Combination therapy of multispecific antibodies against CD40 and CD137 with anti-PD1 Ab and chemotherapy

Abstract

The present invention relates to combination treatments that use a binding agent that binds to human CD40 and binds to human CD137 in combination with a checkpoint inhibitor, which is an inhibitor of the PD-1 / PD-L1 axis, in particular pembumab, and a 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 a combination therapy that uses an agent that binds to human CD40 and binds to human CD137 in combination with a checkpoint inhibitor and chemotherapy to reduce or prevent the progression of head and neck squamous cell carcinoma (HNSCC) or to treat HNSCC, and the checkpoint inhibitor is an inhibitor of the PD-1 / PD-L1 axis (in particular, pembrolizumab). Background Art

[0002] CD40 is a member of the tumor necrosis factor (TNF) receptor (TNFR) family and is known as a co-stimulatory protein present on a variety of cell types. CD40 is constitutively expressed by antigen-presenting cells (APCs) including 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 wide range of tumor cells.

[0003] Presentation of peptide antigens to antigen-specific CD4 + T cells, together with co-stimulatory signals (from CD80 and / or CD86), leads to CD4 + T cell activation and upregulation of the DC licensing factors CD40 ligand (CD40L) and lymphotoxin-α1β2 (LTα1β2). Expression of CD40L and LT LTα1β2 on activated antigen-specific CD4 + T cells induces signal transduction through CD40 and the LTβ receptor (LTβR), and this licenses DCs to induce CD8 +T cell responses. CD40 signaling leads to the production of interleukin-12 (IL-12) and the 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). Signaling systems that control nuclear factor kappaB (NF-κB) activity respond to almost all TNFR superfamily members. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) also contribute to these events. CD8 + T cell sensitization leads to the upregulation of CD27, 4-1BB, OX40, and glucocorticoid-induced TNFR-related protein (GITR). CD8 + T cell stimulation of these receptors by their cognate TNF superfamily ligands in combination with IL-12 and type I IFN results in robust CD8 + T cell activation, proliferation, and effector functions, as well as CD8 +Formation and maintenance of T cell memory. CD40 antibodies can play different roles: by inducing antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or antibody-dependent cell-mediated phagocytosis (ADCP), inducing cell signaling to induce direct apoptosis or growth arrest, and killing CD40-expressing tumor cells by licensing APCs to stimulate anti-cancer immune responses independent of CD40 expression on tumor cells. Antibodies that bind to CD40 can trigger CD40 on APCs to sensitize effector cytotoxic T lymphocytes (CTLs) and induce the release of IL-2 from these cells, and indirectly activate NK cells. Antibodies that stimulate CD40 have been disclosed in the prior art and include the human IgG2 antibody CP-870,893 (WO 03 / 040170), the humanized IgG1 antibody dacetuzumab (WO 00 / 075348), and the chimeric IgG1 antibody Chi Lob 7 / 4 (US 2009 / 0074711). In addition, antagonistic CD40 antibodies, the human IgG1 antibody lucatumumab (WO 02 / 028481), have been disclosed.

[0004] CD137 (4-1BB) is also a member of the TNFR family. CD137 is CD8 +Costimulatory molecules on T cells, CD4+ T cells, regulatory T cells (Tregs), natural killer T cells (NK(T) cells), B cells, and neutrophils. On T cells, CD137 is not constitutively expressed but is induced after activation of the T-cell receptor (TCR) (e.g., on tumor infiltrating lymphocytes (TILs) (Gros et al., J. Clin Invest 2014;124(5):2246-59)). Stimulation through its natural ligand 4-1BBL or agonist antibodies results in signal transduction using TRAF-2 and TRAF-1 as adaptors. Early signal transduction through CD137 involves a K-63 polyubiquitination reaction, which ultimately leads to the activation of the nuclear factor (NF)-κB and mitogen-activated protein (MAP)-kinase pathways. Signal transduction results in enhanced T-cell costimulation, proliferation, cytokine production, maturation, and prolonged survival of CD8+ T cells. Agonistic antibodies against CD137 have been shown to promote antitumor control of T cells in multiple preclinical models (Murillo et al., Clin Cancer Res 2008;14(21):6895-906). Antibodies that stimulate CD137 can induce the survival and proliferation of T cells, thereby enhancing the antitumor immune response. Antibodies that stimulate CD137 have been disclosed in the prior art and include the human IgG4 antibody urelumab (AU 2004279877) 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". WO 2018 / 011421 provides binding agents, such as bispecific antibodies, that bind to human CD40 and bind to human CD137. Such bispecific antibodies crosslink CD40 on antigen-presenting cells (APCs) with 4-1BB on activated T cells and thereby induce costimulation of both cell types and costimulatory activity in both cell types, which can be used to treat solid tumors.

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

[0007] In tumor-draining lymph nodes and within the tumor microenvironment, 4-1BB is expressed by CD4+ and CD8+ T cell subsets, characterized by the co-expression of multiple TCR-induced molecules (including high levels of programmed cell death 1 (PD-1)) (Groset 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 can contribute to T cell exhaustion and reduce T cell activation after binding of T cells to their ligand programmed cell death 1 ligand 1 (PD-L1) (Yu et al., Eur J Pharmacol 881:173240). The expression of PD-L1 is often upregulated by tumor cells, especially in inflamed tumors (Teng, et al., Cancer Res 75:2139-2145). Thus, tumor cells provide inhibitory signals to activated T cells through which they can 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 diagnosed more than 600,000 times per year worldwide. In 2020, approximately 65,630 new cases of oral, pharyngeal, and laryngeal cancers will occur in the United States during the same period, with an estimated 14,500 deaths (Clinical Practice Guidelines in Oncology, 2nd edition, 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 to 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; however, the median overall survival (mOS) is less than 15 months (Clinical Practice Guidelines in Oncology, 2nd edition, 2021).

[0009] Accordingly, HNSCC remains a highly unmet medical need, and there is further opportunity to improve outcomes with new treatment approaches. SUMMARY OF THE INVENTION

[0010] The inventors unexpectedly found that the following combination enhances the immune response against HNSCC: (i) stimulation with an agent that binds to human CD40 and binds to 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 chemotherapeutic agent (particularly cisplatin or carboplatin) and 5-fluorouracil.

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

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

[0013] In other aspects, the present disclosure provides a kit comprising (i) a binder 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 or carboplatin), and (iv) 5-fluorouracil, and a kit for, e.g., a method for reducing or arresting the progression of HNSCC or treating HNSCC in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A schematic diagram showing the expected mode of action of a CD40×4-1BB bispecific antibody. CD40 is expressed on antigen-presenting cells (APCs) as well as tumor cells. 4-1BB (CD137) is expressed on activated T cells. DuoBoty-CD40×4-1BB (GEN1042 / BNT312) is a bispecific antibody that crosslinks CD40 on antigen-presenting cells (APCs) with 4-1BB on activated T cells, thereby conditionally stimulating these two cell types. Thus, the CD40×4-1BB bispecific antibody can enhance DC licensing, T cell clonal expansion, cytokine production, T cell survival, and T cell- and NK cell-mediated cytotoxicity.

[0015] Figure 2 Shows the MC38 syngeneic tumor model established by subcutaneous inoculation of 1×10 6 MC38 cells into hCD40×h4-1BB dKI C57BL / 6 mice. When the tumors reached 37 mm 3When reaching the average volume, mice were randomized and treated with the chemotherapy regimens of GEN1042-mIgG2a (1 mg / kg, BIW×3), anti-mouse PD-1 antibody (anti-mPD-1; 10 mg / kg, BIW×3), carboplatin (20 mg / kg, BIW×3), and 5-fluorouracil (5-FU, 25 mg / kg, Q3D×5) either alone or in combination. The control group was administered both PBS and 0.9% saline (BIW×3). (A) The data shown are the median tumor volumes of each treatment group (n = 10), where the data were carried forward for animals that reached the termination criteria. The arrows indicate the treatment days. (B) The data shown are the tumor volumes of different treatment groups on day 20, which is the last day when all groups were still intact (the vertical dashed line in A), including the number of mice with complete tumor regression (CR). Mann-Whitney analysis was used to compare the tumor volumes between treatment groups (p<0.05 = *).

[0016] Figure 3A Shows the best change in target lesions of subjects with previously untreated recurrent or metastatic HNSCC treated with chemotherapy, pembrolizumab, and GEN1042. Figure 3B Shows the change in target lesions of the subjects over time, with the data cutoff date being October 3, 2022.

[0017] Figure 4A Shows the best change in target lesions of subjects with previously untreated recurrent or metastatic HNSCC treated with pembrolizumab and GEN1042. Figure 4B Shows the change in target lesions of the subjects over time, with the data cutoff date being October 7, 2022.

[0018] Figure 5 Shows the increase in pro-inflammatory IFNγ in the sera of patients receiving GEN1042+SoC. Serum samples were measured for the circulating levels of IFNγ (interferon γ) at baseline and at multiple time points (days 1, 3, 8, and 15) after the administration of GEN1042+SoC in cycles 1 and 2, and before the dosing of cycle 3. Nine patients have been analyzed and grouped according to the treatment. Interim data limited the mature data at all time points, so the maximum available n for each regimen at any given time point was GEN1042+pembro[5] and GEN1042+chemo+pembro[4]. The IFNγ levels in the serum samples were determined by Meso Scale Discovery (MSD) multiplex immunoassay. Abbreviations: IFN = interferon, 1042 = GEN1042, Chemo = 5FU + carbo / cisplatin, pembro = pembrolizumab, pg = picogram, mL = milliliter, SEM = standard error of the mean, pre = before dosing, SoC = standard of care Test reference range: IFNg (pg / mL) < 11.81; Clinical data cutoff = September 26, 2022

[0019] Figure 6 An increase in TARC in the serum of patients administered GEN1042 + SoC is shown. Serum samples were measured for circulating levels of TARC at baseline and at multiple time points (days 1, 3, 8, and 15) after administration of GEN1042 + SoC in cycles 1 and 2, and before dosing in cycle 3. Seven patients have been analyzed and grouped by treatment. Interim data limited mature data at all time points, so the maximum available n for each 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 = picogram, mL = milliliter, SEM = standard error of the mean, pre = before dosing, DC = dendritic cell, SoC = standard of care Test reference range: TARC (pg / mL) < 513; Clinical data cutoff = September 26, 2022

[0020] Figure 7 shows the trafficking / marginalization of immune cells after administration of GEN 1042 + SoC. Immunophenotyping of peripheral blood was performed on whole blood collected at baseline and at multiple time points (days 1, 3, 8, and 15) after administration of GEN1042 + SoC in cycles 1 and 2, and before dosing in cycle 3. Six patients have been analyzed and grouped by treatment. Interim data limited mature data at all time points, so the maximum available n for each regimen at any given time point was GEN1042 + pembro [3] and GEN1042 + chemo + pembro [3]. The frequency of immune cells in whole blood samples was evaluated by flow cytometry. Figure 7A Results for CD8 T cells are shown, Figure 7B Results for B cells are shown. Abbreviations: 1042 = GEN1042, Chemo = 5FU + carbo / cisplatin, pembro = pembrolizumab, uL = microliter, SEM = standard error of the mean, pre = before dosing, abs = absolute value, SoC = standard of care, Clinical data cutoff = September 26, 2022

[0021] Figure 8 shows the proliferation of T cells after GEN1042 + SoC administration. Immunophenotypic analysis of peripheral blood was performed in whole blood collected at baseline and at multiple time points (days 1, 3, 8, and 15) after GEN1042 + SoC administration in cycles 1 and 2, and before dosing in cycle 3. Seven patients have been analyzed and grouped by treatment. Interim data limited mature data at all time points, so the maximum available n for each regimen at any given time point was GEN1042 + pembro [3] and GEN1042 + chemo + pembro [4]. The frequency of proliferating (% Ki67) T cells in whole blood samples was evaluated by flow cytometry. Figure 8A Results for proliferating (% Ki67) CD8 T cells are shown. Figure 8B Results for proliferating (% Ki67) effector memory CD8 T cells (CD45RA-, CCR7-) are shown. Abbreviations: 1042 = GEN1042, Chemo = 5FU + carbo / cisplatin, pembro = pembrolizumab, uL = microliter, SEM = standard error of the mean, pre = before dosing, abs = absolute value, SoC = standard of care, Tem = T effector memory, Clinical data cutoff = September 26, 2022

[0022] Figure 9 shows the activation of T cells after GEN1042 + SoC administration. Immunophenotypic analysis of peripheral blood was performed in whole blood collected at baseline and at multiple time points (days 1, 3, 8, and 15) after GEN1042 + SoC administration in cycles 1 and 2, and before dosing in cycle 3. Seven patients have been analyzed and grouped by treatment. Interim data limited mature data at all time points, so the maximum available n for each regimen at any given time point was GEN1042 + pembro [3] and GEN1042 + chemo + pembro [4]. The frequency of activated (% 4-1BB) CD8 T cells in whole blood samples was evaluated by flow cytometry. Figure 9A Results for CD8 T cells are shown. Figure 9B Results specific for effector memory CD8 T cells (CD45RA-, CCR7-) are shown. Abbreviations: 1042 = GEN1042, Chemo = 5FU + carbo / cisplatin, pembro = pembrolizumab, uL = microliter, SEM = standard error of the mean, pre = before dosing, abs = absolute value, SoC = standard of care, Tem = T effector memory Clinical data cutoff = September 26, 2022

[0023] Figure 10 Shows the activation of B cells after GEN1042 + SoC administration. Immunophenotyping of peripheral blood was performed on whole blood collected at baseline and at multiple time points (days 1, 3, 8, and 15) after GEN1042 + SoC administration in cycles 1 and 2 and before dosing in cycle 3. Six patients have been analyzed and grouped by treatment. Interim data restricted mature data at all time points, so at any given time point, the maximum available n for each protocol was 1042 + pembro[3] and 1042 + chemo + pembro[3]. The frequency of activated (% 4-1BB+) B cells in whole blood samples was evaluated by flow cytometry. Abbreviations: 1042 = GEN1042, Chemo = 5FU + carbo / cisplatin, pembro = pembrolizumab, uL = microliter, SEM = standard error of the mean, pre = before dosing, abs = absolute value, SoC = standard of care Clinical data cutoff = September 26, 2022 Table 1 - Sequence Detailed Description

[0024] Although the present disclosure is further described in more detail below, it should be understood that the present disclosure is not limited to the specific methods, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing some specific embodiments only and are 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.

[0025] In the following, the elements of the present disclosure will be described in more detail. These elements are listed in specific embodiments, however, it should be understood that they can be combined in any way and in any number to form additional embodiments. The multiple described embodiments and preferred embodiments should not be construed as limiting the present disclosure to the explicitly described embodiments. This specification should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. In addition, unless the context otherwise indicates, any arrangement and combination of all the elements described in this application should be considered to be disclosed by the specification of this application. For example, if in a preferred embodiment of a binder used herein, the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 26 or 34 [IgG1-Fc_FEAR], or consists essentially of the amino acid sequence shown in SEQ ID NO: 26 or 34 [IgG1-Fc_FEAR], or consists of the amino acid sequence shown in SEQ ID NO: 26 or 34 [IgG1-Fc_FEAR], and in another preferred embodiment of a binder used herein, the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 25 or 33 [IgG1-Fc_FEAL], or consists essentially of the amino acid sequence shown in SEQ ID NO: 25 or 33 [IgG1-Fc_FEAL], or consists of the amino acid sequence shown in SEQ ID NO: 25 or 33 [IgG1-Fc_FEAL], then in another preferred embodiment of a binder used herein, the first heavy chain comprises the amino acid sequence shown in SEQ ID NO: 26 or 34 [IgG1-Fc_FEAR], or consists essentially of the amino acid sequence shown in SEQ ID NO: 26 or 34 [IgG1-Fc_FEAR], or consists of the amino acid sequence shown in SEQ ID NO: 26 or 34 [IgG1-Fc_FEAR], and the second heavy chain comprises the amino acid sequence shown in SEQ ID NO: 25 or 33 [IgG1-Fc_FEAL], or consists essentially of the amino acid sequence shown in SEQ ID NO: 25 or 33 [IgG1-Fc_FEAL], or consists of the amino acid sequence shown in SEQ ID NO: 25 or 33 [IgG1-Fc_FEAL].

[0026] Preferably, terms used herein such as ″A multilingual glossary of biotechnological tems:(IUPAC Recommendations)″, HG.W.Leuenberger, B.Nagel, and H Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). as defined in

[0027] Unless otherwise indicated, the practice of the present disclosure will employ conventional chemical, biochemical, cell biological, immunological, and recombinant DNA techniques as described in the literature in this field (see, for example 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 Chemic", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0028] Unless otherwise specified herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "for example") provided herein is merely intended to better illustrate the present disclosure and does not limit the scope of the present disclosure that is otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

[0029] 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. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.

[0030] Throughout the text of this specification, several documents are cited. Whether above or below, each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, guides, etc.) is hereby incorporated by reference in its entirety. Nothing in this text should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of a prior invention. Definitions

[0031] Definitions applicable to all aspects of the present disclosure will be provided below. Unless otherwise indicated, the following terms have the following meanings. Any term not defined herein has its generally recognized meaning in the art.

[0032] Throughout this specification and the appended claims, unless the context requires otherwise, the words "comprising / including" and variations thereof will be understood to imply the inclusion of the stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The term "consisting essentially of" means excluding any other member, integer or step of any substantial significance. The term "comprising / including" encompasses the term "consisting essentially of", and "consisting essentially of" in turn encompasses the term "consisting of". Thus, each time it appears in this application, the term "comprising / including" may be replaced by the term "consisting essentially of" or "consisting of". Similarly, each time it appears in this application, the term "consisting essentially of" may be replaced by the term "consisting of".

[0033] Unless otherwise specified herein or clearly contradicted by the context, nouns and similar references used in the context of describing the present disclosure (especially in the context of the claims) without an article are to be construed to cover both the singular and the plural.

[0034] As used herein, "and / or" is considered a specific disclosure of each of two designated features or components having or not having the other of the two. For example, "X and / or Y" is considered a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, as if each were listed separately herein.

[0035] In the context of the present disclosure, the term "about" denotes the interval of precision that will be understood by a person of ordinary skill in the art to still ensure the technical effect of the feature under discussion. The term generally denotes a deviation 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 ±0.01%, from the specified numerical value. As will be understood by a person of ordinary skill in the art, the specific such deviation of the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biotechnological effects may generally have a greater such deviation than artificial or engineered technical effects.

[0036] 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 comprise synthetic, modified, or non-naturally occurring moieties, particularly non-peptide moieties. Such moieties may, for example, link the desired antigen-binding functional group or region, such as an antibody or an antibody fragment. In one embodiment, the binding agent is a synthetic construct comprising an antigen-binding CDR or variable region.

[0037] As used herein, "immune checkpoint" refers to a regulator of the immune system and, in particular, to co-stimulatory and inhibitory signals that regulate the amplitude and quality of T cell receptor recognition of an antigen. In certain embodiments, the immune checkpoint is an inhibitory signal. In certain embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1 and / or PD-L2. In certain embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86 to displace CD28 binding. In certain embodiments, the inhibitory signal is the interaction between LAG-3 and MHC class II molecules. In certain embodiments, the inhibitory signal is the interaction between TIM-3 and one or more of its ligands, such as galectin-9, PtdSer, HMGB1, and CEACAM1. In certain embodiments, the inhibitory signal is the interaction between one or more KIRs and their ligands. In certain embodiments, the inhibitory signal is the interaction between TIGIT and one or more of its ligands PVR, PVRL2, and PVRL3. In certain embodiments, the inhibitory signal is the interaction between CD94 / NKG2A and HLA-E. In certain embodiments, the inhibitory signal is the interaction between VISTA and its binding partner. In certain embodiments, the inhibitory signal is the interaction between one or more Siglecs and their ligands. In certain embodiments, the inhibitory signal is the interaction between GARP and one or more of its ligands. In certain embodiments, the inhibitory signal is the interaction between CD47 and SIRPα. In certain embodiments, the inhibitory signal is the interaction between PVRIG and PVRL2. In certain embodiments, the inhibitory signal is the interaction between CSF1R and CSF1. In certain embodiments, the inhibitory signal is the interaction between BTLA and HVEM. In certain embodiments, the inhibitory signal is part of the adenosinergic pathway, e.g., the interaction between A2AR and / or A2BR and adenosine produced by CD39 and CD73. In certain embodiments, the inhibitory signal is the interaction between B7-H3 and its receptor and / or B7-H4 and its receptor. In certain embodiments, the inhibitory signal is mediated by IDO, CD20, NOX, or TDO.

[0038] As used herein, the terms "checkpoint inhibitor" (CPI) and "immune checkpoint (ICP) inhibitor" are synonymous. The term refers to a molecule, such as a binder, that reduces, inhibits, interferes with, or negatively regulates, in whole or in part, one or more checkpoint proteins, or reduces, inhibits, interferes with, or negatively regulates, in whole or in part, the expression of one or more checkpoint proteins, such as a molecule, such as a binder, that inhibits an immune checkpoint, particularly an inhibitory signal of an immune checkpoint. In one embodiment, the immune checkpoint inhibitor binds to one or more checkpoint proteins. In one embodiment, the immune checkpoint inhibitor binds to one or more molecules that regulate checkpoint proteins. In one embodiment, the immune checkpoint inhibitor binds to a precursor of one or more checkpoint proteins, for example, at the DNA or RNA level. Any agent that functions as a checkpoint inhibitor according to the present disclosure can be used. As used herein, the term "in part" 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% level, such as the level of inhibition of a checkpoint protein.

[0039] In one embodiment, a checkpoint inhibitor can be any compound that inhibits the inhibitory signals of immune checkpoints, such as any binder, wherein the inhibitory signals are selected from: the interaction between PD-1 and PD-L1 and / or PD-L2 (such a checkpoint inhibitor that inhibits the interaction between PD-1 and PD-L1 and / or PD-L2 is also referred to herein as a PD-1 / PD-L1 checkpoint inhibitor); the interaction between CTLA-4 and CD80 or CD86 to displace CD28 binding; the interaction between LAG-3 and MHC class II molecules; the interaction between TIM-3 and one or more of its ligands, such as galectin-9, PtdSer, HMGB1, and CEACAM1; the interaction between one or several KIRs and their ligands; the interaction between TIGIT and one or more of its ligands PVR, PVRL2, and PVRL3; the interaction between CD94 / NKG2A and HLA-E; the interaction between VISTA and its binding partner; the interaction between one or more Siglecs and their ligands; the interaction between GARP and one or more of its ligands; the interaction between CD47 and SIRPα; the interaction between PVRIG and PVRL2; the interaction between CSF1R and CSF1; the interaction between BTLA and HVEM; a part of the adenosinergic pathway, for example, the interaction between A2AR and / or A2BR and adenosine produced by CD39 and CD73; the interaction between B7-H3 and its receptor and / or the interaction between 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 following: 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 can 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.

[0040] In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 43, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 44.

[0041] 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 comprises: (i) CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 45; (ii) CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 46; and (iii) CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 47; and wherein the light chain variable region comprises: (i) CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 48; (ii) CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 49; and (iii) CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 50.

[0042] In one embodiment of the anti-PD-1 antibody 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.

[0043] 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 comprises: (i) CDR-H1, which comprises the amino acid sequence of SEQ ID NO: 81; (ii) CDR-H2, which comprises the amino acid sequence of SEQ ID NO: 82; and (iii) CDR-H3, which comprises the amino acid sequence of SEQ ID NO: 83; and wherein the light chain variable region comprises: (i) CDR-L1, which comprises the amino acid sequence of SEQ ID NO: 84; (ii) CDR-L2, which comprises the amino acid sequence of SEQ ID NO: 85; and (iii) CDR-L3, which comprises the amino acid sequence of SEQ ID NO: 86.

[0044] In one embodiment of the anti-PD-1 antibody 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 antibody 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.

[0045] In one embodiment, the immune checkpoint inhibitor suitable for the methods disclosed herein is an antibody targeting PD-1 or PD-L1. In a preferred embodiment, the immune checkpoint inhibitor suitable for the methods disclosed herein is pembrolizumab.

[0046] The term "immunoglobulin" refers 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) having a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also referred to as surface immunoglobulins or membrane immunoglobulins and are typically part of the BCR. Soluble immunoglobulins are commonly referred to as antibodies.

[0047] The structure of immunoglobulins has been well characterized. See, e.g., Fundamental Immunology Ch.7 (Paul, W., ed., 2 nd ed. Raven Press, N.Y. (1989). Briefly, immunoglobulins generally comprise several chains linked by disulfide bonds, typically two identical heavy chains and two identical light chains. These chains are mainly composed of immunoglobulin domains or immunoglobulin regions, such as V L or VL (variable light chain) domains / regions, C L or CL (constant light chain) domains / regions, V H or VH (variable heavy chain) domains / regions, and C H or CH (constant heavy chain) domains / regions, C H 1 (CH1), C H 2 (CH2), C H 3 (CH3), and C H4(CH4). The heavy chain constant region is usually 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. The disulfide bonds in the hinge region are part of the interaction between the two heavy chains in the IgG molecule. Each light chain usually consists of VL and CL. The light chain constant region usually consists of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions (or hypervariable loops, which can be sequence - hypervariable and / or structurally defined loop forms), also known as complementarity determining regions (CDR), interspersed with more conserved regions called framework regions (FR). Each VH and VL usually consists of three CDRs and four FRs arranged in the following order from the amino - terminus to the carboxy - terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J.Mol.Biol.196,901 - 917(1987)). Unless otherwise stated or inconsistent with the context, the CDR sequences in this article are identified using DomainGapAlign according to the IMGT rules (Lefranc MP.,Nucleic Acids Research1999;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 stated or inconsistent with the context, the amino acid positions in the constant regions in this disclosure are numbered according to EU - numbering (Edelman et al.,ProcNatl Acad Sci USA.1969May;63(1):78 - 85; Kabat et al.,Sequences of Proteins ofImmunological Interest,Fifth Edition.1991NIH Publication No.91 - 3242).

[0048] There are five types of mammalian immunoglobulin heavy chains, namely, α, δ, ε, γ, and μ, which are the basis for different classes of antibodies (i.e., 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 carboxyl terminus. In mammals, there are two types of light chains, namely, λ and κ. Immunoglobulin chains contain variable and constant regions. The constant regions are substantially conserved within the different isotypes of immunoglobulins, where the variable portions are highly diverse and responsible for antigen recognition.

[0049] The terms "amino acid" and "amino acid residue" are used interchangeably herein and should not be construed as limiting. An amino acid is an organic compound that contains amine (-NH2) and carboxyl (-COOH) functional groups, as well as a side chain (R group) specific to each amino acid. In the context of the present disclosure, amino acids can be classified based on structural and chemical characteristics. Thus, the classes of amino acids can be reflected in one or both of the following tables: Table 2: Major classifications based on the structure and general chemical characteristics of the R group Category Amino acid Acidic residue D and E Basic residue K, R, and H Hydrophilic uncharged residue S, T, N, and Q Aliphatic uncharged residue G, A, V, L, and I Nonpolar uncharged residue C, M, and P Aromatic residue F, Y, and W Table 3: Alternative physical and functional classifications of amino acid residues Category Amino acid Hydroxyl-containing residue S and T Aliphatic residue I, L, V, and M Cycloalkenyl-related residue F, H, W, and Y Hydrophobic residue A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residue D and E Polar residue C, D, E, H, K, N, Q, R, S, and T Positively charged residue H, K, and R Small residue A, C, D, G, N, P, S, T, and V Very small residue A, G, and S Residue involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residue Q, T, K, S, G, P, D, E, and R

[0050] For the purposes of the present disclosure, "variants" of amino acid sequences (peptides, proteins, or polypeptides) include 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, conformers, isomers, allelic variants, species variants, and species homologs, particularly those that occur naturally. The term "variant" particularly includes fragments of amino acid sequences.

[0051] Amino acid insertion variants involve the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of an amino acid sequence variant with an insertion, one or more amino acid residues are inserted into a specific site in the amino acid sequence, but random insertion and appropriate screening of the resulting product are also possible.

[0052] Amino acid addition variants include the amino-terminal and / or carboxyl-terminal fusion of one or more amino acids (e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids).

[0053] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, e.g., the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be at any position in the protein. Amino acid deletion variants that contain deletions at the N-terminus and / or C-terminus of the protein are also referred to as N-terminal and / or C-terminal truncated variants.

[0054] 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. The substitution of one amino acid for another can be classified as a conservative or non-conservative substitution. Preference is given to modifications occurring at positions in the amino acid sequence that are non-conservative between homologous proteins or peptides and / or to the replacement of an amino acid with another amino acid having similar properties. Preferably, the amino acid changes in the peptide and protein variants are conservative amino acid changes, i.e., the replacement of an amino acid with a similarly charged or uncharged amino acid. Conservative amino acid changes involve the replacement of one of the amino acid families associated with its side chain. In the context of the present disclosure, a "conservative substitution" is the replacement of one amino acid with another amino acid having similar structural and / or chemical characteristics, such a replacement of one amino acid residue with another amino acid residue of the same type as defined in either of the two tables above: e.g., leucine can be replaced with isoleucine because both are aliphatic, branched hydrophobes. Similarly, aspartic acid can be replaced with glutamic acid because both are small, negatively charged residues. Naturally occurring amino acids can generally be divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes grouped 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.

[0055] The term "amino acid at a position corresponding to..." and similar expressions used herein refer to the amino acid position numbers in the human IgG1 heavy chain. By aligning with human IgG1, the corresponding amino acid positions in other immunoglobulins can be found. Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is an amino acid or segment that aligns with the other amino acid or segment using a standard sequence alignment program (such as ALIGN, ClustalW or a similar program with generally default settings) and has at least 50%, at least 80%, at least 90% or at least 95% identity with the human IgG1 heavy chain. How to align sequences or segments in a sequence and thereby determine the positions in the sequence that correspond to the amino acid positions according to the present disclosure is considered to be well known in the art.

[0056] In the context of the present disclosure, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either, which has the ability to specifically bind to an antigen (particularly an epitope on the antigen) under normal physiological conditions, preferably having a half-life of a significant period, such as 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 or more days, etc., or any other relevant functionally defined period (e.g., a time sufficient to induce, promote, enhance, and / or regulate a physiological response associated with an antibody binding to an antigen and / or a 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 comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain comprises 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 domains and constant domains, respectively. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of VH are designated HCDR1, HCDR2, and HCDR3 (or CDR-H1, CDR-H2, and CDR-H3), and the CDRs of VL are designated LCDR1, LCDR2, and LCDR3 (or CDR-L1, CDR-L2, and CDR-L3). The heavy and light chain variable regions contain binding domains that interact with the antigen. The constant region of the antibody comprises the heavy chain constant region (CH) and the light chain constant region (CL), wherein CH can be further subdivided into constant domains CH1, the hinge region, and constant domains CH2 and CH3 (arranged in the following order from the amino terminus to the carboxy terminus: CH1, CH2, CH3). The constant region of the 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 a complete immunoglobulin derived from a natural source or a recombinant source, and can be an immunologically active portion of a complete immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules.Antibodies can exist in multiple forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies.

[0057] The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding regions that interact with an antigen. The terms "binding region" and "antigen-binding region" are used interchangeably herein and refer to the region that interacts with an antigen and includes both the VH region and the VL region. Antibodies as used herein include not only monospecific antibodies but also multispecific antibodies, which include multiple, such as two or more, such as three or more, different antigen-binding regions.

[0058] As described above, unless otherwise specified or clearly inconsistent with the context, the term antibody herein includes antibody fragments that are antigen-binding fragments, i.e., antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be carried out by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed by 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, divalent fragments containing two Fab fragments linked by a disulfide bond in 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)), which consist essentially of the VH domain and are also referred to as 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). In addition, although the two domains VL and VH of an Fv fragment are encoded by separate genes, they can be joined together using recombinant methods by a synthetic linker such that they can form a single protein chain, where the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain antibody or single-chain Fv (scFv), see, for example, Bird et al., Science 242 , 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883(1988)). Unless otherwise stated or the context clearly indicates, such single-chain antibodies are encompassed within the term antibody. Although such fragments are generally included within the meaning of antibody, they are each and collectively unique features of the present disclosure, exhibiting different biological properties and utilities. These and other available antibody fragments and bispecific forms of such fragments in the context of the present disclosure are further discussed herein. It should also be understood that unless otherwise specified, the term antibody also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides such as chimeric antibodies and humanized antibodies, and antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen provided by any known technique (e.g., enzymatic cleavage, peptide synthesis, and recombinant techniques).

[0059] The antibodies so produced can have any isotype. The term "isotype" as used herein refers to the immunoglobulin class encoded by the heavy-chain constant region gene (e.g., IgG (such as IgG1, IgG2, IgG3, IgG4), IgD, IgA (such as IgA1, IgA2), IgE, IgM, or IgY). When a specific isotype such as IgG1 is mentioned herein, the term is not limited to a specific isotype sequence such as a specific IgG1 sequence, but is used to indicate that the antibody is more similar in sequence to that isotype such as IgG1 than to other isotypes. Thus, for example, an IgG1 antibody disclosed herein can be a sequence variant of a naturally occurring IgG1 antibody, including variants in the constant region.

[0060] 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 some embodiments herein. The allotype variants commonly found in the population are those named by the letters a, f, n, z, or combinations thereof. In any embodiment herein, the antibody can comprise a heavy-chain Fc region containing the human IgG Fc region. In a further embodiment, the human IgG Fc region comprises human IgG1.

[0061] 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 some 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, such as bispecific antibodies, can be in any form, including any bispecific or multispecific antibody form described hereinafter.

[0062] When used in the context of an antibody, the term "full-length" means that the antibody is not a fragment but contains all of the domains of a particular isotype that are typically found in nature for that isotype, such as the VH, CH1, CH2, CH3, hinge, VL, and CL domains of an IgG1 antibody.

[0063] As used herein, the term "human antibody" is intended to include antibodies having variable and framework regions derived from human germline immunoglobulin sequences and antibodies having 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 in vitro random mutagenesis or site-specific mutagenesis or by in vivo somatic mutation). 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 (e.g., mouse) have been grafted onto human framework sequences.

[0064] The term "chimeric antibody" as used herein refers to an antibody in which the variable region is derived from a non-human species (e.g., derived from a rodent) and the constant region is derived from a different species such as a human. Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a term generally used for different kinds of antibody modification, and the methods of antibody engineering are well known to those skilled in the art. In particular, chimeric antibodies can be produced using standard DNA techniques as 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 recombinant antibodies that have been genetically engineered or enzymatically engineered. The production of chimeric antibodies is within the knowledge of those skilled in the art, and thus, the production of chimeric antibodies can be carried out by other methods than those described herein. Chimeric monoclonal antibodies have been developed for therapeutic applications in humans to reduce the expected antibody immunogenicity of non-human antibodies (e.g., rodent antibodies). They typically may contain non-human (e.g., murine or rabbit) variable regions that are specific for the antigen of interest, and human constant heavy and light chain domains. As described below, the term "variable region" or "variable domain" when used in the context of a chimeric antibody refers to the region that contains both the CDRs and framework regions of the heavy and light chains of the immunoglobulin.

[0065] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody that comprises a human antibody constant domain and a non-human variable domain that has been modified to have a high level of sequence homology with a 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 a homologous human acceptor framework region (FR) (see WO 92 / 22653 and EP 0 629 240). To fully reconstruct the binding affinity and specificity of the parental antibody, it may be necessary to replace framework residues from the parental antibody (i.e., the non-human antibody) with human framework regions (backmutations). Structural homology modeling can assist in identifying amino acid residues in the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody can comprise non-human CDR sequences, a predominantly human framework region optionally comprising one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, additional amino acid modifications that are not necessarily backmutations can be applied to obtain a humanized antibody with preferred characteristics such as affinity and biochemical properties.

[0066] As used herein, a protein that is "derived from" another protein (e.g., a parental protein) means that one or more amino acid sequences of the protein are identical or similar to one or more amino acid sequences in another protein or the parental protein. For example, in an antibody, binding arm, antigen-binding region, or constant region that is derived from another antibody or parental antibody, binding arm, antigen-binding region, or constant region, one or more amino acid sequences are identical or similar to the amino acid sequences of another antibody or parental antibody, binding arm, antigen-binding region, or constant region. Examples of such one or more amino acid sequences include, but are not limited to, those of the VH and VL CDRs and / or one or more or all of the framework regions, VH, VL, CL, hinge, or CH regions. For example, a humanized antibody may be described herein as "derived from" a non-human parental antibody, which means that at least the VL and VH CDR sequences are identical or similar to the VH and VL CDR sequences of the non-human parental antibody. A chimeric antibody may be described herein as "derived from" a non-human parental antibody, which means that typically the VH and VL sequences may be identical or similar to the VH and VL sequences of the non-human parental antibody. Another example is a binding arm or antigen-binding region that may be described herein as "derived from" a particular parental antibody, meaning that the binding arm or antigen-binding region typically contains VH and / or VL CDRs, or VH and / or VL sequences, that are identical or similar to the binding arm or antigen-binding region of the parental antibody. However, as described elsewhere herein, amino acid modifications (such as 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 that are derived from a first protein or parental protein, a "similar" amino acid sequence preferably has at least about 50% sequence identity, 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%.

[0067] Non-human antibodies can be produced in many different species, such as mice, rabbits, chickens, guinea pigs, llamas, and goats.

[0068] Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methods, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). Other techniques for producing monoclonal antibodies can be employed, e.g., viral or oncogenic transformation of B lymphocytes or phage display techniques using antibody gene libraries, and such methods are well known to those skilled in the art.

[0069] Hybridoma production in such non-human species is a very widely recognized procedure. Immunization protocols and techniques for isolating splenocytes from immunized animals / non-human species for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion protocols are also known.

[0070] As used herein, unless the context is contradictory, the term "Fab arm" or "arm" refers to a heavy chain-light chain pair and is used interchangeably herein with "half molecule".

[0071] The term "binding arm comprising an antigen-binding region" means an antibody molecule or fragment comprising an antigen-binding region. Thus, a binding arm can comprise, for example, six VH and VL CDR sequences, VH and VL sequences, Fab or Fab' fragments, or Fab arms.

[0072] As used herein, unless contradictory to the context, the term "Fc region" refers to the antibody region consisting of two Fc sequences of an immunoglobulin heavy chain, wherein the Fc sequences comprise at least a hinge region, CH2 domain, and CH3 domain. In one embodiment, the term "Fc region" as used herein refers to the region comprising at least a hinge region, CH2 region, and CH3 region in the direction from the N-terminus to the C-terminus of the antibody. 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 (such as effector cells) and components of the complement system.

[0073] In the context of the present disclosure, the term "less extent of inducing Fc-mediated effector functions" when used in relation to an antibody (including a multispecific antibody) means that the antibody induces Fc-mediated effector functions to a lesser extent compared to a human IgG1 antibody comprising (i) the same CDR sequences as the antibody, particularly comprising the same first antigen-binding region and second antigen-binding region, and (ii) two heavy chains comprising a human IgG1 hinge region, CH2 region, and CH3 region, such functions being particularly selected from the list of IgG Fc receptor (FcgammaR, FcγR) binding, C1q binding, ADCC, or CDC.

[0074] Fc-mediated effector functions can be measured by binding to FcγR, binding to C1q, or inducing Fc-mediated crosslinking via FcγR.

[0075] 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 as described in Kabat (Kabat, E.A. et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91 - 3242, pp662, 680, 689 (1991)). However, the hinge region can also be any other subtype as described herein.

[0076] 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 to 215 according to the EU numbering as described in Kabat (ibid.). However, the CH1 region can also be any other subtype as described herein.

[0077] 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 to 340 according to the EU numbering as described in Kabat (ibid.). However, the CH2 region can also be any other subtype as described herein.

[0078] 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 to 447 according to the EU numbering as described in Kabat (ibid.). However, the CH3 region can also be any other subtype as described herein.

[0079] In the context of the present disclosure, the term "monovalent antibody" means that an antibody molecule is capable of binding a single antigen molecule and thus is not capable of cross - linking antigens.

[0080] A "CD40 antibody" or "anti - CD40 antibody" is an antibody as described above that specifically binds to the antigen CD40.

[0081] A "CD137 antibody" or "anti - CD137 antibody" is an antibody as described above that specifically binds to the antigen CD137.

[0082] A "CD40xCD137 antibody" or "anti - CD40xCD137 antibody" is a bispecific antibody that comprises 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.

[0083] As used herein, the term "binds" or "is capable of binding" generally refers to binding in the case where an antibody binds to a predetermined antigen or epitope, typically when determined using Bio-Layer Interferometry (BLI), or for example when determined using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument with the antigen as the ligand and the antibody as the analyte, with an affinity corresponding to about 10 -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 M or even less) of K D . The antibody binds to the predetermined antigen with an affinity corresponding to the following K D : having an affinity that is at least 10-fold lower, e.g., at least 100-fold lower, e.g., at least 1,000-fold lower, e.g., at least 10,000-fold lower, e.g., at least 100,000-fold lower than its K D for binding to non-specific antigens (such as BSA, casein) other than the predetermined antigen or closely related antigens. The amount by which the affinity is higher depends on the K D of the antibody, so when the K D of the antibody is very low (i.e., the antibody is highly specific), the degree to which the affinity for the antigen is lower than the affinity for the non-specific antigen can be at least 10,000-fold.

[0084] The term "k d " (sec -1 ) used herein refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also referred to as the k off value.

[0085] The term "K D " (M) used herein refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.

[0086] If two antibodies bind to the same antigen and the same epitope, they have "the same specificity". Whether the antibody to be tested binds to the same epitope as a certain antigen-binding antibody, i.e., whether the antibody binds to the same epitope, can be tested by various methods well-known to those skilled in the art.

[0087] Competition between antibodies can be detected by cross - blocking assays. For example, a competitive ELISA assay can be used as a cross - blocking assay. For example, a target antigen can be coated onto the wells of a microtiter plate, and an antigen - binding antibody and a candidate competing test antibody can be added. The amount of antigen - binding antibody that binds to the antigen in the well is indirectly related to 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 smaller the amount of antigen - binding antibody that binds to the antigen - coated well. The amount of antigen - binding antibody that binds to the well can be measured by labeling the antibody with a detectable or measurable labeling substance.

[0088] An antibody that competes with another antibody (e.g., an antibody comprising heavy and light chain variable regions as described herein) for binding to an antigen, or an antibody that is specific for the antigen of another antibody (e.g., an antibody comprising heavy and light chain variable regions as described herein) can be an antibody comprising a variant of the heavy and / or light chain variable regions as described herein (e.g., modifications in the CDRs as described herein and / or a degree of identity).

[0089] As used herein, an “isolated multispecific antibody” is intended to mean 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 and is substantially free of monospecific antibodies that specifically bind to CD40 or CD137).

[0090] As used herein, the term “monoclonal antibody” refers to a preparation of antibody molecules consisting of a single molecule. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

[0091] When used herein, the term “heterodimeric interaction between a first CH3 region and a second CH3 region” refers to the interaction between the first CH3 region and the second CH3 region in a first CH3 / second CH3 heterodimeric antibody.

[0092] When used herein, the term “homodimeric interaction between a first CH3 region and a second CH3 region” refers to the interaction between the first CH3 region and another first CH3 region in a first CH3 / first CH3 homodimeric antibody and the interaction between the second CH3 region and another second CH3 region in a second CH3 / second CH3 homodimeric antibody.

[0093] When used herein, the term “homodimeric antibody” refers to an antibody comprising two first Fab arms or half - molecules, wherein the amino acid sequences of the Fab arms or half - molecules are the same.

[0094] As used herein, the term "heterodimeric antibody" refers to an antibody that comprises first and second Fab arms or half-molecules, wherein the amino acid sequences of the first and second Fab arms or half-molecules are different. In particular, the CH3 region or antigen-binding region, or both the CH3 region and the antigen-binding regions of the first and second Fab arms / half-molecules are different.

[0095] The term "reducing conditions" or "reducing environment" refers to conditions or an environment in which a substrate (e.g., a cysteine residue in an antibody hinge region) is more likely to be reduced than oxidized.

[0096] The present disclosure also describes multispecific antibodies, such as bispecific antibodies, that comprise functional variants of the VL regions, VH regions, or one or more CDRs of the bispecific antibodies of the examples. In the case of bispecific antibodies, the functional variants of the VL, VH, or CDRs used still allow 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 parental bispecific antibody, and in some cases, such bispecific antibodies may have greater affinity, selectivity, and / or specificity than the parental bispecific antibody.

[0097] Such functional variants generally retain significant sequence identity with the parental bispecific antibody. The percentage identity between two sequences is a function of the number of positions shared by the sequences (i.e., % homology = # of identical positions / # of total positions × 100), taking into account the number of gaps and the length of each gap, which need to be introduced to achieve the best alignment of the two sequences. The percentage 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), 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 percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm.

[0098] In the context of the present disclosure, unless otherwise specified, the following symbols are used to describe mutations: i) an amino acid substitution at a given position is written, for example, as K409R, which means that the lysine at position 409 of the protein is replaced by arginine; and ii) for a particular variant, a specific three-letter code or single-letter code is used, including the codes Xaa and X to represent any amino acid residue. Thus, the replacement of lysine with arginine at position 409 is named: K409R, and the replacement of lysine with any amino acid residue at position 409 is named K409X. The case of deletion of lysine at position 409 is represented by K409*.

[0099] Exemplary variants include variants that differ from the VH and / or VL and / or CDRs of the parental sequence mainly by conservative substitutions; for example, 12, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in the variant is a conservative amino acid residue substitution.

[0100] In the context of the present disclosure, conservative substitutions can be defined as substitutions within the amino acid classes defined in Tables 2 and 3.

[0101] The term "CD40" as used herein refers to CD40, also known as tumor necrosis factor receptor superfamily member 5 (TNFRSF5), which is the receptor for the ligand TNFSF5 / CD40L. CD40 is known to transduce TRAF6-mediated signals and MAP3K8-mediated signals, which activate ERK in macrophages and B cells, leading to induction of immunoglobulin secretion in 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, which has the UniProt accession number P25942. The sequence of human CD40 is also shown in SEQ ID NO:35. The amino acids at positions 1 to 20 of SEQ ID NO:35 correspond to the signal peptide of human CD40; while the amino acids at positions 21 to 193 of SEQ ID NO:35 correspond to the extracellular domain of human CD40; and the remaining portions of the protein; i.e., the amino acids from positions 194 to 215 and 216 to 277 of SEQ ID NO:35 are the transmembrane domain and cytoplasmic domain, respectively.

[0102] As used herein, the term "CD137" refers to CD137 (4-1BB), also known as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is the 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), which has a UniProt accession number of Q07011. The sequence of human CD137 is also shown in SEQ ID NO:37. The amino acids at positions 1 to 23 of SEQ ID NO:37 correspond to the signal peptide of human CD137; the amino acids at positions 24 to 186 of SEQ ID NO:37 correspond to the extracellular domain of human CD137; and the remaining portions of the protein, namely the amino acids from positions 187 to 213 and 214 to 255 of SEQ ID NO:37, are the transmembrane domain and cytoplasmic domain, respectively.

[0103] The "Programmed Death-1 (PD-1)" receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 (also known as CD279) is mainly expressed on previously activated T cells in the body 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, isotypes, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The sequence of human PD-1 is also shown in SEQ ID NO:39. "Programmed Death Ligand-1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T cell activation and cytokine secretion after binding to PD-1. The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isotypes, and species homologs of hPD-L1 (such as rhesus macaque (cynomolgus monkey), Asian 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)), and analogs having at least one common epitope with hPD-L1. The sequence of human PD-L1 is also shown in SEQ ID NO:40, where the amino acids at positions 1 to 18 are predicted to be the signal peptide. The sequence of rhesus macaque (cynomolgus monkey) PD-L1 is also shown in SEQ ID NO:41, where the amino acids at positions 1 to 18 are predicted to be the signal peptide. The term "PD-L2" as used herein includes human PD-L2 (hPD-L2), variants, isotypes, and species homologs of hPD-L2, and analogs having at least one common epitope with hPD-L2. The ligands of PD-1 (PD-L1 and PD-L2) are expressed on the surfaces of antigen-presenting cells (such as dendritic cells or macrophages) and other immune cells. The binding of PD-1 to PD-L1 or PD-L2 results in the downregulation of T cell activation. Cancer cells expressing PD-L1 and / or PD-L2 can shut off PD-1 expression on T cells, which leads to the inhibition of the anti-cancer immune response. The interaction between PD-1 and its ligands results in a reduction in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion of cancer cells. Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1, and this effect is additive when the interaction between PD-1 and PD-L2 is also blocked.

[0104] "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, isotypes, and species homologs of hCTLA-4, and analogs having at least one common epitope with hCTLA-4. CTLA-4 is a homolog of the stimulatory checkpoint protein CD28 and has a much higher binding affinity for CD80 and CD86. CTLA4 is expressed on the surface of activated T cells, and its ligands are expressed on the surface of professional antigen-presenting cells. The binding of CTLA4 to its ligands blocks the co-stimulatory signal of CD28 and generates an inhibitory signal. Thus, CTLA-4 downregulates T cell activation. The sequence of human CTLA-4 is also shown in SEQ ID NO: 42.

[0105] "T cell Immunoreceptor with Ig and ITIM domain (TIGIT, also known as WUCAM or Vstm3)" is an immunoreceptor on T cells and natural killer (NK) cells and binds to PVR (CD155), PVRL2 (CD112; nectin-2), and PVRL3 (CD113; nectin-3) on DCs, macrophages, etc. and regulates T cell-mediated immunity. The term "TIGIT" as used herein includes human TIGIT (hTIGIT), variants, isotypes, and species homologs of hTIGIT, and analogs having at least one common epitope with hTIGIT. The term "PVR" as used herein includes human PVR (hPVR), variants, isotypes, and species homologs of hPVR, and analogs having at least one common epitope with hPVR. The term "PVRL2" as used herein includes human PVRL2 (hPVRL2), variants, isotypes, and species homologs of hPVRL2, and analogs having at least one common epitope with hPVRL2. The term "PVRL3" as used herein includes human PVRL3 (hPVRL3), variants, isotypes, and species homologs of hPVRL3, and analogs having at least one common epitope with hPVRL3.

[0106] "B and T Lymphocyte Attenuator" (BTLA, also known as CD272) is a member of the TNFR family expressed in Th1 (but not Th2 cells). BTLA expression is induced during T cell activation and is particularly expressed on the surface of CD8+ T cells. As used herein, the term "BTLA" includes human BTLA (hBTLA), variants, isotypes, and species homologs of hBTLA, and analogs having at least one common epitope with hBTLA. BTLA expression is gradually downregulated during the 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. As used herein, the term "HVEM" includes human HVEM (hHVEM), variants, isotypes, and species homologs of hHVEM, and analogs having at least one common epitope with hHVEM. The BTLA-HVEM complex negatively regulates the T cell immune response.

[0107] "Killer-cell Immunoglobulin-like Receptor" (KIR) is a receptor for MHC class I molecules on NK T cells and NK cells and is involved in the discrimination between healthy and diseased cells. KIR binds to human leukocyte antigen (HLA) A, B, and C and inhibits normal immune cell activation. As used herein, the term "KIR" includes human KIR (hKIR), variants, isotypes, and species homologs of hKIR, and analogs having at least one common epitope with hKIR. As used herein, the term "HLA" includes variants, isotypes, and species homologs of HLA, and analogs having at least one common epitope with HLA. As used herein, KIR particularly refers to KIR2DL1, KIR2DL2, and / or KIR2DL3.

[0108] "Lymphocyte Activation Gene-3 (LAG-3)", also known as CD223, is an inhibitory receptor associated with 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, resulting in the inhibition of the immune response. LAG-3 is expressed on activated T cells, NK cells, B cells, and DCs. As used herein, the term "LAG-3" includes human LAG-3 (hLAG-3), variants, isotypes, and species homologs of hLAG-3, and analogs having at least one common epitope.

[0109] "T Cell Membrane Protein-3 (TIM-3)", also known as HAVcr-2, is an inhibitory receptor involved in the inhibition of lymphocyte activity by suppressing Th1 cell responses. Its ligand is galectin 9 (GAL9), which is upregulated in various types of cancers. Other TIM-3 ligands include phosphatidylserine (PtdSer), High Mobility Group Protein 1 (HMGB1), and Carcinoembryonic Antigen Related Cell Adhesion Molecule 1 (CEACAM1). As used herein, the term "TIM-3" includes human TIM3 (hTIM-3), variants, isotypes, and species homologs of hTIM-3, and analogs having at least one common epitope. As used herein, the term "GAL9" includes human GAL9 (hGAL9), variants, isotypes, and species homologs of hGAL9, and analogs having at least one common epitope. As used herein, the term "PdtSer" includes variants and analogs having at least one common epitope. As used herein, the term "HMGB1" includes human HMGB1 (hHMGB1), variants, isotypes, and species homologs of hHMGB1, and analogs having at least one common epitope. As used herein, the term "CEACAM1" includes human CEACAM1 (hCEACAM1), variants, isotypes, and species homologs of hCEACAM1, and analogs having at least one common epitope.

[0110] "CD94 / NKG2A" is an inhibitory receptor that is mainly expressed on the surface of natural killer cells and CD8+ T cells. The term "CD94 / NKG2A" as used herein includes human CD94 / NKG2A (hCD94 / NKG2A), variants, isotypes, and species homologs of hCD94 / NKG2A, and analogs having at least one common epitope. The CD94 / NKG2A receptor is a heterodimer that comprises CD94 and NKG2A. It can inhibit NK cell activation and CD8+ T cell function by binding to ligands such as HLA-E. CD94 / NKG2A restricts 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 often expressed in tumor infiltrating cells, while HLA-E is overexpressed in several cancers.

[0111] "Indoleamine 2,3-dioxygenase" (IDO) is a tryptophan catabolic enzyme with immunosuppressive properties. The term "IDO" as used herein includes human IDO (hIDO), variants, isotypes, and species homologs of hIDO, and analogs having at least one common epitope. IDO is the rate-limiting enzyme for tryptophan degradation, catalyzing its conversion to kynurenine. Thus, IDO is involved in the depletion of essential amino acids. It is known to be involved in inhibiting T cells and NK cells, generating and activating Tregs and myeloid-derived suppressor cells, and promoting tumor angiogenesis. IDO is overexpressed in many cancers and has been shown to promote immune system escape of tumor cells and to promote chronic tumor progression when induced by local inflammation.

[0112] As used herein, in the "adenergic pathway" or "adenosine signaling pathway", ATP is converted to adenosine by the ectonucleotidases CD39 and CD73, resulting in inhibitory signaling through the binding of adenosine to one or more 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 and is present at high concentrations in the tumor microenvironment, limiting immune cell infiltration, cytotoxicity, and cytokine production. Thus, adenosine signaling is a strategy by which cancer cells evade clearance by the host immune system. Adenosine signaling through A2AR and A2BR is an important checkpoint in cancer therapy activated by the high adenosine concentrations typically 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 through A2AR and A2BR counteracts T cell receptor-mediated immune cell activation and results in increased numbers of Tregs and decreased activation of DCs and effector T cells. The term "CD39" as used herein includes human CD39 (hCD39), variants, isotypes, and species homologs of hCD39, and analogs having at least one common epitope. The term "CD73" as used herein includes human CD73 (hCD73), variants, isotypes, and species homologs of hCD73, and analogs having at least one common epitope. The term "A2AR" as used herein includes human A2AR (hA2AR), variants, isotypes, and species homologs of hA2AR, and analogs having at least one common epitope. The term "A2BR" as used herein includes human A2BR (hA2BR), variants, isotypes, and species homologs of hA2BR, and analogs having at least one common epitope.

[0113] The "V-domain Ig suppressor of T cell activation" (VISTA, also known as C10orf54) is homologous to PD-L1 but exhibits a unique expression pattern restricted to the hematopoietic compartment. The term "VISTA" as used herein includes human VISTA (hVISTA), variants, isotypes, and species homologs of hVISTA, and analogs having at least one common epitope. VISTA induces T cell suppression and is expressed by leukocytes within tumors.

[0114] Members of the "sialic acid binding immunoglobulin-type lectin" (Siglec) family recognize sialic acid and are involved in distinguishing "self" from "non-self". As used herein, the term "Siglec" includes human Siglec (hSiglec), variants, isotypes, and species homologs of hSiglec, and analogs having at least one common epitope with one or more hSiglecs. The human genome contains 14 Siglecs, several of which are involved in immunosuppression, including but not limited to Siglec-2, Siglec-3, Siglec-7, and Siglec-9. Siglec receptors bind glycans containing sialic acid, but differ in the chemical and spatial distribution of their sialic acid residue recognition sites. Family members also have different expression patterns. A variety of malignancies overexpress one or more Siglecs.

[0115] "CD20" is an antigen expressed on the surface of B cells and T cells. High expression of CD20 is seen in cancers such as B cell lymphoma, hairy cell leukemia, B cell chronic lymphocytic leukemia, and melanoma cancer stem cells. As used herein, the term "CD20" includes human CD20 (hCD20), variants, isotypes, and species homologs of hCD20, and analogs having at least one common epitope.

[0116] "Glycoprotein A repetitions predominant" (GARP) plays a role in immune tolerance and the ability of tumors to escape the patient's immune system. As used herein, the term "GARP" includes human GARP (hGARP), variants, isotypes, and species homologs of hGARP, and analogs having at least one common epitope. GARP is expressed on lymphocytes, including Tregs in peripheral blood and tumor-infiltrating T cells at the tumor site. It can bind to latent "transforming growth factor β" (TGF-β). Disruption of GARP signaling in Treg cells results in reduced tolerance and inhibition of Treg migration to the gut and increased proliferation of cytotoxic T cells.

[0117] "CD47" is a transmembrane protein that binds to the ligand "signal-regulatory protein alpha" (SIRPα). The term "CD47" as used herein includes human CD47 (hCD47), variants, isotypes, and species homologs of hCD47, and analogs having at least one common epitope with hCD47. The term "SIRPα" as used herein includes human SIRPα (hSIRPα), variants, isotypes, and species homologs of hSIRPα, and analogs having at least one common epitope with hSIRPα. CD47 signaling is involved in a series of cellular processes, including apoptosis, proliferation, adhesion, and migration. CD47 is overexpressed in many cancers and serves as a "don't eat me" signal to macrophages. Blocking CD47 signaling with inhibitory anti-CD47 or anti-SIRPα antibodies enables macrophages to phagocytose cancer cells and promotes the activation of cancer-specific T lymphocytes.

[0118] "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 (a TIGIT ligand) are co-overexpressed in several cancers. Blocking the PVRIG signaling pathway leads to enhanced T cell function and CD8+ T cell responses, and thus reduced immunosuppression and enhanced interferon responses. The term "PVRIG" as used herein includes human PVRIG (hPVRIG), variants, isotypes, and species homologs of hPVRIG, and analogs having at least one common epitope with hPVRIG. As used herein, "PVRL2" includes hPVRL2 as defined above.

[0119] 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 CSF1. Blocking CSF1R signaling can functionally reprogram macrophage responses, thereby enhancing antigen presentation and anti-tumor T cell responses. As used herein, the term "CSF1R" includes human CSF1R (hCSF1R), variants, isotypes, and species homologs of hCSF1R, and analogs having at least one common epitope with hCSF1R. As used herein, the term "CSF1" includes human CSF1 (hCSF1), variants, isotypes, and species homologs of hCSF1, and analogs having at least one common epitope with hCSF1.

[0120] "Nicotinamide adenine dinucleotide phosphate NADPH oxidase" refers to the enzymes of the NOX family of myeloid cells that produce immunosuppressive reactive oxygen species (ROS). Five NOX enzymes (NOX1 to NOX5) have been found to be involved in cancer development and immunosuppression. Elevated ROS levels are detected in almost all cancers and promote many aspects of tumor development and progression. ROS produced by NOX inhibits the functions of NK and T cells, and inhibiting NOX in myeloid cells improves the anti-tumor functions of adjacent NK and T cells. As used herein, the term "NOX" includes human NOX (hNOX), variants, isotypes, and species homologs of hNOX, and analogs having at least one common epitope with hNOX.

[0121] Another immune checkpoint that can be targeted according to the present disclosure is the signal mediated by "tryptophan-2,3-dioxygenase" (TDO). TDO represents an alternative pathway to IDO in tryptophan degradation and is involved in immunosuppression. Since tumor cells can catabolize tryptophan through TDO rather than IDO, TDO can represent an additional target for checkpoint blockade. Indeed, several 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), variants, isotypes, and species homologs of hTDO, and analogs having at least one common epitope with hTDO.

[0122] Many immune checkpoints are regulated by interactions between specific receptor-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 exploit these checkpoint pathways to protect themselves from the immune system. Thus, the function of checkpoint proteins is generally to regulate T cell activation, T cell proliferation, and / or T cell function. Immune checkpoint proteins thus regulate and maintain self-tolerance as well as the duration and intensity of physiological immune responses. Many immune checkpoint proteins belong to the B7:CD28 family or the tumor necrosis factor receptor (TNFR) superfamily and, by binding to specific ligands, activate signaling molecules recruited to the cytoplasmic domain (Suzuki et al., 2016, Jap J Clin Onc, 46:191-203).

[0123] As used herein, the term "dysfunctional" refers to immune cells in a state of reduced responsiveness to antigen stimulation. Dysfunction includes 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.

[0124] As used herein, the term "anergy" refers to a state of unresponsiveness to antigen stimulation due to incomplete or insufficient signaling delivered through the T cell receptor (TCR). In the absence of costimulation, antigen stimulation also results in T cell anergy, rendering the cells refractory to subsequent activation by antigen even in the presence of costimulation. The unresponsive state is generally overcome by the presence of IL-2. Anergic T cells do not undergo clonal expansion and / or acquire effector functions.

[0125] As used herein, the term "exhaustion" refers to immune cell exhaustion, such as T cell exhaustion as a state of T cell dysfunction caused by persistent TCR signaling that occurs during many chronic infections and cancers. It is distinguished from anergy in that it is not produced by incomplete or insufficient signaling but rather by persistent signaling. Exhaustion is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from functional effector or memory T cells. Exhaustion prevents optimal control of diseases such as infections and tumors. Exhaustion can be caused by extrinsic negative regulatory pathways (such as immunomodulatory cytokines) as well as cell-intrinsic negative regulatory pathways (inhibitory immune checkpoint pathways such as those described herein).

[0126] "Enhancing T cell function" means inducing, causing, or stimulating T cells to have a sustained or amplified biological function, or renewing or reactivating exhausted or inactivated T cells. Examples of enhancing T cell function include: increased interferon-gamma secretion, increased proliferation, and increased antigen responsiveness (e.g., tumor clearance) from CD8+ T cells, relative to the level prior to the intervention. In one embodiment, the enhancement level 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. The manner of measuring such enhancement is known to those of ordinary skill in the art.

[0127] As used herein, the terms "inhibitory nucleic acid" or "inhibitory nucleic acid molecule" refer to nucleic acid molecules that fully or partially reduce, inhibit, interfere with, or negatively regulate one or more checkpoint proteins, such as DNA or RNA. Inhibitory nucleic acid molecules include, but are not limited to, oligonucleotides, siRNA, shRNA, antisense DNA or RNA molecules, and aptamers (e.g., DNA or RNA aptamers).

[0128] As used herein, the term "oligonucleotide" refers to a nucleic acid molecule capable of reducing protein expression (particularly the expression of checkpoint proteins, such as those described herein). Oligonucleotides are short DNA or RNA molecules, typically containing 2 to 50 nucleotides. Oligonucleotides can be single-stranded or double-stranded. Checkpoint inhibitor oligonucleotides can be antisense oligonucleotides.

[0129] Antisense oligonucleotides are single-stranded DNA or RNA molecules that are complementary to a given sequence, particularly to the sequence of the nucleic acid sequence of a checkpoint protein (or a fragment thereof). Antisense RNA is typically used to block the protein translation of mRNA, e.g., mRNA encoding a checkpoint protein, by binding to the mRNA. Antisense DNA is typically used to target specific complementary (coding or non-coding) RNA. If binding occurs, such DNA / RNA hybrids can be degraded by the enzyme RNase H. In addition, morpholino antisense oligonucleotides can be used for gene knockout in vertebrates. For example, Kryczek et al., 2006 (J Exp Med, 203:871-81) designed a B7-H4-specific morpholino that specifically blocked the expression of B7-H4 in macrophages, resulting in increased T cell proliferation and reduced tumor volume in mice with tumor-associated antigen (TAA)-specific T cells.

[0130] The term "siRNA" or "small interfering RNA" or "small inhibitory RNA" is used interchangeably herein and refers to a double-stranded RNA molecule typically having a length of 20 to 25 base pairs that interferes with the expression of a specific gene having a complementary nucleotide sequence, such as a gene encoding a checkpoint protein. In one embodiment, the siRNA interferes with mRNA and thus blocks translation, such as the translation of an immune checkpoint protein. Transfection of exogenous siRNA can be used for gene knockdown, however, the 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. Modifications and vectors available for stable transfection of siRNA into cells are known in the art. The siRNA sequence can also be modified to introduce a short loop between the two strands, thereby generating "small hairpin RNA" or "shRNA". The shRNA can be processed by Dicer into a functional siRNA. The shRNA has a relatively low degradation rate and turnover rate. Thus, an immune checkpoint inhibitor can be an shRNA.

[0131] The term "aptamer" as used herein refers to a single-stranded nucleic acid molecule, such as DNA or RNA, typically having a length of 25 to 70 nucleotides that is capable of binding to a target molecule, such as a polypeptide. In one embodiment, the aptamer binds to an immune checkpoint protein, such as 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 signal transduction pathway that regulates the expression of an immune checkpoint protein or polypeptide. The generation and therapeutic use of aptamers are well known in the art (see, for example, US 5,475,096).

[0132] The term "small molecule inhibitor" or "small molecule" is used interchangeably herein and refers to a low molecular weight organic compound, typically up to 1000 daltons, that reduces, inhibits, interferes with, or negatively regulates one or more of the checkpoint proteins described above. Such small molecule inhibitors are typically synthesized by organic chemistry, but can also be isolated from natural sources (e.g., plants, fungi, and microorganisms). The small molecular weight allows the small molecule inhibitor to rapidly diffuse across the cell membrane. For example, a variety of A2AR antagonists known in the art are organic compounds having a molecular weight of less than 500 daltons.

[0133] 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).

[0134] As used herein, the term "oncolytic virus" refers to a virus that can selectively replicate in cancer cells or hyperproliferative cells in vitro or in vivo and slow down their growth or induce their death, while having little or no effect on normal cells. The oncolytic virus for delivering an immune checkpoint inhibitor contains an expression cassette encoding an immune checkpoint inhibitor, which is an inhibitory nucleic acid molecule such as siRNA, shRNA, oligonucleotide, antisense DNA or RNA, aptamer, antibody or fragment thereof, or a soluble immune checkpoint protein or fusion. The oncolytic virus preferably has the ability to replicate and the expression cassette is under the control of a viral promoter (e.g., a synthetic early / late poxvirus promoter). Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), rhabdovirus (e.g., picornavirus, e.g., Seneca Valley virus; SVV-001), coxsackievirus, parvovirus, Newcastle disease virus (NDV), herpes simplex virus (HSV; OncoVEX GMCSF), retrovirus (e.g., influenza virus), measles virus, reovirus, Sindbis virus, vaccinia virus, as exemplified in WO 2017 / 209053 (including Copenhagen, Western Reserve, Wyeth strains), and adenovirus (e.g., δ-24, δ-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAd1, H101, AD5 / 3-D24-GMCSF). The production and method of use of recombinant oncolytic viruses containing a soluble form of an immune checkpoint inhibitor are disclosed in WO 2018 / 022831, which is incorporated herein by reference in its entirety. The oncolytic virus can be used as an attenuated virus.

[0135] A "treatment cycle" is defined herein as the time period within the influence of a single dose of the binder increased due to its pharmacodynamics, or in other words, the time period after the binder administered in the subject's body has been substantially cleared. Multiple small doses within a small time window (e.g., within 2 to 24 hours, such as 2 to 12 hours or on the same day) may be equivalent to a larger single dose.

[0136] In the context of the present invention, the term "treatment" and variations thereof or "therapeutic intervention" relate to the management and care of a subject for the purpose of combating a disorder (such as a disease or an ailment). The term is intended to encompass the full spectrum of treatment for a given disorder suffered by a subject, such as administering a therapeutically effective compound to alleviate symptoms or complications, retard the progression of a disease, disorder or condition, alleviate or relieve symptoms and complications, and / or cure or eliminate a disease, disorder or condition as well as prevent a condition, where prevention should be understood as the management and care of an individual for the purpose of combating a disease, condition or disorder and includes administering an active compound to prevent the onset of symptoms or complications. In one embodiment, "treatment" refers to administering an effective amount of a therapeutically active binder of the present disclosure, such as a therapeutically active antibody, for the purpose of alleviating, ameliorating, arresting, or eradicating (curing) symptoms or a disease state.

[0137] Resistance, non-responsiveness, and / or recurrence to treatment with a binder of the present disclosure can be determined according to Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST Criteria v1.1). The RECIST criteria are shown in the following table (LD (longest dimension): longest dimension). Table 4: Definitions of response (RECIST Criteria v1.1)

[0138] "Best overall response" is the best response recorded from the start of treatment until disease progression / recurrence (the smallest measurement recorded since 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 be in disease control. Subjects with NE are considered non-responders. Best overall response is the best response recorded from the start of treatment until disease progression / recurrence (the smallest measurement recorded since the start of treatment will be used as the reference for PD). Subjects with CR, PR, or SD are considered to be in disease control. Subjects with NE are considered non-responders.

[0139] Objective response rate (ORR) is the percentage of all subjects in a study or treatment group that have a partial or complete response to treatment. ORR can be calculated by: adding the number of subjects with CR and the number of subjects with PR, and dividing the resulting sum by the total number of subjects in the treatment group. ORR eval, i.e., the ORR of all evaluable subjects in the study or treatment group, is the percentage of all evaluable subjects in the study or treatment group who have a partial or complete response to the treatment.

[0140] The disease control rate (DCR) is the percentage of all subjects in the study or treatment group who have a complete response, partial response, or stable disease (CR, PR, or SD) to the treatment. The DCR can be calculated as follows: add the number of subjects with CR, the number of subjects with PR, and the number of subjects with SD, and divide the resulting sum by the total number of subjects in the treatment group. DCR eval , i.e., the DCR of all evaluable subjects in the study or treatment group, is the percentage of all evaluable subjects in the study or treatment group who have a complete response, partial response, or stable disease (CR, PR, or SD) to the treatment.

[0141] "Duration of response (DOR)" applies only to subjects whose best overall response is confirmed as CR or PR, and is defined as the time from the first recorded objective tumor response (CR or PR) to the date of the first PD or to death due to underlying cancer.

[0142] "Progression-free survival (PFS)" is defined as the number of days from the first day of cycle 1 to the first recorded progression or to death from any cause.

[0143] "Overall survival (OS)" is defined as the number of days from the first day of cycle 1 to death from any cause. If it is not known that the subject has died, then OS will be censored at the latest date known that the subject was alive (on or before the cut-off date).

[0144] In the context of the present disclosure, the term "treatment regimen" refers to a structured treatment plan designed to improve and maintain health.

[0145] The term "effective amount" or "therapeutically effective amount" refers to an amount that effectively achieves the desired therapeutic result at the required dosage and over a period of time. The therapeutically effective amount of a binding agent (such as an antibody, e.g., a multispecific antibody or a monoclonal antibody) can vary depending on individual factors such as disease state, age, sex, and weight, as well as the ability of the binding agent to elicit the desired response in the individual. The therapeutically effective amount is also an amount in which any toxic or detrimental effects of the binding agent or its fragment are outweighed by the therapeutic beneficial effects. In the case where the response in a patient at an initial dosage is inadequate, a higher dosage can be used (or an effectively higher dosage can be achieved by a different, more local route of administration). In the case where a patient experiences undesirable side effects at a certain dosage, a lower dosage can be used (or an effectively lower dosage can be achieved by a different, more local route of administration).

[0146] As used herein, the term "cancer" includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. "Cancer cells" mean abnormal cells that grow by rapid, uncontrolled cell proliferation and continue to grow after the stimulus that initiated the new growth has ceased.

[0147] 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, bowel cancer, liver cancer, colon cancer, stomach cancer, bowel cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ENT cancer, breast cancer, prostate cancer, penile cancer, uterine cancer, ovarian cancer, and lung cancer and their metastases. Some examples thereof are lung cancer, breast cancer, prostate cancer, colon cancer, renal cell cancer, cervical cancer, or metastases of the above cancer types or tumors.

[0148] The term "cancer" according to the present disclosure also includes cancer metastases. "Metastasis" means the spread of cancer cells from their original site to other parts of the body. The formation of metastases is a complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane to enter body cavities and blood vessels, and then infiltration of the target organ after transport through the blood. Ultimately, the growth of new tumors at the target site, i.e., secondary tumors or metastatic tumors, depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor because tumor cells or components may remain and possess metastatic potential. In one embodiment, the term "metastasis" according to the present disclosure relates to "distant metastasis", which involves metastasis away from the primary tumor and the local lymph node system.

[0149] As used herein, terms such as "reduce", "inhibit", "interfere with", and "negatively regulate" mean the ability to cause an overall reduction in level, such as by 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 include complete inhibition or substantially complete inhibition, i.e., reduction to zero or substantially reduction to zero.

[0150] In one embodiment, terms such as "increase" or "enhance" refer 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%.

[0151] "Physiological pH" as used herein refers to a pH of about 7.5.

[0152] "Weight %" as used in this disclosure refers to weight percentage, which is a unit of concentration for measuring the amount of a substance (in grams (g)), expressed as a percentage of the total weight of the total composition (in grams (g)).

[0153] The term "freeze" refers to the solidification of a liquid, usually accompanied by the removal of heat.

[0154] The term "lyophilize" and variations thereof refer to the freeze-drying of a substance by freezing the substance and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or lower) such that the freezing medium in the substance sublimates directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze-drying" are used interchangeably herein.

[0155] The term "recombinant" in the context of this disclosure means "prepared by genetic modification". In one embodiment, a "recombinant object" in the context of this disclosure is not naturally occurring.

[0156] The term "naturally occurring" as used herein refers to the fact that an object can exist in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus) and can be isolated from a natural source and has not been deliberately modified by a person in the laboratory is naturally occurring. The term "exists in nature" means "present in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature but that may be discovered and / or isolated from natural sources in the future.

[0157] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising about two or more, about three or more, about four or more, about six or more, about eight or more, about ten or more, about thirteen or more, about sixteen or more, about twenty or more, and up to about fifty, about one hundred, or about one hundred and fifty consecutive amino acids joined to one another by peptide bonds. The term "protein" refers to a large peptide, particularly a peptide having at least about one hundred and fifty-one amino acids, but the terms "peptide" and "protein" are generally used synonymously herein.

[0158] A "therapeutic protein", when provided to a subject in a therapeutically effective amount, has a positive or beneficial effect on a disorder or disease state of the subject. In one embodiment, a therapeutic protein has curative or palliative properties and can be administered to improve, alleviate, mitigate, reverse a disease or disorder, delay the onset of a disease or disorder, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein can have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. The term "therapeutic protein" includes intact proteins or peptides and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of a protein. Some examples of therapeutically active proteins include, but are not limited to, antigens for vaccination and immunostimulants (such as cytokines).

[0159] The term "portion" refers to a fraction. For a particular structure such as an amino acid sequence or a protein, the "portion" thereof can refer to a continuous or discontinuous fraction of said structure.

[0160] The terms "portion" and "fragment" are used interchangeably herein and refer to consecutive elements. For example, a portion of a structure (such as an amino acid sequence or a protein) refers to consecutive elements of said structure. When used in the context of a composition, the term "portion" means a part of the composition. For example, a portion of a composition can be any part from 0.1% to 99.9% (such as 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90% or 99%) of said composition.

[0161] When referring to an amino acid sequence (peptide or protein), a "fragment" refers to a part of the amino acid sequence, i.e., a sequence representing an amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5'-end of the open reading frame, provided that the truncated open reading frame contains a start codon for initiating translation. Fragments of an amino acid sequence contain, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. Fragments of an amino acid sequence preferably contain at least 6, particularly 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.

[0162] According to the present disclosure, a part or fragment of a peptide or protein preferably has 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, enzyme activity, interaction with antibodies, and selective binding to nucleic acids. For example, a pharmacological active fragment of a peptide or protein has at least one pharmacological activity of the peptide or protein from which the fragment is derived. A part or fragment of a peptide or protein preferably contains 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 part or fragment of a peptide or protein preferably contains a sequence of up to 8, particularly up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the peptide or protein.

[0163] A "variant" herein means an amino acid sequence that differs from a parental amino acid sequence due to at least one amino acid modification. The parental amino acid sequence can be a naturally occurring or wild type (WT) amino acid sequence, or can be a modified form of a wild type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parental amino acid sequence, e.g., having 1 to about 20 amino acid modifications compared to the parental, and preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0164] "Wild type" or "WT" or "natural" herein means an amino acid sequence that exists 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.

[0165] 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 will 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%. Preferably, the degree of similarity or identity is given for 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 amino acid region that is the full 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 given 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 (in some embodiments, contiguous amino acids). In some embodiments, the degree of similarity or identity is given for the full length of the reference amino acid sequence. Alignment for determining sequence similarity (preferably sequence identity) can be performed using tools known in the art, preferably using optimal sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0166] "Sequence similarity" refers to the percentage of amino acids that are the same or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are the same between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are the same between the sequences.

[0167] The terms "percent identical" and "percent identity" or similar terms are specifically intended to refer to the percentage of nucleotides or amino acids that are identical between the sequences to be compared in an optimal alignment. The percentage is purely statistical, and the differences between two sequences can be, but are not necessarily, randomly distributed over the entire length of the sequences being compared. The comparison of two sequences is typically done by comparing the sequences after they have been optimally aligned over a region or "comparison window" to determine the local regions of the corresponding sequences. The optimal alignment for comparison can be performed manually or by means of the local homology algorithms 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 by means of computer programs using these 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 BLASTN or BLASTP algorithms available on the website of the National Center for Biotechnology Information (NCBI) in the United States (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi) are used to determine the percent identity of two sequences. In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word length set to 28; (iii) a maximum match within the query range set to 0; (iv) a match / mismatch score set to 1, -2; (v) a gap cost set to linear; and (vi) a filter for low-complexity regions being used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) an expectation threshold set to 10; (ii) a word length set to 3; (iii) a maximum match within the query range set to 0; (iv) a matrix set to BLOSUM62; (v) a gap cost set to 11 for existence and 1 for extension; and (vi) a conditional compositional score matrix adjustment.

[0168] The percent identity is obtained by determining the number of positions that are identical in the sequences being compared, dividing that number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0169] In some embodiments, the degree of similarity or identity is given for 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 full length of the reference sequence. For example, if the reference amino acid sequence consists of 200 amino acid residues, the degree of identity is given for 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, contiguous amino acid residues). In some embodiments, the degree of similarity or identity is given for the full length of the reference sequence.

[0170] According to the present disclosure, homologous amino acid sequences show at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and preferably at least 95%, at least 98%, or at least 99% identity of amino acid residues.

[0171] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. For example, the manipulation of DNA sequences for preparing peptides or proteins with substitutions, additions, insertions, or deletions is described in detail in Sambrook et al. (1989). In addition, the peptides and amino acid variants described herein can be readily prepared by means of known peptide synthesis techniques, such as by solid-phase synthesis and similar methods.

[0172] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The terms "functional fragment" or "functional variant" of an amino acid sequence refer to any fragment or variant that exhibits one or more functional properties that are the same as or similar to one or more functional properties of the amino acid sequence from which the fragment or variant is derived (i.e., it is functionally equivalent). With respect to an antigen or antigenic sequence, a particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. The terms "functional fragment" or "functional variant" as used herein particularly refer to such variant molecules or sequences that contain an amino acid sequence that has been altered by one or more amino acids compared to the amino acid sequence of the parental molecule or sequence and that is still capable of performing one or more functions of the parental molecule or sequence (e.g., inducing an immune response). In one embodiment, the modifications in the amino acid sequence of the parental 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 a functional variant can be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parental molecule or sequence. However, in some other embodiments, the immunogenicity of the functional fragment or functional variant can be enhanced compared to the parental molecule or sequence.

[0173] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the source 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 the same as, substantially the same as, or homologous to that particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence can be a variant of that particular sequence or a fragment thereof. For example, one of ordinary skill in the art will understand that antigens suitable for use herein can be altered such that their sequences are different from the naturally occurring or native sequences from which they are derived while still retaining the desired activities of the native sequences.

[0174] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that naturally occurs in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-natural environment, such as in a host cell. In a preferred embodiment, the binding agent used in the present disclosure is in a substantially purified form.

[0175] The term "genetic modification" or simply "modification" includes transfecting cells with nucleic acids. The term "transfection" involves introducing nucleic acids, particularly RNA, into cells. For the purposes of the present disclosure, the term "transfection" also includes introducing nucleic acids into cells or the uptake of nucleic acids by such cells, where the cells can be present in a subject such as a patient. Thus, according to the present disclosure, the cells used to transfect the nucleic acids described herein can be present in vitro or in vivo. For example, the cells can form part of an organ, tissue, and / or organism of a patient. According to the present disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient when the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express the protein it encodes. Since the nucleic acids introduced during transfection generally do not integrate into the nuclear genome, the foreign nucleic acids will be diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids greatly reduce the dilution rate. If it is desired that the transfected nucleic acids actually remain in the genome of the cells and their daughter cells, stable transfection must occur. Such stable transfection can be achieved by using virus-based or transposon-based systems for transfection. Generally, nucleic acids encoding antigens are transiently transfected into cells. RNA can be transfected into cells to transiently express the protein it encodes.

[0176] According to the present disclosure, an analogue of a peptide or protein is a modified form of the peptide or protein from which it is derived and has at least one functional property of the peptide or protein. For example, a pharmacologically active analogue of a peptide or protein has at least one pharmacological activity of the peptide or protein from which the analogue is derived. Such modifications include any chemical modification and include 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, "analogues" of a protein or peptide include those modified forms obtained by glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or binding to an antibody or other cellular ligand. The term "analogue" also extends to all functional chemical equivalents of the proteins and peptides.

[0177] As used herein, "activation" or "stimulation" refers to the state of an immune effector cell (e.g., a T cell) that has been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with the initiation of a signaling pathway, induced cytokine production, and detectable effector function. The term "activated immune effector cell" particularly refers to an immune effector cell that is undergoing cell division.

[0178] The term "sensitization" refers to the process in which immune effector cells such as T cells first come into contact with their specific antigen and differentiate into effector cells such as effector T cells.

[0179] The term "clonal expansion" or "expansion" refers to a process in which a particular entity multiplies. In the context of the present disclosure, this term is preferably used in the context of an immune response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cells that recognize the antigen expand. Preferably, clonal expansion results in the differentiation of immune effector cells.

[0180] An "antigen" according to the present disclosure encompasses any substance that will elicit an immune response and / or any substance that an immune response or immune mechanism (such as a cellular response) is directed against. This also includes cases where the antigen is processed into antigenic peptides and the immune response or immune mechanism is directed against one or more antigenic peptides, particularly if presented in the context of MHC molecules. In particular, an "antigen" refers to any substance that specifically reacts with an antibody or a T lymphocyte (T cell), preferably a peptide or a protein. According to the present disclosure, the term "antigen" includes any molecule that contains at least one epitope (such as a T cell epitope). Preferably, an antigen in the context of the present disclosure is a molecule that optionally induces an immune response after processing, and the immune response preferably specifically targets the antigen (including cells expressing the antigen). In one embodiment, the antigen is a disease-related antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0181] According to the present disclosure, any suitable antigen can be used as a candidate for an immune response, where the immune response can be both a humoral immune response and a cellular immune response. In some embodiments of the present disclosure, the antigen is preferably presented by a cell, preferably an antigen-presenting cell (in the case of MHC molecules), which results in an immune response against the antigen. The antigen is preferably a product that corresponds to or is derived from a naturally occurring antigen. Such a naturally occurring antigen can include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen can also be a tumor antigen. According to the present disclosure, the antigen can correspond to a naturally occurring product, such as a viral protein, or a portion thereof.

[0182] The term "disease-related antigen" is used in its broadest sense and refers to any antigen that is related to a disease. A disease-related antigen is a molecule that contains epitopes that will stimulate the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-related antigens include pathogen-related antigens, i.e., antigens related to microbial infections, usually microbial antigens (such as bacterial or viral antigens), or antigens related to cancer, usually tumors, such as tumor antigens.

[0183] In a preferred embodiment, the antigen is a tumor antigen, i.e., a part of a tumor cell, in particular those that are mainly intracellular or are tumor cell surface antigens. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, e.g., an antigen derived from a virus, a bacterium, a single-celled organism or a parasite, such as a viral antigen (e.g., viral ribonucleoprotein or capsid protein). In particular, the antigen should be presented by MHC molecules, which results in the modulation of the immune system, in particular the activation of cells, preferably CD4+ and CD8+ lymphocytes, in particular by modulating the activity of the T cell receptor.

[0184] The term "tumor antigen" refers to a component of a cancer cell that can be derived from the cytoplasm, cell surface or nucleus. In particular, it refers to those antigens that are produced intracellularly or are surface antigens on tumor cells. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin and fetal sulfoglycoprotein, α2-H-ferritin and γ-fetoprotein, as well as a variety of viral tumor antigens. According to the present disclosure, the tumor antigen preferably comprises any antigen that is characteristic of a tumor or cancer and is characteristic of a tumor or cancer cell in terms of type and / or expression level.

[0185] 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. The viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0186] 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. The bacterial antigen can be derived from the cell wall or cytoplasmic membrane of the bacterium.

[0187] The term "epitope" refers to the antigenic determinant in a molecule (e.g., an antigen), i.e., a part or fragment of the molecule that is recognized by the immune system, e.g., by an antibody, a T cell or a B cell, especially when presented in the context of an MHC molecule. In one embodiment, an "epitope" means a protein determinant capable of specifically binding an antibody. Epitopes are usually composed of surface groups of the molecule such as amino acids or sugar side chains and usually have specific three-dimensional structural features as well as specific charge features. The difference between conformational and non-conformational epitopes is that in the presence of a denaturing solvent, the binding to the former is lost, but the binding to the latter is not. An epitope can contain amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, e.g., amino acid residues that are effectively blocked or covered by a specific antigen-binding peptide (in other words, amino acid residues are within the footprint of a specific antigen-binding peptide).

[0188] An epitope of a protein preferably comprises a continuous or discontinuous portion of the protein and preferably has a length of about 5 to about 100 amino acids, preferably about 5 to about 50, more preferably about 8 to about 30, most preferably about 10 to about 25 amino acids. For example, the length of the epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. Particularly preferred in the context of the present disclosure are epitopes that are T cell epitopes.

[0189] Terms such as "epitope", "antigenic fragment", "immunogenic peptide" and "antigenic peptide" are used interchangeably herein and preferably refer to an incomplete representation of an antigen that preferably is capable of eliciting an immune response against the antigen or a cell expressing or containing and preferably presenting the antigen. Preferably, the term refers to an immunogenic portion of an antigen. Preferably, it is the portion of the antigen that is recognized (i.e., specifically bound) by a T cell receptor, particularly if presented in the context of an MHC molecule. Certain preferred immunogenic portions bind to MHC class I or class II molecules. The term "epitope" refers to a part or fragment of a molecule (such as an antigen) that is recognized by the immune system. For example, an epitope can be recognized by a T cell, a B cell or an antibody. An epitope of an antigen can comprise a continuous or discontinuous portion of the antigen and can have a length of about 5 to about 100, such as about 5 to about 50, more preferably about 8 to about 30, most preferably about 10 to about 25 amino acids. For example, the length of the epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. In one embodiment, the length of the epitope is from about 10 amino acids to about 25 amino acids. The term "epitope" includes T cell epitopes.

[0190] 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" include class I MHC and class II MHC molecules and refer to a gene complex present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen-presenting cells or diseased cells in an immune response, where the 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 cell surface and display 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 MHC class I / peptide complexes, the bound peptides are typically about 8 to about 10 amino acids in length, although longer or shorter peptides can also be effective. In the case of class II MHC / peptide complexes, the bound peptides are typically about 10 to about 25 amino acids in length, and particularly about 13 to about 18 amino acids in length, although longer or shorter peptides can be effective.

[0191] Peptides and protein antigens can be from 2 to 100 amino acids in length, including for example 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids or 50 amino acids. In some embodiments, the peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.

[0192] A peptide or protein antigen can be any peptide or protein that is capable of inducing or enhancing the ability of the immune system to generate an antibody and T cell response against the peptide or protein.

[0193] In one embodiment, the vaccine antigen, i.e., the antigen that when inoculated into a subject induces an immune response, is recognized by immune effector cells. Preferably, if recognized by immune effector cells, the vaccine antigen is capable of inducing the stimulation, sensitization, and / or expansion of immune effector cells carrying an antigen receptor that recognizes the vaccine antigen in the presence of appropriate co-stimulatory signals. In the context of the embodiments of the present disclosure, 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 MHC. In one embodiment, when the TCR is expressed by and / or present on a T cell, its binding to an antigen presented by a cell such as an antigen-presenting cell results in the stimulation, sensitization, and / or expansion of the T cell. In one embodiment, when the TCR is expressed by and / or present on a T cell, its binding to an antigen presented on a diseased cell results in the cytolysis and / or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors such as perforin and granzyme.

[0194] In one embodiment, the antigen receptor is an antibody or a 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.

[0195] The term “expressed on the cell surface” or “associated with the cell surface” means that a molecule (e.g., an antigen) associates with and is located in the cytoplasmic membrane, where at least a portion of the molecule faces the extracellular space of the cell and is accessible, for example, from the outside of the cell by an antibody located outside the cell. In this case, the portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association can be direct or indirect. For example, the association can be through one or more transmembrane domains, one or more lipid anchors, or by interaction with any other protein, lipid, carbohydrate, or other structure that can be present on the outer leaflet of the cytoplasmic membrane. For example, a molecule associated with the cell surface can be a transmembrane protein having an extracellular portion, or can be a protein that associates with the cell surface by interaction with another protein that is a transmembrane protein.

[0196] “Cell surface” or “surface of the cell” is used in its ordinary meaning in the art and thus includes the exterior of the cell that is accessible by binding of other molecules through proteins. If an antigen is located on the surface of the cell and is accessible, for example, by binding of an antigen-specific antibody added to the cell, the antigen is expressed on the surface of the cell.

[0197] In the context of the present disclosure, the term "extracellular portion" or "extracellular domain" refers to a part of a molecule (e.g., a protein) that faces the extracellular space of a cell and is preferably accessible from the exterior of the cell (e.g., by a binding molecule located outside the cell, such as an antibody). Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0198] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs) (CD8+ T cells) including cytolytic T cells. The term "antigen-specific T cell" or similar terms refer to a T cell that recognizes an antigen targeted by the T cell, particularly when presented on the surface of an antigen-presenting cell or a diseased cell (e.g., a cancer cell) in the context of an MHC molecule, and preferably exerts the effector function of the T cell. A T cell is considered specific for an antigen if it kills a target cell expressing the antigen. T cell specificity can be evaluated using any of a variety of standard techniques, such as within a chromium release assay or a proliferation assay. Alternatively, the synthesis of lymphokines (e.g., interferon γ) can be measured. In certain embodiments of the present disclosure, RNA (particularly mRNA) encodes at least one epitope.

[0199] The term "target" shall mean a substance that is the target of an immune response (e.g., a cellular immune response), such as a cell or tissue. 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 an antigen and preferably presents the antigen in the context of class I MHC.

[0200] "Antigen processing" refers to the degradation of an antigen into processed products that are fragments of the antigen (e.g., a protein degraded into peptides), and the association (e.g., by binding) of one or more of these fragments with MHC molecules for presentation by a cell (preferably an antigen-presenting cell) to a specific T cell.

[0201] "Antigen-responsive CTL" means a CD8 + T cell that is responsive to an antigen or a peptide derived from the antigen presented by class I MHC on the surface of an antigen-presenting cell.

[0202] According to the present disclosure, CTL responsiveness can include a sustained calcium flux, cell division, cytokine (e.g., IFN-γ and TNF-α) production, upregulation of activation markers (e.g., CD44 and CD69), and specific cytolytic killing of target cells expressing a tumor antigen. CTL responsiveness can also be determined using an artificial reporter that precisely indicates CTL responsiveness.

[0203] The terms "immune response" and "immunological response" are used interchangeably herein in their conventional meanings and refer to the overall bodily response to an antigen, and preferably refer to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the terms "immune response to" or "immunological response against" with respect to a substance (such as an antigen, cell, or tissue) refer to an immune response against the substance, such as a cellular response. An immune response may include one or more reactions selected from the following: production of antibodies against one or more antigens and expansion of antigen-specific T lymphocytes (preferably CD4 + and CD8 + T lymphocytes, more preferably CD8 + T lymphocytes), which can be detected in various proliferation or cytokine production assays in vitro.

[0204] In the context of the present disclosure, the terms "induce an immune response" and "elicit an immune response" and similar terms refer to the induction of an immune response, preferably the induction of a cellular immune response, a humoral immune response, or both. An immune response can be protective / preventive / prophylactic and / or therapeutic. An immune response can be directed against any immunogen or antigen or antigenic peptide, preferably against a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (such as influenza virus (A, B, or C), CMV, or RSV)). In this context, "induce" can mean that there is no immune response against a specific antigen or pathogen prior to induction, but it can also mean that there is a certain level of immune response against a specific antigen or pathogen prior to induction, and after induction, the immune response is enhanced. Thus, in this context, "induce an immune response" also includes "enhance an immune response". Preferably, after inducing an immune response in an individual, the individual is protected from developing a disease, such as an infectious disease or a cancerous disease, or the disease condition is improved by inducing an immune response.

[0205] The terms "cellular immune response", "cellular response", "cell-mediated immunity" or similar terms are intended to include cellular responses against cells characterized by expressing an antigen and / or presenting the antigen with class I or class II MHC. Cellular responses are related to cells called T cells or T lymphocytes, which act as "helpers" or "killers". Helper T cells (also called CD4 + T cells) play a central role in regulating the immune response, and killer cells (also called cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs) kill cells (such as diseased cells).

[0206] The term "humoral immune response" refers to a process in a living organism in which antibodies are produced in response to a substance and the organism, and the antibodies ultimately neutralize and / or eliminate the substance and the organism. The specificity of the antibody response is mediated by the membrane-associated receptors of T and / or B cells that bind a single specific antigen. After binding the appropriate antigen and receiving a variety of other activation signals, B lymphocytes divide, which results in memory B cells and clones of plasma cells that secrete antibodies, each clone producing antibodies that recognize the same epitope of the antigen recognized by its antigen receptor. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigen. When re-exposed to the specific antigen, these lymphocytes provide the cellular basis for memory and result in a rapid increase in the antibody response.

[0207] The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and involve the following procedures: administering to the individual one or more immunogens or antigens or derivatives thereof as described herein (especially in the form of RNA (especially mRNA) encoding them), and stimulating an immune response against the one or more immunogens or antigens or cells characterized by presenting the one or more immunogens or antigens.

[0208] "Cells characterized by presenting an antigen" or "antigen-presenting cells" or "MHC molecules presenting an antigen on the surface of an antigen-presenting cell" or similar expressions mean cells that present an antigen or antigen peptide directly or after processing in the case of MHC molecules (preferably MHC class I and / or MHC class II molecules, most preferably MHC class I molecules), such as diseased cells (especially tumor cells or infected cells) or antigen-presenting cells.

[0209] In the context of the present disclosure, the term "transcription" relates to the process in which the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA (especially mRNA) can be translated into a peptide or protein.

[0210] The term "expression" as used herein is defined as the transcription and / or translation of a specific nucleotide sequence. With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell in which the chain of its mRNA directs the assembly of an amino acid sequence to produce a peptide or protein.

[0211] The term "optional" or "optionally" as used herein means that the subsequently described event, condition or situation may or may not occur, and the description includes instances in which the event, condition or situation occurs and instances in which the event, condition or situation does not occur.

[0212] "Endogenous" as used herein refers to any substance that is from or produced within an organism, cell, tissue or system.

[0213] The terms "linked", "fused", or "fusion / fusant" as used herein are used interchangeably. These terms refer to joining together two or more elements or components or domains.

[0214] The term "disease" (also referred to herein as "disorder") refers to an abnormal condition that affects an individual's body. A disease is generally interpreted as a medical condition associated with specific symptoms and signs. A disease can be caused by factors initially from external sources, such as an infectious disease, or a disease can be caused by internal dysfunction, such as an autoimmune disease. In humans, "disease" is generally 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 in contact with the individual. Broadly speaking, a disease sometimes includes injury, disability, disorder, syndrome, infection, isolated symptoms, abnormal behavior, and atypical variations in structure and function, while in other cases and for other purposes these may be considered distinguishable categories. A disease generally affects an individual not only physically but also emotionally, since the infection and experience of many diseases can alter an individual's view of life and the individual's personality.

[0215] The term "therapeutic treatment" involves any treatment that improves the health condition and / or extends (increases) the lifespan of an individual. The treatment can eliminate a disease in an individual, prevent or slow the onset of a disease in an individual, inhibit or slow the development of a disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual currently suffering from or previously having suffered from a disease.

[0216] The term "preventive treatment" or "preventative treatment" involves any treatment aimed at preventing the occurrence of a disease in an individual. The terms "preventive treatment" or "preventative treatment" are used interchangeably herein. Similarly, in the context of disease progression (such as the progression of a tumor or cancer), the term "method for prevention" involves any method aimed at preventing disease progression in an individual.

[0217] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or other mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) or any other non-mammal (including birds (chicken), fish, or any other animal species) that may have a disease or disorder (e.g., cancer, infectious disease) or be predisposed to a disease or disorder (e.g., cancer, infectious disease), but may or may not have the disease or disorder or may require a prophylactic intervention (e.g., vaccination) or may require an intervention (e.g., by protein replacement). In many embodiments, the individual is a human. Unless otherwise indicated, the terms "individual" and "subject" do not refer to a specific age and thus encompass adults, the elderly, children, and neonates. In some embodiments of the present disclosure, an "individual" or "subject" is a "patient".

[0218] The term "patient" refers to an individual or subject being treated, particularly an individual or subject having a disease. Aspects and embodiments of the present disclosure

[0219] In a first aspect, the present disclosure provides a binder for use in reducing or arresting the progression of head and neck squamous cell carcinoma (HNSCC) or treating HNSCC in a subject, the method comprising administering to the subject (i) a binder, (ii) a checkpoint inhibitor, which 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 chemotherapeutic agent and (b) 5-fluorouracil, wherein the binder comprises a first binding region that binds to CD40 and a second binding region that binds to CD137.

[0220] As demonstrated in the present disclosure, the following combination enhances the immune response: (i) stimulation with a binder that binds human CD40 and binds human CD137, (ii) inhibition of the PD-1 / PD-L1 axis, and (iii) chemotherapy based on a combination of a platinum-based chemotherapeutic agent and 5-fluorouracil. Without being bound by any theory, the rationale behind this unexpected finding is as follows: CD137 is co-expressed on PD-1 + cells. Thus, blocking the PD-L1 / PD-1 signal and co-stimulating via CD137 can synergistically enhance T cell effector function and improve the duration of the response. By conditional activation of CD40 and CD137, binders targeting CD40 and CD137 induce potent anti-tumor activity through enhanced T cell sensitization, production of cytokines and chemokines, and expansion and survival of T cells that have encountered the antigen. It is expected that the PD-(L)1 pathway is activated during sensitization and during continuous antigen exposure, which can reduce the magnitude of the immune response induced by binders targeting CD40 and CD137.

[0221] Based on the results of the KEYNOTE-048 (KN-048) trial, pembrolizumab or pembrolizumab in combination with platinum and 5-fluorouracil (5-FU) has become the global standard of care (SOC) for patients with previously untreated recurrent or metastatic HNSCC. In the KN-048 trial, patients were randomly assigned to receive pembrolizumab, pembrolizumab in combination with platinum (cisplatin or carboplatin) and 5-FU, or the EXTREME regimen (cisplatin or carboplatin + 5-FU + cetuximab).

[0222] In KN-048, in the pembrolizumab + platinum + 5-FU group compared to the EXTREME regimen, overall survival (OS) benefits were observed respectively in 1L HNSCC patients with PD-L1 CPS ≥1 (median OS was 13.6 months vs. 10.4 months); however, there was no improvement in ORR (36.4% vs. 35.7%) and PFS (5.1 months vs. 5.0 months) between the treatment groups (EMA assessment report, 2019). Based on the available data from the GCT1042-01 study as of the data cut-off date, adding an agent that binds to CD40 and CD137 (specifically GEN1042) as disclosed herein to the combination of the pembrolizumab + platinum + 5-FU regimen showed an improved ORR (66.7% vs. 36.4%) compared to platinum + 5-FU + pembrolizumab alone.

[0223] Thus, in one embodiment, the binder used in accordance with the first aspect of the present disclosure provides improved ORR compared to SOC, e.g., compared to a dosing regimen of pembrolizumab alone, a platinum-based chemotherapeutic agent, and 5-fluorouracil in combination, or compared to a dosing regimen of the binder and pembrolizumab in combination. For example, each of the binder, pembrolizumab, and chemotherapeutic combination can be administered at a dose that provides improved ORR compared to standard of care, e.g., compared to a dosing regimen of pembrolizumab alone, a platinum-based chemotherapeutic agent, and 5-fluorouracil in combination, or compared to a dosing regimen of the binder and pembrolizumab in combination. In some of the above embodiments, the ORR can be increased to 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%, or at least 95%.

[0224] As an alternative or in addition, in one embodiment, the binder used in accordance with the first aspect of the present disclosure provides improved ORR compared to SOC eval , e.g., compared to a dosing regimen of pembrolizumab alone, a platinum-based chemotherapeutic agent, and 5-fluorouracil in combination, or compared to a dosing regimen of the binder and pembrolizumab in combination. For example, each of the binder, pembrolizumab, and chemotherapeutic combination can be administered at a dose that provides improved ORR eval compared to standard of care, e.g., compared to a dosing regimen of pembrolizumab alone, a platinum-based chemotherapeutic agent, and 5-fluorouracil in combination, or compared to a dosing regimen of the binder and pembrolizumab in combination. In some of the above embodiments, the ORR evalIt can be increased to 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% or 100%.

[0225] As an alternative or in addition, in one embodiment, the binder used in accordance with the first aspect of the present disclosure provides an increased DCR compared to SOC, for example, compared to a dosing regimen of a combination of pembrolizumab alone, a platinum-based chemotherapeutic agent, and 5-fluorouracil, or compared to a dosing regimen of a combination of the binder and pembrolizumab alone. For example, each of the binder, pembrolizumab, and chemotherapy combination can be administered at a dose that provides an increased DCR compared to standard of care, for example, compared to a dosing regimen of a combination of pembrolizumab alone, a platinum-based chemotherapeutic agent, and 5-fluorouracil, or compared to a dosing regimen of a combination of the binder and pembrolizumab alone. In some of the above embodiments, the DCR can be increased to 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% or at least 95%.

[0226] As an alternative or supplement, in one embodiment, the binder used in accordance with the first aspect of the present disclosure provides an improved DCR compared to SOC eval , for example, compared to a dosing regimen of a combination of pembrolizumab alone, a platinum-based chemotherapeutic agent, and 5-fluorouracil, or compared to a dosing regimen of a combination of the binder and pembrolizumab alone. For example, each of the binder, pembrolizumab, and chemotherapeutic combination may be administered at a dose that provides an improved DCR eval , for example, compared to a dosing regimen of a combination of pembrolizumab alone, a platinum-based chemotherapeutic agent, and 5-fluorouracil, or compared to a dosing regimen of a combination of the binder and pembrolizumab alone. In some of the above embodiments, the DCR eval can be increased to 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% or 100%. Binder that binds to CD40 and CD137

[0227] In one embodiment, CD40 is human CD40, particularly human CD40 comprising the sequence shown in SEQ ID NO:36. In one embodiment, CD137 is human CD137, particularly human CD137 comprising the sequence shown in SEQ ID NO:38. In one embodiment, CD40 is human CD40 and CD137 is human CD137. In one embodiment, CD40 is human CD40 comprising the sequence shown in SEQ ID NO:36, and CD137 is human CD137 comprising the sequence shown in SEQ ID NO:38.

[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) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 7 or 9, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 8 or 10; and b) The second antigen-binding region that binds to human CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 17 or 19, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18 or 20.

[0229] In one embodiment of the binding agent according to the first aspect, a) The first binding region that binds to human CD40 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, 2, and 3 respectively, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 4, 5, and 6 respectively; and b) The second antigen-binding region that binds to human CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 11, 12, and 13 respectively, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 14, 15, and 16 respectively.

[0230] In one embodiment of the binding agent according to the first aspect, a) The first binding region that binds to human CD40 comprises a heavy chain variable region (VH) and a light chain variable region (VL) region, wherein the heavy chain variable region (VH) comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 7 or 9, and the light chain variable region (VL) comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 8 or 10; b) The second binding region that binds to human CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL) region, wherein the heavy chain variable region (VH) comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 17 or 19, and the light chain variable region (VL) comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with SEQ ID NO: 18 or 20.

[0231] In one embodiment of the binding agent according to the first aspect, a) The first binding region that binds to human CD40 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 7 or 9, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 8 or 10; And b) The second binding region that binds to human CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL) region, wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 17 or 19, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 18 or 20.

[0232] In one embodiment of the binding agent according to the first aspect, a) The first binding region that binds to human CD40 comprises a heavy chain variable region (VH) and a light chain variable region (VL) region, wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 10; And b) The second binding region that binds to human CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 20.

[0233] In particular, the binding agent can be an antibody, such as a multispecific antibody, such as a bispecific antibody. The binding agent can also be in the form of a full-length antibody or an antibody fragment.

[0234] More preferably, the binding agent is a human antibody or a humanized antibody.

[0235] Each variable region may contain three complementarity-determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).

[0236] The complementarity-determining regions (CDR) and framework regions (FR) may be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0237] In one embodiment of the first aspect, the binder comprises: i) a polypeptide containing the first heavy-chain variable region (VH) and the first heavy-chain constant region (CH), and ii) a polypeptide containing the second heavy-chain variable region (VH) and the second heavy-chain constant region (CH).

[0238] In one embodiment of the first aspect, the binder comprises: i) a polypeptide containing the first light-chain variable region (VL) and also containing the first light-chain constant region (CL), and ii) a polypeptide containing the second light-chain variable region (VL) and also containing the second light-chain constant region (CL).

[0239] In one embodiment of the first aspect, the binder is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises: i) a polypeptide containing the first heavy-chain variable region (VH) and the first heavy-chain constant region (CH), and ii) a polypeptide containing the first light-chain variable region (VL) and the first light-chain constant region (CL); and the second binding arm comprises: iii) a polypeptide containing the second heavy-chain variable region (VH) and the second heavy-chain constant region (CH), and iv) a polypeptide containing the second light-chain variable region (VL) and the second light-chain constant region (CL).

[0240] In one embodiment of the first aspect, the binder comprises i) a first heavy chain and a first light chain containing the antigen-binding region capable of binding to CD40, the first heavy chain comprising a first heavy-chain constant region, and the first light chain comprising a first light-chain constant region; and ii) a second heavy chain and a second light chain containing the antigen-binding region capable of binding to CD137, the second heavy chain comprising a second heavy-chain constant region, and the second light chain comprising a second light-chain constant region.

[0241] The first heavy chain constant region (CH) and the second heavy chain constant region (CH) may each comprise one or more of a heavy chain constant 1 (CH1) region, a hinge region, a heavy chain constant 2 (CH2) region, and a heavy chain constant 3 (CH3) region, and preferably comprise at least the hinge region, CH2 region, and CH3 region.

[0242] The first heavy chain constant region (CH) and the second heavy chain constant region (CH) may each comprise a CH3 region, wherein the two CH3 regions comprise asymmetric mutations. Asymmetric mutations mean that the sequences of the first CH3 region and the second CH3 region contain amino acid substitutions at different positions. For example, one of the first CH3 region and the second CH3 region contains a mutation at a position corresponding to position 405 in the human IgG1 heavy chain according to EU numbering, and the other of the first CH3 region and the second CH3 region contains a mutation at a position corresponding to position 409 in the human IgG1 heavy chain according to EU numbering.

[0243] In the first heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering may have been replaced, and in the second heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering may have been replaced. In some specific embodiments, the first heavy chain and the second heavy chain are not replaced at the same position (i.e., the first heavy chain and the second heavy chain contain asymmetric mutations).

[0244] In one embodiment of the binder according to the first aspect, (i) in the first heavy chain constant region (CH), the amino acid at a position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L, and in the second heavy chain constant region (CH), the amino acid at a position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R, or (ii) in the first heavy chain, the amino acid at a position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R, and in the second heavy chain, the amino acid at a position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L.

[0245] In one embodiment of the first aspect, compared to another antibody comprising the same first antigen-binding region and second antigen-binding region and two heavy chain constant regions (CH) comprising a human IgG1 hinge region, CH2 region, and CH3 region, the binder induces a lower degree of Fc-mediated effector function.

[0246] In a specific embodiment of the binder according to the first aspect, the first heavy chain constant region (CH) and the second heavy chain constant region (CH) are modified such that the degree of Fc-mediated effector function induced by the antibody is lower compared to an antibody that is identical except for comprising unmodified first and second heavy chain constant regions (CH). In particular, the unmodified first heavy chain constant region (CH) and second heavy chain constant region (CH) each or both comprise the amino acid sequence shown in SEQ ID NO: 21 or 29, consist of the amino acid sequence shown in SEQ ID NO: 21 or 29, or consist essentially of the amino acid sequence shown in SEQ ID NO: 21 or 29.

[0247] The Fc-mediated effector function can be determined by measuring the binding of the binder to an Fcγ receptor, binding to C1q, or inducing crosslinking of Fcγ receptors mediated by Fe. In particular, the Fc-mediated effector function can be determined by measuring the binding of the binder to C1q.

[0248] The first and second heavy chain constant regions of the binder have been modified such that the binding of C1q to the antibody is reduced compared to a wild-type antibody, preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%, wherein C1q binding is preferably determined by ELISA.

[0249] In an embodiment of the binder according to the first aspect, in at least one of the first heavy chain constant region (CH) and the second heavy chain constant region (CH), one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in the human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively.

[0250] In an embodiment of the binder according to the first aspect, in the first and second heavy chains, the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering can be F and E, respectively.

[0251] In particular, in the first and second heavy chain constant regions (HC), the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering can be F, E, and A, respectively.

[0252] In one embodiment of the binder 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 heavy chain constant region and the second heavy chain constant region are F and E, respectively, where: (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.

[0253] In one embodiment of the binder according to the first aspect, the positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain according to EU numbering in both the first heavy chain constant region and the second heavy chain constant region are F, E and A, respectively, where: (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.

[0254] In one embodiment of the binder according to the first aspect, the constant region of the first heavy chain and / or the second heavy chain comprises an amino acid sequence selected from: a) the sequence shown in SEQ ID NO:21 or SEQ ID NO:29 [IgG1-FC]; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids have been deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions, such as 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).

[0255] In one embodiment of the binder according to the first aspect, the constant region of the first heavy chain or the second heavy chain, for example the constant region of the second heavy chain, comprises an amino acid sequence selected from, or consists essentially of, or consists of an amino acid sequence selected from: a) The sequence [IgG1-F405L] shown in SEQ ID NO:22 or SEQ ID NO:30; b) A subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids have been deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) A sequence having at most 9 substitutions, such as at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, 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).

[0256] In one embodiment of the binding agent according to the first aspect, the constant region of the first heavy chain or the second heavy chain, for example, the constant region of the first heavy chain, comprises an amino acid sequence selected from, or consists essentially of, or consists of an amino acid sequence selected from: a) The sequence [IgG1-F409R] shown in SEQ ID NO:23 or SEQ ID NO:31; b) A subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids have been deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) A sequence having at most 10 substitutions, such as at most 9 substitutions, at most 8 substitutions, at most 7 substitutions, at most 6 substitutions, 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).

[0257] In one embodiment of the binding agent according to the first aspect, the constant region of the first heavy chain and / or the second heavy chain comprises an amino acid sequence selected from, or consists essentially of, or consists of an amino acid sequence selected from: a) The sequence [IgG1-Fc_FEA] shown in SEQ ID NO:24 or SEQ ID NO:32; b) A subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids have been deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) A sequence having at most 7 substitutions, such as at most 6 substitutions, 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).

[0258] In one embodiment of the binder according to the first aspect, the constant region of the first heavy chain and / or the second heavy chain, such as the constant region of the second heavy chain, comprises an amino acid sequence selected from the following, or consists essentially of an amino acid sequence selected from the following, or consists of an amino acid sequence selected from the following: a) The sequence shown in SEQ ID NO:25 or SEQ ID NO:33 [IgG1-Fc_FEAL]; b) A subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids have been deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) A sequence having at most 6 substitutions, such as 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).

[0259] In one embodiment of the binder according to the first aspect, the constant region of the first heavy chain and / or the second heavy chain, such as the constant region of the first heavy chain, comprises an amino acid sequence selected from the following, or consists essentially of an amino acid sequence selected from the following, or consists of an amino acid sequence selected from the following: a) The sequence shown in SEQ ID NO:26 or SEQ ID NO:34 [IgG1-Fc_FEAR]; b) A subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids have been deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) A sequence having at most 6 substitutions, such as 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).

[0260] In one embodiment of the first aspect, the binder comprises a kappa (κ) light chain constant region.

[0261] In one embodiment of the first aspect, the binder comprises a lambda (λ) light chain constant region.

[0262] In one embodiment of the binder 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.

[0263] In one embodiment of the binder 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.

[0264] In one embodiment of the binder 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.

[0265] In one embodiment of the binder according to the first aspect, the kappa (κ) light chain comprises an amino acid sequence selected from: a) the sequence shown in SEQ ID NO:27; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids have been 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 substitutions, at most 3, at most 2 or at most 1 substitution compared to the amino acid sequence defined in a) or b).

[0266] In one embodiment of the binder according to the first aspect, the lambda (λ) light chain comprises an amino acid sequence selected from: a) the sequence shown in SEQ ID NO:28; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids have been 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 substitutions, at most 3, at most 2 or at most 1 substitution compared to the amino acid sequence defined in a) or b).

[0267] The binder (especially an antibody) according to the first aspect is an isotype selected from IgG1, IgG2, IgG3 and IgG4. In particular, the binder can be a full-length IgG1 antibody. In a preferred embodiment of the first aspect, the binder (especially an antibody) is the IgG1m(f) allotype.

[0268] In one embodiment, the binder is the antibody GEN1042 as disclosed herein (i.e., the antibody comprises the humanized VH and VL sequences described in Table 1, a human κ light chain, and a human IgG1 heavy chain; the CD40-binding arm has been generated with a human IgG1 heavy chain containing the following amino acid mutations: L234F, L235E, D265A, and F405L (FEAL), where the amino acid position numbering is according to EU numbering (corresponding to SEQ ID NO:33); the CD137-binding arm has been generated with a human IgG1 heavy chain containing the following amino acid mutations: L234F, L235E, D265A, and K409R (FEAR), where the amino acid position numbering is according to EU numbering (corresponding to SEQ ID NO:34)).

[0269] Preferably, the binder is administered in a suitable amount, i.e., for example, the amount of the binder administered in each dose and / or treatment cycle, which can induce intracellular signaling when it binds to CD137 expressed on another cell. Thus, a suitable amount of the binder according to the present disclosure is capable of trans-activating two different cells. In humans, CD40 is expressed on many cells, including antigen-presenting cells (APCs), such as dendritic cells, while CD137 is expressed on T cells and other cells. Thus, a binder that binds to CD40 and CD137 in a suitable amount according to the present disclosure is capable of binding to APCs and T cells expressing these receptors simultaneously. Without being bound by theory, the binder can thus (i) mediate cell-cell interactions between APCs and T cells through receptor binding, and (ii) activate both CD40 and CD137 simultaneously, which is mainly induced by cross-linking and receptor aggregation after cell-cell interactions and does not necessarily rely on the agonistic activity of the parental monospecific bivalent antibody. Thus, these trans-activating binders exert co-stimulatory activity in the context of APC:T cell interactions and can trigger T cell responses against tumor cells. Thus, this mechanism of action can reflect natural T cell activation by activated APCs through antigen presentation, allowing the presentation of multiple tumor-specific antigens from APCs to T cells. Without being limited by theory, the co-stimulatory activity can provide one or more of the following: (i) only specific T cells are activated (i.e., those cells in contact with APCs), as opposed to any T cells; (ii) exhausted T cells are re-activated through strong co-stimulation triggered by activated APCs and CD137; and (iii) T cells are sensitized by antigen presentation induced by activated APCs and simultaneous triggering of CD137.

[0270] The amount of the binder administered in each dose and / or treatment cycle can be particularly within a certain range, wherein 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 binder binds to both CD40 and CD137.

[0271] In some preferred embodiments, the amount of the binder administered (e.g., in each dose and / or each treatment cycle) is about 50 to 150 mg / day (e.g., about 60 to 140 mg / day, about 70 to 130 mg / day, about 80 to 120 mg / day, about 90 to 110 mg / day, or about 95 to 105 mg / day, such as about 100 mg / day) or about 0.62 to 1.88 mg / kg body weight / day (e.g., about 0.75 to 1.75 mg / kg body weight / day, about 0.87 to 1.63 mg / kg body weight / day, 1.00 to 1.50 mg / kg body weight / day, 1.12 to 1.38 mg / kg body weight / day, or 1.18 to 1.31 mg / kg body weight / day, such as about 1.25 mg / kg body weight / day).

[0272] In some preferred embodiments, the amount of the binder administered (e.g., in each dose and / or each treatment cycle) is 335×10 -9 to 1020×10 -9 mol / day (e.g., about 400×10 -9 to 950×10 -9 mol / day, about 470×10 -9 to 880×10 -9 mol / day, about 540×10 -9 to 810×10 -9 mol / day, about 600×10 -9 to 750×10 -9 mol / day, or about 640×10 -9 to 710×10 -9 mol / day, such as about 675×10 -9 mol / day) or about 4.1×10 -9 to 12.7×10 -9 mol / kg body weight / day (e.g., 5.0×10 -9 to 11.9×10 -9 mol / kg body weight / day, 5.8×10 -9 to 11.0×10 -9 mol / kg body weight / day, 6.7×10 -9 to 10.1×10-9 mol / kg body weight / day, 7.5×10 -9 to 9.4×10 -9 mol / kg body weight / day or 8.0×10 -9 to 8.9×10 -9 mol / kg body weight / day, such as about 8.4 mol / kg body weight / day).

[0273] According to these embodiments, based on a median body weight of 80 kg for the subject to whom the binder is administered, the dose defined in mg / kg can be converted to a flat dose, and vice versa.

[0274] The binder can be administered in any manner and by any route known in the art. In a preferred embodiment, the binder is administered systemically, such as parenterally, particularly intravenously.

[0275] The binder can be administered in the form of any suitable pharmaceutical composition as described herein. In a preferred embodiment, the binder is administered in the form of an infusion.

[0276] The binder can be administered before, simultaneously with, or after an inhibitor of the PD-1 / PD-L1 axis (PD-1 / PD-L1 checkpoint inhibitor).

[0277] In one embodiment, the binder is administered before the PD-1 / PD-L1 checkpoint inhibitor. For example, the interval between the end of the administration of the binder and the start of the administration of the PD-1 / PD-L1 checkpoint inhibitor can be at least about 10 minutes, such as 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, and up to about 12 hours, such as 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.

[0278] In one embodiment, the binder is administered after the administration of a PD-1 / PD-L1 checkpoint inhibitor. For example, the interval between the end of the administration of the checkpoint PD-1 / PD-L1 inhibitor and the start of the administration of the binder can be at least about 10 minutes, such as 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, and up to about 12 hours, such as 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.

[0279] In one embodiment, the binder is administered concurrently with the PD-1 / PD-L1 checkpoint inhibitor. For example, a composition containing both drugs can be used to administer the binder and the PD-1 / PD-L1 checkpoint inhibitor. Alternatively, the binder can be administered to one limb of the subject while the PD-1 / PD-L1 checkpoint inhibitor can be administered to another limb of the subject. PD-1 / PD-L1 checkpoint inhibitor

[0280] In one embodiment, the immune checkpoint inhibitor suitable for the methods disclosed herein is an antagonist of the inhibitory signal of the PD-1 / PD-L1 axis, such as an antibody targeting, for example, 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 according to the present disclosure are described herein.

[0281] In one embodiment, the immune checkpoint inhibitor blocks the inhibitory signal associated with the immune checkpoint. In one embodiment, the immune checkpoint inhibitor is an antibody or fragment thereof that disrupts or inhibits the inhibitory signal transduction associated with the immune checkpoint. In one embodiment, the immune checkpoint inhibitor is a small molecule inhibitor that disrupts or inhibits the inhibitory signal transduction. In one embodiment, the immune checkpoint inhibitor is a peptide-based inhibitor that disrupts or inhibits the inhibitory signal transduction. In one embodiment, the immune checkpoint inhibitor is an inhibitory nucleic acid molecule that disrupts or inhibits the inhibitory signal transduction.

[0282] As described herein, inhibiting or blocking inhibitory immune checkpoint signaling results in preventing or reversing immunosuppression and the establishment or enhancement of T cell immunity against cancer cells. In one embodiment, as described herein, inhibition of immune checkpoint signaling reduces or suppresses immune system dysfunction. In one embodiment, as described herein, inhibition of immune checkpoint signaling reduces the degree of dysfunction of dysfunctional immune cells. In one embodiment, as described herein, inhibition of immune checkpoint signaling reduces the degree of dysfunction of dysfunctional T cells.

[0283] In one embodiment, a PD-1 / PD-L1 immune checkpoint inhibitor prevents the interaction between the checkpoint blocker protein PD-1 and PD-L1 or PD-L2.

[0284] The PD-1 / PD-L1 immune checkpoint inhibitor can be an antibody, an antigen-binding fragment thereof, or a construct comprising an antibody moiety that includes an antigen-binding fragment with the desired specificity. Antibodies or antigen-binding fragments thereof are as described herein. The antibody or antigen-binding fragment as an immune checkpoint inhibitor particularly includes an antibody or antigen-binding fragment thereof that binds to an immune checkpoint protein (e.g., an immune checkpoint receptor or an immune checkpoint receptor ligand). As described herein, the antibody or antigen-binding fragment can also be conjugated to additional moieties. In particular, the antibody or antigen-binding fragment thereof is a chimeric antibody, a humanized antibody, or a human antibody. Preferably, the immune checkpoint inhibitor antibody or antigen-binding fragment thereof is an antagonist of an immune checkpoint receptor or an antagonist of an immune checkpoint receptor ligand.

[0285] In a preferred embodiment, the antibody as an immune checkpoint inhibitor is an isolated antibody.

[0286] In one embodiment, the PD-1 / PD-L1 immune checkpoint inhibitor is an antibody, fragment thereof, or construct that blocks 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) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NOs: 81, 82, and 83, respectively, and the light chain variable region (VL) comprises the CDR1, CDR2, and CDR3 sequences shown 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 and a light chain variable region, wherein the heavy chain variable region comprises 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 the light chain variable region comprises 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 thereof, or construct comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 87 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 88. In one embodiment, such an antibody, fragment thereof, or construct comprises a heavy chain and a light chain, wherein 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 ID: 90.

[0287] The PD-1 / PD-L1 immune checkpoint inhibitor can be an inhibitory nucleic acid molecule, such as an oligonucleotide, siRNA, shRNA, antisense DNA or RNA molecule, and an aptamer (e.g., a DNA or RNA aptamer), particularly an antisense oligonucleotide. In one embodiment, the PD-1 / PD-L1 immune checkpoint inhibitor siRNA interferes with mRNA and thus blocks translation, such as the translation of immune checkpoint proteins.

[0288] The PD-1 / PD-L1 checkpoint inhibitor can also be in the form of the molecule (or variant thereof) itself in a soluble form, such as soluble PD-L1 or a PD-L1 fusion.

[0289] In the context of the present disclosure, more than one checkpoint inhibitor can be used, wherein the more than one checkpoint inhibitor targets different checkpoint pathways or the same checkpoint pathway. Preferably, the more than one checkpoint inhibitor are different checkpoint inhibitors. Preferably, if 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.

[0290] In one embodiment, a 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, a PD-1 / PD-L1 checkpoint inhibitor is an inhibitor of the PD-1 signaling pathway. In certain embodiments, a checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 inhibitor. In certain embodiments, a checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 ligand inhibitor, such as a PD-L1 inhibitor or a PD-L2 inhibitor. In a preferred embodiment, a checkpoint inhibitor of the PD-1 signaling pathway is an antibody, an antigen-binding portion thereof or a construct thereof that disrupts or inhibits the interaction between the PD-1 receptor and one or more of its ligands PD-L1 and / or PD-L2. Antibodies that bind to PD-1 and disrupt or inhibit the interaction between PD-1 and one or more of its ligands are known in the art. In certain embodiments, the antibody, an antigen-binding portion thereof or a construct thereof binds specifically to PD-1. In certain embodiments, the antibody, an antigen-binding portion thereof or a construct thereof binds specifically to PD-L1 and disrupts or inhibits its interaction with PD-1, thereby enhancing immune activity. In certain embodiments, the antibody, an antigen-binding portion thereof or a construct thereof binds specifically to PD-L2 and disrupts or inhibits its interaction with PD-1, thereby enhancing immune activity.

[0291] Exemplary PD-1 inhibitors include, but are not limited to, anti-PD-1 antibodies such as BGB-A317 (BeiGene; see US 8,735,553, WO 2015 / 35606, and US2015 / 0079109), lambrolizumab (e.g., disclosed as hPD109A and its humanized derivatives h409A1, h409A16, and h409A17 in WO2008 / 156712), AB137132 (Abcam), EH12.2H7 and RMP1-14 (#BE0146; Bioxcell Lifesciences Pvt. LTD.), MIH4 (Affymetrix eBioscience), nivolumab (OPDIVO, BMS-936558; Bristol Myers Squibb; see U.S. Patent No. 8,008,449, WO 2013 / 173223, WO 2006 / 121168), pembrolizumab (KEYTRUDA; MK-3475; Merck; see WO 2008 / 156712), pidilizumab (CT-011; CureTech; see Hardy et al., 1994, Cancer Res., 54(22):5793-6 and WO 2009 / 101611), PDR001 (Novartis; see WO 2015 / 112900), MEDI0680 (AMP-514; AstraZeneca; see WO 2012 / 145493), TSR-042 (see WO 2014 / 179664), cemiplimab (REGN-2810; Regeneron; H4H7798N; see US2015 / 0203579 and WO 2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., 2007, J. Hematol. Oncol. 70:136), AMP-224 (GSK-2661380; see Li et al., 2016, Int J Mol Sci 17(7):1151 and WO 2010 / 027827 and WO 2011 / 066342), PF-06801591 (Pfizer), tislelizumab (BGB-A317; BeiGene; see WO 2015 / 35606, U.S. Patent No.9,834,606 and US2015 / 0079109), BI 754091, SHR-1210 (see WO2015 / 085847), and antibodies 17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 as described in WO2006 / 121168, INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015 / 085847), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see W02014 / 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 WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics; see WO 2017 / 1983046), IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825 and WO 2017 / 133540), cetrelimab (JNJ-63723283; JNJ-3283; see Calvo et al., J. Clin. Oncol. 36, no. 5_suppl (2018) 58), genolimzumab (CBT-501; see Patelet al., J. ImmunoTher. Cancer, 2017, 5 (Suppl 2):P242), sasanlimab (PF-06801591; see Youssef et al., Proc. Am. Assoc. Cancer Res. Ann. Meeting 2017; CancerRes2017; 77(13Suppl):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 WO 2017 / 106656; Naing et al., J. Clin. Oncol. 34, no. 15_suppl (2016) 3060 - 3060), BCD - 100 (JSC BIOCAD, Russia; see WO 2018 / 103017), balstilimab (AGEN2034; see WO 2017 / 040790), sintilimab (IBI - 308; see WO 2017 / 024465 and WO 2017 / 133540), ezabenlimab (BI - 754091; see US2017 / 334995; Johnson et al., J. Clin. Oncol. 36, no.5_suppl(2018)212-212), zimberelimab (GLS-010; see WO 2017 / 025051), LZM-009 (see US 2017 / 210806), AK-103 (see WO 2017 / 071625, WO 2017 / 166804 and WO 2018 / 036472), retifanlimab (MGA-012; see WO 2017 / 019846), Sym-021 (see WO 2017 / 055547), CS1003 (see CN107840887), the anti-PD-1 antibody IgG1-PD1 disclosed herein (i.e., comprising the VH sequence defined in SEQ ID NO:43, the VL sequence defined in SEQ ID NO:44, the Fc sequence defined in SEQ ID NO:61 and the κ sequence defined in SEQ ID NO:27), such as the anti-PD-1 antibodies described in, for example, US 7,488,802, US 8,008,449, US 8,168,757, WO 03 / 042402, WO 2010 / 089411 (also discloses anti-PD-L1 antibodies), WO 2010 / 036959, WO 2011 / 159877 (also discloses antibodies against TIM-3), WO 2011 / 082400, WO 2011 / 161699, WO 2009 / 014708, WO 03 / 099196, WO 2009 / 114335, WO 2012 / 145493 (also discloses antibodies against PD-L1), WO 2015 / 035606, WO 2014 / 055648 (also discloses anti-KIR antibodies), US2018 / 0185482 (also discloses anti-PD-L1 antibodies and anti-TIGIT antibodies), US 8,008,449, US 8,779,105, US 6,808,710, US 8,168,757, US 2016 / 0272708 and US 8,354,509, such as in, for example, Shaabani et al., 2018, Expert Op Ther Pat., small molecule antagonists targeting the PD-1 signaling pathway as disclosed in 28(9):665-678 and Sasikumar and Ramachandra, 2018, BioDrugs, 32(5):481-497, such as siRNAs targeting PD-1 as disclosed in, for example, WO 2019 / 000146 and WO 2018 / 103501, soluble PD-1 proteins as disclosed in WO 2018 / 222711, and oncolytic viruses containing soluble forms of PD-1 as described in, for example, WO 2018 / 022831.

[0292] 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, BI 754091, or SHR-1210. In one embodiment, the PD-1 inhibitor is IgG1-PD1 as disclosed herein.

[0293] In certain embodiments, the PD-1 / PD-L1 inhibitory immunomodulator is an anti-PD-1 antibody or antigen-binding fragment thereof that comprises the complementarity-determining regions (CDRs) of one of the aforementioned anti-PD-1 antibodies or antigen-binding fragments, such as an anti-PD-1 antibody or antigen-binding fragment selected from 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 the CDRs of IgG1-PD1.

[0294] In some embodiments, the CDRs of anti-PD-1 antibodies are described using the Kabat numbering scheme (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242).

[0295] In certain embodiments, the PD-1 / PD-L1 inhibitory immunomodulator is an anti-PD-1 antibody or an antigen-binding fragment thereof that comprises the heavy-chain variable region and the light-chain variable region of one of the foregoing anti-PD-1 antibodies or antigen-binding fragments, such as an anti-PD-1 antibody or antigen-binding fragment thereof selected from 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.

[0296] In certain embodiments, the PD-1 / PD-L1 inhibitory immunomodulator is an anti-PD-1 antibody or an antigen-binding fragment thereof selected from 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.

[0297] In certain embodiments, the PD-1 / PD-L1 inhibitory immunomodulator is pembrolizumab or an antigen-binding fragment thereof.

[0298] The anti-PD-1 antibodies of the present disclosure are preferably monoclonal and can be multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab’) fragments, fragments generated from a Fab expression library, and PD-1 binding fragments of any of the foregoing. 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 can be of 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 molecules.

[0299] In certain embodiments of the present disclosure, the anti-PD-1 antibody is an antigen-binding fragment as described herein (e.g., a human antigen-binding fragment) and includes, but is not limited to, Fab, Fab’, and F(ab’)2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and fragments containing V L or V H domains. Antigen-binding fragments, including single-chain antibodies, can contain separate variable regions or variable regions combined with all or a portion of the following: hinge region, CHl, CH2, CH3, and CL domains. The present disclosure also includes antigen-binding fragments that contain any combination of variable regions with hinge regions, CH1, CH2, CH3, and CL domains. In some embodiments, the anti-PD-1 antibody or its antigen-binding fragment is an anti-PD-1 antibody or its antigen-binding fragment of human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.

[0300] The anti-PD-1 antibodies disclosed herein can be monospecific, bispecific, trispecific, or of higher multispecificity. Multispecific antibodies can be specific for different epitopes of PD-1 or can be specific for both PD-1 and a heterologous protein. See, e.g., PCT Publication WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt, et al., 1991, J. Immunol. 147:6069; U.S. Pat. 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.

[0301] The anti-PD-1 antibodies disclosed herein can be described or characterized in terms of the specific 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 those described by: 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 Plückthun 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 can vary depending on the scheme used for identification. In some embodiments, the CDR of a given antibody or region thereof (e.g., its variable region) or a single specific CDR (e.g., CDR-H1, CDR-H2, CDR-H3) should be understood to encompass the CDR(s) defined (or specified) by any of the foregoing schemes. For example, as defined by any of the foregoing schemes, where a stated specific CDR (e.g., CDR-H3) contains the amino acid sequence of the corresponding CDR in the amino acid sequence of a given V H or V L region, it should be understood that such a CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region. Schemes for identifying specific CDRs can be specified, such as the CDRs defined by the Kabat, Chothia, AbM, or IMGT methods.

[0302] 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 as described in Lefranc, M.P et al., Dev.Comp.Immunol., 2003, 27, 55-77.

[0303] In some embodiments, the anti-PD-1 antibodies disclosed herein contain the CDRs of the antibody nivolumab. See WO2006 / 121168. In some embodiments, the Kabat numbering scheme (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242) is used to describe the CDRs of the antibody nivolumab. This disclosure encompasses anti-PD-1 antibodies or derivatives thereof that contain a heavy chain variable domain or a light chain variable domain, the variable domain comprising (a) a group of three CDRs, wherein the group of CDRs is from the monoclonal antibody nivolumab, and (b) a group of four framework regions, wherein the group of framework regions is different from the group of framework regions in the monoclonal antibody nivolumab, and wherein the anti-PD-1 antibody or derivative thereof binds to PD-1. In certain embodiments, the anti-PD-1 antibody is nivolumab.

[0304] In some embodiments, the anti-PD-1 antibodies disclosed herein comprise the CDRs of the antibody pembrolizumab. See WO2008 / 156712. In some embodiments, the Kabat numbering scheme (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242) is used to describe the CDRs of the antibody pembrolizumab. This disclosure encompasses anti-PD-1 antibodies or derivatives thereof comprising a heavy chain variable domain or a light chain variable domain, the variable domain comprising (a) a group of three CDRs, wherein the group of CDRs is from the monoclonal antibody pembrolizumab, and (b) a group of four framework regions, wherein the group of framework regions is different from the group of framework regions in the monoclonal antibody pembrolizumab, and wherein the anti-PD-1 antibody or derivative thereof binds to PD-1. In certain embodiments, the anti-PD-1 antibody is pembrolizumab.

[0305] The anti-PD-1 antibodies disclosed herein can also be described or characterized in terms of their binding affinity to PD-1 (e.g., human PD-1). Preferred binding affinities include having a dissociation constant or Kd less than 5x10 -2 M, 10 -2 M, 5x10 -3 M, 10 -3 M, 5x10 -4 M, 10 -4 M, 5x10 -5 M, 10 -5 M, 5x10 -6 M, 10 -6 M, 5x10 - 7 M, 10 -7 M, 5x10 -8 M, 10 -8 M, 5x10 -9 M, 10 -9 M, 5x10 -10 M, 10 -10 M, 5x10 -11 M, 10 -11 M, 5x10 -12 M, 10 -12 M, 5x10 -13 M, 10 -13 M, 5x10 -14 M, 10 -14 M, 5x10-15 M, or 10 -15 Those of M.

[0306] The anti-PD-1 antibody also includes modified derivatives and constructs, namely, by covalently linking any type of molecule to the antibody such that the covalent linkage does not prevent the antibody from binding to PD-1. For example, and not by way of limitation, anti-PD-1 antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cell ligand or other protein, etc. Any of a variety of chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis with tunicamycin, etc. Additionally, the derivative or construct can include one or more non-classical amino acids.

[0307] Exemplary PD-1 ligand inhibitors are PD-L1 inhibitors and PD-L2 inhibitors, and include but are not limited to anti-PD-L1 antibodies such as MEDI4736 (durvalumab; AstraZeneca; see WO 2011 / 066389), MSB-0010718C (see US 2014 / 0341917), YW243.55.S70 (see WO 2010 / 077634 and SEQ ID NO: 20 of US 8,217,149), MIH1 (Affymetrix eBioscience; see EP 3230319), MDX-1105 (Roche / Genentech; see WO2013019906 and US 8,217,149), STI-1014 (Sorrento; see W02013 / 181634), CK-301 (Checkpoint Therapeutics), KN035 (3D Med / Alphamab; see Zhang et al., 2017, CellDiscov. 3:17004), atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267; see US 9,724,413), BMS-936559 (Bristol Myers Squibb; see US 7,943,743, WO 2013 / 173223), avelumab (bavencio; see US2014 / 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), the anti-PD-L1 antibodies disclosed in US 7,943,743, including 3G10, 12A4 (also known as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, as in WO 2010 / 077634, US 8,217,149, WO 2010 / 036959, WO 2010 / 077634, WO 2011 / 066342, US 8,217,149, US 7,943,743, WO 2010 / 089411, US 7,635,757, US 8,217,149, US2009 / 0317368, WO 2011 / 066389, WO2017 / 034916, WO2017 / 020291, WO2017 / 020858, WO2017 / 020801, WO2016 / 111645, WO2016 / 197367, WO2016 / 061142, WO2016 / 149201, WO2016 / 000619, WO2016 / 160792, WO2016 / 022630, WO2016 / 007235, WO2015 / 179654, WO2015 / 173267, WO2015 / 181342, WO2015 / 109124, WO 2018 / 222711, WO2015 / 112805, WO2015 / 061668, WO2014 / 159562, WO2014 / 165082, WO2014 / 100079 the anti-PD-L1 antibody described in

[0308] In one embodiment, the PD-L1 inhibitor is atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267; see US 9,724,413).

[0309] In certain embodiments, the PD-1 / PD-L1 inhibitory immunomodulator is an anti-PD-L1 antibody or an antigen-binding fragment thereof that comprises the complementarity-determining regions (CDRs) of one of the aforementioned anti-PD-L1 antibodies or antigen-binding fragments, such as the CDRs of atezolizumab or an antigen-binding fragment thereof.

[0310] In some embodiments, the Kabat numbering scheme (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242) is used to describe the CDRs of the anti-PD-L1 antibody.

[0311] In certain embodiments, the PD-1 / PD-L1 inhibitory immunomodulator is an anti-PD-L1 antibody or antigen-binding fragment thereof that comprises the heavy chain variable region and the light chain variable region of one of the anti-PD-L1 antibodies or antigen-binding fragments described above, such as the heavy chain variable region and the light chain variable region of atezolizumab or an antigen-binding fragment thereof.

[0312] The anti-PD-L1 antibodies of the present disclosure are preferably monoclonal antibodies and can be multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab’) fragments, fragments produced by a Fab expression library, and PD-L1 binding fragments of any of the foregoing. 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 can be of 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 molecules.

[0313] In certain embodiments of the present disclosure, the anti-PD-1 antibody is an antigen-binding fragment as described herein (e.g., a human antigen-binding fragment) and includes, but is not limited to, Fab, Fab’, and F(ab’)2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and fragments that contain V L or V H domains. Antigen-binding fragments, including single-chain antibodies, can contain individual variable regions or variable regions combined with all or a portion of the following: hinge region, CH1, CH2, CH3, and CL domains. The present disclosure also encompasses antigen-binding fragments that contain any combination of variable regions with hinge regions, CH1, CH2, CH3, and CL domains. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is an anti-PD-1 antibody or antigen-binding fragment thereof of a human, mouse (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.

[0314] The anti-PD-L1 antibodies disclosed herein can be monospecific, bispecific, trispecific, or higher multispecific. Multispecific antibodies can be specific for different epitopes of PD-L1 or can be specific for both PD-L1 and a heterologous protein. See, e.g., PCT Publication WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt, et al., 1991, J. Immunol. 147: 6069; U.S. Pat. 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.

[0315] The anti-PD-L1 antibodies disclosed herein can be described or specified 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 those described by: 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 Plückthun 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 can vary depending on the scheme used for identification. In some embodiments, a CDR of a given antibody or region thereof (e.g., its variable region) or a single specific CDR (e.g., CDR-H1, CDR-H2, CDR-H3) should be understood to encompass the CDR(s) defined (or specified) by any of the foregoing schemes. For example, as defined by any of the foregoing schemes, where a particular CDR (e.g., CDR-H3) is stated to contain the amino acid sequence of the corresponding CDR in the amino acid sequence of a given V H or V L region, it should be understood that such a CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region. Schemes for identifying a particular CDR can be specified, such as CDRs defined by the Kabat, Chothia, AbM, or IMGT methods.

[0316] In some embodiments, the numbering of 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 as described in Lefranc, M.P. et al., Dev. Comp. Immunol., 2003, 27, 55-77.

[0317] In some embodiments, the anti-PD-L1 antibodies disclosed herein contain the CDRs of the antibody atezolizumab. See US 9,724,413. In some embodiments, the Kabat numbering scheme (Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242) is used to describe the CDRs of the antibody atezolizumab. This disclosure encompasses anti-PD-L1 antibodies or derivatives thereof that contain a heavy chain variable domain or a light chain variable domain, wherein the variable domain contains (a) a group of three CDRs, wherein the group of CDRs is from the monoclonal antibody atezolizumab, and (b) a group of four framework regions, wherein the group of framework regions is different from the group of framework regions in the monoclonal antibody atezolizumab, and wherein the anti-PD-L1 antibody or derivative thereof binds to PD-L1. In certain embodiments, the anti-PD-L1 antibody is atezolizumab.

[0318] The anti-PD-L1 antibodies disclosed herein can also be described or characterized in terms of their binding affinity for PD-L1 (e.g., human PD-L1). Preferred binding affinities include having a dissociation constant or Kd less than 5 × 10-2 M, 10 -2 M, 5x10 -3 M, 10 -3 M, 5x10 -4 M, 10 -4 M, 5x10 -5 M, 10 -5 M, 5x10 -6 M, 10 -6 M, 5x10 - 7 M, 10 -7 M, 5x10 -8 M, 10 -8 M, 5x10 -9 M, 10 -9 M, 5x10 -10 M, 10 -10 M, 5x10 -11 M, 10 -11 M, 5x10 -12 M, 10 -12 M, 5x10 -13 M, 10 -13 M, 5x10 -14 M, 10 -14 M, 5x10 -15 M, or 10 -15 Those of M.

[0319] The anti-PD-L1 antibody also comprises modified derivatives and constructs, i.e., by covalently linking any type of molecule to the antibody such that the covalent linkage does not prevent the antibody from binding to PD-L1. For example, and not by way of limitation, anti-PD-L1 antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cell ligand or other protein, etc. Any of a variety of chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis with tunicamycin, etc. Additionally, the derivative or construct can comprise one or more non-classical amino acids.

[0320] According to the present disclosure, the PD-1 / PD-L1 immune checkpoint inhibitor is an inhibitor of inhibitory checkpoint proteins but preferably not an inhibitor of stimulatory checkpoint proteins.

[0321] In a preferred embodiment, the PD-1 / PD-L1 immune checkpoint inhibitor is an antibody, particularly an antagonistic or blocking antibody, which disrupts or inhibits the inhibitory PD-1 immune checkpoint signaling pathway (the interaction of PD-1 with one or more of its ligands, such as PD-L1 and / or PD-L2). In a preferred embodiment, the PD-1 / PD-L1 immune checkpoint inhibitor is an antibody, particularly an antagonistic or blocking antibody, which disrupts or inhibits the interaction between PD-1 and PD-L1.

[0322] Checkpoint inhibitors (such as PD-1 / PD-L1 checkpoint inhibitors) can be administered in the form of a nucleic acid (such as a DNA or RNA molecule) encoding an immune checkpoint inhibitor (e.g., an inhibitory nucleic acid molecule or an antibody or a fragment thereof). For example, as described herein, an antibody can be delivered in an expression vector. The nucleic acid molecule can be delivered by itself, for example, in the form of a plasmid or an mRNA molecule, or complexed with a delivery vehicle, such as a liposome, a lipid complex, or a nucleic acid-lipid particle. Checkpoint inhibitors can also be administered by an oncolytic virus containing an expression cassette encoding the checkpoint inhibitor. Checkpoint inhibitors can also be administered by administering endogenous or allogeneic cells capable of expressing the checkpoint inhibitor, for example, in the form of a cell-based therapy.

[0323] In one embodiment, the cell-based therapy includes genetically modified cells. In one embodiment, the genetically modified cells express an immune checkpoint inhibitor, such as those described herein. In one embodiment, the genetically modified cells express an immune checkpoint inhibitor that is an inhibitory nucleic acid molecule, such as siRNA, shRNA, oligonucleotide, antisense DNA or RNA, aptamer, antibody or a fragment thereof, or a soluble immune checkpoint protein or fusion. The genetically modified cells can also express additional substances that enhance T cell function. Such substances are known in the art. Cell-based therapies for inhibiting immune checkpoint signaling are disclosed, for example, in WO 2018 / 222711, which is incorporated herein by reference in its entirety.

[0324] Preferably, a checkpoint inhibitor (e.g., a PD-1 / PD-L1 checkpoint inhibitor) is administered in a suitable amount, i.e., for example, in each dose and / or treatment cycle, the amount of the checkpoint inhibitor administered can reduce, inhibit, interfere with, or negatively regulate one or more checkpoint proteins in whole or in part, or can reduce, inhibit, interfere with, or negatively regulate the expression of one or more checkpoint proteins in whole or in part. Thus, a suitable amount of a checkpoint inhibitor according to the present disclosure can reduce, inhibit, interfere with, or negatively regulate one or more checkpoint proteins in whole or in part, or can reduce, inhibit, interfere with, or negatively regulate the expression of one or more checkpoint proteins in whole or in part. Accordingly, the checkpoint inhibitor preferably blocks inhibitory signals associated with immune checkpoints, which results in preventing or reversing immunosuppression and establishing or enhancing T cell immunity against cancer cells.

[0325] The amount of the checkpoint inhibitor administered in each dose and / or treatment cycle can be particularly within a certain range, wherein 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 checkpoint inhibitor binds to the checkpoint protein.

[0326] In some embodiments, for example, the amount of the PD-1 / PD-L1 checkpoint inhibitor administered in each dose and / or each treatment cycle is about 100 to 300 mg / day (e.g., about 120 to 280 mg / day, about 140 to 260 mg / day, about 160 to 240 mg / day, about 180 to 220 mg / day, or about 190 to 210 mg / day, such as 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, such as about 2.50 mg / kg body weight / day).

[0327] In some preferred embodiments, for example, the amount of pembrolizumab administered per dose and / or per treatment cycle is about 150 to 250 mg / day (such as 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, such as about 200 mg / day) or about 1.87 to 3.13 mg / kg body weight / day (such as 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, such as about 2.50 mg / kg body weight / day).

[0328] In some preferred embodiments, for example, the amount of pembrolizumab administered per dose and / or per treatment cycle is about 1020×10 -9 to 1710×10 -9 mol / day (such as about 1090×10 -9 to 1640×10 -9 mol / day, about 1160×10 -9 to 1570×10 -9 mol / day, about 1230×10 -9 to 1500×10 -9 mol / day, about 1295×10 -9 to 1435×10 - 9 mol / day or about 1330×10 -9 to 1400×10 -9 mol / day, such as about 1365×10 -9 mol / day) or about 12.7×10 -9 to 21.4×10 -9 mol / kg body weight / day (such as 13.6×10 -9 to 20.5×10 -9 mol / kg body weight / day, 14.5×10 -9 to 19.6×10 -9 mol / kg body weight / day, 15.3×10 -9 to 18.8×10 -9 mol / kg body weight / day, 16.1×10 -9 to 18.0×10 - 9 mol / kg body weight / day or 16.6×10 -9 to 17.5×10 -9mol / kg body weight / day, such as about 17.1 mol / kg body weight / day).

[0329] Checkpoint inhibitors (such as PD-1 / PD-L1 checkpoint inhibitors) can 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, such as parenterally, particularly intravenously.

[0330] Checkpoint inhibitors (such as PD-1 / PD-L1 checkpoint inhibitors) can be administered in the form of any suitable pharmaceutical composition as described herein. In a preferred embodiment, the checkpoint inhibitor is administered in the form of an infusion. Additional therapeutic agent

[0331] In addition to the binder and the PD-1 / PD-L1 checkpoint inhibitor, the treatment regimen according to the first aspect of the present disclosure further includes administering to the subject a chemotherapy combination comprising (a) a platinum-based chemotherapeutic agent and (b) 5-fluorouracil.

[0332] The platinum-based chemotherapeutic agent and 5-fluorouracil can 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 platinum-based chemotherapeutic agent to be used. In some preferred embodiments, the platinum-based chemotherapeutic agent and 5-fluorouracil are administered systemically, such as parenterally, particularly intravenously.

[0333] In one embodiment, the platinum-based compound is selected from platinum-based compounds commonly used in the treatment of tumors or cancers, particularly HNSCC, such as cisplatin, oxaliplatin, and carboplatin.

[0334] In one embodiment, the platinum-based chemotherapeutic 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.

[0335] In some preferred embodiments, for example, the amount of cisplatin administered per dose and / or per treatment cycle is about 50 to 150 mg / m 2 / day, such as about 60 to 140 mg / m 2 / day, about 70 to 130 mg / m 2 / day, about 80 to 120 mg / m 2 / day, about 90 to 110 mg / m 2 / day or about 95 to 105 mg / m 2 / day, such as about 100 mg / m 2 / day.

[0336] In some preferred embodiments, for example, the amount of carboplatin administered per dose and / or per treatment cycle is from AUC = about 4 to AUC = about 6, preferably AUC = about 5.

[0337] The carboplatin dose can be calculated using the following equation (Calvert 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 / minute) × (GFR + 25) where GFR is the glomerular filtration rate, which can be estimated by calculating the creatinine clearance (CrCl) using the following equation (Cockcroft-Gault equation; see also, Cockcroft DW, et al., Nephron. (1976); 16:31-41): CrCl (female; mL / minute) = 0.85 x CrCl (male)

[0338] In one embodiment, a minimum serum creatinine of 0.7 mg / dL is used (particularly if the subject has an abnormally low serum creatinine level, such as in the case of an elderly or cachectic subject).

[0339] In one embodiment, adjusted body weight is used (particularly if the subject is overweight or obese), where the adjusted body weight can be calculated as follows: Adjusted body weight (kg) = ideal body weight (IBW) + 0.4 × (total body weight (TBW) - IBW)

[0340] Additional information regarding the Calvert equation is known to the person skilled in the art (see, e.g., U.S. Food and Drug Administration. Carboplatin dosing. Available at: https: / / wayback.archive-it.org / 7993 / 20170113081146 / http: / / www.fda.gov / AboutFDA / CentersOffices / OfficeofMedicalProductsandTobacco / CDER / ucm228974.htm. Revised November 27, 2015; “Updated FAQ’s for dosing of carboplatin” [newsletter], Philadelphia, PA: Gynecologic Oncology Group Newsletter; Spring 2011. 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., BrJ 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; which is incorporated by reference in its entirety).

[0341] In one embodiment, the maximum dose of carboplatin is limited to the desired AUC to avoid potential toxicity due to over-dosing. The maximum dose can be based on an estimated GFR capped at 125 mL / minute (especially for subjects with normal renal function). Thus, in one embodiment, (i) the maximum carboplatin dose for AUC = 4 is 600 mg (= (125 + 25) × 4); (ii) for AUC = 5 is 750 mg (= (125 + 25) × 5); or (iii) for AUC = 6 is 900 mg (= (125 + 25) × 6).

[0342] In some embodiments, for example, the amount of carboplatin administered per dose and / or per treatment cycle is about 300 to 600 mg / day (for AUC = 4), about 350 to 750 mg / day (for AUC = 5), or about 90 to 900 mg / day (for AUC = 6). In some embodiments, for example, the amount of carboplatin administered per dose and / or per treatment cycle is about 3.7 to 7.5 mg / kg body weight / day (for AUC = 4), about 4.3 to 9.4 mg / kg body weight / day (for AUC = 5), or about 5.0 to 11.3 mg / kg body weight / day (for AUC = 6).

[0343] In some preferred embodiments, for example, the amount of 5-fluorouracil administered per dose and / or per treatment cycle is about 500 to 1500 mg / m 2 / day, such as about 600 to 1400 mg / m 2 / day, about 700 to 1300 mg / m 2 / day, about 800 to 1200 mg / m 2 / day, about 900 to 1100 mg / m 2 / day, or about 950 to 1050 mg / m 2 / day, such as about 1000 mg / m 2 / day. Subject and tumor or cancer to be treated

[0344] The subject to be treated according to the present disclosure is preferably a human subject.

[0345] The tumor or cancer to be treated is head and neck squamous cell carcinoma (HNSCC).

[0346] More than 600,000 cases of HNSCC are diagnosed worldwide each year. In 2020, approximately 65,630 new cases of oral, pharyngeal, and laryngeal cancers will occur in the United States during the same period, and an estimated 14,500 deaths (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 to 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 programmed death ligand 1 (PD-L1) combined positive score (CPS) ≥20 or ≥1) are recommended 1L regimens; however, the median overall survival (mOS) is less than 15 months (NCCN, 2021b). Therefore, HNSCC remains a highly unmet medical need, and there is further opportunity to improve outcomes with new treatment approaches.

[0347] In one embodiment, recurrent or metastatic HNSCC confirmed by histology or cytology is considered incurable by local treatment.

[0348] In one embodiment, the subject has not had prior anti-cancer treatment for recurrent or metastatic disease (e.g., systemic anti-cancer treatment administered in a recurrent or metastatic setting). Anti-cancer treatment completed more than 6 months prior to signing the consent form is permitted if the systemic treatment was given as part of a multimodal treatment for locally advanced disease.

[0349] In one embodiment, eligible primary tumor locations are the oropharynx, oral cavity, hypopharynx, and larynx.

[0350] In one embodiment, the subject does not have a primary tumor site in the nasopharynx (any histology).

[0351] In one embodiment, the subject has a tumor programmed death ligand 1 (PD-L1) combined positive score (CPS) (e.g., tumor PD-L1 immunohistochemistry (IHC) CPS) ≥1, preferably ≥1 and ≤19, which can be determined by local (preferably FDA-approved test) or central laboratory testing (central testing is required for the amplification phase). In one embodiment, the subject has a tumor PD-L1 CPS (e.g., tumor PD-L1 IHC CPS) ≥20.

[0352] 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.

[0353] In one embodiment, the subject (e.g., a human subject (e.g., a human subject having 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 treatment for recurrent or metastatic disease. In one embodiment, the subject (e.g., a human subject (e.g., a human subject having a PD-L1 CPS ≥ 1 and ≤ 19 or a PD-L1 CPS ≥ 20)) has not received prior treatment with any anti-cancer treatment or prior treatment with a checkpoint inhibitor and any anti-cancer treatment. Treatment regimen

[0354] The binding agent, the PD-1 / PD-L1 checkpoint inhibitor (especially pembrolizumab), and the chemotherapeutic agent (i.e., a platinum-based chemotherapeutic agent (such as cisplatin or carboplatin) and 5-fluorouracil) can be administered by any suitable means, such as intravenous administration, intra-arterial administration, subcutaneous administration, intradermal administration, intramuscular administration, intra-articular administration, or intratumoral administration.

[0355] 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.

[0356] In one embodiment, the binding agent is administered in 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 in at least 6 treatment cycles, such as in at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or until the end of treatment.

[0357] In one embodiment, the PD-1 / PD-L1 checkpoint inhibitor (especially pembrolizumab) is administered to a subject particularly by systemic administration. Preferably, the PD-1 / PD-L1 checkpoint inhibitor (especially pembrolizumab) is administered to the subject by intravenous injection or infusion.

[0358] In one embodiment, the PD-1 / PD-L1 checkpoint inhibitor (especially pembrolizumab) is administered in 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 (especially pembrolizumab) is administered in at least 6 treatment cycles, e.g., in at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or administered until the end of treatment.

[0359] In one embodiment, the chemotherapeutic agent (i.e., platinum-based chemotherapeutic agent (such as cisplatin or carboplatin) and 5-fluorouracil) is administered to a subject particularly by systemic administration. Preferably, the chemotherapeutic agent is administered to the subject by intravenous injection or infusion.

[0360] In one embodiment, the chemotherapeutic agent is administered in at least one treatment cycle (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 treatment cycles). For example, the platinum-based chemotherapeutic agent can be carboplatin and can be administered in at least one treatment cycle (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 treatment cycles). Alternatively, the platinum-based chemotherapeutic agent can be cisplatin and can be administered in at least one treatment cycle (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 treatment cycles).

[0361] In one embodiment, the binder, a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab), a platinum-based chemotherapeutic agent, and 5-fluorouracil are each administered to the subject by systemic administration (preferably, by intravenous injection or infusion). In one embodiment, the binder and the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered in at least one treatment cycle, preferably in 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, and the platinum-based chemotherapeutic agent and 5-fluorouracil are administered in at least the first treatment cycle, such as in at least the first and second treatment cycles, preferably only in the first 6 treatment cycles (which means that the administration of the platinum-based chemotherapeutic agent and 5-fluorouracil is stopped after completion of the first 6 treatment cycles).

[0362] In one embodiment, each treatment cycle is about two weeks (14 days), three weeks (21 days), or four weeks (28 days), preferably three weeks (21 days).

[0363] In a particular embodiment, each dose is administered or infused every two weeks (1Q2W), every three weeks (1Q3W), or every four weeks (1Q4W), preferably every three weeks (1Q3W).

[0364] In some embodiments, a dose of the binder is administered every three weeks (1Q3W). In some embodiments, a dose of the binder is administered on day 1 of each treatment cycle. In some embodiments, a dose of the binder is administered on day 1 of each treatment cycle, wherein each treatment cycle is three weeks. In some embodiments, a dose of the binder is administered in at least 6 treatment cycles, such as in at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or until the end of treatment, wherein the binder is administered on day 1 of each treatment cycle, and each treatment cycle is three weeks.

[0365] In some embodiments, a dose of a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered every three weeks (1Q3W). In some embodiments, a dose of a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered on day 1 of each treatment cycle. In some embodiments, a dose of a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered on day 1 of each treatment cycle, wherein each treatment cycle is three weeks. In some embodiments, a dose of a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered in at least 6 treatment cycles, such as in at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or administered until the end of treatment, wherein the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) is administered on day 1 of each treatment cycle and each treatment cycle is three weeks.

[0366] In some embodiments, a dose of a binder and a dose of a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered every three weeks (1Q3W). In some embodiments, a dose of a binder and a dose of a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered on day 1 of each treatment cycle. In some embodiments, a dose of a binder and a dose of a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered on day 1 of each treatment cycle, wherein each treatment cycle is three weeks. In some embodiments, a dose of a binder and a dose of a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered in at least 6 treatment cycles, such as in at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or administered until the end of treatment, wherein the binder and the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered on day 1 of each treatment cycle and each treatment cycle is three weeks.

[0367] In some embodiments, a chemotherapeutic combination (cisplatin and 5-fluorouracil or carboplatin and 5-fluorouracil) is administered at least every three weeks (1Q3W).

[0368] In some embodiments, a platinum-based chemotherapeutic agent is administered once every three weeks (1Q3W). In some embodiments, a platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles). In some embodiments, a platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles), wherein each treatment cycle is three weeks. In some embodiments, a platinum-based chemotherapeutic agent is administered on day 1 of at least the first and second treatment cycles (e.g., only the first 6 treatment cycles), wherein each treatment cycle is three weeks.

[0369] In some embodiments, 5-fluorouracil is administered at least once every three weeks (1Q3W), e.g., over four days in the first week. In some embodiments, 5-fluorouracil is administered on day 1 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles), e.g., on days 1, 2, 3, and 4 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles). In some embodiments, 5-fluorouracil is administered on day 1 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles), e.g., on days 1, 2, 3, and 4 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles), wherein each treatment cycle is three weeks. In some embodiments, 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 6 treatment cycles), wherein each treatment cycle is three weeks.

[0370] In some embodiments, a platinum-based chemotherapeutic agent is administered once every three weeks (1Q3W), and 5-fluorouracil is administered at least once every three weeks (1Q3W), for example, over four days in the first week. In some embodiments, a platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles), and 5-fluorouracil is administered at least on day 1 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles), for example, on days 1, 2, 3, and 4 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles). In some embodiments, a platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles), and 5-fluorouracil is administered at least on day 1 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles), for example, on days 1, 2, 3, and 4 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles), wherein each treatment cycle is three weeks. In some embodiments, a platinum-based chemotherapeutic agent is administered on day 1 of at least the first and second treatment cycles (e.g., only the first 6 treatment cycles), and 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 6 treatment cycles), wherein each treatment cycle is three weeks.

[0371] In some embodiments, a binder, a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab), and a platinum-based chemotherapeutic agent are each administered once every three weeks (1Q3W), and 5-fluorouracil (1Q3W) is administered at least once every three weeks (1Q3W), for example, over four days in the first week.

[0372] In some embodiments, a binder and a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are each administered on day 1 of each treatment cycle, a platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles), and 5-fluorouracil is administered at least on day 1 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles), for example, on days 1, 2, 3, and 4 of at least the first treatment cycle (e.g., at least the first and second treatment cycles, e.g., only the first 6 treatment cycles).

[0373] In some embodiments, a dose of the binder and a dose of a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered on day 1 of each treatment cycle, a dose of a platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (such as at least the first and second treatment cycles, such as only the first 6 treatment cycles), and a dose of 5-fluorouracil is administered on day 1 of at least the first treatment cycle (such as at least the first and second treatment cycles, such as only the first 6 treatment cycles), for example, on days 1, 2, 3, and 4 of at least the first treatment cycle (such as at least the first and second treatment cycles, such as only the first 6 treatment cycles), wherein each treatment cycle is three weeks.

[0374] In some embodiments, a dose of the binder and a dose of a PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered in at least 6 treatment cycles, such as in at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or until the end of treatment, wherein the binder and the PD-1 / PD-L1 checkpoint inhibitor (particularly pembrolizumab) are administered on day 1 of each treatment cycle; a dose of a platinum-based chemotherapeutic agent is administered on day 1 of at least the first and second treatment cycles (such as only the first 6 treatment cycles); and a dose of 5-fluorouracil is administered on days 1, 2, 3, and 4 of at least the first and second treatment cycles (such as only the first 6 treatment cycles), wherein each treatment cycle is three weeks.

[0375] In each of the above embodiments, the dose of the binder can be any dose of the binder as specified herein, such as 50 to 150 mg / day (such as about 60 to 140 mg / day, about 70 to 130 mg / day, about 80 to 120 mg / day, about 90 to 110 mg / day, or about 95 to 105 mg / day, such as about 100 mg / day) or about 0.62 to 1.88 mg / kg body weight / day (such as about 0.75 to 1.75 mg / kg body weight / day, about 0.87 to 1.63 mg / kg body weight / day, 1.00 to 1.50 mg / kg body weight / day, 1.12 to 1.38 mg / kg body weight / day, or 1.18 to 1.31 mg / kg body weight / day, such as about 1.25 mg / kg body weight / day).

[0376] In each of the above embodiments, the dose of the PD-1 / PD-L1 checkpoint inhibitor can be any dose of the PD-1 / PD-L1 checkpoint inhibitor as specified herein, such as about 100 to 300 mg / day (such as about 120 to 280 mg / day, about 140 to 260 mg / day, about 160 to 240 mg / day, about 180 to 220 mg / day or about 190 to 210 mg / day, such as about 200 mg / day) or about 1.25 to 3.75 mg / kg body weight / day (such as 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, such as about 2.50 mg / kg body weight / day). For example, in each of the above embodiments, the dose of pembrolizumab can be any dose of pembrolizumab as specified herein, such as 150 to 250 mg / day (such as 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, such as about 200 mg / day) or about 1.87 to 3.13 mg / kg body weight / day (such as 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, such as about 2.50 mg / kg body weight / day), or about 1020×10 -9 to 1710×10 -9 mol / day (such as about 1090×10 -9 to 1640×10 -9 mol / day, about 1160×10 -9 to 1570×10 -9 mol / day, about 1230×10 -9 to 1500×10 -9 mol / day, about 1295×10 -9 to 1435×10 -9 mol / day or about 1330×10 -9 to 1400×10 -9 mol / day, such as about 1365×10 -9 mol / day) or about 12.7×10 -9 to 21.4×10 -9 mol / kg body weight / day (such as 13.6×10 -9 to 20.5×10 - 9mol / kg body weight / day, 14.5×10 -9 to 19.6×10 -9 mol / kg body weight / day, 15.3×10 -9 to 18.8×10 -9 mol / kg body weight / day, 16.1×10 -9 to 18.0×10 -9 mol / kg body weight / day or 16.6×10 -9 to 17.5×10 -9 mol / kg body weight / day, such as about 17.1 mol / kg body weight / day).

[0377] In each of the above embodiments, the dose of the platinum-based chemotherapeutic agent can be any dose of carboplatin as specified herein or any dose of cisplatin as specified herein. For example, the dose of carboplatin can be AUC = about 4 to AUC = about 6, preferably AUC = about 5 or about 300 to 600 mg / day (for AUC = 4), about 350 to 750 mg / day (for AUC = 5) or about 400 to 900 mg / day (for AUC = 6) or about 3.7 to 7.5 mg / kg body weight / day (for AUC = 4), about 4.3 to 9.4 mg / kg body weight / day (for AUC = 5) or about 5.0 to 11.3 mg / kg body weight / day (for AUC = 6). Similarly, the dose of cisplatin can be about 50 to 150 mg / m 2 / day, such as about 60 to 140 mg / m 2 / day, about 70 to 130 mg / m 2 / day, about 80 to 120 mg / m 2 / day, about 90 to 110 mg / m 2 / day or about 95 to 105 mg / m 2 / day, such as about 100 mg / m 2 / day.

[0378] In each of the above embodiments, the dose of 5-fluorouracil can be any dose of 5-fluorouracil as specified herein, for example, about 500 to 1500 mg / m 2 / day, such as about 600 to 1400 mg / m 2 / day, about 700 to 1300 mg / m 2 / day, about 800 to 1200 mg / m 2 / day, about 900 to 1100 mg / m 2 / day or about 950 to 1050 mg / m 2 / day, such as about 1000 mg / m 2 / day.

[0379] Each dose can be administered or infused in at least 30 minutes, such as in at least 60 minutes, at least 90 minutes, at least 120 minutes, or at least 240 minutes.

[0380] The binder and the PD-1 / PD-L1 checkpoint inhibitor (especially pembrolizumab) can be administered simultaneously. In an alternative preferred embodiment, the binder and the PD-1 / PD-L1 checkpoint inhibitor (especially pembrolizumab) are administered separately. In some embodiments, the binder is administered before the PD-1 / PD-L1 checkpoint inhibitor.

[0381] In some embodiments, the chemotherapy combination (platinum-based chemotherapeutic agent and 5-fluorouracil) is administered after the binder.

[0382] The binder, the PD-1 / PD-L1 checkpoint inhibitor, the platinum-based chemotherapeutic agent, and 5-fluorouracil can be administered in any suitable form (e.g., administered naked by itself). However, preferably, the binder, the PD-1 / PD-L1 checkpoint inhibitor, the 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 binder and the PD-1 / PD-L1 checkpoint inhibitor are administered in the form of separate pharmaceutical compositions (i.e., one pharmaceutical composition for the binder and one pharmaceutical composition for the PD-1 / PD-L1 checkpoint inhibitor), preferably, the binder, the PD-1 / PD-L1 checkpoint inhibitor, the platinum-based chemotherapeutic agent, and 5-fluorouracil are administered in the form of separate pharmaceutical compositions (i.e., one pharmaceutical composition for the binder, one pharmaceutical composition for the PD-1 / PD-L1 checkpoint inhibitor, and at least one pharmaceutical composition for the chemotherapy combination, such as one pharmaceutical composition for the platinum-based chemotherapeutic agent and one pharmaceutical composition for 5-fluorouracil).

[0383] Combinations or pharmaceutical combinations may be formulated with a carrier, excipient, and / or diluent, and any other components suitable for pharmaceutical combinations, including known adjuvants, according to conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th ed., Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. The pharmaceutically acceptable carrier or diluent, and any known adjuvants and excipients, should be suitable for the binder and / or checkpoint inhibitor and / or one or more additional therapeutic agents (if present) and the chosen mode of administration. Suitability of the carrier and other components of the pharmaceutical combination is determined based on lack of a significant negative impact on the desired biological properties of the chosen compound or pharmaceutical combination (e.g., less than a significant effect on antigen binding (10% or less relative inhibition, 5% or less relative inhibition, etc.)).

[0384] Combinations, particularly at least one pharmaceutical combination of a pharmaceutical combination of a binder, a PD-1 / PD-L1 checkpoint inhibitor, and a chemotherapy combination, may include a diluent, filler, salt, buffer, detergent (e.g., a nonionic detergent such as Tween-20 or Tween-80), stabilizer (e.g., a sugar or protein-free amino acid), preservative, solubilizer, and / or other materials suitable for inclusion in a pharmaceutical combination.

[0385] 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).

[0386] A pharmaceutical carrier, excipient, or diluent may be selected according to the intended route of administration and standard pharmaceutical practice.

[0387] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, absorption delaying agents, etc., which are physiologically compatible with the active compounds, particularly the binders, PD-1 / PD-L1 checkpoint inhibitors, platinum-based chemotherapeutic agents, and 5-fluorouracil used herein.

[0388] Examples of suitable aqueous and non-aqueous carriers for (pharmaceutical) compositions include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as 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), carboxymethyl cellulose colloidal solutions, tragacanth, and injectable organic esters (such as ethyl oleate), and / or various buffers. Other carriers are well known in the pharmaceutical art.

[0389] 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 in pharmaceutical active substances is known in the art. The use of any conventional media or agent is contemplated in the (pharmaceutical) composition provided it is not incompatible with the active compound.

[0390] As used herein, the term "excipient" refers to a substance that may be present in the (pharmaceutical) compositions of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents or coloring agents.

[0391] The term "diluent" relates to diluents and / or thinning agents. In addition, the term "diluent" includes any one or more of fluids, liquids or solid suspending agents and / or mixed media. Some examples of suitable diluents include ethanol, glycerol and water.

[0392] (Pharmaceutical) compositions may also contain pharmaceutically acceptable antioxidants, such as (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, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal-chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0393] (Pharmaceutical) compositions may also contain isotonic agents in the composition, such as sugars, polyols, such as mannitol, sorbitol, glycerol or sodium chloride.

[0394] (The pharmaceutical) composition may also contain one or more excipients suitable for the selected route of administration, such as preservatives, wetting agents, emulsifying agents, dispersing agents, antiseptics or buffering agents, which may increase the shelf life or effectiveness of the composition. The compositions used herein can be prepared with carriers that will 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 in combination with waxes), or other substances 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.

[0395] "Pharmaceutically acceptable salts" include, for example, acid addition salts, which may be formed, for example, by using a pharmaceutically acceptable acid, such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. In addition, suitable pharmaceutically acceptable salts may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); ammonium (NH4 +);and salts formed with suitable organic ligands (e.g., quaternary ammonium and amine cations formed using counter anions such as halides, hydroxide, carboxylates, sulfates, phosphates, nitrates, alkyl sulfonates, and aryl sulfonates). Exemplary examples of pharmaceutically acceptable salts include, but are not limited to: acetate, adipate, alginate, arginine salt, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, dextrorotatory camphorsulfonate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluconate, glucuronate, glucuronate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinol, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, hydroxynaphthoate, iodide, isobutyrate, isothiocyanate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methyl sulfate, mucate, 2-naphthalenesulfonate, naphthalenesulfonate, nicotinate, nitrate, N-methylglucamine salt, oleate, oxalate, pamoate (pamoate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, phthalate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, suberate, succinate, tannate, tartrate, theoclate, toluenesulfonate, triethiodide, undecanoate, valerate, etc. (see, e.g., S.M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 66, pp. 1-19 (1977)). Non-pharmaceutically acceptable salts can be used to prepare pharmaceutically acceptable salts and are included in the present disclosure.

[0396] In one embodiment, the binder, PD-1 / PD-L1 checkpoint inhibitor, platinum-based chemotherapeutic agent, and 5-fluorouracil used herein can 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 active pharmaceutical substances is known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Other active or therapeutic compounds can also be incorporated into the compositions.

[0397] Injectable pharmaceutical compositions generally must be sterile and stable under the conditions of preparation and storage. The compositions can be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters, such as ethyl oleate. For example, fluidity can be maintained by using coatings (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants. In many cases, it will be preferable to include in the composition isotonic agents, such as sugars, polyols (such as glycerol, mannitol, sorbitol) or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition agents that delay absorption, such as monostearates and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into a suitable solvent, such as one or a combination of the ingredients listed above as appropriate, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients, such as those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of the preparation methods are vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0398] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into a suitable solvent, such as one or a combination of the ingredients listed above as appropriate, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients, such as those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of the preparation methods are vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0399] In a second aspect, the present disclosure provides methods for reducing or arresting the progression of HNSCC or treating HNSCC in a subject, the methods comprising administering to the subject (i) a binder, (ii) a PD-1 / PD-L1 checkpoint inhibitor (in particular pembrolizumab), and (iii) a chemotherapy combination comprising a platinum-based chemotherapeutic agent (in particular cisplatin or carboplatin) and 5-fluorouracil, wherein the binder comprises a first binding region that binds to CD40 and a second binding region that binds to CD137. Embodiments disclosed herein with respect to the first aspect (in particular with respect to the binder, the PD-1 / PD-L1 checkpoint inhibitor, the platinum-based chemotherapeutic agent, 5-fluorouracil, the doses to be administered thereof, the treatment regimens thereof, and the subject) are also applicable to the methods of the second aspect.

[0400] In another aspect, the present disclosure provides a kit comprising (i) a binder 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 (in particular pembrolizumab), (iii) a platinum-based chemotherapeutic agent (in particular cisplatin and / or carboplatin), and (iv) 5-fluorouracil, and, for example, kits for use in methods for reducing or arresting the progression of HNSCC or treating HNSCC in a subject. Embodiments disclosed herein with respect to the first aspect (in particular with respect to the binder, the PD-1 / PD-L1 checkpoint inhibitor, the platinum-based chemotherapeutic agent, 5-fluorouracil, the doses to be administered thereof, and the treatment regimens thereof) are also applicable to the kits of the other aspect. In one embodiment, the kit comprises at least three containers, wherein one container comprises the binder (either by itself or in the form of a (pharmaceutical) composition), a second container comprises the PD-1 / PD-L1 checkpoint inhibitor (either by itself or in the form of a (pharmaceutical) composition), and a third container comprises the platinum-based chemotherapeutic agent and / or 5-fluorouracil (either by itself or in the form of a (pharmaceutical) composition). Preferably, the kit comprises at least four containers, wherein one container comprises the binder (either by itself or in the form of a (pharmaceutical) composition), a second container comprises the PD-1 / PD-L1 checkpoint inhibitor (either by itself or in the form of a (pharmaceutical) composition), a third container comprises the platinum-based chemotherapeutic agent, such as carboplatin and / or cisplatin (either by itself or in the form of one or two (pharmaceutical) compositions), and a fourth container comprises 5-fluorouracil (either by itself or in the form of a (pharmaceutical) composition)

[0401] In another aspect, the present disclosure provides a pharmaceutical kit for use in a method of reducing or preventing the progression of HNSCC or treating HNSCC in a subject. The embodiments disclosed herein with respect to the first aspect (particularly with respect to the binder, the PD-1 / PD-L1 checkpoint inhibitor, the platinum-based chemotherapeutic agent, 5-fluorouracil, the dosage to be administered thereof, its treatment regimen, and the subject) and / or the second aspect are also applicable to the pharmaceutical kit used in another aspect.

[0402] Citations of documents and studies herein are not intended to admit that any of the foregoing is relevant prior art. All statements regarding the content of these documents are based on information available to the applicant and do not constitute any admission as to the correctness of the content of these documents.

[0403] This description (including the following examples) is provided to enable a person of ordinary skill in the art to make and use various embodiments. The description of specific devices, techniques, and applications is provided only as an example. Various modifications to the examples described herein will be apparent to a person of ordinary skill 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. Thus, the various embodiments are not intended to be limited to the examples described and illustrated herein, but are consistent with the scope of the claims. Embodiments listed item by item

[0404] 1. A binder for use in a method of reducing or preventing the progression of head and neck squamous cell carcinoma (HNSCC) or treating HNSCC in a subject, the method comprising administering to the subject a binder, pembrolizumab, and a chemotherapeutic combination comprising a platinum-based chemotherapeutic agent and 5-fluorouracil, wherein the binder comprises a first binding region that binds to CD40 and a second binding region that binds to CD137.

[0405] 2. The binder for the use according to item 1, wherein the binder, pembrolizumab, and the chemotherapeutic combination are administered in at least one treatment cycle, and each treatment cycle is three weeks (21 days).

[0406] 2a. A binder for the application according to any one of the preceding items, wherein the binder and pembrolizumab are administered in at least 2 treatment cycles, for example, in 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.

[0407] 2b. A binder for the application according to any one of the preceding items, wherein the binder and pembrolizumab are administered in at least 6 treatment cycles, for example, in at least 12, at least 18, at least 24, at least 30 or at least 35 treatment cycles, or administered until the end of treatment.

[0408] 3. A binder for the application according to any one of the preceding items, wherein one dose of the binder and one dose of pembrolizumab are administered every three weeks (1Q3W).

[0409] 4. A binder for the application according to any one of the preceding items, wherein one dose of the binder and one dose of pembrolizumab are administered on the first day of each treatment cycle.

[0410] 5. A binder for the application according to any one of the preceding items, wherein the binder is administered before pembrolizumab.

[0411] 5a. A binder for the application according to any one of items 1 to 4, wherein the binder is administered simultaneously with the administration of pembrolizumab.

[0412] 5b. A binder for the application according to any one of items 1 to 4, wherein the binder is administered after the administration of pembrolizumab.

[0413] 6. A binder for the application according to any one of the preceding items, wherein one dose of the chemotherapy combination is administered at least every three weeks (1Q3W) for at least the first treatment cycle.

[0414] 6a. A binder for the application according to item 6, wherein one dose of the chemotherapy combination is administered at least every three weeks (1Q3W) for at least the first treatment cycle and the second treatment cycle, preferably for the first 6 treatment cycles.

[0415] 7. The binder for the application according to any one of the foregoing items, wherein one dose of a platinum-based chemotherapeutic agent is administered every three weeks (1Q3W) for at least the first treatment cycle, and one dose of 5-fluorouracil is administered at least every three weeks (1Q3W), for example, administered over four days in the first week, for at least the first treatment cycle.

[0416] 7a. The binder for the application according to item 7, wherein one dose of a platinum-based chemotherapeutic agent is administered every three weeks (1Q3W) for at least the first treatment cycle and the second treatment cycle, preferably for the first 6 treatment cycles, and one dose of 5-fluorouracil is administered at least every three weeks (1Q3W), for example, administered over four days in the first week, for at least the first treatment cycle and the second treatment cycle, preferably for the first 6 treatment cycles.

[0417] 8. The binder for the application according to any one of the foregoing items, wherein one dose of a platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle, and one dose of 5-fluorouracil is administered at least on day 1 of at least the first treatment cycle, for example, administered on days 1, 2, 3, and 4 of at least the first treatment cycle.

[0418] 8a. The binder for the application according to any one of the foregoing items, wherein the chemotherapeutic combination is administered after the administration of the binder and / or after the administration of pembrolizumab.

[0419] 9. The binder for the application according to any one of the foregoing items, wherein the objective response rate (ORR) is increased compared to the standard of care, for example, compared to the administration regimen of only pembrolizumab and the chemotherapeutic combination.

[0420] 10. The binder for the application according to any one of the foregoing items, wherein the ORR is increased to at least 40%, preferably at least 50%, more preferably at least 60%, for example, at least 70%, at least 80%, at least 90% or at least 95%.

[0421] 11. The binder for the application according to any one of the foregoing items, wherein the disease control rate (DCR) is increased compared to the standard of care, for example, compared to the administration regimen of only pembrolizumab and the chemotherapeutic combination.

[0422] 12. The binder for the application according to any one of the foregoing items, wherein the DCR is increased to at least 40%, preferably at least 50%, more preferably at least 60%, for example, at least 70%, at least 80%, at least 90% or at least 95%.

[0423] 13. The binder for the application according to any one of the foregoing items, wherein each of the binder, pembrolizumab, and the chemotherapeutic combination is administered at a dose that increases the ORR compared to the standard of care (for example, compared to the administration regimen of only pembrolizumab and the chemotherapeutic combination).

[0424] 14. The binder for the application according to any one of the preceding items, wherein each of the binder, pembrolizumab, and the chemotherapy combination is administered at a dose that increases the ORR 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%.

[0425] 15. The binder for the application according to any one of the preceding items, wherein each of the binder, pembrolizumab, and the chemotherapy combination is administered at a dose that increases the DCR compared to the standard of care (e.g., compared to the administration regimen of the pembrolizumab and chemotherapy combination alone).

[0426] 16. The binder for the application according to any one of the preceding items, wherein each of the binder, pembrolizumab, and the chemotherapy combination is 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%.

[0427] 17. The binder for the application according to any one of the preceding items, wherein the platinum-based chemotherapeutic agent is carboplatin or cisplatin.

[0428] 18. The binder for the application according to any one of the preceding items, wherein the chemotherapy combination is cisplatin and 5-fluorouracil.

[0429] 19. The binder for the application according to any one of the preceding items, wherein the chemotherapy combination is carboplatin and 5-fluorouracil.

[0430] 20. The binder for the application according to any one of the preceding items, wherein the binder is administered at a dose of about 50 to 150 mg / day, preferably about 100 mg / day.

[0431] 21. The binder for the application 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.

[0432] 22. The binder for the application according to any one of the preceding items, wherein when the platinum-based chemotherapeutic agent is carboplatin, it is administered at a dose of AUC = about 4 to 6, preferably AUC = about 5, or when the platinum-based chemotherapeutic agent is cisplatin, it is administered at a dose of about 50 to 150 mg / m 2 / day, preferably about 100 mg / m 2 / day.

[0433] 23. The binder for the application according to any one of the preceding items, wherein 5-fluorouracil is administered at a dose of about 500 to 1500 mg / m 2 / day, preferably about 1000 mg / m 2administered at a dose of / per day.

[0434] 24. The binder for the application according to any one of the preceding items, wherein the binder is administered at a dose of about 100 mg / day, pembrolizumab is administered at a dose of about 200 mg / day, and when the platinum-based chemotherapeutic agent is carboplatin, it is administered at a dose of AUC = about 5, or when the platinum-based chemotherapeutic agent is cisplatin, it is administered at a dose of about 100 mg / m 2 / per day, and 5-fluorouracil is administered at a dose of about 1000 mg / m 2 / per day.

[0435] 25. The binder for the application according to any one of the preceding items, wherein: (i) About 100 mg / day of the binder and about 200 mg / day of pembrolizumab are administered on the first day of each treatment cycle, and each treatment cycle is three weeks (1Q3W); (ii) The platinum-based chemotherapeutic agent is administered on the first day of at least the first and second treatment cycles, wherein when the platinum-based chemotherapeutic agent is carboplatin, it is administered at a dose of AUC = about 5, or when the platinum-based chemotherapeutic agent is cisplatin, it is administered at a dose of about 100 mg / m 2 / per day; and (iii) About 1000 mg / m 2 / per day of 5-fluorouracil is administered on the first, second, third, and fourth days of at least the first and second treatment cycles.

[0436] 26. The binder for the application according to any one of the preceding items, wherein the binder, pembrolizumab, the platinum-based chemotherapeutic agent, and 5-fluorouracil are administered for 6 treatment cycles, and then only the binder and pembrolizumab are further administered for at least one treatment cycle.

[0437] 27. The binder for the application according to any one of the preceding items, wherein any one or all of the binder, pembrolizumab, and the chemotherapy combination are administered systemically, preferably intravenously.

[0438] 28. The binder for the application according to any one of the preceding items, wherein the subject is a human subject.

[0439] 29. The binder for the application according to any one of the preceding items, wherein the subject has not received previous treatment with checkpoint inhibitors and / or anticancer therapy for recurrent or metastatic disease.

[0440] 30. The binder for the application according to any one of the preceding items, wherein the subject has not received previous treatment with any anticancer therapy or with checkpoint inhibitors and any anticancer therapy.

[0441] 31. The binder for the application according to any one of the preceding items, wherein the combined positive score (CPS) of PD-L1 of the subject is ≥1, for example, PD-L1 CPS ≥1 and ≤19 or PD-L1 CPS ≥20.

[0442] 32. The binder for the application according to any one of the preceding items, wherein CD40 is human CD40, particularly human CD40 comprising the sequence shown in SEQ ID NO: 36, and / or CD137 is human CD137, particularly human CD137 comprising the sequence shown in SEQ ID NO: 38.

[0443] 33. The binder for the application according to any one of the preceding items, wherein: a) The first binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 7 or 9, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 8 or 10; And b) The second antigen-binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 17 or 19, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 18 or 20.

[0444] 34. The binder for the application according to any one of the preceding items, wherein: a) The first binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, 2 and 3 respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 4, 5 and 6 respectively; And b) The second antigen-binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 11, 12 and 13 respectively, and the light chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 14, 15 and 16 respectively.

[0445] 35. The binder for the application according to any one of the preceding items, wherein: a) The first binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity with SEQ ID NO: 7 or 9, and the light chain variable region (VL) comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity with SEQ ID NO: 8 or 10; b) The second binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity with SEQ ID NO: 17 or 19, and the light chain variable region (VL) comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity with SEQ ID NO: 18 or 20.

[0446] 36. The binder for the application according to any one of the preceding items, wherein: a) The first binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 7 or 9, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 8 or 10; And b) The second binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 17 or 19, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 18 or 20.

[0447] 37. The binder for the application according to any one of the preceding items, wherein: a) The first binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 9, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 10; And b) The second binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 20.

[0448] 38. The binder for the application according to any one of the foregoing items, wherein the binder is a multispecific antibody, such as a bispecific antibody.

[0449] 39. The binder for the application according to any one of the foregoing items, wherein the binder is in the form of a full-length antibody or an antibody fragment.

[0450] 40. The binder for the application 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).

[0451] 41. The binder for the application according to item 40, wherein the complementarity-determining regions and the framework regions are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0452] 42. The binder for the application according to any one of items 33 to 41, comprising: i) a polypeptide comprising, consisting of, or consisting essentially of: the first heavy-chain variable region (VH) and the first heavy-chain constant region (CH), and ii) a polypeptide comprising, consisting of, or consisting essentially of: the second heavy-chain variable region (VH) and the second heavy-chain constant region (CH).

[0453] 43. The binder for the application according to any one of items 33 to 42, comprising: i) a polypeptide containing the first light-chain variable region (VL) and further containing the first light-chain constant region (CL), and ii) a polypeptide containing the second light-chain variable region (VL) and further containing the second light-chain constant region (CL).

[0454] 44. The binder for the application according to any one of items 33 to 43, wherein the binder is an antibody comprising a first binding arm and a second binding arm, wherein: The first binding arm comprises: i) a polypeptide comprising, consisting of, or consisting essentially of: the first heavy-chain variable region (VH) and the first heavy-chain constant region (CH), and ii) a polypeptide comprising, consisting of, or consisting essentially of: the first light-chain variable region (VL) and the first light-chain constant region (CL); And the second binding arm comprises: iii) a polypeptide comprising, consisting of, or consisting essentially of: the second heavy-chain variable region (VH) and the second heavy-chain constant region (CH), and iv) a polypeptide comprising, consisting of, or consisting essentially of: the second light-chain variable region (VL) and the second light-chain constant region (CL).

[0455] 45. The binder for the application according to any one of the preceding items, comprising: i) a first heavy chain and a first light chain containing the antigen-binding region capable of binding to CD40, and ii) a second heavy chain and a second light chain containing the antigen-binding region capable of binding to CD137.

[0456] 46. The binder for the application according to any one of the preceding items, wherein the binder comprises: i) a first heavy chain and a first light chain containing the antigen-binding region capable of binding to CD40, the first heavy chain comprising a first heavy chain constant region, and the first light chain comprising a first light chain constant region; and ii) a second heavy chain and a second light chain containing the antigen-binding region capable of binding to CD137, the second heavy chain comprising a second heavy chain constant region, and the second light chain comprising a second light chain constant region.

[0457] 47. The binder for the application according to any one of items 42 to 46, wherein each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) comprises one or more of a heavy chain constant 1 (CH1) region, a hinge region, a heavy chain constant 2 (CH2) region, and a heavy chain constant 3 (CH3) region, preferably comprising at least a hinge region, a CH2 region, and a CH3 region.

[0458] 48. The binder for the application according to any one of items 42 to 47, wherein each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) comprises a CH3 region, and wherein the two CH3 regions comprise an asymmetric mutation.

[0459] 49. The binder for the application according to any one of items 42 to 48, wherein in the first heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering has been replaced, and in the second heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering has been replaced, and wherein the first heavy chain and the second heavy chain are not replaced at the same position.

[0460] 50. A binder for the application according to item 49, wherein (i) in the first heavy chain constant region (CH), the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L, and in the second heavy chain constant region (CH), the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R, or (ii) in the first heavy chain, the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R, and in the second heavy chain, the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L.

[0461] 51. A binder for the application according to any one of the preceding items, wherein the degree of Fc-mediated effector function induced by the binder is lower compared to another antibody comprising the same first antigen-binding region and second antigen-binding region and two heavy chain constant regions (CH) comprising the human IgG1 hinge region, CH2 region, and CH3 region.

[0462] 52. A binder for the application according to item 51, wherein the first heavy chain constant region (CH) and the second heavy chain constant region (CH) are modif...

Claims

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

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

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

4. 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 every three weeks (1Q3W).

5. 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 binder for use according to any one of the preceding claims, wherein the binder is administered before the inhibitor of the checkpoint PD-1 / PD-L1 axis.

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

8. The binder for use according to any one of the preceding claims, wherein one dose of the platinum-based chemotherapeutic agent is administered every three weeks (1Q3W) for at least the first treatment cycle, and one dose of 5-fluorouracil is administered at least every three weeks (1Q3W), for example, over four days in the first week, for at least the first treatment cycle.

9. The binder 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 at least on day 1 of at least the first treatment cycle, for example, on days 1, 2, 3, and 4 of at least the first treatment cycle.

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

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

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

13. The binder for the application according to any one of the preceding claims, wherein the DCR is increased 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%.

14. The binder for the application according to any one of the preceding claims, wherein each of the binder, the inhibitor of the checkpoint PD-1 / PD-L1 axis, and the chemotherapy combination is administered at a dose that increases the ORR compared to the standard of care, such as compared to the administration regimen of only the inhibitor of the checkpoint PD-1 / PD-L1 axis, such as pembrolizumab, and the chemotherapy combination.

15. The binder for the application according to any one of the preceding claims, wherein each of the binder, the inhibitor of the checkpoint PD-1 / PD-L1 axis, and the chemotherapy combination is administered at a dose that increases the ORR 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%.

16. The binder for the application according to any one of the preceding claims, wherein each of the binder, the inhibitor of the checkpoint PD-1 / PD-L1 axis, and the chemotherapy combination is administered at a dose that increases the DCR compared to the standard of care, such as compared to the administration regimen of only the inhibitor of the checkpoint PD-1 / PD-L1 axis, such as pembrolizumab, and the chemotherapy combination.

17. The binder for the application according to any one of the preceding claims, wherein each of the binder, the inhibitor of the checkpoint PD-1 / PD-L1 axis, and the chemotherapy combination is 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%.

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

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

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

21. The binder for the application according to any one of the preceding claims, wherein the binder is administered at a dose of about 50 to 150 mg / day, preferably about 100 mg / day.

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

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

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

25. The binder for the application according to any one of the preceding claims, wherein the binder is administered at a dose of about 100 mg / day, the inhibitor of the checkpoint PD-1 / PD-L1 axis is administered at a dose of about 200 mg / day, when the platinum-based chemotherapeutic agent is carboplatin, it is administered at a dose of AUC = about 5, or when the platinum-based chemotherapeutic agent is cisplatin, it 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 / day.

26. The binder for the application according to any one of the preceding claims, wherein: (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, and each treatment cycle is three weeks (1Q3W); (ii) Administering the platinum-based chemotherapeutic agent on day 1 of at least a first treatment cycle and a second treatment cycle, wherein when the platinum-based chemotherapeutic agent is carboplatin, it is administered at a dose of AUC = about 5, or when the platinum-based chemotherapeutic agent is cisplatin, it is administered at a dose of about 100 mg / m 2 / day; and (iii) Administer approximately 1000 mg / m 2 / day of 5-fluorouracil on days 1, 2, 3, and 4 of at least the first and second treatment cycles.

27. The binder for the 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 chemotherapeutic agent and 5-fluorouracil are administered for 6 treatment cycles, and then only the binder and the inhibitor of the checkpoint PD-1 / PD-L1 axis are further administered for at least one treatment cycle.

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

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

30. The binder for the use according to any one of the preceding claims, wherein the subject has not received prior treatment with checkpoint inhibitors and / or anticancer treatment for recurrent or metastatic disease.

31. The binder for the use according to any one of the preceding claims, wherein the subject has not received prior treatment with any anticancer treatment or with checkpoint inhibitors and any anticancer treatment.

32. The binder for the use according to any one of the preceding claims, wherein the combined positive score (CPS) of PD-L1 of the subject is ≥1, for example, PD-L1 CPS ≥1 and ≤19 or PD-L1 CPS ≥20.

33. The binder for the use according to any one of the preceding claims, wherein CD40 is human CD40, especially human CD40 comprising the sequence shown in SEQ ID NO: 36, and / or CD137 is human CD137, especially human CD137 comprising the sequence shown in SEQ ID NO:

38.

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

35. The binder for the use according to any one of the preceding claims, wherein: a) The first binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprising the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 1, 2 and 3 respectively, and the light chain variable region (VL) comprising the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NOs: 4, 5 and 6 respectively; and b) The second antigen-binding region comprises a heavy-chain variable region (VH) and a light-chain variable region (VL), the heavy-chain variable region (VH) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 11, 12 and 13 respectively, and the light-chain variable region (VL) comprises the CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 14, 15 and 16 respectively.

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

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

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

20.

39. The binder for the application according to any one of the preceding claims, wherein the binder is a multispecific antibody, such as a bispecific antibody.

40. The binder for the application according to any one of the preceding claims, wherein the binder is in the form of a full-length antibody or an antibody fragment.

41. The binder for the application 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. The binder for the application according to claim 41, wherein the complementarity-determining regions and the framework regions are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

43. The binder for the application according to any one of claims 34 to 42, comprising: i) a polypeptide comprising, consisting of, or consisting essentially of: the first heavy-chain variable region (VH) and the first heavy-chain constant region (CH), and ii) a polypeptide comprising, consisting of, or consisting essentially of: the second heavy-chain variable region (VH) and the second heavy-chain constant region (CH).

44. The binder for the application according to any one of claims 34 to 43, comprising: i) a polypeptide containing the first light-chain variable region (VL) and also containing the first light-chain constant region (CL), and ii) a polypeptide containing the second light-chain variable region (VL) and also containing the second light-chain constant region (CL).

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

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

47. The binder for the application according to any one of the preceding claims, wherein the binder comprises: i) a first heavy chain and a first light chain containing the antigen-binding region capable of binding to CD40, the first heavy chain comprising a first heavy-chain constant region, and the first light chain comprising a first light-chain constant region; and ii) a second heavy chain and a second light chain containing the antigen-binding region capable of binding to CD137, the second heavy chain comprising a second heavy-chain constant region, and the second light chain comprising a second light-chain constant region.

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

49. A binder for the application according to any one of claims 43 to 48, wherein each of the first heavy chain constant region (CH) and the second heavy chain constant region (CH) comprises a CH3 region, and wherein the two CH3 regions comprise an asymmetric mutation.

50. A binder for the application according to any one of claims 43 to 49, wherein in the first heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering has been replaced, and in the second heavy chain constant region (CH), at least one amino acid at a position corresponding to a position selected from T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering has been replaced, and wherein the first heavy chain and the second heavy chain are not replaced at the same position.

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

52. A binder for the application according to any one of the preceding claims, wherein the binder induces a lower degree of Fc-mediated effector function compared to another antibody comprising the same first antigen-binding region and second antigen-binding region and two heavy chain constant regions (CH) comprising a human IgG1 hinge region, a CH2 region, and a CH3 region.

53. The binder for the application according to claim 52, wherein the first heavy chain constant region (CH) and the second heavy chain constant region (CH) are modified such that the antibody induces a lower degree of Fc-mediated effector function compared to an antibody that is identical except for comprising unmodified first and second heavy chain constant regions (CH).

54. The binder for the application according to claim 53, wherein each of the unmodified first heavy chain constant region (CH) and the second heavy chain constant region (CH) comprises the amino acid sequence shown in SEQ ID NO: 21 or 29.

55. The binder for the application according to 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 Fcγ receptor cross-linking.

56. The binder for the application according to claim 55, wherein the Fc-mediated effector function is measured by binding to C1q.

57. The binder for the application according to any one of claims 52 to 56, wherein the first heavy chain constant region and the second heavy chain constant region have been modified such that, compared to the wild-type antibody, the binding of C1q to the antibody is reduced, preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or 100%, wherein C1q binding is preferably determined by ELISA.

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

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

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

61. The binder for the application according to any one of claims 58 to 60, wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E, respectively, in both the first heavy chain constant region and the second heavy chain constant region, and wherein (i) the position corresponding to position 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 position K409 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is R, or (ii) the position corresponding to position 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 position F405 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is L. A binder for the use according to any one of claims 58 to 61, wherein the positions corresponding to positions L234, L235 and D265 in the human IgG1 heavy chain according to EU numbering in both the first heavy chain constant region and the second heavy chain constant region are F, E and A respectively, and wherein (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. A binder for the use according to any one of claims 43 to 62, wherein the constant region of the first heavy chain and / or the second heavy chain comprises an amino acid sequence selected from the following, or consists essentially of an amino acid sequence selected from the following, or consists of an amino acid sequence selected from the following: a) the sequence shown in SEQ ID NO: 21 or 29 [IgG1-FC]; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids have been deleted starting from the N-terminus or the C-terminus of the sequence defined in a); and c) a sequence having at most 10 substitutions, such as 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). A binder for the use according to any one of claims 43 to 62, wherein the constant region of the first heavy chain or the second heavy chain, for example the constant region of the second heavy chain, comprises an amino acid sequence selected from the following, or consists essentially of an amino acid sequence selected from the following, or consists of an amino acid sequence selected from the following: a) the sequence shown in SEQ ID NO: 22 or 30 [IgG1-F405L]; b) a subsequence of the sequence in a), for example a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids have been deleted starting from the N-terminus or the C-terminus of the sequence defined in a); and c) a sequence having at most 9 substitutions, such as 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). A binder for the use according to any one of claims 43 to 62, wherein the constant region of the first heavy chain or the second heavy chain, for example the constant region of the first heavy chain, comprises an amino acid sequence selected from the following, or consists essentially of an amino acid sequence selected from the following, or consists of an amino acid sequence selected from the following: a) the sequence shown in SEQ ID NO: 23 or 31 [IgG1-F409R]; b) A subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids have been deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) A sequence having at most 10 substitutions, such as 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).

66. The binder for the application according to any one of claims 43 to 62, wherein the constant region of the first heavy chain and / or the second heavy chain comprises an amino acid sequence selected from the following, or consists essentially of an amino acid sequence selected from the following, or consists of an amino acid sequence selected from the following: a) The sequence shown in SEQ ID NO: 24 or 32 [IgG1-Fc_FEA]; b) A subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids have been deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) A sequence having at most 7 substitutions, such as 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).

67. The binder for the application according to any one of claims 43 to 66, wherein the constant region of the first heavy chain and / or the second heavy chain, for example, the constant region of the second heavy chain, comprises an amino acid sequence selected from the following, or consists essentially of an amino acid sequence selected from the following, or consists of an amino acid sequence selected from the following: a) The sequence shown in SEQ ID NO: 25 or 33 [IgG1-Fc_FEAL]; b) A subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids have been deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) A sequence having at most 6 substitutions, such as at most 5 substitutions, 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).

68. The binder for the application according to any one of claims 43 to 67, wherein the constant region of the first heavy chain and / or the second heavy chain, for example, the constant region of the first heavy chain, comprises an amino acid sequence selected from the following, or consists essentially of an amino acid sequence selected from the following, or consists of an amino acid sequence selected from the following: a) The sequence shown in SEQ ID NO: 26 or 34 [IgG1-Fc_FEAR]; b) A subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids have been deleted starting from the N-terminus or C-terminus of the sequence defined in a); and c) A sequence having at most 6 substitutions, such as at most 5 substitutions, 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).

69. The binder for the application according to any one of the preceding claims, wherein the binder comprises a kappa (κ) light chain constant region.

70. The binder for the application according to any one of the preceding claims, wherein the binder comprises a lambda (λ) light chain constant region.

71. The binder for the application 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. The binder for the application 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. The binder for the application 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. The binder for the application according to any one of claims 69 to 73, wherein the kappa (κ) light chain comprises an amino acid sequence selected from: a) The sequence shown in SEQ ID NO:27; b) A subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids have been 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 substitutions, at most 7 substitutions, at most 6 substitutions, 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).

75. The binder for the application according to any one of claims 70 to 74, wherein the lambda (λ) light chain comprises an amino acid sequence selected from: a) The sequence shown in SEQ ID NO:28; b) A subsequence of the sequence in a), for example, a subsequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids have been 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 substitutions, at most 7 substitutions, at most 6 substitutions, 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).

76. The binder for the application according to any one of the preceding claims, wherein the binder is an isotype selected from IgG1, IgG2, IgG3 and IgG4.

77. The binder for the application according to any one of the preceding claims, wherein the binder is a full-length IgG1 antibody.

78. The binder for the application according to any one of the preceding claims, wherein the binder is an antibody of the IgG1m(f) allotype.

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

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

81. The method of claim 79 or 80, wherein the binder and / or the object and / or the administration regimen are defined according to any one of claims 1 to 78.

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