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WO2025056180A1 - Procédés de traitement faisant appel à des agents se liant à epcam et cd137 en combinaison avec des antagonistes de liaison à l'axe pd-1 - Google Patents

Procédés de traitement faisant appel à des agents se liant à epcam et cd137 en combinaison avec des antagonistes de liaison à l'axe pd-1 Download PDF

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Publication number
WO2025056180A1
WO2025056180A1 PCT/EP2023/075492 EP2023075492W WO2025056180A1 WO 2025056180 A1 WO2025056180 A1 WO 2025056180A1 EP 2023075492 W EP2023075492 W EP 2023075492W WO 2025056180 A1 WO2025056180 A1 WO 2025056180A1
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Prior art keywords
binding
heavy chain
region
seq
antigen
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Inventor
Ugur Sahin
Sina FELLERMEIER-KOPF
Andrea IMLE
Maren KÖHNE
Annieck DIKS
Esther Cornelia Wilhelmina Breij
Kristel KEMPER
Andreea IOAN-FACSINAY
Patricia GARRIDO CASTRO
Lars GUELEN
David Satijn
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Biontech SE
Genmab AS
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Biontech SE
Genmab AS
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Priority to PCT/EP2023/075492 priority Critical patent/WO2025056180A1/fr
Priority to PCT/EP2024/075677 priority patent/WO2025056778A1/fr
Priority to ARP240102454A priority patent/AR133823A1/es
Priority to TW113135136A priority patent/TW202528358A/zh
Publication of WO2025056180A1 publication Critical patent/WO2025056180A1/fr
Pending legal-status Critical Current
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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • 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
    • C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
    • 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
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • EpCAM Epithelial cell adhesion molecule
  • EpCAM is a transmembrane glycoprotein mediating Ca 2+ -independent homotypic cell-cell adhesion in epithelia. EpCAM is also involved in cell signaling, migration, proliferation, and differentiation. Additionally, EpCAM has oncogenic potential via its capacity to upregulate c-Myc, E-FABP, and cyclins A & E. EpCAM can be used as diagnostic marker for various cancers. Also, EpCAM appears to play a role in tumorigenesis and metastasis of carcinomas, and, thus, it can also act as a potential prognostic marker and as a potential target for immunotherapeutic strategies.
  • the present disclosure provides a kit comprising, in one or more vials, (i) a binding agent comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137, or nucleic acid encoding the binding agent, (ii) a PD-1 axis binding antagonist or a nucleic acid encoding the PD-1 binding antagonist, and optionally (iii) one or more therapeutic agents.
  • the kit further comprises instructions for the use of the kit in treating or preventing a disease or disorder in a subject.
  • the binding agent or encoding nucleic acid and the PD-1 binding antagonist or encoding nucleic acid are in separate vials.
  • An aspect of the present disclosure provides a binding agent comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137 for use in a method for treating or preventing a disease or disorder in a subject, said method comprising administering the binding agent to the subject in combination with a PD-1 axis binding antagonist, wherein the administration of the binding agent is prior to, simultaneously with, or after administration of the PD-1 axis binding antagonist.
  • An aspect of the present disclosure provides for use of (i) a binding agent comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137 and (ii) a PD-1 axis binding antagonist for the manufacture of a medicament for treating or preventing a disease or disorder in a subject.
  • the disease or disorder a tumor, solid tumor or cancer.
  • the embodiments relating to the method of treatment aspect are equally applicable to the aspects concerning the use of the binding agent and PD-1 axis binding antagonist.
  • An aspect of the present disclosure provides for a medical preparation comprising the binding agent and the PD-1 axis binding antagonist as defined in the present disclosure.
  • the medical preparation is for use in any of the methods of treatment disclosed in the present disclosure.
  • Fig 1 shows the relative EpCAM expression on T84, DiFi, HPAF-II, NCI-N87, Calu-3, NCI-H747, and A549 tumor cell lines as determined by flow cytometry. Data shown is A geometric mean fluorescence intensity (gMFI) for each tumor cell line calculated as: Geomean fluorescence intensity (APC) of the EpCAM antibody - Geomean fluorescence (APC) of the non-binding control antibody.
  • APC geometric mean fluorescence intensity
  • Figure 2 Binding of bivalent EpCAM antibodies and their monovalent counterparts to DiFi, HPAF-II or A549 cells.
  • Binding of monovalent and bivalent EpCAM antibodies was analysed using CHO-S cells transiently transfected with full length human, cynomolgus monkey or mouse EpCAM. As negative control, binding to non-transfected CHO-WT cells was evaluated. Data are presented as geomean fluorescence intensity (gMFI) R-PE values of two technical replicates ⁇ SD.
  • Figure 4 Induction of 4-lBB-dependent luciferase activity by an EpCAMx4-lBB bispecific antibody in a cell-based reporter assay.
  • HEK293_NKF_h4-lBB_gfpJuc reporter cells were either co-cultured with EpCAM-expressing OV-90-SC12 cells, or cultured alone (medium), in the presence of serial dilutions of BsIgGl-CD137-005-FEAR/EpCAM-323-A3-FEAL overnight. Luciferase activity was quantified by luminescence measurements. Fold-induction of luciferase activity relative to cultures without antibody (dotted line) is shown. Error bars depict SD of duplicate wells. Data from one representative experiment out of three are shown.
  • Figure 5 Enhancement of PBMC proliferation in vitro by EpCAMx4-lBB bispecific antibodies in PBMC-DiFi cell cocultures.
  • EpCAMx4-lBB bispecific antibodies were tested in an in vitro PBMC proliferation assay using EpCAM-expressing DiFi cells in co-culture with CFSE-labeled PBMCs.
  • Cells were cultured in the presence of anti-CD3 (0.1 ⁇ g/mL ) with or without the addition of EpCAMx4-lBB or EpCAMxbl2 bispecific antibodies or non-binding control IgGl-bl2-FEAL at the indicated concentrations for 96 h.
  • the number of CFSE-positive cells was extracted and used to calculate the division index. Data shown are the mean division index ⁇ SD of duplicate measurements obtained from one representative experiment.
  • Figure 6 Enhancement of CD4+ and CD8+ T-cell proliferation in vitro by EpCAMx4-lBB bispecific antibodies in PBMC-DiFi cell co-cultures.
  • EpCAMx4-lBB bispecific antibodies were tested in an in vitro PBMC proliferation assay using EpCAM-expressing T84, DiFi, HPAF-II, NCI-N87, Calu-3 or NCI-H747 tumor cells in co-culture with CFSE-labeled PBMCs.
  • Cells were cultured in the presence of anti-CD3 (0.1 ⁇ g/mL ) and EpCAMx4-lBB or EpCAMxbl2 bispecific antibodies or non- binding control IgGl-bl2-FEAL at the indicated concentrations for 96 h.
  • the number of CFSE-positive cells was evaluated as a measure of absolute PBMC counts by flow cytometry and the division index was calculated.
  • EpCAMx4-lBB bispecific antibodies were tested in an in vitro PBMC proliferation assay using EpCAM-expressing DiFi tumor cells in co-culture with CFSE-labeled human PBMCs.
  • Cells were cultured in the presence of anti-CD3 (0.1 pg/mL) and EpCAM-FERLx4-lBB-FEAR (closed diamonds), EpCAM-FEARx4-lBB-FEAL (closed squares), bl2- FERLx4-lBB-FEAR (left-half-closed triangles), EpCAM-FERLxbl2-FEAR (right-half-closed triangles) bispecific antibodies or non-binding control bl2-FERLxbl2-FEAR (open triangles) at the indicated concentrations for 96 h.
  • Cells were cultured in the presence of anti-CD3 (0.1 ⁇ g/mL ; dotted line) and bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2- FEAR or with the combination of bs!gGl-EpCAM-A37-FERL/bl2-FEAR and bsIgGl-bl2-FERL/CD137-HC7LC2-FEAR at the indicated concentrations for 96 h.
  • the percentage of CD4+ and CD8+ T cells expressing CD25 (A) or 4-1BB (B), as well as the geometric mean fluorescence intensity (FI) in the CD25+ or 4-1BB+ population was determined by flow cytometry. Data shown are mean ⁇ SD of duplicate measurements obtained from one representative experiment.
  • EpCAMx4-lBB bispecific antibody bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR was compared to the combination of bsIgGl-EpCAM-A37-FERL/bl2-FEAR and bsIgGl-bl2-FERL/CD137-009-HC7LC2-FEAR in an in vitro proliferation assay using EpCAM-expressing DiFi tumor cells in co-culture with CTV-labeled cancer patient-derived PBMC.
  • Cells were cultured in the presence of anti-CD3 (0.1 ⁇ g/mL ; dotted line) and bsIgGl-EpCAM-A37- FERL/CD137-009-HC7LC2-FEAR or with the combination of bsIgGl-EpCAM-A37-FERL/bl2-FEAR and bsIgGl-bl2- FERL/CD137-009-HC7LC2-FEAR at the indicated concentrations for 96 h.
  • CD4+ and CD8+ T cell proliferation was evaluated by flow cytometry analysis of CTV label dilution. Data shown are mean percentage divided cells ⁇ SD of duplicate measurements obtained from one experiment.
  • FIG. 16 An EpCAMx4-lBB bispecific antibody enhances CD107a and GzmB expression by CD8* ⁇ T cells PBMC-derived CD8 + T cells expressing a CLDN6-specific TCR were co-cultured with MDA-MB-231_hCLDN6_hEpCAM tumor cells for 2 d in the presence of bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR or control antibodies. CD107a and GzmB expression by CD8 + T cells were analyzed by flow cytometry.
  • A Representative flow cytometry plots.
  • B-C Dose-response curves of CD107a (C) and GzmB (D) expression levels.
  • FIG. 17 An EpCAMx4-lBB bispecific antibody enhances CD8* T-cell mediated cytotoxicity towards tumor cells PBMC-derived CD8 + T cells expressing a CLDN6-specific TCR were co-cultured with MDA-MB-231_hCLDN6_hEpCAM tumor cells for 5 to 6 d in the presence of bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR or control antibodies. Cell index values were derived from impedance measurements conducted at 2 to 3 h intervals. (A) Cell index curves in co-cultures from one representative donor are shown. Symbols represent average cell index values of duplicate wells.
  • EpCAMx4-lBB bispecific antibodies with various combinations of Fc-inertness mutations (FER/FEA, FEA/FEA, FER/FER)
  • monoclonal antibodies IgGl-EpCAM-A37-FERL and IgGl-CD137-009- HC7LC2-FEAR was analyzed by SPR: FcyRIa, FcyRIIa-H131, FcyRIIa-R131, FcyRIIb, FcyRIIIa-F158, and FcyRIIIa-V158.
  • the antibody IgGl-bl2 (wild-type Fc) was included as a positive control for FcyR binding.
  • Healthy donor PBMCs were cocultured with EpCAM-expressing (EpCAM+) DiFi or CAL27 tumor cells (at a ratio of 16:1) in the presence of an anti-CD3 antibody (0.1 ⁇ g/mL ) and treated with bsIgGl-EpCAM-A37-FERL/CD137-009- HC7LC2-FEAR, 1 ⁇ g/mL pembroiizumab (either alone or in combination) or control antibodies (bsIgGl-EpCAM-A37- FERL/bl2-FEAR and bs!gGl-bl2-FERL/CD137-009-HC7LC2-FEAR [3 ⁇ g/mL of each antibody] or IgG4 [1 ⁇ g/mL ]) for 96 h in order to determine IFNy secretion.
  • EpCAM+ EpCAM-expressing
  • DiFi CAL27 tumor cells
  • Healthy donor PBMCs were cocultured with EpCAM-expressing (EpCAM+) DiFi or CAL27 tumor cells (at a ratio of 16:1) in the presence of an anti-CD3 antibody (0.1 ⁇ g/mL ) and treated with bsIgGl-EpCAM-A37-FERL/CD137-009- HC7LC2-FEAR, 1 ⁇ g/mL IgGl-PDl (either alone or in combination) or control antibodies (bsIgGl-EpCAM-A37- FERL/bl2-FEAR and bsIgGl-bl2-FERL/CD137-009-HC7LC2-FEAR [3 ⁇ g/mL of each antibody] or IgGl-bl2-FERR [1 pg/mL]) for 96 h in order to determine IFNy secretion.
  • EpCAM-expressing (EpCAM+) DiFi or CAL27 tumor cells at a ratio of 16:1
  • an anti-CD3 antibody 0.1
  • 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.
  • 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 VL or VL (variable light chain) domain/region, C L or CL (constant light chain) domain/region, V H or VH (variable heavy chain) domain/region, and the CH or CH (constant heavy chain) domains/regions 04 (CHI), C H 2 (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
  • the present disclosure is not limited to CDR sequences only determined according to the IMGT rules.
  • the HCDR1, HCDR2, and HCDR3 sequences of a heavy chain variable region (VH), e.g., SEQ ID NO: 1 or SEQ ID NO: 11, or the LCDR1, LCDR2, and LCDR3 sequences of a light chain variable region (VL), e.g., SEQ ID NO: 5 or SEQ ID NO: 15, shall encompass those CDR sequences which are determined by any method for determining CDR sequences, for example according to the IMGT rules or the Kabat rules.
  • sequences shall also be included that represent an overlap of CDR sequences that are determined by different methods for determining CDR sequences, e.g., according to the IMGT rules and the Kabat rules.
  • the following table shows the results of determining CDR sequences according to the IMGT rules and the Kabat rules in relation to SEQ ID NO: 21 and SEQ ID NO: 22, and also shows the overlap of the determined CDR sequences.
  • the following table shows the results of determining CDR sequences according to the IMGT rules and the Kabat rules in relation to SEQ ID NO: 25 and SEQ ID NO: 26, and also shows the overlap of the determined CDR sequences.
  • the following table shows the results of determining CDR sequences according to the IMGT rules and the Kabat rules in relation to SEQ ID NO: 27 and SEQ ID NO: 28, and also shows the overlap of the determined CDR sequences.
  • immunoglobulin heavy chains There are five types of mammalian immunoglobulin heavy chains, i.e., a, ⁇ , e, ⁇ , 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?) 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 of R group
  • Table 3 Alternative Physical and Functional Classifications of Amino Acid Residues
  • variants of an amino acid sequence comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants.
  • variant includes all mutants, splice variants, posttranslationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring.
  • variant includes, in particular, fragments of an amino acid sequence.
  • Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence.
  • 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.
  • a "conservative substitution” is a substitution of one amino acid with another amino acid having similar structural and/or chemical characteristics, such substitution of one amino acid residue for another amino acid residue of the same class as defined in any of the two tables above: for example, leucine may be substituted with isoleucine as they are both aliphatic, branched hydrophobes. Similarly, aspartic acid may be substituted with glutamic acid since they are both small, negatively charged residues.
  • Naturally occurring amino acids may also be generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
  • conservative amino acid substitutions include substitutions within the following groups:
  • amino acid corresponding to position refers to an amino acid position number in a human IgGl heavy chain. Corresponding amino acid positions in other immunoglobulins may be found by alignment with human IgGl.
  • an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings and has at least 50%, at least 80%, at least 90%, or at least 95% identity to a human IgGl heavy chain. It is considered well- known in the art how to align a sequence or segment in a sequence and thereby determine the corresponding position in a sequence to an amino acid position according to the present disclosure.
  • antibody in the context of the present disclosure refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen (in particular an epitope on an antigen), typically under physiological conditions, preferably with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity).
  • the term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • the term “antibody” includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies and combinations of any of the foregoing.
  • Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL).
  • the variable regions and constant regions are also referred to herein as variable domains and constant domains, respectively.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the CDRs of a VH are termed HCDR1, HCDR2 and HCDR3 (or CDR-H1, CDR-H2 and CDR-H3)
  • the CDRs of a VL are termed LCDR1, LCDR2 and LCDR3 (or CDR-L1, CDR-L2 and CDR-L3).
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of an antibody comprise the heavy chain constant region (CH) and the light chain constant region (CL), wherein CH can be further subdivided into constant domain CHI, a hinge region, and constant domains CH2 and CH3 (arranged from amino-terminus to carboxy-terminus in the following order: CHI, CH2, CH3).
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system such as Clq.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoactive portions of intact immunoglobulins.
  • Antibodies are typically tetramers of immunoglobulin molecules.
  • Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)z, as well as single chain antibodies and humanized antibodies.
  • variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen.
  • binding region and “antigen-binding region” are used herein interchangeably and refer to the region which interacts with the antigen and comprises both a VH region and a VL region.
  • An antibody as used herein comprises not only monospecific antibodies, but also multispecific antibodies which comprise multiple, such as two or more, e.g., three or more, different antigen-binding regions.
  • 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.
  • antigen-binding fragments encompassed within the term "antibody” include (i) a Fab' or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, or a monovalent antibody as described in WO 2007/059782 (Genmab); (ii) F(ab’) 2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CHI domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol.
  • 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.
  • fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present disclosure, exhibiting different biological properties and utility.
  • 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
  • antigen-binding fragments provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.
  • 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.
  • 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.
  • CH3 region refers to the CH3 region of an immunoglobulin heavy chain.
  • the CH3 region of a human IgGl antibody corresponds to amino acids 341-447 according to the EU numbering as set forth in Kabat ⁇ ibid).
  • the CH3 region may also be any of the other subtypes as described herein.
  • PFS progression-free survival
  • 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 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.
  • Physiological pH refers to a pH of 7.5 or 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)-
  • 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
  • recombinant in the context of the present disclosure means "made through genetic engineering". In one embodiment, a “recombinant object” in the context of the present disclosure is not occurring naturally.
  • peptide comprises oligo- and polypeptides and refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds.
  • protein refers to large peptides, in particular peptides having at least about 151 amino acids, but the terms "peptide” and “protein” are used herein usually as synonyms.
  • a “therapeutic protein” has a positive or advantageous effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount.
  • 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 immunostimulants such as cytokines.
  • a fragment of an amino acid sequence comprises, e.g., at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence.
  • a fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.
  • 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 adds 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.
  • wild type or “WT” or “native” herein is meant an amino acid sequence that is found in nature, including allelic variations.
  • a wild-type amino acid sequence, peptide or protein has an amino acid sequence that has not been intentionally modified.
  • the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence.
  • 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.
  • the alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS: meedle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • Sequence similarity indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions.
  • Sequence identity between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.
  • 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 optima! 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.
  • percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov/Blast.cgi).
  • 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 filter 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.
  • Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.
  • 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.
  • amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation.
  • the manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example.
  • the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.
  • 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.
  • binding agents one particular function is one or more binding 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., binding 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.
  • function 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 binding agents 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 e.g, antibody, described herein is isolated.
  • An "isolated binding agent” as used herein is intended to refer to a binding agent which is substantially free of other binding agents having different antigenic specificities.
  • an isolated bispecific binding agent that specifically binds to EpCAM and CD137 is substantially free of monospecific antibodies that specifically bind to EpCAM or CD137.
  • 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.
  • the RNA described herein may have modified nucleosides.
  • the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.
  • RNA with a 5'-cap or 5'-cap analog may be achieved by in vitro transcription, in which the 5'-cap is co-transcriptionally expressed into the RNA strand, or may be attached to RNA post-transcriptionally using capping enzymes.
  • the RNA comprises a capO, capl, or cap2.
  • capO means the structure "m 7 GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'.
  • capl means the structure "m 7 GpppNm", wherein Nm is any nucleoside bearing an OCH3 moiety at position 2'.
  • cap2 means the structure "m 7 GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH3 moiety at position 2'.
  • RNA according to the present disclosure comprises a 5'-UTR and/or a 3'-UTR.
  • the term "untranslated region" or “UTR” relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule.
  • An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5 -UTR) and/or 3' (downstream) of an open reading frame (3'-UTR).
  • a 5 -UTR if present, is located at the 5' end, upstream of the start codon of a protein-encoding region.
  • a 5'-UTR is downstream of the 5'-cap (if present), e.g. directly adjacent to the 5'-cap.
  • a 3'-UTR if present, is located at the 3' end, downstream of the termination codon of a protein-encoding region, but the term "3 -UTR" does preferably not include the poly(A) sequence.
  • the 3'-UTR is upstream of the poly(A) sequence (if present), e.g. directly adjacent to the poly(A) sequence.
  • the RNA according to the present disclosure comprises a 3'-poly(A) sequence.
  • poly(A) sequence or “poly-A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3' end of an RNA molecule.
  • Poly(A) sequences are known to those of skill in the art and may follow the 3' UTR in the RNAs described herein.
  • the poly(A) sequence may be of any length.
  • a poly(A) sequence comprises or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides, and, in particular, about 110 nucleotides.
  • the poly(A) sequence only consists of A nucleotides.
  • the poly(A) sequence essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, and U), as disclosed in WO 2016/005324 Al, hereby incorporated by reference. Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • a poly(A) cassette present in the coding strand of DNA that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g. 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency.
  • no nucleotides other than A nucleotides flank a poly(A) sequence at its 3' end, i.e., the poly(A) sequence is not masked or followed at its 3' end by a nucleotide other than A.
  • transcription relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be translated into peptide or protein.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • RNA such as mRNA encodes a protein if translation of the RNA produces the protein in a cell or other biological system.
  • RNA may be naked or packaged, e.g., formulated in particles such as protein and/or lipid particles, e.g., lipid nanoparticles.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • expression is defined as the transcription and/or translation of a particular nucleotide sequence.
  • Fc regions may have at their C-terminus a lysine.
  • the origin of this lysine is a naturally occurring sequence found in humans from which these Fc regions are derived.
  • this terminal lysine can be cleaved off by proteolysis by endogenous carboxypeptidase(s), resulting in a constant region having the same sequence but lacking the C-terminal lysine.
  • the DNA encoding this terminal lysine can be omitted from the sequence such that antibodies are produced without the lysine.
  • Antibodies produced from nucleic acid sequences that either do, or do not encode a terminal lysine are substantially identical in sequence and in function since the degree of processing of the terminal lysine is typically high when e.g. using antibodies produced in CHO-based production systems (Dick, L.W. et al. Biotechnol. Bioeng. 2008;100: 1132-1143).
  • proteins in accordance with the disclosure, such as antibodies can be generated with or without encoding or having a terminal lysine.
  • the present disclosure provides a method for treating or preventing a disease or disorder in a subject, said method comprising (i) administering to the subject binding agent comprising a first antigen-binding region binding to EpCAM and a second antigen-binding region binding to CD137, and (ii) administering to the subject a PD(L)-1 axis binding antagonist, wherein the administration of the binding agent is prior to, simultaneously with, or after administration of the PD(L)-1 axis binding antagonist.
  • the EpCAM is human EpCAM.
  • the CD137 is human CD137.
  • human EpCAM comprises the sequence set forth in SEQ ID NO: 59.
  • human CD137 comprises the sequence set forth in SEQ ID NO: 62.
  • said first antigen-binding region binding to EpCAM binds to EpCAM expressed on tumor cells.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR3 sequence comprising the sequence as set forth in SEQ ID NO: 4.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR2 sequence comprising the sequence as set forth in SEQ ID NO: 3.
  • said first antigen- binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR1 sequence comprising the sequence as set forth in SEQ ID NO: 2.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 2, 3, and 4, respectively.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 64, 65, and 66, respectively.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 74, 3, and 66, respectively.
  • VH heavy chain variable region
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR3 sequence comprising the sequence as set forth in SEQ ID NO: 8. In some embodiments, said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR2 sequence comprising the sequence as set forth in SEQ ID NO: 7. In some embodiments, said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR1 sequence comprising the sequence as set forth in SEQ ID NO: 6.
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 6, 7, and 8, respectively.
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 67, 68, and 8, respectively.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR3 sequence and a light chain variable region (VL) comprising a LCDR3 sequence, wherein the HCDR3 sequence comprises the sequence as set forth in SEQ ID NO: 4, and the LCDR3 sequence comprises the sequence as set forth in SEQ ID NO: 8.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 2, 3, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 6, 7, and 8, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 64, 65, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 67, 68, and 8, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 74, 3, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 6, 7, and 8, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 1 and/or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 5.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 1.
  • VH heavy chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the sequence as set forth in SEQ ID NO: 1.
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 5.
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising the sequence as set forth in SEQ ID NO: 5.
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 1 and the VL comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 5.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 80% identity to the sequence as set forth in SEQ ID NO: 1 and the VL comprises a sequence having at least 80% identity to the sequence as set forth in SEQ ID NO: 5,
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 90% identity to the sequence as set forth in SEQ ID NO: 1 and the VL comprises a sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 5.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 95% identity to the sequence as set forth in SEQ ID NO: 1 and the VL comprises a sequence having at least 95% identity to the sequence as set forth in SEQ ID NO: 5.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 1 and the VL comprises the sequence as set forth in SEQ ID NO: 5.
  • said first antigen-binding region binding to EpCAM comprises heavy and light chain variable regions of an antibody which competes for EpCAM binding with and/or has the specificity for EpCAM of an antibody comprising a heavy chain variable region (VH) and/or a light chain variable region (VL) as set forth above.
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising a HCDR3 sequence comprising the sequence as set forth in SEQ ID NO: 14. In some embodiments, said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising a HCDR2 sequence comprising the sequence as set forth in SEQ ID NO: 13. In some embodiments, said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising a HCDR1 sequence comprising the sequence as set forth in SEQ ID NO: 12.
  • said second antigen- binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 12, 13, and 14, respectively.
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 69, 70, and 71, respectively.
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 75, 13, and 71, respectively.
  • VH heavy chain variable region
  • said second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising a LCDR3 sequence comprising the sequence as set forth in SEQ ID NO: 18. In some embodiments, said second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising a LCDR2 sequence comprising the sequence as set forth in SEQ ID NO: 17. In some embodiments, said second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising a LCDR1 sequence comprising the sequence as set forth in SEQ ID NO: 16.
  • said second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 16, 17 and 18, respectively.
  • said second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 72, 73 and 18, respectively.
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising a HCDR3 sequence and a light chain variable region (VL) comprising a LCDR3 sequence, wherein the HCDR3 sequence comprises the sequence as set forth in SEQ ID NO: 14, and the LCDR3 sequence comprises the sequence as set forth in SEQ ID NO: 18.
  • VH heavy chain variable region
  • VL light chain variable region
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 12, 13, and 14, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprises the sequences as set forth in SEQ ID NO: 16, 17 and 18, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 69, 70, and 71, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprises the sequences as set forth in SEQ ID NO: 72, 73 and 18, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 75, 13, and 71, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprises the sequences as set forth in SEQ ID NO: 16, 17 and 18, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 11 and/or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 15.
  • VH heavy chain variable region
  • VL light chain variable region
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 11.
  • VH heavy chain variable region
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising the sequence as set forth in SEQ ID NO: 11.
  • VH heavy chain variable region
  • said second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 15.
  • VL light chain variable region
  • said second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising the sequence as set forth in SEQ ID NO: 15.
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 11 and the VL comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 15.
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 80% identity to the sequence as set forth in SEQ ID NO: 11 and the VL comprises a sequence having at least 80% identity to the sequence as set forth in SEQ ID NO: 15.
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 90% identity to the sequence as set forth in SEQ ID NO: 11 and the VL comprises a sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 15.
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 95% identity to the sequence as set forth in SEQ ID NO: 11 and the VL comprises a sequence having at least 95% identity to the sequence as set forth in SEQ ID NO: 15.
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 11 and the VL comprises the sequence as set forth in SEQ ID NO: 15.
  • said second antigen-binding region binding to CD137 comprises heavy and light chain variable regions of an antibody which competes for CD137 binding with and/or has the specificity for CD137 of an antibody comprising a heavy chain variable region and/or a light chain variable region as set forth above.
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR3 sequence as set forth in SEQ ID NO: 4
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising a HCDR3 sequence as set forth in SEQ ID NO: 14.
  • a) said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR3 sequence as set forth in SEQ ID NO: 8
  • said second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising a LCDR3 sequence as set forth in SEQ ID NO: 18.
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR3 sequence as set forth in SEQ ID NO: 4, and a light chain variable region (VL) comprising a LCDR3 sequence as set forth in SEQ ID NO: 8, and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising a HCDR3 sequence as set forth in SEQ ID NO: 14, and a light chain variable region (VL) comprising a LCDR3 sequence, as set forth in SEQ ID NO: 18.
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 2, 3, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 6, 7, and 8, respectively; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 12, 13, and 14, respectively, and the LCDR
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 64, 65, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 67, 68, and 8, respectively; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 69
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 74, 3, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 6, 7, and 8, respectively; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 75, 13, and 71
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 1 and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 5; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 11 and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 15.
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 1 and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 5; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 11 and the VL comprises an amino acid sequence as set
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 1 and the VL comprises the sequence as set forth in SEQ ID NO: 5; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 11 and the VL comprises the sequence as set forth in SEQ ID NO: 15.
  • the binding agent is an antibody comprising a first binding arm comprising said first antigen- binding region binding to EpCAM and a second binding arm comprising said second antigen-binding region binding to CD137, wherein the first binding arm comprises i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), wherein the first VH comprises first HCDR1, HCDR2, and HCDR3 sequences and the first VL comprises first LCDR1, LCDR2, and LCDR3 sequences, wherein the first HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 2, 3, and 4, respectively, and the first LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 6, 7, and 8, respectively; and the second binding arm comprises iii) a polypeptide comprising
  • the binding agent comprises i) a first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 9, ii) a first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 10, iii) a second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 19; and iv) a second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 20.
  • the binding agent comprises i) a first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9, ii) a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 10, iii) a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19; and iv) a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 20.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR3 sequence comprising the sequence as set forth in SEQ ID NO: 78. In some embodiments, said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR2 sequence comprising the sequence as set forth in SEQ ID NO: 77. In some embodiments, said first antigen- binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR1 sequence comprising the sequence as set forth in SEQ ID NO: 76.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 76, 77, and 78, respectively.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 82, 83, and 84, respectively.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 87, 77, and 84, respectively.
  • VH heavy chain variable region
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR3 sequence comprising the sequence as set forth in SEQ ID NO: 81. In some embodiments, said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR2 sequence comprising the sequence as set forth in SEQ ID NO: 80. In some embodiments, said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR1 sequence comprising the sequence as set forth in SEQ ID NO: 79.
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 79, 80, and 81, respectively.
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 85, 86, and 81, respectively.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR3 sequence and a light chain variable region (VL) comprising a LCDR3 sequence, wherein the HCDR3 sequence comprises the sequence as set forth in SEQ ID NO: 78, and the LCDR3 sequence comprises the sequence as set forth in SEQ ID NO: 81.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 87, 77, and 84, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 79, 80, and 81, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 21, positions 1 to 116 and/or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 22, positions 1 to 112.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the sequence as set forth in SEQ ID NO: 21, positions 1 to 116.
  • VH heavy chain variable region
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 22, positions 1 to 112.
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising the sequence as set forth in SEQ ID NO: 22, positions 1 to 112.
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 21, positions 1 to 116 and the VL comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 22, positions 1 to 112.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 80% identity to the sequence as set forth in SEQ ID NO: 21, positions 1 to 116 and the VL comprises a sequence having at least 80% identity to the sequence as set forth in SEQ ID NO: 22, positions 1 to 112.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 90% identity to the sequence as set forth in SEQ ID NO: 21, positions 1 to 116 and the VL comprises a sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 22, positions 1 to 112.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 95% identity to the sequence as set forth in SEQ ID NO: 21, positions 1 to 116 and the VL comprises a sequence having at least 95% identity to the sequence as set forth in SEQ ID NO: 22, positions 1 to 112.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 21, positions 1 to 116 and the VL comprises the sequence as set forth in SEQ ID NO: 22, positions 1 to 112.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises heavy and light chain variable regions of an antibody which competes for EpCAM binding with and/or has the specificity for EpCAM of an antibody comprising a heavy chain variable region (VH) and/or a light chain variable region (VL) as set forth above.
  • VH heavy chain variable region
  • VL light chain variable region
  • said second antigen-binding region binding to CD137 is as set forth above.
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR3 sequence as set forth in SEQ ID NO: 78
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising a HCDR3 sequence as set forth in SEQ ID NO: 14.
  • a) said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR3 sequence as set forth in SEQ ID NO: 81
  • said second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising a LCDR3 sequence as set forth in SEQ ID NO: 18.
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR3 sequence as set forth in SEQ ID NO: 78, and a light chain variable region (VL) comprising a LCDR3 sequence as set forth in SEQ ID NO: 81
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising a HCDR3 sequence as set forth in SEQ ID NO: 14, and a light chain variable region (VL) comprising a LCDR3 sequence, as set forth in SEQ ID NO: 18.
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 76, 77, and 78, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 79, 80, and 81, respectively; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO:
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 82, 83, and 84, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 85, 86, and 81, respectively; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO:
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 87, 77, and 84, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 79, 80, and 81, respectively; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 21, positions 1 to 116 and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 22, positions 1 to 112; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 11 and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 15.
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 21, positions 1 to 116 and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 22, positions 1 to 112; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 21, positions
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 21, positions 1 to 116 and the VL comprises the sequence as set forth in SEQ ID NO: 22, positions 1 to 112; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 11 and the VL comprises the sequence as set forth in SEQ ID NO: 15.
  • the binding agent is an antibody comprising a first binding arm comprising said first antigen- binding region binding to EpCAM and a second binding arm comprising said second antigen-binding region binding to CD137, wherein the first binding arm comprises i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), wherein the first VH comprises first HCDR1, HCDR2, and HCDR3 sequences and the first VL comprises first LCDR1, LCDR2, and LCDR3 sequences, wherein the first HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 76, 77, and 78, respectively, and the first LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 79, 80, and 81, respectively; and the second binding arm comprises i
  • the binding agent comprises i) a first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 21, ii) a first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 22, iii) a second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 19; and iv) a second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 20.
  • the binding agent comprises i) a first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21, ii) a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 22, iii) a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19; and iv) a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 20.
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR3 sequence comprising the sequence as set forth in SEQ ID NO: 90. In some embodiments, said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR2 sequence comprising the sequence as set forth in SEQ ID NO: 89. In some embodiments, said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR1 sequence comprising the sequence as set forth in SEQ ID NO: 88.
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 88, 89, and 90, respectively.
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 91, 92, and 90, respectively.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR3 sequence and a light chain variable region (VL) comprising a LCDR3 sequence, wherein the HCDR3 sequence comprises the sequence as set forth in SEQ ID NO: 4, and the LCDR3 sequence comprises the sequence as set forth in SEQ ID NO: 90.
  • VH heavy chain variable region
  • VL light chain variable region
  • the HCDR3 sequence comprises the sequence as set forth in SEQ ID NO: 4
  • the LCDR3 sequence comprises the sequence as set forth in SEQ ID NO: 90.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 2, 3, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 88, 89, and 90, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 64, 65, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 91, 92, and 90, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 74, 3, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 88, 89, and 90, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 25, positions 1 to 115 and/or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 26, positions 1 to 111.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 25, positions 1 to 115.
  • VH heavy chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the sequence as set forth in SEQ ID NO: 25, positions 1 to 115.
  • VH heavy chain variable region
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 26, positions 1 to 111.
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising the sequence as set forth in SEQ ID NO: 26, positions 1 to 111.
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 25, positions 1 to 115 and the VL comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth In SEQ ID NO: 26, positions 1 to 111.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 80% identity to the sequence as set forth in SEQ ID NO: 25, positions 1 to 115 and the VL comprises a sequence having at least 80% identity to the sequence as set forth in SEQ ID NO: 26, positions 1 to 111.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 90% identity to the sequence as set forth in SEQ ID NO: 25, positions 1 to 115 and the VL comprises a sequence having at least 90% identity to the sequence as set forth in SEQ ID NO: 26, positions 1 to 111.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 95% identity to the sequence as set forth in SEQ ID NO: 25, positions 1 to 115 and the VL comprises a sequence having at least 95% identity to the sequence as set forth in SEQ ID NO: 26, positions 1 to 111.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 25, positions 1 to 115 and the VL comprises the sequence as set forth in SEQ ID NO: 26, positions 1 to 111.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises heavy and light chain variable regions of an antibody which competes for EpCAM binding with and/or has the specificity for EpCAM of an antibody comprising a heavy chain variable region (VH) and/or a light chain variable region (VL) as set forth above.
  • said second antigen-binding region binding to CD137 is as set forth above.
  • a) said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR3 sequence as set forth in SEQ ID NO: 90
  • said second antigen-binding region binding to CD137 comprises a light chain variable region (VL) comprising a LCDR3 sequence as set forth in SEQ ID NO: 18.
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR3 sequence as set forth in SEQ ID NO: 4, and a light chain variable region (VL) comprising a LCDR3 sequence as set forth in SEQ ID NO: 90
  • said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising a HCDR3 sequence as set forth in SEQ ID NO: 14, and a light chain variable region (VL) comprising a LCDR3 sequence, as set forth in SEQ ID NO: 18.
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 2, 3, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 88, 89, and 90, respectively; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 12, 13, and 14, respectively
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 64, 65, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 91, 92, and 90, respectively; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO:
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 74, 3, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 88, 89, and 90, respectively; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 75
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 25, positions 1 to 115 and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 26, positions 1 to 111; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 11 and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 15.
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 25, positions 1 to 115 and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 26, positions 1 to 111; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 25, positions
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 25, positions 1 to 115 and the VL comprises the sequence as set forth in SEQ ID NO: 26, positions 1 to 111; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 11 and the VL comprises the sequence as set forth in SEQ ID NO: 15.
  • the binding agent is an antibody comprising a first binding arm comprising said first antigen- binding region binding to EpCAM and a second binding arm comprising said second antigen-binding region binding to CD137, wherein the first binding arm comprises i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), wherein the first VH comprises first HCDR1, HCDR2, and HCDR3 sequences and the first VL comprises first LCDR1, LCDR2, and LCDR3 sequences, wherein the first HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 2, 3, and 4, respectively, and the first LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 88, 89, and 90, respectively; and the second binding arm comprises iii
  • the binding agent comprises i) a first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 25, ii) a first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 26, iii) a second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 19; and iv) a second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 20.
  • the binding agent comprises i) a first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 25, ii) a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 26, iii) a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19; and iv) a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 20.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR3 sequence comprising the sequence as set forth in SEQ ID NO: 4. In some embodiments, said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR2 sequence comprising the sequence as set forth in SEQ ID NO: 93. In some embodiments, said first antigen- binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a HCDR1 sequence comprising the sequence as set forth in SEQ ID NO: 2.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 2, 93, and 4, respectively.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 64, 96, and 66, respectively.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 74, 93, and 66, respectively.
  • VH heavy chain variable region
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR3 sequence comprising the sequence as set forth in SEQ ID NO: 8. In some embodiments, said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR2 sequence comprising the sequence as set forth in SEQ ID NO: 95. In some embodiments, said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a LCDR1 sequence comprising the sequence as set forth in SEQ ID NO: 94.
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 94, 95, and 8, respectively.
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the LCDR1, LCDR2 and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 97, 98, and 8, respectively.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 2, 93, and 4, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 94, 95, and 8, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 74, 93, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 94, 95, and 8, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 27, positions 1 to 115 and/or a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 28, positions 1 to 108.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 27, positions 1 to 115.
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the sequence as set forth in SEQ ID NO: 27, positions 1 to 115.
  • VH heavy chain variable region
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 28, positions 1 to 108.
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a light chain variable region (VL) comprising the sequence as set forth in SEQ ID NO: 28, positions 1 to 108.
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 27, positions 1 to 115 and the VL comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the sequence as set forth in SEQ ID NO: 28, positions 1 to 108.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 80% identity to the sequence as set forth in SEQ ID NO: 27, positions 1 to 115 and the VL comprises a sequence having at least 80% identity to the sequence as set forth in SEQ ID NO: 28, positions 1 to 108.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a sequence having at least at least 95% identity to the sequence as set forth in SEQ ID NO: 27, positions 1 to 115 and the VL comprises a sequence having at least 95% identity to the sequence as set forth in SEQ ID NO: 28, positions 1 to 108.
  • VH heavy chain variable region
  • VL light chain variable region
  • said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 27, positions 1 to 115 and the VL comprises the sequence as set forth in SEQ ID NO: 28, positions 1 to 108.
  • VH heavy chain variable region
  • VL light chain variable region
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 64, 96, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 97, 98, and 8, respectively; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO:
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 74, 93, and 66, respectively, and the LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 94, 95, and 8, respectively; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3 sequences, wherein the HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 75
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 27, positions 1 to 115 and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 28, positions 1 to 108; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) comprising the HCDR1, HCDR2, and HCDR3 sequences of SEQ ID NO: 11 and a light chain variable region (VL) comprising the LCDR1, LCDR2, and LCDR3 sequences of SEQ ID NO: 15.
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 27, positions 1 to 115 and the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 28, positions 1 to 108; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence as set forth in SEQ ID NO: 27, positions
  • a) said first antigen-binding region binding to EpCAM comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 27, positions 1 to 115 and the VL comprises the sequence as set forth in SEQ ID NO: 28, positions 1 to 108; and b) said second antigen-binding region binding to CD137 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the sequence as set forth in SEQ ID NO: 11 and the VL comprises the sequence as set forth in SEQ ID NO: 15.
  • the binding agent is an antibody comprising a first binding arm comprising said first antigen- binding region binding to EpCAM and a second binding arm comprising said second antigen-binding region binding to CD137, wherein the first binding arm comprises i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), wherein the first VH comprises first HCDR1, HCDR2, and HCDR3 sequences and the first VL comprises first LCDR1, LCDR2, and LCDR3 sequences, wherein the first HCDR1, HCDR2, and HCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 2, 93, and 4, respectively, and the first LCDR1, LCDR2, and LCDR3 sequences comprise the sequences as set forth in SEQ ID NO: 94, 95, and 8, respectively; and the second binding arm comprises iii
  • the binding agent comprises i) a first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 27, ii) a first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 28, iii) a second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 19; and iv) a second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 20.
  • the binding agent comprises i) a first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 27, ii) a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 28, iii) a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19; and iv) a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 20.
  • said first antigen-binding region binding to EpCAM comprises heavy and light chain variable regions of an antibody which competes for EpCAM binding with and/or has the specificity for EpCAM of an antibody comprising a heavy chain variable region or a light chain variable region, or a combination thereof of a first antigen- binding region binding to EpCAM as set forth above and said second antigen-binding region binding to CD137 comprises heavy and light chain variable regions of an antibody which competes for CD137 binding with and/or has the specificity for CD137 of an antibody comprising a heavy chain variable region or a light chain variable region, or a combination thereof of a second antigen-binding region binding to CD137 as set forth above.
  • a variable region comprises three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).
  • said complementarity determining regions and said framework regions are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the binding agent is in the format of a full-length antibody or an antibody fragment.
  • the binding agent is a multispecific such as a bispecific binding agent.
  • the binding agent is a multispecific such as a bispecific antibody.
  • the binding agent comprises (i) a first heavy chain variable region (VH) and a first light chain variable region (VL), wherein said first heavy chain variable region (VH) and said first light chain variable region (VL) form said first antigen-binding region binding to EpCAM; and
  • the binding agent comprises i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL), wherein said first heavy chain variable region (VH) and said first light chain variable region (VL) form said first antigen-binding region binding to EpCAM; and ii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL), wherein said second heavy chain variable region (VH) and said second light chain variable region (VL) form said second antigen-binding region binding to CD137.
  • the binding agent is an antibody comprising a first binding arm comprising said first antigen- binding region binding to EpCAM and a second binding arm comprising said second antigen-binding region binding to CD137, wherein the first binding arm comprises i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising a first light chain variable region (VL) and a first light chain constant region (CL); and the second binding arm comprises iii) a polypeptide comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH), and iv) a polypeptide comprising a second light chain variable region (VL) and a second light chain constant region (CL).
  • the first binding arm comprises i) a polypeptide comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a polypeptide comprising a first light chain variable region (V
  • the binding agent is an antibody comprising a first binding arm comprising said first antigen- binding region binding to EpCAM and a second binding arm comprising said second antigen-binding region binding to CD137, wherein the first binding arm comprises i) a first heavy chain comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a first light chain comprising a first light chain variable region (VL) and a first light chain constant region (CL); and the second binding arm comprises i) a second heavy chain comprising a second heavy chain variable region (VH) and a second heavy chain constant region (CH); and ii) a second light chain comprising a second light chain variable region (VL) and a second light chain constant region (CL).
  • the first binding arm comprises i) a first heavy chain comprising a first heavy chain variable region (VH) and a first heavy chain constant region (CH), and ii) a first light chain comprising a first light chain variable region (VL)
  • the first binding arm is derived from a full-length antibody. In some embodiments, the first binding arm is derived from a monoclonal antibody. In some embodiments, the first binding arm is derived from a full-length IgGl, A (lambda) or IgGl, K (kappa) antibody. In some embodiments, the second binding arm is derived from a full-length antibody. In some embodiments, the second binding arm is derived from a monoclonal antibody. In some embodiments, the second binding arm is derived from a full-length IgGl, A (lambda) or IgGl, K (kappa) antibody.
  • the first and second binding arms are derived from full-length antibodies, such as from full-length IgGl, A (lambda) or IgGl, K (kappa) antibodies. In some embodiments, the first and second binding arms are derived from monoclonal antibodies.
  • each of the first and second heavy chain constant regions comprises one or more of a constant heavy chain 1 (CHI) region, a hinge region, a constant heavy chain 2 (CH2) region and a constant heavy chain 3 (CH3) region, preferably at least a hinge region, a CH2 region and a CH3 region.
  • CHI constant heavy chain 1
  • CH2 constant heavy chain 2
  • CH3 constant heavy chain 3
  • each of the first and second heavy chain constant regions comprises a CH3 region and wherein the two CH3 regions comprise asymmetrical mutations.
  • said first heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgGl heavy chain according to EU numbering has been substituted
  • said second heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgGl heavy chain according to EU numbering has been substituted, and wherein said first and said second heavy chains are not substituted in the same positions.
  • the 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 constant region (CH)
  • 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 constant region (CH).
  • said binding agent induces Fc-mediated effector function to a lesser extent compared to another antibody comprising the same first and second antigen binding regions and two heavy chain constant regions (CHs) comprising human IgGl hinge, CH2 and CH3 regions.
  • CHs heavy chain constant regions
  • said first and second heavy chain constant regions (CHs) are modified so that the antibody induces Fc-mediated effector function to a lesser extent compared to an antibody which is identical except for comprising non-modified first and second heavy chain constant regions (CHs).
  • each of said non-modified first and second heavy chain constant regions comprises the amino acid sequence set forth in SEQ ID NO: 47.
  • said Fc-mediated effector function is measured by binding to Fey receptors, binding to Clq, or induction of Fc-mediated crosslinking of Fey receptors.
  • said Fc-mediated effector function is measured by binding to Clq.
  • said first and second heavy chain constant regions have been modified so that binding of Clq to said antibody is reduced compared to a wild-type antibody, preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, wherein Clq binding is preferably determined by ELISA.
  • one or more amino acids in the positions corresponding to positions L234, L235, D265, N297, P331, and G236 in a human IgGl heavy chain according to EU numbering are not L, L, D, N, P, and G, respectively.
  • the positions corresponding to positions L234 and L235 in a human IgGl heavy chain according to EU numbering are F and E, respectively, in said first and second heavy chains.
  • the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering are F, E, and A, respectively, in said first and/or second heavy chain constant regions (HCs) and/or the positions corresponding to positions L234, L235, and G236 in a human IgGl heavy chain according to EU numbering are F, E, and R, respectively, in said first and/or second heavy chain constant regions (HCs).
  • the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering are F, E, and A, respectively, in one of said first and second heavy chain constant regions and the positions corresponding to positions L234, L235, and G236 in a human IgGl heavy chain according to EU numbering are F, E, and R, respectively, in the other of said first and second heavy chain constant regions.
  • the positions corresponding to positions L234 and L235 in a human IgGl heavy chain according to EU numbering of both the first and second heavy chain constant regions are F and E, respectively, and wherein (i) the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is R, and the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the second heavy chain constant region is L.
  • the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering are F, E, and A, respectively, in said second heavy chain constant region (HC) and the positions corresponding to positions L234, L235, and G236 in a human IgGl heavy chain according to EU numbering are F, E, and R, respectively, in said first heavy chain constant region (HC), and wherein (i) the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is R, and the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the second heavy chain constant region is L.
  • the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering are F, E, and A, respectively, in said second heavy chain constant region (HC) and the positions corresponding to positions L234, L235, and G236 in a human IgGl heavy chain according to EU numbering are F, E, and R, respectively, in said first heavy chain constant region (HC), and wherein the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the second heavy chain constant region is R.
  • the constant region of said first and/or second heavy chain comprises an amino acid sequence an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 47.
  • a) the constant region of said first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 54; and b) the constant region of said second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 52.
  • said binding agent comprises a kappa (K) light chain constant region.
  • said binding agent comprises a lambda (X) light chain constant region.
  • said first light chain constant region is a kappa (K) light chain constant region or a lambda (A) light chain constant region.
  • said second light chain constant region is a kappa (K) light chain constant region or a lambda (A) light chain constant region.
  • said first light chain constant region and said second light chain constant region are kappa (K) light chain constant regions
  • said first light chain constant region and said second light chain constant region are lambda (A) light chain constant regions
  • said first light chain constant region is a kappa (K) light chain constant region and said second light chain constant region is a lambda (A) light chain constant region, or
  • said first light chain constant region is a lambda (A) light chain constant region and said second light chain constant region is a kappa (K) light chain constant region.
  • the kappa (K) light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 55.
  • the lambda (A) light chain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 56.
  • the binding agent is of an isotype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.
  • the isotype is selected from the group consisting of human IgGl, human IgG2, human IgG3 and human IgG4.
  • the binding agent is a full-length IgGl antibody.
  • the binding agent is an antibody of the IgGlm(f) allotype.
  • the binding agent disclosed herein can in principle be an antibody of any isotype.
  • the choice of isotype typically will be guided by the desired Fc-mediated effector functions, such as ADCC induction, or the requirement for an antibody devoid of Fc-mediated effector function ("inert" antibody).
  • Exemplary isotypes are IgGl, IgG2, IgG3, and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used.
  • the effector function of the antibodies described herein may be changed by isotype switching to, e.g., an IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses.
  • both heavy chains of an antibody described herein are of the IgGl isotype, for instance an IgGlK.
  • the heavy chain may be modified in the hinge and/or CH3 region as described elsewhere herein.
  • each of the antigen-binding regions comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein said variable regions each comprise three CDR sequences, CDR1, CDR2 and CDR3, respectively, and four framework sequences, FR1, FR2, FR3 and FR4, respectively.
  • the antibody comprises two heavy chain constant regions (CH), and two light chain constant regions (CL).
  • the binding agent is a full-length antibody, such as a full-length IgGl antibody.
  • the binding agent e.g., a bispecific antibody, comprises two half-molecules each comprising an antigen-binding region.
  • bispecific antibodies are known in the art, and were reviewed by Kontermann; Drug Discov Today, 2015 Jul;20(7):838-47 and; MAbs, 2012 Mar-Apr;4(2): 182-97. All these formats are encompassed herein._A bispecific antibody according to the present disclosure is not limited to any particular bispecific format or method of producing it.
  • the binding agent described herein is a diabody or a cross-body.
  • the binding agent is a bispecific antibody obtained via a controlled Fab-arm exchange (such as described in WO20U131746 (Genmab)).
  • binding agents include but are not limited to (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, wherein full length IgG antibodies are fused to extra Fab fragment or parts of Fab fragment; (iv) Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constant-domains, Fc regions or parts thereof; (v) Fab fusion molecules, wherein different Fab-fragments are fused together, fused to heavy-chain constant-domains, Fc regions or parts thereof; and (vi) ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g.
  • IgG-like molecules with complementary CH3 domain molecules include but are not limited to the Triomab/Quadroma molecules (Trion Pharma/Fresenius Biotech; Roche, WO2011069104), the so-called Knobs- into-Holes molecules (Genentech, WO9850431), CrossMAbs (Roche, WO2011117329) and the electrostatically- matched molecules (Amgen, EP1870459 and W02009089004; Chugai, US201000155133; Oncomed, W02010129304), the LUZ-Y molecules (Genentech, Wranik et al. J. Biol. Chem.
  • IgG-like dual targeting molecules examples include but are not limited to Dual Targeting (DT)-Ig molecules (W02009058383), Two-in-one Antibody (Genentech; Bostrom, et al 2009. Science 323, 1610-1614.), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star, W02008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs.
  • DT Dual Targeting
  • W02009058383 Two-in-one Antibody
  • Genentech Bostrom, et al 2009. Science 323, 1610-1614.
  • Cross-linked Mabs Karmanos Cancer Center
  • mAb2 F-Star, W02008003116
  • Zybody molecules Zyngenia; LaFleur et al. MAbs.
  • IgG fusion molecules include but are not limited to Dual Variable Domain (DVD)-Ig molecules (Abbott, US7,612,181), Dual domain double head antibodies (Unilever; Sanofi Aventis, W020100226923), IgG-like Bispecific molecules (ImClone/Eli Lilly, Lewis et al.
  • DVD Dual Variable Domain
  • Ts2Ab Medlmmune/AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92
  • BsAb molecules Zymogenetics, W020101U625), HERCULES molecules (Biogen pie, US007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN 102250246) and TvAb molecules (Roche, WO2012025525, W02012025530).
  • Fc fusion molecules include but are not limited to ScFv/Fc Fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6): 1179-88), SCORPION molecules (Emergent BioSolutions/Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract # 5465); Zymogenetics/BMS, W02010111625), Dual Affinity Retargeting Technology (Fc-DART) molecules (MacroGenics, WO2008157379, W02010080538) and Dual(ScFv)2-Fab molecules (National Research Center for Antibody Medicine - China).
  • Fab fusion bispecific antibodies include but are not limited to F(ab)2 molecules (Medarex/AMGEN; Deo et al J Immunol. 1998 Feb 15;160(4):1677-86.), Dual-Action or Bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323, 1610-1614.), Dock-and-Lock (DNL) molecules (ImmunoMedics, W02003074569, W02005004809), Bivalent Bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec 15;165(12):7050-7.) and Fab-Fv molecules (UCB-Celltech, WO 2009040562 Al).
  • ScFv-, diabody-based and domain antibodies include but are not limited to Bispecific T Cell Engager (BITE) molecules (Micromet, W02005061547), Tandem Diabody molecules (TandAb) (Affimed) Le Gall et al., Protein Eng Des Sei. 2004 Apr;17(4):357-66.), Dual Affinity Retargeting Technology (DART) molecules (MacroGenics, WO2008157379, W02010080538), Single-chain Diabody molecules (Lawrence, FEBS Lett.
  • BITE Bispecific T Cell Engager
  • TandAb Tandem Diabody molecules
  • DART Dual Affinity Retargeting Technology
  • TCR-like Antibodies AIT, ReceptorLogics
  • Human Serum Albumin ScFv Fusion Merrimack, W02010059315
  • COMBODY molecules Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667- 75.
  • dual targeting nanobodies Ablynx, Hmila et al., FASEB J. 2010
  • dual targeting heavy chain only domain antibodies
  • the bispecific antibody of the disclosure comprises a first Fc sequence comprising a first CH3 region, and a second Fc sequence comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO2011131746 and W02013060867 (Genmab), which are hereby incorporated by reference.
  • a stable bispecific EpCAMxCD137 antibody can be obtained at high yield using a particular method on the basis of one homodimeric starting EpCAM antibody and one homodimeric starting CD137 antibody containing only a few, conservative, asymmetrical mutations in the CH3 regions.
  • Asymmetrical mutations mean that the sequences of said first and second CH3 regions contain amino acid substitutions at non-identical positions.
  • the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first CH3 region which has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409 in a human IgGl heavy chain, and a second CH3 region which has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405, 407 and 409 in a human IgGl heavy chain, and wherein the first and second CH3 regions are not substituted in the same positions.
  • the bispecific antibody as defined in any of the embodiments disclosed herein comprises the sequences of said first and second CH3 regions containing asymmetrical mutations, i.e. mutations at different positions in the two CH3 regions, e.g. a mutation at position 405 in one of the CH3 regions and a mutation at position 409 in the other CH3 region.
  • the mutation at position 405 is F405L.
  • the mutation at position 409 is K409R.
  • the bispecific antibody comprises a first and second heavy chain, wherein each of said first and second heavy chains comprises at least a hinge region, a CH2 and a CH3 region, wherein (i) the amino acid in the position corresponding to F405 in human IgGl heavy chain is L in said first heavy chain, and the amino acid in the position corresponding to K409 in human IgGl heavy chain is R in said second heavy chain, or (ii) the amino acid in the position corresponding to K409 in human IgGl heavy chain is R in said first heavy chain, and the amino acid in the position corresponding to F405 in human IgGl heavy chain is L in said second heavy chain.
  • Another strategy to promote formation of heterodimers over homodimers is a "knob-into-hole” strategy in which a protuberance is introduced on a first heavy- chain polypeptide and a corresponding cavity in a second heavy-chain polypeptide, such that the protuberance can be positioned in the cavity at the interface of these two heavy chains so as to promote heterodimer formation and hinder homodimer formation.
  • protuberances are constructed by replacing small amino-acid side-chains from the interface of the first polypeptide with larger side chains.
  • Compensatory "cavities" of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino-acid side-chains with smaller ones (US patent 5,731,168).
  • EP1870459 Chougai
  • W02009089004 Amgen
  • EP1870459 Chougai
  • W02009089004 Amgen
  • one or more residues that make up the CH3-CH3 interface in both CH3 domains are replaced with a charged amino acid such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable.
  • W02007110205 Merck
  • W02007110205 describe yet another strategy, wherein differences between IgA and IgG CH3 domains are exploited to promote heterodimerization.
  • bispecific antibodies Another in vitro method for producing bispecific antibodies has been described in WO2008119353 (Genmab), wherein a bispecific antibody is formed by "Fab-arm” or "half-molecule” exchange (swapping of a heavy chain and attached light chain) between two monospecific IgG4- or IgG4-like antibodies upon incubation under reducing conditions.
  • the resulting product is a bispecific antibody having two Fab arms which may comprise different sequences.
  • a preferred method for preparing bispecific EpCAMxCD137 antibodies of the present disclosure includes the methods described in WO2011131746 and W02013060867 (Genmab) comprising the following steps: a) providing a first antibody comprising an Fc region, said Fc region comprising a first CH3 region; b) providing a second antibody comprising a second Fc region, said Fc region comprising a second CH3 region, wherein the first antibody is a EpCAM antibody and the second antibody is a CD137 antibody, or vice versa; wherein the sequences of said first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions; c) incubating said first antibody together with said second antibody under reducing conditions; and d) obtaining said bispecific EpCAMxCD137 antibody.
  • a method for producing an antibody comprising the steps of: a) culturing a host cell producing a first antibody comprising an antigen-binding region capable of binding to human EpCAM as defined herein and purifying said first antibody from the culture; b) culturing a host cell producing a second antibody comprising an antigen-binding region capable of binding to human CD137 as defined herein and purifying said second antibody from the culture; c) incubating said first antibody together with said second antibody under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide-bond isomerization, and d) obtaining said bispecific antibody.
  • the said first antibody together with said second antibody are incubated under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide-bond isomerization, wherein the heterodimeric interaction between said first and second antibodies in the resulting heterodimeric antibody is such that no Fab-arm exchange occurs at 0.5 mM GSH after 24 hours at 37°C.
  • step c) the heavy-chain disulfide bonds in the hinge regions of the parent antibodies are reduced and the resulting cysteines are then able to form inter heavy-chain disulfide bonds with cysteine residues of another parent antibody molecule (originally with a different specificity).
  • the reducing conditions in step c) comprise the addition of a reducing agent, e.g.
  • step c) comprises restoring the conditions to become non- reducing or less reducing, for example by removal of a reducing agent, e.g. by desalting.
  • the sequences of the first and second CH3 regions of the homodimeric starting antibodies are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. More details on these interactions and how they can be achieved are provided in WO2011131746 and W02013060867 (Genmab), which are hereby incorporated by reference in their entirety.
  • a stable bispecific EpCAMxCD137 antibody can be obtained at high yield using the above method on the basis of two homodimeric starting antibodies which bind EpCAM and CD137, respectively, and contain only a few, asymmetrical mutations in the CH3 regions.
  • Asymmetrical mutations mean that the sequences of said first and second CH3 regions contain amino acid substitutions at non-identical positions.
  • the binding agent according to the present disclosure comprises, in addition to the antigen- binding regions, an Fc region consisting of the Fc sequences of the two heavy chains.
  • the first and second Fc sequences may each be of any isotype, including, but not limited to, IgGl, IgG2, IgG3 and IgG4, and may comprise one or more mutations or modifications.
  • each of the first and second Fc sequences is of the IgGl isotype or derived therefrom, optionally with one or more mutations or modifications.
  • each of the first and second Fc sequences is of the IgG4 isotype or derived therefrom, optionally with one or more mutations or modifications.
  • one of the Fc sequences is of the IgGl isotype and the other of the IgG4 isotype, or is derived from such respective isotypes, optionally with one or more mutations or modifications.
  • one or both Fc sequences are effector-function-deficient.
  • the Fc sequence(s) may be of an IgGl isotype, or a non-IgGl isotype, e.g. IgG2, IgG3, or IgG4, which has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated.
  • effector functions includes any functions mediated by components of the immune system that result, for example, in the killing of diseased cells such as tumor cells, or in the inhibition of tumor growth and/or inhibition of tumor development, including inhibition of tumor dissemination and metastasis.
  • the effector functions in the context of the present disclosure are T cell mediated effector functions.
  • Such functions comprise ADCC, ADCP or CDC.
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • cytotoxic effector cell through a nonphagocytic process, characterised by the release of the content of cytotoxic granules or by the expression of cell death-inducing molecules.
  • ADCC is independent of the immune complement system that also lyses targets but does not require any other cell.
  • ADCC is triggered through interaction of target-bound antibodies (belonging to IgG or IgA or IgE classes) with certain Fc receptors (FcRs), glycoproteins present on the effector cell surface that bind the Fc region of immunoglobulins (Ig).
  • ADCC effectsor cells that mediate ADCC include natural killer (NK) cells, monocytes, macrophages, neutrophils, eosinophils and dendritic cells.
  • NK natural killer
  • ADCC is a rapid effector mechanism whose efficacy is dependent on a number of parameters (density and stability of the antigen on the surface of the target cell; antibody affinity and FcR-binding affinity).
  • ADCC involving human IgGl, the most used IgG subclass for therapeutic antibodies, is highly dependent on the glycosylation profile of its Fc portion and on the polymorphism of Fey receptors.
  • ADCP Antibody-dependent cellular phagocytosis
  • ADCP is one crucial mechanism of action of many antibody therapies. It is defined as a highly regulated process by which antibodies eliminate bound targets via connecting its Fc domain to specific receptors on phagocytic cells, and eliciting phagocytosis. Unlike ADCC, ADCP can be mediated by monocytes, macrophages, neutrophils, and dendritic cells, through FcyRIIa, FcyRI, and FcyRIIIa, of which FcyRIIa (CD32a) on macrophages represent the predominant pathway.
  • Complement-dependent cytotoxicity CDC
  • CDC is another cell-killing method that can be directed by antibodies.
  • IgM is the most effective isotype for complement activation.
  • IgGl and IgG3 are also both very effective at directing CDC via the classical complement- activation pathway.
  • the formation of antigen-antibody complexes results in the uncloaking of multiple Clq binding sites in close proximity on the C H 2 domains of participating antibody molecules such as IgG molecules (Clq is one of three subcomponents of complement Cl).
  • these uncloaked Clq binding sites convert the previously low-affinity Clq-IgG interaction to one of high avidity, which triggers a cascade of events involving a series of other complement proteins and leads to the proteolytic release of the effector-cell chemotactic/activating agents C3a and C5a.
  • the complement cascade ends in the formation of a membrane attack complex, which creates pores in the cell membrane that facilitate free passage of water and solutes into and out of the cell.
  • Antibodies described herein may comprise modifications in the Fc region. When an antibody comprises such modifications, it may become an inert, or non-activating, antibody.
  • the inertness of an Fc region of a humanized or chimeric EpCAM or CD137 antibody is advantageously tested using the antibody in a monospecific format.
  • variants can be constructed to make the Fc region of an antibody inactive for interactions with Fey (gamma) receptors and Clq for therapeutic antibody development. Examples of such variants are described herein.
  • said antibody comprises a first and a second heavy chain, wherein one or both heavy chains are modified so that the antibody induces Fc-mediated effector function to a lesser extent relative to an antibody which is identical, except for comprising non-modified first and second heavy chains.
  • Said Fc-mediated effector function may be measured by determining, by binding to Fey receptors, by binding to Clq, or by induction of Fc-mediated cross-linking of FcRs.
  • the heavy and light chain constant sequences have been modified so that binding of Clq to said antibody is reduced compared to an unmodified antibody by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, wherein Clq binding is determined by ELISA.
  • amino acids in the Fc region that play a dominant role in the interactions with Clq and the Fey receptors may be modified.
  • amino acid positions that may be modified, e.g. in an IgGl isotype antibody, include positions L234, L235, G236, D265 and P331. Combinations thereof, such as L234F/L235E/D265A, or L234F/L235E/G236R can cause a profound decrease in binding to human CD64, CD32, CD16 and Clq. Such modifications and their effect are described, e.g., in WO2022189667.
  • L234F and L235E amino acid substitutions can result in Fc regions with abrogated interactions with Fey receptors and Clq (Canfield et al., 1991, J. Exp. Med. (173): 1483-91; Duncan et al., 1988, Nature (332):738-40).
  • the amino acids in the positions corresponding to L234 and L235 may be F and E, respectively.
  • a D265A or G236R amino acid substitution can decrease binding to all Fey receptors and prevent ADCC (Shields et al., 2001, J. Biol. Chem. (276):6591-604; Wilkinson et al., 2021, PloS one (16(12)) e0260954).
  • the amino acid in the position corresponding to D265 may be A or the amino acid in the position corresponding to G236 may be R.
  • the amino acids in the positions corresponding to positions L234 and L235 in a human IgGl heavy chain are F and E, respectively.
  • the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain are F, E, and A, respectively.
  • the amino acids in the positions corresponding to positions L234, L235, and G236 in a human IgGl heavy chain are F, E, and R, respectively.
  • the L234F-L235E-D265A non-activating mutations have been shown to have an excellent safety profile and ability to strongly suppress Fc-mediated effector function. Nevertheless, it was observed that for IgGl antibodies that are potent inducers of complement-dependent cytotoxicity (CDC), harboring the FEA mutations can show some residual CDC. Furthermore, it was observed that recombinantly expressed antibodies with the FEA format may exhibit increased glycosylation heterogeneity as a result of additional processing of their N-glycans as compared with a wild-type IgGl Fc region and these antibodies were also shown to be more susceptible to aggregation induced by low pH conditions.
  • CDC complement-dependent cytotoxicity
  • the mutations L234F, L235E and G236R are combined (also referred to herein as FER, or FER format) in IgGl antibodies this resulted in an improved inert format capable of avoiding potential residual CDC activity, providing wild-type like glycosylation and having improved tolerance to low pH conditions.
  • the FER format is a highly advantageous non-activating antibody format that is well suitable for clinical development and clinical use.
  • this FER inert format may also be combined in a heterodimeric format with respect to inert format substitutions, for example, a bispecific antibody may be composed of one chain carrying the inert format substitutions, whereas the other chain may comprise different inert format substitutions, e.g. FEA.
  • the FER inert format is well suitable to be combined e.g. with existing candidate antibodies which have already undergone development for clinical use without the need to redesign and redo all the assays required, thereby allowing to quickly generate bispecific antibodies therewith utilizing technologies such as controlled Fab-arm exchange.
  • technologies such as controlled Fab-arm exchange.
  • the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain are F, E, and A, respectively and in the other of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and G236 in a human IgGl heavy chain, are F, E, and R, respectively.
  • the binding agent is a bispecific antibody comprising a first and second heavy chain, wherein the positions corresponding to positions L234 and L235 in a human IgGl heavy chain according to EU numbering of both the first heavy chain and the second heavy chain are F and E, respectively, and wherein (i) the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain is L, and the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the first heavy chain is R, and the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the second heavy chain is L.
  • the binding agent is a bispecific antibody comprising a first and second heavy chain, wherein
  • Antibody variants having the combination of three amino acid substitutions L234F, L235E and D265A and in addition the K409R or the F405L mutation are herein termed with the suffix "FEAR” or "FEAL”, respectively.
  • Antibody variants having the combination of three amino acid substitutions L234F, L235E and G236R and in addition the K409R or the F405L mutation are herein termed with the suffix "FERR” or "FERL”, respectively.
  • the bispecific antibody described herein comprises:
  • the binding agents or antibodies described herein are linked or conjugated to one or more therapeutic moieties, such as a cytokine, an immune-suppressant, an immune-stimulatory molecule and/or a radioisotope.
  • therapeutic moieties such as a cytokine, an immune-suppressant, an immune-stimulatory molecule and/or a radioisotope.
  • conjugates are referred to herein as “immunoconjugates” or “drug conjugates”.
  • Immunoconjugates which include one or more cytotoxins are referred to as "immunotoxins”.
  • the first and/or second Fc sequence is conjugated to a drug or a prodrug or contains an acceptor group for the same.
  • acceptor group may e.g. be an unnatural amino acid.
  • EpCAM is human EpCAM, in particular human EpCAM comprising the sequence set forth in SEQ ID NO: 59.
  • CD137 is human CD137, in particular human CD137 comprising the sequence set forth in SEQ ID NO: 62.
  • EpCAM is human EpCAM and CD137 is human CD137.
  • EpCAM is human EpCAM comprising the sequence set forth in SEQ ID NO: 59 and CD137 is human CD137 comprising the sequence set forth in SEQ ID NO: 62.
  • the first binding region binding to human EpCAM comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 1, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 5; and b) the second binding region binding to human CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 11, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 15.
  • the first binding region binding to human EpCAM comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 2, 3, and 4, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 6, 7, and 8, respectively; and
  • the second binding region binding to human CD137 comprises a heavy chain variable region (VH) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 12, 13, and 14, respectively, and a light chain variable region (VL) comprising the CDR1, CDR2, and CDR3 sequences set forth in: SEQ ID NO: 16, 17, and 18, respectively.
  • the first binding region binding to human EpCAM comprises a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 9 and a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 10
  • the second binding region binding to human CD137 comprises a heavy chain comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 19 and a light chain comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 20.
  • the first binding region binding to human EpCAM comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10
  • the second binding region binding to human CD137 comprises a heavy chain comprising the amino add sequence set forth in SEQ ID NO: 19 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20.
  • the first binding region binding to human EpCAM comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and a light chain variable region (VL) region comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 5, and b) the second binding region binding to human CD137 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 11 and a light chain variable region (VL) region and comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to SEQ ID NO: 15.
  • VH heavy chain variable region
  • VL light chain variable region
  • the first binding region binding to human EpCAM comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 5
  • the second binding region binding to human CD137 comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region (VL) region comprising the amino acid sequence set forth in SEQ ID NO: 15.
  • the binding agent may in particular be an antibody, such as a multispecific antibody, e.g., a bispecific antibody. Also, the binding agent may be in the format of a full-length antibody or an antibody fragment.
  • the binding agent is a human antibody or a humanized antibody.
  • Each variable region may comprise three complementarity determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4).
  • CDR1, CDR2, and CDR3 complementarity determining regions
  • FR1, FR2, FR3, and FR4 framework regions
  • CDRs complementarity determining regions
  • FRs framework regions
  • the binding agent is an antibody comprising a first binding arm and a second binding arm
  • the first binding arm comprises i) a polypeptide comprising said first heavy chain variable region (VH) and said first heavy chain constant region (CH), and ii) a polypeptide comprising said first light chain variable region (VL) and said first light chain constant region (CL);
  • the second binding arm comprises iii) a polypeptide comprising said second heavy chain variable region (VH) and said second heavy chain constant region (CH), and iv) a polypeptide comprising said second light chain variable region (VL) and said second light chain constant region (CL).
  • the binding agent comprises i) a first heavy chain and light chain comprising said antigen- binding region capable of binding to EpCAM, the first heavy chain comprising a first heavy chain constant region and the first light chain comprising a first light chain constant region; and ii) a second heavy chain and light chain comprising said antigen-binding region capable of binding CD137, the second heavy chain comprising a second heavy chain constant region and the second light chain comprising a second light chain constant region.
  • Each of the first and second heavy chain constant regions 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.
  • said first heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgGl heavy chain according to EU numbering may have been substituted
  • said second heavy chain constant region (CH) at least one of the amino acids in a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgGl heavy chain according to EU numbering may have been substituted.
  • the first and the second heavy chains are not substituted in the same positions (i.e., the first and the second heavy chains contain asymmetrical mutations).
  • 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 (CHs) are modified so that the antibody induces Fc-mediated effector function to a lesser extent compared to an antibody which is identical except for comprising non-modified first and second heavy chain constant regions (CHs).
  • each or both of said non-modified 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: 47.
  • 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 first and second heavy chain constant regions of the binding agent may have been modified so that binding of Clq to said antibody is reduced compared to a wild-type antibody, preferably reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, wherein Clq binding is preferably determined by ELISA.
  • Clq binding is preferably determined by ELISA.
  • in at least one of said first and second heavy chain constant regions (CHs) one or more amino acids in the positions corresponding to positions L234, L235, G236, D265, N297, and P331 in a human IgGl heavy chain according to Ell numbering, are not L, L, G, D, N, and P, respectively.
  • 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.
  • positions corresponding to positions L234, L235, and G236 in a human IgGl heavy chain according to EU numbering may be F, E, and R, respectively, in said first and/or second heavy chain constant regions (HCs) and/or the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering may be F, E, and A, respectively, in said first and/or second heavy chain constant regions (HCs).
  • the positions corresponding to positions L234, L235, and G236 in a human IgGl heavy chain according to EU numbering may be F, E, and R, respectively, in one of said first and second heavy chain constant regions (HCs) and the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering may be F, E, and A, respectively, in the other of said first and second heavy chain constant regions (HCs).
  • the positions corresponding to positions L234, L235, and G236 in a human IgGl heavy chain according to EU numbering may be F, E, and R, respectively, in said first heavy chain constant region (HCs) and the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering may be F, E, and A, respectively, in said second heavy chain constant regions (HCs)
  • the positions corresponding to positions L234 and L235 in a human IgGl heavy chain according to EU numbering of both the first and second heavy chain constant regions are F and E, respectively, wherein (i) the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is L, and the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the second heavy chain constant region is R, or (ii) the position corresponding to K409 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is R, and the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the second heavy chain constant region is L.
  • the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering of both the first and second heavy chain constant regions are F, E, and A, respectively
  • the positions corresponding to positions L234, L235, and G236 in a human IgGl heavy chain according to EU numbering of both the first and second heavy chain constant regions are F, E, and R, respectively
  • the positions corresponding to positions L234, L235, and D265 in a human IgGl heavy chain according to EU numbering of one of the first and second heavy chain constant regions are F, E, and A, respectively
  • the positions corresponding to positions L234, L235, and G236 in a human IgGl heavy chain according to EU numbering of the other of the first and second heavy chain constant regions are F, E, and R, respectively, wherein (i) the position corresponding to F405 in a human IgGl heavy chain according to EU numbering of the first heavy chain constant region is L,
  • the binding agent comprises a lambda (A) light chain constant region.
  • the first light chain constant region is a kappa (K) light chain constant region or a lambda (A) light chain constant region.
  • the second light chain constant region is a lambda (A) light chain constant region or a kappa (K) 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 kappa (K) light chain constant region.
  • the first light chain constant region is a lambda (A) light chain constant region and the second light chain constant region is a lambda (A) 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 (A) light chain constant region or the first light chain constant region is a lambda (A) light chain constant region and the second light chain constant region is a kappa (K) light chain constant region.
  • the binding agent in particular, antibody
  • the binding agent 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.
  • Immuno checkpoint refers to regulators of the immune system, and, in particular, co-stimulatory and inhibitory signals that regulate the amplitude and quality of T-cell activity.
  • the immune checkpoint is an inhibitory signal.
  • the inhibitory signal is the interaction between PD-1 and PD-L1 and/or PD-1 and PD-L2.
  • PD-1 is expressed predominantly on previously activated T cells in vivo, and binds to two ligands, PD-L1 and PD- L2.
  • 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.
  • “Programmed Death Ligand-1 (PD-L1)” is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2).
  • PD-L1 as used herein includes human PD-L1 (hPD-Ll), variants, isoforms, and species homologs of hPD-Ll, and analogs having at least one common epitope with hPD-Ll.
  • PD-L2 as used herein includes human PD-L2 (hPD-L2), variants, isoforms, and species homologs of hPD-L2, and analogs having at least one common epitope with hPD-L2.
  • the interaction between PD-1 and its ligands results in a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and immune evasion by the cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1, and the effect is additive when the interaction of PD-1 with PD- L2 is blocked as well.
  • the term "immune checkpoint modulator” or “checkpoint modulator” refers to a molecule or to a compound that modulates the function of one or more checkpoint proteins. Immune checkpoint modulators are typically able to modulate self-tolerance and/or the amplitude and/or the duration of the immune response. Preferably, the immune checkpoint modulator modulates the function of one or more human checkpoint proteins and is, thus, a "human checkpoint modulator”. Specifically, the human checkpoint modulator is an immune checkpoint inhibitor.
  • immune checkpoint inhibitor or “checkpoint inhibitor” refers to a molecule that totally or partially reduces, inhibits, interferes with or negatively modulates one or more checkpoint proteins or that totally or partially reduces, inhibits, interferes with or negatively modulates expression of one or more checkpoint proteins.
  • the immune checkpoint inhibitor binds to one or more checkpoint proteins. In certain embodiments, the immune checkpoint inhibitor binds to one or more molecules regulating checkpoint proteins.
  • the immune checkpoint inhibitor prevents inhibitory signals associated with the immune checkpoint.
  • the immune checkpoint inhibitor is an antibody, or fragment thereof that disrupts inhibitory signaling associated with the immune checkpoint.
  • the immune checkpoint inhibitor is a small molecule inhibitor that disrupts inhibitory signaling.
  • the immune checkpoint inhibitor is a peptide-based inhibitor that disrupts inhibitory signaling.
  • the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimic, that prevents the interaction between checkpoint blocker proteins.
  • inhibiting or blocking of inhibitory immune checkpoint signaling results in preventing or reversing immune-suppression and establishment or enhancement of T-cell immunity.
  • 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 inhibitory immunoregulator is a component of the PD- 1/PD-L1/PD-L2 signaling pathway.
  • the inhibitory immunoregulator is a PD(L)-1 axis binding antagonist.
  • PD-1 axis binding antagonist refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with either one or more of its binding partners, such as PD-1, PD-L1 or PD-L2, so as to remove T-cell dysfunction resulting from signaling on the PD-1 signaling axis - with a result being to restore or enhance T-cell function (e.g., proliferation, cytokine production, target cell killing).
  • a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist.
  • the PD-1 axis binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners.
  • the PD-1 axis binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2.
  • PD-1 axis binding antagonists include anti-PD-1, anti-PD-Ll, and anti-PD-L2 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2.
  • a PD-1 axis binding antagonist reduces the negative co- stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-1 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition).
  • the PD-1 axis binding antagonist is an anti-PD-1 antibody. Specific examples of PD-1 axis binding antagonists are provided, infra.
  • PD-1 binding antagonist refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD- L1 and PD-L2.
  • the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners.
  • the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2.
  • PD-1 binding antagonists include anti-PD-1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD- L2.
  • a PD-1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-1 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition).
  • the PD-1 binding antagonist is an anti-PD-1 antibody. Specific examples of PD-1 binding antagonists are provided, infra.
  • PD-L1 binding antagonist refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L1 with either one or more of its binding partners, such as PD-1, B7-1.
  • a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners.
  • the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1 and/or B7-1.
  • the PD-L1 binding antagonists include anti-PD-Ll antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1, B7-1.
  • a PD-L1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L1 so as to render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition).
  • a PD-L1 binding antagonist is an anti-PD-Ll antibody. Specific examples of PD-L1 binding antagonists are provided, infra.
  • PD-L2 binding antagonist refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1.
  • a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners.
  • the PD-L2 binding antagonist inhibits binding of PD-L2 to PD-1.
  • the PD-L2 antagonists include anti-PD-L2 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1.
  • a PD-L2 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L2 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition).
  • a PD-L2 binding antagonist is an immunoadhesin.
  • a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.
  • Alternative names for "PD-1” include CD279 and SLEB2.
  • Alternative names for "PD-L1” include B7-H1, B7-4, CD274, and B7-H.
  • Alternative names for"PD-L2" include B7-DC, Btdc, and CD273.
  • PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2.
  • the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner(s).
  • the PD-1 ligand binding partners are PD-L1 and/or PD-L2.
  • the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner(s).
  • PD-L1 binding partner(s) are PD-1 and/or B7-1.
  • the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner(s).
  • a PD-L2 binding partner is PD-1.
  • the antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • the PD-1 axis binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).
  • an anti-PD-1 antibody e.g., a human antibody, a humanized antibody, or a chimeric antibody.
  • Exemplary PD-1 axis binding antagonists include, without limitation, anti-PD-1 antibodies such as BGB-A317 (BeiGene; see US 8,735,553, WO 2015/35606 and US 2015/0079109), cemiplimab (Regeneron; see WO 2015/112800) and lambrolizumab (e.g., disclosed as hPD109A and its humanized derivatives h409Al, h409A16 and h409A17 in WO2008/156712), AB137132 (Abeam), EH12.2H7 and RMP1-14 (#BE0146; Bioxcell Lifesciences Pvt.
  • anti-PD-1 antibodies such as BGB-A317 (BeiGene; see US 8,735,553, WO 2015/35606 and US 2015/0079109), cemiplimab (Regeneron; see WO 2015/112800) and lambrolizumab (e.g., disclosed as hPD109A and its humanized
  • JS001 TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., 2007, J. Hematol. Oncol. 70: 136
  • AMP-224 GSK-2661380; cf.
  • STI-1110 Suddeno Therapeutics; see WO 2014/194302), AGEN2034 (Agenus; see WO 2017/040790), MGA012 (Macrogenics; see WO 2017/19846), IBI308 (Innovent; see WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540), anti-PD-1 antibodies as described, e.g., in US 7,488,802, US 8,008,449, US 8,168,757, WO 03/042402, WO 2010/089411 (further disclosing anti-PD-Ll antibodies), WO 2010/036959, WO 2011/159877 (further disclosing antibodies against TIM-3), WO 2011/082400, WO 2011/161699, WO 2009/014708, WO 03/099196, WO 2009/114335, WO 2012/145493 (further disclosing antibodies against PD-
  • the anti-PD-1 antibody comprises nivolumab (OPDIVO; BMS-936558), pembrolizumab (KEYTRUDA; MK-3475), cemiplimab (LIBTAYO, REGN2810), pidilizumab (CT-011), spartalizumab (PDR001), MEDI0680 (AMP-514), dostarlimab (TSR-042), cetrelimab (JNJ 63723283), toripalimab (JSOO1), AMP-224 (GSK- 2661380), PF-06801591, tislelizumab (BGB-A317), ABBV-181, BI 754091, or SHR-1210.
  • the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4).
  • Nivolumab also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006/121168.
  • the anti-PD-1 antibody comprises a heavy chain and a light chain sequence depicted in SEQ ID NOS: 119 and 120, respectively.
  • the anti-PD-1 antibody comprises the six CDR sequences from SEQ ID NO: 119 and SEQ ID NO: 120 (e.g., the three heavy chain CDRs from SEQ ID NO: 119 and the three light chain CDRs from SEQ ID NO: 120). In some embodiments, the anti-PD-1 antibody comprises the heavy chain variable domain from SEQ ID NO: 119 and the light chain variable domain from SEQ ID NO: 120. In some embodiments, the anti-PD-1 antibody comprises: a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 129, and (b) a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 130.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PD-1 antibody comprises: (a) a heavy chain variable region (VH) that comprises a CDR-1 comprising an amino acid sequence of GITFSNSG (SEQ ID NO: 131), a CDR-2 comprising an amino acid sequence of IWYDGSKR (SEQ ID NO: 132), and a CDR-3 comprising an amino acid ATNDDY (SEQ ID NO: 133), and (b) a light chain variable region (VL) that comprises a CDR-1 comprising an amino acid sequence of QSVSSY (SEQ ID NO: 134), a CDR-2 comprising an amino acid sequence of DAS (SEQ ID NO: 135), and a CDR-3 comprising an amino acid sequence of QQSSNWPRT (SEQ ID NO: 136).
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PD-1 antibody is nivolumab which may be administered at a dose of 240 mg intravenously.
  • Nivolumab may be given intravenously according to institutional guidelines, published guidelines and the respective product prescribing information, and dosed according to this protocol.
  • the anti-PD-1 antibody is pembrolizumab (CAS Registry Number: 1374853-91-4).
  • Pembrolizumab (Merck), also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009/114335.
  • the anti-PD-1 antibody comprises a heavy chain and a light chain sequence depicted in SEQ ID NOS: 117 and 118, respectively.
  • the anti-PD-1 antibody comprises the six CDR sequences from SEQ ID NO: 117 and SEQ ID NO: 118(e.#., the three heavy chain CDRs from SEQ ID NO: 117 and the three light chain CDRs from SEQ ID NO: 118). In some embodiments, the anti-PD-1 antibody comprises the heavy chain variable domain from SEQ ID NO: 117 and the light chain variable domain from SEQ ID NO: 118. In some embodiments, the anti-PD-1 antibody comprises: a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 115, and (b) a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 116.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PD-1 antibody comprises: (a) a heavy chain variable region (VH) that comprises a CDR-1 comprising an amino acid sequence of GYTFTNYY (SEQ ID NO: 109), a CDR-2 comprising an amino acid sequence of INPSNGGT (SEQ ID NO: 110), and a CDR-3 comprising an amino acid ARRDYRFDMGFDY (SEQ ID NO: 111), and (b) a light chain variable region (VL) that comprises a CDR-1 comprising an amino acid sequence of KGVSTSGYSY (SEQ ID NO: 112), a CDR-2 comprising an amino acid sequence of LAS (SEQ ID NO: 113), and a CDR-3 comprising an amino acid sequence of QHSRDLPLT (SEQ ID NO: 114).
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PD-1 antibody is pembrolizumab which may be administered at a dose of 200 mg intravenously.
  • Pembrolizumab may be given intravenously according to institutional guidelines, published guidelines and the respective product prescribing information, and dosed according to this protocol.
  • the anti-PD-1 antibody is IgGl-PDl. In some embodiments, the anti-PD-1 antibody comprises a heavy chain and a light chain sequence depicted in SEQ ID NOS: 107 and 108, respectively.
  • the anti-PD-1 antibody comprises the six CDR sequences from SEQ ID NO: 107 and SEQ ID NO: 108 (e.g., the three heavy chain CDRs from SEQ ID NO: 107 and the three light chain CDRs from SEQ ID NO: 108). In some embodiments, the anti-PD-1 antibody comprises the heavy chain variable domain from SEQ ID NO: 107 and the light chain variable domain from SEQ ID NO: 108. In some embodiments, the anti-PD-1 antibody comprises: a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 105, and (b) a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 106.
  • VH heavy chain variable region
  • VL light chain variable region
  • the anti-PD-1 antibody comprises: (a) a heavy chain variable region (VH) that comprises a CDR-1 comprising an amino acid sequence of GFSLYSYN (SEQ ID NO: 99), a CDR-2 comprising an amino acid sequence of ISGGTIG (SEQ ID NO: 100), and a CDR-3 comprising an amino acid ARAFYDDYDYNV (SEQ ID NO: 101), and (b) a light chain variable region (VL) that comprises a CDR-1 comprising an amino acid sequence of QSVYGNNQ (SEQ ID NO: 102), a CDR-2 comprising an amino acid sequence of QAS (SEQ ID NO: 103), and a CDR- 3 comprising an amino acid sequence of AGGYSSSSDTT (SEQ ID NO: 104).
  • VH heavy chain variable region
  • the anti-PD-1 antibody is IgGl-PDl which may be administered at a dose of 200 mg intravenously.
  • IgGl-PDl may be given intravenously according to institutional guidelines, published guidelines and the respective product prescribing information, and dosed according to this protocol.
  • the anti-PD-1 antibody comprises cemiplimab.
  • the anti-PD-1 antibody comprises an antibody comprising a heavy chain and a light chain sequence depicted in SEQ ID NOS: 121 and 122, respectively.
  • the immune checkpoint inhibitor comprises an antibody comprising the six CDR sequences from SEQ ID NO: 121 and SEQ ID NO: 122 (e.g., the three heavy chain CDRs from SEQ ID NO: 121 and the three light chain CDRs from SEQ ID NO: 122). In certain embodiments, the immune checkpoint inhibitor comprises an antibody comprising the heavy chain variable domain from SEQ ID NO: 121 and the light chain variable domain from SEQ ID NO: 122.
  • the immune checkpoint inhibitor comprises an antibody comprising: (a) a heavy chain variable region (VH) that comprises a CDR-1 comprising the amino acid sequence FTFSNFG (SEQ ID NO: 137), a CDR-2 comprising the amino acid sequence ISGGGRDT (SEQ ID NO: 138), and a CDR-3 comprising the amino acid sequence VKWGNIYFDY (SEQ ID NO: 139), and (b) a light chain variable region (VL) that comprises a CDR-1 comprising the amino acid sequence LSINTF (SEQ ID NO: 140), a CDR-2 comprising the amino acid sequence AAS (SEQ ID NO: 141), and a CDR-3 comprising the amino acid sequence QQSSNTPFT (SEQ ID NO: 142).
  • the anti-PD-1 antibody is MEDI-0680 (AMP-514; AstraZeneca).
  • MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.
  • the anti-PD-1 antibody is PDR001 (CAS Registry No. 1859072-53-9; Novartis).
  • PDR001 is a humanized IgG4 anti-PDl antibody that blocks the binding of PD-L1 and PD-L2 to PD-1.
  • the anti-PD-1 antibody is REGN2810 (Regeneron).
  • REGN2810 is a human anti-PDl antibody also known as LIBTAYO® and cemiplimab-rwlc.
  • the anti-PD-1 antibody is BGB-108 (BeiGene). In some embodiments, the anti-PD-1 antibody is BGB-A317 (BeiGene).
  • the anti-PD-1 antibody is JS-001 (Shanghai Junshi).
  • JS-001 is a humanized anti-PDl antibody.
  • the anti-PD-1 antibody is STI-A1110 (Sorrento).
  • STI-A1110 is a human anti-PDl antibody.
  • the anti-PD-1 antibody is INCSHR-1210 (Incyte).
  • INCSHR-1210 is a human IgG4 anti-PDl antibody.
  • the anti-PD-1 antibody is PF-06801591 (Pfizer).
  • the anti-PD-1 antibody is TSR-042 (also known as ANB011; Tesaro/AnaptysBio).
  • the anti-PD-1 antibody is AM0001 (ARMO Biosciences).
  • the PD(L)-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
  • the PD(L)-1 binding antagonist is AMP-224.
  • AMP-224 (CAS Registry No. 1422184-00-6; GlaxoSmithKline/Medlmmune), also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in W02010/027827 and WO2011/066342.
  • the PD-1 binding antagonist is a peptide or small molecule compound.
  • the PD-1 binding antagonist is AUNP-12 (PierreFabre/Aurigene). See, e.g., WO2012/168944, WO2015/036927, WO2015/044900, W02015/033303, WO2013/ 144704, WO2013/132317, and WO20U/161699.
  • the PD-L1 binding antagonist is an anti-PD-Ll antibody.
  • a variety of anti-PD-Ll antibodies are contemplated and described herein.
  • the isolated anti-PD-Ll antibody can bind to a human PD-L1, for example a human PD-L1 as shown in UniProtKB/Swiss-Prot Accession No.Q9NZQ7.1, or a variant thereof.
  • the anti-PD-Ll antibody is capable of inhibiting binding between PD- L1 and PD-1 and/or between PD-L1 and B7-1.
  • the anti-PD-Ll antibody is a monoclonal antibody.
  • the anti-PD-Ll antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (FabOz fragments.
  • the anti-PD-Ll antibody is a humanized antibody.
  • the anti-PD-Ll antibody is a human antibody. Examples of anti-PD-Ll antibodies useful for the methods of this disclosure, and methods for making thereof are described in PCT patent application WO 2010/077634 Al and US Patent No. 8,217,149, which are incorporated herein by reference.
  • Exemplary PD-L1 binding antagonists include, without limitation, anti-PD-Ll antibodies such as MEDI4736 (durvalumab; AstraZeneca; see WO 2011/066389), MSB-0010718C (see US 2014/0341917), YW243.55.S70 (see SEQ ID NO: 20 of WO 2010/077634 and US 8,217,149), MIH1 (Affymetrix eBioscience; cf.
  • anti-PD-Ll antibodies such as MEDI4736 (durvalumab; AstraZeneca; see WO 2011/066389), MSB-0010718C (see US 2014/0341917), YW243.55.S70 (see SEQ ID NO: 20 of WO 2010/077634 and US 8,217,149), MIH1 (Affymetrix eBioscience; cf.
  • the anti-PD-Ll antibody comprises atezolizumab (TECENTRIQ; RG7446; MPDL3280A; R05541267), durvalumab (MEDI4736), BMS-936559, avelumab (bavencio), lodapolimab (LY3300054), CX-072 (Proclaim-CX-072), FAZ053, KN035, or MDX-1105.
  • the PD-L2 binding antagonist is an anti-PD-L2 antibody.
  • a variety of anti-PD-L2 antibodies are contemplated and described herein.
  • the isolated anti-PD-L2 antibody can bind to a human PD-L2, for example a human PD-L2 as shown in UniProtKB/Swiss-Prot Accession NO.Q9BQ51, or a variant thereof.
  • the anti-PD-L2 antibody is capable of inhibiting binding between PD-L2 and PD-1.
  • the anti-PD-L2 antibody is a monoclonal antibody.
  • the anti-PD-L2 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments.
  • the anti-PD-L2 antibody is a humanized antibody.
  • the anti-PD-L2 antibody is a human antibody. Examples of anti-PD-L2 antibodies useful for the methods of this disclosure, and methods for making thereof are described in PCT patent application WO 2019/158645 Al, WO 2021/197358 Al and WO 2023/166210 Al, which are incorporated herein by reference.
  • Exemplary PD-L2 binding antagonists include, without limitation, anti-PD-L2 antibodies such as OT17B10 (ThermoFischer), MAB1224 (bio-techne), PDL2/1850 (Antibodies.com EUROPE).
  • PD-1 axis binding antagonists such as anti-PD-1 antibodies, anti-PD-Ll antibodies, and anti-PD-L2 antibodies 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 PD-1 axis binding antagonist to be used.
  • PD-1 axis binding antagonists may be administered in the form of any suitable pharmaceutical composition as described herein.
  • PD-1 axis binding antagonists such as anti-PD-1 antibodies, anti-PD-Ll antibodies and anti-PD-L2 antibodies may be administered in the form of nucleic acid, such DNA or RNA, encoding a PD-1 axis binding antagonist such as anti-PD-1 antibody, anti-PD-Ll antibody, or anti-PD-L2 antibody.
  • antibodies can be delivered encoded in expressing nucleic acids, 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 any other nucleic- acid particle such as nucleic-acid lipid particle.
  • PD-1 axis binding antagonists such as anti-PD-1 antibodies, anti-PD- Ll antibodies, and anti-PD-L2 antibodies may also be administered via an oncolytic virus comprising an expression cassette encoding the PD-1 axis binding antagonist.
  • the PD-1 axis binding antagonist is an anti-PD-1, anti-PD-Ll or anti-PD-L2 antibody or antigen-binding fragment thereof comprising the complementary determining regions (CDRs) of one of the anti- PD-1, PD-L1 or PD-L2 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 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.
  • CDRs complementary determining regions
  • the CDRs of the anti-PD-1, PD-L1 or PD-L2 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 binding antagonist 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.
  • Anti-PD-1, anti-PD-Ll or PD-L2 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, PD-L1, PD-L2 binding fragments of any of the above.
  • an anti-PD-1, anti-PD-Ll or PD-L2 antibody described herein binds specifically to PD-1, PD-L1 or PD- L2, respectively, (e.g., human PD-1).
  • the immunoglobulin molecules of the disclosure can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • 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-1, anti-PD-Ll or anti-PD-L2 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 V L or V H 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, PD-L1 or PD-L2 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, PD-L1 or PD-L2 antibodies disclosed herein may be monospecific, bispecific, trispecific or of greater multi specificity. Multispecific antibodies may be specific for different epitopes of PD-1, PD-L1 or PD-L2 or may be specific for one or more of PD-1, PD-L1 or PD-L2 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 a!., 1991, J. Immunol. 147:60 69; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547 1553.
  • the anti-PD-1, PD-L1 or PD-L2 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 a!., J. Mol. Biol.
  • a CDR or individual specified CDRs e.g., CDR-H1, CDR-H2, CDR-H3
  • a given antibody or region thereof e.g., variable region thereof
  • 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 V L 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, PD-L1 or PD-L2 antibodies or antigen-binding fragments thereof provided herein are according to the IMGT numbering scheme as described in Lefranc, M. P. etal., Dev. Comp. Immunol., 2003, 27, 55-77.
  • the anti-PD-1, PD-L1 or PD-L2 antibodies disclosed herein comprise the CDRs of the antibody nivolumab. See WO 2006/121168.
  • the CDRs of the antibody nivolumab are delineated using the Kabat numbering scheme (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NTH Publication No. 91-3242).
  • the present disclosure encompasses an anti-PD-1 antibody or derivative thereof comprising a heavy or light chain variable domain, said variable domain comprising (a) a set of three CDRs, in which said set of CDRs are from the monoclonal antibody nivolumab, and (b) a set of four framework regions, in which said set of framework regions differs from the set of framework regions in the monoclonal antibody nivolumab, and in which said anti-PD-1 antibody or derivative thereof binds to PD-1.
  • Anti-PD-1, PD-L1 or PD-L2 antibodies disclosed herein may also be described or specified in terms of their binding affinity to PD-1, PD-L1 or PD-L2, respectively ⁇ e.g., human PD-1).
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5 xl0‘ 2 M, 10' 2 M, 5xl0‘ 3 M, 10 3 M, 5xl0 -4 M, 10' 4 M, 5xl0‘ 5 M, 10’ 5 M, 5xl0 ⁇ 6 M, 10' 6 M, 5xl0 7 M, IO 7 M, SxlO’ 8 M, 10' 8 M, 5x 10 9 M, IO’ 9 M, 5x 10 10 M, IO' 10 M, 5x10 11 M, IO' 11 M, 5xl0‘ 12 M, IO’ 12 M, 5xl0‘ 13 M, IO’ 13 M, 5xl0 14 M, IO 14 M, 5xl0 15
  • the anti-PD-1, PD-L1 or PD-L2 antibodies also include derivatives and constructs that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to PD-1, PD-L1 or PD-L2.
  • the anti-PD-1, PD-L1 or PD-L2 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 binding agent and PD-1 axis binding antagonist disclosed herein may be used in the treatment of tumors or cancer.
  • the tumor or cancer is a tumor or cancer expressing EpCAM.
  • EpCAM is human EpCAM, in particular human EpCAM comprising the sequence set forth in SEQ ID NO: 59 and the PD-1 axis binding antagonist is an anti-PD-1 antibody or binding fragment thereof.
  • the binding agent to be used in the treatment or prevention of tumors or cancer is a binding agent that comprises a first binding region binding to human EpCAM that comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 9 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 10, and a second binding region binding to human CD137 that comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 20.
  • this binding agent is used in combination with an anti-PD-1 antibody.
  • the subject to be treated according to the present disclosure is preferably a human subject.
  • the tumor or cancer to be treated is a solid tumor or cancer.
  • the tumor or cancer may be a metastatic tumor or cancer.
  • the tumor or cancer may be selected from the group consisting of cholangiocarcinoma (bile duct cancer), Gastric/gastroesophageal junction (GEJ) cancer, melanoma, ovarian cancer, lung cancer ⁇ e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, head and neck cancer, gastric cancer, breast cancer, renal cancer, urothelial cancer, bladder cancer, esophageal cancer, pancreatic cancer, hepatic cancer, thymoma and thymic carcinoma, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndromes, endometrial cancer, prostate cancer, penile cancer, cervical cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma and mesothelio
  • the tumor or cancer is selected from the group consisting of cholangiocarcinoma (bile duct cancer), Gastric/gastroesophageal junction (GEJ) cancer, lung cancer (e.g. non-small cell lung cancer (NSCLC), urothelial cancer (cancer of the bladder, ureter, urethra, or renal pelvis), endometrial cancer (EC), breast cancer (e.g. triple negative breast cancer (TNBC)), squamous cell carcinoma of the head and neck (SCCHN) (e.g. cancer of the oral cavity, pharynx or larynx) and cervical cancer.
  • cholangiocarcinoma bile duct cancer
  • GEJ Gastric/gastroesophageal junction
  • lung cancer e.g. non-small cell lung cancer (NSCLC), urothelial cancer (cancer of the bladder, ureter, urethra, or renal pelvis
  • endometrial cancer EC
  • breast cancer e.
  • the tumor or cancer to be treated is a non-central nervous system (CNS) tumor or cancer, such as a non-CNS malignant tumor.
  • CNS central nervous system
  • the tumor or cancer to be treated is a solid tumor or cancer.
  • the tumor or cancer to be treated is a non-CNS solid tumor or cancer, such as a non-CNS solid malignant tumor.
  • the amount of binding agent and/or PD-1 axis binding antagonist administered in each dose and/or in each treatment cycle is about 0.3-5 mg/kg body weight or about 25-400 mg in total; and/or about 2.1 x 10' 9 - 3.4 x 10’ 8 mol/kg body weight or about 1.7 x 10‘ 7 - 2.7 x 10' 6 mol in total.
  • the dose defined in mg/kg may be converted to flat dose, and vice versa, based on the median body weight of the subjects to whom the binding agent is administered being 80 kg.
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.3-4.0 mg/kg body weight or about 25-320 mg in total; and/or about 2.1 x 10’ 9 - 2.7 x 10' 8 mol/kg body weight or about 1.7 x 10‘ 7 - 2.2 x 10' 6 mol in total.
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.38-4.0 mg/kg body weight or about 30-320 mg in total; and/or about 2.6 x 10’ 9 - 2.7 x 10’ 8 mol/kg body weight or about 2.4 x 10’ 7 - 2.2 x IO -6 mol in total.
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.5-3.3 mg/kg body weight or about 40-260 mg in total; and/or about 3.4 x 10‘ 9 - 2.2 x 10' 8 mol/kg body weight or about 2.7 x 10' 7 - 1.8 x 10' 6 mol in total.
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.6-2.5 mg/kg body weight or about 50-200 mg in total; and/or about 4.3 x 10' 9 - 1.7 x IO -8 mol/kg body weight or about 3.4 x 10' 7 - 1.4 x 10 ® mol in total.
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.8-1.8 mg/kg body weight or about 60-140 mg in total; and/or about 5.1 x IO -9 - 1.2 x 10 ® mol/kg body weight or about 4.1 x IO -7 - 9.5 x 10’ 7 mol in total.
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.9-1.8 mg/kg body weight or about 70-140 mg in total; and/or about 6.0 x 10" 9 - 1.2 x 10’ 8 mol/kg body weight or about 4.8 x 10 ⁇ 7 - 9.5 x 10 7 mol in total.
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 1-1.5 mg/kg body weight or about 80-120 mg in total; and/or about 6.8 x IO 9 - 1.0 x IO -8 mol/kg body weight or about 5.5 x 10‘ 7 - 8.2 x 10 ⁇ 7 mol in total.
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0,8-1.5 mg/kg body weight or about 65-120 mg in total; and/or about 5.5 x 10' 9 - 1.0 x 10’ 8 mol/kg body weight or about 4.4 x 10' 7 - 8.2 x 10‘ 7 mol in total.
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be about 0.9-1.3 mg/kg body weight or about 70-100 mg in total; and/or about 6.0 x IO -9 - 8.5 x IO 9 mol/kg body weight or about 4.8 x 10' 7 - 6.8 x 10 7 mol in total. about 0.9-1.1 mg/kg body weight or about 75-90 mg in total; and/or about 6.4 x 10‘ 9 - 7.7 x 10‘ 9 mol/kg body weight or about 5.1 x IO -7 - 6.1 x 10 7 mol in total.
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.3-4.0 mg/kg body weight or 25-320 mg in total; and/or
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.38-
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.5-
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.6-
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.8-
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.9-
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 1-1.5 mg/kg body weight or 80-120 mg in total; and/or
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 1.1-
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 1.2-
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0,8-
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.9-
  • the amount of binding agent administered in each dose and/or in each treatment cycle may in particular be 0.9- 1.1 mg/kg body weight or 75-90 mg in total; and/or
  • the amount of binding agent administered in each dose and/or in each treatment cycle may be about 1.1 mg/kg body weight or about 80 mg in total; and/or about 6.8 x 10 9 mol/kg body weight or about 5.5 x 10 ⁇ 7 mol in total.
  • the amount of binding agent administered in each dose and/or in each treatment cycle may be any amount of binding agent administered.
  • the amount of binding agent administered in each dose and/or in each treatment cycle is about 1.25 mg/kg body weight or about 100 mg in total; and/or about 8.5 x 10' 9 mol/kg body weight or about 6.8 x 10 7 mol in total.
  • the binding agent and/or PD-1 axis binding antagonist may be administered in any manner and by any route known in the art.
  • the binding agent and/or PD-1 axis binding antagonist is administered systemically, such as parenterally, in particular intravenously.
  • the binding agent and/or PD-1 axis binding antagonist may be administered in the form of any suitable pharmaceutical composition as described herein.
  • the binding agent and/or PD-1 axis binding antagonist is administered in the form of an infusion.
  • the binding agent and/or PD-1 axis binding antagonist may be administered by using intravenous (IV) infusion, such as by intravenous infusion over a minimum of 30 minutes, such as over a minimum of 60 minutes e.g., by using intravenous infusion over 30 to 120 minutes.
  • IV intravenous
  • the binding agent and/or PD-1 axis binding antagonist is administered by using intravenous (IV) infusion over 30 minutes.
  • binding agent and/or PD-1 axis binding antagonist described herein may be administered perse or in the form of nucleic acid encoding the binding agent and/or PD-1 axis binding antagonist.
  • the binding agent and/or PD-1 axis binding antagonist or nucleic acid encoding the binding agent and/or PD-1 axis binding antagonist may be administered in any suitable form (e.g., naked as such or packaged in the form of suitable particles comprising the binding agent or nucleic acid). However, it is preferred that the binding agent and/or PD-1 axis binding antagonist or nucleic acid, are administered in the form of any suitable pharmaceutical composition as described herein.
  • the binding agent and/or PD-1 axis binding antagonist may be co-administered together with one or more additional therapeutic agents.
  • the one or more additional therapeutic agents comprise one or more chemotherapeutic agents, in particular, those chemotherapeutic agents which are commonly used in the treatment of a tumor or cancer as described herein.
  • the one or more chemotherapeutic agents include platinum-based compounds (e.g., cisplatin, oxaliplatin, and carboplatin), taxane-based compounds (e.g., paclitaxel and nab-paclitaxel), nucleoside analogs (e.g., 5-fluorouracil and gemcitabine), and combinations thereof (e.g., cisplatin/carboplatin + 5-fluorouracil or nab-paclitaxel + gemcitabine).
  • platinum-based compounds e.g., cisplatin, oxaliplatin, and carboplatin
  • taxane-based compounds e.g., paclitaxel and nab-paclitaxel
  • nucleoside analogs e.g.,
  • 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 nucleic acid and/or, if present, 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.
  • a composition in particular a pharmaceutical composition, 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
  • compositions of the present disclosure may comprise one or more pharmaceutically acceptable carriers, excipients and/or diluents.
  • pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).
  • compositions can be selected with regards to the intended route of administration and standard pharmaceutical practice.
  • carrier refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition.
  • a carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy- propylene copolymers.
  • the pharmaceutical composition of the present disclosure includes isotonic saline.
  • 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
  • Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.
  • 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), but
  • 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.
  • Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poly-ortho esters, and polylactic acid alone or with a wax, or other materials well known in the art. Methods for the preparation of such formulations are generally known to those skilled in the art, see e.g. Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • “Pharmaceutically acceptable salts” comprise, for example, acid addition salts which may, for example, be formed by using a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.
  • a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.
  • 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).
  • 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 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 non-aqueous solvent or dispersion medium containing for instance water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients e.g. from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Sterile injectable solutions may be prepared by incorporating the active compounds in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • examples of methods of preparation are vacuum-drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the binding agent and/or PD-1 axis binding antagonist is formulated in a composition or formulation comprising histidine, sucrose and Polysorbate-80, and having a pH from about 5 to about 6, such as from 5 to 6.
  • the binding agent and/or PD-1 axis binding antagonist may be in a composition or formulation comprising about 20 mM histidine, about 250 mM Sucrose, about 0.02% Polysorbate-80, and having a pH of about 5.5, such as a composition or formulation comprising 20 mM histidine, 250 mM Sucrose, 0.02% Polysorbate-80, and having a pH of 5.5.
  • the formulation may in particular embodiments comprise about 10 to about 30 mg binding agent/mL, such as 10-30 mg binding agent/mL, in particular about 20 mg binding agent/mL, such as 20 mg binding agent/mL.
  • the binding agent and/or PD-1 axis binding antagonist may be provided in a composition as defined above and may then be diluted in 0.9% NaCI (saline) prior to administration.
  • compositions according to the present disclosure are generally applied in a “pharmaceutically effective amount” and in “a pharmaceutically acceptable preparation”.
  • pharmaceutically acceptable refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.
  • VH and VL sequences of UBS-54 have been disclosed in patent no EP 1 479 696 Al (Crucell Holland BV), and the antibodies based on these sequences are referred to herein as "IgGl-EpCAM-UBS-54".
  • VH and VL sequences of A37 have been disclosed in patent no EP 1 479 696 Al (Crucell Holland BV), and the antibodies based on these sequences are referred to herein as "IgGl-EpCAM-A37”.
  • VH and VL sequences of C52 have been disclosed in EP 1 479 696 Al (Crucell Holland BV), and the antibodies based on these sequences are referred to herein as "IgGl- EpCAM-C52".
  • CD137 antibody clone 005 as described in Example 1 and Table 1 of W02018/011421 (Genmab A/S; BioNTech AG). CD137 antibody clone 005 is referred to herein as "IgGl-CD137-005".
  • CD137 antibody clone 009 as described in Example 1 and Table 1 of WO2018/011421 (Genmab A/S; BioNTech AG). CD137 antibody done 009 is referred to herein as "IgGl-CD137-009".
  • CD137-009 humanized antibody (HC7 and LC2) as described in Example 7 and Table 1 of W02018/011421 (Genmab A/S; BioNTech AG).
  • Name 4-1BB-009-HC7LC2 is referred to herein as "IgGl-CD137-009-HC7LC2".
  • Antibody sequences were cloned into pcDNA3.3 expression vectors (Invitrogen, US) and expressed as IgGl, K or IgGl, A, with or without Fc-silencing and/or DuoBody® technology amino add substitutions in the Fc domain (see below). All antibodies were produced under serum-free conditions by co-transfecting relevant heavy and light chain expression vectors in Expi293FTM cells (Thermo Fisher Scientific, US; cat. no. A14527) using ExpiFectamineTM 293 (Thermo Fisher Scientific; cat. no. A14525), essentially as described by the manufacturer.
  • Bispecific antibodies were generated in vitro using the DuoBody® platform technology, i.e. 2-MEA-induced controlled Fab-arm exchange (cFAE) as described in WO2011147986, WO2011131746 and W02013060867 (Genmab), Labrijn et al., PNAS 2013, 110: 5145-50 and Gramer et al., MAbs 2013, 5: 962- 973.
  • cFAE 2-MEA-induced controlled Fab-arm exchange
  • IgGl molecules carrying a single mutation in the CH3 domain were generated: in one parental IgGl antibody the F405L mutation, in the other parental IgGl antibody the K409R mutation.
  • the parental IgGl antibodies included substitutions that result in an Fc domain that is unable to interact with IgG Fc receptors (Fc gamma receptors) and/or complement factors such as Clq: L234F, L235E, D265A (FEA; US 2015/0337049) or L234F, L235E, G236R (FER).
  • Fc gamma receptors IgG Fc receptors
  • complement factors such as Clq: L234F, L235E, D265A (FEA; US 2015/0337049) or L234F, L235E, G236R (FER).
  • L234F, L235E, D265A, and K409R FEAR L234F, L235E, G236R, and F405L: FERL
  • Mouse bispecific antibodies were generated in vitro by 2-MEA-induced controlled Fab-arm exchange (cFAE) as described in Labrijn et al., Sci Rep 2017, 7:2476.
  • cFAE 2-MEA-induced controlled Fab-arm exchange
  • mouse IgG2a molecules (IgG2amm) carrying double matched point mutations in the CH3 domain were generated: in one parental IgG2amm antibody the F405L/R411T (LT) mutations, in the other parental IgG2amm antibody the T370K/K409R (KR) mutations.
  • the parental IgG2amm antibodies included substitutions that result in an Fc domain that is unable to interact with IgG Fc receptors (Fc gamma receptors) and/or complement factors such as Clq: L234A, L235A as described in Labrijn et al., Sci Rep 2017, 7:2476.
  • L234A, L235A, F405L/R411T AALT L234A, L235A, T370K/K409R: AAKR
  • the heavy chain (HC) and light chain (LC) sequences of the parental antibodies are set forth in the following SEQ ID NOs:
  • IgG2amm-EpCAM-323-A3-AAKR SEQ ID NO: 39 (HC) and SEQ ID NO: 40 (LC).
  • the two parental antibodies were mixed in equal molar ratios in PBS buffer (Phosphate Buffered Saline; 8.7 mM HPO4 2 ', 1.8 mM H2PO4 , 163.9 mN Na+, 140.3 mM CI-, pH 7.4).
  • PBS buffer Phosphate Buffered Saline; 8.7 mM HPO4 2 ', 1.8 mM H2PO4 , 163.9 mN Na+, 140.3 mM CI-, pH 7.4
  • 2- mercaptoethylamine-HCI (2-MEA) was added to a final concentration of 75 mM and the reaction mixture was incubated at 31°C for 5 h.
  • the 2-MEA was removed by dialysis into PBS buffer using 10 kDa molecular-weight cutoff Slide-A-Lyzer carriages (Thermo Fisher Scientific) according to the manufacturer's protocol.
  • IgGl-bl2 is an HIV-1 gpl2O specific antibody (Barbas, CF. J Mol Biol. 1993 Apr 5; 230(3):812-23) that is used in some of the examples as negative, non-binding control antibody for monospecific and bispecific antibodies.
  • the heavy chain (HC) and light chain (LC) sequences of the parental antibodies are set forth in the following SEQ ID NOs;
  • IgG2amm-bl2-AAKR SEQ ID NO: 46 (HC) and SEQ ID NO: 45 (LC).
  • Target binding affinity of EpCAM-specific antibodies was determined by label-free biolayer interferometry (BLI) on an Octet HTX instrument (ForteBio).
  • HIS1K biosensors (ForteBio, cat. no. 18-5120) were pre-conditioned by exposure to 10 mM glycine (Sigma-Aldrich, cat. no. 15527) buffer pH 1.5 for 5 s, followed by neutralization in Sample Diluent (ForteBio, cat. no. 18-1104) for 5 s; both steps were repeated 2 times.
  • HIS1K biosensors (ForteBio, cat. no. 18-5120) were pre-conditioned by exposure to 10 mM glycine (Sigma-Aldrich, cat. no. 15527) buffer pH 1.5 for 5 s, followed by neutralization in Sample Diluent (ForteBio, cat. no. 18-1104) for 5 s; both steps were repeated 2 times.
  • the anti-Penta-HIS biosensors were loaded with 100 nM (2.8 ) recombinant human EpCAM ⁇ g/mL (R&D systems, cat no 9277-EP) or recombinant cynomolgus monkey EpCAM (R&D systems, cat no. 10451-EP) for 600 s. After a baseline measurement in Sample Diluent (300 s), the association (200 s) and dissociation (1,000 s) of the functionally monovalent antibodies was determined using a concentration range of 1.56 - 100 nM, with two- fold dilution steps in Sample Diluent.
  • Functionally monovalent EpCAM antibodies are bispecific molecules that contains one EpCAM-specific Fab arm and a non-binding control Fab arm (based on HIV gpl20-specific bl2 antibody described in Example 1), thereby ensuring that binding to EpCAM in this assay is functionally monovalent.
  • the molecular mass based on the amino acid sequence of the used antibodies 146.0 - 148.6 kDa was used for calculations. For each antibody a reference sensor was used, which was incubated with Sample Diluent instead of antibody.
  • Data were acquired using Data Acquisition Software V12.0 (ForteBio) and analyzed with Data Analysis Software vl2 (ForteBio). Data traces were corrected per antibody by subtraction of the reference sensor. The Y-axis was aligned to the last 10 s of the baseline. Interstep Correction alignment to dissociation and Savitzky-Golay filtering were applied. Data traces with a response ⁇ 0.05 nm were excluded from analysis. The data was fitted with the 1:1 Global Full fit model, using a window of interest for association of 200 s and dissociation times set at 50 s and 200 s (indicated in Table 4 and Table 5).
  • the dissociation time that was used for the reported data was chosen based upon visual inspection of the curve- fits and highest R 2 value. Data corresponding to curve fits with a R 2 ⁇ 0.98 were excluded from analyses.
  • Ab refers to the equilibrium dissociation constant of the antibody-antigen interaction, and is obtained by dividing Aa by k&.
  • Aa (sec 1 ) refers to the dissociation rate constant of the antibody-antigen interaction. This is sometimes also referred to as the value or off-rate,
  • ka (M -1 x sec 1 ) refers to the association rate constant of the antibody-antigen interaction. This is sometimes also referred to as the k on value or on-rate.
  • Table 4 shows the association rate constant k, (1/Ms), dissociation rate constant fa (1/s) and equilibrium dissociation constant Ko (M) of bispecific, functionally monovalent EpCAM antibodies for recombinant human EpCAM, as determined by biolayer interferometry. Affinities of the antibodies to human EpCAM ranged from 3.2 - 55 nM.
  • Table 5 shows the association rate constant fa (1/Ms), dissociation rate constant fa (1/s) and equilibrium dissociation constant Ko (M) of the EpCAM antibodies for cynomolgus monkey EpCAM, as determined with biolayer interferometry. Only BisGl-bl2-FEAL/EpCAM-A37-FEAR showed reliable results for binding to cynomolgus monkey EpCAM, with an affinity of 41 nM.
  • Table 4 Binding affinities of bispecific, functionally monovalent EpCAM antibodies to human EpCAM extracellular domain as determined by label-free biolayer interferometry.
  • Table 5 Binding affinities of bispecific, functionally monovalent EpCAM antibodies to cynomolgus monkey EpCAM extracellular domain as determined by label-free biolayer interferometry.
  • Example 3 Cross-block of EpCAM antibodies as determined by biolayer interferometry
  • Antibody cross-block analysis in classical sandwich format was performed by BLI on an Octet HTX instrument (ForteBio). Experiments were carried out while shaking at 1,000 RPM and at 30°C.
  • Amine Reactive 2nd Generation (AR2G) biosensors were activated for 300 s with a solution of 20 mM EDC (N-[3-Dimethylaminopropyl]-N'-ethylcarbodiimide hydrochloride; ForteBio, 18-1033) and 10 mM s-NHS (N-Hydroxysulfosuccinimide sodium salt; ForteBio, 18-1067).
  • the activated AR2G sensors were loaded with 20 ⁇ g/m fLirst EpCAM antibody in 10 mM Sodium Acetate pH 6.0 (ForteBio, cat. no. 18-1070) for 600 s and quenched with 1 M ethanolamine pH 8.5 (ForteBio cat. no. 18-1071) for 300 s.
  • the first column shows the immobilized antibodies (first antibody) and the first row shows the antibodies in solution (second antibody). Corrected association responses of the antibodies in solution are shown. Cross-block of antibodies is indicated by the dark grey color.
  • Example 4 Expression of EpCAM on human tumor cell lines
  • EpCAM surface expression levels were evaluated using flow cytometry on the following human tumor cell lines: T84 (lung metastasis of colon adenocarcinoma; ATCC, cat.no. CLL-248), DiFi (rectal carcinoma; kindly provided by the Thomas Valerius Christian Albrechts University, Kiel, Germany), HPAF-II (pancreatic adenocarcinoma; ATCC, cat.no. CRL-1997), NCI-N87 (gastric carcinoma; ATCC, cat.no. CRL-5822), Calu-3 (non-small cell lung carcinoma; ATCC, cat.no.
  • NCI- H747 colonal adenocarcinoma; ATCC, cat.no. CLL-252
  • A549 non-small cell lung adenocarcinoma; ATCC, cat.no. CLL-185.
  • Figure 1 shows the relative EpCAM expression on tumor cell lines, as determined using flow cytometry, confirming EpCAM expression on all tested cell lines.
  • the tumor cell lines were ranked based on EpCAM expression as following: T84 > DiFi > HPAF-II > NCI-N87 > Calu-3 > NCI-H747 » A549.
  • EpCAM-binding arm Binding to the EpCAM-expressing human tumor cell lines DiFi, HPAF-II and A549 by bivalent EpCAM antibodies or bispecific antibodies carrying one EpCAM-binding arm was analyzed by flow cytometry. Cells (3-5xl0 4 cells/well) were incubated in polystyrene 96-well round-bottom plates (Greiner bio-one, cat. no.
  • FIG 2 shows that monospecific, bivalent (monoclonal) EpCAM antibodies (A) IgGl-EpCAM-A37-FEAR, (B) IgGl- EpCAM-C52-FEAR and (C) IgGl-EpCAM-UBS-54-FEAR display dose-dependent binding to human EpCAM-expressing DiFi, HPAF-II and A549 tumor cells.
  • Bispecific, monovalent EpCAMxbl2 antibodies (A) bsIgGl-bl2-FEALxEpCAM- A37-FEAR, (B) bsIgGl-bl2-FEALxEpCAM-C52-FEAR and (C) bsIgGl-bl2-FEALxEpCAM-UBS-54-FEAR display binding at higher antibody concentrations compared to IgGl-EpCAM-A37-FEAR, IgGl-EpCAM-C52-FEAR or IgGl- EpCAM-UBS-54-FEAR, respectively.
  • the maximum binding to DiFi, HPAF-II and A549 tumor cells was comparable to that of their respective monospecific, bivalent EpCAM counterparts.
  • EpCAM antibody IgGl-323-A3-FEAR displayed dose-dependent binding to DiFi and HPAF-II tumor cells (D).
  • the capacity to bind DiFi tumor cells was studied for two bispecific EpCAMx4-lBB antibodies that contained the same variable domains but differed in their Fc-domain: one containing an Fc-domain harboring the L234F/L235E/D265A (FEA) inertness mutations in both heavy chains (HC), and one containing the FEA inertness mutations in the HC of the 4-lBB-binding arm and the L234F/L235E/G236R (FER) inertness mutations in the HC of the EpCAM-binding arm.
  • the two bispecific antibodies showed comparable binding as demonstrated by the EC50 values (3.841 nM vs 3.961 nM, respectively; Figure 2E).
  • Chinese Hamster Ovary (CHO)-S cells (1.0 x 10 6 cells/mL; ThermoFisher Scientific, cat. no. R800-07) were cultured in FreestyleTM CHO Expression Medium (ThermoFisher Scientific, cat. no. 12651022).
  • FreestyleTM CHO Expression Medium (ThermoFisher Scientific, cat. no. 12651022).
  • ⁇ tgh/em mLedium was supplemented with 50 units penicillin and 50 pg/mL streptomycin (Pen/Strep; Lonza, cat. no. DE17-603E).
  • Transfection was performed using Freestyle MaxTM transfection reagent (ThermoFisher Scientific, cat. no. 16447100) and OptiPROTM SFM medium (ThermoFisher Scientific, cat. no. 12309019), according to the manufacturer's instructions. After transfection, cells were incubated 24 h at 37°C, 85% humidity, 8% COz while shaking, and then frozen in FreestyleTM CHO Expression Medium supplemented with 10% DMSO until further use.
  • Freestyle MaxTM transfection reagent ThermoFisher Scientific, cat. no. 16447100
  • OptiPROTM SFM medium ThermoFisher Scientific, cat. no. 12309019
  • EpCAM antibodies Binding of EpCAM antibodies to CHO-S cells transiently transfected with full length human, cynomolgus monkey or mouse EpCAM was analyzed by flow cytometry.
  • Cells were thawed in Roswell Memorial Park Institute 1640 medium (RPMI; Lonza, cat. no. 12-115F) supplemented with 10% Donor Bovine Serum with Iron (DBSI; Life technologies, cat. no. 20371). Cells were incubated (3 x 10 4 cells/well) in polystyrene 96-well round-bottom plates (Greiner bio- one, cat. no.
  • EpCAM antibodies range 0.0001 to 50 in 5-fold dilution steps
  • 50 pL PBS/0.1% BSA/0.02% azide FACS buffer
  • FACS buffer 50 pL R-Phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab')z (1:500 in FACS buffer; Jackson ImmunoResearch Laboratories, cat. no. 109-116-098) at 4°C for 30 min.
  • Figure 3A shows dose-dependent binding of bivalent EpCAM-binding antibodies IgGl-EpCAM-UBS-54-FEAR, IgGl- EpCAM-A37-FEAR, IgGl-EpCAM-C52-FEAR and IgGl-EpCAM-323-A3 to CHO-S cells transfected with full length human, cynomolgus monkey and mouse EpCAM.
  • Figure 3B shows dose-dependent binding of monovalent EpCAM-binding antibodies bs!gGl-bl2-FEAL/EpCAM-UBS-54-FEAR, bs!gGl-EpCAM-A37-FERL/bl2-FEAR, bsIgGl- bl2-FEAL/EpCAM-C52-FEAR, and bs!gGl-bl2-FEAL/EpCAM-323-A3-FEAR to CHO-S cells transfected with full length human, cynomolgus monkey and mouse EpCAM.
  • no binding of either monovalent or bivalent EpCAM antibodies to non-transfected CHO-WT cells was observed.
  • Example 7 Cell-based reporter assay to determine 4-1BB agonist activity of an EpCAM X4-1BB bispecific antibody and the dependence of agonist activity on target crosslinking.
  • a reporter cell line expressing luciferase under the control of NF-KB response elements (HEK293_NFK_ gfpjuc cells) was transduced with human 4-1BB (HEK293_NFK_ h4-lBB_gfp_luc cells).
  • the reporter assay was conducted either using the reporter cell line alone, or in co-culture with EpCAM-expressing OV-90-SC12 cells.
  • HEK293_NFK_h4-lBB_gfp_luc cells were generated by transposon-mediated transfection of human 4-1BB into HEK293_NFK_gfpJuc cells (BioCat GmbH, cat. no. TR860A-1) followed by blasticidin selection. Cells were seeded in 96-well white flat-bottom plates (Fisher Scientific, cat. no. 10072151) at 3xl0 4 cells/well and cultured overnight in DMEM (Thermo Fisher Scientific, cat. no. 31966-047) supplemented with 10% non heat-inactivated FBS (Biochrom, cat. no. S0115).
  • Example 8 Capacity to induce CD4+ and CD8+ T-cell proliferation in PBMC - tumor cell co-cultures by EpCAMx4-lBB bispecific antibodies containing inertness mutations
  • EpCAMx4-lBB bispecific antibodies to increase PBMC proliferation through 4-1BB co-stimulation was assessed in in vitro co-cultures of EpCAM-expressing tumor cell lines and primary human peripheral blood mononuclear cells (PBMCs), which were isolated from healthy human donor huffy coats (Sanquin, Amsterdam, The Netherlands) using a Ficoll gradient (Corning; lymphocyte separation medium, cat. no. 25-072-CI).
  • PBMCs peripheral blood mononuclear cells
  • EpCAMx4-lBB bispecific antibodies with an Fc-domain containing the FEA inertness mutations in both heavy chains (HC)
  • EpCAMx4-lBB bispecific antibodies with an Fc- domain containing the FEA inertness mutations in the HC of the 4-lBB-binding arm, and the FER inertness mutations in the HC of the EpCAM-binding arm.
  • EpCAM-expressing tumor cells (DiFi, T84, HPAF-II, NCI-N87, Calu-3 or NCI-H747; as described herein above) were seeded at a density of 6,250 cells/well into polystyrene 96-well flat- bottom plates (Greiner bio-one, cat. no. 655180) in T-cell medium (Iscove's Modified Dulbecco's Medium [IMDM; Lonza, cat. no. 12-722F] supplemented with 5% Human Serum [Sanquin Blood Bank, cat. no. B0626] and 1% Pen/Strep, Lonza, cat. no.
  • PBMCs were labelled with 0.5 pM CellTraceTM CFSE (carboxyfluorescein succinimidyi ester; ThermoFisher Scientific; cat. no. C34554A) in PBS (phosphate-buffered saline, Hyclone, cat. no. SH3A383003) at 37°C for 20 min. After washing, resting the PBMCs for 0.5-1.5 h in T-cell medium and washing again, the PBMCs were mixed with anti-CD3 antibody (0.1 pg/mL; clone UCHT1; Stemcell, cat. no.
  • Bispecific EpCAMx4-lBB, EpCAMxbl2 or non-binding control antibodies were added (final concentration serial dilution: 8 - 25,000 ng/mL in 5-fold dilutions [Figure 5 and 7], or at a single concentration of 10 [ Figure 6]) and plates ⁇ g/mL were incubated at 37°C for 96 h. Next, 150 pL supernatants containing PBMCs were transferred to 96-well round- bottom plates.
  • PBMCs were stained with 50 pL BD HorizonTM Fixable Viability Stain 510 (FVS510; 1:5000 dilution in PBS; BD Biosciences; cat. no. 564406) for 20 min at RT protected from light.
  • FVS510 Fluorescence-Coupled Device
  • CD4+ and CD8+ T-cell subsets cells were additionally stained with brilliant violet (BV)785-labeled anti-CD19 (clone SJ25C1, 1:50; BioLegend, cat. no. 363028), APC-eF780- labeled anti-CD4 (clone OKT4, 1:50; eBioscience, cat. no.
  • AF700-labeled anti-CD8 (clone RPA-T8, 1:100; BioLegend, cat. no. 301028), and BV605-labeled anti-CD14 (clone: M5E2, 1:200; BioLegend, cat. no. 301834) antibodies in 50 pL FACS buffer at 4°C for 30 min protected from light. After washing the cells, cells were resuspended in 30-60 pL FACS buffer (PBS [Lonza, BE17-517Q] supplemented with 0.1% bovine serum albumin [BSA, fraction V, Roche, cat, no. 10735086001], 0.02% NaN3 [Bio-world, cat. no.
  • the cells were additionally stained with brilliant violet (BV)785-labeled anti-CD19 (clone SJ25C1, 1:80), APC-eF780-labeled anti-CD4 (clone OKT4, 1:100), AF700-labeled anti-CD8 (clone RPA-T8, 1:100; BioLegend, cat. no. 301028), and PE-Cy7-labeled anti-CD14 (clone: M5E2; 1:50; BD, cat. no. 557742) antibodies in 50 pL FACS buffer at 4°C for 30 min protected from light.
  • BV brilliant violet
  • PBMC proliferation cells were gated for lymphocytes (based on FSC/SSC) and viable cells (FVS510- population).
  • PBMCs were gated for lymphocytes (based on FSC/SSC), viable cells (FVS510- population), T cells (CD14- CD19- population) and finally CD4+ or CD8+ T cells (CD4+CD8- or CD4-CD8+ populations, respectively).
  • CFSE- stained cells were measured to assess PBMC, CD4+ or CD8+ T-cell proliferation.
  • the Division Index representing the average number of divisions per cell, was calculated using Microsoft Office Excel and visualized using Graphpad Prism, using the following formula: Z (x,/2i*i) / Z (xi/2i), where i is the number of the peak with the undivided peak is 0, the first divided is 1 etc., and Xi is the number of cells in the corresponding peak.
  • EpCAMx4-lBB bispecific antibodies to increase T-cell proliferation through 4-1BB co-stimulation was assessed in in vitro co-cultures of the EpCAM-expressing DiFi tumor cell line and PBMCs.
  • Figure 5 shows that bsIgGl-CD137-009-HC7LC2-FEAL/EpCAM-UBS54-FEAR (A), bs!gGl-CD137-009-HC7LC2-FEAL/EpCAM-A37-FEAR (B), bs!gGl-CD137-009-HC7LC2-FEAL/EpCAM-C52-FEAR (C) and bs!gGl-CD137-009-HC7LC2-FEAL/EpCAM-323- A3-FEAR (D) enhanced dose-dependent proliferation of CFSE-labeled cells (shown as increase in the division index).
  • EpCAMxbl2 antibodies bsIgGl-bl2-FEAL/EpCAM-UBS54-FEAR (A), bs!gGl-bl2-FEAL/EpCAM-A37- FEAR (B), bs!gGl-bl2-FEAL/EpCAM-C52-FEAR (C) and bs!gGl-bl2-FEAL/EpCAM-323-A3-FEAR (D) did not enhance proliferation, as the division index was comparable to isotype control antibody IgGl-bl2-FEAL.
  • EpCAMx4-lBB bispecific antibodies were evaluated by gating specifically on these T-cell subsets.
  • Figure 6 shows that the EpCAMx4-lBB bispecific antibodies bs!gGl-CD137-009-HC7LC2-FEAL/EpCAM-UBS54-FEAR, bs!gGl-CD137-009-HC7LC2-FEAL/EpCAM-A37-FEAR and bsIgGl-CD137-009-HC7LC2-FEAL/EpCAM-C52-FEAR enhanced proliferation of both (A) CD4+ and (B) CD8+ T cells (shown as increase in the division index).
  • EpCAMxbl2 antibodies bs!gGl-bl2- FEAL/EpCAM-UBS54-FEAR, bs!gGl-bl2-FEAL/EpCAM-A37-FEAR, bsIgGl- bl2-FEAL/EpCAM-C52-FEAR did not affect CD4+ or CD8+ T-cell proliferation, as the division index was comparable to isotype control antibody IgGl- bl2-FEAL.
  • FIG. 7 shows that EpCAMx4-lBB bispecific antibodies bsIgGl-CD137-009-HC7LC2-FEAL/EpCAM-A37-FEAR and bs!gGl-CD137-009-HC7LC2- FEAL/EpCAM-323-A3-FEAR enhanced PBMC proliferation (shown as increase in the division index) in the presence of T84, DiFi, HPAF-II, NCI-N87, Caiu-3 or NCI-H747 tumor cells.
  • EpCAMxbl2 antibodies bs!gGl-bl2-FEAL/EpCAM-A37-FEAR and bs!gGl-bl2-FEAL/EpCAM-323-A3-FEAR did not affect proliferation in any of the PBMC-tumor cell co-cultures, as the division index was comparable to isotype control antibody IgGl-bl2- FEAL.
  • EpCAMx-4-lBB bsAb were able to increase proliferation of activated PBMC in all tumor cells lines tested, with no clear impact of EpCAM expression in the range tested here.
  • EpCAMx4-lBB bispecific antibodies either with an Fc-domain containing the FEA mutations in both HCs, or with an Fc-domain with the FEA mutations in one HC and the FER mutations in the other HC, to enhance proliferation of activated CD4+ and CD8+ T cells was compared (Figure 8).
  • EpCAMx4-lBB bispecific antibodies enhanced proliferation of activated PBMCs in co-cultures with tumor cells with a range of EpCAM expression levels, including proliferation of both CD4+ and CD8+ T cells.
  • the capacity to enhance T-cell proliferation was comparable for EpCAMx4-lBB bispecific antibodies that either contained the FEA inertness mutations in both HCs or that contained the FEA inertness mutations in one HC and the FER inertness mutations in the other HC.
  • Example 9 Ex vivo TIL expansion assay to determine the capacity of EpCAMx4-lBB bispecific antibodies to induce proliferation of tumor-infiltrating T cells and NK cells.
  • ex vivoTLL expansion assays were conducted using surgically resected primary tumor tissue from human non-small cell lung cancer patients.
  • Cryopreserved tumor tissue fragments were thawed and dissected into fragments of approximately 1 mm 3 .
  • the fragments were seeded in 24-well plates (Greiner, cat. no. 662160; 2 fragments per well, 12-16 wells per condition) in X-VIVOTM 15 medium (Lonza, cat. no. BE02-060Q) supplemented with 2% human serum albumin (CLS Behring, PZN-00504775), 1% penicillin/streptomycln (Thermo Fisher Scientific, cat. no. 15140-122), 1% amphotericin B (Thermo Fisher Scientific, cat. no.
  • interleukin-2 Proleukin-S, Novartis Pharma GmbH, PZN-02238131
  • Bispecific antibodies BsIgGl-CD137-009-HC7LC2-FEAL/EpCAM-323-A3-FEAR or BsIgGl- CD137-009-HC7LC2-FEAL/EpCAM-A37-FEAR
  • the ⁇ g/mL fragments were cultured for 3 days. Fresh culture medium containing IL-2 and antibodies (same concentrations as above) was added on day 3. During the remaining culture period, TIL growth and formation of TIL microclusters were regularly monitored through a microscope.
  • Example 10 Treatment of MC38 tumor-bearing mice to determine in vivo anti-tumor activity of an EpCAMx4-lBB bispecific antibody.
  • MC38_hEpCAM human EpCAM-transgenic mice bearing human EpCAM-overexpressing MC38 tumors.
  • MC38_hEpCAM cells were generated by retroviral transduction of human EpCAM into MC38 cells (Kerafast, cat. no. ENH204-FP).
  • mice Age and gender-matched mice were subcutaneously inoculated with 5xl0 5 MC38_hEpCAM cells in 100 pL PBS (Thermo Fisher Scientific, cat. no. 141-902-50). T umor growth was evaluated at least twice per week using a caliper and tumor volumes were calculated from caliper measurements as ([length] x [width] 2 ) I 2, where length is the longest tumor dimension and width is the longest tumor dimension perpendicular to the length.
  • mice When tumors reached a median size of approximately 30 mm 3 , treatment with BsIgG2amm-EpCAM-323-A3-AALT/m4-lBB-3H3- AAKR or the isotype control antibody IgG2amm-bl2-AAKR was commenced.
  • Mice were treated with 100 pg of the respective antibody intraperitoneally in 200 pL PBS on days 12, 17, 21, 24, 28, and 31 after tumor inoculation. Body weight of mice was measured three times per week. Individual animals were euthanized when the tumor volume exceeded 1,500 mm 3 or when mice reached other humane endpoints (e.g. body weight loss >20%, ulceration of tumors (>75%) or occurrence of clinical signs of illness).
  • EpCAMx4-lBB bispecific antibodies to enhance CD4+ and CD8+ T-cell proliferation and activation was assessed in in vitro co-cultures of EpCAM-expressing DiFi tumor cells and PBMCs, which were purified from healthy human donor buffy coats (Sanquin, Amsterdam, The Netherlands).
  • BV785-labeled anti-CD19 (clone SJ25C1, 1:80; BioLegend, cat. no. 363028), APC-eF780-labeled anti-CD4 (clone OKT4, 1:100; eBioscience, cat. no. 47-0048-42), AF700-labeled anti-CD8 (clone RPA-T8, 1:100; BioLegend, cat. no. 301028), and PE-Cy7-labeled anti-CD14 (clone: M5E2, 1:50; BD Biosciences, cat. no. 557742) antibodies in 50 pL FACS buffer at 4°C for 30 min protected from light.
  • CD4+ or CD8+ T cells were resuspended in 80 pL FACS buffer and were acquired on a BD FACSCelestaTM Cell Analyzer (BD Biosciences) and analyzed using FlowJo software.
  • PBMCs were gated for lymphocytes and single cells (based on FSC/SSC), viable cells (FVS510- population), T cells (CD14-CD19- population) and finally CD4+ or CD8+ T cells (CD4+CD8- or CD4-CD8+ populations, respectively).
  • CTV-stained cells were measured to assess CD4+ or CD8+ T-cell proliferation. The percentage divided cells were determined as the percentage of cells that had undergone cell division (based on CTV-dilution) out of the total CD4+ or CD8+ T-cell population. Expansion index values were calculated as follows:
  • BV785- labeled anti-CD19 (clone SJ25C1, 1:80; BioLegend, cat. no. 363028), APC-eF780-labeled anti-CD4 (clone OKT4, 1:100; eBioscience, cat. no. 47-0048-42), AF700-labeled anti-CD8 (clone RPA-T8, 1:100; BioLegend, cat. no. 301028), APC-labeled anti-4-lBB (clone: 4B4-1, 1: 40, Biolegend, cat. no. 309810), PE-labeled anti-CD25 (clone: M-A251, 1:200, Biolegend, cat. no.
  • CD4+ or CD8+ T cells were gated for lymphocytes and single cells (based on FSC/SSC), viable cells (FVS575- population), T cells (CD14-CD19- population) and finally CD4+ or CD8+ T cells (CD4+CD8- or CD4-CD8+ populations, respectively). CTV-stained cells were measured to assess CD4+ or CD8+ T-cell proliferation. As the expansion index could not be calculated for these experiments, the percentage of divided cells were reported instead and were determined as the percentage of cells that had undergone cell division (based on CTV-dilution) out of the total CD4+ or CD8+ T-celi population. % CD25+ or % 4-1BB+ cells or geomean fluorescent intensity (FI) of the CD25+ or 4-1BB+ population were determined within the CD4+ and CD8+ T-cell populations.
  • EpCAMx4-lBB bispecific antibodies to enhance CD4+ and CD8+ T-cell proliferation and activation was assessed in in vitro co-cultures of EpCAM-expressing DiFi tumor cells and PBMCs. Proliferation and activation of T cells treated with EpCAMx4-lBB bispecific antibodies was compared to EpCAM-specific and 4-lBB-specific antibodies (monovalent or bivalent, either alone or in combination).
  • Figure 11 shows that bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR enhanced proliferation of CTV-labeled CD4+ and CD8+ T cells compared to bivalent monoclonal antibodies IgGl-EpCAM-A37-FERL and IgGl-CD137-009- HC7LC2-FEAR (either alone or in combination), as shown by an increase in the expansion index. Across all PBMC donors tested, bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR consistently showed the highest increase in CD4+ and CD8+ T-cell proliferation (data not shown).
  • Figure 12 shows that bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEARdose-dependently enhanced proliferation of CTV-labeled CD4+ and CD8+ T cells, as shown by an increase in the percentage of divided cells.
  • the combination of monovalent EpCAM- and 4-lBB-specific antibodies bsIgGl-EpCAM-A37-FERL/bl2-FEAR and bsIgGl-bl2-FERL/CD137-HC7LC2-FEAR did not enhance CD4+ or CD8+ T-cell proliferation, as the percentage of divided cells was comparable to anti-CD3-stimulated PBMCs that were co-cultured with DiFi tumor cells without addition of antibody.
  • Figure 13 shows that bsIgGl-EpCAM-A37-FERiyCD137-009-HC7LC2-FEAR dose-dependently enhanced activation of CD4+ and CD8+ T cells, as shown by the increase in expression of the T-cell activation markers CD25 and 4- 1BB. Both the percentage of CD4+ and CD8+ T cells expressing CD25 and 4-1BB, as well as the geometric mean FI of the CD25+ or 4-1BB+ population were increased by bs!gGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR.
  • the combination of the monovalent EpCAM- and 4-lBB-specific antibodies bsIgGl-EpCAM-A37-FERL/bl2- FEAR and bsIgGl-bl2-FERL/CD137-HC7LC2-FEAR did not enhance activation of CD4+ and CD8+ T cells, as the percentage of cells expressing CD25 and 4-1BB as well as the geomean FI of the CD25+ or 4-1BB+ population were comparable to anti-CD3-stimulated PBMCs that were co-cultured with DiFi tumor cells without addition of antibody.
  • EpCAMx4-lBB bispecific antibody showed enhanced CD4+ and CD8+ T-cell proliferation and activation in PBMC-tumor cell co-cultures compared to the combination of bivalent or monovalent EpCAM- and 4- IBB-specific antibodies.
  • Example 12 Effect of EpCAMx4-lBB bispecific antibodies on T-cell proliferation and activation in PBMC - tumor cell co-cultures using cancer patient-derived PBMCs
  • EpCAMx4-lBB bispecific antibodies to enhance CD4+ and CD8+ T-cell proliferation and activation was assessed in in vitro co-cultures of EpCAM-expressing DiFi tumor cells and cancer patient-derived PBMCs (Discovery Life Sciences). These experiments were essentially performed as described in Example 8, with the following exceptions.
  • PBMCs were derived from a colorectal cancer patient receiving chemotherapy and other treatments and exhibited stable disease at the time of blood collection. PBMCs were labelled with 0.5 pM CellTraceTM Violet (CTV; 1: 10,000, ThermoFisher Scientific; cat. no. C34557) in PBS at 37°C for 20 min protected from light.
  • CTV CellTraceTM Violet
  • PE-labeled anti-CD25 (clone: M-A251, 1:200, Biolegend, cat. no. 356104) and PE-Cy7-labeled anti-CD14 (clone: M5E2, 1:50; BD Biosciences, cat. no. 557742) antibodies in 50 pL FACS buffer at 4°C for 30 min protected from light. After washing, the cells were resuspended in 80 pL FACS buffer and were acquired on a BD LSRFortessa FACS (BD Biosciences, USA) and analyzed using FlowJo software.
  • PBMCs were gated for lymphocytes and single cells (based on FSC/SSC), viable cells (FVS510- population), T cells (CD14-CD19- population) and finally CD4+ or CD8+ T cells (CD4+CD8- or CD4-CD8+ populations, respectively).
  • CTV-stained cells were measured to assess CD4+ or CD8+ T-cell proliferation.
  • the percentage divided cells were determined as the percentage of cells that had undergone cell division (based on CTV-dilution) out of the total CD4+ or CD8+ T-cell population.
  • % CD25+ or % 4-1 BB+ cells or geomean fluorescence intensity (FI) of the CD25+ or 4-1BB+ population were determined within the CD4+ and CD8+ T-cell populations.
  • EpCAMx4-lBB bispecific antibodies to enhance CD4+ and CD8+ T-cell proliferation and activation was assessed in in vitro co-cultures of EpCAM-expressing DiFi tumor cells and colorectal cancer patient-derived PBMCs and compared to the combination of monovalent EpCAM-specific and 4-lBB-specific antibodies.
  • Figure 14 shows that bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEARdose-dependently enhanced proliferation of CTV-labeled CD4+ and CD8+ T cells, as shown by an increase in the percentage of divided cells.
  • the combination of monovalent EpCAM- and 4-lBB-specific antibodies bsIgGl-EpCAM-A37-FERL/bl2-FEAR and bs!gGl-bl2-FERL/CD137-009-HC7LC2-FEAR did not enhance CD4+ or CD8+ T-cell proliferation at 3 pg/mL.
  • Figure 15 shows that bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR dose-dependently enhanced activation of CD4+ and CD8+ T cells, as shown by the increase in T-cell activation markers CD25 and 4-1BB. Both the percentage of CD4+ and CD8+ T cells expressing CD25 or 4-1BB, as well as the geometric mean FI in the CD25+ or 4-1BB+ population were increased by bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR.
  • EpCAMx4-lBB bispecific antibody showed enhanced CD4+ and CD8+ T-cell proliferation and activation in co-cultures of cancer patient-derived PBMCs and DiFi tumor cells compared to the combination of monovalent EpCAM- and 4-lBB-specific antibodies.
  • Example 13 In vitro killing assay to determine the capacity of EpCAMx4-lBB bispecific antibodies to enhance T-cell mediated cytotoxicity towards tumor cells
  • the effect of DuoBody EpCAMx4-lBB on the cytotoxic activity of human CD8 + T cells was investigated in co-culture with tumor cells as target cells.
  • Expression of the cytotoxicity-associated molecules Granzyme B (GzmB) and CD107a by the CD8 + T cells was assessed by flow cytometry.
  • Target cell killing was evaluated by real-time cell analysis using the xCELLigence system.
  • the human breast cancer cell line MDA-MB-231 (ATCC®, HTB-26TM) was stably transduced with the model antigen claudin-6 and with human EpCAM (MDA-MB-231_hCLDN6_hEpCAM cells) and served as a target cell line.
  • MDA-MB-231_hCLDN6_hEpCAM cells were seeded at 1.5 x 10 4 cells per well in 96-well flat-bottom plates (Greiner, Cat no. # 655180) and xCELLigence E-plates (Agilent, Cat no. # 05232368001) in 100 pL Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Cat no. # 31966-047) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, Cat no. # F7524). Cells were allowed to settle for 30 min at RT.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • the 96-well flat bottom plates (for flow cytometry analysis) and E-plates (for real-time cell analysis) were incubated for 1 d in the incubator or the xCELLigence real-time cell analysis instrument (Acea Biosciences), respectively (37°C, 5% CO2 in both cases).
  • CD8 + T cells were purified from PBMCs derived from HLA-A*02:01 positive healthy human donors by magnetic- activated cell sorting (MACS) using CD8 microbeads (Miltenyi Biotec, Cat no. # 130-045-201).
  • the purified CD8 + T cells were electroporated with RNA encoding the alpha and beta chains of a CLDN6-specific TCR (TCR#12a, TCR#12£; 10 pg RNA each) at 500 V, 3 ms and 1 pulse.
  • prewarmed IMDM GlutaMAX Life Technologies GmbH, Cat no. # 31980030
  • PHS One Lambda Inc., Cat no. # A25761
  • Electroporated T cells were transferred to a 6-well plate and incubated overnight (37°C, 5% CO2). Electroporation efficiency was >85% as determined by flow cytometry on the next day.
  • CD8 + T cells were resuspended in DMEM with 10% FBS.
  • the CD8 + T cells were added to the wells (both 96-well plate and E-plate) containing the MDA-MB-231_hCLDN6_hEpCAM cells seeded on the previous day. 7.5 x 10 4 CD8 + T cells were added per well, resulting in an effectontarget ratio of 5: 1.
  • MDA-MB-231_hCLDN6_hEpCAM cells were cultured without the addition of T cells.
  • bsIgGl-EpCAM-A37- FERL/CD137-009-HC7LC2-FEAR or control antibodies were diluted in DMEM with 10% FBS and added to the wells.
  • Fc-inert monovalent bispecific antibodies containing one EpCAM-specific Fab arm or one 4-lBB-specific Fab arm paired with one non-binding Fab arm bsIgGl-EpCAM-A37-FERL/bl2-FEAR and bsIgGl-bl2-FERLxCD137- 009-HC7LC2-FEAR, respectively
  • Fc-inert non-binding antibody bsIgGl-bl2-FERL/bl2-FEAR were used.
  • bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR was tested over a final concentration range of 0.0032 to 3.2 ⁇ g/mL, whereas the control antibodies were tested at a single final concentration of 3.2 pg/mL.
  • the final volume was 200 pL/well.
  • a fluorochrome-labeled anti-CD107a antibody (Biolegend, Cat no. # 328611; final dilution 1:2,500) was added to the wells of the 96-well plate but not to the E-plate.
  • Cells in the 96-well plate were cultured in an incubator (37°C, 5% COz) for 2 d.
  • 20 pL of Golgi-Plug (BD Biosciences, Cat no. # 555029, final dilution 1:1,100) was added.
  • the cells were stained with fluorochrome-labeled anti-CD8o antibody (BD Biosciences, Cat no.
  • Cells in the E-plate were cultured in an xCELLigence real-time cell analysis instrument for 5 to 6 d without disturbance, with impedance measurements at 2 to 3-hour intervals. The impedance measurements were expressed as cell index values. Cell indices correlate with the total area of adherent tumor-cell monolayers. Therefore, lower cell indices in T-cell/tumor-cell co-cultures as compared to tumor cells only indicate tumor cell killing. Cell indices for each condition were normalized to the time point at which T-cell/tumor-cell co-cultures were initiated. Normalized cell index curves over time were visualized using GraphPad Prism.
  • AUC area under the curve
  • Figure 16A shows that CD8 + T cells upregulated CD107a and GzmB expression when co-cultured with tumor cells expressing their cognate antigen.
  • Figure 16 shows that bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR dose- dependently enhanced CD107a and GzmB expression, whereas the monovalent control antibodies bsIgGl-EpCAM- A37-FERL/bl2-FEAR and bsIgGl-bl2-FERL/CD137-009-HC7LC2-FEAR did not increase expression levels over the non-binding control antibody bsIgGl-bl2-FERL/bl2-FEAR.
  • bsIgGl- EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR concentrations of 0.2 and 0.8 the increase in CD107a an ⁇ dg/mL GzmB expression levels was statistically significant compared to both monovalent control antibodies.
  • Figure 17 shows that Antigen-stimulated CD8 + T cells exhibited cytotoxic activity against MDA-MB- 231_hCLDN6_hEpCAM cells, as indicated by lower cell index values of CD8 + T-cell/tumor-cell co-cultures treated with the non-binding control antibody bsIgGl-bl2-FERL/bl2-FEAR as compared to tumor cells cultured alone.
  • bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR dose-dependently enhanced CD8 + T-cell mediated cytotoxic activity compared to bsIgG-bl2-FERL/bl2-FEAR, with the maximum effect observed at concentrations >0.2 pg/mL.
  • the monovalent control antibodies bsIgGl-EpCAM-A37-FERL/bl2-FEAR and bsIgGl-bl2-FERL/CD137- 009-HC7LC2-FEAR had no considerable effect on cytotoxic activity.
  • the increase ⁇ ign/m cyLtotoxic activity was statistically significant compared to both monovalent control antibodies.
  • ECso values for CD107a and GzmB expression by CDfF T cells were not determined, because no sigmoidal dose-response curve was obtained.
  • EC50 half-maximal effective concentration
  • GMFI geometric mean fluorescence intensity
  • GzmB granzyme B
  • N/A not applicable
  • SD standard deviation.
  • Binding of EpCAMx4-lBB bispecific antibodies with various Fc-inertness mutations (FER/FEA, FEA/FEA, FER/FER) to immobilized human recombinant FcyR variants (FcyRIa, FcyRIIa, FcyRIIb, and FcyRIIIa) was analyzed by SPR using a Biacore 8K SPR system.
  • Biacore Series S Sensor Chips CM5 (Cytiva, cat. no. 29104988) were covalently coated with anti-histidine (His) antibody using amine coupling and His-capture kits (Cytiva, cat. no. BR100050 and cat. no. 29234602) according to the manufacturer's instructions.
  • antibodies (IgGl-EpCAM-A37-FERL, IgGl-CD137-009-HC7LC2-FEAR, bs!gGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR, bs!gGl-CD137-009-HC7LC2-FEAL/EpCAM-A37-FEAR, bs!gGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FERR, IgGl-bl2) were injected to generate binding curves, using antibody ranges as indicated in Table 9. Each sample that was analyzed on a surface with captured FcyR (active surface) was also analyzed on a parallel flow cell without captured FcyR (reference surface), which was used for background correction.
  • the surface was regenerated using 10 mN Glycine-HCI pH 1.5 (Cytiva, cat. no. BR100354). Sensorgrams were generated using Biacore Insight Evaluation software (Cytiva) and a four-parameter logistic fit was applied on endpoint measurements (binding plateau versus post-capture baseline) to calculate relative binding of the EpCAMx4-lBB bispecific antibodies against the IgGl-bl2 antibody that has a wild-type Fc region.
  • FcRn neonatal Fc receptor
  • the antibody concentration curve for IgGl-bl2 was 0-100 nM, while for bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR, IgGl-EpCAM-A37-FERL and IgGl-CD137-009-HC7LC2-FEAR it was 0-500 nM. Experiments were performed three times at pH 6.0 (under acidic conditions, at which IgG binds to FcRn) and two times at pH 7.4 (binding to IgG is not expected at this pH).
  • Figure 18 shows that no binding of bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR, bsIgGl-CD137-009- HC7LC2-FEAL/EpCAM-A37-FEAR, bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FERR to FcyRIa, FcyRIIa (either the H131 or R131 variant), FcyRIIb and FcyRIIIa (either the F158 or V158 variant) was detected.
  • monoclonal antibodies IgGl-EpCAM-A37-FERL and IgGl-CD137-009-HC7LC2-FEAR also did not show any binding to these FcyRs.
  • the positive control antibody IgGl-bl2 showed dose-dependent binding to all tested FcyRs.
  • Table 10 shows that binding of bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR, IgGl-EpCAM-A37-FERL and IgGl-CD137-009-HC7LC2-FEAR to FcRn was retained with mean binding affinities (Ab) of 312 nM, 230 nM and 375 nM, respectively.
  • EpCAMx4-lBB bispecific antibodies containing different inertness mutations (FER/FEA, FEA/FEA or FER/FER), showed binding to FcyRs by SPR, while binding to FcRn was retained.
  • a toxicology assessment for bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR was performed in cynomolgus monkeys. Toxicity was assessed by examining clinical signs, body weight, ECGs, toxicokinetics, immunophenotyping, clinical chemistry parameters (including lipase and amylase), gross necropsy, and histopathology. Preliminary results indicate that doses of up to 50 mg/kg were well tolerated when bsIgGl-EpCAM- A37-FERL/CD137-009-HC7LC2-FEAR was given intravenously up to 5 times in weekly intervals.
  • Example 16 Effect of an EpCAMx4-lBB bispecific antibody combined with anti-PD-1 antibodies on IFNy secretion in PBMC-tumor cell cocultures
  • PBMCs peripheral blood mononuclear cells
  • EpCAM-expressing tumor cell lines DiFi or CAL27 were seeded at a density of 6,250 cells/well into polystyrene 96-well flat-bottom plates (Greiner bio-one, cat. no. 655180) in T-cell medium (Iscove's Modified Dulbecco's Medium [IMDM; Lonza, cat. no. 12-722F] supplemented with 5% Human Serum [Sanquin Blood Bank, cat. no.
  • PBMC-tumor cell cocultures were incubated with bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR (0.00137-3.0 pg/mL), pembrolizumab (0.01 or 1 ; Merc ⁇ kg/ SmhLarp & Dohme, cat. no. AE22003937; WO 2008/156712), IgGl-PDl (0.01 or 1 ⁇ g/)m, oLr combinations of bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR and pembrolizumab or IgGl-PDl at 37°C for 96 h.
  • Predicted synergy effect was defined as the excess of observed effect over expected effect (the maximum of the single agent treatment values) as calculated by the highest single agent (HSA) reference model (Berenbaum M.C., 1989, Pharmacol Rev 41:93-141), with synergy scores of >10 suggesting potentiation of the response in the combination compared to the single agent treatment.
  • bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR dose-dependently increased secretion of IFNy compared to the combination of monovalent EpCAM -specific and 4- IBB-specific control antibodies (bsIgGl-EpCAM-A37-FERL/bl2-FEAR and bsIgGl-bl2-FERL/CD137-009-HC7LC2- FEAR; Figure 19).
  • Pembrolizumab increased secretion of IFNy compared to the IgG4 isotype control.
  • IgGl-PDl increased secretion of IFNy compared to the IgGl-bl2-FERR isotype control ( Figure 20).
  • Table 12 HSA synergy analysis oflFNy secretion induced by the combination of bs!gGl-EpCAM-A37-FERL/CD137- 009-HC7LC2-FEAR and pembroHzumab in PBMC-CAL27 tumor cell cocultures
  • Synergy scores >10 are indicated in gray shade.
  • Example 17 Ex r/VoTIL expansion assay to determine the capacity of combining an EpCAMx4-lBB bispecific antibody with an anti-PD-(L)l antibody to induce proliferation of tumor-infiltrating T cells
  • ex wVoTIL assays were conducted using surgically resected primary tumor tissue from colorectal cancer (CRC) patients.
  • Freshly resected tumor tissue was dissected into fragments of approximately 1 mm 3 .
  • the fragments were seeded in 24-well plates in culture medium containing 50 U/mL recombinant human IL-2 as described in Example 9.
  • ag/ cmomL bination of bsIgGl-EpCAM-A37-FERL/CD137-009-HC7LC2-FEAR and an anti-PD-1 antibody were added to the wells.
  • the fragments were cultured for 3 days. Fresh culture medium containing IL-2 and antibodies (same concentrations as above) was added on day 3. During the remaining culture period, TIL expansion and formation of TIL microclusters were regularly monitored by microscopic inspection. On Days 6 and 8, fresh culture medium containing IL-2 (33 U/mL) was added. On day 10, cells were pooled by transferring contents of two identically treated wells of the original 24-well plate into one well of a fresh 6-well plate (Greiner, cat. no. 657185). Fresh culture medium containing IL-2 (15 U/mL) was added. On day 14, cells were harvested for flow cytometry analysis.

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Abstract

La présente invention concerne l'utilisation d'un agent de liaison qui se lie à EpCAM et à CD137 en combinaison avec un antagoniste de liaison à l'axe PD-1 dans le traitement ou la prévention d'une tumeur ou d'un cancer, ou pour empêcher la progression d'une tumeur ou d'un cancer.
PCT/EP2023/075492 2023-09-15 2023-09-15 Procédés de traitement faisant appel à des agents se liant à epcam et cd137 en combinaison avec des antagonistes de liaison à l'axe pd-1 Pending WO2025056180A1 (fr)

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PCT/EP2024/075677 WO2025056778A1 (fr) 2023-09-15 2024-09-13 Procédés de traitement utilisant des agents se liant à epcam et cd137 en combinaison avec des antagonistes de liaison à l'axe pd-1
ARP240102454A AR133823A1 (es) 2023-09-15 2024-09-13 Métodos de tratamiento que utilizan agentes de unión a epcam y cd137 en combinación con antagonistas de unión al eje pd-1
TW113135136A TW202528358A (zh) 2023-09-15 2024-09-16 利用結合至epcam及cd137之藥劑與pd-1軸結合拮抗劑組合的治療方法

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