WO2024115725A1

WO2024115725A1 - Multispecific antibody against cd40 and cd137 in combination therapy with anti-pd1 ab and chemotherapy

Info

Publication number: WO2024115725A1
Application number: PCT/EP2023/083904
Authority: WO
Inventors: Ugur Sahin; Michelle NIEWOOD; Homer ADAMS III; Brandon Higgs; Tahamtan Ahmadi
Original assignee: Biontech SE; Genmab AS
Current assignee: Biontech SE; Genmab AS
Priority date: 2022-12-01
Filing date: 2023-12-01
Publication date: 2024-06-06
Anticipated expiration: 2025-06-01
Also published as: CN120302979A; AU2023401158A1; IL320806A; EP4626446A1

Abstract

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

Description

MULTISPECIFIC ANTIBODY AGAINST CD40 AND CD137 IN COMBINATION THERAPY WITH ANTI-PD1 AB AND CHEMOTHERAPY

Technical Field

The present invention relates to combination therapy using a binding agent that binds to human CD40 and to human CD 137 in combination with a checkpoint inhibitor which is an inhibitor of the PD-l/PD- L1 axis (in particular pembrolizumab) and chemotherapy to reduce or prevent progression of head and neck squamous cell carcinoma (HNSCC) or treat HNSCC.

Background

CD40 is a member of the tumor necrosis factor (TNF) receptor (TNFR) family and is known as a costimulatory protein found on a diversity 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.

The presentation of peptide antigens in the context of MHC class II molecules to antigen-specific CD4⁺ T cells, together with co-stimulatory signals (from CD80 and/or CD86), results in the activation of CD4⁺ T cells and the up-regulation of the DC licensing factors CD40 ligand (CD40L) and lymphotoxin-aip2 (LTaip2). Expression of CD40L and LT LTaip2 on activated antigen-specific CD4+ T cells induces signaling through CD40 and the LTp receptor (LTpR), and this licenses DCs to induce CD8⁺ T-cell responses. CD40 signaling results in the production of interleukin- 12 (IL-12) and the up-regulation of CD70, CD86, 4-1BB ligand (4-1BBL), 0X40 ligand (OX40L) and GITR ligand (GITRL), whereas LTpR signaling leads to the production of type I interferons (IFNs). The signaling system that controls the activity of nuclear factor kappaB (NF-KB) is responsive to virtually all TNFR superfamily members. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) also contribute to these events. Priming of CD8⁺ T cells by MHC class I-restricted peptides results in the up-regulation of CD27, 4-1BB, 0X40 and glucocorticoid-induced TNFR-related protein (GITR). Stimulation of these receptors on CD8⁺ T cells by their cognate TNF superfamily ligands, in combination with IL-12 and type I IFNs, results in robust CD8⁺ T cell activation, proliferation and effector function, as well as the formation and maintenance of CD8⁺ T cell memory. CD40 antibodies can exert different actions: CD40-expressing tumor cell kill by induction of antibody -dependent cell- mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or antibody-dependent cell- mediated phagocytosis (ADCP), induction of cell signaling to induce direct apoptosis or growth arrest, but also, independent of CD40 expression on the tumor cells, through licensing of APCs to stimulate an anti-cancer immune response. Antibodies binding to CD40 can trigger CD40 on APCs to prime effector cytotoxic T lymphocytes (CTLs) and induce release of IL-2 by these cells, and indirectly activate NK cells. Antibodies stimulating CD40 have been disclosed in the prior art, and include CP-870,893, a human IgG2 antibody (WO 03/040170); dacetuzumab, a humanized IgGl antibody (WO 00/075348) and Chi Lob 7 /4, a chimeric IgGl antibody (US 2009/0074711). Furthermore, an antagonistic CD40 antibody has been disclosed, lucatumumab, a human IgGl antibody (WO 02/028481).

CD137 (4-1BB) is also a member of the TNFR family. CD137 is a co-stimulatory molecule on CD8⁺ and 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 induced upon T-cell receptor (TCR) activation (for example, on tumor infiltrating lymphocytes (TILs) (Gros et al., J. Clin Invest 2014;124(5):2246-59)). Stimulation via its natural ligand 4-1BBL or agonist antibodies leads to signaling using TRAF-2 and TRAF-1 as adaptors. Early signaling by CD137 involves K-63 poly- ubiquitination reactions that ultimately result in activation of the nuclear factor (NF)-KB and mitogen-activated protein (MAP)-kinase pathways. Signaling leads to increased T cell co-stimulation, proliferation, cytokine production, maturation and prolonged CD8+ T-cell survival. Agonistic antibodies against CD137 have been shown to promote anti-tumor control by T cells in various pre- clinical models (Murillo et al., Clin Cancer Res 2008;14(21):6895-906). Antibodies stimulating CD137 can induce survival and proliferation of T cells, thereby enhancing the anti-tumor immune response. Antibodies stimulating CD 137 have been disclosed in the prior art, and include urelumab, a human IgG4 antibody (AU 2004279877) and utomilumab, a human IgG2 antibody (Fisher et al., 2012, Cancer Immunol. Immunother. 61: 1721-1733).

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, binding human CD40 and binding human CD137. Such bispecific antibodies crosslink CD40 on antigen presenting cells (APCs) with 4- IBB on activated T cells, and thereby induce conditional stimulation of and co-stimulatory activity in both cell types useful for the treatment of solid tumors.

PD-1, CTLA4, PD-L1, TIM-3, KIR or LAG-3 are inhibitory checkpoint molecules regulating the immune system and enabling self-tolerance. At the same time inhibitory checkpoint molecules are ideal targets for cancer immunotherapy.

In tumor-draining lymph nodes and within the tumor microenvironment, 4- IBB is expressed by a subset of CD4+ and CD8+ T cells that are characterized by the co-expression of multiple TCR-inducible molecules including high levels of programmed cell death 1 (PD-1) (Gros et al., J. Clin Invest 2014;124(5):2246-59; Seifert et al., Cancers (Basel) 12; Simoni et al., Nature 557: 575-579). Upregulation of PD-1 on T cells can contribute to T-cell exhaustion and reduce T-cell activation upon binding to its ligand programmed cell death 1 ligand 1 (PD-L1) (Yu et al., Eur J Pharmacol 881: 173240). PD-L1 expression is often upregulated by tumor cells, particularly in inflamed tumors (Teng, et al., Cancer Res 75: 2139-2145). Thereby, the tumor cells provide an inhibitory signal to the activated T cells through which they can evade T-cell mediated cytotoxicity. Antibodies that block the PD-l/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).

Head and neck squamous cell carcinoma (HNSCC) is a diagnosis that is made over 600.000 times a year worldwide. In 2020, approximately 65,630 new cases of oral cavity, pharyngeal, and laryngeal cancers and an estimated 14,500 deaths will occur over the same period in the US (Clinical Practice Guidelines in Oncology, version 2, 2021). Tobacco use, alcohol use, and human papillomavirus (HPV) infection increase the risk of developing HNSCC. Patients with locally HPV positive HNSCC have improved treatment outcomes compared with patients with HPV negative disease. For patients with recurrent or metastatic HNSCC, pembrolizumab/platinum (cisplatin or carboplatin)/5-FU and pembrolizumab monotherapy are recommended IL regimens; however, the median overall survival (mOS) is less than 15 months (Clinical Practice Guidelines in Oncology, version 2, 2021).

Therefore, HNSCC remains an area of high unmet medical need and further opportunity exists to improve outcomes with novel treatment approaches.

Summary

The present inventors have surprisingly found that a combination of (i) stimulation with a binding agent binding human CD40 and binding human CD137; (ii) inhibition of the checkpoint PD-1/PD-L1 axis (in particular using pembrolizumab); and (iii) chemotherapy using a combination comprising a platinumbased chemotherapeutic agent (in particular, cisplatin or carboplatin) and 5 -fluorouracil amplifies the immune response against HNSCC.

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

In a second aspect, the present disclosure provides a method for reducing or preventing progression of HNSCC or treating HNSCC in a subject, said method comprising administering to said subject (i) the binding agent, (ii) an 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 binding agent comprises a first binding region binding to CD40 and a second binding region binding to CD137.

In further aspect, the present disclosure provides a kit comprising (i) a binding agent comprising a first binding region binding to CD40 and a second binding region binding to CD137, (ii) a PD-l/PD- Llcheckpoint inhibitor (in particular, pembrolizumab), (iii) a platinum-based chemotherapeutic agent (in particular, cisplatin and/or carboplatin), and (iv) 5 -fluorouracil, as well as such as kit for use in a method for reducing or preventing progression of HNSCC or treating HNSCC in a subject.

Brief description of the Figures

Fig. 1 shows a schematic representation of the anticipated mode of action of the CD40x4-lBB bispecific antibody. CD40 is expressed on antigen-presenting cells (APCs) as well as on tumor cells. 4-1BB (CD137) is expressed on activated T cells. DuoBody -CD40x4-lBB (GEN1042/BNT312) is a bispecific antibody that crosslinks CD40 on antigen presenting cells (APCs) with 4- IBB on activated T cells, thereby conditionally stimulating both cell types. Thereby CD40x4-lBB bispecific antibodies may enhance DC licencing, T-cell clonal expansion, cytokine production, T-cell survival and T-cell andNK- cell-mediated cytotoxicity.

Fig. 2 shows the MC38 syngeneic tumor model that was established by subcutaneous inoculation of 1 x 10⁶ MC38 cells into hCD40xh4-lBB dKI C57BL/6 mice. When tumors reached an average volume of 37 mm³, mice were randomized and treated with GEN1042-mIgG2a (1 mg/kg, BIW^X3), an antimouse PD-1 antibody (anti-mPD-1; 10 mg/kg, BIW^X3), chemotherapy regimen of carboplatin (20 mg/kg, BIW^X3) and 5 -fluorouracil (5-FU, 25 mg/kg, Q3D^X5) either alone or in combination. The control group was dosed with both PBS and 0.9% saline (BIW^X3). (A) Data shown are the median tumor volume per treatment group (n=10) with data carried forward for animals that reached termination criteria. Arrows indicate days of treatment. (B) Data shown are the tumor volumes of the different treatment groups on Day 20, the last day all groups were still intact (vertical dotted line in A), including the number of mice with complete tumor regression (CR). Mann- Whitney analysis was used to compare tumor volumes between treatment groups (p < 0.05 = *).

Fig. 3A shows the best change in target lesions for subjects with previously untreated recurrent or metastatic HNSCC treated with chemotherapy, pembrolizumab and GEN1042. Fig. 3B shows the change in target lesions over time for said subjects. Data cutoff date was October 3, 2022.

Fig. 4A shows best change in target lesions for subjects with previously untreated recurrent or metastatic HNSCC treated with pembrolizumab and GEN 1042. Fig. 4B shows the change in target lesions over time for said subjects. Data cutoff date was October 7, 2022.

Fig. 5 shows an increase of pro-inflammatory IFNg in patient sera of GEN1042 + SoC administration. Circulating levels of IFNg (interferon gamma) were measured in serum samples at baseline, and at multiple timepoints post administration of GEN1042+SoC in cycle 1 and cycle 2 (days 1, 3, 8, and 15) as well as pre-dose for cycle 3. 9 patients have been analyzed, grouped by treatment. Interim data limits mature data at all timepoints, therefore maximal n available per regimen at any given timepoint were GEN1042+pembro [5] and GEN1042+chemo+pembro [4], IFNg levels in serum samples were determined by Meso Scale Discovery (MSD) multiplex immune assay. Abbreviations: IFN = interferon, 1042 = GEN1042, Chemo = 5FU+carbo/cisplatin, pembro = pembrolizumab, pg = picograms, mL = milliliters, SEM = standard error of means, pre = pre-dose, SoC = standard of care

Test reference range: IFNg (pg/mL) < 11.81; Clinical data cutoff = September 26, 2022

Fig. 6 shows an increase TARC in patient sera of GEN1042 + SoC administration. Circulating levels of TARC were measured in serum samples at baseline, and at multiple timepoints post administration of GEN1042+SoC in cycle 1 and cycle 2 (days 1, 3, 8, and 15) as well as pre-dose for cycle 3. 7 patients have been analyzed, grouped by treatment. Interim data limits mature data at all timepoints, therefore maximal n available per regimen at any given timepoint were GEN1042+pembro [3] and GEN1042+chemo+pembro [4], TARC levels in serum samples were determined by Meso Scale Discovery (MSD) multiplex immune assay.

Abbreviations: TARC = Thymus and activation regulated chemokine, 1042 = GEN1042, Chemo = 5FU+carbo/cisplatin, pembro = pembrolizumab, pg = picograms, mL = milliliters, SEM = standard error of means, pre = pre-dose, DC = dendritic cell, SoC = standard of care

Test reference range: TARC (pg/mL) < 513;Clinical data cutoff = September 26, 2022

Fig. 7 shows the trafficking/margination of immune cells post GEN 1042 + SoC administration. Immunophenotyping of peripheral blood was conducted in whole blood collected at baseline and at multiple timepoints post administration of GEN1042+SoC in cycle 1 and cycle 2 (days 1, 3, 8 and 15) and pre-dose for cycle 3. 6 patients have been analyzed, grouped by treatment. Interim data limits mature data at all timepoints, therefore maximal n available per regimen at any given timepoint were GEN1042+pembro [3] and GEN1042+chemo+pembro [3], The frequency immune cells was assessed in whole blood samples by flow cytometry. Figure 7A shows the results for CD8 T-cells. Figure 7B shows the results for B-cells.

Abbreviations: 1042 = GEN1042, Chemo = 5FU+carbo/cisplatin, pembro = pembrolizumab, uL = microliters, SEM = standard error of means, pre = pre-dose, abs = absolute, SoC = standard of care Clinical data cutoff = September 26, 2022

Fig. 8 shows the proliferation of T cells post GEN1042+SoC administration. Immunophenotyping of peripheral blood was conducted in whole blood collected at baseline and at multiple timepoints post administration of GEN1042+SoC in cycle 1 and cycle 2 (days 1, 3, 8 and 15) and pre-dose for cycle 3. 7 patients have been analyzed, grouped by treatment. Interim data limits mature data at all timepoints, therefore maximal n available per regimen at any given timepoint were GEN1042+pembro [3] and GEN1042+chemo+pembro [4], The frequency of proliferating (%Ki67) T cells was assessed in whole blood samples by flow cytometry. Figure 8A shows the results for proliferating (%Ki67) CD8 T-cells. Figure 8B shows the results for proliferating (%Ki67) effector memory CD8 T cells (CD45RA-, CCR7-).

Abbreviations: 1042 = GEN1042, Chemo = 5FU+carbo/cisplatin, pembro = pembrolizumab, uL = microliters, SEM = standard error of means, pre = pre-dose, abs = absolute, SoC = standard of care, Tem = T effector memory

Clinical data cutoff = September 26, 2022

Fig. 9 shows the activation of T cells post GEN1042+SoC administration. Immunophenotyping of peripheral blood was conducted in whole blood collected at baseline and at multiple timepoints post administration of GEN1042+SoC in cycle 1 and cycle 2 (days 1, 3, 8 and 15) and pre-dose for cycle 3. 7 patients have been analyzed, grouped by treatment. Interim data limits mature data at all timepoints, therefore maximal n available per regimen at any given timepoint were GEN1042+pembro [3] and GEN1042+chemo+pembro [4], The frequency of activated (%4-lBB) CD8 T cells was assessed in whole blood samples by flow cytometry. Figure 9A shows the results for CD8 T-cells. Figure 9B shows the results specifically for effector memory CD8 T cells (CD45RA-, CCR7-).

Clinical data cutoff = September 26, 2022

Fig. 10 shows B cell activation post GEN1042+SoC administration. Immunophenotyping of peripheral blood was conducted in whole blood collected at baseline and at multiple timepoints post administration of GEN1042+SoC in cycle 1 and cycle 2 (days 1, 3, 8 and 15) and pre-dose for cycle 3. 6 Patients have been analyzed, grouped by treatment. Interim data limits mature data at all timepoints, therefore maximal n available per regimen at any given timepoint were 1042+pembro [3] and 1042+chemo+pembro [3], The frequency of activated (%4-lBB+) B cells was assessed in whole blood samples by flow cytometry.

Abbreviations: 1042 = GEN1042, Chemo = 5FU+carbo/cisplatin, pembro = pembrolizumab, uL = microliters, SEM = standard error of means, pre = pre-dose, abs = absolute, SoC = standard of care Clinical data cutoff = September 26, 2022 Table 1 - Sequences

SEQ ID NO: 63 HCDR1 (MAB- 19-0618) intersection of Kabat and IMGT

SEQ ID NO: 64 HCDR1 (MAB- 19-0618) Kabat

SEQ ID NO: 45 HCDR1 (MAB- 19-0618) IMGT

SEQ ID NO: 65 HCDR2 (MAB- 19-0618) intersection of Kabat and IMGT (= IMGT)

SEQ ID NO: 66 HCDR2 (MAB- 19-0618) Kabat

SEQ ID NO: 47 HCDR3 (MAB- 19-0618) intersection of Kabat and IMGT (= Kabat) SEQ ID NO: 67 HCDR3 (MAB- 19-0618) IMGT

SEQ ID NO: 48 LCDR1 (MAB-19-0618) intersection of Kabat and IMGT (= IMGT)

SEQ ID NO: 68 LCDR1 (MAB-19-0618) Kabat SEQ ID NO: 49 LCDR2 (MAB- 19-0618) intersection of Kabat and IMGT (= IMGT)

SEQ ID NO: 69 LCDR2 (MAB- 19-0618) Kabat

5 SEQ ID NO: 50 LCDR3 (MAB- 19-0618) intersection = Kabat = IM GT

Detailed Description of the Invention

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

In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise. For example, if in a preferred embodiment of the binding agent used herein the first heavy chain comprises or consists essentially of or consists of an amino acid sequence set forth in SEQ ID NO: 26 or 34 [IgGl-Fc_FEAR] and in another preferred embodiment of the binding agent used herein the second heavy chain comprises or consists essentially of or consists of an amino acid sequence set forth in SEQ ID NO: 25 or 33 [IgGl-Fc_FEAL], then in a further preferred embodiment of the binding agent used herein the first heavy chain comprises or consists essentially of or consists of an amino acid sequence set forth in SEQ ID NO: 26 or 34 [IgGl-Fc_FEAR] and the second heavy chain comprises or consists essentially of or consists of an amino acid sequence set forth in SEQ ID NO: 25 or 33 [IgGl-

Fc FEAL],

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

The practice of the present disclosure will employ, unless otherwise indicated, conventional chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser/Heathcook, "Organische Chemie", VCH, 1990; Beyer/Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey /Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Rompp Chemie Lexikon, Falbe/Regitz (Hrsg.), Georg Thieme Verlag Stutgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to beter illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

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 individual value is incorporated into the specification as if it were individually recited herein.

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

Definitions

In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a 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 other members, integers or steps of any essential significance. The term "comprising" encompasses the term "consisting essentially of' which, in turn, encompasses the term "consisting of'. Thus, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of' or "consisting of'. Likewise, at each occurrence in the present application, the term "consisting essentially of' may be replaced with the term "consisting of'. The terms "a", "an" and "the" and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

Where used herein, "and/or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and/or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein.

In the context of the present disclosure, the term "about" denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±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%, and for example ±0.01%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.

The term "binding agent" in the context of the present disclosure refers to any agent capable of binding to desired antigens. In certain embodiments of the present disclosure, the binding agent is an antibody, antibody fragment, or construct thereof. The binding agent may also comprise synthetic, modified or non-naturally occurring moieties, in particular non-peptide moieties. Such moieties may, for example, link desired antigen-binding functionalities or regions such as antibodies or antibody fragments. In one embodiment, the binding agent is a synthetic construct comprising antigen-binding CDRs or variable regions.

As used herein, "immune checkpoint" refers to regulators of the immune system, and, in particular, costimulatory 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, HMGB 1 and CEAC AM 1. In certain embodiments, the inhibitory signal is the interaction between one or several 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(s). 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 SIRPa. 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.

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

In one embodiment, the checkpoint inhibitor can be any compound, such as any binding agent, which inhibits the inhibitory signal of an immune checkpoint, wherein the inhibitory signal is selected from the group consisting of: the interaction between PD-1 and PD-L1 and/or PD-L2 (such a checkpoint inhibitor which inhibits the interaction between PD-1 and PD-L1 and/or PD-L2 is also called a PD- 1/PD-L1 checkpoint inhibitor herein); 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(s); 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 SIRPa; the interaction between PVRIG and PVRL2; the interaction between CSF1R and CSF1; the interaction between BTLA and HVEM; part of the adenosinergic pathway, e.g., the interaction between A2AR and/or A2BR and adenosine, produced by CD39 and CD73; the interaction between B7-H3 and its receptor and/or B7-H4 and its receptor; an inhibitory signal mediated by IDO, CD20, NOX or TDO. In one embodiment, the checkpoint inhibitor is at least one selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, KIR inhibitors, LAG-3 inhibitors, TIGIT inhibitors, VISTA inhibitors, and GARP inhibitors. In one embodiment, the checkpoint inhibitor may be a blocking antibody, such as a PD-1 blocking antibody, a CTLA4 blocking antibody, a PD-L1 blocking antibody, a PD-L2 blocking antibody, a TIM- 3 blocking antibody, a KIR blocking antibody, a LAG-3 blocking antibody, a TIGIT blocking antibody, a VISTA blocking antibody, or a GARP blocking antibody. Examples of a PD-1 blocking antibody include pembrolizumab, nivolumab, cemiplimab, and spartalizumab. Examples of a CTLA4 blocking antibody include ipilimumab and tremelimumab. Examples of a PD-L1 blocking antibody include atezolizumab, durvalumab, and avelumab.

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

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) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 45;

(ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46; and

(iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 47; and wherein the light chain variable region comprises:

(i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48;

(ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 49; and

(iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 50.

In one embodiment of the anti-PD-1 antibodies described herein, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 43 and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 44. 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) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 81 ;

(ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 82; and

(iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 83; and wherein the light chain variable region comprises:

(i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 84;

(ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 85; and

(iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 86.

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

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

The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, preferably to antigen receptors such as antibodies or the B cell receptor (BCR). The immunoglobulins are characterized by a structural domain, i.e., the immunoglobulin domain, having a characteristic immunoglobulin (Ig) fold. The term encompasses membrane bound immunoglobulins as well as soluble immunoglobulins. Membrane bound immunoglobulins are also termed surface immunoglobulins or membrane immunoglobulins, which are generally part of the BCR. Soluble immunoglobulins are generally termed antibodies.

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, typically two identical heavy chains and two identical light chains which are linked via disulfide bonds. These chains are primarily composed of immunoglobulin domains or regions, such as the V_L or VL (variable light chain) domain/region, CL or CL (constant light chain) domain/region, VH or VH (variable heavy chain) domain/region, and the CH or CH (constant heavy chain) domains/regions CHI (CHI), CH2 (CH2), CH3 (CH3), and CH4 (CH4). The heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3. The hinge region is the region between the CHI and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interactions between two heavy chains in an IgG molecule. Each light chain typically is comprised of a VL and a CL. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to IMGT rules using DomainGapAlign (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); see also internet http address www.imgt.org. Unless otherwise stated or contradicted by context, reference to amino acid positions in the constant regions in the present disclosure is according to the EU-numbering (Edelman et al., Proc Natl Acad Sci USA. 1969 May;63(l):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).

There are five types of mammalian immunoglobulin heavy chains, i.e., a, 5, e, y, and p which account for the different classes of antibodies, i.e., IgA, IgD, IgE, IgG, and IgM. As opposed to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins comprise a transmembrane domain and a short cytoplasmic domain at their carboxy-terminus. In mammals there are two types of light chains, i.e., lambda and kappa. The immunoglobulin chains comprise a variable region and a constant region. The constant region is essentially conserved within the different isotypes of the immunoglobulins, wherein the variable part is highly divers and accounts for antigen recognition.

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

Table 2: Main classification based on structure and general chemical characterization ofR group

Table 3: Alternative Physical and Functional Classifications of Amino Acid Residues

For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide, protein or polypeptide) comprise 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, posttranslationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" includes, in particular, fragments of an amino acid sequence.

Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.

Amino acid addition variants comprise amino- and/or carboxy -terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and/or C-terminal end of the protein are also called N-terminal and/or C-terminal truncation variants.

Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Substitution of one amino acid for another may be classified as a conservative or non-conservative substitution. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and/or to replacing amino acids with other ones having similar properties. Preferably, amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. In the context of the present disclosure, a "conservative substitution" is a substitution of one amino acid with another amino acid having similar structural and/or chemical characteristics, such substitution of one amino acid residue for another amino acid residue of the same class as defined in any of the two tables above: for example, leucine may be substituted with isoleucine as they are both aliphatic, branched hydrophobes. Similarly, aspartic acid may be substituted with glutamic acid since they are both small, negatively charged residues. Naturally occurring amino acids may also be generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly 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.

The term "amino acid corresponding to position... " and similar expressions as used herein refer to an amino acid position number in a human IgGl heavy chain. Corresponding amino acid positions in other immunoglobulins may be found by alignment with human IgGl. Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings and has at least 50%, at least 80%, at least 90%, or at least 95% identity to a human IgGl heavy chain. It is considered well-known in the art how to align a sequence or segment in a sequence and thereby determine the corresponding position in a sequence to an amino acid position according to the present disclosure.

The term "antibody" (Ab) in the context of the present disclosure refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen (in particular an epitope on an antigen) under typical physiological conditions, preferably with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four 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 (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies and combinations of any of the foregoing. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The variable regions 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 regions of hypervariability, termed complementarity determining regions (CD Rs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of a VH are termed HCDR1, HCDR2 and HCDR3 (or CDR-H1, CDR-H2 and CDR-H3), the CDRs of a VL are termed LCDR1, LCDR2 and LCDR3 (or CDR-L1, CDR-L2 and CDR-L3). The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of an antibody comprise the heavy chain constant region (CH) and the light chain constant region (CL), wherein CH can be further subdivided into constant domain CHI, a hinge region, and constant domains CH2 and CH3 (arranged from amino-terminus to carboxy -terminus in the following order: CHI, CH2, CH3). The constant regions of the antibodies may 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 Clq. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.

The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The terms "binding region" and "antigen-binding region" are used herein interchangeably and refer to the region which interacts with the antigen and comprises both a VH region and a VL region. An antibody as used herein comprises not only monospecific antibodies, but also multispecific antibodies which comprise multiple, such as two or more, e.g., three or more, different antigen-binding regions.

As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, i.e., retain the ability to specifically bind to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include (i) a Fab’ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, or a monovalent antibody as described in WO 2007/059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CHI domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., Nature 341. 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov;21(l 1 ) :484- 90); (vi) camelid or Nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan;5(l):ll l-24); and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al. , Science 242. 423-426 (1988) and Huston et al. , PNAS USA 85. 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present disclosure, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific formats of such fragments, are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody -like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

An antibody as generated can possess any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (for instance IgG (such as IgGl, IgG2, IgG3, IgG4), IgD, IgA (such as IgAl, IgA2), IgE, IgM, or IgY) that is encoded by heavy chain constant region genes. When a particular isotype, e.g. IgGl, is mentioned herein, the term is not limited to a specific isotype sequence, e.g. a particular IgGl sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g. IgGl, than to other isotypes. Thus, e.g. an IgGl antibody disclosed herein may be a sequence variant of a naturally-occurring IgGl antibody, including variations in the constant regions.

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

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, said different antigen-binding regions bind different epitopes on the same antigen. However, in preferred embodiments, said different antigen-binding regions bind different target antigens. In one embodiment, the multispecific antibody is a "bispecific antibody" or "bs". A multispecific antibody, such as a bispecific antibody, can be of any format, including any of the bispecific or multispecific antibody formats described herein below.

The term "full-length" when used in the context of an antibody indicates that the antibody is not a fragment, but contains all of the domains of the particular isotype normally found for that isotype in nature, e.g. the VH, CHI, CH2, CH3, hinge, VL and CL domains for an IgGl antibody.

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

The term "chimeric antibody" as used herein, refers to an antibody wherein the variable region is derived from a non-human species (e.g. derived from rodents) and the constant region is derived from a different species, such as human. Chimeric antibodies may be generated by antibody engineering. "Antibody engineering" is a term used generically for different kinds of modifications of antibodies, and processes for antibody engineering are well-known for the skilled person. In particular, a chimeric antibody may be generated by 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, the chimeric antibody may be a genetically or an enzymatically engineered recombinant antibody. It is within the knowledge of the skilled person to generate a chimeric antibody, and thus, generation of the chimeric antibody may be performed by other methods than those described herein. Chimeric monoclonal antibodies for therapeutic applications in humans are developed to reduce anticipated antibody immunogenicity of non-human antibodies, e.g. rodent antibodies. They may typically contain non-human (e.g. murine or rabbit) variable regions, which are specific for the antigen of interest, and human constant antibody heavy and light chain domains. The terms "variable region" or "variable domain" as used in the context of chimeric antibodies, refer to a region which comprises the CD Rs and framework regions of both the heavy and light chains of an immunoglobulin, as described below.

The term "humanized antibody" as used herein, refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody complementarity -determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see WO 92/22653 and EP 0 629 240). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non- human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties.

As used herein, a protein which is "derived from" another protein, e.g., a parent protein, means that one or more amino acid sequences of the protein are identical or similar to one or more amino acid sequences in the other or parent protein. For example, in an antibody, binding arm, antigen-binding region, constant region, or the like which is derived from another or a parent antibody, binding arm, antigen-binding region, or constant region, one or more amino acid sequences are identical or similar to those of the other or parent antibody, binding arm, antigen-binding region, or constant region. Examples of such one or more amino acid sequences include, but are not limited to, 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 can be described herein as "derived from" a non-human parent antibody, meaning that at least the VL and VH CDR sequences are identical or similar to the VH and VL CDR sequences of said non- human parent antibody. A chimeric antibody can be described herein as being "derived from" a non- human parent antibody, meaning that typically the VH and VL sequences may be identical or similar to those of the non-human parent antibody. Another example is a binding arm or an antigen-binding region which may be described herein as being "derived from" a particular parent antibody, meaning that said binding arm or antigen-binding region typically comprises identical or similar VH and/or VL CDRs, or VH and/or VL sequences to the binding arm or antigen-binding region of said parent antibody. As described elsewhere herein, however, amino acid modifications such as mutations can be made in the CDRs, constant regions or elsewhere in the antibody, binding arm, antigen-binding region or the like, to introduce desired characteristics. When used in the context of one or more sequences derived from a first or parent protein, a "similar" amino acid sequence preferably has a sequence identity of at least about 50%, such as 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%.

Non-human antibodies can be generated in a number of different species, such as mouse, rabbit, chicken, guinea pig, llama and goat.

Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, 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 libraries of antibody genes, and such methods are well known to a person skilled in the art.

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

When used herein, unless contradicted by context, the term "Fab-arm" or "arm" refers to one heavy chain-light chain pair and is used interchangeably with "half molecules" herein.

The term "binding arm comprising an antigen-binding region" means an antibody molecule or fragment that comprises an antigen-binding region. Thus, a binding arm can comprise, e.g., the six VH and VL CDR sequences, the VH and VL sequences, a Fab or Fab' fragment, or a Fab-arm.

When used herein, unless contradicted by context, the term "Fc region" refers to an antibody region consisting of the two Fc sequences of the heavy chains of an immunoglobulin, wherein said Fc sequences comprise at least a hinge region, a CH2 domain, and a CH3 domain. In one embodiment, the term "Fc region", as used herein, refers to a region comprising, in the direction from the N- to C-terminal end of the antibody, at least a hinge region, a CH2 region and a CH3 region. An Fc region of the antibody may 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.

In the context of the present disclosure, the term "induce Fc-mediated effector function to a lesser extent" used in relation to an antibody, including a multispecific antibody, means that the antibody induces Fc- mediated effector functions, such function in particular being selected from the list of IgG Fc receptor (FcgammaR, FcyR) binding, Clq binding, ADCC or CDC, to a lesser extent compared to a human IgGl antibody comprising (i) the same CDR sequences, in particular comprising the same first and second antigen-binding regions, as said antibody and (ii) two heavy chains comprising human IgGl hinge, CH2 and CH3 regions.

Fc-mediated effector function may be measured by binding to FcyRs, binding to Clq, or induction of Fc-mediated cross-linking via FcyRs.

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 IgGl antibody corresponds to amino acids 216-230 according to the EU numbering as set forth 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, pp 662,680,689 (1991). However, the hinge region may also be any of the other subtypes as described herein.

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

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 IgGl antibody corresponds to amino acids 231-340 according to the EU numbering as set forth in Kabat (ibid). However, the CH2 region may also be any of the other subtypes as described herein.

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 IgGl antibody corresponds to amino acids 341-447 according to the EU numbering as set forth in Kabat (ibid). However, the CH3 region may also be any of the other subtypes as described herein.

The term "monovalent antibody" means in the context of the present disclosure that an antibody molecule is capable of binding a single molecule of the antigen, and thus is not capable of antigen crosslinking.

A "CD40 antibody" or "anti-CD40 antibody" is an antibody as described above, which binds specifically to the antigen CD40. A "CD137 antibody" or "anti-CD137 antibody" is an antibody as described above, which binds specifically to the antigen CD 137.

A "CD40xCD137 antibody" or "anti-CD40xCD137 antibody" is a bispecific antibody, which comprises two different antigen-binding regions, one of which binds specifically to the antigen CD40 and one of which binds specifically to the antigen CD 137.

As used herein, the terms "binding" or "capable of binding" in the context of the binding of an antibody to a predetermined antigen or epitope typically is a binding with an affinity corresponding to a K_D of about 10'⁷ M or less, such as about 10'⁸M or less, such as about 10'⁹ M or less, about IO ⁰ M or less, or about 10⁴¹ M or even less, when determined using Bio-Layer Interferometry (BLI) or, for instance, when determined using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte. The antibody binds to the predetermined antigen with an affinity corresponding to a K_D that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its K_D for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is higher is dependent on the K_D of the antibody, so that when the K_D of the antibody is very low (that is, the antibody is highly specific), then the degree to which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000-fold.

The term "kj" (sec ), as used herein, refers to the dissociation rate constant of a particular antibodyantigen interaction. Said value is also referred to as the k_Off value.

The term "K_D" (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody -antigen interaction.

Two antibodies have the "same specificity" if they bind to the same antigen and to the same epitope. Whether an antibody to be tested recognizes the same epitope as a certain antigen-binding antibody, i.e., the antibodies bind to the same epitope, may be tested by different methods well known to a person skilled in the art.

The competition between the antibodies can be detected by a cross-blocking assay. For example, a competitive ELISA assay may be used as a cross-blocking assay. E.g., target antigen may be coated on the wells of a microtiter plate and antigen-binding antibody and candidate competing test antibody may be added. The amount of the antigen-binding antibody bound to the antigen in the well indirectly correlates with the binding ability of the candidate competing test antibody that competes therewith for binding to the same epitope. Specifically, the larger the affinity of the candidate competing test antibody is for the same epitope, the smaller the amount of the antigen-binding antibody bound to the antigen- coated well. The amount of the antigen-binding antibody bound to the well can be measured by labeling the antibody with detectable or measurable labeling substances.

An antibody competing for binding to an antigen with another antibody, e.g., an antibody comprising heavy and light chain variable regions as described herein, or an antibody having the specificity for an antigen of another antibody, e.g., an antibody comprising heavy and light chain variable regions as described herein, may be an antibody comprising variants of said heavy and/or light chain variable regions as described herein, e.g. modifications in the CDRs and/or a certain degree of identity as described herein.

An "isolated multispecific antibody" as used herein is intended to refer to a multispecific antibody which is substantially free of other antibodies having different antigenic specificities (for instance an isolated bispecific antibody that specifically binds to CD40 and CD137 is substantially free of monospecific antibodies that specifically bind to CD40 or CD 137).

The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

When used herein the term "heterodimeric interaction between the first and second CH3 regions" refers to the interaction between the first CH3 region and the second CH3 region in a first-CH3/second-CH3 heterodimeric antibody.

When used herein the term "homodimeric interactions of the first and second CH3 regions" refers to the interaction between a first CH3 region and another first CH3 region in a first-CH3/first-CH3 homodimeric antibody and the interaction between a second CH3 region and another second CH3 region in a second-CH3/second-CH3 homodimeric antibody.

When used herein the term "homodimeric antibody" refers to an antibody comprising two first Fab-arms or half-molecules, wherein the amino acid sequence of said Fab-arms or half-molecules is the same.

When used herein the term "heterodimeric antibody" refers to an antibody comprising a first and a second Fab-arm or half-molecule, wherein the amino acid sequence of said first and second Fab-arms or half-molecules are different. In particular, the CH3 region, or the antigen-binding region, or the CH3 region and the antigen-binding region of said first and second Fab-arms/half-molecules are different. The term "reducing conditions" or "reducing environment" refers to a condition or an environment in which a substrate, such as a cysteine residue in the hinge region of an antibody, is more likely to become reduced than oxidized.

The present disclosure also describes multispecific antibodies, such as bispecific antibodies, comprising functional variants of the VL regions, VH regions, or one or more CDRs of the bispecific antibodies of the examples. A functional variant of a VL, VH, or CDR used in the context of a bispecific antibody still allows 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 the specificity /selectivity of the parent bispecific antibody and in some cases such a bispecific antibody may be associated with greater affinity, selectivity and/or specificity than the parent bispecific antibody.

Such functional variants typically retain significant sequence identity to the parent bispecific antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (/. e. , % homology = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g. be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm.

In the context of the present disclosure, unless otherwise indicated, the following notations are used to describe a mutation: i) substitution of an amino acid in a given position is written as e.g. K409R which means a substitution of a lysine in position 409 of the protein with an arginine; and ii) for specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue. Thus, the substitution of lysine with arginine in position 409 is designated as: K409R, and the substitution of lysine with any amino acid residue in position 409 is designated as K409X. In case of deletion of lysine in position 409 it is indicated by K409*.

Exemplary variants include those which differ from the VH and/or VL and/or CDRs of the parent sequences mainly by conservative substitutions; for example, 12, such as 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative amino acid residue replacements. In the context of the present disclosure, conservative substitutions may be defined by substitutions within the classes of amino acids as defined in tables 2 and 3.

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

The term "CD137" as used herein, refers to CD137 (4-1BB), also referred to as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is the receptor for the ligand TNFSF9/4-1BBL. CD137 (4-1BB) is believed to be involved in T-cell activation. Other synonyms for CD137 include, but are not limited to, 4-1BB ligand receptor, CDwl37, T-cell antigen 4-1BB homolog and T-cell antigen ILA. In one embodiment, CD137 (4-1BB) is human CD137 (4-1BB), having UniProt accession number Q07011. The sequence of human CD137 is also shown in SEQ ID NO: 37. Amino acids 1-23 of SEQ ID NO: 37 correspond to the signal peptide of human CD137; while amino acids 24-186 of SEQ ID NO: 37 correspond to the extracellular domain of human CD137; and the remainder of the protein, i.e. from amino acids 187-213 and 214-255 of SEQ ID NO: 37 are transmembrane and cytoplasmic domain, respectively.

The "Programmed Death-1 (PD-1)" receptor refers to an immuno-inhibitory receptor belonging to the CD28 family. PD-1 (also known as CD279) is expressed predominantly on previously activated T cells in vivo, and binds to two ligands, PD-L1 (also known as B7-H1 or CD274) and PD-L2 (also known as B7-DC or CD273). The term "PD-1" as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, 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 two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1" as used herein includes human PD-L1 (hPD-Ll), variants, isoforms, and species homologs of hPD-Ll, such as macaque (cynomolgus monkey), African elephant, wild boar and mouse PD-L1 (cf., e.g., Genbank accession no. NP 054862.1, XP 005581836, XP 003413533, XP 005665023 and NP 068693, respectively), and analogs having at least one common epitope with hPD-Ll. The sequence of human PD-L1 is also shown in SEQ ID NO: 40, wherein amino acids 1-18 are predicted to be a signal peptide. The sequence of macaque (cynomolgus monkey) PD-L1 is also shown in SEQ ID NO: 41, wherein amino acids 1-18 are predicted to be a signal peptide. The term "PD-L2" as used herein includes human PD-L2 (hPD-L2), variants, isoforms, 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 surface of antigen-presenting cells, such as dendritic cells or macrophages, and other immune cells. Binding of PD-1 to PD-L1 or PD-L2 results in downregulation of T cell activation. Cancer cells expressing PD-L1 and/or PD-L2 are able to switch off T cells expressing PD-1 what results in suppression of the anticancer immune response. The interaction between PD-1 and its ligands results in a decrease in tumor infiltrating lymphocytes, a decrease in T cell receptor mediated proliferation, and immune evasion by the cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well.

"Cytotoxic T Lymphocyte Associated Antigen-4 (CTLA-4)" (also known as CD 152) is a T cell surface molecule and is a member of the immunoglobulin superfamily. This protein downregulates the immune system by binding to CD80 (B7-1) and CD86 (B7-2). The term "CTLA-4" as used herein includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, and analogs having at least one common epitope with hCTLA-4. CTLA-4 is a homolog of the stimulatory checkpoint protein CD28 with 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. Binding of CTLA 4 to its ligands prevents the co-stimulatory signal of CD28 and produces an inhibitory signal. Thus, CTLA-4 downregulates T cell activation. The sequence of human CTLA-4 is also shown in SEQ ID NO: 42.

"T cell Immunoreceptor with Ig and ITIM domains" (TIGIT, also known as WUCAM or Vstm3) is an immune receptor on T cells and natural killer (NK) cells and binds to PVR (CD 155) on DCs, macrophages etc., and PVRL2 (CD112; nectin-2) and PVRL3 (CD113; nectin-3) and regulates T cell- mediated immunity. The term "TIGIT" as used herein includes human TIGIT (hTIGIT), variants, isoforms, and species homologs of hTIGIT, and analogs having at least one common epitope with hTIGIT. The term "PVR" as used herein includes human PVR (hPVR), variants, isoforms, and species homologs of hPVR, and analogs having at least one common epitope with hPVR. The term "PVRL2" as used herein includes human PVRL2 (hPVRL2), variants, isoforms, and species homologs of hPVRL2, and analogs having at least one common epitope with hPVRL2. The term "PVRL3" as used herein includes human PVRL3 (hPVRL3), variants, isoforms, and species homologs of hPVRL3, and analogs having at least one common epitope with hPVRL3. "B and T Lymphocyte Attenuator" (BTLA, also known as CD272) is a TNFR family member expressed in Thl but not Th2 cells. BTLA expression is induced during activation of T cells and is in particular expressed on surfaces of CD8+ T cells. The term "BTLA" as used herein includes human BTLA (hBTLA), variants, isoforms, and species homologs of hBTLA, and analogs having at least one common epitope with hBTLA. BTLA expression is gradually downregulated during differentiation of human CD8+ T cells to effector cell phenotype. Tumor-specific human CD8+ T cells express high levels of BTLA. BTLA binds to "Herpesvirus entry mediator" (HVEM, also known as TNFRSF14 or CD270) and is involved in T cell inhibition. The term "HVEM" as used herein includes human HVEM (hHVEM), variants, isoforms, and species homologs of hHVEM, and analogs having at least one common epitope with hHVEM. BTLA-HVEM complexes negatively regulate T cell immune responses.

"Killer-cell Immunoglobulin-like Receptors" (KIRs) are receptors for MHC Class I molecules onNK T cells and NK cells that are involved in differentiation between healthy and diseased cells. KIRs bind to human leukocyte antigen (HLA) A, B and C, what suppresses normal immune cell activation. The term "KIRs" as used herein includes human KIRs (hKIRs), variants, isoforms, and species homologs of hKIRs, and analogs having at least one common epitope with a hKIR. The term "HLA" as used herein includes variants, isoforms, and species homologs of HLA, and analogs having at least one common epitope with a HLA. KIR as used herein in particular refers to KIR2DL1, KIR2DL2, and/or KIR2DL3.

"Lymphocyte Activation Gene-3 (LAG-3)" (also known as CD223) is an inhibitory receptor associated with inhibition of lymphocyte activity by binding to MHC class II molecules. This receptor enhances the function of Treg cells and inhibits CD8+ effector T cell function leading to immune response suppression. LAG-3 is expressed on activated T cells, NK cells, B cells and DCs. The term "LAG-3" as used herein includes human LAG-3 (hLAG-3), variants, isoforms, and species homologs of hLAG-3, and analogs having at least one common epitope.

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

"CD94/NKG2A" is an inhibitory receptor predominantly expressed on the surface of natural killer cells and of CD8+ T cells. The term "CD94/NKG2A" as used herein includes human CD94/NKG2A (hCD94/NKG2A), variants, isoforms, and species homologs of hCD94/NKG2A, and analogs having at least one common epitope. The CD94/NKG2A receptor is a heterodimer comprising CD94 and NKG2A. It suppresses NK cell activation and CD8+ T cell function, probably by binding to ligands such as HLA- E. CD94/NKG2A restricts cytokine release and cytotoxic response of natural killer cells (NK cells), natural killer T cells (NK-T cells) and T cells (ot/p and y/8). NKG2A is frequently expressed in tumor infiltrating cells and HLA-E is overexpressed in several cancers.

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

In the "adenosinergic pathway" or "adenosine signaling pathway" as used herein ATP is converted to adenosine by the ectonucleotidases CD39 and CD73 resulting in inhibitory signaling through adenosine binding by one or more of the inhibitory adenosine receptors "Adenosine A2A Receptor" (A2AR, also known as AD0RA2A) and "Adenosine A2B Receptor" (A2BR, also known as AD0RA2B). Adenosine is a nucleoside with immunosuppressive properties and is present in high concentrations in the tumor microenvironment restricting immune cell infiltration, cytotoxicity and cytokine production. Thus, adenosine signaling is a strategy of cancer cells to avoid host immune system clearance. Adenosine signaling through A2AR and A2BR is an important checkpoint in cancer therapy that is activated by 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 activation of immune cells 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, isoforms, and species homologs of hCD39, and analogs having at least one common epitope. The term "CD73" as used herein includes human CD73 (hCD73), variants, isoforms, and species homologs of hCD73, and analogs having at least one common epitope. The term "A2AR" as used herein includes human A2AR (hA2AR), variants, isoforms, and species homologs of hA2AR, and analogs having at least one common epitope. The term "A2BR" as used herein includes human A2BR (hA2BR), variants, isoforms, and species homologs of hA2BR, and analogs having at least one common epitope.

"V-domain Ig suppressor of T cell activation" (VISTA, also known as C10orf54) bears homology to PD-L1 but displays a unique expression pattern restricted to the hematopoietic compartment. The term "VISTA" as used herein includes human VISTA (h VISTA), variants, isoforms, 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.

The "Sialic acid binding immunoglobulin type lectin" (Siglec) family members recognize sialic acids and are involved in distinction between "self¹ and "non-self". The term "Siglecs" as used herein includes human Siglecs (hSiglecs), variants, isoforms, and species homologs of hSiglecs, and analogs having at least one common epitope with one or more hSiglecs. The human genome contains 14 Siglecs of which several are involved in immunosuppression, including, without limitation, Siglec-2, Siglec-3, Siglec-7 and Siglec-9. Siglec receptors bind glycans containing sialic acid, but differ in their recognition of the linkage regiochemistry and spatial distribution of sialic residues. The members of the family also have distinct expression patterns. A broad range of malignancies overexpress one or more Siglecs.

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

"Glycoprotein A repetitions predominant" (GARP) plays a role in immune tolerance and the ability of tumors to escape the patient's immune system. The term "GARP" as used herein includes human GARP (hGARP), variants, isoforms, 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 tumor sites. It probably binds to latent "transforming growth factor " (TGF- ). Disruption of GARP signaling in Tregs cells results in decreased tolerance and inhibits migration of Tregs to the gut and increased proliferation of cytotoxic T cells.

"CD47" is a transmembrane protein that binds to the ligand "signal-regulatory protein alpha" (SIRPa). The term "CD47" as used herein includes human CD47 (hCD47), variants, isoforms, and species homologs of hCD47, and analogs having at least one common epitope with hCD47. The term "SIRPa" as used herein includes human SIRPa (hSIRPa), variants, isoforms, and species homologs of hSIRPa, and analogs having at least one common epitope with hSIRPa. CD47 signaling is involved in a range of cellular processes including apoptosis, proliferation, adhesion and migration. CD47 is overexpressed in many cancers and functions as "don’t eat me" signal to macrophages. Blocking CD47 signaling through inhibitory anti-CD47 or anti-SIRPa antibodies enables macrophage phagocytosis of cancer cells and fosters the activation of cancer-specific T lymphocytes.

"Poliovirus receptor related immunoglobulin domain containing" (PVRIG, also known as CD112R) binds to "Poliovirus receptor-related 2" (PVRL2). PVRIG and PVRL2 are overexpressed in a number of cancers. PVRIG expression also induces TIGIT and PD-1 expression and PVRL2 and PVR (a TIGIT ligand) are co-overexpressed in several cancers. Blockade of the PVRIG signaling pathway results in increased T cell function and CD8+ T cell responses and, therefore, reduced immune suppression and elevated interferon responses. The term "PVRIG" as used herein includes human PVRIG (hPVRIG), variants, isoforms, and species homologs of hPVRIG, and analogs having at least one common epitope with hPVRIG. "PVRL2" as used herein includes hPVRL2, as defined above.

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

"Nicotinamide adenine dinucleotide phosphate NADPH oxidase" refers to an enzyme of the NOX family of enzymes of myeloid cells that generate immunosuppressive reactive oxygen species (ROS). Five NOX enzymes (N0X1 to N0X5) have been found to be involved in cancer development and immunosuppression. Elevated ROS levels have been detected in almost all cancers and promote many aspects of tumor development and progression. NOX produced ROS dampens NK and T cell functions and inhibition of NOX in myeloid cells improves anti-tumor functions of adjacent NK cells and T cells. The term "NOX" as used herein includes human NOX (hNOX), variants, isoforms, and species homologs of hNOX, and analogs having at least one common epitope with hNOX.

Another immune checkpoint that can be targeted according to the disclosure is the signal mediated by "tryptophan-2, 3 -dioxygenase" (TDO). TDO represents an alternative route to IDO in tryptophan degradation and is involved in immune suppression. Since tumor cells may catabolize tryptophan via TDO instead of IDO, TDO may represent an additional target for checkpoint blockade. Indeed, several cancer cell lines have been found to upregulate TDO and TDO may complement IDO inhibition. The term "TDO" as used herein includes human TDO (hTDO), variants, isoforms, and species homologs of hTDO, and analogs having at least one common epitope with hTDO.

Many of the immune checkpoints are regulated by interactions between specific receptor and 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 being attacked by the immune system. Hence, the function of checkpoint proteins is typically the regulation of T cell activation, T cell proliferation and/or T cell function. Immune checkpoint proteins thus regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses. Many of the immune checkpoint proteins belong to the B7:CD28 family or to the tumor necrosis factor receptor (TNFR) super family and, by binding to specific ligands, activate signaling molecules that are recruited to the cytoplasmic domain (Suzuki et al., 2016, Jap J Clin One, 46:191-203).

The term "dysfunctional", as used herein, refers to an immune cell that is in a state of reduced immune responsiveness to antigen stimulation. Dysfunctional includes unresponsive to antigen recognition and impaired capacity to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2) and/or target cell killing.

The term "anergy ", as used herein, refers to the state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signals delivered through the T cell receptor (TCR). T cell anergy can also result upon stimulation with antigen in the absence of co-stimulation, resulting in the cell becoming refractory to subsequent activation by the antigen even in the context of co-stimulation. The unresponsive state can often be overridden by the presence of IL-2. Anergic T cells do not undergo clonal expansion and/or acquire effector functions.

The term "exhaustion", as used herein, refers to immune cell exhaustion, such as T cell exhaustion as a state of T cell dysfunction that arises from sustained TCR signaling that occurs during many chronic infections and cancer. It is distinguished from anergy in that it arises not through incomplete or deficient signaling, but from sustained signaling. Exhaustion is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of diseases (e.g., infection and tumors). Exhaustion can result from both extrinsic negative regulatory pathways (e.g., immunoregulatory cytokines) as well as cell intrinsic negative regulatory pathways (inhibitory immune checkpoint pathways, such as described herein). "Enhancing T cell function" means to induce, cause or stimulate a T cell to have a sustained or amplified biological function, or renew or reactivate exhausted or inactive T cells. Examples of enhancing T cell function include increased secretion of y-interferon from CD8+ T cells, increased proliferation, increased antigen responsiveness (e.g., tumor clearance) relative to such levels before the intervention. In one embodiment, the level of enhancement is as 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. Manners of measuring this enhancement are known to one of ordinary skill in the art.

The term "inhibitory nucleic acid" or "inhibitory nucleic acid molecule" as used herein refers to a nucleic acid molecule, e.g., DNA or RNA, that totally or partially reduces, inhibits, interferes with or negatively modulates one or more checkpoint proteins. Inhibitory nucleic acid molecules include, without limitation, oligonucleotides, siRNA, shRNA, antisense DNA or RNA molecules, and aptamers (e.g., DNA or RNA aptamers).

The term "oligonucleotide" as used herein refers to a nucleic acid molecule that is able to decrease protein expression, in particular expression of a checkpoint protein, such as the checkpoint proteins described herein. Oligonucleotides are short DNA or RNA molecules, typically comprising from 2 to 50 nucleotides. Oligonucleotides maybe single-stranded or double-stranded. A checkpoint inhibitor oligonucleotide may be an antisense-oligonucleotide.

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

The terms "siRNA" or "small interfering RNA" or "small inhibitory RNA" are used interchangeably herein and refer to a double-stranded RNA molecule with a typical length of 20-25 base pairs that interferes with expression of a specific gene, such as a gene coding for a checkpoint protein, with a complementary nucleotide sequence. In one embodiment, siRNA interferes with mRNA therefore blocking translation, e.g., translation of an immune checkpoint protein. Transfection of exogenous siRNA may be used for gene knockdown, however, the effect maybe only transient, especially in rapidly dividing cells. Stable transfection may be achieved, e.g., by RNA modification or by using an expression vector. Useful modifications and vectors for stable transfection of cells with siRNA are known in the art. siRNA sequences may also be modified to introduce a short loop between the two strands resulting in a "small hairpin RNA" or "shRNA". shRNA can be processed into a functional siRNA by Dicer. shRNA has a relatively low rate of degradation and turnover. Accordingly, the immune checkpoint inhibitor may be a shRNA.

The term "aptamer" as used herein refers to a single -stranded nucleic acid molecule, such as DNA or RNA, typically in a length of 25-70 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 the immune checkpoint proteins described herein. For example, an aptamer according to the disclosure can specifically bind to an immune checkpoint protein or polypeptide, or to a molecule in a signaling pathway that modulates the expression of an immune checkpoint protein or polypeptide. The generation and therapeutic use of aptamers is well known in the art (see, e.g., US 5,475,096).

The terms "small molecule inhibitor" or "small molecule" are used interchangeably herein and refer to a low molecular weight organic compound, usually up to 1000 daltons, that totally or partially reduces, inhibits, interferes with, or negatively modulates one or more checkpoint proteins as described above. Such small molecular inhibitors are usually synthesized by organic chemistry, but may also be isolated from natural sources, such as plants, fungi, and microbes. The small molecular weight allows a small molecule inhibitor to rapidly diffuse across cell membranes. For example, various A2AR antagonists known in the art are organic compounds having a molecular weight below 500 daltons.

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

The term "oncolytic virus" as used herein, refers to a virus capable of selectively replicating in and slowing the growth or inducing the death of a cancerous or hyperproliferative cell, either in vitro or in vivo, while having no or minimal effect on normal cells. An oncolytic virus for the delivery of an immune checkpoint inhibitor comprises an expression cassette that may encode an immune checkpoint inhibitor that is an inhibitory nucleic acid molecule, such as a siRNA, shRNA, an oligonucleotide, antisense DNA or RNA, an aptamer, an antibody or a fragment thereof or a soluble immune checkpoint protein or fusion. The oncolytic virus preferably is replication competent and the expression cassette is under the control of a viral promoter, e.g., synthetic early /late poxvirus promoter. Exemplary oncolytic viruses include vesicular stomatitis virus (VSV), rhabdoviruses (e.g., picomaviruses such as Seneca Valley virus; SW-001), coxsackievirus, parvovirus, Newcastle disease virus (NDV), herpes simplex virus (HSV; OncoVEX GMCSF), retroviruses (e.g., influenza viruses), measles virus, reovirus, Sinbis virus, vaccinia virus, as exemplarily described in WO 2017/209053 (including Copenhagen, Western Reserve, Wyeth strains), and adenovirus (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx- 015, ColoAdl, H101, AD5/3-D24-GMCSF). Generation of recombinant oncolytic viruses comprising a soluble form of an immune checkpoint inhibitor and methods for their use are disclosed in WO 2018/022831, herein incorporated by reference in its entirety. Oncolytic viruses can be used as attenuated viruses.

"Treatment cycle" is herein defined as the time period, within the effects of separate dosages of the binding agent add on due to its pharmacodynamics, or in other words the time period after the subject's body is essentially cleared from the administrated biding agent. Multiple small doses in a small time window, e.g. within 2-24 few hours, such as 2-12 hours or on the same day, might be equal to a larger single dose.

In the present context, the term "treatment", "treating" or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications. In one embodiment, "treatment" refers to the administration of an effective amount of a therapeutically active binding agent, such as of a therapeutically active antibody, of the present disclosure with the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states.

The resistance to, failure to respond to and/or relapse from treatment with a binding agent of the present disclosure may be determined according to the Response Evaluation Criteria in Solid Tumors; version 1.1 (RECIST Criteria vl.l). The RECIST Criteria are set forth in the table below (LD: longest dimension).

Table 4: Definition ofiResponse (RECIST Criteria vl.l)

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

The objective response rate (ORR) is the percentage of all subjects in a study or treatment group who have either a partial or complete response to the treatment. The ORR can be calculated by adding the numbers of subjects having CR and the numbers of subjects having PR and dividing the resulting sum by the total number of subjects in the treatment group. The ORR_evai, i.e., the ORR of all evaluable subjects in a study or treatment group, is the percentage of all evaluable subjects in a study or treatment group who have either a partial or complete response to the treatment.

The disease control rate (DCR) is the percentage of all subjects in a study or treatment group who have either a complete response, a partial response, or a stable disease to the treatment (CR, PR or SD). The DCR can be calculated by adding the numbers of subjects having CR, the numbers of subjects having PR, and the numbers of subjects having SD and dividing the resulting sum by the total number of subjects in the treatment group. The DCR_evai, i.e., the DCR of all evaluable subjects in a study or treatment group, is the percentage of all evaluable subjects in a study or treatment group who have either a complete response, a partial response, or a stable disease to the treatment (CR, PR or SD). "Duration of response (DOR)" only applies to subjects whose confirmed best overall response is CR or PR and is defined as the time from the first documentation of objective tumor response (CR or PR) to the date of first PD or death due to underlying cancer.

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

"Overall survival (OS)" is defined as the number of days from Day 1 in Cycle 1 to death due to any cause. If a subject is not known to have died, then OS will be censored at the latest date the subject was known to be alive (on or before the cut-off date).

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

The term "effective amount" or "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of a binding agent, such as an antibody, like a multispecific antibody or monoclonal antibody, may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the binding agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the binding agent or a fragment thereof, are outweighed by the therapeutically beneficial effects. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used. In case that unwanted side effects occur in a patient with a dose, lower doses (or effectively lower doses achieved by a different, more localized route of administration) may be used.

As used herein, the term "cancer" includes a disease characterized by aberrantly regulated cellular growth, proliferation, differentiation, adhesion, and/or migration. By "cancer cell" is meant an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease.

The term "cancer" according to the present disclosure comprises leukemias, seminomas, melanomas, sarcomas, myelomas, teratomas, lymphomas, mesotheliomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, renal cancer, urothelial cancer, adrenal cancer, adrenocortical cancer, thyroid cancer, blood cancer, skin cancer, cancer of the brain, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestine cancer, head and neck cancer, gastric cancer, gastrointestinal cancer, lymph node cancer, esophagus cancer, colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, penile cancer, cancer of the uterus, ovarian cancer and lung cancer and the metastases thereof. Examples thereof are lung carcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas, renal cell carcinomas, cervical carcinomas, or metastases of the cancer types or tumors described above.

The term "cancer" according to the present disclosure also comprises cancer metastases. By "metastasis" is meant the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumor, i.e. a secondary tumor or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the present disclosure relates to "distant metastasis" which relates to a metastasis which is remote from the primary tumor and the regional lymph node system.

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

Terms such as "increase" or "enhance" in one embodiment relate to an increase or enhancement by 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%.

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

As used in the present disclosure, "% by weight" refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g) expressed as a percent of the total weight of the total composition in grams (g).

The term "freezing" relates to the solidification of a liquid, usually with the removal of heat.

The term "lyophilizing" or "lyophilization" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze-drying" are used herein interchangeably.

The term "recombinant" in the context of the present disclosure means "made through genetic engineering". In one embodiment, a "recombinant object" in the context of the present disclosure is not occurring naturally.

The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term "found 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 in the future from a natural source.

According to the present disclosure, the term "peptide" comprises oligo- and polypeptides and refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "protein" refers to large peptides, in particular peptides having at least about 151 amino acids, but the terms "peptide" and "protein" are used herein usually as synonyms.

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

The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or protein the term "portion" thereof may designate a continuous or a discontinuous fraction of said structure.

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

"Fragment", with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C -terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5 '-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, e.g., at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.

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 has been derived. Such functional properties comprise a pharmacological activity, the interaction with other peptides or proteins, an enzymatic activity, the interaction with antibodies, and the selective binding of nucleic acids. E.g., a pharmacological active fragment of a peptide or protein has at least one of the pharmacological activities of the peptide or protein from which the fragment has been derived. A part or fragment of a peptide or protein preferably comprises a sequence of at least 6, in particular 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 comprises a sequence of up to 8, in particular 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.

By "variant" herein is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid modifications, and preferably from 1 to about 10 or from 1 to about 5 amino acid modifications compared to the parent.

By "wild type" or "WT" or "native" herein is meant an amino acid sequence that is found in nature, including allelic variations. A wild type amino acid sequence, peptide or protein has an amino acid sequence that has not been intentionally modified. Preferably the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said 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%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably 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 continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS: meedle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

"Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov/Blast.cgi). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the fdter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.

Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference amino acid sequence consists of 200 amino acid residues, the degree of identity is 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 continuous amino acid residues. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

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

The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example. Furthermore, the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.

In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to antigens or antigenic sequences, one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., inducing an immune response. In one embodiment, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

An amino acid sequence (peptide, protein or polypeptide) "derived from" a designated amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.

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

The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acid. The term "transfection" relates to the introduction of nucleic acids, in particular RNA, into a cell. For purposes of the present disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient. Thus, according to the present disclosure, a cell for transfection of a nucleic acid described herein can be present in vitro or in vivo, e.g. the cell can form part of an organ, a tissue and/or an organism of a patient. According to the present disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection. Generally, nucleic acid encoding antigen is transiently transfected into cells. RNA can be transfected into cells to transiently express its coded protein.

According to the present disclosure, an analog of a peptide or protein is a modified form of said peptide or protein from which it has been derived and has at least one functional property of said peptide or protein. E.g., a pharmacological active analog of a peptide or protein has at least one of the pharmacological activities of the peptide or protein from which the analog has been derived. Such modifications include any chemical modification and comprise single or multiple substitutions, deletions and/or additions of any molecules associated with the protein or peptide, such as carbohydrates, lipids and/or proteins or peptides. In one embodiment, "analogs" of proteins or peptides include those modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protective/blocking groups, proteolytic cleavage or binding to an antibody or to another cellular ligand. The term "analog" also extends to all functional chemical equivalents of said proteins and peptides.

"Activation" or "stimulation", as used herein, refers to the state of an immune effector cell such as T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with initiation of signaling pathways, induced cytokine production, and detectable effector functions. The term "activated immune effector cells" refers to, among other things, immune effector cells that are undergoing cell division.

The term "priming" refers to a process wherein an immune effector cell such as a T cell has its first contact with its specific antigen and causes differentiation into effector cells such as effector T cells.

The term "clonal expansion" or "expansion" refers to a process wherein a specific entity is multiplied. In the context of the present disclosure, the term is preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cell recognizing said antigen is amplified. Preferably, clonal expansion leads to differentiation of the immune effector cells. An "antigen" according to the present disclosure covers any substance that will elicit an immune response and/or any substance against which an immune response or an immune mechanism such as a cellular response is directed. This also includes situations wherein the antigen is processed into antigen peptides and an immune response or an immune mechanism is directed against one or more antigen peptides, in particular if presented in the context of MHC molecules. In particular, an "antigen" relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T- lymphocytes (T-cells). According to the present disclosure, the term "antigen" comprises any molecule which comprises at least one epitope, such as a T cell epitope. Preferably, an antigen in the context of the present disclosure is a molecule which, optionally after processing, induces an immune reaction, which is preferably specific for the antigen (including cells expressing the antigen). In one embodiment, an antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such antigen.

According to the present disclosure, any suitable antigen may be used, which is a candidate for an immune response, wherein the immune response may be both a humoral as well as a cellular immune response. In the context of some embodiments of the present disclosure, the antigen is preferably presented by a cell, preferably by an antigen presenting cell, in the context of MHC molecules, which results in an immune response against the antigen. An antigen is preferably a product which corresponds to or is derived from a naturally occurring antigen. Such naturally occurring antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens or an antigen may also be a tumor antigen. According to the present disclosure, an antigen may correspond to a naturally occurring product, for example, a viral protein, or a part thereof.

The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule which contains epitopes that will stimulate a host's immune system to make a cellular antigen-specific immune response and/or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens which are associated with infection by microbes, typically microbial antigens (such as bacterial or viral antigens), or antigens associated with cancer, typically tumors, such as tumor antigens.

In a preferred embodiment, the antigen is a tumor antigen, i.e., a part of a tumor cell, in particular those which primarily occur intracellularly or as surface antigens of tumor cells. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, e.g., from a virus, bacterium, unicellular organism, or parasite, for example a viral antigen such as viral ribonucleoprotein or coat protein. In particular, the antigen should be presented by MHC molecules which results in modulation, in particular activation of cells of the immune system, preferably CD4⁺ and CD8⁺ lymphocytes, in particular via the modulation of the activity of a T-cell receptor. The term "tumor antigen" refers to a constituent of cancer cells which may be derived from the cytoplasm, the cell surface or the cell nucleus. In particular, it refers to those antigens which are produced intracellularly or as surface antigens on tumor cells. For example, tumor antigens include the carcinoembryonal antigen, al -fetoprotein, isoferritin, and fetal sulphoglycoprotein, a2-H-ferroprotein and y-fetoprotein, as well as various virus tumor antigens. According to the present disclosure, a tumor antigen preferably comprises any antigen which is characteristic for tumors or cancers as well as for tumor or cancer cells with respect to type and/or expression level.

The term "viral antigen" refers to any viral component having antigenic properties, i.e., being able to provoke an immune response in an individual. The viral antigen may be a viral ribonucleoprotein or an envelope protein.

The term "bacterial antigen" refers to any bacterial component having antigenic properties, i.e. being able to provoke an immune response in an individual. The bacterial antigen may be derived from the cell wall or cytoplasm membrane of the bacterium.

The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by antibodies T cells or B cells, in particular when presented in the context of MHC molecules. In one embodiment, "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope may comprise amino acid residues directly involved in the binding and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked or covered by the specifically antigen-binding peptide (in other words, the amino acid residue is within the footprint of the specifically antigen-binding peptide).

An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably between about 5 and about 100, preferably between about 5 and about 50, more preferably between about 8 and about 0, most preferably between about 10 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. It is particularly preferred that the epitope in the context of the present disclosure is a T cell epitope. Terms such as "epitope", "fragment of an antigen", "immunogenic peptide" and "antigen peptide" are used interchangeably herein and preferably relate to an incomplete representation of an antigen which is preferably capable of eliciting an immune response against the antigen or a cell expressing or comprising and preferably presenting the antigen. Preferably, the terms relate to an immunogenic portion of an antigen. Preferably, it is a portion of an antigen that is recognized (/.e., specifically bound) by a T cell receptor, in particular if presented in the context of MHC molecules. Certain preferred immunogenic portions bind to an MHC class I or class II molecule. 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, the epitope may be recognized by T cells, B cells or antibodies. An epitope of an antigen may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, such as between about 5 and about 50, more preferably between about 8 and about 30, most preferably between about 8 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, an epitope is between about 10 and about 25 amino acids in length. The term "epitope" includes T cell epitopes.

The term "T cell epitope" refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" includes MHC class I and MHC class II molecules and relates to a complex of genes which is present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells. The proteins encoded by the MHC are expressed on the surface of cells, and display both selfantigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T cell. In the case of class I MHC/peptide complexes, the binding peptides are typically about 8 to about 10 amino acids long although longer or shorter peptides may be effective. In the case of class II MHC/peptide complexes, the binding peptides are typically about 10 to about 25 amino acids long and are in particular about 13 to about 18 amino acids long, whereas longer and shorter peptides may be effective.

The peptide and protein antigen can be 2 to 100 amino acids, 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 length. In some embodiments, a peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.

The peptide or protein antigen can be any peptide or protein that can induce or increase the ability of the immune system to develop antibodies and T cell responses to the peptide or protein. In one embodiment, vaccine antigen, i.e., an antigen whose inoculation into a subject induces an immune response, is recognized by an immune effector cell. Preferably, the vaccine antigen if recognized by an immune effector cell is able to induce in the presence of appropriate co-stimulatory signals, stimulation, priming and/or expansion of the immune effector cell carrying an antigen receptor recognizing the vaccine antigen. 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, an antigen is presented by a diseased cell (such as tumor cell or an infected cell). In one embodiment, an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC. In one embodiment, binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and/or expansion of said T cells. In one embodiment, binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented on diseased cells results in cytolysis and/or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g., perforins and granzymes.

In one embodiment, an antigen receptor is an antibody or B cell receptor which binds to an epitope in an antigen. In one embodiment, an antibody or B cell receptor binds to native epitopes of an antigen.

The term "expressed on the cell surface" or "associated with the cell surface" means that a molecule such as an antigen is associated with and located at the plasma membrane of a cell, wherein at least a part of the molecule faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by antibodies located outside the cell. In this context, a part is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association may be direct or indirect. For example, the association may be by one or more transmembrane domains, one or more lipid anchors, or by the interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane protein.

"Cell surface" or "surface of a cell" is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. An antigen is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by, e.g., antigen-specific antibodies added to the cells.

The term "extracellular portion" or "exodomain" in the context of the present disclosure refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or a fragment thereof. 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) which comprise cytolytic T cells. The term "antigenspecific T cell" or similar terms relate to a T cell which recognizes the antigen to which the T cell is targeted, in particular when presented on the surface of antigen presenting cells or diseased cells such as cancer cells in the context of MHC molecules and preferably exerts effector functions of T cells. T cells are considered to be specific for antigen if the cells kill target cells expressing an antigen. T cell specificity may be evaluated using any of a variety of standard techniques, for example, within a chromium release assay or proliferation assay. Alternatively, synthesis of lymphokines (such as interferon-y) can be measured. In certain embodiments of the present disclosure, the RNA (in particular mRNA) encodes at least one epitope.

The term "target" shall mean an agent such as a cell or tissue which is a target for an immune response such as a cellular immune response. Targets include cells that present an antigen or an antigen epitope, i.e., a peptide fragment derived from an antigen. In one embodiment, the target cell is a cell expressing an antigen and preferably presenting said antigen with class I MHC.

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

By "antigen-responsive CTL" is meant a CD8⁺ T-cell that is responsive to an antigen or a peptide derived from said antigen, which is presented with class I MHC on the surface of antigen presenting cells.

According to the present disclosure, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFNy and TNFa, up-regulation of activation markers such as CD44 and CD69, and specific cytolytic killing of tumor antigen expressing target cells. CTL responsiveness may also be determined using an artificial reporter that accurately indicates CTL responsiveness.

The terms "immune response" and "immune reaction" are used herein interchangeably in their conventional meaning and refer to an integrated bodily response to an antigen and preferably refers to a cellular immune response, a humoral immune response, or both. According to the present disclosure, the term "immune response to" or "immune response against" with respect to an agent such as an antigen, cell or tissue, relates to an immune response such as a cellular response directed against the agent. An immune response may comprise one or more reactions selected from the group consisting of developing 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 may be detected in various proliferation or cytokine production tests in vitro.

The terms "inducing an immune response" and "eliciting an immune response" and similar terms in the context of the present disclosure refer to the induction of an immune response, preferably the induction of a cellular immune response, a humoral immune response, or both. The immune response may be protective/preventive/prophylactic and/or therapeutic. The immune response may be directed against any immunogen or antigen or antigen 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)). "Inducing" in this context may mean that there was no immune response against a particular antigen or pathogen before induction, but it may also mean that there was a certain level of immune response against a particular antigen or pathogen before induction and after induction said immune response is enhanced. Thus, "inducing the immune response" in this context also includes "enhancing the immune response". Preferably, after inducing an immune response in an individual, said individual is protected from developing a disease such as an infectious disease or a cancerous disease or the disease condition is ameliorated by inducing an immune response.

The terms "cellular immune response", "cellular response", "cell-mediated immunity" or similar terms are meant to include a cellular response directed to cells characterized by expression of an antigen and/or presentation of an antigen with class I or class II MHC. The cellular response relates to cells called T cells or T lymphocytes which act as either "helpers" or "killers". The helper T cells (also termed CD4⁺ T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8⁺ T cells or CTLs) kill cells such as diseased cells.

The term "humoral immune response" refers to a process in living organisms wherein antibodies are produced in response to agents and organisms, which they ultimately neutralize and/or eliminate. The specificity of the antibody response is mediated by T and/or B cells through membrane-associated receptors that bind antigen of a single specificity. Following binding of an appropriate antigen and receipt of various other activating signals, B lymphocytes divide, which produces memory B cells as well as antibody secreting plasma cell clones, each producing antibodies that recognize the identical antigenic epitope as was recognized by its antigen receptor. Memory B lymphocytes remain dormant until they are subsequently activated by their specific antigen. These lymphocytes provide the cellular basis of memory and the resulting escalation in antibody response when re-exposed to a specific antigen.

The terms "vaccination" and "immunization" describe the process of treating an individual for therapeutic or prophylactic reasons and relate to the procedure of administering one or more immunogen(s) or antigen(s) or derivatives thereof, in particular in the form of RNA (especially mRNA) coding therefor, as described herein to an individual and stimulating an immune response against said one or more immunogen(s) or antigen(s) or cells characterized by presentation of said one or more immunogen(s) or antigen(s).

By "cell characterized by presentation of an antigen" or "cell presenting an antigen" or "MHC molecules which present an antigen on the surface of an antigen presenting cell" or similar expressions is meant a cell such as a diseased cell, in particular a tumor cell or an infected cell, or an antigen presenting cell presenting the antigen or an antigen peptide, either directly or following processing, in the context of MHC molecules, preferably MHC class I and/or MHC class II molecules, most preferably MHC class I molecules.

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

The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence. With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

The term "optional" or "optionally" as used herein means that the subsequently described event, circumstance or condition may or may not occur, and that the description includes instances where said event, circumstance, or condition occurs and instances in which it does not occur.

As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

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

The term "disease" (also referred to as "disorder" herein) refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one's perspective on life, and one's personality.

The term "therapeutic treatment" relates to any treatment which improves the health status and/or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and/or decrease the recurrence in an individual who currently has or who previously has had a disease.

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

The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate), or any other nonmammal-animal, including birds (chicken), fish or any other animal species that can be afflicted with or is susceptible to a disease or disorder (e.g., cancer, infectious diseases) but may or may not have the disease or disorder, or may have a need for prophylactic intervention such as vaccination, or may have a need for interventions such as by protein replacement. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In embodiments of the present disclosure, the "individual" or "subject" is a "patient".

The term "patient" means an individual or subject for treatment, in particular a diseased individual or subject. Aspects and embodiments of the present disclosure

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

As demonstrated in the present disclosure, a combination of (i) stimulation with a binding agent binding human CD40 and binding 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 amplifies the immune response. Without being bound to any theory, the rational behind this surprising finding could be as follows: CD137 is co-expressed on PD-1⁺ cells. Thus, blockade of PD-L1/PD-1 signals and costimulation through CD137 can synergize to enhance T-cell effector functions and improve the duration of the response. Through conditional activation of CD40 and CD137, a binding agent targeting CD40 and CD137 induces potent anti-tumor activity through enhanced T-cell priming, cytokine and chemokine production, and expansion and survival of antigen-experienced T cells. The PD-(L)1 pathway is expected to be activated during priming as well as during continuous antigen exposure, which may reduce the magnitude of the immune response induced by the binding agent targeting CD40 and CD137.

Pembrolizumab or pembrolizumab in combination with platinum and 5 -fluorouracil (5-FU) became the global Standard of Care (SOC) for patients with previously untreated recurrent or metastatic HNSCC based on the results of the KEYNOTE-048 (KN-048) trial. In the KN-048 trial, patients were randomized to receive pembrolizumab, pembrolizumab with platinum (cisplatin or carboplatin) and 5- FU, or the EXTREME regimen (cisplatin or carboplatin + 5-FU + cetuximab).

In KN-048, overall survival (OS) benefit was observed in IL HNSCC patients with PD-L1 CPS >1 in the pembrolizumab+platinum+5-FU arm vs. the EXTREME regimen respectively (median OS was 13.6 months vs. 10.4 months, respectively); 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 GCT 1042-01 study as of the data cutoff, the addition of a binding agent binding to CD40 and CD137 as disclosed herein (in particular, GEN1042) to the combination of pembrolizumab+platinum+5-FU regimen appears to improve ORR vs. platinum + 5-FU + pembrolizumab alone (66.7% vs. 36.4%). Thus, in one embodiment, the binding agent for use according to the first aspect of the present disclosure provides an ORR which is increased compared to the SOC, such as compared to an administration regimen of a combination of pembrolizumab, platinum-based chemotherapeutic agent and 5 -fluorouracil only, or compared to an administration regimen of a combination of the binding agent and pembrolizumab only. For example, each of the binding agent, pembrolizumab, and the chemotherapy combination may be administered in a dose to increase the ORR compared to the standard of care, such as compared to an administration regimen of a combination of pembrolizumab, platinum-based chemotherapeutic agent and 5 -fluorouracil only, or compared to an administration regimen of a combination of the binding agent and pembrolizumab only. In some of the above embodiments, the ORR may 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%.

Alternatively, or additionally, in one embodiment, the binding agent for use according to the first aspect of the present disclosure provides an ORR_eVai which is increased compared to the SOC, such as compared to an administration regimen of a combination of pembrolizumab, platinum-based chemotherapeutic agent and 5 -fluorouracil only, or compared to an administration regimen of a combination of the binding agent and pembrolizumab only. For example, each of the binding agent, pembrolizumab, and the chemotherapy combination may be administered in a dose to increase the ORRevai compared to the standard of care, such as compared to an administration regimen of a combination of pembrolizumab, platinum-based chemotherapeutic agent and 5 -fluorouracil only, or compared to an administration regimen of a combination of the binding agent and pembrolizumab only. In some of the above embodiments, the ORR_eVai may 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%. Alternatively, or additionally, in one embodiment, the binding agent for use according to the first aspect of the present disclosure provides an DCR which is increased compared to the SOC, such as compared to an administration regimen of a combination of pembrolizumab, platinum-based chemotherapeutic agent and 5 -fluorouracil only, or compared to an administration regimen of a combination of the binding agent and pembrolizumab only. For example, each of the binding agent, pembrolizumab, and the chemotherapy combination may be administered in a dose to increase the DCR compared to the standard of care, such as compared to an administration regimen of a combination of pembrolizumab, platinumbased chemotherapeutic agent and 5 -fluorouracil only, or compared to an administration regimen of a combination of the binding agent and pembrolizumab only. In some of the above embodiments, the DCR may 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%.

Alternatively, or additionally, in one embodiment, the binding agent for use according to the first aspect of the present disclosure provides an DCR_evai which is increased compared to the SOC, such as compared to an administration regimen of a combination of pembrolizumab, platinum-based chemotherapeutic agent and 5 -fluorouracil only, or compared to an administration regimen of a combination of the binding agent and pembrolizumab only. For example, each of the binding agent, pembrolizumab, and the chemotherapy combination may be administered in a dose to increase the DCRevai compared to the standard of care, such as compared to an administration regimen of a combination of pembrolizumab, platinum-based chemotherapeutic agent and 5 -fluorouracil only, or compared to an administration regimen of a combination of the binding agent and pembrolizumab only. In some of the above embodiments, the DCR_evai may 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%. Binding, agent binding to CD40 and CD137

In one embodiment, CD40 is human CD40, in particular human CD40 comprising the sequence set forth in SEQ ID NO: 36. In one embodiment, CD137 is human CD137, in particular human CD137 comprising the sequence set forth 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 set forth in SEQ ID NO: 36, and CD137 is human CD137 comprising the sequence set forth in SEQ ID NO: 38.

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

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

In one embodiment of the binding agent according to the first aspect, a) the first binding region binding to human CD40 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 7 or 9 and a light chain variable region (VL) region and comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or 10; b) the second binding region binding to human CD 137 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 17 or 19 and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 18 or 20.

In one embodiment of the binding agent according to the first aspect, a) the first binding region binding to human CD40 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 7 or 9 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; and b) the second binding region binding to human CD 137 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 18 or 20.

In one embodiment of the binding agent according to the first aspect, a) the first binding region binding to human CD40 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 10; and b) the second binding region binding to human CD 137 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 20.

The binding agent may in particular be an antibody, such as a multispecific antibody, e.g., a bispecific antibody. Also, the binding agent may be in the format of a full-length antibody or an antibody fragment.

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

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

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

In one embodiment of the first aspect, the binding agent comprises i) a polypeptide comprising said first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising said second heavy chain variable region (VH) and a second heavy chain constant region (CH).

In one embodiment of the first aspect, the binding agent comprises i) a polypeptide comprising said first light chain variable region (VL) and further comprising a first light chain constant region (CL), and ii) a polypeptide comprising said second light chain variable region (VL) and further comprising a second light chain constant region (CL).

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

In one embodiment of the first aspect, the binding agent comprises i) a first heavy chain and light chain comprising said 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 light chain comprising said antigen-binding region capable of binding CD137, the second heavy chain comprising a second heavy chain constant region and the second light chain comprising a second light chain constant region.

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

Each of the first and second heavy chain constant regions (CHs) may comprise a CH3 region, wherein the two CH3 regions comprise asymmetrical mutations. Asymmetrical mutations mean that the sequences of said first and second CH3 regions contain amino acid substitutions at non-identical positions. For example, one of said first and second CH3 regions contains a mutation at the position corresponding to position 405 in a human IgGl heavy chain according to EU numbering, and the other of said first and second CH3 regions contains a mutation at the position corresponding to position 409 in a human IgGl heavy chain according to EU numbering.

In said first heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgGl heavy chain according to EU numbering may have been substituted, and in said second heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgGl heavy chain according to EU numbering may have been substituted. In particular embodiments, the first and the second heavy chains are not substituted in the same positions (i.e., the first and the second heavy chains contain asymmetrical mutations).

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

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

In one particular embodiment of the binding agent according to the first aspect, said first and second heavy chain constant regions (CHs) are modified so that the antibody induces Fc-mediated effector function to a lesser extent compared to an antibody which is identical except for comprising nonmodified first and second heavy chain constant regions (CHs). In particular, each or both of said nonmodified first and second heavy chain constant regions (CHs) may comprise, consists of or consist essentially of the amino acid sequence set forth in SEQ ID NO: 21 or 29.

The Fc-mediated effector function may be determined by measuring binding of the binding agent to Fey receptors, binding to Clq, or induction of Fc-mediated cross-linking of Fey receptors. In particular, the Fc-mediated effector function may be determined by measuring binding of the binding agent to Clq.

The first and second heavy chain constant regions of the binding agent may have been modified so that binding of Clq to said 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 Clq binding is preferably determined by ELISA.

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

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

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

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

In one embodiment of the binding agent according to the first aspect, the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering of both the first and second heavy chain constant regions are F, E, and A, respectively, wherein (i) the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the first heavy chain is R, and the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the second heavy chain is L. In one embodiment of the binding agent according to the first aspect, the constant region of said first and/or second heavy chain comprises an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 29 [IgGl-FC]; b) a subsequence of the sequence in a), such as a subsequence wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/have deleted, starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at the most 10 substitutions, such as at the most 9 substitutions, at the most 8, at the most 7, at the most 6, at the most 5, at the most 4, at the most 3, at the most 2 or at the most 1 substitution compared to the amino acid sequence defined in a) or b).

In one embodiment of the binding agent according to the first aspect, the constant region of said first or second heavy chain, such as the second heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 30 [IgGl-F405L]; b) a subsequence of the sequence in a), such as a subsequence wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/have deleted, starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at the most 9 substitutions, such as at the most 8, at the most 7, at the most 6, at the most 5, at the most 4, at the most 3, at the most 2 or at the most 1 substitution compared to the amino acid sequence defined in a) or b).

In one embodiment of the binding agent according to the first aspect, the constant region of said first or second heavy chain, such as the first heavy chain comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 23 or 31 [IgGl-F409R]; b) a subsequence of the sequence in a), such as a subsequence wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/have deleted, starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at the most 10 substitutions, such as at the most 9 substitutions, at the most 8, at the most 7, at the most 6, at the most 5, at the most 4 substitutions, at the most 3, at the most 2 or at the most 1 substitution compared to the amino acid sequence defined in a) or b).

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

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

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

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

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

In one embodiment of the binding agent according to the first aspect, the first light chain constant region is a kappa (K) light chain constant region or a lambda (X) light chain constant region. In one embodiment of the binding agent according to the first aspect, the second light chain constant region is a lambda (X) light chain constant region or a kappa (K) light chain constant region.

In one embodiment of the binding agent according to the first aspect, the first light chain constant region is a kappa (K) light chain constant region and the second light chain constant region is a lambda (X) light chain constant region or the first light chain constant region is a lambda (X) light chain constant region and the second light chain constant region is a kappa (K) light chain constant region.

In one embodiment of the binding agent according to the first aspect, the kappa (K) light chain comprises an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 27; b) a subsequence of the sequence in a), such as a subsequence wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/have been deleted, starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at the most 10 substitutions, such as at the most 9 substitutions, at the most 8, at the most 7, at the most 6, at the most 5, at the most 4 substitutions, at the most 3, at the most 2 or at the most 1 substitution, compared to the amino acid sequence defined in a) or b).

In one embodiment of the binding agent according to the first aspect, the lambda (X) light chain comprises an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 28; b) a subsequence of the sequence in a), such as a subsequence wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/have been deleted, starting from the N-terminus or C-terminus of the sequence defined in a); and c) a sequence having at the most 10 substitutions, such as at the most 9 substitutions, at the most 8, at the most 7, at the most 6, at the most 5, at the most 4 substitutions, at the most 3, at the most 2 or at the most 1 substitution, compared to the amino acid sequence defined in a) or b).

The binding agent (in particular, antibody) according to the first aspect is of an isotype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4. In particular, the binding agent may be a full-length IgGl antibody. In preferred embodiments of the first aspect, the binding agent (in particular, antibody) is of the IgGlm(f) allotype.

In one embodiment, the binding agent is the antibody GEN1042 as disclosed herein (i.e., the antibody contains humanized VH and VL sequences, the human kappa light chain, and a human IgGl heavy chain described in Table 1; the CD40 binding arm has been produced with the human IgGl heavy chain containing the following amino acid mutations: L234F, L235E, D265A and F405L (FEAL), wherein the amino acid position number is according to EU numbering (corresponding to SEQ ID NO: 33); the CD137 binding arm has been produced with a human IgGl heavy chain containing the following amino acid mutations: L234F, L235E, D265A and K409R (FEAR), wherein the amino acid position number is according to EU numbering (correspond to SEQ ID NO: 34)).

Preferably, the binding agent is administered in a suitable amount, i.e., the amount of binding agent administered, e.g., in each dose and/or treatment cycle, may induce intracellular signaling when binding to CD 137 expressed on another cell. Thus, a binding agent in a suitable amount according to the present disclosure is able to trans-activate two different cells. In humans, CD40 is expressed on a number of cells including antigen-presenting cells (APCs), such as dendritic cells, whereas CD137 is expressed on T cells and other cells. Thus, a binding agent binding to CD40 and CD137 in a suitable amount according to the present disclosure is able to bind simultaneously to an APC and a T cell expressing these receptors. Without being bound by theory, a binding agent may thus (i) mediate cell-to-cell interaction between APCs and T cells by receptor binding and (ii) activate both CD40 and CD137 at once, which is primarily induced by cross-linking and receptor clustering upon cell-to-cell interaction and not necessarily dependent on agonistic activity of the parental monospecific bivalent antibodies. Thus, these transactivating binding agent exert co-stimulatory activity in the context of APC:T cell interactions, and can elicit a T cell response against tumor cells. As such, this mechanism of action can reflect natural T-cell activation via antigen-presentation by activated APCs, allowing for the presentation of a variety of tumor-specific antigens by the APCs to T cells. Without being limited to theory, the costimulatory activity may provide for one or more of (i) only specific T cells being activated (i.e., those that are in contact with an APC) as opposed to any T cell; (ii) re-activation of exhausted T cells, by strong costimulation via activated APCs and CD137 triggering; and (iii) the priming of T cells by inducing antigen presentation by activated APCs and at the same time triggering CD137.

The amount of binding agent administered in each dose and/or treatment cycle may in particular be in a 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%, most preferably more than 50% of said binding agents bind to both, CD40 and CD137.

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

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

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

The binding agent may be administered in any manner and by any route known in the art. In a preferred embodiment, the binding agent is administered systemically, such as parenterally, in particular intravenously.

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

The binding agent can be administered prior to, simultaneously with, or after administration of the inhibitor of the PD-1/PD-L1 axis (PD-1/PD-L1 checkpoint inhibitor).

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

In one embodiment, the binding agent is administered simultaneously with the PD-1/PD-L1 checkpoint inhibitor. For example, the binding agent and the PD-1/PD-L1 checkpoint inhibitor may be administered using a composition comprising both drugs. Alternatively, the binding agent may be administered into one extremity of the subject, and the PD-1/PD-L1 checkpoint inhibitor may be administered into another extremity of the subject.

PD-1/PD-L1 checkpoint inhibitor

In one embodiment, the immune checkpoint inhibitor suitable for use in the methods disclosed herein, is an antagonist of inhibitory signals of the PD-1/PD-L1 axis, e.g., an antibody which targets, 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. Further immune checkpoint proteins that can be targeted according the disclosure are described herein.

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

Inhibiting or blocking of inhibitory immune checkpoint signaling, as described herein, results in preventing or reversing immune-suppression and establishment or enhancement of T cell immunity against cancer cells. In one embodiment, inhibition of immune checkpoint signaling, as described herein, reduces or inhibits dysfunction of the immune system. In one embodiment, inhibition of immune checkpoint signaling, as described herein, renders dysfunctional immune cells less dysfunctional. In one embodiment, inhibition of immune checkpoint signaling, as described herein, renders a dysfunctional T cell less dysfunctional. In one embodiment, the PD-1/PD-L1 immune checkpoint inhibitor prevents the interaction between the checkpoint blocker proteins PD-1 and PD-L1 or PD-L2.

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

In a preferred embodiment, an antibody that is an immune checkpoint inhibitor is an isolated antibody.

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

The PD-1/PD-L1 immune checkpoint inhibitor may be an inhibitory nucleic acid molecule, such as an oligonucleotide, siRNA, shRNA, an antisense DNA or RNA molecule, and an aptamer (e.g., DNA or RNA aptamer), in particular an antisense-oligonucleotide. In one embodiment, the PD-1/PD-L1 immune checkpoint inhibitor being siRNA interferes with mRNA therefore blocking translation, e.g., translation of an immune checkpoint protein.

The PD-1/PD-L1 checkpoint inhibitor may also be in the form of the soluble form of the molecules (or variants thereof) themselves, e.g., a soluble PD-L1 or PD-L1 fusion.

In the context of the disclosure, more than one checkpoint inhibitor can be used, wherein the more than one checkpoint inhibitors are targeting distinct checkpoint pathways or the same checkpoint pathway. Preferably, the more than one checkpoint inhibitors are distinct checkpoint inhibitors. Preferably, if more than one distinct checkpoint inhibitor is used, in particular at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 distinct checkpoint inhibitors are used, preferably 2, 3, 4 or 5 distinct checkpoint inhibitors are used, more preferably 2, 3 or 4 distinct checkpoint inhibitors are used, even more preferably 2 or 3 distinct checkpoint inhibitors are used and most preferably 2 distinct checkpoint inhibitors are used.

In one embodiment, the PD-1/PD-L1 checkpoint inhibitor (or inhibitory immunoregulator or immune checkpoint blocker) is a component of the PD-1/PD-L1 or PD-1/PD-L2 signaling pathway. Accordingly, in one embodiment of the disclosure, the PD-1/PD-L1 checkpoint inhibitor is an inhibitor of the PD-1 signaling pathway. In certain embodiments, the checkpoint inhibitor of the PD-1 signaling pathway is a PD-1 inhibitor. In certain embodiments, the 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, the 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 which 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, antigen-binding portion thereof or a construct thereof binds specifically to PD-1. In certain embodiments, the antibody, antigen-binding portion thereof or a construct thereof binds specifically to PD-L1 and disrupts or inhibits its interaction with PD-1, thereby increasing immune activity. In certain embodiments, the antibody, antigen-binding portion thereof or a construct thereof binds specifically to PD-L2 and disrupts or inhibits its interaction with PD-1, thereby increasing immune activity.

Exemplary PD-1 inhibitors include, without limitation, anti-PD-1 antibodies such as BGB-A317 (BeiGene; see US 8,735,553, WO 2015/35606 and US 2015/0079109), lambrolizumab (e.g., disclosed as hPD109A and its humanized derivatives h409Al, h409A16 and h409A17 in WO2008/156712), AB137132 (Abeam), EH12.2H7 and RMP1-14 (#BE0146; Bioxcell Lifesciences Pvt. LTD.), MIH4 (Affymetrix eBioscience), nivohimab (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; of. US 2015/0203579 and WO 2015/112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., 2007, J. Hematol. Oncol. 70: 136), AMP-224 (GSK-2661380; cf. 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 US 2015/0079109), BI 754091, SHR-1210 (see WO2015/085847), and antibodies 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4 as described in WO 2006/121168, 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 2017/19846), 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 Patel et al., J. ImmunoTher. Cancer, 2017, 5(Suppl 2):P242), sasanlimab (PF-06801591; see Youssef et al., Proc. Am. Assoc. Cancer Res. Ann. Meeting 2017; Cancer Res 2017;77(13 Suppl) Abstract), toripalimab (JS-001; see US 2016/0272708), camrelizumab (SHR- 1210; INCSHR-1210; see US 2016/376367; 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 US 2017/334995; 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-PDl -antibody IgGl-PDl disclosed herein (i.e., comprising the VH sequence as defined in SEQ ID NO: 43, the VL sequence as defined in SEQ ID NO: 44, the Fc sequence as defined in SEQ ID NO: 61, and the kappa sequence as defined in SEQ ID NO: 27), anti-PD-1 antibodies as described, e.g., in US 7,488,802, US 8,008,449, US 8,168,757, WO 03/042402, WO 2010/089411 (further disclosing anti-PD-Ll antibodies), WO 2010/036959, WO 2011/159877 (further disclosing antibodies against TIM-3), WO 2011/082400, WO 2011/161699, WO 2009/014708, WO 03/099196, WO 2009/114335, WO 2012/145493 (further disclosing antibodies against PD-L1), WO 2015/035606, WO 2014/055648 (further disclosing anti-KIR antibodies), US 2018/0185482 (further disclosing anti-PD-Ll 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, small molecule antagonists to the PD-1 signaling pathway as disclosed, e.g., in Shaabani et al., 2018, Expert Op Ther Pat., 28(9):665-678 and Sasikumar and Ramachandra, 2018, BioDrugs, 32(5):481-497, siRNAs directed to PD-1 as disclosed, e.g., in WO 2019/000146 and WO 2018/103501, soluble PD-1 proteins as disclosed in WO 2018/222711 and oncolytic viruses comprising a soluble form of PD-1 as described, e.g., in WO 2018/022831.

In a certain embodiment, 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 IgGl-PDl as disclosed herein.

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

In some embodiments, the CDRs of the anti-PD-1 antibody are delineated 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).

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

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

In certain embodiments, the PD-1/PD-L1 inhibitory immunoregulator is pembrolizumab or an antigenbinding fragment thereof. Anti-PD-1 antibodies of the disclosure are preferably monoclonal, and may be multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, and PD-1 binding fragments of any of the above. In some embodiments, an anti-PD-1 antibody described herein binds specifically to PD-1 (e.g., human PD-1). The immunoglobulin molecules of the disclosure can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

In certain embodiments of the disclosure, the anti-PD-1 antibodies are antigen-binding fragments (e.g., human antigen-binding fragments) as described herein and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, CH3 and CL domains. Also included in the present disclosure are antigen-binding fragments comprising any combination of variable region(s) with a hinge region, CHI, CH2, CH3 and CL domains. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or chicken.

The anti-PD-1 antibodies disclosed herein may be monospecific, bispecific, trispecific or of greater multi specificity. Multispecific antibodies may be specific for different epitopes of PD-1 or may be specific for both PD-1 as well as for a heterologous protein. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., 1991, J. Immunol. 147:60 69; 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.

The anti-PD-1 antibodies disclosed herein may be described or specified in terms of the particular CD Rs they comprise. 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); MacCalhim 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. , "IM GT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003; 27(l):55-77 ("IMGT" numbering scheme); Honegger A and Pliickthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001;309(3):657-70, ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272, ("AbM" numbering scheme). The boundaries of a given CDR may vary depending on the scheme used for identification. In some embodiments, a CDR or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof (e.g., variable region thereof) should be understood to encompass a (or the specific) CDR as defined by any of the aforementioned schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VH or VL region amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes. The scheme for identification of a particular CDR or CDRs may be specified, such as the CDR as defined by the Kabat, Chothia, AbM or IMGT method.

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

In some embodiments, the anti-PD-1 antibodies disclosed herein comprise the CDRs of the antibody nivolumab. See WO 2006/121168. In some embodiments, the CDRs of the antibody nivohimab are delineated 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). The present disclosure encompasses an anti-PD-1 antibody or derivative thereof comprising a heavy or light chain variable domain, said variable domain comprising (a) a set of three CDRs, in which said set of CDRs are from the monoclonal antibody nivolumab, and (b) a set of four framework regions, in which said set of framework regions differs from the set of framework regions in the monoclonal antibody nivolumab, and in which said anti-PD-1 antibody or derivative thereof binds to PD-1. In certain embodiments, the anti-PD-1 antibody is nivolumab.

In some embodiments, the anti-PD-1 antibodies disclosed herein comprise the CDRs of the antibody pembrolizumab. See WO 2008/156712. In some embodiments, the CDRs of the antibody pembrolizumab are delineated 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). The present disclosure encompasses an anti-PD-1 antibody or derivative thereof comprising a heavy or light chain variable domain, said variable domain comprising (a) a set of three CDRs, in which said set of CDRs are from the monoclonal antibody pembrolizumab, and (b) a set of four framework regions, in which said set of framework regions differs from the set of framework regions in the monoclonal antibody pembrolizumab, and in which said anti-PD-1 antibody or derivative thereof binds to PD-1. In certain embodiments, the anti-PD-1 antibody is pembrolizumab. Anti-PD-1 antibodies disclosed herein may also be described or specified in terms of their binding affinity to PD-1 (e.g., human PD-1). Preferred binding affinities include those with a dissociation constant or Kd less than 5 xlO* M, 10* M, 5x10* M, 10* M, 5x 10* M, 10* M, 5x10* M, 10* M, 5x10* M, 10* M, 5x10* M, 10* M, 5x10* M, 10*M, 5x10* M, 10* M, 5x10*° M, 10*° M, SxlO*¹ M, 10*¹ M, 5xl0*² M, IO*² M, 5xl0*³ M, 10*³ M, 5xl0*⁴ M, 10*⁴ M, 5xl0*⁵ M, or 10*⁵ M.

The anti-PD-1 antibodies also include derivatives and constructs that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to PD-1. For example, but not by way of limitation, the anti-PD-1 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 cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative or construct may contain one or more non-classical amino acids.

Exemplary PD-1 ligand inhibitors are PD-L1 inhibitors and PD-L2 inhibitors and include, without limitation, anti-PD-Ll antibodies such as MEDI4736 (durvalumab; AstraZeneca; see WO 2011/066389), MSB-0010718C (see US 2014/0341917), YW243.55.S70 (see SEQ ID NO: 20 of WO 2010/077634 and US 8,217,149), MIH1 (Affymetrix eBioscience; cf. EP 3 230 319), MDX-1105 (Roche/Genentech; see W02013019906 and US 8,217,149) STI-1014 (Sorrento; see W02013/181634), CK-301 (Checkpoint Therapeutics), KN035 (3D Med/Alphamab; see Zhang et al., 2017, Cell Discov. 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; cf. US 2014/0341917), LY3300054 (Eli Lilly Co.), CX-072 (Proclaim-CX-072; also called CytomX; see W02016/149201), FAZ053, KN035 (see W02017020801 and WO2017020802), MDX-1105 (see US 2015/0320859), anti-PD-Ll antibodies disclosed in US 7,943,743, including 3G10, 12A4 (also referred to as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, anti-PD-Ll antibodies as described 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, US 2009/0317368, WO

2011/066389, WO2017/034916, WO2017/020291, WO2017/020858, WO2017/020801, WO2016/111645, WO2016/197367, W02016/061142, WO2016/ 149201, WO2016/000619, WO2016/160792, WO2016/022630, W02016/007235, WO2015/ 179654, WO2015/173267, WO2015/181342, W02015/109124, WO 2018/222711, WO2015/112805, WO2015/061668, WO2014/159562, WO2014/165082, W02014/100079. In a certain embodiment, the PD-L1 inhibitor is atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267; see US 9,724,413).

In certain embodiments, the PD-1/PD-L1 inhibitory immunoregulator is an anti-PD-Ll antibody or antigen-binding fragment thereof comprising the complementary determining regions (CD Rs) of one of the anti-PD-Ll antibodies or antigen-binding fragments described above, such as the CDRs of atezolizumab or an antigen-binding fragment thereof.

In some embodiments, the CDRs of the anti-PD-Ll antibody are delineated 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).

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

Anti-PD-Ll antibodies of the disclosure are preferably monoclonal, and may be multispecific, human, humanized 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 above. In some embodiments, an anti-PD-Ll antibody described herein binds specifically to PD-L1 (e.g., human PD- Ll). The immunoglobulin molecules of the disclosure can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

In certain embodiments of the disclosure, the anti-PD-L 1 antibodies are antigen-binding fragments (e.g. , human antigen-binding fragments) as described herein and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, CH3 and CL domains. Also included in the present disclosure are antigen-binding fragments comprising any combination of variable region(s) with a hinge region, CHI, CH2, CH3 and CL domains. In some embodiments, the anti-PD-Ll antibodies or antigen-binding fragments thereof are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or chicken. The anti-PD-Ll antibodies disclosed herein may be monospecific, bispecific, trispecific or of greater multi specificity. Multispecific antibodies may be specific for different epitopes of PD-L1 or may be specific for both PD-L1 as well as for a heterologous protein. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., 1991, J. Immunol. 147:60 69; 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.

The anti-PD-Ll antibodies disclosed herein may be described or specified in terms of the particular CDRs they comprise. 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. , "IM GT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003; 27(l):55-77 ("IMGT" numbering scheme); Honegger A and Pliickthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001;309(3):657-70, ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272, ("AbM" numbering scheme). The boundaries of a given CDR may vary depending on the scheme used for identification. In some embodiments, a CDR or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof (e.g., variable region thereof) should be understood to encompass a (or the specific) CDR as defined by any of the aforementioned schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given V_H or VL region amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes. The scheme for identification of a particular CDR or CDRs may be specified, such as the CDR as defined by the Kabat, Chothia, AbM or IMGT method.

In some embodiments, numbering of amino acid residues in CDR sequences of anti-PD-Ll antibodies or antigen-binding fragments thereof provided herein are according to the IMGT numbering scheme as described in Lefranc, M. P. et al., Dev. Comp. Immunol., 2003, 27, 55-77.

In some embodiments, the anti-PD-Ll antibodies disclosed herein comprise the CDRs of the antibody atezolizumab. See US 9,724,413. In some embodiments, the CDRs of the antibody atezolizumab are delineated 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). The present disclosure encompasses an anti-PD-Ll antibody or derivative thereof comprising a heavy or light chain variable domain, said variable domain comprising (a) a set of three CDRs, in which said set of CDRs are from the monoclonal antibody atezolizumab, and (b) a set of four framework regions, in which said set of framework regions differs from the set of framework regions in the monoclonal antibody atezolizumab, and in which said anti-PD-Ll antibody or derivative thereof binds to PD-L1. In certain embodiments, the anti-PD-Ll antibody is atezolizumab.

Anti-PD-Ll antibodies disclosed herein may also be described or specified in terms of their binding affinity to PD-L1 (e.g., human PD-L1). Preferred binding affinities include those with a dissociation constant or Kd less than 5 xlO* M, 10* M, 5x10* M, 10* M, 5x 10* M, 10* M, 5x10* M, 10* M, 5x10* M, 10* M, 5x10* M, 10* M, 5x10* M, 10*M, 5x10* M, 10* M, 5xl0-¹⁰ M, 10*° M, SxlO*¹ M, 10*¹ M, 5xl0*² M, IO*² M, 5xl0*³ M, 10*³ M, 5xl0*⁴ M, 10*⁴ M, 5xl0*⁵ M, or 10*⁵ M.

The anti-PD-Ll antibodies also include derivatives and constructs that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to PD-L1. For example, but not by way of limitation, the anti-PD-Ll 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 cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative or construct may contain one or more non-classical amino acids.

According to the disclosure, the PD-1/PD-L1 immune checkpoint inhibitor is an inhibitor of an inhibitory checkpoint protein but preferably not an inhibitor of a stimulatory checkpoint protein.

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

Checkpoint inhibitors, such as the PD-1/PD-L1 checkpoint inhibitors, may be administered in the form of nucleic acid, such DNA or RNA molecules, encoding an immune checkpoint inhibitor, e.g., an inhibitory nucleic acid molecule or an antibody or fragment thereof. For example, antibodies can be delivered encoded in expression vectors, as described herein. Nucleic acid molecules can be delivered as such, e.g., in the form of a plasmid or mRNA molecule, or complexed with a delivery vehicle, e.g., a liposome, lipoplex or nucleic-acid lipid particles. Checkpoint inhibitors may also be administered via an oncolytic virus comprising an expression cassette encoding the checkpoint inhibitor. Checkpoint inhibitors may also be administered by administration of endogeneic or allogeneic cells able to express a checkpoint inhibitor, e.g., in the form of a cell based therapy.

In one embodiment, the cell based therapy comprises genetically engineered cells. In one embodiment, the genetically engineered cells express an immune checkpoint inhibitor, such as described herein. In one embodiment, the genetically engineered cells express an immune checkpoint inhibitor that is an inhibitory nucleic acid molecule, such as a siRNA, shRNA, an oligonucleotide, antisense DNA or RNA, an aptamer, an antibody or a fragment thereof or a soluble immune checkpoint protein or fusion. Genetically engineered cells may also express further agents that enhance T cell function. Such agents are known in the art. Cell based therapies for the use in inhibition of immune checkpoint signaling are disclosed, e.g., in WO 2018/222711, herein incorporated by reference in its entirety.

Preferably, the checkpoint inhibitor, such as the PD-1/PD-L1 checkpoint inhibitor, is administered in a suitable amount, i.e., the amount of checkpoint inhibitor administered, e.g., in each dose and/or treatment cycle, may totally or partially reduce, inhibit, interfere with or negatively modulate one or more checkpoint proteins or may totally or partially reduce, inhibit, interfere with or negatively modulate expression of one or more checkpoint proteins. Thus, a checkpoint inhibitor in a suitable amount according to the present disclosure is able to totally or partially reduce, inhibit, interfere with or negatively modulate one or more checkpoint proteins or is able to totally or partially reduce, inhibit, interfere with or negatively modulate expression of one or more checkpoint proteins. Therefore, the checkpoint inhibitors preferably prevent inhibitory signals associated with the immune checkpoint resulting in preventing or reversing immune-suppression and establishment or enhancement of T cell immunity against cancer cells.

The amount of checkpoint inhibitor administered in each dose and/or treatment cycle may in particular be in a 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%, most preferably more than 50% of said checkpoint inhibitors bind to the checkpoint protein.

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

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

In preferred embodiments, the amount of pembrolizumab administered, e.g., in each dose and/or in each treatment cycle, is about 1020 x 10'⁹ - 1710 x 10'⁹ mol/day (such as about 1090 x 10'⁹ - 1640 x 10'⁹ mol/day, about 1160 x 10'⁹ - 1570 x 10'⁹ mol/day, about 1230 x 10'⁹ - 1500 x 10'⁹ mol/day, about 1295 x 10"⁹ - 1435 x 10'⁹ mol/day, or about 1330 x 10'⁹ - 1400 x 10'⁹ mol/day, e.g., about 1365 x 10'⁹ mol/day) or about 12.7 x 10'⁹ - 21.4 x 10'⁹ mol/kg body weight/day (such as 13.6 x 10'⁹ - 20.5 x 10'⁹ mol/kg body weight/day, 14.5 x 10'⁹ - 19.6 x 10'⁹ mol/kg body weight/day, 15.3 x 10'⁹ - 18.8 x 10'⁹ mol/kg body weight/day, 16.1 x 10'⁹ - 18.0 x 10'⁹ mol/kg body weight/day, or 16.6 x 10'⁹ - 17.5 x 10'⁹ mol/kg body weight/day, e.g., about 17.1 mol/kg body weight/day).

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

Checkpoint inhibitors, such as the PD-1/PD-L1 checkpoint inhibitors, may 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 agents

Besides the binding agent and the PD-1/PD-L1 checkpoint inhibitor, the treatment regimen according to the first aspect of the present disclosure further comprises administering to the subject a chemotherapy combination comprising (a) a platinum -based chemotherapeutic agent and (b) 5 -fluorouracil.

The platinum-based chemotherapeutic agent and the 5 -fluorouracil may be administered in any manner and by any route known in the art. The mode and route of administration will depend on the type of platinum-based chemotherapeutic agent to be used. In a preferred embodiment, the platinum-based chemotherapeutic agent as well as 5 -fluorouracil are administered systemically, such as parenterally, in particular intravenously.

In one embodiment, the platinum-based compound is selected from platinum-based compounds commonly used in the treatment of a tumor or cancer, in particular HNSCC, such as cisplatin, oxaliplatin, and carboplatin.

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.

In preferred embodiments, the amount of cisplatin administered, e.g., in each dose and/or in each treatment cycle, is about 50-150 mg/m²/day, such as about 60-140 mg/m²/day, about 70-130 mg/m²/day, about 80-120 mg/m²/day, about 90-110 mg/m²/day, or about 95-105 mg/m²/day, e.g., about 100 mg/m²/day.

In preferred embodiments, the amount of carboplatin administered, e.g., in each dose and/or in each treatment cycle, is AUC = about 4 to AUC = about 6, preferably AUC = about 5.

The carboplatin dosing can be calculated by using the following equation (Calvert equation; cf., also Calvert AH, et al, J Clin Oncol. (1989); 7:1748-1756):

Carboplatin Dose (mg) = Target area under the curve (AUC mg/mL/min) x (GFR + 25) wherein GFR is the glomerular filtration rate which can be estimated by calculated creatinine clearance (CrCl) using the following equation (Cockcroft-Gault equation; cf., also Cockcroft DW, et al., Nephron. (1976); 16:31-41):

, _{T /} (140 - age) x (weight in kg) CrCl (male; mL/min)= -

72 x serum creatinine (mg/dL)

CrCl (female; mL/min) = 0.85 x CrCl (male)

In one embodiment, a minimum serum creatine of 0.7 mg/dL is used (in particular, if subjects have an abnormally low serum creatinine level, such as elderly or cachectic subjects).

In one embodiment, an adjusted body weight is used (in particular, if subjects have overweight or are obese), wherein the adjusted body weight can be calculated as follows:

Adjusted body weight (kg) = ideal body weight (IBW) + 0.4 x (total body weight [TBW] - IBW) Further information about the Calvert equation are known to the skilled person (of., e.g., US Food & Drug Administration. Carboplatin dosing. Available at: https://wayback.archive- it.org/7993/20170113081146/http://www.fda.gov/AboutFDA/CentersOffices/OfficeofMedicalProduct sandTobacco/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., Br J Cancer. (1990); 62(2):257-262; Mann JR, et al., Med Pediatr Oncol. (1998); 20(4):217-227; Schwartz GJ, et al., J Am Soc Nephrol. (2009); 20(3):629-637; which are all incorporated by reference).

In one embodiment, the maximum carboplatin dose is capped for the desired AUC to avoid potential toxicity due to overdosing. The maximum dose may be based on a GFR estimate that is capped at 125 mL/min (in particular for subjects with normal renal function). Thus, in one embodiment, the maximum carboplatin dose for (i) AUC = 4 is 600 mg (= (125+25)x4); (ii) AUC = 5 is 750 mg (= (125+25)x5); or (iii) AUC =6 is 900 mg (= (125+25)x6).

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

In preferred embodiments, the amount of 5 -fluorouracil administered, e.g., in each dose and/or in each treatment cycle, is about 500-1500 mg/m²/day, such as about 600-1400 mg/m²/day, about 700-1300 mg/m²/day, about 800-1200 mg/m²/day, about 900-1100 mg/m²/day, or about 950-1050 mg/m²/day, e.g., about 1000 mg/m²/day.

Subject and tumor or cancer to be treated

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

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

Over 600,000 cases of HNSCC are diagnosed annually worldwide. In 2020, approximately 65,630 new cases of oral cavity, pharyngeal, and laryngeal cancers and an estimated 14,500 deaths will occur over the same period in the US (NCCN, 2021b). Tobacco use, alcohol use, and human papillomavirus (HPV) infection increase the risk of developing HNSCC. Patients with locally HPV-positive HNSCC have improved treatment outcomes compared with patients with HPV-negative disease. For patients with recurrent or metastatic HNSCC, pembrolizumab/platinum (cisplatin or carboplatin)/5-FU and pembrolizumab monotherapy (for patients with PD-L1 combined positive score [CPS] > 20 or > 1) are recommended IL regimens; however, the median overall survival (mOS) is less than 15 months (NCCN, 2021b). Therefore, HNSCC remains an area of high unmet medical need and further opportunity exists to improve outcomes with novel treatment approaches.

In one embodiment, histologically or cytologically-confirmed recurrent or metastatic HNSCC is considered incurable by local therapies.

In one embodiment, the subject has not had prior anti-cancer therapy for recurrent or metastatic disease (such as systemic anti-cancer therapy administered in the recurrent or metastatic setting). Anti-cancer therapy which was completed more than 6 months prior to signing consent if given as part of multimodal treatment for locally advanced disease is allowed.

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

In one embodiment, the subject does not have a primary tumor site of nasopharynx (any histology).

In one embodiment, the subject has tumor PD-L1 combined positive score (CPS) (such as tumor PD-L1 IHC CPS) > 1, preferably > 1 and < 19 (which may be determined by local (preferably an FDA-approved test) or central laboratory testing (central testing is mandated for the expansion phase)). In one embodiment, the subject has tumor PD-L1 CPS (such as tumor PD-L1 IHC CPS) > 20.

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

In one embodiment, the subject, e.g., the human subject (such as the 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 therapy for recurrent or metastatic disease. In one embodiment, the subject, e.g., the human subject (such as the 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 therapy or with a checkpoint inhibitor and any anti-cancer therapy.

Treatment regimen The binding agent, the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab), and the chemotherapeutic agents (i.e., a platinum-based chemotherapeutic agent (such as cisplatin or carboplatin) and 5 -fluorouracil) can be administered by any suitable way, such as intravenously, intraarterially, subcutaneously, intradermally, intramuscularly, intranodally, or intratumorally.

In one embodiment of the first aspect, the binding agent is in particular administered to the subject 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.

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 at least 12, at least 18, at least 24, at least 30, or at least 35 treatment cycles, or until the end of the treatment.

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

In one embodiment, the PD-1/PD-L1 checkpoint inhibitor (in particular, 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 (in particular, pembrolizumab) is administered 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 the treatment.

In one embodiment, the chemotherapeutic agents (i.e., a platinum-based chemotherapeutic agent (such as cisplatin or carboplatin) and 5 -fluorouracil) are in particular administered to the subject by systemic administration. Preferably, the chemotherapeutic agents are administered to the subject by intravenous injection or infusion. In one embodiment, the chemotherapeutic agents are 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).

In one embodiment, the binding agent, the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab), the 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 binding agent and the PD- 1/PD-L 1 checkpoint inhibitor (in particular, 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 the treatment, and the platinum-based chemotherapeutic agent and 5 -fluorouracil are administered in at least the first treatment cycle, such as at least in the first and second treatment cycle, preferably only in the first 6 treatment cycles (which means that administration of both the platinum -based chemotherapeutic agent and 5 -fluorouracil is discontinued after completion of the first 6 treatment cycles).

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).

In particular embodiments, each dose is administered or infused every second week (1Q2W), every third week (1Q3W) or every fourth week (1Q4W), preferably every third week (1Q3W).

In some embodiments, one dose of the binding agent is administered every third week (1Q3W). In some embodiments, one dose of the binding agent is administered on day 1 of each treatment cycle. In some embodiments, one dose of the binding agent is administered on day 1 of each treatment cycle, wherein each treatment cycle is three weeks. In some embodiments, one dose of the binding agent is administered 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 the treatment, wherein the binding agent is administered on day 1 of each treatment cycle and each treatment cycle is three weeks.

In some embodiments, one dose of the PD- 1/PD-L 1 checkpoint inhibitor (in particular, pembrolizumab) is administered every third week (1Q3W). In some embodiments, one dose of the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) is administered on day 1 of each treatment cycle. In some embodiments, one dose of the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) is administered on day 1 of each treatment cycle, wherein each treatment cycle is three weeks. In some embodiments, one dose of the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) is administered 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 the treatment, wherein the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) is administered on day 1 of each treatment cycle and each treatment cycle is three weeks.

In some embodiments, one dose of the binding agent and one dose of the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) are administered every third week (1Q3W). In some embodiments, one dose of the binding agent and one dose of the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) are administered on day 1 of each treatment cycle. In some embodiments, one dose of the binding agent and one dose of the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) are administered on day 1 of each treatment cycle, wherein each treatment cycle is three weeks. In some embodiments, one dose of the binding agent and one dose of the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) are administered 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 the treatment, wherein the binding agent and the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) are administered on day 1 of each treatment cycle and each treatment cycle is three weeks.

In some embodiments, one dose of the chemotherapy combination (cisplatin and 5 -fluorouracil or carboplatin and 5 -fluorouracil) is administered at least every third week (1Q3W).

In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered every third week (1Q3W). In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (such as of at least the first and second treatment cycle, e.g., of only the first 6 treatment cycles). In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (such as of at least the first and second treatment cycle, e.g., of only the first 6 treatment cycles), wherein each treatment cycle is three weeks. In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first and second treatment cycle (such as of only the first 6 treatment cycles), wherein each treatment cycle is three weeks.

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

In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered every third week (1Q3W) and one dose of 5 -fluorouracil is administered at least every third week (1Q3W), such as over four days in the first week. In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (such as of at least the first and second treatment cycle, e.g., of only the first 6 treatment cycles) and one dose of 5- fluorouracil is administered at least on day 1 of at least the first treatment cycle (such as of at least the first and second treatment cycle, e.g., of only the first 6 treatment cycles), such as on days 1, 2, 3, and 4 of at least the first treatment cycle (such as of at least the first and second treatment cycle, e.g., of only the first 6 treatment cycles). In some embodiments, one dose of the platinum -based chemotherapeutic agent is administered on day 1 of at least the first treatment cycle (such as of at least the first and second treatment cycle, e.g., of only the first 6 treatment cycles) and one dose of 5 -fluorouracil is administered at least on day 1 of at least the first treatment cycle (such as of at least the first and second treatment cycle, e.g., of only the first 6 treatment cycles), such as on days 1, 2, 3, and 4 of at least the first treatment cycle (such as of at least the first and second treatment cycle, e.g., of only the first 6 treatment cycles), wherein each treatment cycle is three weeks. In some embodiments, one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first and second treatment cycle (such as of only the first 6 treatment cycles) and one dose of 5 -fluorouracil is administered on days 1, 2, 3, and 4 of at least the first and second treatment cycle (such as of only the first 6 treatment cycles), wherein each treatment cycle is three weeks.

In some embodiments, one dose of the binding agent, one dose of the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab), and one dose of the platinum-based chemotherapeutic agent are administered every third week (1Q3W) and one dose of 5 -fluorouracil is administered at least every third week (1Q3W), such as over four days in the first week.

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

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

In some embodiments, one dose of the binding agent and one dose of the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) are administered 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 the treatment, wherein the binding agent and the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) are administered on day 1 of each treatment cycle; one dose of the platinum-based chemotherapeutic agent is administered on day 1 of at least the first and second treatment cycle (such as of only the first 6 treatment cycles); and one dose of 5 -fluorouracil is administered on days 1, 2, 3, and 4 of at least the first and second treatment cycle (such as of only the first 6 treatment cycles), wherein each treatment cycle is three weeks.

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

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

In each of the above embodiments, a dose of the platinum-based chemotherapeutic agent may be any dose of carboplatin as specified herein or any dose of cisplatin as specified herein. E.g., the dose of carboplatin may be AUC = about 4 to AUC = about 6, preferably AUC = about 5 or about 300-600 mg/day (for AUC = 4), about 350-750 mg/day (for AUC = 5), or about 400-900 mg/day (for AUC = 6) or about 3.7-7.5 mg/kg body weight/day (for AUC = 4), about 4.3-9.4 mg/kg body weight/day (for AUC = 5), or about 5.0-11.3 mg/kg body weight/day (for AUC = 6). Likewise, the dose of cisplatin may be about 50-150 mg/m²/day, such as about 60-140 mg/m²/day, about 70-130 mg/m²/day, about 80-120 mg/m²/day, about 90-110 mg/m²/day, or about 95-105 mg/m²/day, e.g., about 100 mg/m²/day.

In each of the above embodiments, a dose of 5 -fluorouracil may be any dose of 5 -fluorouracil as specified herein, e.g., about 500-1500 mg/m²/day, such as about 600-1400 mg/m²/day, about 700-1300 mg/m²/day, about 800-1200 mg/m²/day, about 900-1100 mg/m²/day, or about 950-1050 mg/m²/day, e.g., about 1000 mg/m²/day

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

The binding agent and the PD-1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) may be administered simultaneously. In an alternative preferred embodiment, the binding agent and the PD- 1/PD-L1 checkpoint inhibitor (in particular, pembrolizumab) are administered separately. In some embodiments, the binding agent is administered prior to the administration of the PD-1/PD-L1 checkpoint inhibitor. In some embodiments, the chemotherapy combination (platinum-based chemotherapeutic agent and 5- fluorouracil) is administered after the administration of the binding agent.

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

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

A composition, in particular the pharmaceutical composition of the binding agent, the pharmaceutical composition of the PD-1/PD-L1 checkpoint inhibitor, and the at least one pharmaceutical composition of the chemotherapy combination, may include diluents, fillers, salts, buffers, detergents (e.g., a nonionic detergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition. 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).

Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, antioxidants and absorption-delaying agents, and the like that are physiologically compatible with the active compound(s), in particular the binding agent, the PD-1/PD-L1 checkpoint inhibitor, the platinum-based chemotherapeutic agent, and 5 -fluorouracil as used herein.

Examples of suitable aqueous and non-aqueous carriers which may be employed in the (pharmaceutical) compositions include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, com oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Other carriers are well known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the (pharmaceutical) compositions is contemplated.

The term "excipient" as used herein refers to a substance which may be present in a (pharmaceutical) composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

The term "diluent" relates a diluting and/or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol, glycerol and water

A (pharmaceutical) composition may also comprise pharmaceutically acceptable antioxidants for instance (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

A (pharmaceutical) composition may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the composition.

A (pharmaceutical) composition may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the composition. The composition as used herein may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and micro- encapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polygly colic acid, collagen, poly -ortho esters, and poly lactic acid alone or with a wax, or other materials well known in the art. Methods for the preparation of such formulations are generally known to those skilled in the art, see e.g. Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

"Pharmaceutically acceptable salts" comprise, for example, acid addition salts which may, for example, be formed 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. Furthermore, 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 counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrative examples of pharmaceutically acceptable salts include, but are not limited to, acetate, adipate, alginate, arginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, hydroxynaphthoate, iodide, isobutyrate, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2- naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylghicamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, phthalate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, suberate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like (see, for example, S. M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 66, pp. 1-19 (1977)). Salts which are not pharmaceutically acceptable may be used for preparing pharmaceutically acceptable salts and are included in the present disclosure.

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

Pharmaceutical compositions for injection must typically be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, micro-emulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be an aqueous or a nonaqueous solvent or dispersion medium containing for instance water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile -fdtered solution thereof. Sterile injectable solutions may be prepared by incorporating the active compounds in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum-drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In a second aspect, the present disclosure provides a method for reducing or preventing progression of HNSCC or treating HNSCC in a subject, said method comprising administering to said subject (i) the binding agent, (ii) an 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 binding agent comprises a first binding region binding to CD40 and a second binding region binding to CD 137. The embodiments disclosed herein with respect to the first aspect (in particular regarding the binding agent, the PD-1/PD-L1 checkpoint inhibitor, the platinum-based chemotherapeutic agent, 5 -fluorouracil, their doses to be administered, their treatment regimens, and the subject) also apply to the method of the second aspect.

In further aspect, the present disclosure provides a kit comprising (i) a binding agent comprising a first binding region binding to CD40 and a second binding region binding 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, as well as such as kit for use in a method for reducing or preventing progression of HNSCC or treating HNSCC in a subject. The embodiments disclosed herein with respect to the first aspect (in particular regarding the binding agent, the PD-l/PD- L1 checkpoint inhibitor, the platinum-based chemotherapeutic agent, 5 -fluorouracil, their doses to be administered and their treatment regimens) also apply to the kit of the further aspect. In one embodiment, the kit comprises at least three containers, wherein one thereof contains the binding agent (as such or in the form of a (pharmaceutical) composition), the second container contains the PD-1/PD-L1 checkpoint inhibitor (as such or in the form of a (pharmaceutical) composition), and the third container contains the platinum -based chemotherapeutic agent and/or 5 -fluorouracil (as such or in the form of (a) (pharmaceutical) composition(s)). It is preferred that the kit comprises at least four containers, wherein one thereof contains the binding agent (as such or in the form of a (pharmaceutical) composition), the second container contains the PD-1/PD-L1 checkpoint inhibitor (as such or in the form of a (pharmaceutical) composition), the third container contains the platinum-based chemotherapeutic agent, e.g., carboplatin and/or cisplatin (as such or in the form of one or two (pharmaceutical) compositions), and the fourth container contains 5 -fluorouracil (as such or in the form of a (pharmaceutical) composition).

In another aspect, the present disclosure provides a kit of the further aspect for use in a method for reducing or preventing progression of HNSCC or treating HNSCC in a subject. The embodiments disclosed herein with respect to the first aspect (in particular regarding the binding agent, the PD-l/PD- L1 checkpoint inhibitor, the platinum-based chemotherapeutic agent, 5 -fluorouracil, their doses to be administered, their treatment regimens, and the subject) and/or the second aspect also apply to the kit for use of the another aspect.

Citation of documents and studies referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the contents of these documents.

The description (including the following examples) is presented to enable a person of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those 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 herein and shown, but are to be accorded the scope consistent with the claims.

Itemized embodiments

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

2. The binding agent for use of item 1, wherein the binding agent, pembrolizumab, and the chemotherapy combination are administered in at least one treatment cycle, each treatment cycle being three weeks (21 days).

2a. The binding agent for use of any one of the preceding items, wherein the binding agent and pembrolizumab are administered in at least 2 treatment cycles, such as 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.

2b. The binding agent for use of any one of the preceding items, wherein the binding agent and pembrolizumab are administered 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 the treatment.

3. The binding agent for use of any one of the preceding items, wherein one dose of the binding agent and one dose of pembrolizumab are administered every third week (1Q3W).

4. The binding agent for use of any one of the preceding items, wherein one dose of the binding agent and one dose of pembrolizumab are administered on day 1 of each treatment cycle.

5. The binding agent for use of any one of the preceding items, wherein the binding agent is administered prior to the administration of pembrolizumab.

5a. The binding agent for use of any one of items 1-4, wherein the binding agent is administered simultaneously with the administration of pembrolizumab.

5b. The binding agent for use of any one of items 1-4, wherein the binding agent is administered after the administration of pembrolizumab.

6. The binding agent for use of any one of the preceding items, wherein one dose of the chemotherapy combination is administered at least every third week (1Q3W) for at least the first treatment cycle.

6a. The binding agent for use of item 6, wherein one dose of the chemotherapy combination is administered at least every third week (1Q3W) for at least the first and the second treatment cycle, preferably for the first 6 treatment cycles.

7. The binding agent for use of any one of the preceding items, wherein one dose of the platinumbased chemotherapeutic agent is administered every third week (1Q3W) for at least the first treatment cycle and one dose of 5 -fluorouracil is administered at least every third week (1Q3W), such as over four days in the first week for at least the first treatment cycle. 7a. The binding agent for use of item 7, wherein one dose of the platinum-based chemotherapeutic agent is administered every third week (1Q3W) for at least the first and the second treatment cycle, preferably for the first 6 treatment cycles, and one dose of 5 -fluorouracil is administered at least every third week (1Q3W), such as over four days in the first week for at least the first and second treatment cycle, preferably for the first 6 treatment cycles.

8. The binding agent for use of any one of the preceding items, wherein one dose of the platinumbased 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, such as on days 1, 2, 3, and 4 of at least the first treatment cycle.

8a. The binding agent for use of any one of the preceding items, wherein the chemotherapy combination is administered after the administration of the binding agent and/or after the administration of pembrolizumab.

9. The binding agent for use of any one of the preceding items, wherein the objective response rate (ORR) is increased compared to the standard of care, such as compared to an administration regimen of pembrolizumab and the chemotherapy combination only.

10. The binding agent for use of any one of the preceding items, wherein the ORR 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%.

11. The binding agent for use of any one of the preceding items, wherein the disease control rate (DCR) is increased compared to the standard of care, such as compared to an administration regimen of pembrolizumab and the chemotherapy combination only.

12. The binding agent for use of any one of the preceding items, 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%.

13. The binding agent for use of any one of the preceding items, wherein each of the binding agent, pembrolizumab, and the chemotherapy combination is administered in a dose to increase the ORR compared to the standard of care, such as compared to an administration regimen of pembrolizumab and the chemotherapy combination only. 14. The binding agent for use of any one of the preceding items, wherein each of the binding agent, pembrolizumab, and the chemotherapy combination is administered in a dose to increase 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%.

15. The binding agent for use of any one of the preceding items, wherein each of the binding agent, pembrolizumab, and the chemotherapy combination is administered in a dose to increase the DCR compared to the standard of care, such as compared to an administration regimen of pembrolizumab and the chemotherapy combination only.

16. The binding agent for use of any one of the preceding items, wherein each of the binding agent, pembrolizumab, and the chemotherapy combination is administered in a dose to increase 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%.

17. The binding agent for use of any one of the preceding items, wherein the platinum-based chemotherapeutic agent is carboplatin or cisplatin.

18. The binding agent for use of any one of the preceding items, wherein the chemotherapy combination is cisplatin and 5 -fluorouracil.

19. The binding agent for use of any one of the preceding items, wherein the chemotherapy combination is carboplatin and 5 -fluorouracil.

20. The binding agent for use of any one of the preceding items, wherein the binding agent is administered at a dose of about 50-150 mg/day, preferably about 100 mg/day.

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

22. The binding agent for use of any one of the preceding items, wherein when the platinum-based chemotherapeutic agent is carboplatin, it is administered at a dose of AUC = about 4-6, preferably AUC = about 5, or when the platinum-based chemotherapeutic agent is cisplatin, it is administered at a dose of about 50-150 mg/m²/day, preferably about 100 mg/m²/day.

23. The binding agent for use of any one of the preceding items, wherein 5 -fluorouracil is administered at a dose of about 500-1500 mg/m²/day, preferably about 1000 mg/m²/day. 24. The binding agent for use of any one of the preceding items, wherein the binding agent is administered at a dose of about 100 mg/day, pembrolizumab 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/m²/day, and 5 -fluorouracil is administered at a dose of about 1000 mg/m²/day.

25. The binding agent for use of any one of the preceding items, wherein:

(i) about 100 mg/day of the binding agent and about 200 mg/day of pembrolizumab are administered on day 1 of each treatment cycle, each treatment cycle being three weeks (1Q3W);

(ii) the platinum-based chemotherapeutic agent is administered on day 1 of at least the first and 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/m²/day; and

(iii) about 1000 mg/m²/day of 5 -fluorouracil is administered on days 1, 2, 3, and 4 of at least the first and second treatment cycle.

26. The binding agent for use of any one of the preceding items, wherein the binding agent, pembrolizumab, the platinum -based chemotherapeutic agent, and 5 -fluorouracil are administered for 6 treatment cycles and then only the binding agent and pembrolizumab are further administered for at least one treatment cycle.

27. The binding agent for use of any one of the preceding items, wherein any one or all of the binding agent, pembrolizumab and the chemotherapy combination is/are administered systemically, preferably intravenously.

28. The binding agent for use of any one of the preceding items, wherein the subject is a human subject.

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

30. The binding agent for use of any one of the preceding items, wherein the subject has not received prior treatment with any anti-cancer therapy or with a checkpoint inhibitor and any anti-cancer therapy. 31. The binding agent for use of any one of the preceding items, wherein the subject has a PD-L1 combined positive score (CPS) > 1, such as a PD-L1 CPS > 1 and < 19 or a PD-L1 CPS > 20.

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

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

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

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

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

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

38. The binding agent for use of any one of the preceding items, wherein the binding agent is a multispecific antibody, such as a bispecific antibody.

39. The binding agent for use of any one of the preceding items, wherein the binding agent is in the format of a full-length antibody or an antibody fragment.

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

41. The binding agent for use of item 40, wherein said complementarity determining regions and said framework regions are arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

42. The binding agent for use of any one of items 33-41, which comprises i) a polypeptide comprising, consisting of or consisting essentially of, said first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising, consisting of or consisting essentially of, said second heavy chain variable region (VH) and a second heavy chain constant region (CH).

43. The binding agent for use of any one of items 33-42, which comprises i) a polypeptide comprising said first light chain variable region (VL) and further comprising a first light chain constant region (CL), and ii) a polypeptide comprising said second light chain variable region (VL) and further comprising a second light chain constant region (CL).

44. The binding agent for use of any one of items 33-43, wherein the binding agent is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises i) a polypeptide comprising said first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising said first light chain variable region (VL) and a first light chain constant region (CL); and the second binding arm comprises iii) a polypeptide comprising said second heavy chain variable region (VH) and a second heavy chain constant region (CH), and iv) a polypeptide comprising said second light chain variable region (VL) and a second light chain constant region (CL).

45. The binding agent for use of any one of the preceding items, which comprises i) a first heavy chain and light chain comprising said antigen-binding region capable of binding to CD40, and ii) a second heavy chain and light chain comprising said antigen-binding region capable of binding CD137.

46. The binding agent for use of any one of the preceding items, wherein said binding agent comprises i) a first heavy chain and light chain comprising said 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 light chain comprising said antigen-binding region capable of binding CD137, the second heavy chain comprising a second heavy chain constant region and the second light chain comprising a second light chain constant region.

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

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

49. The binding agent for use of any one of items 42-48, wherein in said first heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting ofT366, L368, K370, D399, F405, Y407, and K409 in a human IgGl heavy chain according to EU numbering has been substituted, and in said second heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgGl heavy chain according to EU numbering has been substituted, and wherein said first and said second heavy chains are not substituted in the same positions.

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

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

52. The binding agent for use of item 51 , wherein said first and second heavy chain constant regions (CHs) are modified so that the antibody induces Fc-mediated effector function to a lesser extent compared to an antibody which is identical except for comprising non-modified first and second heavy chain constant regions (CHs).

53. The binding agent for use of item 52, wherein each of said non-modified first and second heavy chain constant regions (CHs) comprises the amino acid sequence set forth in SEQ ID NO: 21 or 29.

54. The binding agent for use of item 52 or 53, wherein said Fc-mediated effector function is measured by binding to Fey receptors, binding to Clq, or induction of Fe-mediated crosslinking of Fey receptors.

.55 The binding agent for use of item 54, wherein said Fc-mediated effector function is measured by binding to Clq.

56. The binding agent for use of any one of items 51-55, wherein said first and second heavy chain constant regions have been modified so that binding of Clq to said 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 Clq binding is preferably determined by ELISA.

57. The binding agent for use of any one of items 42-56, wherein in at least one of said first and second heavy chain constant regions (CH), one or more amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgGl heavy chain according to EU numbering, are not L, L, D, N, and P, respectively.

58. The binding agent for use of item 57, wherein the positions corresponding to positions L234 and L235 in a human IgGl heavy chain according to EU numbering are F and E, respectively, in said first and second heavy chains.

59. The binding agent for use of item 57 or 58, wherein the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering are F, E, and A, respectively, in said first and second heavy chain constant regions (HCs).

60. The binding agent for use of any one of items 57-59, wherein the positions corresponding to positions L234 and L235 in a human IgGl heavy chain according to EU numbering of both the first and second heavy chain constant regions are F and E, respectively, and wherein (i) the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is R, and the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the second heavy chain is L.

61. The binding agent for use of any one of items 57-60, wherein the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering of both the first and second heavy chain constant regions are F, E, and A, respectively, and wherein (i) the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the first heavy chain is R, and the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the second heavy chain is L.

62. The binding agent for use of any one of items 42-61, wherein the constant region of said first and/or second heavy chain comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 21 or 29 [IgGl-FC]; b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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 substitutions or at most 1 substitution, compared to the amino acid sequence defined in a) or b).

63. The binding agent for use of any one of items 42-61, wherein the constant region of said first or second heavy chain, such as the second heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 22 or 30 [IgGl-F405L]; b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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, 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). 64. The binding agent for use of any one of items 42-61, wherein the constant region of said first or second heavy chain, such as the first heavy chain comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 23 or 31 [IgGl-F409R]; b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or

10 consecutive amino acids has/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).

65. The binding agent for use of any one of items 42-61, wherein the constant region of said first and/or second heavy chain comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 24 or 32 [IgGl-Fc_FEA]; b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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).

66. The binding agent for use of any one of items 42-65, wherein the constant region of said first and/or second heavy chain, such as the second heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 25 or 33 [IgGl-Fc_FEAL]; b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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).

67. The binding agent for use of any one of items 42-66, wherein the constant region of said first and/or second heavy chain, such as the first heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 26 or 34 [IgGl-Fc_FEAR]; b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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).

68. The binding agent for use of any one of the preceding items, wherein said binding agent comprises a kappa (K) light chain constant region.

69. The binding agent for use of any one of the preceding items, wherein said binding agent comprises a lambda (X) light chain constant region.

70. The binding agent for use of any one of the preceding items, wherein said first light chain constant region is a kappa (K) light chain constant region or a lambda (X) light chain constant region.

71. The binding agent for use of any one of the preceding items, wherein said second light chain constant region is a lambda (X) light chain constant region or a kappa (K) light chain constant region.

72. The binding agent for use of any one of the preceding items, wherein said first light chain constant region is a kappa (K) light chain constant region and said second light chain constant region is a lambda (X) light chain constant region or said first light chain constant region is a lambda (X) light chain constant region and said second light chain constant region is a kappa (K) light chain constant region.

73. The binding agent for use of any one of items 68-72, wherein the kappa (K) light chain comprises an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 27, b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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).

74. The binding agent for use of any one of items 69-73, wherein the lambda (X) light chain comprises an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 28, b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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).

75. The binding agent for use of any one of the preceding items, wherein the binding agent is of an isotype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

76. The binding agent for use of any one of the preceding items, wherein the binding agent is a full- length IgGl antibody.

77. The binding agent for use of any one of the preceding items, wherein the binding agent is an antibody of the IgGlm(f) allotype.

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

79. The method of item 78, wherein the binding agent and/or the subject and/or the administration regimen is/are as defined in any one of items 1-77.

Further aspects of the present disclosure are disclosed herein.

Examples

Example 1: Antitumor activity of GEN1042-mIgG2a in combination with PD-1 blockade and platinum-based chemotherapy in human CD40 and 4-1 BB double knock-in mice

GEN1042 shows no binding to cells transiently overexpressing mouse CD40 or 4-1BB. Therefore, to assess antitumor activity of GEN 1042 in vivo, C57BL/6 mice engineered to express the extracellular domain of human CD40 (hCD40) and 4-1BB (h4-lBB) from the mouse CD40 and 4-1BB gene loci, respectively, (hCD40xh4-lBB double knock-in [dKI] mice) were treated with the GEN1042 mouse surrogate GEN1042-mIgG2a, a chimeric antibody that contains CD40- and 4-lBB-specific Fab arms identical to GEN1042 and a murine, inert Fc.

Objective

To investigate the anti-tumor activity of GEN1042-mIgG2a antibody either alone or in combination with chemotherapy (carboplatin/5 -fluorouracil [5-FU]), checkpoint inhibitors (anti-mouse PD-1 [anti- mPD-1]) or both using syngeneic MC38 mouse colorectal tumors implanted in hCD40xh4-lBB dKI mice.

Methods

MC38 mouse colon cancer cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C, 5% CO2. MC38 cells were harvested from a cell culture growing in log-phase and quantified.

All animal experiments were performed at Crown Bioscience Inc. and approved by their Institutional Animal Care and Use Committee (IACUC) prior to execution. Animals were housed and handled in accordance with good animal practice as defined by the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

MC38 cells (1 x 10⁶ tumor cells in 100 pL PBS) were injected subcutaneously in the right lower flank of female hCD40xh4-lBB dKI mice (Shanghai Model Organisms Center, Inc., cat. no. NM -HU-200256; C57BL/6-Cd40eml(hCD40)Tnfrsf9tm2(TNFRSF9)Smoc; age 9-11 weeks at start of experiment). Tumor growth was evaluated three times per week using a caliper. Tumor volumes (mm³) were calculated from caliper measurements as ([length] x [width]²)/2, where the length is the longest tumor dimension and the width is the longest tumor dimension perpendicular to the length.

Mice were randomized into groups (10 mice per group) with equal average tumor volume prior to treatment (37 mm³). On treatment days, the mice were injected intraperitoneally (IP) with GEN1042- m!gG2a (SEQ IDs 77, 78, 79, 80), anti-mPD-1 (clone RMP1-14, BioXcell, cat. no. BP0146), a chemotherapy regimen of carboplatin (Qilu Pharmaceutical, cat. no. BB2J2003) and 5-FU (Shanghai Xudong Haipu Pharmaceutical, cat. no. FA220702) either alone or in combination. The control group was dosed with both PBS and 0.9% saline. For combination treatments, anti-mPD-1 was injected first, followed by GEN1042-mIgG2a with carboplatin and 5-FU thereafter, if applicable (Table 5), in separate injections with a volume of 10 pL/g body weight each and 30 min to 2 hours in between. The mice were monitored daily for clinical signs of illness. Body weight measurements were performed three times a week after randomization. The antibodies and combinations thereof were well-tolerated, as mice showed minimal body weight loss (<20%) upon treatment. The experiment ended for the individual mice when the tumor volume exceeded 1500 mm³ or when the animals reached other humane endpoints. The mice showed no signs of illness, but one mouse in the 10 mg/kg anti-mPD-1 treatment group was found dead. The cause of this death was undetermined.

The difference in tumor volumes between the groups was compared using a nonparametric Mann- Whitney analysis (in GraphPad Prism) on the last day that all groups were still intact (i.e., until the first tumor-related death in the study, i.e., Day 20).

Table 5. Treatment groups and dosing regimen

^a For combination treatments, anti-mPD-1 was injected first, followed by GEN1042-mIgG2a with carboplatin and 5-FU thereafter, if applicable, in separate injections with 30 min to 2 hours in between. Abbreviations: 5-FU = 5 -fluorouracil; BIW><3 = two doses per week for three weeks; IP = intraperitoneally; mlgG = mouse immunoglobulin G; mPD-1 = mouse programmed cell death protein 1; N/A = not applicable; PBS = phosphate -buffer saline; Q3D*5 = five times one dose every three days.

Results

Rapid tumor outgrowth was observed in MC38-bearing hCD40xh4-lBB dKI mice treated with PBS (Figure 2). In mice treated with GEN1042-mIgG2a or anti-mPD-1 as single agents delayed tumor outgrowth was observed (Figure 2). Carboplatin and 5-FU were administered at non-therapeutic doses based on monotherapy studies (data not shown), but combined chemotherapy treatment also delayed tumor outgrowth as compared to the control-treated mice. However, responses remained incomplete as none of the animals treated with GEN1042-mIgG2a, anti-mPD-1 or the chemotherapy combination completely rejected the implanted tumors. Combining chemotherapy with anti-mPD-1 blockade did not further delay tumor outgrowth, but partnership with GEN1042-mIgG2a led to complete tumor regression in 1/10 mice at Day 22 post-treatment initiation (Figure 2), suggesting a slight advantage of this regimen. Similar growth inhibition was also observed after treatment with the combination of mGEN1042-IgG2a and anti-mPD-1 blockade, resulting in complete tumor regression in 1/10 mice at Day 13. In mice treated with the quadruple combination of GEN1042-mIgG2a, anti-mPD-1 and carboplatin/5-FU chemotherapy, tumor outgrowth was significantly reduced compared to GEN1042- m!gG2a as a single agent (p<0.05, Mann-Whitney, Figure 2) and three complete responders were observed (from Day 18 onwards), suggesting that quadruple combination of GEN1042-mIgG2a with anti-mPD-1 and chemotherapy combination might be more potent than the combination of GEN 1042- m!gG2a with either anti-mPD-1 or carboplatin/5-FU chemotherapy.

These results provide rationale for evaluating the quadruple combination of GEN 1042 with an anti-PD- 1 antibody and platinum-based chemotherapy to further amplify the anti-tumor immune response in cancer patients to produce durable and deep clinical responses and enhance survival.

Example 2: Clinical trial design

Trial design

This is a first-in-human, open-label, multicenter phase 1/2 trial of GEN 1042 in subjects with solid malignant tumors. The trial consists of 4 parts: a GEN 1042 monotherapy dose escalation (phase la), a GEN1042 monotherapy expansion (phase 2a), a combination therapy safety run-in (phase lb), and a combination therapy expansion (phase 2).

The dose escalation for monotherapy (phase la) will evaluate GEN1042 in subjects with non-central nervous system (CNS) solid malignant tumors to determine the MTD or maximum administered dose and/or RP2D. The phase lb safety run-in will evaluate the GEN1042 monotherapy RP2D from dose escalation in combination with 1 or more therapies in select tumor types following as described in detail below. The RP2D of GEN 1042 determined during the safety run-in will be further evaluated in phase 2.

Treatment schedule

Safety Run-In for Combination Therapy - Phase lb

The “3+3” design is conventional for phase 1 oncology studies. The combination safety run-in part of this trial will follow “3+3” to allow the safety of each dose of GEN1042 administered +/- pembrolizumab +/- chemotherapy in 3 subjects to be assessed before treating additional subjects with the same or next doses. Subjects will not be randomized; they will be assigned to the cohort that is being filled at the time the subject is ready to enter the trial. In detail, safety combination cohorts will receive GEN 1042 + pembrolizumab OR GEN 1042 + chemotherapy +/- pembrolizumab. Enrollment in phase lb will begin after the RP2D of GEN1042 monotherapy has been determined from the dose escalation part (phase la).

Three parallel regimens are planned during the combination safety run-in:

1. GEN1042 + pembrolizumab Q3W; continued treatment until PD, undue toxicity, withdrew consent or up to 35 cycles (total 2 years)

• NSCLC (CPI-naive, PD-L1 expressing, TPS >1% per local lab testing)

• HNSCC (CPI-naive, PD-L1 expressing, CPS >1 per local lab testing)

• Melanoma (CPI-naive, regardless of PD-L1 expressing)

2. GEN1042 + pembrolizumab + cis-/carboplatin + 5-FU Q3W for 6 cycles followed by GEN1042 + pembrolizumab Q3W; continued treatment until PD, undue toxicity, withdrew consent or up to additional 29 cycles (total 2 years)

• HNSCC (CPI-naive, PD-L1 expressing, CPS >1 per local lab testing)

3. PDAC (Regardless of PD-L1 expression):

• Regimen 3a: GEN 1042 Q3 W+ gemcitabine + nab-paclitaxel 2Q3 W for a total of 8 cycles

• Regimen 3b: GEN1042 + pembrolizumab Q3W+ gemcitabine + nab-paclitaxel 2Q3W for 8 cycles followed by GEN 1042 + pembrolizumab Q3W continued treatment until PD, undue toxicity, withdrew consent or up to an additional 27 cycles (total 2 years)

The above respective safety combination regimen will be evaluated following a 3+3 dose de-escalation design. Cohorts of 3-6 subjects will be entered sequentially into de-escalating dosage tiers. The starting dose for GEN1042 is 100 mg 1Q3W (DL1) for regimens 1, 2 and 3a. The next dose level will be 60 mg (DL2) of GEN1042. The dose of GEN1042 determined through Regimen 3a will be the starting dose for Regimen 3b. The approved dose of pembrolizumab and chemotherapy will be used following the SOC practice.

• Each regimen will be tested starting with three subjects using the RP2D GEN1042 dose from phase la (100 mg 1Q3W) for regimens 1, 2 and 3a, OR using the safe and tolerable dose from Regimen 3a for Regimen 3b, administered in combination with the designated therapy(ies) shown above.

• If none of the three subjects in a cohort experiences a DLT, the regimen will be deemed safe, and this regimen will be further tested in the expansion arm.

• If one of the first three subjects experiences a DLT, three more subjects will be treated at the same dose level. If at most one out of six subjects experienced DLT, the regimen will also be considered safe to move forward into the expansion part. • If at least two subjects among a cohort of three to six subjects experience DLTs (i.e., >33% of subjects with a DLT at that dose level), de-escalate to the next lower dose level, e.g., 60 mg of GEN 1042.

• The goal in Regimen 3 is to identify a safe and tolerable dose of GEN 1042 in combination with pembrolizumab, gemcitabine and nab-paclitaxel.

Doses below the highest dose level which is deemed safe in the dose escalation part (e.g., 300 mg, 30 mg, etc.) may also be tested in the expansion part upon agreement between the investigator and the sponsor. If there is no tolerable dose identified, the safety run-in and its associated expansion arm will be terminated.

DLT will be evaluated after each subject completed DLT observation period, that is 1 cycle (21 days) for GEN 1042 + pembrolizumab OR 2 cycles (21 days) for GEN 1042 + chemotherapy +/- pembrolizumab, respectively. Determination of RP2D of GEN 1042 for the combination regimen will be based on the totality of the data, taking into consideration the DLT and the overall safety profde, antitumor activity, PK, and biomarker data if available.

Expansion Part - Combination Therapy Cohorts - Phase 2

For the combination therapy expansion, the selected RP2D GEN 1042 dose determined from phase lb combination safety run-in will be administered, in combination with 1 or more therapies as shown below. The combination therapy will be administered as the IL treatment setting. Five parallel arms in four indications are planned.

Table 6:

Abbreviations: lL=first line; 2Q3W= Twice every 3 weeks (weekly for 2 weeks followed by a week of rest); 5- FU=5-fluorouracil; Cis/Carbo=cisplatin/carboplatin; CPI=checkpoint inhibitor; CPS=combined positive score; HSNCC=head and neck squamous cell carcinoma; NSCLC=non-small-cell lung cancer; PD=progressive disease; PDAC=pancreatic ductal adenocarcinoma; PD-Ll=programmed death-ligand 1; Q3W=every 3 weeks; TPS=tumor proportion score.

Table 7: Dosage and Administration

Abbreviations: 5-FU=5-fluorouracil; Q3W=every 3 weeks. a. Starting from Cycle 7, subjects without disease progression will receive GEN1042 + pembrolizumab Q3W continuously b. Starting from Cycle 9, subjects without disease progression will receive GEN1042 + pembrolizumab Q3W continuously

For combination therapy expansion, treatment will be administered in the order presented below: Pembrolizumab infusion will be administered first followed by GEN 1042 followed by SOC chemotherapy. Based on convenience for the subject and institution, the gap between drugs can range from 30 min to 2 hours (meal breaks, short walks, managing infusion-related reactions [IRRs], etc.) as long as the start times of every component of combination regimens are duly recorded. Generation of bispecific antibodies

The bispecific anti-CD40 anti-4- IBB (herein after referred to as GEN1042 or DuoBody -CD40x4- IBB) was produced with the humanized VH and VL sequences, the human kappa light chain, and a human IgGl heavy chain described in Table 1. The CD40 binding arm has been produced with the human IgGl heavy chain containing the following amino acid mutations: L234F, L235E, D265A and F405L (FEAL), wherein the amino acid position number is according to EU numbering (corresponding to SEQ ID NO: 33). The CD 137 binding arm has been produced with a human IgGl heavy chain containing the following amino acid mutations: L234F, L235E, D265A and K409R (FEAR), wherein the amino acid position number is according to EU numbering (correspond to SEQ ID NO: 34).

Bispecific IgGl antibodies were generated by Fab-arm-exchange under controlled reducing conditions. The basis for this method is the use of complementary CH3 domains, which promote the formation of heterodimers under specific assay conditions as described in WO2011/131746. The F405L and K409R EU numbering) mutations were introduced into the relevant antibodies to create antibody pairs with complementary CH3 domains.

To generate bispecific antibodies, the two parental complementary antibodies, each antibody at a final concentration of 0.5 mg/ml, were incubated with 75 mM 2-mercaptoethylamine-HCI (2-MEA) in a total volume of 100 pL PBS at 31 °C for 5 hours. The reduction reaction was stopped by removing the reducing agent 2-MEA using spin columns (Microcon centrifugal filters, 30k, Millipore) according to the manufacturer's protocol.

Inclusion criteria for combination therapy

The subjects must be > 18 years of age; have measurable disease according to RECIST 1.1; life expectancy > 3 months; have an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0-1; adequate organ, bone marrow, liver, coagulation, and renal function; and not received prior therapy with an anti-PD-1, anti-PD-Ll, or anti-programmed death-ligand 2 agent or with an agent directed to another stimulatory or co-inhibitory T-cell receptor (eg, CTLA-4, OX-40, CD40 or 4-1BB). Additional criteria for each cohort are as follows:

• Melanoma a. Histologically confirmed unresectable Stage III or Stage IV melanoma, as per American Joint Committee on Cancer (AJCC; version 8) staging system. Primary ocular or mucosal melanoma is excluded. b. No prior systemic anticancer therapy for unresectable or metastatic melanoma. c. With a known tumor BRAF mutation status as per local standard testing (preferably an FDA- approved test). d. For subjects with BRAF V600E mutant melanoma, the following additional criteria should be met: i. Lactate dehydrogenase < local upper limit of normal ii. No clinically significant tumor related symptoms in the judgment of the investigator iii. Absence of rapidly progressing metastatic melanoma in the judgment of the investigator

• NSCLC a. Have a histologically confirmed diagnosis of Stage IV metastatic or recurrent NSCLC (AJCC version 8), with no prior systemic anticancer therapy given as primary therapy for advanced or metastatic disease. b. Tumor does not have an actionable EGFR activating mutation or ALK translocation. For subjects who are known to have a tumor of predominantly squamous histology, molecular testing for EGFR mutation and ALK translocation will not be required if this is per local SOC. c. Tumor demonstrates PD-L1 expression in >1% of tumor cells (TPS >1%) as assessed by immunohistochemistry (IHC) determined by a local SOC testing (preferably an FDA-approved test) or at a central laboratory. Central laboratory testing is mandated for the expansion phase.

• HNSCC a. Histologically or cytologically-confirmed recurrent or metastatic HNSCC that is considered incurable by local therapies. b. Subjects should not have had prior systemic therapy administered in the recurrent or metastatic setting. Systemic therapy which was completed more than 6 months prior to signing consent if given as part of multimodal treatment for locally advanced disease is allowed. c. The eligible primary tumor locations are oropharynx, oral cavity, hypopharynx, and larynx. d. Subjects should not have a primary tumor site of nasopharynx (any histology). e. Have tumor PD-L 1 IHC CPS > 1 per local (preferably an FDA-approved test) or central laboratory testing (central testing is mandated for the expansion phase). f. Human papillomavirus (HPV) pl 6 test results available per local SOC for participants with oropharyngeal disease. Note: Oral cavity, hypopharynx, and larynx cancer are not required to undergo HPV testing by pl 6 IHC as by convention these tumor locations are assumed to be HPV negative. PDAC a. Histologically or cytologically confirmed metastatic pancreatic adenocarcinoma. Pancreatic endocrine cancer is excluded. b. No actionable gene alterations such as BRCA 1/2 or PALB2 mutations c. No previous radiotherapy, surgery, chemotherapy, or investigational therapy for the treatment of metastatic disease d. If a subject has had adjuvant/neoadjuvant therapy and/or therapy for locally advanced disease (chemotherapy for non-metastatic pancreatic cancer in combination with or without radiation therapy), all toxicities must have returned to baseline or < grade 1.

Preliminary results

GEN1042 + pembrolizumab + cisplatin or carboplatin + 5-FU

Male and female subjects aged 18 years and older with previously untreated recurrent or metastatic oral cavity, oropharyngeal, hypopharyngeal, or laryngeal head and neck squamous cell carcinoma (HNSCC) with program death ligand 1 (PD-L1) combined positivity score (CPS) of >1 were administered GEN 1042 (CD40x4-lBB) + Standard of Care (SOC) pembrolizumab + Investigator’s Choice of cisplatin or carboplatin + 5 -fluorouracil (5-FU) 1Q3W for 6 cycles, followed by GEN 1042 + pembrolizumab 1Q3W for up to an additional 29 cycles (total 2 years). Subjects were treated until Progressive Disease (PD), undue toxicity, withdrawal of consent. All subjects treated must have had measurable disease. Tumor response was assessed every 6 weeks according to RECIST 1.1.

The combination of GEN 1042 + pembrolizumab + cisplatin or carboplatin + 5-FU was generally well- tolerated in all patients dosed. No Dose Limiting Toxicities (DLTs) were observed, and related treatment-emergent adverse events (TEAEs) were mostly grade 1 or grade 2 per Common Terminology Criteria for Adverse Events (CTCAE) v.5.0. Serious-treatment related TEAEs were observed in 28.6% of subjects. Figure 3A represents best percent change in target lesions for all subjects with previously untreated recurrent or metastatic HNSCC dosed with GEN 1042 + pembrolizumab + cisplatin or carboplatin +5-FU as of the data cutoff (DCO) of October 3, 2022. At the time of the DCO, 4 of 6 subjects treated were evaluable for response. Of the response evaluable subjects, 2 subjects experienced a confirmed complete response (CR), and 2 subjects experienced a confirmed partial response (PR). Two subjects not shown in Figure 3A were considered not evaluable (NE); one subject died due to COVID prior to the first response assessment and 1 subject withdrew consent prior to the first response assessment. One additional subject was treated in the study but had not yet reached the first on-study disease assessment at the time of the DCO. Objective Response Rate (ORR) for all subjects in the eligible analysis set was 66.7% [95% CI: (22.3, 95.7)] and the Disease Control Rate (DCR) was 66.7% [95% CI: (22.3, 95.7)] (Table 8). The ORR_evai was 100%. Change in target lesion response over time for all subjects dosed with GEN1042 + pembrolizumab + cisplatin or carboplatin + 5-FU at the time of the DCO is represented in Figure 3B. All subjects evaluable for response at the time of the DCO had responses ongoing at Week 18 or beyond.

GEN1042 + pembrolizumab + cisplatin or carboplatin + 5-FU has been well-tolerated and showed initial antitumor activity in subjects with previously untreated recurrent or metastatic HNSCC.

GEN1042 + pembrolizumab

GEN1042 was also evaluated in combination with pembrolizumab in male and female subjects aged 18 years and older with previously untreated recurrent or metastatic oral cavity, oropharyngeal, hypopharyngeal, or laryngeal head and neck squamous cell carcinoma (HNSCC) with program death ligand 1 (PD-L1) combined positivity score (CPS) results of >20. Subjects were administered GEN1042 (CD40x4-lBB) + Standard of Care (SOC) pembrolizumab 1Q3W for up to 35 cycles (total 2 years). Subjects were treated until Progressive Disease (PD), undue toxicity, withdrawal of consent. Tumor response was assessed every 6 weeks according to RECIST 1.1 (Eisenhauer et al., Eur J Cancer (2009); 45, 228-247).

The combination of GEN 1042 + pembrolizumab was generally well-tolerated in all patients dosed. No DLTs were observed in subjects treated with the combination of GEN 1042 + pembrolizumab and related treatment-emergent adverse events (TEAEs) were mostly grade 1 or grade 2 per CTCAE v.5.0. At the time of the DCO of October 7, 2022, 4 of 6 subjects treated were evaluable for response. Figure 4A represents best percent change in target lesions for all subjects with previously untreated recurrent or metastatic HNSCC dosed with GEN1042 + pembrolizumab. All 4 response evaluable subjects experienced a decrease in target lesions at their first post-baseline disease assessment; however, disease progression was observed in 3 of the 4 subjects at the subsequent assessment. All 4 subjects experienced a Best Overall Response of stable disease (SD) per RECIST vl.l. Two additional subjects that were dosed with GEN 1042 + pembrolizumab were considered not evaluable (NE); one subject withdrew consent prior to the first on-study response assessment and one subject discontinued due to grade 4 hypertransaminasemia and no response data was available for the subject at the time of the DCO. Change in target lesion response over time for all subjects dosed with GEN1042 + pembrolizumab is represented in Figure 4B. The confirmed Objective Response Rate (ORR) for all subjects in the eligible analysis set was 0.00% [95% CI: (0, 45.9)] and the Disease Control Rate (DCR) was 66.7% [95% CI: (22.3, 95.7)] (Table 8).

Table 8: Summary of Confirmed Objective Response Rate for Subjects with Previously Untreated Recurrent or Metastatic HNSCC Treated with Chemotherapy and Pembrolizumab and GEN1042 - Eligible Analysis Set

Pembro = Pembrolizumab, Cis = Cisplatin, Carbo = Carboplatin, 5-FU = 5-Flourouracil Summaries are n (%) unless otherwise specified. Eligible set used.

CI: Confidence Interval; CR: Complete Response; NE: Not Evaluable; PD: Progressive Disease; PR: Partial Response; SD:

Stable Disease. Objective Response = CR+PR; Disease Control = CR+PR+SD.

[1] Exact Binomial Confidence Interval.

Data Cut-off (DCO) date: October 3, 2022, Data extraction date: October 18, 2022

Example 3: Pharmacodynamic evaluation of GEN1042+SoC in peripheral blood of IL, CPI naive, HNSCC patients

To investigate the biological activity of lOOmg GEN 1042+ SoC (pembrolizumab + cisplatin or carboplatin + 5-FU) in IL, CPI naive, HNSCC patients, blood and serum samples were collected at baseline and at multiple timepoints on treatment. Based on the mechanism of action of GEN 1042, it was anticipated that dose levels with biological activity will modulate circulating levels of interferon-y (IFN- y) and IFN-y -inducible macrophage/dendritic cell soluble factor, thymus and activation-regulated chemokine (TARC), as well as induce proliferation/activation of peripheral CD8 T cells similar to previous observations from latter line pan-tumor monotherapy treatments.

To determine serum levels of IFN-y and TARC, samples were collected from patients at baseline and at multiple timepoints post administration of GEN1042 in cycle 1 and cycle 2 (days 1, 3, 8, and 15) as well as pre-dose at cycle 3. Serum levels of IFN-y and TARC were measured by a Meso Scale Discovery (MSD) multiplex immune-assay (cat. no. K15209G) following the manufacturer’s instructions.

Administration of lOOmg of GEN1042+SoC to cancer patients resulted in modulation of circulating levels of IFN-y (Figure 5) and TARC (Figure 6). Levels of IFN-y exceeded the normal reference range (<11.81 pg/mL), indicative of T cell activation, in the first two cycles for at least 1 patient in both the GEN1042+Pembro and GEN1042+Chemo+Pembro treatment groups. Peak induction occurred 2-14 days post dosing. TARC levels consistently exceeded the normal reference range (<513 pg/mL), indicative of dendritic cell/myeloid cell activation, in the first two cycles for at least 1 patient in both the GEN1042+Pembro and GEN1042+Chemo+Pembro treatment groups. Peak induction occurred 2-7 days post dosing. To measure peripheral modulation of immune cells subsets, immunophenotyping of peripheral blood was conducted in whole blood collected in EDTA tubes at baseline and at multiple timepoints post GEN1042+SoC administration in cycle 1 and cycle 2 (days 1, 3, 8 and 15) as well as pre-dose at cycle 3. 100 pL of whole blood was added to fluorochrome-conjugated monoclonal antibodies that bind specifically to cell surface antigens: [T cell panel] CD45RA-FITC (clone L48, BD Biosciences cat. no. 335039), CCR7-BV510 (clone 3D12, BD Biosciences, cat. no. 563449), CD8-PerCP-Cy5.5 (clone RPA- T8, BD Biosciences, cat. no. 560662), CD4-PE (clone SK3, BD Biosciences, cat. no. 345769), CD45-BV605 (clone HI30, BD Biosciences cat. no. 564047), CD19-PE-Cy7 (clone Sj2SCl, BD Biosciences, cat. no. 341113), CD3-APC-H7 (clone SK7, BD Biosciences, cat. no. 560176) and 4-1BB- AF647 (clone 4B4-1, Biolegend, cat. no. 309824) or [BNKcell panel] CD56-FITC (clone NCAM16.2, BD Biosciences cat. no. 345811), CD45-BV605 (clone HI30, BD Biosciences, cat. no. 564047), CD38- APC-R7 (clone HIT2, BD Biosciences, cat. no. 564979), HLA-DR-PE (clone L243, BD Biosciences, cat. no. 347367), CD16-PerCP-Cy5.5 (clone 3G8, BD Biosciences cat. no. 338440), CD19-PE-Cy7 (clone Sj2SCl, BD Biosciences, cat. no. 341113), CD3-APC-H7 (clone SK7, BD Biosciences, cat. no. 560176) and 4-1BB-AF647 (clone 4B4-1, Biolegend, cat. no. 309824) and CD86-BV421 (clone 2331, BD Biosciences, cat. no. 562432) or CD86-APC (clone 2331, BD Biosciences, cat. no. 555660). After incubation on ice, the stained samples were treated with FACS Lysing Solution (BD Biosciences, Catalog No 349202) to lyse erythrocytes. Excess antibody and cell debris were removed by washing with Stain Buffer (BD Biosciences, cat. no. 554656). Following lyse/wash, cells were fixed and permeabilized by incubation with Permeabilizing Solution 2 buffer (BD Biosciences, cat. no. 340973). Next, cells were washed and resuspended in Stain Buffer and incubated on ice with antibody to Ki67- BV421 (clone B56, BD Biosciences, cat. no. 562899) to detect proliferating cells. After incubation, excess antibody was removed by washing with Stain Buffer. Cells were resuspended in Stain Buffer and acquired on a BD FACSCanto™ flow cytometer (Becton Dickinson) within 1 hour of staining.

GEN1042+SoC treatment elicited transient trafficking/margination of CD8 T cells (Figure 7A) and B cells (Figure 7B) post dosing indicative of 4-1BB and CD40 target engagement, respectively. This was observed consistently with either GEN1042+Pembro or GEN1042+Chemo+Pembro treatment.

GEN1042+SoC elicited proliferation and activation of total CD8+ T cells (Figures 8 A, 9 A) and CD8+ effector memory T cells (Figures 8B, 9B) as measured by an increase in the frequency of %Ki67+ and %4-lBB+ populations, respectively. Furthermore, B cells exhibited activation post dosing as characterized by an increase in the frequency of %4-lBB+ populations (Figure 10) indicative of CD40 engagement and B cell stimulation. Peak immunophenotypic changes occurred approximately 7 days post dosing.

Conclusion

GEN1042+SoC elicited pharmacodynamics in IL HNSCC CPI naive patients, characterized by modulation of immune effector cells and soluble factors critical for the generation of antitumor immune responses. Similar pharmacodynamics were observed in GEN1042 monotherapy 2L+ advanced solid tumor patients suggesting that GEN 1042 -mediated immunomodulation is retained in the combination setting. Analysis notes

Pharmacodynamic assessments, including changes in circulating levels of cytokines, chemokines and immune cell populations, were conducted using blood samples from patients with advanced solid tumors enrolled in the dose escalation phase of an open-label, multi-center safety trial of GEN 1042 (NCT04083599). Data cut off for analyses (DCO) from clinical was September 26, 2022 and for our CRO databases September 8, 2022.

Claims

CLAIMS A binding agent for use in a method for reducing or preventing progression of head and neck squamous cell carcinoma (HNSCC) or treating HNSCC in a subject, said method comprising administering to said subject the binding agent, 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 binding agent comprises a first binding region binding to CD40 and a second binding region binding to CD 137. The binding agent for use of claim 1, wherein the inhibitor of the checkpoint PD-1/PD-L1 axis is pembrolizumab. The binding agent for use of claim 1 or 2, wherein the binding agent, 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). The binding agent for use of any one of the preceding claims, wherein one dose of the binding agent and one dose of the inhibitor of the checkpoint PD-1/PD-L1 axis are administered every third week (1Q3W). The binding agent for use of any one of the preceding claims, wherein one dose of the binding agent and one dose of the inhibitor of the checkpoint PD-1/PD-L1 axis are administered on day 1 of each treatment cycle. The binding agent for use of any one of the preceding claims, wherein the binding agent is administered prior to the administration of the inhibitor of the checkpoint PD-1/PD-L1 axis. The binding agent for use of any one of the preceding claims, wherein one dose of the chemotherapy combination is administered at least every third week ( 1 Q3 W) for at least the first treatment cycle. The binding agent for use of any one of the preceding claims, wherein one dose of the platinumbased chemotherapeutic agent is administered every third week (1Q3W) for at least the first treatment cycle and one dose of 5 -fluorouracil is administered at least every third week (1Q3W), such as over four days in the first week for at least the first treatment cycle. The binding agent for use of any one of the preceding claims, wherein one dose of the platinumbased 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, such as on days 1, 2, 3, and 4 of at least the first treatment cycle. The binding agent for use of any one of the preceding claims, wherein the objective response rate (ORR) is increased compared to the standard of care, such as compared to an administration regimen of an inhibitor of the checkpoint PD-1/PD-L1 axis, e.g., pembrolizumab, and the chemotherapy combination only. The binding agent for use of any one of the preceding claims, wherein the ORR 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%. The binding agent for use of any one of the preceding claims, wherein the disease control rate (DCR) is increased compared to the standard of care, such as compared to an administration regimen of an inhibitor of the checkpoint PD-1/PD-L1 axis, e.g., pembrolizumab, and the chemotherapy combination only. The binding agent for use of 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%. The binding agent for use of any one of the preceding claims, wherein each of the binding agent, the inhibitor of the checkpoint PD-1/PD-L1 axis, and the chemotherapy combination is administered in a dose to increase the ORR compared to the standard of care, such as compared to an administration regimen of an inhibitor of the checkpoint PD-1/PD-L1 axis, e.g., pembrolizumab, and the chemotherapy combination only. The binding agent for use of any one of the preceding claims, wherein each of the binding agent, the inhibitor of the checkpoint PD-1/PD-L1 axis, and the chemotherapy combination is administered in a dose to increase 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%. The binding agent for use of any one of the preceding claims, wherein each of the binding agent, the inhibitor of the checkpoint PD-1/PD-L1 axis, and the chemotherapy combination is administered in a dose to increase the DCR compared to the standard of care, such as compared to an administration regimen of an inhibitor of the checkpoint PD-1/PD-L1 axis, e.g., pembrolizumab, and the chemotherapy combination only. The binding agent for use of any one of the preceding claims, wherein each of the binding agent, the inhibitor of the checkpoint PD-1/PD-L1 axis, and the chemotherapy combination is administered in a dose to increase 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%. The binding agent for use of any one of the preceding claims, wherein the platinum-based chemotherapeutic agent is carboplatin or cisplatin. The binding agent for use of any one of the preceding claims, wherein the chemotherapy combination is cisplatin and 5 -fluorouracil. The binding agent for use of any one of the preceding claims, wherein the chemotherapy combination is carboplatin and 5 -fluorouracil. The binding agent for use of any one of the preceding claims, wherein the binding agent is administered at a dose of about 50-150 mg/day, preferably about 100 mg/day. The binding agent for use of any one of the preceding claims, wherein the inhibitor of the checkpoint PD-1/PD-L1 axis is administered at a dose of about 150-250 mg/day, preferably about 200 mg/day. The binding agent for use of any one of the preceding claims, wherein when the platinum-based chemotherapeutic agent is carboplatin, it is administered at a dose of AUC = about 4-6, preferably AUC = about 5, or when the platinum-based chemotherapeutic agent is cisplatin, it is administered at a dose of about 50-150 mg/m²/day, preferably about 100 mg/m²/day. The binding agent for use of any one of the preceding claims, wherein 5 -fluorouracil is administered at a dose of about 500-1500 mg/m²/day, preferably about 1000 mg/m²/day. The binding agent for use of any one of the preceding claims, wherein the binding agent 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/m²/day, and 5- fluorouracil is administered at a dose of about 1000 mg/m²/day. The binding agent for use of any one of the preceding claims, wherein:

(i) about 100 mg/day of the binding agent 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, each treatment cycle being three weeks (1Q3W);

(iii) about 1000 mg/m²/day of 5 -fluorouracil is administered on days 1, 2, 3, and 4 of at least the first and second treatment cycle. The binding agent for use of any one of the preceding claims, wherein the binding agent, 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 binding agent and the inhibitor of the checkpoint PD-1/PD-L1 axis are further administered for at least one treatment cycle. The binding agent for use of any one of the preceding claims, wherein any one or all of the binding agent, the inhibitor of the checkpoint PD-1/PD-L1 axis, and the chemotherapy combination is/are administered systemically, preferably intravenously. The binding agent for use of any one of the preceding claims, wherein the subject is a human subject. The binding agent for use of any one of the preceding claims, wherein the subject has not received prior treatment with a checkpoint inhibitor and/or an anti -cancer therapy for recurrent or metastatic disease. The binding agent for use of any one of the preceding claims, wherein the subject has not received prior treatment with any anti-cancer therapy or with a checkpoint inhibitor and any anti -cancer therapy. The binding agent for use of any one of the preceding claims, wherein the subject has a PD-L1 combined positive score (CPS) > 1, such as a PD-L1 CPS > 1 and < 19 or a PD-L1 CPS > 20. The binding agent for use of any one of the preceding claims, wherein CD40 is human CD40, in particular human CD40 comprising the sequence set forth in SEQ ID NO: 36, and/or CD137 is human CD 137, in particular human CD 137 comprising the sequence set forth in SEQ ID NO: 38. The binding agent for use of any one of the preceding claims, wherein a) the first binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 7 or 9, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 8 or 10; and b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 17 or 19, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 18 or 20. The binding agent for use of any one of the preceding claims, wherein a) the first binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 1, 2, and 3, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 4, 5, and 6, respectively; and b) the second antigen-binding region comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 11, 12, and 13, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 14, 15, and 16, respectively. The binding agent for use of any one of the preceding claims, wherein a) the first binding region comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 7 or 9 and a light chain variable region (VL) region and comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or 10; b) the second binding region comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 25 100% sequence identity to SEQ ID NO: 17 or 19 and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 18 or 20. The binding agent for use of any one of the preceding claims, wherein a) the first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 7 or 9 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 8 or 10; and b) the second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 17 or 19 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 18 or 20. The binding agent for use of any one of the preceding claims, wherein a) the first binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 10; and b) the second binding region comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 20. The binding agent for use of any one of the preceding claims, wherein the binding agent is a multispecific antibody, such as a bispecific antibody. The binding agent for use of any one of the preceding claims, wherein the binding agent is in the format of a full-length antibody or an antibody fragment. The binding agent for use of any one of claims 34-40, wherein each variable region comprises three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4). The binding agent for use of claim 41, wherein said complementarity determining regions and said framework regions are arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The binding agent for use of any one of claims 34-42, which comprises i) a polypeptide comprising, consisting of or consisting essentially of, said first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising, consisting of or consisting essentially of, said second heavy chain variable region (VH) and a second heavy chain constant region (CH). The binding agent for use of any one of claims 34-43, which comprises i) a polypeptide comprising said first light chain variable region (VL) and further comprising a first light chain constant region (CL), and ii) a polypeptide comprising said second light chain variable region (VL) and further comprising a second light chain constant region (CL). The binding agent for use of any one of claims 34-44, wherein the binding agent is an antibody comprising a first binding arm and a second binding arm, wherein the first binding arm comprises i) a polypeptide comprising said first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising said first light chain variable region (VL) and a first light chain constant region (CL); and the second binding arm comprises iii) a polypeptide comprising said second heavy chain variable region (VH) and a second heavy chain constant region (CH), and iv) a polypeptide comprising said second light chain variable region (VL) and a second light chain constant region (CL). The binding agent for use of any one of the preceding claims, which comprises i) a first heavy chain and light chain comprising said antigen-binding region capable of binding to CD40, and ii) a second heavy chain and light chain comprising said antigen-binding region capable of binding CD 137. The binding agent for use of any one of the preceding claims, wherein said binding agent comprises i) a first heavy chain and light chain comprising said 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 light chain comprising said antigen-binding region capable of binding CD 137, the second heavy chain comprising a second heavy chain constant region and the second light chain comprising a second light chain constant region. The binding agent for use of any one of claims 43-47, wherein each of the first and second heavy chain constant regions (CH) comprises one or more of a constant heavy chain 1 (CHI) region, a hinge region, a constant heavy chain 2 (CH2) region and a constant heavy chain 3 (CH3) region, preferably at least a hinge region, a CH2 region and a CH3 region. The binding agent for use of any one of claims 43-48, wherein each of the first and second heavy chain constant regions (CHs) comprises a CH3 region and wherein the two CH3 regions comprise asymmetrical mutations. The binding agent for use of any one of claims 43-49, wherein in said first heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting ofT366, L368, K370, D399, F405, Y407, and K409 in a human IgGl heavy chain according to EU numbering has been substituted, and in said second heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgGl heavy chain according to EU numbering has been substituted, and wherein said first and said second heavy chains are not substituted in the same positions. The binding agent for use of claim 50, wherein (i) the amino acid in the position corresponding to F405 in a human IgGl heavy chain according to EU numbering is L in said first heavy chain constant region (CH), and the amino acid in the position corresponding to K409 in a human IgGl heavy chain according to EU numbering is R in said second heavy chain constant region (CH), or (ii) the amino acid in the position corresponding to K409 in a human IgGl heavy chain according to EU numbering is R in said first heavy chain, and the amino acid in the position corresponding to F405 in a human IgGl heavy chain according to EU numbering is L in said second heavy chain. The binding agent for use of any of the preceding claims, wherein said binding agent induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen binding regions and two heavy chain constant regions (CHs) comprising human IgGl hinge, CH2 and CH3 regions. The binding agent for use of claim 52, wherein said first and second heavy chain constant regions (CHs) are modified so that the antibody induces Fc-mediated effector function to a lesser extent compared to an antibody which is identical except for comprising non-modified first and second heavy chain constant regions (CHs). The binding agent for use of claim 53, wherein each of said non-modified first and second heavy chain constant regions (CHs) comprises the amino acid sequence set forth in SEQ ID NO: 21 or 29. The binding agent for use of claim 53 or 54, wherein said Fc-mediated effector function is measured by binding to Fey receptors, binding to Clq, or induction of Fe-mediated crosslinking of Fey receptors. The binding agent for use of claim 55, wherein said Fc-mediated effector function is measured by binding to Clq. The binding agent for use of any one of claims 52-56, wherein said first and second heavy chain constant regions have been modified so that binding of Clq to said 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 Clq binding is preferably determined by ELISA. The binding agent for use of any one of claims 43-57, wherein in at least one of said first and second heavy chain constant regions (CH), one or more amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgGl heavy chain according to EU numbering, are not L, L, D, N, and P, respectively. The binding agent for use of claim 58, wherein the positions corresponding to positions L234 and L235 in a human IgGl heavy chain according to EU numbering are F and E, respectively, in said first and second heavy chains. The binding agent for use of claim 58 or 59, wherein the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering are F, E, and A, respectively, in said first and second heavy chain constant regions (HCs). The binding agent for use of any one of claims 58-60, wherein the positions corresponding to positions L234 and L235 in a human IgGl heavy chain according to EU numbering of both the first and second heavy chain constant regions are F and E, respectively, and wherein (i) the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is R, and the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the second heavy chain is L. The binding agent for use of any one of claims 58-61, wherein the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering of both the first and second heavy chain constant regions are F, E, and A, respectively, and wherein (i) the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the first heavy chain is R, and the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the second heavy chain is L. The binding agent for use of any one of claims 43-62, wherein the constant region of said first and/or second heavy chain comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 21 or 29 [IgGl-FC]; b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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 substitutions or at most 1 substitution, compared to the amino acid sequence defined in a) or b). The binding agent for use of any one of claims 43-62, wherein the constant region of said first or second heavy chain, such as the second heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 22 or 30 [IgGl-F405L]; b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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, 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). The binding agent for use of any one of claims 43-62, wherein the constant region of said first or second heavy chain, such as the first heavy chain comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 23 or 31 [IgGl-F409R]; b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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). The binding agent for use of any one of claims 43-62, wherein the constant region of said first and/or second heavy chain comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 24 or 32 [IgGl-Fc_FEA]; b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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). The binding agent for use of any one of claims 43-66, wherein the constant region of said first and/or second heavy chain, such as the second heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 25 or 33 [IgGl-Fc_FEAL]; b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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). The binding agent for use of any one of claims 43-67, wherein the constant region of said first and/or second heavy chain, such as the first heavy chain, comprises or consists essentially of or consists of an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 26 or 34 [IgGl-Fc_FEAR]; b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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). The binding agent for use of any one of the preceding claims, wherein said binding agent comprises a kappa (K) light chain constant region. The binding agent for use of any one of the preceding claims, wherein said binding agent comprises a lambda (X) light chain constant region. The binding agent for use of any one of the preceding claims, wherein said first light chain constant region is a kappa (K) light chain constant region or a lambda (X) light chain constant region. The binding agent for use of any one of the preceding claims, wherein said second light chain constant region is a lambda (X) light chain constant region or a kappa (K) light chain constant region. The binding agent for use of any one of the preceding claims, wherein said first light chain constant region is a kappa (K) light chain constant region and said second light chain constant region is a lambda (X) light chain constant region or said first light chain constant region is a lambda (X) light chain constant region and said second light chain constant region is a kappa (K) light chain constant region. The binding agent for use of any one of claims 69-73, wherein the kappa (K) light chain comprises an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 27, b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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). The binding agent for use of any one of claims 70-74, wherein the lambda (X) light chain comprises an amino acid sequence selected from the group consisting of a) the sequence set forth in SEQ ID NO: 28, b) a subsequence of the sequence in a), such as a subsequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids has/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). The binding agent for use of any one of the preceding claims, wherein the binding agent is of an isotype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4. The binding agent for use of any one of the preceding claims, wherein the binding agent is a full-length IgGl antibody. The binding agent for use of any one of the preceding claims, wherein the binding agent is an antibody of the IgGlm(f) allotype. A method for reducing or preventing progression of head and neck squamous cell carcinoma (HNSCC) or treating HNSCC in a subject, said method comprising administering to said subject a binding agent, 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 binding agent comprises a first binding region binding to CD40 and a second binding region binding to CD 137. The method of claim 79, wherein the inhibitor of the checkpoint PD-1/PD-L1 axis is pembrolizumab. The method of claim 79 or 80, wherein the binding agent and/or the subject and/or the administration regimen is/are as defined in any one of claims 1-78.