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EP4626446A1 - Anticorps multispécifique contre cd40 et cd137 en polythérapie avec un anticorps anti-pd1 et une chimiothérapie - Google Patents

Anticorps multispécifique contre cd40 et cd137 en polythérapie avec un anticorps anti-pd1 et une chimiothérapie

Info

Publication number
EP4626446A1
EP4626446A1 EP23817404.9A EP23817404A EP4626446A1 EP 4626446 A1 EP4626446 A1 EP 4626446A1 EP 23817404 A EP23817404 A EP 23817404A EP 4626446 A1 EP4626446 A1 EP 4626446A1
Authority
EP
European Patent Office
Prior art keywords
binding agent
heavy chain
binding
region
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23817404.9A
Other languages
German (de)
English (en)
Inventor
Ugur Sahin
Michelle NIEWOOD
Homer ADAMS III
Brandon Higgs
Tahamtan Ahmadi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biontech SE
Genmab AS
Original Assignee
Biontech SE
Genmab AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Biontech SE, Genmab AS filed Critical Biontech SE
Publication of EP4626446A1 publication Critical patent/EP4626446A1/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • 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.
  • 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.
  • 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.
  • CD40L CD40 ligand
  • LTaip2 lymphotoxin-aip2
  • 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).
  • IL-12 interleukin- 12
  • 4-1BB ligand 4-1BB ligand
  • OX40L 0X40 ligand
  • GITRL GITR ligand
  • IFNs type I interferons
  • 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.
  • 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.
  • GITR glucocorticoid-induced TNFR-related protein
  • 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.
  • ADCC antibody -dependent cell- mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ADCP antibody-dependent cell- mediated phagocytosis
  • 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.
  • CTLs effector cytotoxic T lymphocytes
  • 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.
  • TCR T-cell receptor
  • TILs tumor infiltrating lymphocytes
  • CD137 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.
  • NF nuclear factor
  • MAP mitogen-activated protein
  • 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).
  • 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.
  • 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).
  • PD-1 programmed cell death 1
  • 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).
  • HNSCC Head and neck squamous cell carcinoma
  • 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).
  • HNSCC remains an area of high unmet medical need and further opportunity exists to improve outcomes with novel treatment approaches.
  • 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.
  • a platinumbased chemotherapeutic agent in particular, cisplatin or carboplatin
  • 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-
  • 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%.
  • 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.
  • checkpoint inhibitor CPI
  • ICP immune checkpoint
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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.
  • 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).
  • V L or VL variable light chain
  • CL or CL constant light chain domain/region
  • VH or VH variable heavy chain domain/region
  • 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).
  • CDRs complementarity determining regions
  • immunoglobulin heavy chains 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.
  • the heavy chains of membrane or surface immunoglobulins comprise a transmembrane domain and a short cytoplasmic domain at their carboxy-terminus.
  • 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.
  • 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 (-NH 2 ) and carboxyl (-COOH) functional groups, along with a side chain (R group) specific to each amino acid.
  • 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
  • Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence.
  • 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 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.
  • 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.
  • 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.
  • conservative amino acid substitutions include substitutions within the following groups:
  • 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.
  • 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)).
  • single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context.
  • antibody 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.
  • mAbs monoclonal antibodies
  • antibody -like polypeptides such as chimeric antibodies and humanized antibodies
  • 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.
  • 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.
  • 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.
  • chimeric antibody 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.
  • 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.
  • the chimeric antibody may be a genetically or an enzymatically engineered recombinant antibody.
  • 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.
  • humanized antibody 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.
  • CDRs complementarity -determining regions
  • FR homologous human acceptor framework region
  • 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.
  • 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.
  • a protein which is "derived from" another 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.
  • a parent protein e.g., 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a "similar" amino acid sequence 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
  • fusion procedures are also known.
  • binding arm comprising an antigen-binding region means an antibody molecule or fragment that comprises an antigen-binding region.
  • 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.
  • 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.
  • Fc region 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.
  • 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.
  • IgG Fc receptor FcgammaR, FcyR
  • Fc-mediated effector function may be measured by binding to FcyRs, binding to Clq, or induction of Fc-mediated cross-linking via FcyRs.
  • CH2 region refers to the CH2 region of an immunoglobulin heavy chain.
  • 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).
  • the CH2 region may also be any of the other subtypes as described herein.
  • 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.
  • 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.
  • 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' 8 M or less, such as about 10' 9 M or less, about IO 0 M or less, or about 10 41 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.
  • BLI Bio-Layer Interferometry
  • SPR surface plasmon resonance
  • 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.
  • a non-specific antigen e.g., BSA, casein
  • 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.
  • 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 O ff value.
  • 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.
  • a competitive ELISA assay may be used as a cross-blocking assay.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • substitution of lysine with arginine in position 409 is designated as: K409R
  • substitution of lysine with any amino acid residue in position 409 is designated as K409X.
  • 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.
  • conservative substitutions may be defined by substitutions within the classes of amino acids as defined in tables 2 and 3.
  • CD40 refers to CD40, also referred to as tumor necrosis factor receptor superfamily member 5 (TNFRSF5), which is the receptor for the ligand TNFSF5/CD40L.
  • TNFRSF5 tumor necrosis factor receptor superfamily member 5
  • 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.
  • 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 "Programmed Death-1 (PD-1)" receptor refers to an immuno-inhibitory receptor belonging to the CD28 family.
  • PD-1 also known as CD279
  • PD-L1 also known as B7-H1 or CD274
  • 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.
  • 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.
  • 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).
  • PtdSer phosphatidyl serine
  • HMGB1 High Mobility Group Protein 1
  • CEACAM1 Carcinoembryonic Antigen Related Cell Adhesion Molecule 1
  • 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.
  • GAL9 as used herein includes human GAL9 (hGAL9), variants, isoforms, and species homologs of hGAL9, and analogs having at least one common epitope.
  • PdtSer as used herein includes variants and analogs having at least one common epitope.
  • HMGB1 as used herein includes human HMGB1 (hHMGBl), variants, isoforms, and species homologs of hHMGBl, and analogs having at least one common epitope.
  • 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).
  • NK cells natural killer cells
  • NK-T cells natural killer T cells
  • T cells ot/p and y/8.
  • NKG2A is frequently expressed in tumor infiltrating cells and HLA-E is overexpressed in several cancers.
  • IDO 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.
  • 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.
  • CD39 as used herein includes human CD39 (hCD39), variants, isoforms, and species homologs of hCD39, and analogs having at least one common epitope.
  • CD73 as used herein includes human CD73 (hCD73), variants, isoforms, and species homologs of hCD73, and analogs having at least one common epitope.
  • A2AR as used herein includes human A2AR (hA2AR), variants, isoforms, and species homologs of hA2AR, and analogs having at least one common epitope.
  • 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.
  • VISTA 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.
  • Siglec The "Sialic acid binding immunoglobulin type lectin” family members recognize sialic acids and are involved in distinction between “self 1 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.
  • GARP Glycoprotein A repetitions predominant
  • CD47 is a transmembrane protein that binds to the ligand “signal-regulatory protein alpha” (SIRPa).
  • SIRPa signal-regulatory protein alpha
  • 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.
  • PVRIG Polyovirus receptor related immunoglobulin domain containing
  • CD112R Polypeptide-binds to "Poliovirus receptor-related 2"
  • 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.
  • PVRIG 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.
  • CSF1R colony-stimulating factor 1 pathway
  • 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.
  • 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.
  • 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.
  • NADPH oxidase refers to an enzyme of the NOX family of enzymes of myeloid cells that generate immunosuppressive reactive oxygen species (ROS).
  • NOX reactive oxygen species
  • 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.
  • 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.
  • TDO tumor-derived antigenase
  • 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.
  • TDO includes human TDO (hTDO), variants, isoforms, and species homologs of hTDO, and analogs having at least one common epitope with hTDO.
  • immune checkpoint proteins mediate immune checkpoint signaling.
  • 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.
  • 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.
  • 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).
  • Dysfunctional 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.
  • T cell effector functions such as proliferation, cytokine production (e.g., IL-2) and/or target cell killing.
  • T cell anergy 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.
  • exhaust 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).
  • extrinsic negative regulatory pathways e.g., immunoregulatory cytokines
  • 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.
  • 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.
  • 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.
  • 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).
  • oligonucleotide 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.
  • morpholino antisense oligonucleotides can be used for gene knockdowns in vertebrates.
  • 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.
  • TAA tumor associated antigen
  • 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.
  • 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.
  • aptamer 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.
  • the aptamer binds to an immune checkpoint protein such as the immune checkpoint proteins described herein.
  • 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).
  • 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.
  • 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.
  • various A2AR antagonists known in the art are organic compounds having a molecular weight below 500 daltons.
  • 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).
  • cells e.g., T lymphocytes, dendritic cells, or stem cells
  • 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).
  • 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 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.
  • 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 "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 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 eva i 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 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 eva i 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.
  • PFS progression-free survival
  • OS Global System for Mobile Communications
  • treatment regimen refers to a structured treatment plan designed to improve and maintain health.
  • an 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.
  • cancer includes a disease characterized by aberrantly regulated cellular growth, proliferation, differentiation, adhesion, and/or migration.
  • 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.
  • cancer also comprises cancer metastases.
  • 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.
  • the growth of a new tumor i.e. a secondary tumor or metastatic tumor
  • Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop metastatic potential.
  • 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 “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 refers to a pH of about 7.5.
  • % 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).
  • freeze relates to the solidification of a liquid, usually with the removal of heat.
  • lyophilizing 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.
  • surrounding pressure e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less
  • naturally occurring refers to the fact that an object can be found in nature.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • part and fragment are used interchangeably herein and refer to a continuous element.
  • a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure.
  • the term “part” means a portion of the composition.
  • 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 is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame.
  • 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.
  • 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.
  • 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.
  • WT wild type
  • 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.
  • 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.
  • 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.
  • the degree of similarity or identity is given for the entire length of the reference amino acid sequence.
  • sequence similarity preferably sequence identity
  • 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.
  • Sequnce identity between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.
  • % identical and % identity 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a fragment or variant of an amino acid sequence 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.
  • one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived.
  • 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.
  • 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.
  • immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.
  • amino acid sequence "derived from” a designated amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • an antigen relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies or T- lymphocytes (T-cells).
  • the term "antigen” comprises any molecule which comprises at least one epitope, such as a T cell epitope.
  • 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).
  • 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.
  • 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.
  • 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.
  • an antigen may correspond to a naturally occurring product, for example, a viral protein, or a part thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • epitope refers to a part or fragment of a molecule such as an antigen that is recognized by the immune system.
  • 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.
  • epitope includes T cell epitopes.
  • 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.
  • 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 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.
  • an antigen is presented by a diseased cell (such as tumor cell or an infected cell).
  • an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC.
  • 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.
  • 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.
  • 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.
  • the term refers to one or more extracellular loops or domains or a fragment thereof.
  • 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.
  • the RNA in particular mRNA
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • cellular immune response means 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
  • the killer cells also termed cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs kill cells such as diseased cells.
  • 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.
  • 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.
  • expression as used herein is defined as the transcription and/or translation of a particular nucleotide sequence.
  • 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.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • disease 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.
  • 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.
  • terapéutica 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.
  • 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.
  • 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.
  • the individual is a human being.
  • the terms “individual” and “subject” do not denote a particular age, and thus encompass adults, elderlies, children, and newborns.
  • the "individual” or “subject” is a "patient”.
  • patient means an individual or subject for treatment, in particular a diseased individual or subject.
  • 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.
  • HNSCC head and neck squamous cell carcinoma
  • 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.
  • CD137 is co-expressed on PD-1 + cells.
  • 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.
  • 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.
  • OS overall survival
  • 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.
  • 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 49%, at
  • 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.
  • 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.
  • 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 80%, at least 8
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • the binding agent is a human antibody or a humanized antibody.
  • 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).
  • Each of the first and second heavy chain constant regions 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.
  • 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
  • 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.
  • 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)
  • 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)
  • 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
  • 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 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.
  • CHs heavy chain constant regions
  • said first and second heavy chain constant regions 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).
  • 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.
  • the Fc-mediated effector function may be determined by measuring binding of the binding agent to Clq.
  • 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.
  • the constant region of said first 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: 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).
  • 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).
  • 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: 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).
  • the binding agent comprises a kappa (K) light chain constant region.
  • the binding agent comprises a lambda (X) light chain constant region.
  • 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.
  • 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).
  • 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.
  • the binding agent may be a full-length IgGl antibody.
  • the binding agent (in particular, antibody) is of the IgGlm(f) allotype.
  • 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.
  • a binding agent in a suitable amount according to the present disclosure is able to trans-activate two different cells.
  • CD40 is expressed on a number of cells including antigen-presenting cells (APCs), such as dendritic cells
  • APCs antigen-presenting cells
  • CD137 is expressed on T cells and other cells.
  • 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.
  • 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, 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).
  • the amount of binding agent administered 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/
  • the binding agent may be administered in the form of any suitable pharmaceutical composition as described herein.
  • 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).
  • the binding agent is administered prior to the administration of the PD-1/PD-L1 checkpoint inhibitor.
  • 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.
  • the binding agent is administered after the administration of the PD-1/PD-L1 checkpoint inhibitor.
  • 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.
  • the binding agent is administered simultaneously with the PD-1/PD-L1 checkpoint inhibitor.
  • the binding agent and the PD-1/PD-L1 checkpoint inhibitor may be administered using a composition comprising both drugs.
  • 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.
  • the immune checkpoint inhibitor prevents inhibitory signals associated with the immune checkpoint.
  • the immune checkpoint inhibitor is an antibody, or fragment thereof that disrupts or inhibits inhibitory signaling associated with the immune checkpoint.
  • the immune checkpoint inhibitor is a small molecule inhibitor that disrupts or inhibits inhibitory signaling.
  • the immune checkpoint inhibitor is a peptide-based inhibitor that disrupts or inhibits inhibitory signaling.
  • the immune checkpoint inhibitor is an inhibitory nucleic acid molecule that disrupts or inhibits inhibitory signaling.
  • Inhibiting or blocking of inhibitory immune checkpoint signaling results in preventing or reversing immune-suppression and establishment or enhancement of T cell immunity against cancer cells.
  • inhibition of immune checkpoint signaling reduces or inhibits dysfunction of the immune system.
  • inhibition of immune checkpoint signaling renders dysfunctional immune cells less dysfunctional.
  • inhibition of immune checkpoint signaling renders a dysfunctional T cell less dysfunctional.
  • 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.
  • antibodies or antigen-binding fragments thereof are chimerized, humanized or human antibodies.
  • immune checkpoint inhibitor antibodies or antigen-binding fragments thereof are antagonists of immune checkpoint receptors or of immune checkpoint receptor ligands.
  • an antibody that is an immune checkpoint inhibitor is an isolated antibody.
  • 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.
  • 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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.
  • the PD-1 inhibitor is IgGl-PDl as disclosed herein.
  • 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.
  • CD Rs complementary determining regions
  • 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).
  • 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.
  • 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.
  • 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.
  • an anti-PD-1 antibody described herein binds specifically to PD-1 (e.g., human PD-1).
  • 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.
  • antigen-binding fragments comprising any combination of variable region(s) with a hinge region, CHI, CH2, CH3 and CL domains.
  • 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.
  • a particular CDR e.g., a CDR-H3
  • a CDR-H3 contains the amino acid sequence of a corresponding CDR in a given VH or VL region amino acid sequence
  • 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.
  • 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.
  • 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.
  • the PD-L1 inhibitor is atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267; see US 9,724,413).
  • 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.
  • CD Rs complementary determining regions
  • 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).
  • 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.
  • 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.
  • isotype e.g., IgG, IgE, IgM, IgD, IgA and IgY
  • class e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2
  • subclass of immunoglobulin molecule e.g., IgG, IgE, IgM, IgD, IgA and IgY
  • subclass of immunoglobulin molecule e.g., IgG, IgE, IgM, IgD, IgA and IgY
  • subclass of immunoglobulin molecule e.g
  • 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.
  • antigen-binding fragments comprising any combination of variable region(s) with a hinge region, CHI, CH2, CH3 and CL domains.
  • 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.
  • 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.
  • a particular CDR e.g., a CDR-H3
  • a CDR-H3 contains the amino acid sequence of a corresponding CDR in a given V H or VL region amino acid sequence
  • 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.
  • 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.
  • the anti-PD-Ll antibodies disclosed herein comprise the CDRs of the antibody atezolizumab. See US 9,724,413.
  • 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.
  • 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, 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.
  • 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.
  • 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.
  • 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)).
  • 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.
  • an immune checkpoint inhibitor e.g., an inhibitory nucleic acid molecule or an antibody or fragment thereof.
  • 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.
  • the cell based therapy comprises genetically engineered cells.
  • the genetically engineered cells express an immune checkpoint inhibitor, such as described herein.
  • 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.
  • the checkpoint inhibitor such as the PD-1/PD-L1 checkpoint inhibitor
  • 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.
  • 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.
  • 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).
  • 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).
  • the amount of pembrolizumab administered 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
  • 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.
  • the checkpoint inhibitor is administered in the form of an infusion.
  • 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.
  • the platinum-based chemotherapeutic agent as well as 5 -fluorouracil are administered systemically, such as parenterally, in particular intravenously.
  • 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.
  • the platinum-based chemotherapeutic agent is carboplatin or cisplatin.
  • the chemotherapy combination is cisplatin and 5-fluorouracil. In another embodiment, the chemotherapy combination is carboplatin and 5-fluorouracil.
  • the amount of cisplatin administered is about 50-150 mg/m 2 /day, such as about 60-140 mg/m 2 /day, about 70-130 mg/m 2 /day, about 80-120 mg/m 2 /day, about 90-110 mg/m 2 /day, or about 95-105 mg/m 2 /day, e.g., about 100 mg/m 2 /day.
  • 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):
  • 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)
  • Total body weight [TBW] - IBW 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.
  • 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).
  • the amount of 5 -fluorouracil administered is about 500-1500 mg/m 2 /day, such as about 600-1400 mg/m 2 /day, about 700-1300 mg/m 2 /day, about 800-1200 mg/m 2 /day, about 900-1100 mg/m 2 /day, or about 950-1050 mg/m 2 /day, e.g., about 1000 mg/m 2 /day.
  • HNSCC 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.
  • mOS median overall survival
  • the PD-1/PD-L1 checkpoint inhibitor in particular, pembrolizumab
  • the PD-1/PD-L1 checkpoint inhibitor is in particular administered to the subject by systemic administration.
  • the PD-1/PD-L1 checkpoint inhibitor in particular, pembrolizumab
  • 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.
  • 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).
  • 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
  • a dose of 5 -fluorouracil may be any dose of 5 -fluorouracil as specified herein, e.g., about 500-1500 mg/m 2 /day, such as about 600-1400 mg/m 2 /day, about 700-1300 mg/m 2 /day, about 800-1200 mg/m 2 /day, about 900-1100 mg/m 2 /day, or about 950-1050 mg/m 2 /day, e.g., about 1000 mg/m 2 /day
  • 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.
  • 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.
  • detergents e.g., a nonionic detergent, such as Tween-20 or Tween-80
  • stabilizers e.g., sugars or protein-free amino acids
  • preservatives e.g., sugars or protein-free amino acids
  • solubilizers e.g., sugars or protein-free amino acids
  • Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example
  • compositions 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.
  • aqueous and non-aqueous carriers examples 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.
  • 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.
  • excipient refers to a substance which may be present in a (pharmaceutical) composition of the present disclosure but is not an active ingredient.
  • excipients include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.
  • diluting and/or thinning agent relates a diluting and/or thinning agent.
  • 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.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butyl
  • a (pharmaceutical) composition may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the composition.
  • 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.
  • 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.
  • 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 (NH 4 + ); 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).
  • alkali metal salts e.g., sodium or potassium salts
  • alkaline earth metal salts e.g., calcium or magnesium salts
  • ammonium NH 4 +
  • suitable organic ligands e.g., quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl s
  • 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, he
  • 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.
  • 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.
  • 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.
  • 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).
  • binding agent for use of any one of the preceding items, wherein the binding agent is administered prior to the administration of pembrolizumab.
  • 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%.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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/m 2 /day, and 5 -fluorouracil is administered at a dose of about 1000 mg/m 2 /day.
  • 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.
  • 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.
  • 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.
  • 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).
  • phase la 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.
  • CNS non-central nervous system
  • 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.
  • NSCLC CPI-naive, PD-L1 expressing, TPS >1% per local lab testing
  • HNSCC CPI-naive, PD-L1 expressing, CPS >1 per local lab testing
  • HNSCC CPI-naive, PD-L1 expressing, CPS >1 per local lab testing
  • Doses below the highest dose level which is deemed safe in the dose escalation part 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.
  • lL first line
  • Pembrolizumab infusion will be administered first followed by GEN 1042 followed by SOC chemotherapy.
  • 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.
  • 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.
  • 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.
  • 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:
  • NSCLC • 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.
  • IHC immunohistochemistry
  • HNSCC 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).
  • HNSCC program death ligand 1
  • PPS program death ligand 1
  • GEN 1042 CD40x4-lBB
  • SOC Standard of Care
  • pembrolizumab 5 -fluorouracil
  • 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.
  • ORR Objective Response Rate
  • DCR Disease Control Rate
  • 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 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).
  • 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 Partial Response
  • Example 3 Pharmacodynamic evaluation of GEN1042+SoC in peripheral blood of IL, CPI naive, HNSCC patients
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 4-1BB-AF647 clone 4B4-1, Biolegend, cat. no. 309824
  • CD86-BV421 clone 2331, BD Biosciences, cat. no.
  • 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.
  • 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.
  • 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

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Abstract

La présente invention concerne une polythérapie utilisant un agent de liaison qui se lie au CD40 humain et au CD137 humain en combinaison avec un inhibiteur de point de contrôle qui est un inhibiteur de l'axe PD-1/PD-L1 (en particulier le pembrolizumab) et une chimiothérapie pour réduire ou prévenir la progression d'un carcinome épidermoïde de la tête et du cou (CETEC) ou traiter le CETEC.
EP23817404.9A 2022-12-01 2023-12-01 Anticorps multispécifique contre cd40 et cd137 en polythérapie avec un anticorps anti-pd1 et une chimiothérapie Pending EP4626446A1 (fr)

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