatin or carboplatin) and 5-fluorouracil) are administered to the subject, particularly by systemic administration. Preferably, the chemotherapeutic agents are administered to the subject by intravenous injection or infusion.

[0355] In one embodiment, the chemotherapeutic agent is administered for at least one treatment cycle (e.g., at least two, at least three, at least four, at least five, or at least six treatment cycles). For example, the platinum-based chemotherapeutic agent may be carboplatin and may be administered for at least one treatment cycle (e.g., at least two, at least three, at least four, at least five, or at least six treatment cycles). Alternatively, the platinum-based chemotherapeutic agent may be cisplatin and may be administered for at least one treatment cycle (e.g., at least two, at least three, at least four, at least five, or at least six treatment cycles).

[0356] In one embodiment, the binding agent, PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab), platinum-based chemotherapy, and 5-fluorouracil are each administered to a subject by systemic administration, preferably by intravenous injection or infusion. In one embodiment, the binding agent and PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered for at least one treatment cycle, preferably at least six treatment cycles, e.g., at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or until the end of treatment, and the platinum-based chemotherapy and 5-fluorouracil are administered for at least the first treatment cycle, e.g., at least the first and second treatment cycles, preferably only the first six treatment cycles (this means that administration of both the platinum-based chemotherapy and 5-fluorouracil is discontinued after completion of the first six treatment cycles).

[0357] In one embodiment, each treatment cycle is about 2 weeks (14 days), 3 weeks (21 days), or 4 weeks (28 days), preferably 3 weeks (21 days).

[0358] In certain embodiments, each dose is administered or infused every two weeks (1Q2W), every three weeks (1Q3W), or every four weeks (1Q4W), preferably every three weeks (1Q3W).

[0359] In some embodiments, one dose of the binding agent is administered every three weeks (1Q3W). In some embodiments, one dose of the binding agent is administered on day 1 of each treatment cycle. In some embodiments, one dose of the binding agent is administered on day 1 of each treatment cycle, each treatment cycle being 3 weeks long. In some embodiments, one dose of the binding agent is administered for at least 6 treatment cycles, for example, at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or until the end of treatment, where the binding agent is administered on day 1 of each treatment cycle, each treatment cycle being 3 weeks long.

[0360] In some embodiments, one dose of the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered every three weeks (1Q3W). In some embodiments, one dose of the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered on day 1 of each treatment cycle. In some embodiments, one dose of the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered on day 1 of each treatment cycle, each treatment cycle being three weeks long. In some embodiments, one dose of the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered for at least six treatment cycles, for example, at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or until the end of treatment, in which case the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered on day 1 of each treatment cycle, each treatment cycle being three weeks long.

[0361] In some embodiments, one dose of the binder and one dose of the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered every three weeks (1Q3W). In some embodiments, one dose of the binder and one dose of the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered on day 1 of each treatment cycle. In some embodiments, one dose of the binder and one dose of the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered on day 1 of each treatment cycle, and each treatment cycle is 3 weeks long. In some embodiments, one dose of the binding agent and one dose of the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered for at least 6 treatment cycles, e.g., at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or until the end of treatment, where the binding agent and the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered on day 1 of each treatment cycle, and where each treatment cycle is 3 weeks long.

[0362] In some embodiments, one dose of the chemotherapy combination (cisplatin and 5-fluorouracil or carboplatin and 5-fluorouracil) is administered at least every three weeks (1Q3W).

[0363] In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered every three weeks (1Q3W). In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (e.g., on day 1 of at least the first and second treatment cycles, for example, only the first six treatment cycles). In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (e.g., on day 1 of at least the first and second treatment cycles, for example, only the first six treatment cycles), where each treatment cycle is three weeks. In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first and second treatment cycles (e.g., on day 1 of only the first six treatment cycles), where each treatment cycle is three weeks.

[0364] In some embodiments, one dose of 5-fluorouracil is administered at least every three weeks (1Q3W), e.g., administered over four days during the first week. In some embodiments, one dose of 5-fluorouracil is administered on at least day 1 of at least a first treatment cycle (e.g., on day 1 of at least the first and second treatment cycles, e.g., only the first six treatment cycles), e.g., on days 1, 2, 3, and 4 of at least a first treatment cycle (e.g., on at least the first and second treatment cycles, e.g., only the first six treatment cycles). In some embodiments, one dose of 5-fluorouracil is administered on at least day 1 of at least a first treatment cycle (e.g., on day 1 of at least a first and second treatment cycles, e.g., only the first six treatment cycles), e.g., on days 1, 2, 3, and 4 of at least a first treatment cycle (e.g., on at least a first and second treatment cycles, e.g., only the first six treatment cycles), where each treatment cycle is 3 weeks. In some embodiments, one dose of 5-fluorouracil is administered on days 1, 2, 3, and 4 of at least a first and second treatment cycle (e.g., only the first six treatment cycles), where each treatment cycle is 3 weeks.

[0365] In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered every three weeks (1Q3W), and one dose of 5-fluorouracil is administered at least every three weeks (1Q3W), e.g., administered over four days during the first week. In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (e.g., on day 1 of at least the first and second treatment cycles, e.g., only the first six treatment cycles), and one dose of 5-fluorouracil is administered on at least day 1 of at least the first treatment cycle (e.g., on day 1 of at least the first and second treatment cycles, e.g., only the first six treatment cycles), e.g., on days 1, 2, 3, and 4 of at least the first treatment cycle (e.g., on at least the first and second treatment cycles, e.g., only the first six treatment cycles). In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (e.g., on day 1 of at least the first and second treatment cycles, e.g., on only the first six treatment cycles), and one dose of 5-fluorouracil is administered on at least day 1 of at least the first treatment cycle (e.g., on day 1 of at least the first and second treatment cycles, e.g., on only the first six treatment cycles), e.g., on days 1, 2, 3, and 4 of at least the first treatment cycle (e.g., on at least the first and second treatment cycles, e.g., on only the first six treatment cycles), where each treatment cycle is 3 weeks long. In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first and second treatment cycles (e.g., on day 1 of only the first six treatment cycles), and one dose of 5-fluorouracil is administered on days 1, 2, 3, and 4 of at least the first and second treatment cycles (e.g., only the first six treatment cycles), where each treatment cycle is 3 weeks long.

[0366] In some embodiments, one dose of the binding agent, one dose of the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab), and one dose of the platinum-based chemotherapy agent are administered every three weeks (1Q3W), and one dose of 5-fluorouracil is administered at least every three weeks (1Q3W), for example, over four days during the first week.

[0367] In some embodiments, one dose of the binding agent and one dose of the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered on day 1 of each treatment cycle, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (e.g., on day 1 of at least the first and second treatment cycles, e.g., of only the first 6 treatment cycles), and one dose of 5-fluorouracil is administered on at least day 1 of at least the first treatment cycle (e.g., on day 1 of at least the first and second treatment cycles, e.g., of only the first 6 treatment cycles), e.g., on days 1, 2, 3, and 4 of at least the first treatment cycle (e.g., of at least the first and second treatment cycles, e.g., of only the first 6 treatment cycles).

[0368] In some embodiments, one dose of the binding agent and one dose of the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered on day 1 of each treatment cycle, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least a first treatment cycle (e.g., on day 1 of at least a first and second treatment cycle, e.g., of only the first six treatment cycles), and one dose of 5-fluorouracil is administered on at least day 1 of at least a first treatment cycle (e.g., on day 1 of at least a first and second treatment cycle, e.g., of only the first six treatment cycles), e.g., on days 1, 2, 3, and 4 of at least a first treatment cycle (e.g., of at least a first and second treatment cycle, e.g., of only the first six treatment cycles), wherein each treatment cycle is 3 weeks.

[0369] In some embodiments, one dose of the binding agent and one dose of the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered for at least six treatment cycles, e.g., at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or until the end of treatment, where the binding agent and the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered on day 1 of each treatment cycle; one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first and second treatment cycles (e.g., on day 1 of only the first six treatment cycles); and one dose of 5-fluorouracil is administered on days 1, 2, 3, and 4 of at least the first and second treatment cycles (e.g., only the first six treatment cycles), where each treatment cycle is 3 weeks long.

[0370] In each of the above embodiments, the dosage of the binding agent may be any dosage of the binding agent specified herein, for example, 50-150 mg / day (e.g., about 60-140 mg / day, about 70-130 mg / day, about 80-120 mg / day, about 90-110 mg / day, or about 95-105 mg / day, e.g., about 100 mg / day), or about 0.62-1.88 mg / kg body weight / day (e.g., about 0.75-1.75 mg / kg body weight / day, about 0.87-1.63 mg / kg body weight / day, 1.00-1.50 mg / kg body weight / day, 1.12-1.38 mg / kg body weight / day, or 1.18-1.31 mg / kg body weight / day, e.g., about 1.25 mg / kg body weight / day).

[0371] In each of the above embodiments, the dose of the PD-1 / PD-L1 checkpoint inhibitor can be any dose specified herein for a PD-1 / PD-L1 checkpoint inhibitor, for example, about 100-300 mg / day (e.g., about 120-280 mg / day, about 140-260 mg / day, about 160-240 mg / day, about 180-220 mg / day, or about 190-210 mg / day). 0 mg / day, e.g., about 200 mg / day), or about 1.25 to 3.75 mg / kg body weight / day (e.g., about 1.50 to 3.50 mg / kg body weight / day, about 1.75 to 3.25 mg / kg body weight / day, about 2.0 to 3.0 mg / kg body weight / day, about 2.25 to 2.75 mg / kg body weight / day, or about 2.37 to 2.63 mg / kg body weight / day, e.g., about 2.50 mg / kg body weight / day). For example, in each of the above embodiments, the dose of pembrolizumab can be any dose specified herein for pembrolizumab, for example, 150 to 250 mg / day (e.g., about 160 to 240 mg / day, about 170 to 230 mg / day, about 180 to 220 mg / day, about 190 to 210 mg / day, or about 195 to 205 mg / day, e.g., about 200 mg / day). , or about 1.87 to 3.13 mg / kg body weight / day (e.g., about 1.75 to 3.00 mg / kg body weight / day, about 2.12 to 2.88 mg / kg body weight / day, about 2.25 to 2.75 mg / kg body weight / day, about 2.37 to 2.63 mg / kg body weight / day, or about 2.43 to 2.56 mg / kg body weight / day, e.g., about 2.50 mg / kg body weight / day), or about 1020 x 10 -9 ~1710×10 -9 mol / day (e.g., approximately 1090 × 10 -9 ~1640×10 -9 mol / day, approximately 1160×10 -9 ~1570×10 -9 mol / day, approximately 1230×10 -9 ~1500×10 -9 mol / day, approximately 1295×10 -9 ~1435×10 -9 mol / day, or approximately 1330 × 10 -9 ~1400×10 -9 mol / day, e.g., about 1365 × 10 -9mol / day), or approximately 12.7 × 10 -9 ~21.4×10 -9 mol / kg body weight / day (e.g., 13.6 × 10 -9 ~20.5×10 -9 mol / kg body weight / day, 14.5 × 10 -9 ~19.6×10 -9 mol / kg body weight / day, 15.3 × 10 -9 ~18.8×10 -9 mol / kg body weight / day, 16.1 × 10 -9 ~18.0×10 -9 mol / kg body weight / day, or 16.6 × 10 -9 ~17.5×10 -9 mol / kg body weight / day, for example, about 17.1 mol / kg body weight / day).

[0372] In each of the above embodiments, the dose of the platinum-based chemotherapeutic agent may be any of the doses specified herein for carboplatin or any of the doses specified herein for cisplatin. For example, the dose of carboplatin may be AUC=about 4 to AUC=about 6, preferably AUC=about 5, or may be about 300-600 mg / day (if AUC=4), about 350-750 mg / day (if AUC=5), or about 400-900 mg / day (if AUC=6), or may be about 3.7-7.5 mg / kg body weight / day (if AUC=4), about 4.3-9.4 mg / kg body weight / day (if AUC=5), or about 5.0-11.3 mg / kg body weight / day (if AUC=6). Similarly, the dose of cisplatin may be about 50-150 mg / m 2 / day, e.g., about 60-140 mg / m 2 / day, about 70~130mg / m 2 / day, about 80~120mg / m 2 / day, about 90~110mg / m 2 / day, or approximately 95-105 mg / m 2 / day, e.g., about 100 mg / m 2 / day may be possible.

[0373] In each of the above embodiments, the dose of 5-fluorouracil can be any dose specified herein for 5-fluorouracil, for example, about 500 to 1500 mg / m 2 / day, e.g., about 600-1400 mg / m 2 / day, about 700~1300mg / m 2 / day, about 800~1200mg / m 2 / day, about 900~1100mg / m 2 / day, or approximately 950-1050 mg / m 2 / day, e.g., about 1000 mg / m 2 / day may be possible.

[0374] Each dose may be administered or infused over a minimum of 30 minutes, such as a minimum of 60 minutes, a minimum of 90 minutes, a minimum of 120 minutes or a minimum of 240 minutes.

[0375] The binding agent and the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) may be administered simultaneously. In an alternative preferred embodiment, the binding agent and the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered separately. In some embodiments, the binding agent is administered prior to administration of the PD-1 / PD-L1 checkpoint inhibitor.

[0376] In some embodiments, a chemotherapy combination (platinum-based chemotherapy agent and 5-fluorouracil) is administered after administration of the binding agent.

[0377] The binding agent, PD-1 / PD-L1 checkpoint inhibitor, platinum-based chemotherapeutic agent, and 5-fluorouracil can be administered in any suitable form (e.g., naked per se). However, it is preferred that the binding agent, PD-1 / PD-L1 checkpoint inhibitor, platinum-based chemotherapeutic agent, and 5-fluorouracil are administered in the form of any suitable pharmaceutical composition described herein. In one embodiment, at least the binding agent and the PD-1 / PD-L1 checkpoint inhibitor are administered in the form of separate pharmaceutical compositions (i.e., one pharmaceutical composition for the binding agent and one pharmaceutical composition for the PD-1 / PD-L1 checkpoint inhibitor), preferably the binding agent, PD-1 / PD-L1 checkpoint inhibitor, platinum-based chemotherapeutic agent, and 5-fluorouracil are administered in the form of separate pharmaceutical compositions (i.e., one pharmaceutical composition for the binding agent, one pharmaceutical composition for the PD-1 / PD-L1 checkpoint inhibitor, and at least one pharmaceutical composition for the chemotherapy combination, e.g., one pharmaceutical composition for the platinum-based chemotherapeutic agent and one pharmaceutical composition for 5-fluorouracil).

[0378] The composition or pharmaceutical composition may be prepared as described in Remington: The Science and Practice of Pharmacy, 19 thThe carrier, excipient, and / or diluent, as well as any other components suitable for pharmaceutical compositions, such as known adjuvants, may be formulated according to conventional techniques, such as those disclosed in "Therapeutic Agents for the Development of Novel Antigens," Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. The pharmaceutically acceptable carrier or diluent, as well as any known adjuvants and excipients, should be suitable for the binding agent and / or checkpoint inhibitor, and / or one or more additional therapeutic agents, if present, and the selected mode of administration. The suitability of the carrier and other components of the pharmaceutical composition is determined based on the lack of a significant negative effect on the desired biological properties of the selected compound or pharmaceutical composition (e.g., less than a substantial effect on antigen binding [e.g., 10% or less relative inhibition, 5% or less relative inhibition, etc.]).

[0379] The compositions, particularly the pharmaceutical compositions of the binding agent, the pharmaceutical compositions of the PD-1 / PD-L1 checkpoint inhibitor, and at least one of the pharmaceutical compositions of the chemotherapy combination, may include diluents, bulking agents, salts, buffers, detergents (e.g., non-ionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugar- or protein-free amino acids), preservatives, solubilizers, and / or other materials suitable for inclusion in a pharmaceutical composition.

[0380] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0381] Pharmaceutical carriers, excipients, or diluents can be selected according to the intended route of administration and standard pharmaceutical practice.

[0382] Pharmaceutically acceptable carriers include any suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents, etc. that are physiologically compatible with the active compounds used herein, particularly the binding agents, PD-1 / PD-L1 checkpoint inhibitors, platinum-based chemotherapeutic agents, and 5-fluorouracil.

[0383] Examples of suitable aqueous and non-aqueous carriers that may be employed in the (pharmaceutical) compositions include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solution, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical field.

[0384] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutical active substances is well known in the art.Except insofar as any conventional media or agent is incompatible with the active compound, its use in the (pharmaceutical) composition is anticipated.

[0385] The term "excipient" as used herein refers to a substance that may be present in the (pharmaceutical) composition of the present disclosure, but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0386] The term "diluent" refers to diluting and / or thinning a drug. Furthermore, the term "diluent" includes any one or more of fluids, liquids or solids suspending and / or mixing media. Examples of suitable diluents include ethanol, glycerol, and water.

[0387] The (pharmaceutical) composition may also contain a pharmaceutically acceptable antioxidant, examples of which include, for example, (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0388] The (pharmaceutical) composition may also comprise isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol or sodium chloride in the composition.

[0389] The (pharmaceutical) composition may also contain one or more adjuvants appropriate for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersing agents, preservatives, or buffers, which can enhance the shelf life or effectiveness of the composition. The compositions used herein may be prepared using carriers that are expected to protect the compound from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, alone or with waxes, or other materials known in the art. Methods for preparing such formulations are generally known to those skilled in the art, see, for example, *Sustained and Controlled Release Drug Delivery Systems*, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0390] "Pharmaceutically acceptable salts" include, for example, acid addition salts, which can be formed by using pharmaceutically acceptable acids such as, for example, hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid. Further suitable pharmaceutically acceptable salts include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); ammonium (NH +); and salts formed with suitable organic ligands (e.g., quaternary ammonium and amine cations formed using counteranions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkylsulfonates, and arylsulfonates).Illustrative examples of pharmaceutically acceptable salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentaneproline, cyclopentanediol ... Pionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, Hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isobutyrate, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methyl sulfate, mucate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate Examples of suitable salts include hydroxybenzoates, pamoates (embonates), palmitates, pantothenates, pectinates, persulfates, 3-phenylpropionates, phosphates / diphosphates, phthalates, picrates, pivalates, polygalacturonates, propionates, salicylates, stearates, sulfates, suberates, succinates, tannates, tartrates, teoclates, tosylates, triethyl iodides, undecanoates, valerates, and the like (see, for example, S.M. Berge et al., "Pharmaceutical Salts," J. Pharm. Sci., 66, pp. 1-19 (1977)). Pharmaceutically unacceptable salts may be used to prepare pharmaceutically acceptable salts, and are included in the disclosure of the present invention.

[0391] In one embodiment, the binding agents, PD-1 / PD-L1 checkpoint inhibitors, platinum-based chemotherapeutic agents, and 5-fluorouracil used herein may be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Other active or therapeutic compounds may also be incorporated into the compositions.

[0392] Pharmaceutical compositions for injections are typically sterile and must be stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier may be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition substances that delay absorption, such as monostearate salts and gelatin. Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent, for example, with one or combination of the above-listed components as needed, and then sterilizing by microfiltration.Generally, dispersion is prepared by incorporating active compound into sterile medium that contains basic dispersion medium and other necessary components, for example, from the above-listed components.For the sterile powder that is used to prepare sterile injectable solution, the example of preparation method is vacuum drying and freeze-drying (lyophilization), which can obtain the powder of active ingredient plus any additional desired components from the solution that has been previously sterilized and filtered.

[0393] Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent with one or combination of above-listed components as needed, and then sterilized by microfiltration.Generally, dispersion is prepared by incorporating active compound into sterile medium that contains basic dispersion medium and other components required from above-listed.For the sterile powder that is used to prepare sterile injectable solution, the example of preparation method is vacuum drying and freeze-drying (lyophilization), which can obtain the powder of active ingredient plus any additional desired components from the solution that has been previously sterilized and filtered.

[0394] In a second aspect, the present disclosure provides a method for reducing or preventing the progression of or treating HNSCC in a subject, comprising administering to the subject (i) a binding agent, (ii) a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab), and (iii) a chemotherapy combination comprising a platinum-based chemotherapy agent (particularly cisplatin or carboplatin) and 5-fluorouracil, wherein the binding agent comprises a first binding domain that binds CD40 and a second binding domain that binds CD137. The embodiments disclosed herein with respect to the first aspect (particularly with respect to the binding agent, PD-1 / PD-L1 checkpoint inhibitor, platinum-based chemotherapy agent, 5-fluorouracil, their administered doses, their treatment regimens, and the subject) also apply to the method of the second aspect.

[0395] In a further aspect, the present disclosure provides a kit comprising (i) a binding agent comprising a first binding region that binds CD40 and a second binding region that binds CD137, (ii) a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab), (iii) a platinum-based chemotherapeutic agent (particularly cisplatin and / or carboplatin), and (iv) 5-fluorouracil, as well as a kit for use in a method, e.g., for reducing or preventing the progression of HNSCC in a subject or for treating HNSCC. The embodiments disclosed herein with respect to the first aspect (particularly with respect to the binding agent, PD-1 / PD-L1 checkpoint inhibitor, platinum-based chemotherapeutic agent, 5-fluorouracil, their administered doses, and their treatment regimens) also apply to the kit of the further aspect. In one embodiment, the kit comprises at least three containers, one of which contains the binding agent (by itself or in the form of a (pharmaceutical) composition), a second container contains the PD-1 / PD-L1 checkpoint inhibitor (by itself or in the form of a (pharmaceutical) composition), and a third container contains a platinum-based chemotherapeutic agent and / or 5-fluorouracil (by itself or in the form of a (pharmaceutical) composition). Preferably, the kit comprises at least four containers, one of which contains the binding agent (by itself or in the form of a (pharmaceutical) composition), a second container contains the PD-1 / PD-L1 checkpoint inhibitor (by itself or in the form of a (pharmaceutical) composition), a third container contains a platinum-based chemotherapeutic agent, such as carboplatin and / or cisplatin (by itself or in the form of one or two (pharmaceutical) compositions), and a fourth container contains 5-fluorouracil (by itself or in the form of a (pharmaceutical) composition).

[0396] In another aspect, the present disclosure provides a kit of a further aspect for use in a method for reducing or preventing the progression of, or treating, HNSCC in a subject. Also, embodiments disclosed herein with respect to the first aspect (particularly with respect to the binding agent, PD-1 / PD-L1 checkpoint inhibitor, platinum-based chemotherapeutic agent, 5-fluorouracil, their administered doses, their treatment regimens, and the subject) and / or the second aspect apply to the kit for use of the other aspects.

[0397] Citation of documents and works referenced herein is not intended as an admission that any of the foregoing is relevant prior art. All statements regarding the contents of these documents are based on information available to applicant and are not to be construed as any admission regarding the contents of these documents.

[0398] The description (including the following examples) is presented to enable those skilled in the art to make and use various embodiments. Descriptions of specific devices, techniques, and applications are provided merely as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Accordingly, the various embodiments are not intended to be limited to the examples described and shown herein, but are to be accorded the scope consistent with the claims.

[0399] Itemized implementation examples 1. A binding agent for use in a method for reducing or preventing the progression of head and neck squamous cell carcinoma (HNSCC) in a subject or for treating HNSCC, said method comprising administering to said subject a combination of a binding agent, pembrolizumab, and chemotherapy comprising a platinum-based chemotherapy agent and 5-fluorouracil, wherein the binding agent comprises a first binding region that binds CD40 and a second binding region that binds CD137.

[0400] 2. The binder for use according to item 1, wherein the combination of binder, pembrolizumab, and chemotherapy is administered in at least one treatment cycle, each treatment cycle being 3 weeks (21 days).

[0401] 2a. The binder for use according to any one of the preceding items, wherein the binder and pembrolizumab are administered for at least two treatment cycles, e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, or at least 35 treatment cycles.

[0402] 2b. The binder for use according to any one of the preceding items, wherein the binder and pembrolizumab are administered for at least 6 treatment cycles, such as at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or until the end of treatment.

[0403] 3. The binder for use according to any one of the preceding items, wherein one dose of binder and one dose of pembrolizumab are administered every three weeks (1Q3W).

[0404] 4. The binder for use according to any one of the preceding items, wherein one dose of binder and one dose of pembrolizumab are administered on day 1 of each treatment cycle.

[0405] 5. The binder for use according to any one of the preceding items, wherein the binder is administered prior to administration of pembrolizumab.

[0406] 5a. The binding agent for use according to any one of items 1 to 4, which is administered simultaneously with the administration of pembrolizumab.

[0407] 5b. The binding agent for use according to any one of items 1 to 4, which is administered after administration of pembrolizumab.

[0408] 6. The binding agent for use according to any one of the preceding items, wherein one dose of the chemotherapy combination is administered at least every three weeks (1Q3W) during at least the first treatment cycle.

[0409] 6a. The binding agent for use according to item 6, wherein one dose of the chemotherapy combination is administered at least every three weeks (1Q3W) during at least the first and second treatment cycles, preferably during the first six treatment cycles.

[0410] 7. The binding agent for use according to any one of the preceding items, wherein one dose of the platinum-based chemotherapy agent is administered every three weeks (1Q3W) during at least the first treatment cycle, and one dose of 5-fluorouracil is administered at least every three weeks (1Q3W), such as over four days in the first week during at least the first treatment cycle.

[0411] 7a. The binder for use according to item 7, wherein one dose of the platinum-based chemotherapy agent is administered every three weeks (1Q3W) during at least the first and second treatment cycles, preferably during the first six treatment cycles, and one dose of 5-fluorouracil is administered at least every three weeks (1Q3W) during at least the first and second treatment cycles, preferably during the first six treatment cycles, for example administered over four days during the first week.

[0412] 8. The binder for use according to any one of the preceding items, wherein one dose of the platinum-based chemotherapy agent is administered on day 1 of at least the first treatment cycle, and one dose of 5-fluorouracil is administered on at least day 1 of at least the first treatment cycle, such as on days 1, 2, 3, and 4 of at least the first treatment cycle.

[0413] 8a. The binder for use according to any one of the preceding items, wherein the chemotherapy combination is administered after administration of the binder and / or after administration of pembrolizumab.

[0414] 9. The binding agent for use according to any one of the preceding items, wherein the objective response rate (ORR) is increased compared to standard of care, for example compared to a dosing regimen of the combination of pembrolizumab and chemotherapy alone.

[0415] 10. The binder for use according to any one of the preceding items, wherein the ORR is increased by at least 40%, preferably at least 50%, more preferably at least 60%, such as at least 70%, at least 80%, at least 90%, or at least 95%.

[0416] 11. The binding agent for use according to any one of the preceding items, wherein the disease control rate (DCR) is increased compared to standard treatment, for example compared to a dosing regimen of only the combination of pembrolizumab and chemotherapy.

[0417] 12. A binder for use according to any one of the preceding items, wherein the DCR is increased by at least 40%, preferably at least 50%, more preferably at least 60%, such as at least 70%, at least 80%, at least 90%, or at least 95%.

[0418] 13. The binder for use according to any one of the preceding items, wherein the combination of binder, pembrolizumab, and chemotherapy is each administered at a dose that increases the ORR compared to standard of care, e.g., compared to a dosing regimen of the combination of pembrolizumab and chemotherapy alone.

[0419] 14. The binder for use according to any one of the preceding items, wherein the combination of binder, pembrolizumab, and chemotherapy is each administered at a dose that increases the ORR by at least 40%, preferably at least 50%, more preferably at least 60%, such as at least 70%, at least 80%, at least 90%, or at least 95%.

[0420] 15. The binder for use according to any one of the preceding items, wherein the combination of binder, pembrolizumab, and chemotherapy is each administered at a dose that increases DCR compared to standard of care, for example compared to a dosing regimen of the combination of pembrolizumab and chemotherapy alone.

[0421] 16. The binder for use according to any one of the preceding items, wherein the combination of binder, pembrolizumab, and chemotherapy is each administered at a dose that increases the DCR to at least 40%, preferably at least 50%, more preferably at least 60%, such as at least 70%, at least 80%, at least 90%, or at least 95%.

[0422] 17. The binding agent for use according to any one of the preceding items, wherein the platinum-based chemotherapy agent is carboplatin or cisplatin.

[0423] 18. The binder for use according to any one of the preceding items, wherein the chemotherapy combination is cisplatin and 5-fluorouracil.

[0424] 19. The binder for use according to any one of the preceding items, wherein the chemotherapy combination is carboplatin and 5-fluorouracil.

[0425] 20. The binder for use according to any one of the preceding items, which is administered at a dose of about 50 to 150 mg / day, preferably about 100 mg / day.

[0426] 21. The binding agent for use according to any one of the preceding items, wherein pembrolizumab is administered at a dose of about 150 to 250 mg / day, preferably about 200 mg / day.

[0427] 22. When the platinum-based chemotherapeutic agent is carboplatin, the carboplatin is administered at a dose of AUC=about 4-6, preferably AUC=about 5; or when the platinum-based chemotherapeutic agent is cisplatin, the cisplatin is administered at a dose of about 50-150 mg / m 2 / day, preferably about 100 mg / m 2 The binding agent for use according to any one of the preceding items, administered in a dose of 100 mg / day.

[0428] 23. 5-Fluorouracil is approximately 500-1500 mg / m 2 / day, preferably about 1000 mg / m 2 The binding agent for use according to any one of the preceding items, administered in a dose of 100 mg / day.

[0429] 24. When the binder is administered at a dose of about 100 mg / day and pembrolizumab is administered at a dose of about 200 mg / day, and the platinum-based chemotherapy agent is carboplatin, the carboplatin is administered at a dose of AUC=about 5, or when the platinum-based chemotherapy agent is cisplatin, the cisplatin is administered at a dose of about 100 mg / m 2 / day, and 5-fluorouracil is administered at a dose of approximately 1000 mg / m 2 The binding agent for use according to any one of the preceding items, administered in a dose of 100 mg / day.

[0430] 25.(i) Approximately 100 mg / day of binder and approximately 200 mg / day of pembrolizumab are administered on day 1 of each treatment cycle, and each treatment cycle is 3 weeks long (1Q3W); (ii) a platinum-based chemotherapy agent is administered on at least day 1 of the first and second treatment cycles, and if the platinum-based chemotherapy agent is carboplatin, the carboplatin is administered at a dose of AUC=about 5, or if the platinum-based chemotherapy agent is cisplatin, the cisplatin is administered at a dose of about 100 mg / m 2 Administered at a dose of / day; (iii) Approximately 1000mg / m 2 5-fluorouracil / day is administered on days 1, 2, 3, and 4 of at least the first and second treatment cycles.

[0431] 26. The binder for use according to any one of the preceding items, wherein the binder, pembrolizumab, platinum-based chemotherapy agent, and 5-fluorouracil are administered for six treatment cycles, and then the binder and pembrolizumab alone are administered for at least one further treatment cycle.

[0432] 27. The binder for use according to any one of the preceding items, wherein any one or all of the combination of binder, pembrolizumab and chemotherapy is administered systemically, preferably intravenously.

[0433] 28. The binding agent for use according to any one of the preceding items, wherein the subject is a human subject.

[0434] 29. The binding agent for use according to any one of the preceding items, wherein the subject has not received prior treatment with a checkpoint inhibitor and / or an anti-cancer therapy for recurrent or metastatic disease.

[0435] 30. The binding agent for use according to any one of the preceding items, wherein the subject has not received prior treatment with any anti-cancer therapy, or prior treatment with a checkpoint inhibitor and any anti-cancer therapy.

[0436] 31. The binding agent for use according to any one of the preceding items, wherein the subject has a PD-L1 combined positive score (CPS) ≧1, such as a PD-L1 CPS ≧1 and ≦19 or a PD-L1 CPS ≧20.

[0437] 32. The binding agent for use according to any one of the preceding items, wherein CD40 is human CD40, in particular human CD40 comprising the sequence set forth in SEQ ID NO: 36, and / or CD137 is human CD137, in particular human CD137 comprising the sequence set forth in SEQ ID NO: 38.

[0438] 33. a) the first binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 7 or 9, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 8 or 10; b) The binding agent for use according to any one of the preceding items, wherein the second antigen-binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 17 or 19, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18 or 20.

[0439] 34. a) the first binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; b) The binding agent for use according to any one of the preceding items, wherein the second antigen-binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 11, 12, and 13, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively.

[0440] 35. a) the first binding region comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 7 or 9, and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or 10; b) The binding agent for use according to any one of the preceding items, wherein the second binding region comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 17 or 19, and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 18 or 20.

[0441] 36. a) the first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 7 or 9 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; b) The binding agent for use according to any one of the preceding items, wherein the second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 18 or 20.

[0442] 37. a) the first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 10; b) The binding agent for use according to any one of the preceding items, wherein the second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0443] 38. The binding agent for use according to any one of the preceding items, which is a multispecific antibody, such as a bispecific antibody.

[0444] 39. The binding agent for use according to any one of the preceding items, which is in the form of a full-length antibody or an antibody fragment.

[0445] 40. The binding agent for use according to any one of items 33 to 39, wherein each variable region comprises three complementarity-determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).

[0446] 41. A binding agent for use according to item 40, wherein the complementarity determining regions and the framework regions are arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0447] 42.i) a polypeptide comprising, consisting of, or consisting essentially of said first heavy chain variable region (VH) and first heavy chain constant region (CH); and ii) a polypeptide comprising, consisting of, or consisting essentially of said second heavy chain variable region (VH) and second heavy chain constant region (CH). 42. A binder for use according to any one of items 33 to 41, comprising:

[0448] 43.i) a polypeptide comprising said first light chain variable region (VL) and further comprising a first light chain constant region (CL), and ii) a polypeptide comprising the second light chain variable region (VL) and further comprising a second light chain constant region (CL). 43. A binder for use according to any one of items 33 to 42, comprising:

[0449] 44. An antibody comprising a first binding arm and a second binding arm; The first binding arm comprises: i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH); and ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL). Includes; The second binding arm is iii) a polypeptide comprising the second heavy chain variable region (VH) and a second heavy chain constant region (CH); and iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL). 44. A binder for use according to any one of items 33 to 43, comprising:

[0450] 45.i) a first heavy chain and a light chain comprising said antigen-binding region capable of binding to CD40, and ii) a second heavy chain and a light chain comprising said antigen-binding region capable of binding to CD137; 2. A binder for use according to any one of the preceding items, comprising:

[0451] 46.i) a first heavy chain and a light chain comprising the antigen-binding region capable of binding to CD40, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; and ii) a second heavy chain and a light chain comprising said antigen-binding region capable of binding to CD137, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region. 2. A binder for use according to any one of the preceding items, comprising:

[0452] 47. The binding agent for use according to any one of items 42 to 46, wherein each of the first and second heavy chain constant regions (CH) comprises one or more of the constant heavy chain 1 (CH1) region, the hinge region, the constant heavy chain 2 (CH2) region and the constant heavy chain 3 (CH3) region, preferably at least the hinge region, the CH2 region and the CH3 region.

[0453] 48. The binding agent for use according to any one of items 42 to 47, wherein each of the first and second heavy chain constant regions (CHs) comprises a CH3 region, and the two CH3 regions comprise asymmetric mutations.

[0454] 49. The binding agent for use according to any one of items 42 to 48, wherein in the first heavy chain constant region (CH) at least one amino acid is substituted at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering, and in the second heavy chain constant region (CH) at least one amino acid is substituted at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering, and wherein the first and second heavy chains do not have substitutions at the same positions.

[0455] 50. The binding agent for use according to item 49, wherein (i) the amino acid at the position corresponding to F405 in human IgG1 heavy chain according to EU numbering is L in the first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 in human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in human IgG1 heavy chain according to EU numbering is R in the first heavy chain and the amino acid at the position corresponding to F405 in human IgG1 heavy chain according to EU numbering is L in the second heavy chain.

[0456] 51. The binding agent for use according to any one of the preceding items, which induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CHs) comprising a human IgG1 hinge, CH2 and CH3 regions.

[0457] 52. The binding agent for use according to item 51, wherein the first and second heavy chain constant regions (CHs) have been modified so as to induce Fc-mediated effector function to a lesser extent compared to an otherwise identical antibody comprising unmodified first and second heavy chain constant regions (CHs).

[0458] 53. The binding agent for use according to item 52, wherein each of the unmodified first and second heavy chain constant regions (CHs) comprises an amino acid sequence set forth in SEQ ID NO: 21 or 29.

[0459] 54. The binding agent for use according to item 52 or 53, wherein the Fc-mediated effector function is measured by binding to an Fcγ receptor, binding to C1q, or induction of Fe-mediated cross-linking of an Fcγ receptor.

[0460] ...

Claims

1. 1. A binding agent for use in a method for reducing or preventing the progression of, or treating, head and neck squamous cell carcinoma (HNSCC) in a subject, the method comprising administering to the subject a combination of the binding agent, an inhibitor of the checkpoint PD-1 / PD-L1 axis, and chemotherapy comprising a platinum-based chemotherapy agent and 5-fluorouracil, wherein the binding agent comprises a first binding region that binds to CD40 and a second binding region that binds to CD137.

2. 2. The binding agent for use according to claim 1, wherein the inhibitor of the checkpoint PD-1 / PD-L1 axis is pembrolizumab.

3. 3. The binder for use according to claim 1 or 2, wherein the combination of binder, inhibitor of the checkpoint PD-1 / PD-L1 axis, and chemotherapy is administered in at least one treatment cycle, each treatment cycle being 3 weeks (21 days).

4. 10. The binder for use according to any one of the preceding claims, wherein one dose of binder and one dose of inhibitor of the checkpoint PD-1 / PD-L1 axis are administered every three weeks (1Q3W).

5. 10. The binder for use according to any one of the preceding claims, wherein one dose of the binder and one dose of the inhibitor of the checkpoint PD-1 / PD-L1 axis are administered on day 1 of each treatment cycle.

6. The binding agent for use according to any one of the preceding claims, which is administered prior to the administration of an inhibitor of the checkpoint PD-1 / PD-L1 axis.

7. 10. The binder for use according to any one of the preceding claims, wherein one dose of the chemotherapy combination is administered at least every three weeks (1Q3W) during at least the first treatment cycle.

8. 10. The binding agent for use according to any one of the preceding claims, wherein one dose of the platinum-based chemotherapy agent is administered every three weeks (1Q3W) during at least a first treatment cycle, and one dose of 5-fluorouracil is administered at least every three weeks (1Q3W), such as over four days during the first week during at least a first treatment cycle.

9. 10. The binding agent for use according to any one of the preceding claims, wherein one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle, and one dose of 5-fluorouracil is administered on at least day 1 of at least the first treatment cycle, such as on days 1, 2, 3, and 4 of at least the first treatment cycle.

10. 10. The binding agent for use according to any one of the preceding claims, wherein the objective response rate (ORR) is increased compared to standard of care, such as compared to a dosing regimen of only a combination of an inhibitor of the checkpoint PD-1 / PD-L1 axis, such as pembrolizumab, and chemotherapy.

11. 10. The binder for use according to any one of the preceding claims, wherein the ORR is increased by at least 40%, preferably by at least 50%, more preferably by at least 60%, such as by at least 70%, at least 80%, at least 90%, or at least 95%.

12. 10. The binding agent for use according to any one of the preceding claims, wherein the disease control rate (DCR) is increased compared to standard of care, such as compared to a dosing regimen of only a combination of an inhibitor of the checkpoint PD-1 / PD-L1 axis, such as pembrolizumab, and chemotherapy.

13. 10. The binder for use according to any one of the preceding claims, wherein the DCR is increased by at least 40%, preferably at least 50%, more preferably at least 60%, such as at least 70%, at least 80%, at least 90%, or at least 95%.

14. 10. The binder for use according to any one of the preceding claims, wherein the combination of binder, inhibitor of the checkpoint PD-1 / PD-L1 axis, and chemotherapy is each administered at a dose that increases ORR compared to standard of care, e.g., compared to a dosing regimen of the inhibitor of the checkpoint PD-1 / PD-L1 axis, e.g., pembrolizumab, and the combination of chemotherapy alone.

15. 10. The binder for use according to any one of the preceding claims, wherein the combination of binder, inhibitor of the checkpoint PD-1 / PD-L1 axis, and chemotherapy is each administered at a dose that increases the ORR by at least 40%, preferably at least 50%, more preferably at least 60%, such as at least 70%, at least 80%, at least 90%, or at least 95%.

16. 10. The binding agent for use according to any one of the preceding claims, wherein the combination of the binding agent, the inhibitor of the checkpoint PD-1 / PD-L1 axis, and the chemotherapy are each administered at a dose that increases the DCR compared to standard of care, e.g., compared to a dosing regimen of the inhibitor of the checkpoint PD-1 / PD-L1 axis, e.g., pembrolizumab, and the combination of the chemotherapy alone.

17. 10. The binder for use according to any one of the preceding claims, wherein the combination of binder, inhibitor of the checkpoint PD-1 / PD-L1 axis, and chemotherapy is each administered at a dose that increases the DCR by at least 40%, preferably at least 50%, more preferably at least 60%, such as at least 70%, at least 80%, at least 90%, or at least 95%.

18. 10. The binding agent for use according to any one of the preceding claims, wherein the platinum-based chemotherapeutic agent is carboplatin or cisplatin.

19. The binding agent for use according to any one of the preceding claims, wherein the chemotherapy combination is cisplatin and 5-fluorouracil.

20. The binder for use according to any one of the preceding claims, wherein the chemotherapy combination is carboplatin and 5-fluorouracil.

21. The binder for use according to any one of the preceding claims, administered in a dose of about 50-150 mg / day, preferably about 100 mg / day.

22. The binding agent for use according to any one of the preceding claims, wherein the inhibitor of the checkpoint PD-1 / PD-L1 axis is administered in a dose of about 150-250 mg / day, preferably about 200 mg / day.

23. When the platinum-based chemotherapeutic agent is carboplatin, the carboplatin is administered at a dose of about AUC=4-6, preferably about AUC=5, or when the platinum-based chemotherapeutic agent is cisplatin, the cisplatin is administered at a dose of about 50-150 mg / m ２ / day, preferably about 100 mg / m ２ 10. The binder for use according to any one of the preceding claims, administered in a dose of 0.1 mg / day.

24. 5-fluorouracil at about 500 to 1500 mg / m ２ / day, preferably about 1000 mg / m ２ 10. The binder for use according to any one of the preceding claims, administered in a dose of 0.1 mg / day.

25. When the binding agent is administered at a dose of about 100 mg / day, and the inhibitor of the checkpoint PD-1 / PD-L1 axis is administered at a dose of about 200 mg / day, and the platinum-based chemotherapeutic agent is carboplatin, the carboplatin is administered at a dose of about AUC=5, or when the platinum-based chemotherapeutic agent is cisplatin, the cisplatin is administered at a dose of about 100 mg / m ２ / day, and 5-fluorouracil is administered at a dose of about 1000 mg / m ２ 10. The binder for use according to any one of the preceding claims, administered in a dose of 0.1 mg / day.

26. (i) about 100 mg / day of the binder and about 200 mg / day of the inhibitor of the checkpoint PD-1 / PD-L1 axis are administered on day 1 of each treatment cycle, wherein each treatment cycle is three weeks long (1Q3W); (ii) a platinum-based chemotherapeutic agent is administered on at least day 1 of the first and second treatment cycles, and if the platinum-based chemotherapeutic agent is carboplatin, the carboplatin is administered at a dose of AUC=about 5, or if the platinum-based chemotherapeutic agent is cisplatin, the cisplatin is administered at a dose of about 100 mg / m ２ / day; (iii) Approximately 1000mg / m ２ 10. The method of claim 9, wherein 5-fluorouracil / day is administered on days 1, 2, 3, and 4 of at least the first and second treatment cycles.

27. 10. The binder for use according to any one of the preceding claims, wherein the binder, the inhibitor of the checkpoint PD-1 / PD-L1 axis, the platinum-based chemotherapy agent, and 5-fluorouracil are administered for six treatment cycles, followed by at least one further treatment cycle in which only the binder and the inhibitor of the checkpoint PD-1 / PD-L1 axis are administered.

28. 10. The binding agent for use according to any one of the preceding claims, wherein any one or all of the combination of binding agent, inhibitor of the checkpoint PD-1 / PD-L1 axis, and chemotherapy are administered systemically, preferably intravenously.

29. 10. The binding agent for use according to any one of the preceding claims, wherein the subject is a human subject.

30. 10. The binding agent for use according to any one of the preceding claims, wherein the subject has not received prior treatment with a checkpoint inhibitor and / or an anti-cancer therapy for recurrent or metastatic disease.

31. 10. The binding agent for use according to any one of the preceding claims, wherein the subject has not received prior treatment with any anti-cancer therapy, or prior treatment with a checkpoint inhibitor and any anti-cancer therapy.

32. 10. The binding agent for use according to any one of the preceding claims, wherein the subject has a PD-L1 combined positive score (CPS) > 1, such as a PD-L1 CPS > 1 and < 19 or a PD-L1 CPS > 20.

33. 2. The binding agent for use according to any one of the preceding claims, wherein CD40 is human CD40, in particular human CD40 comprising the sequence set forth in SEQ ID NO: 36, and / or CD137 is human CD137, in particular human CD137 comprising the sequence set forth in SEQ ID NO:

38.

34. a) the first binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 7 or 9, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 8 or 10; b) The binding agent for use according to any one of the preceding claims, wherein the second antigen-binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 17 or 19, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18 or 20.

35. a) the first binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; b) The binding agent for use according to any one of the preceding claims, wherein the second antigen-binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 11, 12, and 13, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively.

36. a) the first binding region comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 7 or 9, and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or 10; b) The binding agent for use according to any one of the preceding claims, wherein the second binding region comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 17 or 19, and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 18 or 20.

37. a) the first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 7 or 9 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; b) The binding agent for use according to any one of the preceding claims, wherein the second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 18 or 20.

38. a) the first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 10; b) The binding agent for use according to any one of the preceding claims, wherein the second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO:

20.

39. 10. The binding agent for use according to any one of the preceding claims, which is a multispecific antibody, such as a bispecific antibody.

40. Binding agent for use according to any one of the preceding claims in the form of a full length antibody or an antibody fragment.

41. 41. The binding agent for use according to any one of claims 34 to 40, wherein each variable region comprises three complementarity determining regions (CDR1, CDR2 and CDR3) and four framework regions (FR1, FR2, FR3 and FR4).

42. 42. The binding agent for use according to claim 41, wherein the complementarity determining regions and the framework regions are arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

43. i) a polypeptide comprising, consisting of, or consisting essentially of said first heavy chain variable region (VH) and first heavy chain constant region (CH); and ii) a polypeptide comprising, consisting of, or consisting essentially of said second heavy chain variable region (VH) and second heavy chain constant region (CH).

43. A binder for use according to any one of claims 34 to 42, comprising:

44. i) a polypeptide comprising said first light chain variable region (VL) and further comprising a first light chain constant region (CL); and ii) a polypeptide comprising said second light chain variable region (VL) and further comprising a second light chain constant region (CL). A binder for use according to any one of claims 34 to 43, comprising:

45. an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises: i) a polypeptide comprising the first heavy chain variable region (VH) and the first heavy chain constant region (CH); and ii) a polypeptide comprising the first light chain variable region (VL) and the first light chain constant region (CL). Including; The second binding arm is iii) a polypeptide comprising the second heavy chain variable region (VH) and a second heavy chain constant region (CH); and iv) a polypeptide comprising the second light chain variable region (VL) and the second light chain constant region (CL). A binder for use according to any one of claims 34 to 44, comprising:

46. i) a first heavy chain and a light chain comprising said antigen-binding region capable of binding to CD40; and ii) a second heavy chain and a light chain comprising said antigen-binding region capable of binding to CD137; 10. A binder for use according to any one of the preceding claims, comprising:

47. i) a first heavy chain and a light chain comprising said antigen-binding region capable of binding to CD40, wherein the first heavy chain comprises a first heavy chain constant region and the first light chain comprises a first light chain constant region; and ii) a second heavy chain and a light chain comprising said antigen-binding region capable of binding to CD137, wherein the second heavy chain comprises a second heavy chain constant region and the second light chain comprises a second light chain constant region.

10. A binder for use according to any one of the preceding claims, comprising:

48. 48. The binding agent for use according to any one of claims 43 to 47, wherein each of the first and second heavy chain constant regions (CH) comprises one or more of the constant heavy chain 1 (CH1) region, the hinge region, the constant heavy chain 2 (CH2) region and the constant heavy chain 3 (CH3) region, preferably at least the hinge region, the CH2 region and the CH3 region.

49. 49. The binding agent for use according to any one of claims 43 to 48, wherein each of the first and second heavy chain constant regions (CHs) comprises a CH3 region, and the two CH3 regions comprise asymmetric mutations.

50. 50. The binding agent for use according to any one of claims 43 to 49, wherein the first heavy chain constant region (CH) has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering, and the second heavy chain constant region (CH) has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering, and wherein the first and second heavy chains do not have substitutions at the same positions.

51. 51. The binding agent for use according to claim 50, wherein (i) the amino acid at the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in the first heavy chain constant region (CH) and the amino acid at the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in the second heavy chain constant region (CH), or (ii) the amino acid at the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering is R in the first heavy chain and the amino acid at the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering is L in the second heavy chain.

52. 10. The binding agent for use according to any one of the preceding claims, which induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen-binding regions and two heavy chain constant regions (CHs) comprising a human IgG1 hinge, CH2 and CH3 regions.

53. 53. The binding agent for use of claim 52, wherein the first and second heavy chain constant regions (CHs) have been modified such that the antibody induces Fc-mediated effector function to a lesser extent compared to an otherwise identical antibody comprising unmodified first and second heavy chain constant regions (CHs).

54. 54. The binding agent for use of claim 53, wherein each of the unmodified first and second heavy chain constant regions (CH) comprises the amino acid sequence set forth in SEQ ID NO: 21 or 29.

55. 55. The binding agent for use of claim 53 or 54, wherein the Fc-mediated effector function is measured by binding to an Fcγ receptor, binding to C1q, or induction of Fe-mediated cross-linking of an Fcγ receptor.

56. 56. The binding agent for use of claim 55, wherein the Fc-mediated effector function is measured by binding to C1q.

57. 57. The binding agent for use according to any one of claims 52 to 56, wherein the first and second heavy chain constant regions are modified such that binding of C1q to the antibody is reduced compared to a wild type antibody, preferably by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%; and wherein C1q binding is preferably determined by ELISA.

58. 58. The binding agent for use according to any one of claims 43 to 57, wherein in at least one of the first and second heavy chain constant regions (CH), one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively.

59. 59. The binding agent for use according to claim 58, wherein the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E in the first and second heavy chains, respectively.

60. 60. The binding agent for use according to claim 58 or 59, wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A in the first and second heavy chain constant regions (HC), respectively.

61. 61. The binding agent for use according to any one of claims 58 to 60, wherein the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F and E, respectively, and (i) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering in the second heavy chain is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is R and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering in the second heavy chain is L.

62. 62. The binding agent for use according to any one of claims 58 to 61, wherein the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering in both the first and second heavy chain constant regions are F, E, and A, respectively, and (i) the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering in the first heavy chain constant region is L and the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is R, or (ii) the position corresponding to K409 in a human IgG1 heavy chain according to EU numbering in the first heavy chain is R and the position corresponding to F405 in a human IgG1 heavy chain according to EU numbering in the second heavy chain constant region is L.

63. the constant region of the first and / or second heavy chain comprises: a) the sequence set forth in SEQ ID NO: 21 or 29 [IgG1-FC]; b) a subsequence of the sequence of a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions, for example at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2 substitutions or at most 1 substitution compared to the amino acid sequence defined in a) or b).

63. A binding agent for use according to any one of claims 43 to 62, comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of:

64. the constant region of the first or second heavy chain, e.g., the second heavy chain, comprises: a) the sequence set forth in SEQ ID NO: 22 or 30 [IgG1-F405L]; b) a subsequence of the sequence of a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 9 substitutions, e.g. at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2 substitutions or at most 1 substitution, compared to the amino acid sequence defined in a) or b).

63. A binding agent for use according to any one of claims 43 to 62, comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of:

65. the constant region of the first or second heavy chain, e.g., the first heavy chain, comprises: a) the sequence set forth in SEQ ID NO: 23 or 31 [IgG1-F409R]; b) a subsequence of the sequence of a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions, e.g. at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2 substitutions or at most 1 substitution compared to the amino acid sequence defined in a) or b).

63. A binding agent for use according to any one of claims 43 to 62, comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of:

66. the constant region of the first and / or second heavy chain comprises: a) the sequence set forth in SEQ ID NO: 24 or 32 [IgG1-Fc_FEA]; b) a subsequence of the sequence of a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 7 substitutions, e.g. at most 6 substitutions, at most 5, at most 4, at most 3, at most 2 substitutions or at most 1 substitution compared to the amino acid sequence defined in a) or b).

63. A binding agent for use according to any one of claims 43 to 62, comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of:

67. the constant region of the first and / or second heavy chain, e.g., the second heavy chain, a) the sequence set forth in SEQ ID NO: 25 or 33 [IgG1-Fc_FEAL]; b) a subsequence of the sequence of a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 6 substitutions, e.g. at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions or at most 1 substitution compared to the amino acid sequence defined in a) or b).

67. A binding agent for use according to any one of claims 43 to 66, comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of:

68. the constant region of the first and / or second heavy chain, e.g., the first heavy chain, comprises: a) the sequence set forth in SEQ ID NO: 26 or 34 [IgG1-Fc_FEAR]; b) a subsequence of the sequence of a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 6 substitutions, e.g. at most 5 substitutions, at most 4 substitutions, at most 3 substitutions, at most 2 substitutions or at most 1 substitution compared to the amino acid sequence defined in a) or b).

68. A binding agent for use according to any one of claims 43 to 67, comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of:

69. 10. The binding agent for use according to any one of the preceding claims, comprising a kappa (κ) light chain constant region.

70. 10. The binding agent for use according to any one of the preceding claims, comprising a lambda (λ) light chain constant region.

71. 10. The binding agent for use according to any one of the preceding claims, wherein the first light chain constant region is a kappa (κ) light chain constant region or a lambda (λ) light chain constant region.

72. 10. The binding agent for use according to any one of the preceding claims, wherein the second light chain constant region is a lambda (λ) light chain constant region or a kappa (κ) light chain constant region.

73. 10. The binding agent for use according to any one of the preceding claims, wherein the first light chain constant region is a kappa (κ) light chain constant region and the second light chain constant region is a lambda (λ) light chain constant region, or the first light chain constant region is a lambda (λ) light chain constant region and the second light chain constant region is a kappa (κ) light chain constant region.

74. Kappa (κ) light chains a) the sequence set forth in SEQ ID NO: 27; b) a subsequence of the sequence of a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions, e.g. at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2 substitutions or at most 1 substitution compared to the amino acid sequence defined in a) or b).

74. The binding agent for use according to any one of claims 69 to 73, comprising an amino acid sequence selected from the group consisting of:

75. Lambda (λ) light chains a) the sequence set forth in SEQ ID NO: 28; b) a subsequence of the sequence of a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids are deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions, e.g. at most 9 substitutions, at most 8, at most 7, at most 6, at most 5, at most 4 substitutions, at most 3, at most 2 substitutions or at most 1 substitution compared to the amino acid sequence defined in a) or b).

75. The binding agent for use according to any one of claims 70 to 74, comprising an amino acid sequence selected from the group consisting of:

76. 10. The binding agent for use according to any one of the preceding claims, which is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

77. 10. The binding agent for use according to any one of the preceding claims, which is a full-length IgG1 antibody.

78. 10. The binding agent for use according to any one of the preceding claims, which is an antibody of the IgG1m(f) allotype.

79. 1. A method for reducing or preventing the progression of head and neck squamous cell carcinoma (HNSCC) or for treating HNSCC in a subject, comprising administering to the subject a combination of a binding agent, an inhibitor of the checkpoint PD-1 / PD-L1 axis, and chemotherapy comprising a platinum-based chemotherapy agent and 5-fluorouracil, wherein the binding agent comprises a first binding region that binds CD40 and a second binding region that binds CD137.

80. 80. The method of claim 79, wherein the inhibitor of the checkpoint PD-1 / PD-L1 axis is pembrolizumab.

81. 81. The method of claim 79 or 80, wherein the binding agent and / or the subject and / or the administration regimen is as defined in any one of claims 1 to 78.

Images