WO2026003224A2 - Protéines bispécifiques de liaison à l'antigène (abp) ciblant des molécules de point de contrôle immunitaire et à la fois la sous-famille b1 des récepteurs de type immunoglobuline leucocytaire (lilrb1) et lilrb2; combinaisons et utilisations associées - Google Patents

Protéines bispécifiques de liaison à l'antigène (abp) ciblant des molécules de point de contrôle immunitaire et à la fois la sous-famille b1 des récepteurs de type immunoglobuline leucocytaire (lilrb1) et lilrb2; combinaisons et utilisations associées

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WO2026003224A2
WO2026003224A2 PCT/EP2025/068165 EP2025068165W WO2026003224A2 WO 2026003224 A2 WO2026003224 A2 WO 2026003224A2 EP 2025068165 W EP2025068165 W EP 2025068165W WO 2026003224 A2 WO2026003224 A2 WO 2026003224A2
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antibody
abp
binding
sequence
antigen
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WO2026003224A3 (fr
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Simone FRIEDRICH
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IOmx Therapeutics AG
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IOmx Therapeutics AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • 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
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], 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
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/524—CH2 domain
    • 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
    • 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

  • an LILRB1 and/or ULRB2 antigen binding protein such as an antibody binding to both ULRB1 and/or LILRB2
  • cancerous cells often exploit immune- checkpoints to evade a patient's immune system, such as by preventing immune-recognition or down-regulating a tumour-specific cytotoxic T cell (CTL) response, thereby generating resistance against an immune response (Rabinovich et al 2007, Annu Rev Immunol 25:267; Zitvogel et al 2006, Nat Rev Immunol 6:715).
  • CTL cytotoxic T cell
  • the human leukocyte immunoglobulin-like receptor also known as immunoglobulin-like transcript (ILT) family belongs to the superfamily of paired receptors that have the potential to transmit stimulatory or inhibitory signals according to the presence or absence of tyrosine-based signalling motifs in their cytoplasmic tail.
  • Human ULRs consist of six stimulatory receptors (ULRA1-6) and five inhibitory receptors (ULRBI -5). ULRs are predominately expressed on myeloid and lymphoid cells and some non-immune cells, and the expression patterns are different from receptor to receptor. Polymorphism and copy-number variation contribute to diversity within humans.
  • Receptor engagement results in intracellular phosphorylation of the tyrosine-based motifs within the receptors (ULRB) or on associated adaptor molecules (LILRA).
  • Downstream signalling events can be mediated by phosphatases, such as SHP1, SHP2 and SHIP.
  • LILR activity can result in the upregulation or downregulation of both innate and adaptive immune functions with a range of effects on different cell types.
  • Certain ULRs also play regulatory roles in neuronal activity and osteoclast development.
  • ULRBI is broadly expressed on myeloid cells, as well as B cells and subsets of T cells and natural killer (NK) cells.
  • LILRB2-5 are more restricted to myeloid cells and dendritic cells (DCs).
  • DCs dendritic cells
  • Some of the ligands and signaling pathways for ULRBs have been identified.
  • LILRB1 and ULRB2 are the best characterized receptors, and both bind to classical (HLA-A, HLA-B and HLA-C) and non-classical (HLA-E, HLA-F, HLA-G and HLA-H) MHC class I or HLA class I molecules, as well as to members of the angiopoietin-like protein family.
  • LILRBs Because the immune-suppressive function of ULRBs is similar to that of the classical immune checkpoint proteins, CTLA-4 and PD-1, the interaction between LILRBs and ligands is proposed to serve as immune checkpoints. For example, engagement of LILRB1 and LILRB2 by HLA-G on cancer cells inhibits immune cell activation and generates regulatory T cells (Tregs) and suppressive antigen presenting cells (APCs), which can indirectly support tumour development. Further, the interaction of P2-microglobulin (P2M)-associated MHC class I on cancer cells with LILRB2 on macrophages leads to loss of immune surveillance. Whether the interaction between LILRB2 and ligand also functions as a phagocytosis checkpoint is unknown.
  • P2M P2-microglobulin
  • ULRBs may also represent targets for induction of transplantation tolerance to prevent allograft rejection.
  • LILRBs (especially ULRB2 and LILRB4) are critical for induction of the tolerogenic phenotype of APCs and initiation of the T cell suppression cascade that results in immune tolerance.
  • LILRB1 and ULRB2 can also mediate graft tolerance by binding to HLA-G.
  • ULRBs are expressed by cancer cells and may support malignant transformation and relapse, as well as the activity of cancer stem cells.
  • PD-1 Programmed cell death protein 1
  • CD28 is an inhibitory member of the CD28 family of receptors, that also includes CD28, CTLA-4, ICOS and BTLA.
  • PD-1 is a cell surface receptor and is expressed on activated B cells, T cells, and myeloid cells (Okazaki et al (2002) Curr. Opin. Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol 170:711-8).
  • PD-1 is a monomeric type 1 transmembrane protein, consisting of one immunoglobulin variable-like extracellular domain and a cytoplasmic domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM).
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • ITMS immunoreceptor tyrosine-based switch motif
  • Activated T cells transiently express PDI, but sustained hyperexpression of PDI and its ligand PDL1 promote immune exhaustion, leading to persistence of viral infections, tumor evasion, increased infections and mortality.
  • PD-1 expression is induced by antigen recognition via the T-cell receptor and its expression is maintained primarily through continuous T-cell receptor signaling.
  • PD-1 locus After prolonged antigen exposure, the PD-1 locus fails to be remethylated, which promotes continuous hyperexpression. Blocking the PDI pathway can restore the exhausted T-cell functionality in cancer and chronic viral infections (Sheridan, Nature Biotechnology 30 (2012), 729-730).
  • Monoclonal antibodies to PD-1 have been described, for example, in WO 2003/042402, WO 2004/004771, WO 2004/056875, WO 2004/072286, WO 2004/087196, WO 2006/121168, WO 2006/133396, WO 2007/005874, WO 2008/083174, WO 2008/156712, WO 2009/024531, WO 2009/014708, WO
  • WO 2020/023268 discloses combination therapies comprising a Programmed Death 1 receptor (PD-1) pathway inhibitor, and a Leukocyte Immunoglobulin Like Receptor B (LILRB) signaling inhibitor, and the use of the combination therapies for the treatment of cancer. Further disclosed is the treatment of cancer patients who are refractory to monotherapy with a PD-1 pathway inhibitor.
  • PD-1 Programmed Death 1 receptor
  • LILRB Leukocyte Immunoglobulin Like Receptor B
  • WO 2020/023268 exemplifies that anti-PD- 1 and anti-ULRBl synergistically activated T cells in mixed lymphocyte reaction. Blocking both pathways significantly enhanced T cell activity and IFNy production than blocking each pathway alone.
  • the present invention seeks to provide, in particular, novel therapeutic approaches and methods involving novel compounds; for example, bispecific compounds and bispecific ABPs that reduce the immune-suppressive function of ULRB receptors while not inhibiting the immune-stimulatory function of any of the ULRA receptors, and to combine them with inhibitors of immune checkpoints, such as in particular PD-1 / PD-L1.
  • the invention seeks to provide novel strategies to diagnose, prognose and/or monitor immune cells on the basis of LILRB expression. Accordingly, it is an object of the present invention to provide alternative, improved, simpler, cheaper and/or integrated means or methods that address one or more of these or other problems. Such an object underlying the present invention is solved by the subject matter as disclosed or defined anywhere herein, for example by the subject matter of the itemized embodiments and/or attached claims.
  • the invention is grounded by the surprising finding that certain antigen binding proteins, such as antibodies, of the invention bind selectively and specifically to both ULRB1 and LILRB2, while not binding, or binding relatively less selective and less specific, to any of the ULRA receptors.
  • inventive bispecific molecules are used in the present invention in bispecific and multispecific formats in combination with immune checkpoint inhibitors.
  • the antibodies of the invention maintain cross-specificity with respect to targets ULRB1 and ULRB2 while binding significantly less to the off-target proteins ULRA1 and LILRA3. Maintaining cross specific binding only with respect to the LILRB on-target proteins without off-target binding to ULRAs is one of multiple surprising features of the antibodies of the present invention.
  • the invention relates to a bispecific antigen binding protein (ABP) which with a first antigen binding site specifically binds to LJLRBl and/or LLLRB2 protein (eg to the extra cellular domain (ECD) of LILRB1 and/or LLLRB2 protein) and, optionally, wherein the ABP is able to inhibit the binding of a ligand of LJLRBl and/or LLLRB2 protein or a variant thereof to LILRB1 and/or LLLRB2 protein or a variant thereof, and wherein the bispecific ABP with a second antigen binding site binds to (and preferably inhibits) an immune checkpoint, such as PD-1 / PD-L1.
  • the ABP or biABP, with its first antigen binding site does not specifically binding to, or binds significantly less to, leukocyte immunoglobulin-
  • the invention in a second aspect, relates to a bispecific ABP comprising a first antigen binding site which competes with the first antigen binding site of a bispecific ABP of a first aspect for a binding to LILRB1 and/or LILRB2 protein (eg to the ECD of LJLRBl and/or LLLRB2 protein), wherein the bispecific ABP of the second aspect comprises a second antigen binding site which binds to (and preferably inhibits) an immune checkpoint, such as PD-1 / PD-L1.
  • the invention relates to a bispecific ABP which binds to the same epitope as an bispecific ABP of a first aspect, with regard to their first antigen binding site.
  • the invention in a third aspect, relates to a nucleic acid encoding for a bispecific ABP of the invention or of components thereof, and in related aspects, the invention relates to a nucleic acid construct (NAC) comprising such a nucleic acid, and relates to a host cell comprising a nucleic acid or NAC of the invention.
  • NAC nucleic acid construct
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a bispecific ABP, nucleic acid, NAC or host cell of the invention, or comprising a compound that is a modulator of the expression, function, activity and/or stability of leukocyte immunoglobulin-like receptor subfamily Bl (LJLRBl) and/or LLLRB2 type proteins, and a pharmaceutically acceptable carrier, stabiliser and/or excipient.
  • LJLRBl leukocyte immunoglobulin-like receptor subfamily Bl
  • LLLRB2 LLLRB2
  • the invention relates to a method for the treatment of certain diseases, disorders or conditions in a subject by administering a product to the subject, wherein the product is selected from the list consisting of a bispecific ABP, nucleic acid, NAC and host cell of the invention, or is a compound that is a modulator of the expression, function, activity and/or stability of LJLRBl and/or LLLRB2.
  • the invention relates to a product for use in medicine, and relates to the use of a product for the manufacture of a medicament, wherein the product is selected from the list consisting of an ABP, nucleic acid, NAC or host cell of the invention, or is a compound that is modulator of the expression, function, activity and/or stability of LJLRBl and/or LLLRB2.
  • the invention also relates in other aspects to various methods to produce a recombinant cell line or ABP of the invention, a hybridoma or host cell capable of producing an ABP of the present invention, as well as relating to various determination and/or diagnostic methods or uses, and to kits useful for such determination and/or diagnostic methods, as well as to various methods for identifying and/or charactering compounds, such as those suitable for use in medicine.
  • Figure 3 shows inhibition of the interaction of (A) ULRB1 or (B) ULRB2 and their ligand HLA-G using antibody of the invention clone A-001, and maturated variants thereof.
  • Y-axes Residual Binding (%)
  • Figure 4 shows inhibition of the interaction of (A) ULRB1 or (B) ULRB2 and their ligand HLA-G using antibody of the invention clone A-003 and maturated variants of thereof.
  • Y-axes Residual Binding (%)
  • Figure 5 shows inhibition of the interaction of ULRB1 or ULRB2 and their ligand HLA-G using reference antibodies.
  • A shows inhibition of ULRB1 and HLA-G;
  • B shows inhibition of LILRB2 and HLA-G.
  • Figure 6 shows effects of anti-ULRB2/l blockade on (A - C) polarization of human M2 macrophages in a functional myeloid assay and (D - H) on polarization of human M2 macrophages and cytotoxic T cells in a myeloid suppression assay.
  • Figure 9 depicts BU measurements showing displacement of ULRB2 from HLA-G interaction site and inhibition of new LILRB2 and HLA-G complex formation using antigen binding fragments obtained from antibodies of the invention (A) A-010, (B) A-045, (C) A-047 , (D) A-048, antigen binding fragment obtained from (E) reference molecule Ref062 [NGM707, cross-specific] or (F) assay buffer.
  • C - E show cytokine secretion of antibody treated Ml- like macrophages in the presence of recombinant LILRA3 for GM-CSF (C), IFNg (D) and IL-9 (E).
  • FIG 19 depicts an overview of the construct design of the respective antibody formats including (A) stabilizing VH/VL charge pairs (VL: Q38E/K and VH: Q39E/K), (B) stabilizing VH/VL disulphide bridges (VL: G100C and VH: G44C), (C) combination of VH/VL charge pairs and disulfide bridges and (D) CrossMab2 including charge pair in CH1-CL for stabilization.
  • VH/VL charge pairs VL: Q38E/K and VH: Q39E/K
  • VL stabilizing VH/VL disulphide bridges
  • VL: G100C and VH: G44C stabilizing VH/VL disulphide bridges
  • C combination of VH/VL charge pairs and disulfide bridges
  • D CrossMab2 including charge pair in CH1-CL for stabilization.
  • the ABP or biABP of the invention is not an antibody as disclosed as ADA-011 in WO 2023/225626, more preferably is specifically not an antibody comprising the heavy chain CDR1 to CDR3 domain sequences of ADA-011 as disclosed in WO 2023/225626, and/or is not comprising the light chain CDR1 to CDR3 domain sequences of ADA-011 as disclosed in WO 2023/225626.
  • Anti-PD-1 antibodies are preferably PD-1 inhibitors.
  • PD-1 inhibitors include nivolumab and pembrolizumab.
  • Nivolumab, (OPDIVOTM ) is also known as iMDX- 1106, MDX-1106-04, ONO-4538, or BMS-936558 and has a CAS Registry Number: of 946414-94-4.
  • Nivolumab is a fully human IgG4 monoclonal antibody which specifically blocks PD-1.
  • Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in US 8,008,449 and W02006/121168.
  • Pembrolizumab (KEYTRUDATM) (formerly lambrolizumab), also known as Merck 3745, MK-3475 or SCH-900475, is a humanized IgG4 monoclonal antibody that binds to PD-1.
  • Pembrolizumab is disclosed in Hamid, O. et al., New England Journal of Medicine, 2013, 369(2): 134-44; WO2009/114335; and US 8,354,509. Additional anti-PD-1 antibodies also include pidilizumab (CT-011) and AMP-224. Other anti- PD-1 antibodies are disclosed in US 8,609,089; US 2010028330; and/or US 20120114649.
  • An "antigen binding protein” means a protein that specifically binds to a target antigen, such as to one or more epitope(s) displayed by or present on a target antigen.
  • the antigen of the ABPs or biABPs of the invention is ULRB1 and/or ULRB2; and the ABP or biABP can, optionally bind to one or more extracellular domains of said LILRB1 and/or ULRB2 (such as the epitope(s) can be displayed by or present on one or more extracellular domains of said LILRB1 and/or ULRB2).
  • an antigen binding protein is an antibody (or a fragment thereof), however other forms of antigen binding protein are also envisioned by the invention.
  • the ABP or biABP may be another (non-antibody) receptor protein derived from small and robust non-immunoglobulin "scaffolds", such as those equipped with binding functions for example by using methods of combinatorial protein design (Gebauer 8i Skerra, 2009; Curr Opin Chem Biol, 13:245).
  • non-antibody ABPs or biABPs include: Affibody molecules based on the Z domain of Protein A (Nygren, 2008; FEBS J 275:2668); Affilins based on gamma-B crystalline and/or ubiquitin (Ebersbach et al, 2007; J Mo Biol, 372: 172); Affimers based on cystatin (Johnson et al, 2012; Anal Chem 84:6553); Affitins based on Sac7d from Sulfolobus acidcaldarius (Krehenbrink et al, 2008; J Mol Biol 383: 1058); Alphabodies based on a triple helix coiled coil (Desmet et al, 2014; Nature Comms 5:5237); Anticalins based on lipocalins (Skerra, 2008; FEBS J 275:2677); Avimers based on A domains of various membrane receptors (Silver
  • an ABP or biABP of the present invention that specifically binds ULRB1 and/or LILRB2 (and does not bind, or binds with significantly less affinity, ULRA1 and/or ULRA3)
  • such an ABP or biABP is not a protein being a natural ligand of LILRB1 and/or ULRB2 (in particular, a ligand protein having more than 70%, 80% or 90% sequence identify to the amino acid sequence of HLA-G).
  • epitope includes any determinant capable of being bound by an antigen binding protein, such as an antibody.
  • An epitope is a region of an antigen that is bound by an antigen binding protein that targets that antigen, and when the antigen is a protein, includes specific amino acids that bind the antigen binding protein (such as via an antigen binding domain of said protein).
  • Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three- dimensional structural characteristics, and/or specific charge characteristics.
  • antigen binding proteins specific for a particular target antigen will preferentially recognise an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.
  • extracellular domain refers to the region or regions of the protein which are exposed to the extracellular space and which are typically responsible for ligand binding.
  • Immunoglobulin (Ig) superfamily genes typically have an Immunoglobulin-like ECD, such as a Ig-like C2-type domain.
  • An antigen binding protein is "specific" when it binds to one antigen (such as ULRBl and/or LILRB2; eg human LILRB1 and/or ULRB2, orthologues and other variants thereof) more preferentially (eg, more strongly or more extensively) than it binds to another antigen, preferably an ULRA protein (such as LILRA1 and LILRA3).
  • one antigen such as ULRBl and/or LILRB2; eg human LILRB1 and/or ULRB2, orthologues and other variants thereof
  • an ULRA protein such as LILRA1 and LILRA3
  • ABP or biABP specifically binds
  • the desired antigen eg LILRB1 and/or ULRB2, in particular an ECD of LJLRBl and/or LILRB2
  • other proteins or other molecules
  • LILRB1 and/or ULRB2 in particular an ECD of LJLRBl and/or LILRB2
  • ULRBA leucocyte immunoglobulin-like subfamily A
  • LILRB1 and/or LILRB2 is at least 2-fold, 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, at least 1000-fold, at least 2000-fold, at least 5000-fold, at least 10000-fold, at least 10 5 -fold or even at least 10 5 -fold, most preferably at least 2-fold, compared to its affinity to the other targets (e.g. unrelated proteins such as mouse or human Fc domain, or streptavidin).
  • targets e.g. unrelated proteins such as mouse or human Fc domain, or streptavidin.
  • Leukocyte Ig-like receptors are a family of immunoreceptors expressed predominantly on monocytes and B cells and at lower levels on dendritic cells and natural killer (NK) cells. All members of LIR subfamily B, such as ULRB2, contain a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and have an inhibitory function. Upon engagement of members of LIR subfamily B by MHC class I or other ligands and tyrosine phosphorylation of the ITIM, intracellular protein-tyrosine phosphatases, such as SHP1 (PTPN6), are recruited and an inhibitory signal cascade ensues.
  • ITIM cytoplasmic immunoreceptor tyrosine-based inhibitory motif
  • UR subfamily A e.g., ULRA1
  • ULRA3 One member of subfamily A, ULRA3, lacks a transmembrane region and is presumed to be a soluble receptor (summary by Borges et al., 1997).
  • the human LILRB1 gene is located at chromosomal position 19ql3.42, and has orthologues (eg, is conserved) in many species.
  • the term LILRB1 in some embodiments of the invention may also pertain to variants of the human ULRBl protein having an amino acid sequence that is substantially identical to, or of at least 70%, 75% or 80%, preferably 85%, more preferably at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (such as at least 90% or 95% sequence identity) to, the amino acid sequence shown in any of SEQ ID NOs.
  • ULRBl can mean, as applicable to the context (if not more specifically indicated), an ULRBl protein (such as one described above) or an mRNA molecule encoding such an ULRBl protein.
  • the human LILRB2 gene is located at chromosomal position 19ql3.42, and has orthologues (eg, is conserved) in many species.
  • LILRB2 in some embodiments of the invention may also pertain to variants of the human ULRB2 protein having an amino acid sequence that is substantially identical to, or of at least 70%, 75% or 80%, preferably 85%, more preferably at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity (such as at least 90% or 95% sequence identity) to, the amino acid sequence shown in any of SEQ ID NOs.
  • ULRB2 can mean, as applicable to the context (if not more specifically indicated), an ULRB2 protein (such as one described above) or an mRNA molecule encoding such an ULRB2 protein.
  • an ABP or biABP of the invention that binds to the ECD of human LJLRB1 and/or ULRB2 protein is cross reactive to the ECD of an orthologous protein, such as cross reactive to the ECD of cynomolgus LJLRB1 and/or LILRB2 protein and/or to the ECD of mouse LJLRB1 and/or LILRB2 protein and/or to the ECD of rat LJLRB1 and/or ULRB2 protein.
  • orthologue as used herein means a variant that descends from the same ancestral gene but which is present in another organism due to a speciation event. Orthologues of LJLRB1 and/or LILRB2 are typically expected to retain the same function as (or have a similar function to) human LJLRB1 and/or ULRB2.
  • variant as used herein in the context of a protein means any natural or non-natural version of such protein which comprises one or more amino acid mutations compared to the reference protein, but which shares significant amino acid sequence identity with the reference protein, e.g. at least 70% or 75% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity and most preferably at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity.
  • the variant of the protein possesses and/or maintains at least one function/activity that is the same, essentially the same or similar as the reference protein.
  • Variants of LILRB1 and/or LILRB2 may include orthologues to and natural variants of human LJLRB1 and/or LILRB2.
  • a "functional variant" of LJLRB1 and/or ULRB2 (such as a functional fragment of an LJLRB1 and/or LILRB2 protein) is a variant of the protein of LJLRB1 and/or LILRB2 that provides, possesses and/or maintains one or more of the herein described functions/activities of the non-variant protein of LJLRB1 and/or LILRB2.
  • such functional variant may bind HLA-G protein and/or to suppress T cell (or other immune cell) function/activity as LILRB1 and/or ULRB2 protein, such as having the same, essentially the same or similar specificity and/or function as a receptor as LILRB1 and/or LILRB2 protein.
  • such a functional variant may possess other activities than those possessed by the non-variant ULRB1 and/or LILRB2 protein, as long as, preferably, it provides, possesses and/or maintains at least one function/activity that is the same, essentially the same or similar as LILRB1 and/or LILRB2 protein.
  • a functional variant of ULRB1 and/or ULRB2 may act as an immune checkpoint inhibitor, such as by inhibiting one or more cell-based immune response(s) to a tumour or cancer cell that expresses such functional variant.
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences.
  • Percent identity means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (i.e., an "algorithm”). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.
  • the sequences being compared are typically aligned in a way that gives the largest match between the sequences.
  • One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et aL, 1984, NucL Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI).
  • GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined.
  • the sequences are aligned for optimal matching of their respective amino acid or nucleotide (the "matched span", as determined by the algorithm).
  • a gap opening penalty (which is calculated as 3x the average diagonal, wherein the "average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.
  • a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) may also be used by the algorithm.
  • a preferred method of determining similarity between a protein or nucleic acid and (or between) human ULRB1 and/or ULRB2, a paralogue, orthologue or other variant thereof, is that provided by the Blast searches supported at Uniprot supra (e.g., http://www.uniprot.org/uniprot/Q8NHL6 for ULRB1 and http://www.uniprot.org/uniprot/Q8N423 for LILRB2); in particular for amino acid identity, those using the following parameters: Program: blastp; Matrix: blosum62; Threshold: 10; Filtered: false; Gapped: true; Maximum number of hits reported: 250.
  • Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted if so desired to result in an alignment that spans at least about 10, 15, 20, 25, 30, 35, 40, 45, 50 or other number of contiguous amino acids of the target polypeptide or region thereof.
  • the ULRB1 is human ULRB1, preferably a protein comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, and SEQ ID NO: 389 (in particular, SEQ ID NO. 385), or a protein having no more than two, four, six, eight, or ten, for example no more than one, two or three, such as no more than one, amino acid substitutions, insertions or deletions compared to these sequences.
  • the ULRB2 is human ULRB2, preferably a protein comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, and SEQ ID NO: 393 (in particular, SEQ ID NO. 390), or a protein having no more than two, four, six, eight, or ten, for example no more than one, two or three, such as no more than one, amino acid substitutions, insertions or deletions compared to these sequences.
  • variants of ULRB1 and/or ULRB2 are a protein comprising an amino acid sequence having at least 80%, 85%, 90%, 92% 95% or 97% sequence identity (in particular, at least 92% or 95% sequence identity) to any one of the sequences of SEQ ID NO: 385-393 (preferably of SEQ ID NO: 353 (ULRB1) or SEQ ID NO 358 (LILRB2)).
  • the invention also includes those embodiments where a variant of ULRB1 and/or LILRB2 is selected from the group consisting of an ortholog (or paralog) of ULRB1 and/or ULRB2, and a functional fragment of an LILRB1 and/or ULRB2 protein.
  • a functional fragment of an ULRB1 and/or LILRB2 protein binds to a natural ligand of ULRB1 and/or ULRB2 protein, such as a human leucocyte antigen (HLA) type proteins (such as one described elsewhere herein).
  • HLA human leucocyte antigen
  • An ABP or biABP of the invention may, in particular embodiments, be able to inhibit (eg, inhibits) the interaction between a natural ligand of ULRB1 and/or ULRB2 protein and ULRB1 and/or ULRB2 protein (such as for example to HLA-G).
  • the ABP or biABP is optionally able to inhibit (eg, inhibits) the binding of ULRB1 and/or LILRB2 protein to a ligand of ULRB1 and/or LILRB2 (such as for example HLA-G).
  • the protein sequences of the canonical isoform, and further isoforms of the off targets ULRA1 and ULRA3 can also be derived from the UniProt database.
  • LILRA1 protein sequences and additional information is obtainable under the accession number: 075019.
  • the canonical protein sequence is provided herein as SEQ ID NO: 394.
  • SEQ ID NO: 395 protein sequences and additional information is obtainable under the accession number: 075019.
  • HLA-G refers to a protein called human leukocyte antigen G or HLA-G histocompatibility antigen class G, etc. This protein was first discovered in extravillous trophoblasts (EVT) present at the maternal-fetal interface during pregnancy, and is a heterologous material (membrane-bound HLA-G, such as HLA- Gl, G2, G3 and G4 are present) that is expressed only in the cellular membrane by selective conjugation of HLA-G mRNA, and has a single molecular form in a soluble state to be secreted to the outside of the cells (HLA-G5, G6 and G7 are present as soluble HLA-G). Human HLA-G amino acid sequences and further information can be derived from the UniProt database in the version of December 2022 with the accession number P17693.
  • An ABP or biABP of the invention may, in particular embodiments, be able to inhibit (eg, inhibits) the interaction between a natural ligand of ULRB1 and LILRB2 protein and ULRB1 and LILRB2 protein (such as for example to HLA- G).
  • the ABP or biABP is optionally able to inhibit (eg, inhibits) the binding of LILRB1 and LILRB2 protein to a ligand of ULRB1 and ULRB2 (such as for example HLA-G).
  • the ABP or biABP of the invention further does not inhibit the interaction of ULRA1 and ULRA3 with a natural ligand of ULRA1 and/or ULRA3.
  • the ABP or biABP is a modulator of the expression, function, activity and/or stability of LILRB1 and/or LILRB2, or the variant of LILRB1 and/or LILRB2, such as wherein the ABP or biABP inhibits the expression, function, activity and/or stability of ULRB1 and/or LILRB2, or the variant of LILRB1 and/or ULRB2, or in particular where the ABP or biABP is an inhibitor of the function and/or activity of said ULRB1 and/or ULRB2 or the variant of ULRB1 and/or ULRB2.
  • an ABP or biABP of the invention is an inhibitor of the interaction between ULRB1 and/or LILRB2, or the variant of ULRB1 and/or LILRB2, to its endogenous receptor or ligand, such as to a HLA type protein, in particular an ABP or biABP of the invention is capable of inhibiting (eg, inhibits or is an inhibitor of) the binding of a natural ligand of ULRB1 and/or LILRB2 protein to LILRB1 and/or ULRB2 protein. Accordingly, ABPs or biABPs of the invention can be "modulators".
  • modulator refers to a molecule that changes, modifies or alters one or more characteristics, properties and/or abilities of another molecule or, for example, that changes, modifies or alters an immune response (“immunomodulators”), such as a cell-mediated immune response.
  • a modulator eg, an inhibiting or antagonistic modulator
  • a modulator eg, an activating or agonistic modulator
  • a modulator can enhance or promote, or cause an increase in the magnitude of, expression, function, activity and/or stability, such as a certain activity or function, of a molecule compared to the magnitude of such characteristic, property or ability observed in the absence of the modulator.
  • Certain exemplary characteristics, properties or abilities of a molecule include, but are not limited to, expression, function, activity and/or stability, such as binding ability or affinity, enzymatic activity, and signal transduction; for example, any of the functions or activities of ULRB1 and/or LILRB2 described herein.
  • Modulatory molecules in particular, modulatory ABPs or biABPs can act as "inhibitors" ("antagonists") against a receptor such as ULRB1 and/or ULRB2, such as by impairing (e.g. blocking) ligand engagement to such receptor, eg by inhibiting the interaction between LILRB1 and/or ULRB2 and their ligand.
  • modulatory molecules in particular, modulatory ABPs or biABPs
  • modulatory ABPs or biABPs can act as "activators" ("agonists") for a receptor such as ULRB1 and/or ULRB2, such as by enhancing or promoting function and/or activity of such receptor, for example by triggering the receptor's signalling pathway, such as by mimicking the binding of the endogenous ligand for such receptor.
  • a particular embodiment of a modulator of ULRB1 and/or LILRB2 is an "inhibitor of ULRB1 and/or ULRB2" (or"LILRBl and/or LILRB2 inhibitor”), which meaning includes any moiety that inhibits LILRB1 and/or LILRB2, which can mean inhibition of the expression (eg the amount), function, activity and/or stability of LILRB1 and/or ULRB2, especially of protein of ULRB1 and/or ULRB2.
  • an inhibitor of LILRB1 and/or ULRB2 can reduce the function (and/or activity) of LILRB1 and/or ULRB2 protein, and in another of such embodiments, an inhibitor of ULRB1 and/or LILRB2 can reduce the expression of ULRB1 and/or ULRB2 mRNA and/or protein.
  • ULRB1 and/or LILRB2 inhibitors including those that are ABPs or biABPs of the present invention are described elsewhere herein, including those as may be characterised by the applicable functional and/or structural features set out herein.
  • an ABP or biABP of the invention is one that is capable of specifically binding to (eg which specifically binds to) ULRB1 and/or ULRB2, as well as being capable of inhibiting (eg reducing or blocking) the interaction between ULRB1 and/or LILRB2 protein (or a variant thereof, such as one described above) and its natural ligand (such as a HLA protein, or other ligand).
  • such an ABP or biABP is able to inhibit (eg inhibits) the binding of HLA-G protein (or a variant thereof, such as one described above) to LILRB1 and/or LILRB2 protein (or a variant thereof, such as one described above).
  • Methodologies to determine the interaction (eg binding) between LILRB1 and/or ULRB2 and a HLA protein such as HLA-G protein (or between variants thereof) are known to the person of ordinary skill, and include ELISA assays (such as described in the examples below), and technologies such as inter alia, flow cytometry, surface plasmon resonance, surface acoustic waves and microscale thermophoresis. Such determination methodologies can be used (or adapted) to not only detect the presence of such interaction/binding, but also to measure (eg quantitatively) the degree of binding between the interacting partners ULRB1 and/or ULRB2 and their ligands such as HLA-G proteins (or variants thereof).
  • Such (quantitative) measurement of interaction (binding) may be determined or measured in the presence of a competing (eg inhibiting) ABP or biABP of the invention, and hence the potential of an ABP or biABP of the present invention to inhibit (eg block) such interaction can be measured, and eg reported as an IC50.
  • Such IC50 values may be determined, such as using ELISA methodology (eg, using an assay correspond to, or substantially as, the EUSA described in Example 3), in the presence of a suitable concentration of HLA-G protein (or variant thereof) in solution and with surface-bound ULRB1 and/or LILRB2.
  • the ABP or biABP of the invention (eg one that binds to [one or more epitope(s) displayed by] an extracellular domain(s) of LILRB1 and/or ULRB2, or a paralogue, orthologue or other variant thereof) is capable of inhibiting (eg will inhibit) the binding of a natural ligand of ULRB1 and/or LILRB2 protein or a variant thereof to LILRB1 and/or ULRB2 protein or a variant thereof with an IC50 of lOOnM, 50nM, or preferably 20nM or less, such as 15nM or less, lOnM or less, 5nM or less, 2nM or less, InM or less, 500pM or less, 250pM or less, or lOOpM or less.
  • an ABP or biABP of the invention is capable of inhibiting (eg will inhibit) the binding of HLA-G protein or a variant thereof to LILRB1 and/or ULRB2 protein or a variant thereof with an IC50 of lOnM or less, such as 5nM or less and preferably 2nM or less.
  • a modulator of the invention eg, an ABP or biABP that binds to LILRB1 and/or LILRB2
  • an inhibitor or antagonist may instead or also: • inhibit, impair, reduce or reverse ULRB1 and/or LILRB2-mediated inhibition of a cell-mediated immune response (eg in an in-vitro assay or in a subject, such as one in need thereof); and/or
  • a macrophage such as a tumour associated macrophage (TAM)
  • TAM tumour associated macrophage
  • an immune suppressive phenotype such as an M2 like macrophage or myeloid-derived suppressor cell (MDSC);
  • cell-mediated immune response may include, but is not limited to, a response in a host organism involving, utilising, and/or promoting any one or combinations of T cell maturation, proliferation, activation, migration, infiltration and/or differentiation, and/or the activation/modulation/migration/infiltration of a macrophage, a natural killer cell, a T lymphocyte (or T cell), a helper T lymphocyte, a memory T lymphocyte, a suppressor T lymphocyte, a regulator T lymphocyte, and/or a cytotoxic T lymphocyte (CTL), and/or the production, release, and/or effect of one or more cell-secretable or cell-secreted factor such as a cytokine or autocoid (in particular a pro-inflammatory cytokine), and/or one or more components of any of such processes (such as a cytokine or autocoid, particular a pro-inflammatory cytokine).
  • a cytokine or autocoid in particular a pro-inflammatory cytokin
  • cell-mediated immune response may include a cellular response involving a genetically engineered, in-vitrv cultured, autologous, heterologous, modified, and/or transferred T lymphocyte, or it may include a cell-secretable or cell-secreted factor (such as a cytokine or autocoid, in particular a pro-inflammatory cytokine) produced by genetic engineering.
  • a cell-mediated immune response is preferably not a humoral immune response, such as an immune response involving the release of antibodies.
  • the cell-mediated immune response is an anti-tumour cell-mediated immune response.
  • a cytotoxic cell-mediated immune response such as a cytotoxic T cell exposure
  • the cell mediating the cell-mediated immune response may be mediated by a cell, such as an immune cell, capable of secreting (eg secreting) pro-inflammatory cytokine, such as one selected from the group consisting of: interleukin-1 (IL-1), IL- 2, IL-12, IL-17 and IL- 18, tumour necrosis factor (TNF) [alpha], interferon gamma (IFN-gamma), and granulocyte-macrophage colony stimulating factor.
  • IL-1 interleukin-1
  • IL-12 IL-12
  • IL-17 IL-17
  • TNF tumour necrosis factor
  • IFN-gamma interferon gamma
  • granulocyte-macrophage colony stimulating factor granulocyte-macrophage colony stimulating factor
  • the cell-mediated immune response can be mediated by a pro-inflammatory cytokine-secreting cell, such as a lymphocyte (eg a T cell), in particular a cytotoxic T lymphocyte (CTL), or a natural killer cell (NK cell).
  • a pro-inflammatory cytokine-secreting cell such as a lymphocyte (eg a T cell), in particular a cytotoxic T lymphocyte (CTL), or a natural killer cell (NK cell).
  • a lymphocyte eg a T cell
  • CTL cytotoxic T lymphocyte
  • NK cell natural killer cell
  • the cell-mediated immune response may induce killing of cells associated or involved with a disease, disorder or condition, such as a proliferative disorder (eg a cancer).
  • a disease, disorder or condition such as a proliferative disorder (eg a cancer).
  • Humoral immunity (or “humoral immune response”) will also be readily understood by the person of ordinary skill, and includes an aspect of an immune response that is mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins, and certain antimicrobial peptides. Humoral immunity is so named because it involves substances found in the humors, or body fluids. Its aspects involving antibodies can be termed antibody-mediated immunity.
  • a "subject” includes all mammals, including without limitation humans, but also non-human primates such as cynomolgus monkeys. It also includes dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents (such as mice and rats). It will be appreciated that a particularly preferred subject according to the invention is a human subject, such as a human suffering from (or at risk of suffering from) a disorder, disease or condition, for example a human patient.
  • a modulator of the invention eg, an ABP or biABP that binds to ULRB1 and/or ULRB2
  • an inhibitor or antagonist preferably of an interaction of LILRB1 and/or ULRB2 with a natural ligand thereof, such as HLA-G
  • a modulator of the invention may instead or also:
  • the present invention pertains to bispecific ABP that in their first antigen binding site comprise CDRs as contained in the specific ABP sequences described herein elsewhere (in particular in Table 1). Further, the biABP of the invention comprises at least a second antigen binding site binding to an immune checkpoint. For the biABP of the invention the following applies:
  • the term "monospecific" antibody as used herein denotes an antibody or ABP that has one or more binding sites each of which bind to the same epitope of the same antigen.
  • bispecific means that the antibody or ABP is able to specifically bind to at least two distinct antigenic determinants, for example two binding sites (epitopes) each formed by a pair of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) binding to different antigens or to different epitopes on the same antigen.
  • VH antibody heavy chain variable domain
  • VL antibody light chain variable domain
  • Such a bispecific antibody or ABP may have an 1+1 format.
  • Other bispecific antibody formats are 2+1 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 formats (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope).
  • the bispecific ABP of the invention can be provided in any bispecific format known to the person of skill.
  • the biABP may be a bi-specific, tri-specific, or tetra-specific antibody, in particular a bi-specific antibody is selected from: a bispecific T-cell engager (BiTE) antibody, a dual-affinity retargeting molecule (DART), a CrossMAb antibody, a DutaMabTM antibody, a DuoBody antibody; a Triomab, a TandAb, a bispecific NanoBody, Tandem scFv, a diabody, a single chain diabody, a HSA body, a (scFv)2 HSA Antibody, an scFv-IgG antibody, a Dock and Lock bispecific antibody, a DVD-IgG antibody, a TBTI DVD-IgG, an IgG-fynomer, a Tetravalent bispecific tandem IgG antibody,
  • bispecific and/or bispecific antibodies or biABPs are at least “bivalent” and may be “trivalent” or “multivalent” (e.g. "tetravalent” or "hexavalent”).
  • the antibodies or ABPs of the present invention have two or more binding sites and are bispecific. That is, the antibodies or ABPs may be bispecific even in cases where there are more than two binding sites (i.e. that the antibody is trivalent or multivalent).
  • antigen binding domain or "antigen-binding site” or “paratope” are used as synonyms and refers to the part of the antigen binding protein (ABP) that specifically binds to an antigenic determinant. More particularly, the term “antigen-binding domain” refers the part of an antibody or ABP that comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antigen binding protein may only bind to a particular part of the antigen, which part is termed an epitope.
  • An antigen binding domain may be provided by, for example, one or more variable domains (also called variable regions).
  • an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the antigen binding domain is able to bind to its antigen and block or partly block its function.
  • Antigen binding domains that specifically bind to ULRB1/LILRB2 are defined herein elsewhere.
  • the antigen-binding site is an antibody paratope, preferably wherein the antibody paratope is comprised in an antigen-binding fragment of an antibody, such as comprised in an antibody heavy chain and/or light chain variable domain, preferably wherein the one or more ABP is or comprises a combination of an antibody paratope provided as a full-length antibody, such as an IgG antibody, or provided as an antigen binding fragment of an antibody, such as preferably an F(ab')2-, Fab-, or Fv-fragment, and wherein the fragment is preferably a single-chain (sc) construct, most preferably as a scFab.
  • sc single-chain
  • BiABPs of the invention in some embodiments may be composed of antibody fragments including but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g. scFv); multispecific antibodies formed from antibody fragments and single domain antibodies.
  • scFv single-chain antibody molecules
  • Diabodies are antibody fragments with two antigen binding domains that may be bivalent or bispecific, see, for example, EP 404,097; WO 1993/01161; Hudson et aL, Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003).
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see e.g. U.S. Patent No. 6,248,516 Bl).
  • antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of fulllength antibodies.
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein. Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments containing each the heavy- and light-chain variable domains and also the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab fragment refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CHI) of a heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteins from the antibody hinge region.
  • Fab'-SH are Fab' fragments wherein the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region.
  • cross-Fab fragment or "xFab fragment” or “crossover Fab fragment” refers to a Fab fragment, wherein either the variable regions or the constant regions of the heavy and light chain are exchanged.
  • Two different chain compositions of a crossover Fab molecule are possible and comprised in the bispecific antibodies of the invention: On the one hand, the variable regions of the Fab heavy and light chain are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CHI), and a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL).
  • This crossover Fab molecule is also referred to as CrossFab (VLVH)-
  • the crossover Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CHI).
  • This crossover Fab molecule is also referred to as CrossFab (CLCHI)-.
  • a "single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1 -linker- VL-CL, b) VL-CL-linker-VH-CHI, c) VH-CL-linker-VL-CHI or d) VL-CH1 -linker- VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids.
  • Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CHI domain.
  • these single chain Fab molecules might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g. position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).
  • a "crossover single chain Fab fragment” or “x-scFab” is a is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CHI), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CL-linker-VL-CHI and b) VL-CH1 -linker- VH-CL; wherein VH and VL form together an antigen binding domain which binds specifically to an antigen and wherein said linker is a polypeptide of at least 30 amino acids.
  • these x-scFab molecules might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues (e.g. position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).
  • a "single-chain variable fragment (scFv)" is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.
  • This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker.
  • scFv antibodies are, e.g. described in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96).
  • antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full-length antibodies.
  • Bispecific ABPs and bispecific antibodies of the invention may be provided as heterodimeric antibody-like complexes that produced using the so called “knob in the hole” technology which allows a production of the heterodimer without chain mispairing, for example by using a heavy chain constant 3 (CH3) region with an introduced “protuberance” ("knob”) in one chain thereof and a corresponding introduced “cavity” ("hole”) in the other chain thereof; see US Patent No. 5,821,333, expressly incorporated herein by reference).
  • CH3 domains were shown be used to promote heterodimerization of two non- identical antibody heavy chains as herein described.
  • EU numbering system also called the EU index
  • EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • This approach is known as the "knob-into-hole” technology which is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
  • the method involves introducing a protuberance ("knob”) at the interface of a first polypeptide and a corresponding cavity ("hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity 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 (e.g. tyrosine or tryptophan).
  • 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 (e.g. alanine or threonine).
  • the protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
  • a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain
  • the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain.
  • the subunit of the Fc domain comprising the knob modification additionally comprises the amino acid substitution S354C
  • the subunit of the Fc domain comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
  • a biABP of the invention comprises two antibody binding sites (or domains) which are interconnected at least in part covalently.
  • Such covalent connection is realized using peptide linkers and/or covalent bonds such as disulfide bridges.
  • antibody fragments such as Fab or scFab as described herein elsewhere may be connected via a peptide linker.
  • peptide linker refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or are described herein.
  • Suitable, non-immunogenic linker peptides are, for example, (G4S)n, (SG4)n or G4(SG4)n peptide linkers, wherein "n” is generally a number between 1 and 10, typically between 2 and 8, in particular a G4S)n linker.
  • the isolated ABP, or ABP-containing composition, in (B) is a bispecific ABP comprising two first antigen-binding sites and two second antigen binding sites, wherein (X) the two first antigen binding sites are provided as one F(ab')2 fragment or one IgG antibody, wherein the IgG antibody comprises an antibody hinge-, CH2- and/or CH3-domain, and wherein the two second antibody binding sites are provided as two antibody scFv or scFab fragments, each of which is independently covalently connected to one of the two first antigen-binding sites, preferably via a protein linker, or (Y) the two second antigen binding sites are provided as one F(ab')2 fragment or one IgG antibody, wherein the IgG antibody comprises an antibody hinge-, CH2- and/or CH3-domain, and wherein the two first antibody binding sites are provided as two antibody scFv or scFab fragments, each of which is independently covalently connected to the two
  • One format preferred in context of the present invention is a tetravalent construct (2:2) comprising a homodimer of a single chain construct which combine Fab and/or scFV antibody binding sites of two ABPs (wherein scFv are preferred).
  • a preferred embodiment of a bispecific ABP of the invention is composed of at least one, preferably two, first antibody (heavy/light) chain sequences, and at least one, preferably two, second antibody (heavy/light) chain sequences, wherein at least one, preferably both, of the first antibody (heavy/light) chain sequences comprises in N- to C-terminal direction:
  • (1-2) optionally, one or more antibody heavy chain constant domain sequences, such as a CHI, hinge, CH2 and/or CH3 domain,
  • (1-8) optionally, an antibody heavy chain constant domain sequence; and wherein at least one, preferably both, of the second antibody (heavy/light) chain sequences comprises in N- to C-terminal direction
  • (2-2) an antibody light chain constant domain; wherein [(1-4) to (1-5)] and [(1-7) to (1-8)] can be interchanged, and/or wherein (1-4) and (1-5); and (1-7) and (1-8); can be (both) interchanged.
  • first and or second peptide linker preferably has a length of 0 to 100 amino acids, preferably of 0 to 50 amino acids, optionally, wherein the first and/or second peptide linker comprises one or more iterations of 4GS.
  • Certain preferred embodiments of the invention pertain to the isolated ABP, which is a bispecific ABP, of a format that is composed of at least one, preferably two, first antibody (heavy/light) chain sequences, and at least one, preferably two, second antibody (heavy/light) chain sequences, wherein at least one, preferably both, of the first antibody (heavy/light) chain sequences comprises in N- to C-terminal direction:
  • (1-2) optionally, one or more antibody heavy chain constant domain sequences, such as a CHI, hinge, CH2 and/or CH3 domain,
  • (1-8) optionally, an antibody heavy chain constant domain sequence; and wherein at least one, preferably both, of the second antibody (heavy/light) chain sequences comprises in N- to C-terminal direction
  • (2-2) an antibody light chain constant domain; wherein [(1-4) to (1-5)] and [(1-7) to (1-8)] can be interchanged, and/or wherein (1-4) and (1-5); and (1-7) and (1- 8); can be (both) interchanged.
  • chain pairing domain in context of the present invention shall refer to an amino acid or amino acid sequence that allows for a specific heterodimeric pairing of the first antibody (heavy/light) chain sequence with the second antibody (heavy/light) chain sequence, which avoids homodimeric mispairing or mispairing with other, unrelated, endogeneous antibody chains.
  • chain pairing domains may include domains known as “knobs” or “holes” or positively or negatively charged amino acids.
  • Such chain pairing domains are referred to as donor (knob, or positive) or acceptor (hole, negative) respectively.
  • said chain pairing domain modification is a so-called "knob-into-hole” modification of the ABP of the invention, comprising a "knob” modification in one of the two subunits of the Fc domain and a "hole” modification in the other one of the two subunits of the Fc domain.
  • the invention relates to a bispecific ABP or bispecific antibody comprising a first antigen binding domain that specifically binds to ULRB1/LILRB2 (for example with an antigen binding domain contained in any one of an antibody of table 1) and a second antigen-binding site that specifically binds to an immune checkpoint, such as an antigen binding domain of an ant-PDl or anti-PD-Ll antibody, wherein the first subunit of the Fc domain comprises knobs and the second subunit of the Fc domain comprises holes according to the knobs into holes method.
  • the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).
  • knob-into-hole technology is described e.g. in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
  • the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity 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 (e.g. tyrosine or tryptophan).
  • 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 (e.g. alanine or threonine).
  • the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).
  • the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to Kabat EU index).
  • the bispecific ABP or antibody comprises the mutations R409D and K370E in the CH3 domain of the "knobs chain” and the mutations D399K and E357K in the CH3 domain of the "hole-chain” (numbering according to Kabat EU index).
  • the bispecific ABP or antibody comprises a T366W mutation in the CH3 domain of the "knobs chain” and the mutations T366S, L368A and Y407V in the CH3 domain of the "hole chain” and additionally the mutations R409D and K370E in the CH3 domain of the "knobs chain” and the mutations D399K and E357K in the CH3 domain of the "hole chain” (numbering according to the Kabat EU index).
  • a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g. as described in PCT publication WO 2009/089004.
  • this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable.
  • Apart from the "knob-into-hole technology" other techniques for modifying the CH3 domains of the heavy chains of a bispecific ABP or antibody to enforce heterodimerization are known in the art.
  • the approach described in EP 1870459 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3-domain-interface between both, the first and the second heavy chain.
  • the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain form an interface that is located between the respective antibody CH3 domains, wherein the respective amino acid sequences of the CH3 domain of the first heavy chain and the amino acid sequence of the CH3 domain of the second heavy chain each comprise a set of amino acids that is located within said interface in the tertiary structure of the antibody, wherein from the set of amino acids that is located in the interface in the CH3 domain of one heavy chain a first amino acid is substituted by a positively charged amino acid and from the set of amino acids that is located in the interface in the CH3 domain of the other heavy chain a second amino acid is substituted by a negatively charged amino acid.
  • the bispecific antibody according to this aspect is herein also referred to as "CH3(+/-)-engineered bispecific antibody" (wherein the abbreviation "+/-" stands for the oppositely charged amino acids that were introduced in the respective CH3 domains).
  • the positively charged amino acid is selected from K, R and H, and the negatively charged amino acid is selected from E or D.
  • the positively charged amino acid is selected from K and R, and the negatively charged amino acid is selected from E or D.
  • the positively charged amino acid is K
  • the negatively charged amino acid is E
  • the approach described in WO 2013/157953 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific ABR
  • the amino acid T at position 366 is substituted by K
  • the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index).
  • the amino acid T at position 366 is substituted by K and the amino acid L at position 351 is substituted by K
  • the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index).
  • the amino acid T at position 366 is substituted by K and the amino acid L at position 351 is substituted by K, and in the CH3 domain of the other heavy chain the amino acid L at position 351 is substituted by D (numbering according to Kabat EU index).
  • the amino acid Y at position 349 is substituted by E
  • the amino acid Y at position 349 is substituted by D
  • the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index).
  • the amino acid L at position 368 is substituted by E (numbering according to Kabat EU index).
  • the bispecific, preferably biparatopoc, ABP or antibody is engineered according to WO 2012/058768), i.e. in the CH3 domain of one heavy chain the amino acid L at position 351 is substituted by Y and the amino acid Y at position 407 is substituted by A, and in the CH3 domain of the other heavy chain the amino acid T at position 366 is substituted by V and the amino acid K at position 409 is substituted by F (numbering according to Kabat EU index).
  • the approach described in WO 2011/143545 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody.
  • amino acid modifications in the CH3 domains of both heavy chains are introduced at positions 368 and/or 409 (numbering according to Kabat EU index).
  • the amino acid K or R at position 409 is substituted by a negatively charged amino acid (in one embodiment by E or D, in one preferred embodiment by D) (numbering according to Kabat EU index).
  • the amino acid K at position 439 and/or the amino acid K at position 370 is substituted independently from each other by a negatively charged amino acid (in one embodiment by E or D, in one preferred embodiment by D) (numbering according to Kabat EU index).
  • a bispecific antibody comprising a heavy chain including a C- terminal CH3 domain as specified herein comprises the C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat EU index).
  • a bispecific antibody comprising a heavy chain including a C-terminal CH3 domain, as specified herein comprises a C-terminal glycine residue (G446, numbering according to Kabat EU index).
  • the invention relates to a bispecific antibody or biABP comprising a first antigen binding site that specifically binds to LILRB1/LILRB2 and a second antigen binding site that specifically binds to an immune checkpoint, wherein in one of the antigen binding sites, which may be for example Fab fragments, either the variable domains VH and VL or the constant domains CHI and CL are exchanged.
  • the bispecific antibodies are prepared according to the Crossmab technology. Multispecific antibodies with a domain replacement/exchange in one binding arm (CrossMabVH-VL or CrossMabCH-CL) are described in detail in W02009/080252, W02009/080253 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191. They clearly reduce the byproducts caused by the mismatch of a light chain against a first antigen with the wrong heavy chain against the second antigen (compared to approaches without such domain exchange).
  • a preferred embodiment therefore pertains to a bipABP in a format of a CrossMab Fab , CrossMab VHVL or, preferably, a CrossMab CH1CL , preferably wherein the CrossMab CH1CL bispecific antibody is composed of a first heterodimer, and a second heterodimer, wherein the first heterodimer is composed of:
  • a first antibody chain sequence comprising in N- to C-terminal orientation an antibody heavy chain variable domain sequence comprising at least parts of the first antigen-binding site (in an alternative embodiment of the second antigen-binding site), and an antibody heavy chain constant region (CHI), and antibody hinge sequence, an antibody CH2 and/or CH3 domain; and
  • a second antibody chain sequence comprising in N- to C-terminal orientation an antibody light chain variable domain sequence comprising at least parts of the first antigen-binding site (in an alternative embodiment of the second antigen-binding site), and an antibody light chain constant region (CL); and the second heterodimer is composed of: a third antibody chain sequence comprising in N- to C-terminal orientation an antibody heavy chain variable domain sequence comprising at least parts of the second antigen-binding site (in an alternative embodiment of the first antigen-binding site), and an antibody light chain constant region (CL), an antibody hinge sequence, an antibody CH2 and/or CH3 domain; and
  • a fourth antibody chain sequence comprising in N- to C-terminal orientation an antibody light chain variable domain sequence comprising at least parts of the second antigen-binding site (in an alternative embodiment of the first antigen-binding site), and an antibody heavy chain constant region (CHI); wherein at least the first and the third antibody chain sequences comprise each either a donor-, or acceptor- respectively, antibody chain pairing domain which are capable of mediating a specific pairing of the first antibody chain sequence with the third antibody chain sequence.
  • CHI antibody heavy chain constant region
  • An immune checkpoint molecule in context of the invention may be selected from the group consisting of PD- 1, PD-L1 CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, AXL, ILT2, or ILT3.
  • the bispecific ABP of the invention may instead of a binding site binding to an immune checkpoint, comprise a binding site to an immune costimulatory molecule (such as CD27, CD40, 4-1BB, 0X40, or GITR), or a tumour antigen (such as HER2, EGFR, ErB3, or CD24).
  • exemplary binding agents for use as antigen binding sites directed at immune checkpoints are antibodies or antigen binding fragments thereof which bind to human PD-1 or PD-L1, e.g., a PD-1 or PD-L1 antagonist.
  • An exemplary PD-1 antibody is pembrolizumab (Keytruda®).
  • Other exemplary binding agents include PD-L1 and PD-1 antibodies (or antigen binding fragments thereof) such as durvalumab, nivolumab, cemiplimab (Libtaylo®), avelumab (Bavencio®), durvalumab (Imfinzi®), and atezolizumab (Tecentriq®).
  • An exemplary bispecific antibody on the basis of the antigen binding domain of pembrolizumab is provided herein in Example 13.
  • a bispecific antibody according to the invention in preferred embodiments comprises a first antigen binding site which specifically binds to the extra cellular domain (ECD) of leukocyte immunoglobulin-like receptor subfamily Bl (ULRB1) and ULRB2 protein, wherein the first antigen binding site comprises a CDR-containing sequence according to the following disclosure of ABPs or biABPs.
  • ABPs or biABPs of the invention comprising one or more complementarity determining regions
  • an ABP or biABP of the invention can preferentially comprise at least one complementarity determining region (CDR), such as one from an antibody (in particular from a human antibody), and in particular embodiments the ABP or biABP can comprise a CDR having an amino acid sequence with at least 80%, 85%, 90% or 95% sequence identity to (preferably, at least 90% sequence identity to), or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a CDR sequence set forth in Table 1 herein.
  • CDR complementarity determining region
  • CDR complementarity determining region
  • a framework sequence of an ABP or biABP of the invention may include one or more mutations, for example in order to improve the isoelectric point of the molecules.
  • One preferred mutation of the ABP or biABP of the invention are at position E81, for example is a E81M mutation, in accordance with the Kabat nomenclature.
  • an ABP or biABP can comprise at least one complementarity determining region (CDR).
  • an ABP or biABP of the invention comprises at least one complementarity determining region 3 (CDR3), such as one having an amino acid sequence with at least 80%, 85%, 90% or 95% (preferably at least 90%) sequence identity to, or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from those heavy and light chain CDR3 sequences shown in Table 1 (eg, a sequence selected from the list consisting of SEQ ID Nos: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95,
  • CDR3 complementarity determining region 3
  • An ABP or biABP of the invention may, alternatively or as well as a CDR3 sequence, comprise at least one CDR1, and/or at least one CDR2 (such as one from an antibody, in particular from a human antibody).
  • ABP or biABP of the invention comprises at least one such CDR3, as well as at least one such CDR1 and at least one such CDR2, more preferably where each of such CDRs having an amino acid sequence with at least 80%, 85%, 90% or 95% (preferably at least 90%) sequence identity to, or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from the corresponding (heavy and light chain) CDR1, CD2 and CD3 sequences shown in Table 1.
  • the antibodies of the invention are derived from three parental antibody sequences denoted as A-001, A-002 and A-003.
  • the invention shows an inventive binding profile for the parental sequences, but also for maturated derivative antibody heavy and/or light chain sequences.
  • the invention therefore in specific embodiments also pertains to antibody chain combination of any light chain sequence with any heavy chain sequence, of an antibody of the invention.
  • an ABP or biABP of the invention can be an antibody or an antigen binding fragment thereof.
  • an antibody may be understood in the broadest sense as any immunoglobulin (Ig) that enables binding to its epitope.
  • An antibody as such is a species of an ABP or biABP.
  • Full length "antibodies” or “immunoglobulins” are generally heterotetrameric glycoproteins of about 150 kDa, composed of two identical light and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulphide bond, while the number of disulphide linkages varies between the heavy chain of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulphide bridges.
  • Each heavy chain has an amino terminal variable domain (VH) followed by three carboxy terminal constant domains (CH).
  • Each light chain has a variable N-terminal domain (VL) and a single C-terminal constant domain (CL).
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR 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 variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to cells or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • Other forms of antibodies include heavy-chain antibodies, being those which consist only of two heavy chains and lack the two light chains usually found in antibodies.
  • Heavy-chain antibodies include the hdgG (IgG-like) antibodies of camelids such as dromedaries, camels, llamas and alpacas, and the IgNAR antibodies of cartilaginous fishes (for example sharks).
  • Single-domain antibodies include single-domain antibodies (sdAb, called Nanobody by Ablynx, the developer) being an antibody fragment consisting of a single monomeric variable antibody domain.
  • Single-domain antibodies are typically produced from heavy-chain antibodies, but may also be derived from conventional antibodies.
  • Antibodies can include, for instance, chimeric, humanized, (folly) human, or hybrid antibodies with dual or multiple antigen or epitope specificities, antibody fragments and antibody sub-fragments, e.g., Fab, Fab' or F(ab')2 fragments, single chain antibodies (scFv) and the like (described below), including hybrid fragments of any immunoglobulin or any natural, synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
  • an ABP or biABP of the invention can comprise an antibody heavy chain, or an antigen binding fragment thereof, and/or an antibody light chain, or an antigen binding fragment thereof.
  • an ABP or biABP of the invention can comprise an antibody heavy chain variable region, or an antigen binding fragment thereof, and/or an antibody light chain variable region, or an antigen binding fragment thereof, and in yet further embodiments, an ABP or biABP of the invention can comprise an antibody heavy chain variable region CDR1, CDR2, and CDR3, and/or an antibody light chain variable region CDR1, CDR2, and CDR3.
  • the antibody heavy chain sequence, or the fragment thereof can comprise a CDR3 having at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity to, or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a CDR3 sequence selected from those heavy chain CDR3 sequences shown in Table 1 (eg, a sequence selected from the list consisting of SEQ ID Nos: 3, 11, 19, l, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139, 147, 155, 163, 171, 179, 187, 195, 203, 211, 219, 227, 235, 243, 251, 259, 267, 275, 283, 291, 2
  • the antibody heavy chain sequence, or the fragment thereof can comprise a CDR3 having at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity to, or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a CDR3 sequence selected from those heavy chain CDR3 sequences selected from the list consisting of SEQ ID Nos: 75, 115, and 195, and/or wherein antibody light chain sequence, or the fragment thereof, can comprise a CDR3 having at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity to, or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to a CDR3 sequence selected from those light chain CDR3 sequences selected from those light chain CDR3 sequences selected from
  • ABP or biABP of the invention Any combination of these heavy chain CDR3 and light chain CDR3 are included in ABP or biABP of the invention, in particular the combinations selected from SEQ ID NO: 75 and 287, SEQ ID NO: 115 and 287, SEQ ID NO: 195 and 311 and SEQ ID NO: 195 and 319.
  • the antibody heavy chain sequence, or the fragment thereof can further comprise a CDR1 having at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity to, or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from SEQ ID NOs.
  • an ABP or biABP of the invention comprises an antibody light chain, or an antigen binding fragment thereof, wherein the antibody light chain sequence, or the fragment thereof, further comprises a CDR1 having at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity to, or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from SEQ ID NOs.
  • an ABP or biABP of the invention can comprise an antibody variable chain sequence having at least 80%, 85%, 90%; or 95% (preferably at least 90%) sequence identity to, or having no more than ten, nine, eight, seven, six, five, four, three, two or one, preferably no more than three, two or one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from SEQ ID NOs. 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120,
  • an ABP or biABP of the invention comprises an antigen binding fragment of an antibody, wherein the antigen binding fragment comprises CDR1, CDR2 and CDR3.
  • the CDR1 is selected from those disclosed in Table 1
  • the CDR2 is selected from those disclosed in Table 1
  • the CDR3 is selected from those disclosed in Table 1 (eg, the CDR1, CDR2 and CDR3 are selected from the CDR1, CDR2 and CDR3 sequences having the respective amino acid sequences of SEQ ID Nos.
  • an ABP or biABP of the invention can comprise an antibody heavy chain variable region CDR1, CDR2, and CDR3, and/or an antibody light chain variable region CDR1, CDR2, and CDR3, wherein the CDR1 has an amino acid sequence of a heavy or light chain CDR1 shown in Table 1 (eg has an amino acid sequence selected from the list consisting of SEQ ID No 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237, 241,
  • the ABP or biABP may be an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences, and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences and at least one, preferably both, of the antibody light chain sequences comprise CDR1 to CDR3 sequences in a combination selected from any of the combinations of heavy chain CDRs shown in Table B and/or selected from any of the combinations of light chain CDRs shown in Table B (in each case, combinations CDRs-A-001 to CDRs-A-044); in each case independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • the combination of both the heavy chain CDRs and the light chain CDRs is one selected from a row marked by any one of the combinations CDRs-A-001 to CDRs-A- 044, in each CDR independently optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • Table B preferred combinations of heavy chain CDRs and preferred combinations of light chain CDRs
  • the ABP or biABP may be an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequence, and at least one, preferably two, antibody light chain sequence, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprises a variable region sequence in a combination of heavy and light chain variable domain shown in Table C (eg, selected from any of the variable chain combinations Chains-A-001 to Chains-A-044); in each case independently, optionally with no more than ten, nine, eight, seven, six, five, four, preferably no more than three, two or one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • Table C eg, selected from any of the variable chain combinations Chains-A-001 to Chains-A-044
  • the ABP or biABP of the invention are all characterized by an advantageous and surprising on-target and off- target binding affinity profile.
  • the present invention is some embodiments pertain to ABPs or biABPs binding to ULRB1 and ULRB2, while not binding to LILRA1 and not binding to ULRA3.
  • the ABP or biABP of the invention is an ABP or biABP wherein the binding of the ABP or biABP to any of the on target LILRB1 or ULRB2 is at least 2 or 3 times higher than to any of the off targets ULRA1 or ULRA3, preferably wherein the binding affinity is measured by BU in KD (monovalent) under conditions set out in the example section of this disclosure.
  • the invention provides for the first time, advantageous binding profiles, wherein the binding of the ABP or biABP to any of the on target LILRB1 or ULRB2 is at least 5, or preferably 10 times higher than to any of the off targets LILRA1 or ULRA3, preferably wherein the binding affinity is measured by BLI in KD (monovalent) under conditions set out in the example section of this disclosure.
  • the ABP or biABP has a variable heavy chain sequence that is at least 90%, preferably 95%, 96%, 97%, 98% or 99% identical to the variable heavy chain sequence shown in any one of ABPs A-001 to A-048; and/or wherein, the ABP or biABP has a variable light chain sequence that is at least 90%, preferably 95%, 96%, 97%, 98% or 99% identical to the variable light chain sequence shown in any one of ABPs A-001 to A-048.
  • an ABP or biABP of the invention can comprise a combination of heavy chain CDR1, CDR2 and CDR3 sequences and a combination of light chain CDR1, CDR2 and CDR3 sequences in the combination shown by antibody A-010, such as shown in Table B by row CDRs-A-010 (eg, heavy chain CDR1, CDR2 and CDR3 having a sequence shown by SEQ ID Nos, 73, 74 and 75, respectively, and light chain CDR1, CDR2 and CDR3 having a sequence shown by SEQ ID Nos, 77, 78 and 79, respectively), in each CDR independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • an ABP or biABP of the invention can be an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences, and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences each comprises heavy chain CDR1 to CDR3 sequences in the combination CDRs- A-010 and at least one, preferably both, of the antibody light chain sequences each comprises light chain CDR1 to CDR3 sequences in the combination shown in the row of Table B marked by CDRs-A-010, in each CDR independently, optionally with no more than one amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • an ABP or biABP of the invention can be an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequence, and at least one, preferably two, antibody light chain sequence, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprises a variable region sequence in a combination of heavy and light chain variable domain shown the row of Table B marked by Chains-A-010.
  • the ABP or biABP is able to inhibit the binding of HLA-G protein or a variant thereof to LILRB1 and/or ULRB2 protein or a variant thereof with an IC50 of 20nM or less or lOnM or less, such as 5nM or less, or preferably 2nM or less.
  • IC50s can be determined using the methods described elsewhere herein.
  • an ABP or biABP of the invention can comprise a combination of heavy chain CDR1, CDR2 and CDR3 sequences and a combination of light chain CDR1, CDR2 and CDR3 sequences in the combination shown by antibody A-026, such as shown in Table B by row CDRs-A-026 (eg, heavy chain CDR1, CDR2 and CDR3 having a sequence shown by SEQ ID Nos, 201, 202 and 203, respectively, and light chain CDR1, CDR2 and CDR3 having a sequence shown by SEQ ID Nos 205, 206 and 207, respectively), in each CDR independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • an ABP or biABP of the invention can be an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences, and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences each comprises heavy chain CDR1 to CDR3 sequences in the combination CDRs- A-026 and at least one, preferably both, of the antibody light chain sequences each comprises light chain CDR1 to CDR3 sequences in the combination shown in the row of Table B marked by CDRs-A-026, in each CDR independently, optionally with no more than one amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • an ABP or biABP of the invention can be an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequence, and at least one, preferably two, antibody light chain sequence, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprises a variable region sequence in a combination of heavy and light chain variable domain shown the row of Table B marked by Chains-A-026.
  • the ABP or biABP is able to inhibit the binding of HLA-G protein or a variant thereof to LILRB1 and/or ULRB2 protein or a variant thereof with an IC50 of 20nM or less or lOnM or less, such as 5nM or less, or preferably 2nM or less.
  • IC50s can be determined using the methods described elsewhere herein.
  • an ABP or biABP of the invention can comprise a combination of heavy chain CDR1, CDR2 and CDR3 sequences and a combination of light chain CDR1, CDR2 and CDR3 sequences in the combination shown by antibody A-045, such as shown in Table B by row CDRs-A-045 (eg, heavy chain CDR1, CDR2 and CDR3 having a sequence shown by SEQ ID Nos, 353, 354, and 355, respectively, and light chain CDR1, CDR2 and CDR3 having a sequence shown by SEQ ID Nos, 357, 358, and 359, respectively), in each CDR independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • an ABP or biABP of the invention can be an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences, and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences each comprises heavy chain CDR1 to CDR3 sequences in the combination CDRs-A-045 and at least one, preferably both, of the antibody light chain sequences each comprises light chain CDR1 to CDR3 sequences in the combination shown in the row of Table B marked by CDRs-A-045, in each CDR independently, optionally with no more than one amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • an ABP or biABP of the invention can be an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequence, and at least one, preferably two, antibody light chain sequence, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprises a variable region sequence in a combination of heavy and light chain variable domain shown the row of Table B marked by Chains-A-045.
  • the ABP or biABP is able to inhibit the binding of HLA-G protein or a variant thereof to ULRB1 and/or LILRB2 protein or a variant thereof with an IC50 of 20nM or less or lOnM or less, such as 5nM or less.
  • IC50s can be determined using the methods described elsewhere herein (see Example 3).
  • the ABP or biABP of this embodiment has a binding affinity to ULRB1 and/or ULRB2 as measured in KD (monovalent) of less than 20nM, more preferably of less than lOnM more preferably of less than 5nM, wherein the binding affinity ranges are provided preferably for both binding to ULRB1 and ULRB2, as measured using BU under conditions as described in the examples.
  • the ABP or biABP of this embodiment is preferably a ULRB1 and ULRB2 ABP or biABP.
  • such antibody is further characterized by a significantly lower binding affinity to the counter targets LILRA1 and ULRA3, wherein the binding is with less affinity compared to the binding to ULRB1 and/or ULRB2.
  • the binding affinity as measured in KD (monovalent) of such ABP or biABP of this embodiment to ULRA1 and ULRA3 is higher than 50 nM, more preferably higher lOOnm, more preferably higher than 200 nM, and most preferably higher than 400 nM, also as measured using BLI under conditions described herein in the example section.
  • an ABP or biABP of the invention can comprise a combination of heavy chain CDR1, CDR2 and CDR3 sequences and a combination of light chain CDR1, CDR2 and CDR3 sequences in the combination shown by antibody A-046, such as shown in Table B by row CDRs-A-046 (eg, heavy chain CDR1, CDR2 and CDR3 having a sequence shown by SEQ ID Nos, 361, 362, and 363, respectively, and light chain CDR1, CDR2 and CDR3 having a sequence shown by SEQ ID Nos, 365, 366, and 367, respectively), in each CDR independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • an ABP or biABP of the invention can be an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences, and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences each comprises heavy chain CDR1 to CDR3 sequences in the combination CDRs-A-046 and at least one, preferably both, of the antibody light chain sequences each comprises light chain CDR1 to CDR3 sequences in the combination shown in the row of Table B marked by CDRs-A-046, in each CDR independently, optionally with no more than one amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • an ABP or biABP of the invention can be an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequence, and at least one, preferably two, antibody light chain sequence, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprises a variable region sequence in a combination of heavy and light chain variable domain shown the row of Table B marked by Chains-A-046.
  • the ABP or biABP is able to inhibit the binding of HLA-G protein or a variant thereof to ULRB1 and/or LILRB2 protein or a variant thereof with an IC50 of 20nM or less or lOnM or less, such as 5nM or less.
  • IC50s can be determined using the methods described elsewhere herein (see Example 3).
  • the ABP or biABP of this embodiment has a binding affinity to ULRB1 and/or ULRB2 as measured in KD (monovalent) of less than lOOnM, more preferably of less than 50nM more preferably of less than 15nM, wherein the binding affinity ranges are provided preferably for both binding to ULRB1 and ULRB2, as measured using BU under conditions as described in the examples.
  • the ABP or biABP of this embodiment is preferably a ULRB1 and ULRB2 ABP or biABP.
  • such antibody is further characterized by a significantly lower binding affinity to the counter targets LILRA1 and ULRA3, wherein the binding is with less affinity compared to the binding to ULRB1 and/or ULRB2.
  • the binding affinity as measured in KD (monovalent) of such ABP or biABP of this embodiment to ULRA1 and LILRA3 is higher than 100 nM, more preferably higher 500nm, more preferably higher than 1000 nM, and most preferably higher than 2000 nM, also as measured using BU under conditions described herein in the example section.
  • an ABP or biABP of the invention can comprise a combination of heavy chain CDR1, CDR2 and CDR3 sequences and a combination of light chain CDR1, CDR2 and CDR3 sequences in the combination shown by antibody A-047, such as shown in Table B by row CDRs-A-047 (eg, heavy chain CDR1, CDR2 and CDR3 having a sequence shown by SEQ ID Nos, 369, 370, and 371, respectively, and light chain CDR1, CDR2 and CDR3 having a sequence shown by SEQ ID Nos, 373, 374, and 375, respectively), in each CDR independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • an ABP or biABP of the invention can be an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences, and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences each comprises heavy chain CDR1 to CDR3 sequences in the combination CDRs-A-047 and at least one, preferably both, of the antibody light chain sequences each comprises light chain CDR1 to CDR3 sequences in the combination shown in the row of Table B marked by CDRs-A-047, in each CDR independently, optionally with no more than one amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • an ABP or biABP of the invention can be an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequence, and at least one, preferably two, antibody light chain sequence, wherein the antibody heavy chain sequence and the antibody light chain sequence each comprises a variable region sequence in a combination of heavy and light chain variable domain shown the row of Table B marked by Chains-A-047.
  • the ABP or biABP of this embodiment has a binding affinity to LILRB1 and ULRB2 as measured in KD (monovalent) of less than 20nM, more preferably of less than lOnM more preferably of less than 5nM, wherein the binding affinity ranges are provided preferably for both binding to ULRB1 and ULRB2, as measured using BU under conditions as described in the examples.
  • the ABP or biABP of this embodiment is preferably a ULRB1 and ULRB2 ABP or biABP.
  • isolated refers to a protein that is purified from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research or other use.
  • An isolated ABP or biABP according to the invention may be a recombinant, synthetic or modified (non-natural) ABP or biABP.
  • isolated refers to a nucleic acid or cells that is/are purified from DNA, RNA, proteins or polypeptides or other contaminants (such as other cells) that would interfere with its therapeutic, diagnostic, prophylactic, research or other use, or it refers to a recombinant, synthetic or modified (non-natural) nucleic acid.
  • an isolated ABP or biABP or nucleic acid or cells is/are substantially pure.
  • a "recombinant" protein or nucleic acid is one made using recombinant techniques. Methods and techniques for the production of recombinant nucleic acids and proteins are well known in the art.
  • an ABP or biABP of the invention may bind to (e.g., via one or more epitope(s) displayed by one or more EC domain(s) of) LJLRBl and/or LLLRB2 or a paralogue, orthologue or other variant thereof (such as any LILRB1 and/or LILRB2 or variant described herein) with a KD that is less than 20nM, such as less than about lOnM, 5nM or 2nM (in particular, less than about 1 nM).
  • the ABP or biABP of the invention will bind (e.g.
  • the ABP or biABP of the invention will bind said LILRB1 and/or LLLRB2 or variant with a KD that is less than 10 pM. In a most preferred embodiment, the ABP or biABP of the invention will bind said ULRB1 and/or LILRB2 or variant with a KD that is less than 2 pM.
  • Binding of an ABP or biABP of the invention, such as an antibody of the invention, to a human cell line expressing said LILRB1 and/or ULRB2 or variant may, in some embodiments, occur at an EC50 of less than about lOpg/mL, 5pg/mL, 2pg/mL, lpg/mL, 0.5pg/mL or 0.2pg/mL, preferably with an EC50 of less than 2pg/mL.
  • Binding of an ABP or biABP of the invention, such as an antibody of the invention, to a Cynomolgus cell line expressing an orthologue of said ULRB1 and/or LILRB2 or variant may, in some embodiments, occur at an EC50 of less than about lOpg/mL, 5pg/mL, 2pg/mL, lpg/mL, 0.5pg/mL or 0.2pg/mL, preferably with an EC50 of less than 2pg/mL.
  • an ABP or biABP of the invention may: (i) bind to the ULRB1 and/or LILRB2, or to the variant of LILRB1 and/or ULRB2, with a KD that is less than 20nM, such as less than about lOnM, 5nM or 2nM (in particular, less than about 1 nM), is less than 100 pM, or is less than 10 pM; and/or (ii) binds to a human cell line expressing the LILRB1 and/or ULRB2 or the variant of LILRB1 and/or ULRB2 with an EC50 of less than 2ug/mL.
  • an ABP or biABP of the invention may not bind to LILRB counter targets LILRAs, such as specifically not to LILRA1 and ULRA3, or a orthologue or other variant thereof.
  • the ABP or biABP of the invention binds to one or more, or any of, a ULRA protein (preferably an ECD thereof) with a KD that at least 2 fold higher than the KD of its binding to LILRB1 and/or ULRB2, more preferably with a KD that is at least 3 fold, 4 fold 5 or 10 fold higher.
  • the ABP or biABP of the invention binds to an ULRA protein with a KD of more than lOOnM, preferably more than 50nm, even more preferably of more than 20nM.
  • KD is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i. e., Kd/Ka) and is expressed as a molar concentration (M).
  • KD values for antibodies can be determined using methods well established in the art such as plasmon resonance (BIAcore®), ELISA and KINEXA.
  • a preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a BIAcore® system or by EUSA.
  • Another preferred method of the present invention to determine KD values for ABPs or biABPs of the invention is by bio-layer interferometry (BU), such as using the Octet Red® system.
  • Ka (or “K-assoc”), as used herein, refers broadly to the association rate of a particular antibody-antigen interaction
  • Kd or “K-diss”
  • an ABP or biABP of the invention may compete for binding to ULRB1 and/or ULRB2, or to the variant of LILRB1 and/or ULRB2, with an endogenous LILRB1 and/or ULRB2 ligand or receptor, preferably wherein said endogenous ULRB1 and/or LILRB2 ligand or receptor is HLA-G (or a variant of HLA-G).
  • the ABP or biABP of the invention eg one that binds to [one or more epitope(s) displayed by] an extracellular domain(s) of ULRB1 and/or LILRB2, or a paralogue, orthologue or other variant thereof
  • an ABP or biABP of the invention is capable of inhibiting (eg will inhibit) the binding of HLA-G protein or a variant thereof to ULRB1 and/or ULRB2 protein or a variant thereof with an IC50 of lOnM or less, such as 5nM or less and preferably 2nM or less.
  • the ABP or biABP is an antibody or an antigen binding fragment thereof, and the antibody is a monoclonal antibody, or wherein the antigen binding fragment is a fragment of a monoclonal antibody.
  • the term "monoclonal antibody” or "mAb” as used herein refers to an antibody obtained from a population of substantially identical antibodies based on their amino acid sequence. Monoclonal antibodies are typically highly specific. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (e.g. epitopes) of an antigen, each mAb is typically directed against a single determinant on the antigen.
  • mAbs are advantageous in that they can be synthesized by cell culture (hybridomas, recombinant cells or the like) uncontaminated by other immunoglobulins.
  • the mAbs herein include for example chimeric, humanized or human antibodies or antibody fragments.
  • Monoclonal antibodies in accordance with the present invention may be prepared by methods well known to those skilled in the art. For example, mice, rats or rabbits may be immunized with an antigen of interest together with adjuvant. Splenocytes are harvested as a pool from the animals that are administered several immunisations at certain intervals with test bleeds performed to assess for serum antibody titers. Splenocytes are prepared that are either used immediately in fusion experiments or stored in liquid nitrogen for use in future fusions. Fusion experiments are then performed according to the procedure of Stewart & Fuller, J. Immunol. Methods 1989, 123:45-53.
  • ELISA-positive cultures are cloned either by limiting dilutions or fluorescence-activated cell sorting, typically resulting in hybridomas established from single colonies.
  • the ability of an antibody, including an antibody fragment or subfragment, to bind to a specific antigen can be determined by binding assays known in the art, for example, using the antigen of interest as the binding partner.
  • Phage are "screened” to identify those antibody fragments having affinity for target.
  • certain such processes mimic immune selection through the display of antibody fragment repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to target.
  • high affinity functional neutralizing antibody fragments are isolated.
  • a complete repertoire of human antibody genes may thus be created by cloning naturally rearranged human V genes from peripheral blood lymphocytes (see, e.g., Mullinax et al., Proc Natl Acad Sci (USA), 87: 8095-8099 (1990)) or by generating folly synthetic or semi-synthetic phage display libraries with human antibody sequences (see Knappik et al 2000; J Mol Biol 296:57; de Kruif et al, 1995; J Mol Biol 248): 97).
  • mice are capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of murine immunoglobulin molecules and antibodies.
  • a preferred embodiment of transgenic production of mice and antibodies is disclosed in U.S. Patent Application Serial No. 08/759,620, filed December 3, 1996 and International Patent Application Nos. WO 98/24893, published June 11, 1998 and WO 00/76310, published December 21, 2000. See also Mendez et aL, Nature Genetics, 15:146-156 (1997). Through the use of such technology, fully human monoclonal antibodies to a variety of antigens have been produced.
  • XenoMouse® lines of mice are immunized with an antigen of interest, e.g. ULRB1 and/or ULRB2, lymphatic cells (such as B-cells) are recovered from the hyper-immunized mice, and the recovered lymphocytes are fused with a myeloid-type cell line to prepare immortal hybridoma cell lines.
  • lymphatic cells such as B-cells
  • myeloid-type cell line to prepare immortal hybridoma cell lines.
  • mice are also commercially available: eg, Medarex - HuMab mouse, Kymab - Kymouse, Regeneron - Velocimmune mouse, Kirin -TC mouse, Trianni -Trianni mouse, OmniAb - OmniMouse, Harbour Antibodies - H2L2 mouse, Merus - MeMo mouse. Also are available are “humanised” other species: rats: OmniAb - OmniRat, OMT - UniRat. Chicken: OmniAb - OmniChicken.
  • humanised antibody refers to immunoglobulin chains or fragments thereof (such as Fab, Fab', F(ab')2, Fv, or other antigen-binding sub-sequences of antibodies), which contain minimal sequence (but typically, still at least a portion) derived from non-human immunoglobulin.
  • humanised antibodies are human immunoglobulins (the recipient antibody) in which CDR residues of the recipient antibody are replaced by CDR residues from a non-human species immunoglobulin (the donor antibody) such as a mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • the framework sequence of said antibody or fragment thereof may be a human consensus framework sequence.
  • humanised antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximise antibody performance.
  • the humanised antibody will comprise substantially all of at least one, and typically at least two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanised antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, which (eg human) immunoglobulin constant region may be modified (eg by mutations or glycoengineering) to optimise one or more properties of such region and/or to improve the function of the (eg therapeutic) antibody, such as to increase or reduce Fc effector functions or to increase serum half-life.
  • an immunoglobulin constant region typically that of a human immunoglobulin, which (eg human) immunoglobulin constant region may be modified (eg by mutations or glycoengineering) to optimise one or more properties of such region and/or to improve the function of the (eg therapeutic) antibody, such as to increase or reduce Fc effector functions or to increase serum half-life.
  • Fc modification for example, Fc engineering or Fc enhancement
  • chimeric antibody refers to an antibody whose light and/or heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant regions which are identical to, or homologous to, corresponding sequences of different species, such as mouse and human.
  • variable region genes derive from a particular antibody class or subclass while the remainder of the chain derives from another antibody class or subclass of the same or a different species. It covers also fragments of such antibodies.
  • a typical therapeutic chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species may be used.
  • an ABP or biABP of the invention comprises an antigen binding domain of an antibody wherein the antigen binding domain is of a human antibody.
  • ABP or biABP comprises an antigen binding domain of an antibody or an antigen binding fragment thereof, which is a human antigen binding domain; (ii) the antibody is a monoclonal antibody, or wherein the antigen binding fragment is a fragment of a monoclonal antibody; and (iii) the antibody is a human antibody or a humanised antibody, or wherein the antigen binding fragment is a fragment of a human antibody, a humanised antibody or a chimeric-human antibody.
  • Light chains of human antibodies generally are classified as kappa and lambda light chains, and each of these contains one variable region and one constant domain. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon chains, and these define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • Human IgG has several subtypes, including, but not limited to, IgGl, lgG2, lgG3, and lgG4.
  • Human IgM subtypes include IgM, and lgM2.
  • Human IgA subtypes include IgAl and lgA2.
  • the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains ten or twelve heavy chains and ten or twelve light chains.
  • Antibodies according to the invention may be IgG, IgE, IgD, IgA, or IgM immunoglobulins.
  • the ABP or biABP of the invention is an IgG antibody or fragment thereof. In some embodiments, the ABP or biABP of the invention is an IgE antibody or fragment thereof. In some embodiments, the ABP or biABP of the invention is an IgD antibody or fragment thereof. In some embodiments, the ABP or biABP of the invention is an IgA antibody or fragment thereof. In some embodiments, the ABP or biABP of the invention is an IgM antibody or fragment thereof.
  • the ABP or biABP of the invention is, comprises or is derived from an IgG immunoglobulin or fragment thereof; such as a human, human-derived IgG immunoglobulin, or a rabbit- or rat-derived IgG, and/or an IgG2 immunoglobulin, or fragment thereof.
  • an IgG immunoglobulin or fragment thereof such as a human, human-derived IgG immunoglobulin, or a rabbit- or rat-derived IgG, and/or an IgG2 immunoglobulin, or fragment thereof.
  • the ABP or biABP of the invention comprises or is derived from a rat-derived IgG
  • the ABP or biABP is, comprises or is derived from, a rat IgG2a or IgG2b immunoglobulin.
  • the ABP or biABP of the invention comprises or is derived from a human-derived IgG
  • the ABP or biABP of the invention comprises or is derived from a human IgGl, IgG2 or IgG4
  • the ABP of the invention is, comprises or is derived from a human IgGl, IgG2, or IgG4.
  • an ABP is an antibody wherein the antibody is an IgG, IgE, IgD, IgA, or IgM immunoglobulin; preferably an IgG immunoglobulin.
  • An ABP of the invention where comprising at least a portion of an immunoglobulin constant region (typically that of a human immunoglobulin) may have such (eg human) immunoglobulin constant region modified - for example eg by glycoengineering or mutations - to optimise one or more properties of such region and/or to improve the function of the (eg therapeutic) antibody, such as to increase or reduce Fc effector functions or to increase serum half-life.
  • an immunoglobulin constant region typically that of a human immunoglobulin
  • modified - for example eg by glycoengineering or mutations - to optimise one or more properties of such region and/or to improve the function of the (eg therapeutic) antibody, such as to increase or reduce Fc effector functions or to increase serum half-life.
  • ABPs or biABPs of the invention include antibodies that induce antibody-dependent cytotoxicity (ADCC) of ULRB1 and/or LJLRB2-expressing cells.
  • ADCC antibody-dependent cytotoxicity
  • the ADCC of an anti-LILRBl and/or LILRB2 antibody can be improved by using antibodies that have low levels of or lack fucose.
  • Antibodies lacking fucose have been correlated with enhanced ADCC (antibody- dependent cellular cytotoxicity) activity, especially at low doses of antibody (Shields et ah, 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et ah, 2003, J. Biol. Chem. 278:3466).
  • Methods of preparing fucose-less antibodies or antibodies with reduced fucose levels include growth in rat myeloma YB2/0 cells (ATCC CRL 1662).
  • YB 2/0 cells express low levels of FUT8 mRNA, which encodes an enzyme (.alpha. 1,6- fucosyltransferase) necessary for focosylation of polypeptides.
  • an inhibitor against an enzyme relating to the modification of a sugar chain may be used, including: tunicamycin which selectively inhibits formation of GIcNAc-P-P- Dol which is the first step of the formation of a core oligosaccharide which is a precursor of an N-glycoside-linked sugar chain, castanospermin and W-methyl-l-deoxynojirimycin which are inhibitors of glycosidase I, kifonensine which is an inhibitor of mannosidase I, bromocondulitol which is an inhibitor of glycosidase II, 1 - deoxynojirimycin and 1 ,4-dioxy- 1 ,4-imino-D-mannitol which are inhibitors of mannosidase I, swainsonine which is an inhibitor of mannosidase II and the like.
  • Examples of an inhibitor specific for a glycosyltransferase include deoxy derivatives of substrates against N- acetylglucosamine transferase V (GnTV) and the like. Also, it is known that 1 -deoxynojirimycin inhibits synthesis of a complex type sugar chain and increases the ration of high mannose type and hybrid type sugar chains (Glycobiology series 2 -Destiny of Sugar Chain in Cell, edited by Katsutaka Nagai, Senichiro Hakomori and Akira Kobata, 1993).
  • GLYCART BIOTECHNOLOGY AG (Zurich, CH) has expressed N-acetyl-glucosaminyltransferase III (GnTIII) which catalyses the addition of the bisecting GIcNac residue to the N-linked oligosaccharide, in a Chinese hamster ovary (CHO) cell line, and showed a greater ADCC of IgGl antibody produced (WO 99/54342; WO 03/01 1878; WO 2005/044859).
  • GnTIII N-acetyl-glucosaminyltransferase III
  • W020070166306 is related to the modification of an antibody anti-CD19 containing 60% N-acetylglucosamine bisecting oligosaccharides and 10% non-fucosylated N-acetylglucosamine bisecting oligosaccharides produced in a mammalian human 293T embryonal kidney cells transfected with (i) the cDNA for the anti-CD19 antibody and (ii) the cDNA for the GnTIII enzyme.
  • Herbst et al. generated a humanized IgGl MAb MEDI-551 expressed in a fucosyltransferase-deficient producer CHO cell line This paper does not consider amino acid mutations (Herbst et al., 2010).
  • S. Siberil et al used the rat myeloma YB2/0 cell line to produce a MAb anti RhD with a low fucose content. Whereas the MAb produced in a wild type CHO exhibited a high fucose content (81 %), the same MAb produced in YB2/0 cell exhibiited a lower fucose content (32%). This paper does consider amino acid mutations (Siberil et aL, 2006).
  • an ABP or biABP of the invention may be prepared and/or may have one or more of the characteristics of such glycoengineering (eg afocosylated) approaches/antibodies described above.
  • Alternative methods for increasing ADDC activity for an ABP or biABP of the invention include mutations in an Fc portion of such ABP or biABP, particularly mutations which increase antibody affinity for an Fc-gamma-R receptor.
  • any of the ABPs or biABPs of the invention described above can be produced with different antibody isotypes or mutant isotypes to control the extent of binding to different Fc-gamma receptors.
  • Antibodies lacking an Fc region e.g., Fab fragments
  • Selection of isotype also affects binding to different Fc-gamma receptors.
  • the respective affinities of various human IgG isotypes for the three different Fc-gamma receptors, Fc-gamma-RI, Fc- gamma-RII, and Fc- gamma-RIII, have been determined. (See Ravetch 8i Kinet, Annu. Rev. Immunol.
  • Fc- gamma-RI is a high affinity receptor that binds to IgGs in monomeric form, and the latter two are low affinity receptors that bind IgGs only in multimeric form.
  • IgGl and IgG3 have significant binding activity to all three receptors, IgG4 to Fc-gamma-RI, and IgG2 to only one type of Fc- gamma-RII called IlaLR (see Parren et al., J. Immunol. 148, 695 (1992). Therefore, human isotype IgGl is usually selected for stronger binding to Fc-gamma receptors, and IgG2 or IgG4 is usually selected for weaker binding.
  • Methods for increasing ADCC activity through specific Fc region mutations include the Fc variants comprising at least one amino acid substitution at a position selected from the group consisting of: 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328, 329, 330 and 332, wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat (Kabat et ah, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987).
  • said Fc variants comprise at least one substitution selected from the group consisting of L234D, L234E, L234N, L234Q, L234T, L234H, L234Y, L234I, L234V, L234F, L235D, L235S, L235N, L235Q, L235T, L235H, L235Y, L235I, L235V, L235F, S239D, S239E, S239N, S239Q, S239F, S239T, S239H, S239Y, V240I, V240A, V240T, V240M, F241W, F241L, F241Y, F241E, F241R, F243W, F243L, F243Y, F243R, F243Q, P244H, P245A, P247V, P247G, V262I, V262A, V262T, V26
  • Fc variants can also be selected from the group consisting of V264L, V264I, F241W, F241L, F243W, F243L,
  • F243L/V262I/V264W F241Y/F243Y/V262T/V264T, F241E/F243R/V262E/V264R, F241E/F243Q/V262T/V264E,
  • F241R/F243Q/V262T/V264R F241E/F243Y/V262T/V264R, L328M, L328E, L328F, I332E, L3238M/I332E, P244H, P245A, P247V, W313F, P244H/P245A/P247V, P247G, V264I/I332E, F241E/F243R/V262E/V264R/I332E,
  • mutations on, adjacent, or close to sites in the hinge link region can be made, in all of the isotypes, to reduce affinity for Fc- gamma receptors, particularly Fc-gamma-RI receptor (see, eg US6624821).
  • positions 234, 236 and/or 237 are substituted with alanine and position 235 with glutamate. (See, eg US5624821.)
  • Position 236 is missing in the human IgG2 isotype.
  • Exemplary segments of amino acids for positions 234, 235 and 237 for human IgG2 are Ala Ala Gly, Vai Ala Ala, Ala Ala Ala, Vai Glu Ala, and Ala Glu Ala.
  • a preferred combination of mutants is L234A, L235E and G237A, or is L234A, L235A, and G237A for human isotype IgGl.
  • a particular preferred ABP or biABP of the invention is an antibody having human isotype IgGl and one of these three mutations of the Fc region.
  • Other substitutions that decrease binding to Fc-gamma receptors are an E233P mutation (particularly in mouse IgGl) and D265A (particularly in mouse IgG2a).
  • mutations and combinations of mutations reducing Fc and/or Clq binding are E318A/K320A/R322A (particularly in mouse IgGl), L235A/E318A/K320A/K322A (particularly in mouse IgG2a).
  • residue 241 (Ser) in human IgG4 can be replaced, eg with proline to disrupt Fc binding.
  • Additional mutations can be made to a constant region to modulate effector activity.
  • mutations can be made to the IgGl or IgG2 constant region at A330S, P331S, or both.
  • mutations can be made at E233P, F234V and L235A, with G236 deleted, or any combination thereof.
  • IgG4 can also have one or both of the following mutations S228P and L235E.
  • the use of disrupted constant region sequences to modulate effector function is further described, eg in WO2006118,959 and W02006036291.
  • Additional mutations can be made to the constant region of human IgG to modulate effector activity (see, e.g., W0200603291). These include the following substitutions: (i) A327G, A330S, P331S; (ii) E233P, L234V, L235A, G236 deleted; (iii) E233P, L234V, L235A; (iv) E233P, L234V, L235A, G236 deleted, A327G, A330S, P331S; and (v) E233P, L234V, L235A, A327G, A330S, P331S to human IgGl; or in particular, (vi) L234A, L235E, G237A, A330S and P331S (eg, to human IgGl), wherein the numbering of the residues in the Fc region is that of the EU index as in Kabat. See also W02004029207, incorporated by reference
  • the affinity of an antibody for the Fc-gamma-R can be altered by mutating certain residues of the heavy chain constant region. For example, disruption of the glycosylation site of human IgGl can reduce Fc-gamma-R binding, and thus effector function, of the antibody (see, eg W02006036291).
  • the tripeptide sequences NXS and NXT, where X is any amino acid other than proline, are the enzymatic recognition sites for glycosylation of the N residue. Disruption of any of the tripeptide amino acids, particularly in the CH2 region of IgG, will prevent glycosylation at that site. For example, mutation of N297 of human IgGl prevents glycosylation and reduces Fc-gamma-R binding to the antibody.
  • ADCC and CDC activation of ADCC and CDC is often desirable for therapeutic antibodies
  • an ABP or biABP of the invention unable to activate effector functions is preferential (eg, an ABP or biABP of the invention that is an agnostic modulator).
  • an ABP or biABP of the invention that is an agnostic modulator.
  • IgG4 has commonly been used but this has fallen out of favour in recent years due the unique ability of this sub-class to undergo Fab-arm exchange, where heavy chains can be swapped between IgG4 in vivo as well as residual ADCC activity.
  • Fc engineering approaches can also be used to determine the key interaction sites for the Fc domain with Fc-gamma receptors and Clq and then mutate these positions, such as in an Fc of an ABP or biABP of the invention, to reduce or abolish binding.
  • Fc-gamma receptors and Clq Through alanine scanning Duncan and Winter (1998; Nature 332:738) first isolated the binding site of Clq to a region covering the hinge and upper CH2 of the Fc domain.
  • researchers at Genmab identified mutants K322A, L234A and L235A, which in combination are sufficient to almost completely abolish Fc-gamma-R and Clq binding (Hezareh et al, 2001; J Virol 75:12161).
  • the invention also includes embodiments of the ABPs or biABPs in which such technologies or mutations have been used to reduce effector functions.
  • IgG naturally persists for a prolonged period in (eg human) serum due to FcRn-mediated recycling, giving it a typical half-life of approximately 21 days. Despite this there have been a number of efforts to engineer the pH dependant interaction of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4.
  • ABPs or biABPs of the invention may also be PEGylated.
  • PEGylation ie chemical coupling with the synthetic polymer poly-ethylene glycol (PEG)
  • PEG poly-ethylene glycol
  • ABPs or biABPs of the invention may also be subjected to PASylation, a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins (Schlapschy et al, 2013; Protein Eng Des Sei 26:489; XL-protein GmbH, Germany). Similarily, the XTEN half-life extension technology from Amunix provides another biological alternative to PEGylation (Schellenberger, 2009, Nat Biotechnol. ;27(12): 1186- 90. doi: 10.1038/nbt.l588). Accordingly, the invention also includes embodiments of the ABPs or biABPs in which such technologies or mutations have been used to prolong serum half-life, especially in human serum.
  • Antibody fragments include "Fab fragments", which are composed of one constant and one variable domain of each of the heavy and the light chains, held together by the adjacent constant region of the light chain and the first constant domain (CHI) of the heavy chain. These may be formed by protease digestion, e.g. with papain, from conventional antibodies, but similar Fab fragments may also be produced by genetic engineering. Fab fragments include Fab', Fab and "Fab-SH" (which are Fab fragments containing at least one free sulfhydryl group).
  • Fab' fragments differ from Fab fragments in that they contain additional residues at the carboxy terminus of the first constant domain of the heavy chain including one or more cysteines from the antibody hinge region.
  • Fab' fragments include "Fab'-SH" (which are Fab' fragments containing at least one free sulfhydryl group).
  • antibody fragments include F(ab')2 fragments, which contain two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains ("hinge region"), such that an interchain disulphide bond is formed between the two heavy chains.
  • a F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulphide bond between the two heavy chains.
  • F(ab')2 fragments may be prepared from conventional antibodies by proteolytic cleavage with an enzyme that cleaves below the hinge region, e.g. with pepsin, or by genetic engineering.
  • An “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
  • Single-chain antibodies or “scFv” are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region.
  • An “Fc region” comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulphide bonds and by hydrophobic interactions of the CH3 domains.
  • the ABP or biABP of the invention is an antibody fragment selected from the list consisting of: Fab' Fab, Fab'-SH, Fab-SH, Fv, scFv and F(ab')2.
  • ABPs or biABPs that are fragments of immunoglobulins, such as an antibody fragment
  • those fragments capable of binding to eg an epitope displayed by) the extracellular domain(s) of LILRB1 and/or LILRB2, or a paralogue, orthologue or other variant thereof, such as any epitope or other binding characteristic as described herein: and more preferably said fragment is a modulator (such as an inhibitor or antagonist) of the expression, function, activity and/or stability of ULRB1 and/or LILRB2 or a paralogue, orthologue or other variant of ULRB1 and/or ULRB2.
  • a modulator such as an inhibitor or antagonist
  • an ABP or biABP of the invention is an antibody wherein at least a portion of the framework sequence of said antibody or fragment thereof is a human consensus framework sequence, for example, comprises a human germline-encoded framework sequence.
  • an ABP or biABP of the invention is modified or engineered to increase antibodydependent cellular cytotoxicity (ADCC).
  • ADCC antibodydependent cellular cytotoxicity
  • therapy is synonymous with treating a disease, disorder or condition, which includes reducing symptoms of the disease, disorder or condition, inhibiting progression of the disease, disorder or condition, causing regression of the disease, disorder or condition and/or curing the disease, disorder or condition.
  • an ABP or biABP of the invention may be afocosylated (GlycArt Biotechnology) e.g., in which antibodies are produced in CHO cells in which the endogenous FUT8 gene has been knocked out; or the ABP or biABP may be a "Sugar-Engineered Antibody” (Seattle Genetics), e.g. in which fucose analogues are added to antibody-expressing CHO cells, resulting in a significant reduction in fucosylation.
  • GlycArt Biotechnology e.g., in which antibodies are produced in CHO cells in which the endogenous FUT8 gene has been knocked out
  • the ABP or biABP may be a "Sugar-Engineered Antibody” (Seattle Genetics), e.g. in which fucose analogues are added to antibody-expressing CHO cells, resulting in a significant reduction in fucosylation.
  • fucose analogues are added to antibody-expressing CHO cells,
  • an ABP or biABP of the invention may be PEGylated and/or PASylated, or has an Fc region with a T250Q/M428L, H433K/N434F/Y436 or M252Y/S254T/T256E/H433K/N434F modification.
  • an ABP or biABP of the invention can comprise a chimeric antigen receptor (CAR), and preferably comprises an extracellular antigen binding region, a membrane anchor such as a transmembrane domain, and an intracellular region, for example, an intracellular signalling region.
  • CAR chimeric antigen receptor
  • an ABP or biABP of the invention can comprise at least one antibody constant domain, in particular wherein at least one antibody constant domain is a CHI, CH2, or CH3 domain, or a combination thereof.
  • an ABP or biABP of the invention having antibody constant domain comprises a mutated Fc region, for example for increasing interaction of the Fc region with a Fc receptor (Fc receptor on an immune effector cell (eg Saxena &Wu, 2016; Front Immunol 7:580). Examples and embodiments thereof are described elsewhere herein.
  • effector group means any group, in particular one coupled to another molecule such as an antigen binding protein, that acts as a cytotoxic agent.
  • suitable effector groups are radioisotopes or radionuclides.
  • Other suitable effector groups include toxins, therapeutic groups, or chemotherapeutic groups.
  • suitable effector groups include calicheamicins, auristatins, geldanamycins, alpha-amanitine, pyrrolobenzodiazepines and maytansines.
  • label or “labelling group” refers to any detectable label.
  • labels fall into a variety of classes, depending on the assay in which they are to be detected: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox active moieties; d) optical dyes; enzymatic groups (e.g.
  • a secondary reporter e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.
  • an effector group or a labelling group is coupled to another molecule (such as the ABP) via spacer arms of various lengths to reduce potential steric hindrance.
  • the invention relates to a nucleic acid encoding for an ABP or biABP (or ABD) of the invention (such as one described above) or of components thereof.
  • the component encoded by a nucleic acid of the invention may be all or part of one chain of an antibody of the invention; or the component may be a scFV of said ABP or biABP.
  • the component encoded by such a nucleic acid may be all or part of one or other of the chains of an antibody of the invention; for example, the component encoded by such a nucleic acid may be an ABP or biABP of the invention.
  • the nucleic acids of the invention may also encode a fragment, derivative, mutant, or variant of an ABP or biABP of the invention, and/or represent components that are polynucleotides suitable and/or sufficient for use as hybridisation probes, polymerase chain reaction (PCR) primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense or inhibitory nucleic acids (such as RNAi/siRNA/shRNA or gRNA molecules) for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing.
  • PCR polymerase chain reaction
  • anti-sense or inhibitory nucleic acids such as RNAi/siRNA/shRNA or gRNA molecules
  • a nucleic acid of the invention comprises a nucleic acid having a sequence encoding a heavy or light chain CDR, a combination of heavy and/or light chain CDR1, CDR2 and CDR3 or a heavy or light chain variable domain, in each case as displayed in Table 1, or a functional fragment thereof.
  • a nucleic acid of the invention comprises a nucleic acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%; or 95% (preferably at least 75%) sequence identity to (or having no more than fifty, forty, thirty, twenty, fifteen, ten or five, preferably no more than three, two or one, base substitution(s), insertion(s) or deletion(s), preferably at the third base of a codon of) a nucleic acid sequence selected from the list consisting of SEQ IDS Nos. 9, 10, 19, 20, 29, 30, 39, 40, 49, 50, 59, 60, 69, 70, 79, 80, 89, 90, 99, 100, 109, 110, 119, 120, 129, 130, 139, 140,
  • the nucleic acid according to the invention may be a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof, optionally linked to a polynucleotide to which it is not linked in nature.
  • such nucleic acid may comprise one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 20, in particular between 1 and about 5, or preferably all instances of a particular nucleotide in the sequence) unnatural (e.g. synthetic) nucleotides; and/or such nucleic acid may comprise (e.g. is conjugated to) another chemical moiety, such as a labelling group or an effector group; for example, a labelling group or an effector group as described elsewhere herein.
  • the nucleic acid of the invention may be isolated or substantially pure.
  • the nucleic acid of the invention may be recombinant, synthetic and/or modified, or in any other way non-natural.
  • a nucleic acid of the invention may contain at least one nucleic acid substitution (or deletion) modification (such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 such modifications, in particular between 1 and about 5 such modifications, preferably 2 or 3 such modifications) relative to a product of nature, such as a human nucleic acid.
  • the nucleic acids can be any suitable length, such as about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125,
  • siRNA nucleic acids may, preferably, be between about 15 to about 25 base pairs in length (preferably between about 19 and about 21 base pairs in length);
  • shRNA nucleic acids may, preferably, comprise a 20-30 base pair stem, a loop of at least 4 nucleotides, and a dinucleotide overhang at the 3' end;
  • microRNA may, preferably, be about 22 base pairs in length;
  • an mRNA or DNA sequence encoding an ABP or biABP or a component thereof (such as a heavy or light chain or an IgG antibody) of the invention may, preferably, be between about 500 and 1,500 nucleotides.
  • a nucleic acid encoding a mammalian light chain of an antibody may be between about 630 and about 650 nucleotides, and one encoding a mammalian heavy chain of an antibody may be between about 1,300 and about 1,650 nucleotides.
  • a nucleic acid can comprise one or more additional sequences, for example, regulatory sequences, and/or be part of a larger nucleic acid.
  • the nucleic acids can be single-stranded or doublestranded and can comprise RNA and/or DNA nucleotides, and artificial variants thereof (e.g., peptide nucleic acids).
  • Nucleic acids encoding antibody polypeptides may be isolated from B-cells of mice, rats, llamas, alpacas, chicken or rabbits that have been immunized with an LILRB1 and/or ULRB2 antigen or fragment thereof, such as one or more EC domains (or a polynucleotide encoding and capable of expressing an LILRB1 and/or LILRB2 antigen or fragment thereof).
  • the nucleic acid may be isolated by conventional procedures such as PCR.
  • Changes can be introduced by mutation into the sequence of a nucleic acid of the invention. Such changes, depending on their nature and location in a codon, can lead to changes in the amino acid sequence of a polypeptide (e.g., an antigen binding protein) that it encodes. Mutations can be introduced using any technique known in the art.
  • one or more particular amino acid residues may be changed using, for example, a site- directed mutagenesis protocol.
  • one or more randomly selected residues may be changed using, for example, a random mutagenesis protocol.
  • a mutant polypeptide can be expressed and screened for a desired property. Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues.
  • nucleic acid of the invention may not alter the amino acid sequence of the encoded polypeptide, but may lead to changes to its stability and/or effectiveness of expression of the encoded polypeptide.
  • codon optimisation the expression of a given polypeptide sequence may be improved by utilising the more common codons for a given amino acid that are found for the species in which the nucleotide is to be expressed.
  • codon optimisation and alternative methods (such as optimisation of CpG and G/C content), are described in, for example, Hass et al, 1996 (Current Biology 6:315); WO1996/09378; W02006/015789 and WO 2002/098443).
  • the invention relates to a nucleic acid construct (NAC) comprising at least one nucleic acid of the invention (such as described above).
  • NAC nucleic acid construct
  • Such an NAC can comprise one or more additional features permitting the expression of the encoded ABP or biABP or component of said ABP or biABP (eg the ABD) in a cell (such as in a host cell).
  • NACs of the invention include, but are not limited to, plasmid vectors, viral vectors, mRNA, non-episomal mammalian vectors and expression vectors, for example, recombinant expression vectors.
  • the nucleic acid constructs of the invention can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a cell, such as a host cell, (see below).
  • the nucleic acid constructs of the invention will be, typically, recombinant nucleic acids, and/or may be isolated and/or substantially pure. Recombinant nucleic acids will, typically, be non-natural; particularly if they comprise portions that are derived from different species and/or synthetic, in-vitro or mutagenic methods.
  • an NAC of the invention comprises one or more constructs either of which includes a nucleic acid encoding either a heavy or a light antibody chain.
  • the NAC of the invention comprises two constructs, one of which includes a nucleic acid encoding the heavy antibody chain, the other of which includes a nucleic acid encoding the light antibody chain, such that expression from both constructs can generate a complete antibody molecule.
  • the NAC of the invention comprises a construct which includes nucleic acids encoding both heavy and light antibody chains, such that a complete antibody molecule can be expressed from one construct.
  • an NAC of the invention can comprise a single construct that encodes a single chain which is sufficient to form an ABP or biABP of the invention; for example, if the encoded ABP or biABP is a scFv or a single-domain antibody (such as a camelid antibody).
  • An NAC according to the invention may comprise (or consist of) a mRNA molecule which includes an open reading frame encoding an ABP or biABP of the invention, and for example together with upstream and downstream elements (such as 5' and/or 3' UTRs and/or poly-A stretch) that enables expression of the ABP or biABP, and preferably enhancing stability of the mRNA and/or expression of the ABP or biABP.
  • upstream and downstream elements such as 5' and/or 3' UTRs and/or poly-A stretch
  • UTRs that may be comprised in an mRNA NAC of the invention include: 5'UTR of a TOP gene (WO2013/143699), and/or a histone stem-loop (WO 2013/120629).
  • NACs such as DNA-, retroviral- and mRNA-based NACs of the invention may be used in genetic therapeutic methods in order to treat or prevent diseases of the immune system (see Methods of Treatment below), whereby an NAC that comprises an expressible sequence encoding an ABP or biABP of the invention is administered to the cell or organism (e.g. by transfection).
  • an NAC that comprises an expressible sequence encoding an ABP or biABP of the invention is administered to the cell or organism (e.g. by transfection).
  • mRNA therapeutics for the expression of antibodies is known from W02008/083949.
  • the invention relates to a cell (such as a host cell and/or a recombinant host cell) comprising one or more nucleic acid or NAC of the invention.
  • a cell such as a host cell and/or a recombinant host cell
  • such cell is capable of expressing the ABP or biABP (or component thereof) encoded by said NAC(s).
  • an ABP or biABP of the invention comprises two separate polypeptide chains (e.g.
  • the cell of the invention may comprise a first NAC that encodes (and can express) the heavy chain of such ABP or biABP as well as a second NAC that encodes (and can express) the light chain of such ABP; alternatively, the cell may comprise a single NAC that encodes both chains of such ABP or biABP.
  • a (host) cell of invention may be one of the mammalian, prokaryotic or eukaryotic host cells as described elsewhere herein, in particularly where the cell is a Chinese hamster ovary (CHO) cell.
  • the (host) cell is a human cell; in particular it may be a human cell that has been sampled from a specific individual (eg an autologous human cell).
  • a specific individual eg an autologous human cell
  • such human cell can be propagated and/or manipulated in-vitro so as to introduce a NAC of the present invention.
  • the utility of a manipulated human cell from a specific individual can be to produce an ABP or biABP of the invention, including to reintroduce a population of such manipulated human cells into a human subject, such as for use in therapy.
  • the manipulated human cell may be introduced into the same human individual from which it was first sampled; for example, as an autologous human cell.
  • the human cell that is subject to such manipulation can be of any germ cell or somatic cell type in the body.
  • the donor cell can be a germ cell or a somatic cell selected from the group consisting of fibroblasts, B cells, T cells, dendritic cells, keratinocytes, adipose cells, epithelial cells, epidermal cells, chondrocytes, cumulus cells, neural cells, glial cells, astrocytes, cardiac cells, oesophageal cells, muscle cells, melanocytes, hematopoietic cells, macrophages, monocytes, and mononuclear cells.
  • the donor cell can be obtained from any organ or tissue in the body; for example, it can be a cell from an organ selected from the group consisting of liver, stomach, intestines, lung, pancreas, cornea, skin, gallbladder, ovary, testes, kidneys, heart, bladder, and urethra.
  • the ABPs or biABPs, nucleic acids or NACs (or the cells, such as host cells) of the invention may be formulated into a pharmaceutical composition appropriate to facilitate administration to animals or humans.
  • pharmaceutical composition means a mixture of substances including a therapeutically active substance (such as an ABP or biABP of the invention) for pharmaceutical use.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound that is modulator of the expression, function, activity and/or stability of immunoglobulin superfamily member 11 (ULRB1 and/or ULRB2), or of a variant of ULRB1 and/or ULRB2 and a pharmaceutically acceptable carrier, stabiliser and/or excipient.
  • the LILRB1 and/or ULRB2 modulator is an ABP or biABP of the invention, and/or at least one NAC of the invention, and/or a (host) cell of the invention.
  • a pharmaceutical composition comprising an ABP or biABP of the invention, and/or at least one NAC of the invention, and/or a (host) cell of the invention, and a pharmaceutically acceptable excipient or carrier.
  • the pharmaceutical composition comprises an ABP or biABP of the invention
  • the LILRB1 and/or ULRB2 modulator is an ABP or biABP of the invention (eg an LILRB1 and/or LILRB2-inhibitory ABP or biABP of the invention).
  • the pharmaceutical composition of the invention may comprise between 0.1% and 100% (w/w) active ingredient (for example, an ULRB1 and/or LILRB2 modulator), such as about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 8% 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably between about 1% and about 20%, between about 10% and 50% or between about 40% and 90%.
  • active ingredient for example, an ULRB1 and/or LILRB2 modulator
  • the language "pharmaceutically acceptable" excipient, stabiliser or carrier is intended to include any and all solvents, solubilisers, fillers, stabilisers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary agents can also be incorporated into the compositions.
  • the pharmaceutical composition of (or for use with) the invention is, typically, formulated to be compatible with its intended route of administration.
  • routes of administration include oral, parenteral, e.g., intrathecal, intra-arterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical) and transmucosal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application, as well as comprising a compound of (or for use with) the invention can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine; propylene glycol or other synthetic solvents; anti-bacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulphate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Kolliphor® EL (formerly Cremophor ELTM; BASF, Parsippany, NJ.) or phosphate buffered saline (PBS).
  • the injectable composition should, typically, be sterile and be fluid to the extent that easy syringability exists. It should, typically, be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the requited particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the compound of (or for use with) the invention (e.g., an ULRB1 and/or LILRB2 modulator) in the required amount in an appropriate solvent with one or a combination of ingredients described herein, as required, followed by filtered sterilisation.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those described herein.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions as well as comprising a compound of (or for use with) the invention (eg an ULRB1 and/or ULRB2 inhibitor), generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Stertes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Stertes
  • a glidant such as colloidal silicon dioxide
  • a rectal composition can be any rectally acceptable dosage form including, but not limited to, cream, gel, emulsion, enema, suspension, suppository, and tablet.
  • One preferred dosage form is a suppository having a shape and size designed for introduction into the rectal orifice of the human body.
  • a suppository usually softens, melts, or dissolves at body temperature.
  • Suppository excipients include, but are not limited to, theobroma oil (cocoa butter), glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights, and fatty acid esters of polyethylene glycol.
  • the compounds of (or for use with) the invention are typically delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebuliser.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebuliser.
  • Cells such as immune cells (eg CAR T cells) for use with the invention can be included in pharmaceutical formulations suitable for administration into the bloodstream or for administration directly into tissues or organs.
  • a suitable format is determined by the skilled person (such as a medical practitioner) for each patient, tissue, and organ, according to standard procedures.
  • Suitable pharmaceutically acceptable carriers and their formulation are known in the art (see, e.g. Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980).
  • Such cells when formed in a pharmaceutical composition, are preferably formulated in solution at a pH from about 6.5 to about 8.5.
  • Excipients to bring the solution to isotonicity can also be added, for example, 4.5% mannitol or 0.9% sodium chloride, pH buffered with art-known buffer solutions, such as sodium phosphate.
  • Other pharmaceutically acceptable agents can also be used to bring the solution to isotonicity, including, but not limited to, dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol) or other inorganic or organic solutes.
  • a media formulation is tailored to preserve the cells while maintaining cell health and identity.
  • a premixture including an aqueous solution of anticoagulant (ACD-A), an equal amount of dextrose (50%), and phosphate buffered saline (PBS), or the like is pre-mixed and aliquoted in a volume to typically match or approximate the cellular matrix or environment from which the cell was extracted from the tissue or organ.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fosidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the pharmaceutical compositions can be formulated into ointments, salves, gels, or creams as generally known in the art.
  • the pharmaceutical composition is formulated for sustained or controlled release of a compound of (or for use with) the invention (eg an ULRB1 and/or ULRB2 modulator).
  • a compound of (or for use with) the invention eg an ULRB1 and/or ULRB2 modulator.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
  • Dosage unit form as used herein includes physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the pharmaceutical composition comprising an ULRB1 and/or ULRB2 modulator is in unit dose form of between 10 and lOOOmg LILRB1 and/or LILRB2 modulator. In some embodiments, the pharmaceutical composition comprising an LILRB1 and/or ULRB2 modulator is in unit dose form of between 10 and 200mg LILRB1 and/or LILRB2 modulator. In some embodiments, the pharmaceutical composition comprising an ABP or biABP is in unit dose form of between 200 and 400mg LILRB1 and/or ULRB2 modulator.
  • the pharmaceutical composition comprising an LILRB1 and/or ULRB2 modulator is in unit dose form of between 400 and 600mg ULRB1 and/or ULRB2 modulator. In some embodiments, the pharmaceutical composition comprising an LILRB1 and/or ULRB2 modulator is in unit dose form of between 600 and 800mg ULRB1 and/or LILRB2 modulator. In some embodiments, the pharmaceutical composition comprising an LILRB1 and/or LILRB2 modulator is in unit dose form of between 800 and 100 mg ULRB1 and/or LILRB2 modulator.
  • kits are provided for producing a single-dose administration unit.
  • the kit can contain both a first container having a dried active ingredient and a second container having an aqueous formulation.
  • the kit can contain single and multi-chambered pre-loaded syringes.
  • Toxicity and therapeutic efficacy (eg effectiveness) of such active ingredients can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, eg, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Active agents which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimise potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage of the active ingredients (eg an ULRB1 and/or ULRB2 modulator), such as for use in humans.
  • the dosage of such active ingredients lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilised.
  • the (therapeutically) effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (ie, the concentration of the active ingredients which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful (eg effective) amounts or doses, such as for administration to humans.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • an effective amount of the LJLRBl and/or LLLRB2 modulator or the pharmaceutical composition can be one that will elicit the biological, physiological, pharmacological, therapeutic or medical response of a cell, tissue, system, body, animal, individual, patient or human that is being sought by the researcher, scientist, pharmacologist, pharmacist, veterinarian, medical doctor, or other clinician, eg, lessening of the effects/symptoms of a disorder, disease or condition, such as a proliferative disorder, for example, a cancer or tumour, or killing or inhibiting growth of a cell involved with a proliferative disorder, such as a tumour cell.
  • the effective amount can be determined by standard procedures, including those described below.
  • the effective amount administered at least once to a subject in need of treatment with a LJLRBl and/or LLLRB2 modulator is, typically, between about O.Olmg/kg and about lOOmg/kg per administration, such as between about lmg/kg and about lOmg/kg per administration. In some embodiments, the effective amount administered at least once to said subject of a LJLRBl and/or LLLRB2 modulator is between about O.Olmg/kg and about 0. lmg/kg per administration, between about 0.
  • lmg/kg and about lmg/kg per administration between about lmg/kg and about 5mg/kg per administration, between about 5mg/kg and about lOmg/kg per administration, between about lOmg/kg and about 50mg/kg per administration, or between about 50mg/kg and about lOOmg/kg per administration.
  • a LJLRBl and/or LLLRB2 modulator (or a pharmaceutical composition comprised thereof) will depend on the type of disease to be treated, the severity and course of the disease, whether the LJLRBl and/or LLLRB2 modulator and/or pharmaceutical composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history, age, size/weight and response to the LILRB1 and/or LLLRB2 modulator and/or pharmaceutical composition, and the discretion of the attending physician.
  • the LJLRBl and/or LILRB2 modulator and/or pharmaceutical composition is suitably administered to the patient at one time or over a series of treatments.
  • the total number of administrations for a given course of treatment may consist of a total of about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than about 10 treatments.
  • a treatment may be given once every day (or 2, 3 or 4 times a day) for a week, a month or even several months.
  • the course of treatment may continue indefinitely.
  • the amount of the LILRB1 and/or LLLRB2 modulator and/or pharmaceutical composition administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health, age, size/weight of the patient, the in vivo potency of the LJLRBl and/or LILRB2 modulator and/or pharmaceutical composition, and the route of administration.
  • the initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment.
  • Human dosage can be optimised, e.g., in a conventional Phase I dose escalation study designed to run from relatively low initial doses, for example from about O.Olmg/kg to about 20mg/kg of active ingredient.
  • Dosing frequency can vary, depending on factors such as route of administration, dosage amount and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks.
  • Formulation of an ULRB1 and/or ULRB2 modulator of (or for use with) the present is within the ordinary skill in the art. In some embodiments of the invention such LILRB1 and/or ULRB2 modulator is lyophilised and reconstituted in buffered saline at the time of administration.
  • the LILRB1 and/or ULRB2 modulator and/or pharmaceutical composition of may further result in a reduced relapsing of the disease to be treated or reduce the incidence of drug resistance or increase the time until drug resistance is developing; and in the case of cancer may result in an increase in the period of progression-free survival and/or overall survival.
  • Modulating compounds of LILRB1 and/or LILRB2, in particular inhibiting the interaction of ULRB1 and/or ULRB2 and a natural ligand thereof (such as HLA-G) or of a variant of LILRB1 and/or ULRB2, and/or the ABPs or biABPs, NAC, (host) cells and the pharmaceutical compositions of the invention can be used in various ways to modulate the expression, function, activity and/or stability of the ULRB1 and/or LILRB2 (or variant thereof), including their use in therapy or for prophylaxis.
  • a method of modulating the expression, function, activity and/or stability of of ULRB1 and/or ULRB2, or of a variant of LILRB1 and/or ULRB2 comprising contacting a cell that expresses said LILRB1 and/or ULRB2 or variant with a modulating compound as described above, in particular an ABP or biABP of the invention or an NAC encoding said ABP or biABP.
  • ABP or biABP is a modulator of the expression, function, activity and/or stability of said LILRB1 and/or ULRB2 or variant, thereby the expression, function, activity and/or stability of said ULRB1 and/or ULRB2 or variant is modulated.
  • Such method may be practiced on cells that are present ex-vivo, that is where said cells are contained in receptacles or containers, such as those used in research facilities. Accordingly, in such embodiments such method of the invention can be described as an in-vitro method of modulating the expression, function, activity and/or stability of ULRB1 and/or ULRB2, or of a variant of ULRB1 and/or ULRB2.
  • the method may be practiced using cells within the body, for example an in-vivo method of modulating the expression, function, activity and/or stability of ULRB1 and/or ULRB2, or of a variant of ULRB1 and/or LILRB2.
  • such an in-vitro (or in-vivo) method comprises the inhibition of the function and/or activity of the ULRB1 and/or ULRB2 or variant, when such modulating compound (eg the ABP) is an inhibitor of and/or antagonist of such function and/or activity.
  • modulating compound eg the ABP
  • it further comprises the step of contacting the cell with an immune cell, such as a CTL or TIL.
  • an immune cell such as a CTL or TIL.
  • the ABP or biABP is an antibody, or an antibody fragment, and is an inhibitor or antagonist of the function and/or activity of the ULRB1 and/or ULRB2 or variant.
  • such an in-vitro (or in-vivo) method comprises the activation of the function and/or activity of the ULRB1 and/or LILRB2 or variant, when such modulating compound (eg the ABP) is an activator of and/or agonistof such function and/or activity.
  • modulating compound eg the ABP
  • it further comprises the step of contacting the cell with an immune cell, such as a CTL or TIL.
  • an immune cell such as a CTL or TIL.
  • the ABP or biABP is an antibody, or an antibody fragment, and is an activator and/or agonistof the function and/or activity of the ULRB1 and/or LILRB2 or variant.
  • the method of modulating comprises contacting a cell that expresses said ULRB1 and/or LILRB2 or variant with a modulating compound as described above that is an activator and/or agonist of the function and/or activity of the ULRB1 and/or LILRB2 or variant, and the method mediates any one or combination of at least one of the functional characteristic or effects of the activating or agonistic modulators described herein, in particular as set forth in the section above "Modulators of LILRB1 and/or ULRB2 expression, function, activity and/or stability.
  • the method of modulating comprises contacting a cell that expresses said ULRB1 and/or ULRB2 or variant with a modulating compound as described above that is an inhibitor and/or antagonist of the function and/or activity of the LILRB1 and/or ULRB2 or variant, and the method mediates any one or combination of at least one of the functional characteristic or effects of the inhibitor or antagonist modulators described herein, in particular as set forth in the section above "Modulators of LILRB1 and/or ULRB2 expression, function, activity and/or stability.
  • the modulating compound in particular, an ABP
  • the modulating compound is an inhibitor and/or antagonist of the function and/or activity of the ULRB1 and/or LILRB2 or variant and inhibits the interaction between of a natural ligand of ULRB1 and/or ULRB2 (such as HLA-G) protein or a variant thereof and ULRB1 and/or LILRB2 protein or a variant thereof; that is, such a compound inhibits the binding function and/or activity of the ULRB1 and/or ULRB2 protein or variant thereof.
  • a natural ligand of ULRB1 and/or ULRB2 such as HLA-G protein or a variant thereof
  • ULRB1 and/or LILRB2 protein or a variant thereof a natural ligand of ULRB1 and/or ULRB2
  • the modulating compound (such as one that is an inhibitor or antagonist of expression, function, activity and/or stability of the ULRB1 and/or LILRB2 or variant) for example an ABP or biABP, or an NAC encoding said ABP or biABP, is capable of: (i) modulating the expression, function, activity and/or stability of the LILRB1 and/or LILRB2 or variant; and/or (ii) enhancing a cell-mediated immune response to a mammalian cell, decreases or reduces the resistance of cells (such as tumour cells that express a natural ligand of ULRB1 and/or ULRB2 (for example HLA-G or variant)), to an immune response.
  • cells such as tumour cells that express a natural ligand of ULRB1 and/or ULRB2 (for example HLA-G or variant)
  • the ABP or biABP (such as one that is an inhibitor or antagonist of expression, function, activity and/or stability of the ULRB1 and/or LILRB2 or variant, in particular one that inhibits the binding function and/or activity of the ULRB1 and/or ULRB2 protein to a ligand of ULRB1 and/or ULRB2), or an NAC encoding said ABP or biABP, enhances or increases the sensitivity of cells (such as tumour cells that express a natural ligand of ULRB1 and/or LILRB2 (for example HLA-G or variant)), to an immune response.
  • cells such as tumour cells that express a natural ligand of ULRB1 and/or LILRB2 (for example HLA-G or variant)
  • the term "resistance” refers to an acquired or natural resistance of a cell involved with (eg of or affected by) a disease (eg a proliferative disorder), such as tumour or cancer cell, to a patient's own immune response (such as a cell-mediated immune response), or to immune responses aided by immune therapy such as adoptive T-cell transfer or treatment with checkpoint blockers. Therefore, a resistant cell (eg a resistant tumour or cancer cell) is more likely to escape and survive humoural and/or cellular immune defence mechanisms in a subject having the disorder (such as the tumour or cancer).
  • a disease eg a proliferative disorder
  • a patient's own immune response such as a cell-mediated immune response
  • immune therapy such as adoptive T-cell transfer or treatment with checkpoint blockers. Therefore, a resistant cell (eg a resistant tumour or cancer cell) is more likely to escape and survive humoural and/or cellular immune defence mechanisms in a subject having the disorder (such as the tumour or cancer).
  • a treatment of a resistant proliferative disease, such as tumour/cancer resistance, in context of the invention shall be effective if, compared to a non-treated control, the cell involved with the proliferative disease (such as a cell of the tumour of cancer) becomes more sensitive or susceptible to an immune response (such as a cell- mediated immune response) - in other words will be more likely to be recognised and/or neutralised (for example by cytotoxic processes such as apoptosis) by the subject's immune response.
  • the cell involved with the proliferative disease such as a cell of the tumour of cancer
  • an immune response such as a cell- mediated immune response
  • cell(s) involved with the disease may be resistant against (to) a cell-mediated immune response; and/or such cell(s) may have or display a resistant phenotype.
  • the terms "cellular resistance", “cell resistance” and the like refers to a resistance of the subject cell(s) (such as a tumour or cancer cell) to a cell-mediated immune response, such as a cytotoxic T lymphocyte (CTL) response (eg, the tumour or tumour cell being nonresponsive to, or having reduced or limited response to a CTL targeting a tumour cell).
  • CTL cytotoxic T lymphocyte
  • a tumour cell may show a reduced or limited response when contacted with a CTL specific for an antigen expressed on that tumour cell.
  • a reduced or limited response is a reduction to a 90% cytotoxic T cell response, preferably a reduction to 80%, 70%, 60%, 50% or more preferably a reduction to 40%, 30%, 20% or even less.
  • a subject cell eg a tumour cell
  • a patient's (cell-mediated) immune response may be tested in-vitro by contacting a sample of the subjects such cells (eg autologous tumour cells) with (eg autologous) T-cells and thereafter quantifying the survival/proliferation rate of the (eg) tumour cells.
  • the reduction in (cell-mediated) immune response is determined by comparing cancer samples of the same cancer before and after the resistance is acquired (for example induced by therapy), or by comparing with a cancer sample derived from a different cancer which is known to have no resistance to the CTL.
  • the treatments of the present invention include the sensitisation of cells involved with the proliferative disorder against CTL and therefor to decrease resistance of such cells.
  • a decrease of (eg tumour) cell resistance against CTL is preferably a significant increase of CTL toxicity, preferably a 10% increase, more preferably 20%, 30%, 40%, 50%, 60%, 70%, 80% or more, even more preferably 2 fold increase, 3 fold, 4 fold, 5 fold or more.
  • a resistant phenotype of the cells involved with the proliferative disorder is displayed by such cells when a subject suffering from the proliferative disorder (eg a cancer or tumour) has been previously treated with an (immune)therapy and, for example, such proliferative disorders has progressed despite such prior (immune)therapy.
  • a subject suffering from the proliferative disorder eg a cancer or tumour
  • an (immune)therapy for example, such proliferative disorders has progressed despite such prior (immune)therapy.
  • a class of subject suitable for the various therapeutic methods of the invention can be those whose tumour (or cancer) has progressed (such as has relapsed or recurred, or has not responded to) after prior treatment with a cancer immunotherapy.
  • such prior treatment may be any immunotherapy as described elsewhere herein, including adoptive immune cell transfer (eg TCR or CART cell therapy), an anti-tumour vaccine, an antibody binding to an immune checkpoint molecule (such as CTLA-4, PD-1 or PD-L1).
  • adoptive immune cell transfer eg TCR or CART cell therapy
  • an anti-tumour vaccine an antibody binding to an immune checkpoint molecule (such as CTLA-4, PD-1 or PD-L1).
  • an immune checkpoint molecule such as CTLA-4, PD-1 or PD-L1
  • the subject may suffer from a tumour or cancer, and such cancer may have progressed (such as has relapsed or recurred, or has not responded to) after prior radiotherapy.
  • the immune response is, in particular of such embodiments, a cell-mediated immune response such as one mediated by T-cells including cytotoxic T-cells and/or TILs; and/or the immune response is the lysis and/or killing of the cells, in particular those that express ULRB1 and/or LILRB2 or a variant thereof) that is mediated by cytotoxic T- cells and/or TILs.
  • the immune response is a cytotoxic immune response against cells (such as tumour cells), in particular a cell-mediated cytotoxic immune response such as one mediated by T-cells including cytotoxic T-cells and/or TILs.
  • ULRB1 and/or ULRB2 expressing cells are found in the myeloid compartment and modulate cell-mediated immune responses.
  • the modulating compound as disclosed herein in particular the ABP or biABP (such as one that is an inhibitor or antagonist of expression, function, activity and/or stability of the LILRB1 and/or ULRB2 or variant thereof), or an NAC encoding said ABP or biABP, enhances or increases killing and/or lysis of cells associated with the proliferative disorder, such as tumour cells.
  • the ABP or biABP such as one that is an inhibitor or antagonist of expression, function, activity and/or stability of the LILRB1 and/or ULRB2 or variant thereof
  • an NAC encoding said ABP or biABP enhances or increases killing and/or lysis of cells associated with the proliferative disorder, such as tumour cells.
  • Such effect is brought about via modulation of cells expressing ULRB1 and/or ULRB2 or variant thereof, (such as macrophage cells); which modulate a killing and/or lysis of tumour cells mediated by cytotoxic T-cells and/or TILs, and/or mediated by an enhancement of or increase in the sensitivity of the cells of the immune system, and/or mediated by a decrease in or reduction of the immune inhibitory effects of the cells expressing the ULRB1 and/or ULRB2 or variant thereof on a (cytotoxic) immune response, such an immune response described above.
  • LILRB1 and/or ULRB2 expressing cells brought into contact with the agents of the invention are therefore less immune inhibitory and support a subject's immune response against a proliferative disease to be treated.
  • the cells that express LILRB1 and/or ULRB2 or variant thereof are, in certain of such preferred embodiments, cells of the myeloid compartment, such as macrophages, or alternatively in some instances may be cancer cells or are cells that originated from a tumour cell.
  • Exemplary cancer or tumour cells can be those as described or exemplified elsewhere herein.
  • the modulating compounds in particular the ABP or biABP (such as one that is an inhibitor or antagonist of expression, function, activity and/or stability of ULRB1 and/or ULRB2 or variant thereof, in particular that is an inhibitor of the HLA-G-binding function of the ULRB1 and/or LILRB2 or variant), or an NAC encoding said ABP or biABP, increases T-cell activity and/or survival (and/or increases T-cell proliferation), which in certain embodiments, may lead to an enhancement of a (cytotoxic) immune response mediated by such T-cells.
  • ABP or biABP such as one that is an inhibitor or antagonist of expression, function, activity and/or stability of ULRB1 and/or ULRB2 or variant thereof, in particular that is an inhibitor of the HLA-G-binding function of the ULRB1 and/or LILRB2 or variant
  • an NAC encoding said ABP or biABP increases T-cell activity and/or survival (and/or increases T-cell proliferation), which in
  • the invention relates to a method for the treatment of a disease, disorder or condition in a mammalian subject by administering a product to the subject wherein the product is a modulator of the expression, function, activity and/or stability of immunoglobulin superfamily member 11 (LILRB1 and/or LILRB2), or of a variant of ULRB1 and/or ULRB2.
  • the invention relates to a product for use in medicine, wherein the product is a compound that is modulator of the expression, function, activity and/or stability of immunoglobulin superfamily member 11 (LILRB1 and/or LILRB2), or of a variant of ULRB1 and/or LILRB2.
  • the modulating compound (such as an ABP) is an inhibitor of the binding HLA-G-binding function of ULRB1 and/or ULRB2 or variant thereof; and/or wherein the product is selected from the list consisting of an ABP or biABP, ABD, nucleic acid, NAC or recombinant host cell of the invention, in particular an ABP or biABP of the invention.
  • the invention also relates to method of treating or preventing a disease, disorder or condition in a mammalian subject in need thereof, comprising administering to said subject at least once an effective amount of modulating compound as desired above, or, and in particular administering to said subject at least once an effective amount of the ABP or biABP, the NAC, the (host) cells, or the pharmaceutical composition as described above.
  • the invention also relates to the use of a product of the invention as describe above, or a modulating compound as described above (in particular an ABP or biABP of the invention) for the manufacture of a medicament, in particular for the treatment of a disease, disorder or condition in a mammalian subject, in particular where the disease, disorder or condition is one as set out herein.
  • a product of the invention as describe above, or a modulating compound as described above (in particular an ABP or biABP of the invention) for the manufacture of a medicament, in particular for the treatment of a disease, disorder or condition in a mammalian subject, in particular where the disease, disorder or condition is one as set out herein.
  • treatment in the present invention is meant to include therapy, e.g. therapeutic treatment, as well as prophylactic or suppressive measures for a disease (or disorder or condition).
  • therapy e.g. therapeutic treatment
  • prophylactic or suppressive measures for a disease (or disorder or condition) for example, successful administration of a LILRB1 and/or LILRB2 inhibitor prior to onset of the disease results in treatment of the disease.
  • Treatment also encompasses administration of a ULRB1 and/or ULRB2 inhibitor after the appearance of the disease in order to ameliorate or eradicate the disease (or symptoms thereof).
  • Administration of a LILRB1 and/or LILRB2 inhibitor after onset and after clinical symptoms, with possible abatement of clinical symptoms and perhaps amelioration of the disease also comprises treatment of the disease.
  • Those "in need of treatment” include subjects (such as a human subject) already having the disease, disorder or condition, as well as those prone to or suspected of having the disease, disorder or condition, including those in which the disease, disorder or condition is to be prevented
  • the modulating compound is one described above, and/or is an ABP or biABP, NAC, a (host) cell, or a pharmaceutical composition of the present invention; in particular is an ABP or biABP of the invention, and/or is an inhibitory nucleic acid of the invention.
  • Such a compound can for example in preferred embodiments, be an inhibitor or antagonist of expression, function, activity and/or stability of LILRB1 and/or ULRB2, or of the variant of LILRB1 and/or LILRB2.
  • the compound inhibits the binding of a natural ligand of ULRB1 and/or ULRB2 protein (such as HLA-G or a variant thereof) to ULRB1 and/or LILRB2 protein (or a variant thereof), in particular inhibits the binding of HLA-G protein (or a variant thereof) to human ULRB1 and/or ULRB2 protein (or a variant thereof), such as inhibits the binding between the ECDs of such proteins; preferably wherein such proteins (or variants) and the inhibitions is described as above.
  • a natural ligand of ULRB1 and/or ULRB2 protein such as HLA-G or a variant thereof
  • human ULRB1 and/or ULRB2 protein or a variant thereof
  • Such a compound can, for example, be a compound (such as an ABP or biABP or inhibitors nucleic acid) that has the any one or any combination of the following characteristics
  • ULRB1 and/or (preferably and) ULRB2 e.g. comprising the amino acid sequence of SEQ ID NO: 353 (LILRB1) or SEQ ID NO: 358 (ULRB2), e.g., with a KD 50 nM or less, or of 20nM or less; more preferably of lOnM or less or 5nM or less lack of specific binding to a ULRA protein, such as preferably ULRA1 and/or LILRA3; • stimulates T cell activation, e.g., in a mixed lymphocyte reaction (MLR) assay, as measured by increased T cell proliferation or IFN-gamma secretion, e.g.;
  • MLR mixed lymphocyte reaction
  • the disease, disorder or condition that is characterised by a pathological immune response is characterised by a pathological immune response.
  • the disease, disorder or condition is characterised by expression of a natural ligand of ULRB1 and/or ULRB2, in particular by expression of HLA-G, by cells associated with the disease, disorder or condition, such as cancer cells.
  • the disease, disorder or condition can be associated with the undesired presence of a natural ligand of ULRB1 and/or ULRB2, such as a HLA-G positive cells or cells positive for such a ligand, in particular a disease or condition characterized by ULRB1 and/or ULRB2 positive monocytes and/or macrophages (in particular, TAMs).
  • a subject suffering from, or suspected of suffering from, a disease, disorder or condition is characterised as: (i) having a cancer positive for a natural ligand of ULRB1 and/or ULRB2, such as HLA-G and/or (ii) having ULRB1 and/or LILRB2 positive immune cells, in particular ULRB1 and/or LILRB2 positive monocytes and/or macrophages; and/or (iii) having ULRB1 and/or ULRB2 positive immune cells, in particular ULRB1 and/or ULRB2 positive monocytes (or macrophages); preferably wherein such ULRB1 and/or LILRB2 positive immune cells are present at or associated with the site of a cancer or tumour (such as being present in the tumour bed or tumour micro environment (TME) of such cancer or tumour, in particular with the presence of TAMs and/or MDSCs).
  • a cancer positive for a natural ligand of ULRB1 and/or ULRB2 such as HLA
  • a disorder, disorder or condition treatable by the subject matter of the invention is, in certain alterative embodiments, one characterised by expression of a ligand of ULRB1 and/or ULRB2; in particular, one characterised by such expression that is aberrant, for example over- (or under-) expression or representation or activity of a ligand of ULRB1 and/or ULRB2 (in particular of HLA-G) in a given cell or tissue (such as those cells or tissues involved with the proliferative disease of the subject) compared to that in a healthy subject or a normal cell.
  • the disease, disorder or condition is characterised by expression and/or activity of ULRB1 and/or LILRB2 in an immune cell, such as a macrophage, in particular such cells express mRNA and/or protein of ULRB1 and/or ULRB2, and/or are positive for such ULRB1 and/or ULRB2 expression and/or activity.
  • the disease, disorder or condition is a proliferative disorder (or a condition associated with such disorder or disease), in particular when the product or modulating compound (such as a ABP or biABP, ABD, nucleic acid, NAC or recombinant host cell of the invention, in particular an ABP or biABP of the invention) is an inhibitor and/or antagonist of the expression, function, activity and/or stability of ULRB1 and/or LILRB2 or a variant of LILRB1 and/or LILRB2.
  • a "proliferative disorder” refers to a disorder characterised by abnormal proliferation of cells.
  • a proliferative disorder does not imply any limitation with respect to the rate of cell growth, but merely indicates loss of normal controls that affect growth and cell division. Thus, in some embodiments, cells of a proliferative disorder can have the same cell division rates as normal cells but do not respond to signals that limit such growth.
  • neoplasm or tumour which is an abnormal growth of tissue or cells. Cancer is art understood, and includes any of various malignant neoplasms characterised by the proliferation of cells that have the capability to invade surrounding tissue and/or metastasise to new colonisation sites.
  • Proliferative disorders include cancer, atherosclerosis, rheumatoid arthritis, idiopathic pulmonary fibrosis and cirrhosis of the liver.
  • Non-cancerous proliferative disorders also include hyperproliferation of cells in the skin such as psoriasis and its varied clinical forms, Reiter's syndrome, pityriasis rubra pilaris, and hyperproliferative variants of disorders of keratinization (e.g., actinic keratosis, senile keratosis), scleroderma, and the like.
  • the proliferative disorder is a cancer or tumour, in particular a solid tumour (or a condition associated with such cancer or tumour).
  • Such proliferative disorders include, but are not limited to, head and neck cancer, squamous cell carcinoma, multiple myeloma, solitary plasmacytoma, renal cell cancer, retinoblastoma, germ cell tumours, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumour of the kidney, Ewing Sarcoma, chondrosarcoma, any haemotological malignancy (e.g., chronic lymphoblastic leukemia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, acute myeloblasts leukemia, chronic myeloblastic leukemia, Hodgkin's disease, non- Hodgkin's lymphoma,
  • the various aspects of the invention relate to, for example the ABPs or biABPs of the invention used to detect/diagnose, prevent and/or treat, such proliferative disorders that include but are not limited to carcinoma (including breast cancer, prostate cancer, gastric cancer, lung cancer, colorectal and/ or colon cancer, hepatocellular carcinoma, melanoma), lymphoma (including non-Hodgkin's lymphoma and mycosis fongoides), leukemia, sarcoma, mesothelioma, brain cancer (including glioma), germinoma (including testicular cancer and ovarian cancer), choriocarcinoma, renal cancer, pancreatic cancer, thyroid cancer, head and neck cancer, endometrial cancer, cervical cancer, bladder cancer, or stomach cancer.
  • carcinoma including breast cancer, prostate cancer, gastric cancer, lung cancer, colorectal and/ or colon cancer
  • lymphoma including non-Hodgkin's lymphoma and mycosis
  • the proliferative disease is a cancer, for example lung cancer, breast cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, pancreatic cancer, ovarian cancer, melanoma, myeloma, kidney cancer, head and neck cancer, Hodgkin lymphoma, bladder cancer or prostate cancer, in particular one selected from the list consisting of: melanoma, lung cancer (such as non-small cell lung cancer), bladder cancer (such as urothelial carcinoma), kidney cancer (such as renal cell carcinoma), head and neck cancer (such as squamous cell cancer of the head and neck) and Hodgkin lymphoma.
  • the proliferative disease is melanoma, or lung cancer (such as non-small cell lung cancer).
  • the disease, disorder or condition is a cancer positive for the ligand of ULRB1 and/or LILRB2 (such as HLA-G) and/or is a cancer characterised by the presence of LILRB1 and/or ULRB2 positive immune cells, in particular ULRB1 and/or ULRB2 positive monocytes and/or macrophages and/or is a cancer (or other proliferative disorder) characterised by being resistant and/or refractory to blockade of an immune checkpoint molecule (eg resistant and/or refractory to therapy for blockade of an immune checkpoint molecule), such as blockade using a ligand to an immune checkpoint molecule (as further described below, such as blockade of PD1/CTLA4; analogous to Gao et al, 2017).
  • the disease, disorder or condition can be a proliferative disorder (such as cancer) resistant and/or refractory to PD1/CTLA
  • a preferred embodiment pertains to treatment of a disease, disorder or condition, preferably a proliferative disorder, which is characterized by a resistance to a therapy to a PD-1 / PD-L1 inhibitor therapy, such as a therapy with a anti- PD-1 inhibitory antibody.
  • the disease, disorder or condition is an infectious disease (or a condition associated with such disorder or disease), in particular when the product or modulating compound (such as a ABP or biABP, ABD, nucleic acid, NAC or recombinant host cell of the invention, in particular an ABP or biABP of the invention) is an inhibitor and/or antagonist of the expression, function, activity and/or stability of ULRB1 and/or LILRB2 or a variant of LILRB1 and/or LILRB2.
  • the product or modulating compound such as a ABP or biABP, ABD, nucleic acid, NAC or recombinant host cell of the invention, in particular an ABP or biABP of the invention
  • infectious disease is art recognized, and as used herein includes those diseases, disorders or conditions associated with (eg resulting from or caused by) by any pathogen or agent that infects mammalian cells, preferable human cells.
  • pathogens include bacteria, yeast, fungi, protozoans, mycoplasma, viruses, prions, and parasites.
  • infectious disease examples include( a) viral diseases such as, for example, diseases resulting from infection by an adenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), a poxvirus (e ⁇ g-, an orthopoxvirus such as variola or vaccinia, or molluscum contagiosum), a picornavirus (e.g., rhinovirus or enterovirus), an orthomyxovirus (e.g., influenza virus), a paramyxovirus (e.g., parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), a cononavirus (e.g., SARS), a papovavirus (e.g., papillomaviruses, such as those that cause genital warts, common warts, or plantar warts), a hepadnavirus (e.g., hepati
  • the disease, disorder or condition is one associated with an over-active or immune system or an immune system displaying undesired activity, such as autoimmunity, allergy or inflammatory conditions, in particular for allergy, autoimmunity, transplant rejection, inflammation, graft vs host disease or sepsis (or a condition associated with such diseases, disorders or conditions), in particular when the product or modulating compound (such as a ABP or biABP, ABD, nucleic acid, NAC or recombinant host cell of the invention, in particular an ABP or biABP of the invention) is an activator and/or agonist of the expression, function, activity and/or stability of ULRB1 and/or ULRB2 or a variant of LILRB1 and/or ULRB2.
  • the product or modulating compound such as a ABP or biABP, ABD, nucleic acid, NAC or recombinant host cell of the invention, in particular an ABP or biABP of the invention
  • the subject is a mammal, and may include mice, rats, rabbits, monkeys and humans.
  • the mammalian subject is a human patient.
  • cells involved in the proliferative disorder are resistant to a humoral or cell-mediated immune response.
  • cells involved in the proliferative disorder eg cells of a cancer or tumour
  • are resistant and/or refractory to blockade of an immune checkpoint molecule such as blockade using a ligand to an immune checkpoint molecule, in exemplary instances blockade of PD1/CTLA4 (analogous to Gao et al, 2017).
  • the treatment methods may be applied to a proliferative disorder that has been subjected to prior immunotherapy (such as therapy for blockade of an immune checkpoint molecule, eg blockade of PD1/CTLA4), in particular prior immunotherapy with a ligand to an immune checkpoint molecule.
  • prior immunotherapy such as therapy for blockade of an immune checkpoint molecule, eg blockade of PD1/CTLA4
  • the ULRB1 and/or ULRB2 (eg antagonist) modulator such as an ABP or biABP of the present invention, can be for use in the treatment of a proliferative disorder in a subject in need thereof, and the subject has been subjected to to prior immunotherapy, in particular prior administration of a ligand to an immune checkpoint molecule.
  • the ligand is one that binds to an immune (inhibitory) checkpoint molecule.
  • checkpoint molecule may be one selected from the group consisting of: A2AR, B7-H3, B7-H4, CTLA-4, IDO, KIR, LAG3, PD-1 (or one of its ligands PD-L1 and PD-L2), TIM-3 (or its ligand galectin-9), TIGIT and VISTA.
  • the ligand binds to a checkpoint molecule selected from: CTLA-4, PD-1 and PD-L1.
  • the ligand is an antibody selected from the group consisting of: ipilimumab, nivolumab, pembrolizumab, BGB-A317, atezolizumab, avelumab and durvaluma; in particular an antibody selected from the group consisting of: ipilimumab (YERVOY), nivolumab (OPDIVO), pembrolizumab (KEYTRUDA) and atezolizumab (TECENTRIQ).
  • a method or use in therapy of the present invention eg, one involving an ABP or biABP of the invention
  • any of such other procedures eg, another agent or a cancer immunotherapy, such as a ligand that binds to an immune (inhibitory) checkpoint molecule
  • such method or use being a combination treatment regimen may comprise embodiments where such exposures/administrations are concomitant.
  • such administrations may be sequential; in particular those embodiments where the LILRB1 and/or ULRB2 modulator (eg an ABP or biABP of the invention) is administered before such other procedure.
  • such ULRB1 and/or LILRB2 modulator may be sequentially administered within about 14 days of (eg before) the other procedure, such as within about 10 days, 7 days, 5 days, 2 days or 1 day of (eg before) the other procedure; and further including where the ULRB1 and/or ULRB2 modulator may be sequentially administered within about 48 hours, 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hours, 30 mins, 15 mins or 5 mins of (eg before) the other procedure.
  • the medical uses or compositions are for use in enhancing an immune response in the subject, preferably for use in aiding a cell-mediated immune response in the subject such as the subject's T cell mediated immune response, for example for treating a proliferative disease such as a cancer disease.
  • the treatment can comprise a transfer of cells to the subject, preferably a transfer of immune cells to the subject, more preferably an adoptive T-cell transfer.
  • such cells can be autologous cells of the subject, for example autologous immune cells, such as T-cells, dendritic cells or Natural Killer (NK)-cells, of the subject.
  • autologous immune cells such as T-cells, dendritic cells or Natural Killer (NK)-cells
  • the modulating compound eg ABP or biABP of the invention
  • the modulating compound is an inhibitor or antagonist of expression, function, activity and/or stability of said ULRB1 and/or ULRB2, or the variant of LILRB1 and/or LILRB2, and wherein the inhibition of the expression, function, activity and/or stability of said LILRB1 and/or ULRB2, or the variant of ULRB1 and/or ULRB2, enhances an immune response, preferably enhances a cell-mediated immune response in the subject such as a T-cell mediated immune response in the subject, for example for treating an infectious disease or a proliferative disease such as a cancer disease, in particular where the composition is an ABP or biABP of the invention.
  • the immune response can be enhanced by an increase in T cell activity, proliferation and/or survival, in particular wherein the increase in T cell activity comprises an increase in production of one or more pro-inflammatory cytokines by such T cells (such as TILs).
  • the cytokine is one selected from the group consisting of: interleukin-1 (IL-1), IL- 2, IL-12, IL-17, and IL- 18, tumour necrosis factor (TNF) [alpha], interferon gamma (IFN-gamma), and granulocyte-macrophage colony stimulating factor, such as IL-2 (and/or IL-17 or IFN-gamma).
  • Such an increase in T cell activity, proliferation and/or survival can be associated with the inhibition of the interaction between ULRB1 and/or LILRB2 and their ligand(s), in particular mediated by ULRB1 and/or ULRB2- mediated signaling.
  • Administration of the modulating (eg inhibiting) compound is, in certain embodiments, associated with the inhibition of the interaction between ULRB1 and/or ULRB2 and their ligand(s), in particular mediated by ULRB1 and/or LILRB2-mediated signaling.
  • administering decreases or reduces the resistance of cells (such as tumour cells and/or cells that express ULRB1 and/or ULRB2, or the variant of ULRB1 and/or ULRB2), to an immune response, preferably wherein the compound enhances or increases the sensitivity of cells (such as tumour cells and/or cells that express LILRB1 and/or LILRB2, or the variant of ULRB1 and/or ULRB2), to an immune response.
  • cells such as tumour cells and/or cells that express ULRB1 and/or ULRB2, or the variant of ULRB1 and/or ULRB2
  • the medical uses are for the treatment of a proliferative disorder (such as a cancer described herein) in a mammalian subject in need thereof.
  • a proliferative disorder such as a cancer described herein
  • the subject is a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or preferably a human, for example a human patient.
  • a cell such as (recombinant) host cell or a hybridoma capable of expressing an ABP or biABP as described above.
  • a cell which comprises at least one NAC encoding an ABP or biABP or a component of an ABP or biABP as described above.
  • Cells of the invention can be used in methods provided herein to produce the ABPs or biABPs and/or NACs of the invention.
  • the cell is isolated or substantially pure, and/or is a recombinant cell and/or is a nonnatural cell (i.e., it is not found in, or is a product of, nature), such as a hybridoma.
  • the invention relates to a method of producing a recombinant cell line capable of expressing an ABP or biABP specific for LILRB1 and/or ULRB2, or for a LILRB1 and/or ULRB2 variant, the method comprising the steps of:
  • the invention relates to a method of producing an ABP or biABP specific for ULRB1 and/or ULRB2, or for a ULRB1 and/or LILRB2 variant, the method comprising the steps of:
  • a hybridoma or (host) cell capable of expressing an ABP or biABP according to the invention for example a recombinant cell line comprising at least one genetic construct comprising coding sequence(s) encoding said compound or ABP;
  • DNA molecules encoding the proteins are inserted into an expression vector (or NAC) such that the sequences are operatively linked to transcriptional and translational control sequences.
  • DNA molecules encoding the ABP or biABP can be chemically synthesized. Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs encoding the desired ABP or biABP.
  • Expression vectors include, but are not limited to, plasmids, retroviruses, cosmids, EBV-derived episomes, and the like.
  • expression vector or "NAC” comprises any vector suitable for the expression of a foreign DNA.
  • viral vectors such as adenovirus, vaccinia virus, baculovirus and adeno-associated virus vectors.
  • virus vector is understood to mean both a DNA and a viral particle.
  • phage or cosmid vectors examples include pWE15, M13, AEMBL3, AEMBL4, AFIXII, ADASHII, AZAPII, AgTIO, Agtll, Charon4A and Charon21A.
  • plasmid vectors examples include pBR, pUC, pBluescriptll, pGEM, pTZ and pET groups.
  • shuttle vectors may be used, e.g., vectors which may autonomously replicate in a plurality of host microorganisms such as E. coli and Pseudomonas sp.
  • artificial chromosome vectors are considered as expression vectors.
  • the expression vector and expression control sequences are selected to be compatible with the cell, such as a host cell.
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRepS, D H26S, D HBB, pNMTl, pNMT41, pNMT81, which are available from InvitrogenTM, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Agilent Technologies, pTRES which is available from Clontech, and their derivatives.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors.
  • both DNA sequences are inserted into the same expression vector.
  • Convenient vectors are those that encode a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed, as described above, wherein the CHI and/or upper hinge region comprises at least one amino acid modification of the invention.
  • the constant chain is usually kappa or lambda for the antibody light chain.
  • the recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from a (host) cell.
  • the DNA encoding the antibody chain may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the mature antibody chain DNA.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous peptide from a non-immunoglobulin protein.
  • the DNA sequence encoding the antibody chain may already contain a signal peptide sequence.
  • the recombinant expression vectors carry regulatory sequences including promoters, enhancers, termination and polyadenylation signals and other expression control elements that control the expression of the antibody chains in a (host) cell.
  • promoter sequences are promoters and/or enhancers derived from CMV (such as the CMV Simian Virus 40 (SV40) promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters.
  • promoter sequences are promoters and/or enhancers derived from CMV (such as the CMV Simian Virus 40 (SV40) promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters such as native immunoglobulin and actin promoters.
  • the recombinant expression vectors may also carry sequences that regulate replication of the vector in (host) cells (e.g. origins of replication) and selectable marker genes.
  • Nucleic acid molecules encoding the heavy chain or an antigen-binding portion thereof and/or the light chain or an antigen-binding portion thereof of an antibody of the present invention, and vectors comprising these DNA molecules can be introduced into (host) cells, e.g. bacterial cells or higher eukaryotic cells, e.g. mammalian cells, according to transfection methods well known in the art, including liposome-mediated transfection, polycation-mediated transfection, protoplast fusion, microinjections, calcium phosphate precipitation, electroporation or transfer by viral vectors.
  • the heavy chain and the light chain are present on two vectors which are co-transfected into the (host) cell, preferably a mammalian cell
  • Mammalian cell lines available as hosts for expression are well known in the art and include, inter alia, Chinese hamster ovary (CHO, CHO-DG44, BI-HEX-CHO) cells, NSO, SP2/0 cells, HeLa cells, HEK293 cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells or the derivatives/progenies of any such cell line.
  • Other mammalian cells including but not limited to human, mice, rat, monkey and rodent cells lines, or other eukaryotic cells, including but not limited to yeast, insect and plant cells, or prokaryotic cells such as bacteria may be used.
  • the antibody molecules of the invention are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody molecule in the host cells.
  • the intact antibody (or the antigen-binding fragment of the antibody) can be harvested and isolated using purification techniques well known in the art, e.g., Protein A, Protein G, affinity tags such as glutathione-S-transferase (GST) and histidine tags.
  • purification techniques well known in the art, e.g., Protein A, Protein G, affinity tags such as glutathione-S-transferase (GST) and histidine tags.
  • ABPs or biABPs are preferably recovered from the culture medium as a secreted polypeptide or can be recovered from host cell lysates if for example expressed without a secretory signal. It is necessary to purify the ABP or biABP molecules using standard protein purification methods used for recombinant proteins and host cell proteins in a way that substantially homogenous preparations of the ABP or biABP are obtained.
  • state-of- the art purification methods useful for obtaining the ABP or biABP molecule of the invention include, as a first step, removal of cells and/or particulate cell debris from the culture medium or lysate.
  • the ABP or biABP is then purified from contaminant soluble proteins, polypeptides and nucleic acids, for example, by fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse phase HPLC, Sephadex chromatography, chromatography on silica or on a cation exchange resin.
  • ABPs or biABPs are purified by standard protein A chromatography, e.g., using protein A spin columns (GE Healthcare). Protein purity may be verified by reducing SDS PAGE.
  • ABP or biABP concentrations may be determined by measuring absorbance at 280nm and utilizing the protein specific extinction coefficient.
  • the purified ABP or biABP molecule may be dried, e.g. lyophilized, for therapeutic applications.
  • the method comprises a further step of isolation and/or purification of the ABP or biABP.
  • herein provided is a method of manufacturing a pharmaceutical composition comprising an ABP or biABP as described above, comprising formulating the ABP or biABP isolated by the methods described above into a pharmaceutically acceptable form.
  • a method of manufacturing a pharmaceutical composition comprising an NAC as described above, comprising formulating the NAC prepared by the methods described above into a pharmaceutically acceptable form.
  • the methods of manufacturing a pharmaceutical composition comprise a further step of combining said ABP or biABP and/or NAC with a pharmaceutically acceptable excipient or carrier.
  • the ABP or biABP typically will be labelled with a detectable labelling group before being formulated into a pharmaceutically acceptable form.
  • Various methods for labelling proteins are known in the art and may be used.
  • Suitable labelling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, Ulin, 1251, 1311), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, - galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • the labelling group is coupled to the ABP or biABP via spacer arms of various lengths to reduce potential steric hindrance.
  • the ABP or biABP is a modified antibody and the method comprises a further step of addition of a functional moiety selected from a detectable labelling group or a cytotoxic moiety.
  • ULRB1 and/or ULRB2 can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders and/or conditions associated with the undesired presence of LILRB1 and/or LILRB2-positive cells or cells positive for a variant of LILRB1 and/or ULRB2 and/or associated with cellular resistance against a cell-mediated immune response; and in particular aberrant and/or localised expression/activity of ULRB1 and/or LILRB2 (in particular phosphorylated ULRB1 and/or ULRB2) can be so used.
  • the disease, disorder and/or conditions so detected, diagnosed, or monitored, can be one of those described elsewhere herein.
  • the invention relates to a method for determining the presence or an amount of LILRB1 and/or LILRB2 (or a variant thereof) in a biological sample from a subject, the method comprising the steps of:
  • protein LILRB1 and/or ULRB2 (or a variant thereof) is detected with a ABP or biABP of the invention.
  • a biological sample will (preferably) comprise cells or tissue of the subject, or an extract of such cells or tissue, in particular where such cells are those involved with the proliferative disorder (eg cells of the tumour microenvironment, or immune cells present at the site of the tumour).
  • the tumour or cell thereof may be one or, or derived from, one of the tumours described elsewhere herein.
  • the method will also comprise a step of:
  • such detection and/or determination methods can be practiced as a method of diagnosis, such as a method of diagnosis whether a mammalian subject (such as a human subject or patient) has a disease, disorder or condition (such as one described above), in particular a proliferative disorder such as a cancer or tumour (or has a risk of developing such a disease, disorder or condition) that is associated with the undesired presence of LILRB1 and/or LILRB2-positive cells or cells positive for a variant of LILRB1 and/or LILRB2 and/or that is associated with cellular resistance against a cell-mediated immune response and/or that is associated with (eg aberrant) expression or activity of LILRB1 and/or ULRB2 (or a variant thereof); in particular an immune cell of a (solid) tumour, such as one having cellular resistance against a cell-mediated immune response.
  • a mammalian subject such as a human subject or patient
  • a proliferative disorder such as a cancer or tumour (or has a
  • the cellular resistance against a cell-mediated immune response is cellular resistance against a T cell-mediated immune response.
  • the biological sample is one obtained from a mammalian subject like a human patient.
  • the term "biological sample” is used in its broadest sense and can refer to a bodily sample obtained from the subject (eg, a human patient).
  • the biological sample can include a clinical sample, i.e., a sample derived from a subject.
  • samples can include, but are not limited to: peripheral bodily fluids, which may or may not contain cells, e.g., blood, urine, plasma, mucous, bile pancreatic juice, supernatant fluid, and serum; tissue or fine needle biopsy samples; tumour biopsy samples or sections (or cells thereof), and archival samples with known diagnosis, treatment and/or outcome history.
  • Bio samples may also include sections of tissues, such as frozen sections taken for histological purposes.
  • the term "biological sample” can also encompass any material derived by processing the sample. Derived materials can include, but are not limited to, cells (or their progeny) isolated from the biological sample, nucleic acids and/or proteins extracted from the sample. Processing of the biological sample may involve one or more of, filtration, distillation, extraction, amplification, concentration, fixation, inactivation of interfering components, addition of reagents, and the like.
  • Such detection, determination and/or diagnosis methods can be conducted as an in-vitro method, and can be, for example, practiced using the kit of the present invention (or components thereof).
  • the biological sample is a tissue sample from the subject, such as a sample of a tumour or a cancer from the subject.
  • a sample may contain tumour cells and/or blood cells (eg monocytes and T cells).
  • tumour cells and/or blood cells eg monocytes and T cells.
  • tissue sample may be a biopsy sample of the tumour or a cancer such as a needle biopsy samples, or a tumour biopsy sections or an archival sample thereof.
  • a tissue sample may comprise living, dead or fixed cells, such as from the tumour or a cancer, and such cells may be suspected of expressing (e.g. aberrantly or localised) the applicable biomarker to be determined.
  • the biological sample is a blood sample from the subject, such as a sample of immune cells present in blood (eg monocytes and T cells).
  • a sample of immune cells present in blood eg monocytes and T cells.
  • determination and/or diagnosis method of the invention can comprise, such as in a further step, comparing the detected amount (or activity of) of (eg protein or mRNA of) the applicable biomarker (ie ULRB1 and/or LILRB2 or a variant thereof) with a standard or cut-off value; wherein a detected amount greater than the standard or cut-off value indicates a phenotype (or a risk of developing a phenotype) that is associated with the undesired presence of LILRB1 and/or ULRB2-positive cells (or cells positive for a variant of ULRB1 and/or ULRB2) and/or that it associated with cellular resistance against the cell-mediated immune response in the subject and/or is associated with (eg aberrant) expression or activity of ULRB1 and/or ULRB2 (or the variant) in the subject.
  • Such a standard or cut-off value may be determined from the use of a control assay, or may be pre-determined from one or more values obtained from a study or a plurality of samples having known phenotypes.
  • a cut-off value for a diagnostic test may be determined by the analysis of samples taken from patients in the context of a controlled clinical study, and determination of a cut-off depending on the desired (or obtained) sensitivity and/or specificity of the test.
  • Examples of methods useful in the detection of (such as the presence or absence of, or an amount of) the applicable biomarker include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA), which employ ABP (eg of the present invention) such as an antibody or an antigen-binding fragment thereof, that specifically binds to such applicable biomarker.
  • immunoassays such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA), which employ ABP (eg of the present invention) such as an antibody or an antigen-binding fragment thereof, that specifically binds to such applicable biomarker.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • ABP eg of the present invention
  • a monoclonal antibody or a polyclonal antibody may be employed. Examples of monoclonal antibodies are described elsewhere herein.
  • polyclonal antibody refers to a mixture of antibodies which are genetically different since produced by plasma cells derived from multiple somatic recombination and clonal selection events and which, typically, recognise a different epitope of the same antigen.
  • the presence of the applicable biomarker may be detected by detection of the presence of mRNA that encodes such applicable biomarker, or fragments of such mRNA.
  • Methods to detect the presence of such mRNA (or fragments) can include, PCR (such as quantitative RT-PCR), hybridisation (such as to Illumina chips), nucleic-acid sequencing etc.
  • PCR such as quantitative RT-PCR
  • hybridisation such as to Illumina chips
  • Such methods may involve or comprise steps using one or more nucleic acids as described herein, such as PCR primers or PCR probes, or hybridisation probes, that bind (eg specifically) to such mRNA.
  • the ABP or biABP or nucleic acid typically, will be labelled with a detectable labelling group.
  • labelling groups fall into a variety of classes, depending on the assay in which they are to be detected: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox active moieties; d) optical dyes; enzymatic groups (e.g.
  • a secondary reporter e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.
  • Suitable labelling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, "Tc, U1 ln, 125 l, 131 l), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognised by a secondary reporter (eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • radioisotopes or radionuclides e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, "Tc, U1 ln, 125 l,
  • the labelling group is coupled to the ABP or biABP or nucleic acid via spacer arms of various lengths to reduce potential steric hindrance.
  • Various methods for labelling proteins are known in the art and may be used.
  • the ABP or biABP or nucleic acid may be labelled with a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.).
  • the means eg ABP or biABP or nucleic acid
  • the detection eg detector
  • protein or mRNA of the applicable biomarker eg LJLRBl and/or LLLRB2
  • label or “labelling group” refers to any detectable label, including those described herein.
  • the detection/diagnostic methods of the invention involve an immunohistochemistry (IHC) assay or an immunocytochemistry (IC) assay.
  • IHC immunohistochemistry
  • IC immunocytochemistry
  • IHC and ICC are art recognised, and include the meanings of techniques employed to localise antigen expression that are dependent on specific epitope-antibody interactions.
  • IHC typically refers to the use of tissue sections
  • ICC typically describes the use of cultured cells or cell suspensions.
  • positive staining is typically visualised using a molecular label (eg, one which may be fluorescent or chromogenic). Briefly, samples are typically fixed to preserve cellular integrity, and then subjected to incubation with blocking reagents to prevent non-specific binding of the antibodies. Samples are subsequently typically incubated with primary (and sometimes secondary) antibodies, and the signal is visualised for microscopic analysis.
  • kits such as one for performing the diagnostic methods or the determination methods or the detection methods (or the monitoring or prognostic methods) of the invention, eg, for determining the presence, absence, amount, function, activity and/or expression of the applicable biomarker (ie LJLRBl and/or ULRB2 or a variant thereof) in a sample (eg a biological sample), such as on cells in a sample.
  • the kit comprises an ABP or biABP and/or a nucleic acid as described above and, optionally one or more additional components.
  • an additional component may comprise instructions describing how to use the ABP or biABP or a nucleic acid or kit, for detecting the presence of the applicable biomarker in the sample, such as by detecting binding between the ABP or biABP and protein such applicable biomarker, and/or detecting binding between the nucleic acid and mRNA of such applicable biomarker.
  • Such instructions may consist of a printed manual or computer readable memory comprising such instructions, or may comprise instructions as to identify, obtain and/or use one or more other components to be used together with the kit.
  • the additional component may comprise one or more other claim, component, reagent or other means useful for the use of the kit or practice of a detection method of the invention, including any such claim, component, reagent or means disclosed herein useful for such practice.
  • the kit may further comprise reaction and/or binding buffers, labels, enzymatic substrates, secondary antibodies and control samples, materials or moieties etc.
  • Item l An isolated antigen binding protein (ABP) which specifically binds to the extra cellular domain (ECD) of leukocyte immunoglobulin-like receptor subfamily Bl (LJLRBl) and/or LILRB2 protein and wherein the isolated ABP or biABP comprises at least one complementarity determining region (CDR) and is able to inhibit the binding of LILRB1 and/or LILRB2 to a natural ligand thereof (a natural ligand of LILRB1 and/or LILRB2, such as HLA-G), preferably wherein the ABP or biABP does not bind to, or binds with less affinity to, a leukocyte immunoglobulin-like receptor subfamily A (ULRA) protein, such as ULRA1 and/or LILRA3.
  • ULRA leukocyte immunoglobulin-like receptor subfamily A
  • Item 3 The isolated ABP or biABP of item 1 or 2, wherein the ABP or biABP is an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences, and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences and at least one, preferably both, of the antibody light chain sequences comprise CDR1 to CDR3 sequences in a combination selected from any of the following combinations of heavy and/or light chain CDRs, CDRs-A-001 to CDRs-A-044: in each case independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences.
  • Item 4 The isolated ABP or biABP of any one of items 1 to 3, wherein the ABP or biABP is an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences, and at least one, preferably two, antibody light chain sequences,
  • the antibody heavy chain sequences each comprises heavy chain CDR1 to CDR3 sequences in the combination CDRs-A-010 and at least one, preferably both, of the antibody light chain sequences each comprises light chain CDR1 to CDR3 sequences in the combination CDRs-A-010, in each case independently, optionally with no more than one amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences, and preferably wherein the ABP or biABP specifically binds to ULRB1 and/or LILRB2 protein or a variant thereof with an IC50 of lOnM or less, preferably of about 5nM or less; or
  • the antibody heavy chain sequences each comprises heavy chain CDR1 to CDR3 sequences in the combination CDRs-A-026 and at least one, preferably both, of the antibody light chain sequences each comprises light chain CDR1 to CDR3 sequences in the combination CDRs-A-026, in each case independently, optionally with no more than one amino acid substitution(s), insertion(s) or deletion(s) compared to these sequences, and preferably wherein the ABP or biABP specifically binds to ULRB1 and/or LILRB2 protein or a variant thereof with an IC50 of lOnM or less, preferably of about 5nM or less.
  • Item 5 An isolated ABP or biABP which competes with an ABP or biABP as recited in any one of items 1 to 4 for binding to the ECD of the ULRB1 and/or LILRB2 protein and is able to inhibit the binding of the ULRB1 and/or LILRB2 protein or the variant thereof to a natural ligand of ULRB1 and/or LILRB2, preferably wherein the isolated ABP or biABP.
  • Item 6 The isolated ABP or biABP of any one of items 1 to 5 that specifically binds to LILRB1 and/or LILRB2 with a KD of lOOnM or less (preferably 50nM or less, more preferably lOnM or less); preferably wherein the isolated ABP or biABP binds to LILRA, such as ULRA1 and/or ULRA3, with a KD of 50nM or more (preferably of lOOnM or more, more preferably of 200nM or more).
  • Item 7 The isolated ABP or biABP of any one of items 1 to 6 that (i) inhibits a binding of a natural ligand of ULRB1 and/or LILRB2 to LILRB1 and/or LILRB2, wherein such ligand is preferably HLA-G, and/or (ii) reduces development of immune-suppressive phenotypes, for example in a tumour microenvironment.
  • Item 8 The isolated ABP or biABP of any one of items 1 to 7that is an antibody or an antigen binding fragment thereof, wherein the antibody is a monoclonal antibody, or wherein the antigen binding fragment is a fragment of a monoclonal antibody.
  • Item 9 The isolated ABP or biABP of any one of items 1 to 8 that is an antibody or an antigen binding fragment thereof, wherein the antibody is a human antibody a humanised antibody or a chimeric-human antibody, or wherein the antigen binding fragment is a fragment of a human antibody a humanised antibody or a chimeric-human antibody.
  • Item 10 An isolated nucleic acid encoding for an ABP or biABP, or for an antigen binding fragment or a monomer of an ABP or biABP, wherein the ABP or biABP is one recited in any one of items 1 to 9.
  • Item 11 A recombinant host cell comprising a nucleic acid recited in item 10.
  • Item 12 A pharmaceutical composition comprising: (i) an ABP or biABP recited in any one of items 1 to 9, or
  • nucleic acid recited in item 10 or (iii) a recombinant host cell of item 11, and a pharmaceutically acceptable carrier, stabiliser and/or excipient.
  • Item 13 A product for use in medicine, wherein the product is selected from the list consisting of: (i) an isolated ABP or biABP recited in any one of items 1 to 9, and (ii) an isolated nucleic acid recited in item 10, (iii) a recombinant host cell of item 11, and (iv) a pharmaceutical composition of item 12.
  • Item 14 The product for use in medicine of item 13 wherein the product is for use in the treatment of an infective disorder or a proliferative disorder that is associated with a suppressed cell-mediated immune response due to the expression or activity of LILRB1 and/or ULRB2 or a variant thereof.
  • Item 15 The product for use in medicine of item 14, wherein cells involved in the proliferative disorder express a natural ligand of LILRB1 and/or ULRB2, and wherein such cells induce an immune-suppressive phenotype in immune cells, such as macrophages.
  • Item 16 The product for use in medicine of any one of items 13 to 15, wherein the product is for use in enhancing an immune response in a mammalian subject, preferably for use in promoting immune-activation of a humoral or cell-mediated immune response in the subject, such as promoting pro-inflammatory polarization of macrophages towards an Ml phenotype, for example for treating a proliferative disease, such as a cancer disease, of for treating an infectious disease.
  • Item 17 The product for use in medicine of any one of items 13 to 16, wherein the product is for use in the treatment of a proliferative disorder resistant and/or refractory to PD1/CTLA4 blockade therapy.
  • Item 18 The product for use of any one of items 13 to 17, wherein the product has any one or any combination of the following characteristics: a. specific binding to human ULRB1 and/or (preferably and) LILRB2 (e.g. comprising the amino acid sequence of SEQ ID NO: 353 (LILRB1) and SEQ ID NO: 358 (ULRB2)), e.g., with a KD 50 nM or less, or of 20nM or less; more preferably of lOnM or less or 5nM or less b. lack of specific binding to a ULRA protein, such as preferably LILRA1 and/or ULRA3; c.
  • LILRB2 e.g. comprising the amino acid sequence of SEQ ID NO: 353 (LILRB1) and SEQ ID NO: 358 (ULRB2)
  • LILRB2 e.g. comprising the amino acid sequence of SEQ ID NO: 353 (LILRB1) and SEQ ID NO: 358 (ULRB2)
  • T cell activation e.g., in a mixed lymphocyte reaction (MLR) assay, as measured by increased T cell proliferation or IFN-gamma secretion, e.g.;
  • MLR mixed lymphocyte reaction
  • IFN-gamma secretion e.g.
  • d stimulates differentiation or activation of monocytes into macrophages, e.g., stimulates differentiation of monocytes into pro-inflammatory macrophages, e.g., as shown in an assay described in the Examples;
  • f. has a binding profile as shown in Table 2; g. promotes pro-inflammatory polarization of macrophages towards Ml macrophages; and h. does not induce (or trigger) basophil activation.
  • Item Bl An isolated antigen binding protein (ABP) which specifically binds to the extra cellular domain
  • ECD leukocyte immunoglobulin-like receptor subfamily Bl
  • LILRB1 and LILRB2 protein leukocyte immunoglobulin-like receptor subfamily Bl
  • the isolated ABP or biABP comprises at least one, preferably 3 or 6, complementarity determining regions (CDRs) and is capable of inhibiting the binding of LJLRBl and LILRB2 to a natural ligand thereof (a natural ligand of LILRB1 and/or LILRB2, such as HLA-G), wherein the ABP or biABP does not bind to, or binds with less affinity to (at least 2 times less), LILRA1 and LLLRA3.
  • CDRs complementarity determining regions
  • Item B2 The isolated ABP or biABP of item Bl, wherein the ABP or biABP binds to the ECD of LILRB1 and
  • LLLRB2 with at least 5 times higher, or preferably 10 times higher binding affinity compared the binding of the ABP or biABP to an ECD of LLLRA1 and/or LILRA3, preferably wherein the ABP or biABP binding affinity is determined by biolayer interferometry (BLI).
  • BBI biolayer interferometry
  • Item B3 The isolated ABP or biABP of item Bl or B2, wherein the ABP or biABP binds to the ECD of LJLRBl and/or LJLRB2 with a binding dissociation constant (KD) that is at least 2 times lower, preferably 3 times lower, more preferably 5 times lower, most preferably 10 times lower, compared to the binding KD of the ABP or biABP to an ECD of LILRA1 and/or LJLRA3, wherein the KD is determined by bio-layer interferometry (BLI), preferably under the conditions set out in example 3.
  • BBI bio-layer interferometry
  • Item B4 The isolated ABP or biABP of item B3, wherein the ABP or biABP binds to an ECD of LJLRBl and
  • LLLRB2 with a dissociation constant (KD) that is at least 2 times lower, preferably 3 times lower, more preferably 5 times lower, most preferably 10 times lower, compared to the KD of the ABP or biABP to an ECD of LLLRA1 and LILRA3, wherein the KD is determined by bio-layer interferometry (BLI), preferably under the conditions set out in example 3.
  • KD dissociation constant
  • Item B5 The isolated ABP or biABP of any one of items Bl to B4, wherein ABP or biABP comprises one, preferably two, an antibody heavy chain sequence and one, preferably two, antibody light chain sequence, and wherein the antibody heavy chain sequence and the antibody light chain sequence are derived from one or a combination of the antibody parental clones A-001 to A-003, preferably from A-001 and/or A-003.
  • Item B6 The isolated ABP or biABP of item B5, wherein the ABP or biABP comprises comprises one, preferably two, an antibody heavy chain variable sequence and one, preferably two, antibody light chain variable sequence, wherein the antibody heavy and light chain variable sequence each comprise a sequence that is at least 90%, preferably at least 95%, more preferably at least 96%, 97%, 98%, 99% identical to an antibody heavy or light chain variable sequence shown for any of the parental antibody sequences A-001 to A-003, preferably A-001 and/or A-003.
  • Item B7 The isolated ABP or biABP of any one of items Bl to B6, wherein the ABP or biABP competes for binding to an ECD of LJLRBl and/or LLLRB2, or to ECD of the variant of LJLRBl and/or LLLRB2, with an endogenous LJLRBl and/or LLLRB2 ligand or receptor, preferably wherein said endogenous LJLRBl and/or LLLRB2 ligand or receptor is a HLA-G protein (or a variant of HLA-G)
  • Item B8 The isolated ABP or biABP of item B7, wherein the ABP or biABP is capable of inhibiting the binding of HLA-G protein or a variant thereof to LILRBl and/or LLLRB2 protein or a variant thereof with an IC50 of less than lOOnM, less than 50nM, or preferably 20nM or less, such as 15nM or less, or less than lOnM, as determined by ELISA.
  • Item B9 The isolated ABP or biABP of any one items BItem Bl : to B8, comprising at least one complementarity determining region 3 (CDR3) having an amino acid sequence with at least 90% sequence identity to, or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from SEQ ID Nos. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79,
  • CDR3 complementarity determining region 3
  • Item BIO The isolated ABP or biABP of any one of items BItem Bl: to B9, wherein the ABP or biABP is an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences, and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences and at least one, preferably both, of the antibody light chain sequences comprise CDR1 to CDR3 sequences in a combination selected from any of the following combinations of heavy and/or light chain
  • Item Bll An isolated ABP or biABP which competes with an ABP or biABP as recited in any one of items BItem Bl: to BIO for binding to the ECD of the ULRB1 and/or ULRB2 protein and is able to inhibit the binding of the ULRB1 and/or ULRB2 protein or the variant thereof to a natural ligand of ULRB1 and/or LILRB2, preferably wherein the isolated ABP or biABP.
  • Item B12 The isolated ABP or biABP of any one of items BItem Bl: to Bll that (i) inhibits a binding of a natural ligand of ULRB1 and/or ULRB2 to LILRB1 and/or LILRB2, wherein such ligand is preferably HLA-G, and/or (ii) reduces development of immune-suppressive phenotypes, for example in a tumour microenvironment.
  • Item B13 The isolated ABP or biABP of any one of items BItem Bl: to B12 that is an antibody or an antigen binding fragment thereof, wherein the antibody is a monoclonal antibody, or wherein the antigen binding fragment is a fragment of a monoclonal antibody.
  • Item B14 An isolated nucleic acid encoding for an ABP or biABP, or for an antigen binding fragment or a monomer of an ABP or biABP, wherein the ABP or biABP is one recited in any one of items BItem Bl: to B13.
  • Item B15 A recombinant host cell comprising a nucleic acid recited in item B14.
  • Item B16 A pharmaceutical composition comprising: (I) an ABP or biABP recited in any one of items BItem
  • Bl to B13, or (ii) a nucleic acid recited in item B14, or (iii) a recombinant host cell of item B15, and a pharmaceutically acceptable carrier, stabiliser and/or excipient.
  • Item B17 A product for use in medicine, wherein the product is selected from the list consisting of: (i) an isolated ABP or biABP recited in any one of items BItem Bl: to B13, and (ii) an isolated nucleic acid recited in item BItem B14:, (iii) a recombinant host cell of item B15, and (iv) a pharmaceutical composition of item B16.
  • Item B18 The product for use of item BFehler! Verweissammlung Ober Vietnamese ceremoni ceremony entrench., wherein the use in medicine is a treatment of a proliferative disorder, and wherein cells involved in the proliferative disorder express a natural ligand of ULRB1 and/or ULRB2, and wherein such cells induce an immune-suppressive phenotype in immune cells, such as macrophages, preferably of the subject.
  • Item B19 The product for use of item BItem B17: or B18, wherein the product is for use in enhancing an immune response in a mammalian subject to be treated, preferably for use in promoting immune-activation of a humoral or cell-mediated immune response in the subject, such as promoting pro-inflammatory polarization of macrophages towards an Ml phenotype, for example for treating a proliferative disease, such as a cancer disease, of for treating an infectious disease.
  • a proliferative disease such as a cancer disease
  • Item B20 The product for use of any one of items B17 to B19, wherein the product has any one or any combination of the following characteristics: a. specific binding to human ULRB1 and/or (preferably and) LILRB2 (e.g. comprising the amino acid sequence of SEQ ID NO: 353 (LILRB1) and SEQ ID NO: 358 (ULRB2)), e.g., with a KD 50 nM or less, or of 20nM or less; more preferably of lOnM or less or 5nM or less b. lack of specific binding to a ULRA protein, such as preferably LILRA1 and/or ULRA3; c.
  • LILRB2 e.g. comprising the amino acid sequence of SEQ ID NO: 353 (LILRB1) and SEQ ID NO: 358 (ULRB2)
  • LILRB2 e.g. comprising the amino acid sequence of SEQ ID NO: 353 (LILRB1) and SEQ ID NO: 358 (ULRB2)
  • T cell activation e.g., in a mixed lymphocyte reaction (MLR) assay, as measured by increased T cell proliferation or IFN-gamma secretion, e.g.;
  • MLR mixed lymphocyte reaction
  • IFN-gamma secretion e.g.
  • d stimulates differentiation or activation of monocytes into macrophages, e.g., stimulates differentiation of monocytes into pro-inflammatory macrophages, e.g., as shown in an assay described in the Examples;
  • f. has a binding profile as shown in Table 2; g. promotes pro-inflammatory polarization of macrophages towards Ml macrophages; and h. does not induce (or trigger) basophil activation.
  • ABP bispecific antigen binding protein
  • a first antigen-binding site which specifically binds to the extra cellular domain (ECD) of leukocyte immunoglobulin-like receptor subfamily Bl (ULRB1) and LILRB2 protein and wherein the isolated ABP comprises at least one, preferably 3 or 6, complementarity determining regions (CDRs) and is capable of inhibiting the binding of LILRB1 and LILRB2 to a natural ligand thereof (a natural ligand of ULRB1 and/or ULRB2, such as HLA-G), wherein the ABP does not bind to, or binds with less affinity to (at least 2 times less), ULRAl and LILRA3, and (ii) a second antigen-binding site specifically binding to an immune checkpoint inhibitor, preferably PD1 or PDL1.
  • CDRs complementarity determining regions
  • C-Item 2 The isolated bispecific ABP of C-item 1, wherein the first antigen-binding site binds to the ECD of ULRB1 and ULRB2 with at least 5 times higher, or preferably 10 times higher binding affinity compared the binding of the first antigen-binding site to an ECD of ULRAl and/or ULRA3, preferably wherein the binding affinity of the first antigen-binding site is determined by bio-layer interferometry (BLI).
  • BBI bio-layer interferometry
  • C-Item 3 The isolated bispecific ABP of C-item 1 or 2, wherein the first antigen-binding site binds to the ECD of LILRB1 and/or ULRB2 with a binding dissociation constant (KD) that is at least 2 times lower, preferably 3 times lower, more preferably 5 times lower, most preferably 10 times lower, compared to the binding KD of the first antigenbinding site to an ECD of ULRAl and/or ULRA3, wherein the KD is determined by bio-layer interferometry (BLI), preferably under the conditions set out in example 3.
  • KD binding dissociation constant
  • C-Item 4 The isolated bispecific ABP of C-item 3, wherein the first binding site of the ABP binds to an ECD of ULRB1 and ULRB2 with a dissociation constant (KD) that is at least 2 times lower, preferably 3 times lower, more preferably 5 times lower, most preferably 10 times lower, compared to the KD of the first antigen-binding site to an ECD of ULRAl and ULRA3, wherein the KD is determined by bio-layer interferometry (BU), preferably under the conditions set out in example 3.
  • KD dissociation constant
  • C-Item 5 The isolated bispecific ABP of any one of C-items 1 to 4, wherein first antigen-binding site of the bispecific ABP comprises one, preferably two, antibody heavy chain sequence, and one, preferably two, antibody light chain sequence, and wherein the antibody heavy chain sequence and the antibody light chain sequence are derived from one or a combination of the antibody parental clones A-001 to A-003, preferably from A-001 and/or A-003.
  • C-Item 6 The isolated bispecific ABP of C-item 5, wherein the first antigen-binding site of the bispecific ABP comprises one, preferably two, antibody heavy chain variable sequence, and one, preferably two, antibody light chain variable sequence, wherein the antibody heavy and light chain variable sequences each comprise a sequence that is at least 90%, preferably at least 95%, more preferably at least 96%, 97%, 98%, 99% identical to an antibody heavy or light chain variable sequence shown for any of the parental antibody sequences of antibodies A-001 to A-003, preferably A-001 and/or A-003.
  • C-Item 7 The isolated bispecific ABP of any one of C-items 1 to 6, wherein the first antigen-binding site of the bispecific ABP competes for binding to an ECD of LILRB1 and/or ULRB2, or to ECD of the variant of ULRB1 and/or ULRB2, with an endogenous LILRB1 and/or ULRB2 ligand or receptor, preferably wherein said endogenous LILRB1 and/or LILRB2 ligand or receptor is an HLA-G protein (or a variant of HLA-G).
  • C-Item 8 The isolated bispecific ABP of C-item 7, wherein the first antigen-binding site of the bispecific ABP is capable of inhibiting the binding of HLA-G protein or a variant thereof to ULRB1 and/or LILRB2 protein or a variant thereof with an IC50 of less than lOOnM, less than 50nM, or preferably 20nM or less, such as 15nM or less, or less than lOnM, as determined by ELISA.
  • C-Item 9 The isolated bispecific ABP of any one C-items Item Bl: to 8, wherein the first antigen-binding site of the bispecific ABP comprises at least one complementarity determining region 3 (CDR3) having an amino acid sequence with at least 90% sequence identity to, or having no more than three or two, preferably no more than one amino acid substitution(s), deletion(s) or insertion(s) compared to, a sequence selected from SEQ ID Nos. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127,
  • CDR3 complementarity determining region 3
  • C-Item 10 The isolated ABP of any one of C-items Item Bl: to 9, wherein the first antigen-binding site of the bispecific ABP is an antibody, or an antigen binding fragment thereof, composed of at least one, preferably two, antibody heavy chain sequences, and at least one, preferably two, antibody light chain sequences, wherein at least one, preferably both, of the antibody heavy chain sequences and at least one, preferably both, of the antibody light chain sequences comprise CDR1 to CDR3 sequences in a combination selected from any of the following combinations of heavy and/or light chain CDRs, CDRs-A-001 to CDRs-A-048: in each case independently, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to these sequence
  • C-Item 11 The isolated bispecific ABP of any one of C-items 1 to 10, wherein the first antigen-binding site of the bispecific ABP comprises a heavy chain variable domain sequence comprising: a heavy chain CDR1 of SEQ ID NO: 353, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to this sequence; and a heavy chain CDR2 of SEQ ID NO: 354, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to this sequence; and a heavy chain CDR3 of SEQ ID NO: 355, optionally with no more than three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to this sequence; and and comprises a light chain variable domain sequence comprising a light chain CDR1 of SEQ ID NO: 357, optionally with no more than three or two, preferably
  • C-Item 12 The isolated bispecific ABP of any one of C-items 1 to 11, wherein the first antigen-binding site of the bispecific ABP comprises an antibody heavy chain variable domain sequence of SEQ ID No: 356, optionally with no more than ten, nine, eight, seven, six, five, four, three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to this sequence; and comprises an antibody light chain variable domain sequence of SEQ ID No: 360, optionally with no more than ten, nine, eight, seven, six, five, four, three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to this sequence.
  • SEQ ID No: 356 optionally with no more than ten, nine, eight, seven, six, five, four, three or two, preferably no more than one, amino acid substitution(s), insertion(s) or deletion(s) compared to this sequence
  • SEQ ID No: 360 optionally with no
  • C-Item 13 The isolated bispecific ABP of any one of C-items 1 to 12, wherein the first antigen-binding site of the bispecific ABP binds to the same epitope of an extracellular domain of LJLRBl and/or LLLRB2 as antibody clone A- 045.
  • C-Item 14 The isolated bispecific ABP of any one of C-items 1 to 13, characterized by: a. specifically binding to LJLRBl and LILRB2, wherein the bispecific ABP is characterized by binding the same epitope on LJLRBl and LILRB2 as an antibody of Table 1, preferably A-001, A-003, A-010 or A-045; b. not binding to, or binding with less affinity to, LILRA1; c. not binding to, or binding with less affinity to, LILRA3; d. when bound to LJLRBl and/or LLLRB2 on a human monocyte decrease M2-like polarization of monocyte.
  • C-Item 15 The isolated bispecific ABP of C-item 14, wherein the first antigen-binding site of the bispecific ABP is characterized by not binding to, or binding with less affinity to, LILRA1 and LLLRA2 compared to the binding affinity of the ABP to LJLRBl and LLLRB2.
  • C-Item 16 The isolated bispecific ABP of C-item 14 or 15, further characterized by e. when bound to LILRB1 on a human macrophage induces phagocytosis of the cell.
  • C-Item 17 The isolated bispecific ABP of any one of C-items 14 to 16, wherein the bispecific ABP is further characterized by f. when bound to human LJLRBl and/or LLLRB2 on a human monocyte or macrophage reduces expression of markers of M2-like macrophages, such as CD163, CD206 or CD209.
  • C-Item 18 The isolated bispecific ABP of any one of C-items 14 to 17 wherein the bispecific ABP is further characterized by g. when bound to human LILRB1 and/or LILRB2 on a human monocyte or macrophage increases expression of markers of Ml-like macrophages, such as CD86.
  • C-Item 19 The isolated bispecific ABP of any one of C-items 14 to 18, wherein the bispecific ABP is further characterized by h.
  • ULRB1 and/or ULRB2 on a human monocyte or macrophage induces secretion of pro-inflammatory cytokines, such as increases secretion GM-CSF, IFNg and IL-9; preferably independent of the presence of LILRA1 and/or LILRA3.
  • pro-inflammatory cytokines such as increases secretion GM-CSF, IFNg and IL-9; preferably independent of the presence of LILRA1 and/or LILRA3.
  • C-Item 20 The isolated bispecific ABP of any one of C-items 1 to 19, wherein the bispecific ABP is an inhibitor or antagonist of LILRB1 and LILRB2, preferably of an interaction of LILRB1 and LILRB2 with a natural ligand thereof, such as HLA-G.
  • C-Item 21 The isolated bispecific ABP of any one of C-items 1 to 20, wherein the bispecific ABP when bound to ULRB1 or LILRB2 on a macrophage reduces the polarization into an M2 like macrophage or repolarize an M2 like macrophage.
  • C-Item 22 The isolated bispecific ABP of any one of C-items 1 to 21, wherein the ABP when bound to ULRB1 or ULRB2 on a macrophage reduces the polarization into an M2 like macrophage or repolarize an M2 like macrophage into an Ml like macrophage.
  • C-Item 23 The isolated bispecific ABP of any one of C-items 1 to 22, wherein the bispecific ABP comprises at least one, alternatively two, first antigen-binding site(s), and at least one, alternatively two, second antigen-binding site(s).
  • C-Item 24 The isolated bispecific ABP of any one of C-items 1 to 23, wherein the bispecific ABP comprises two first antigen-binding sites and two second antigen binding sites, wherein (X) the two second antigen binding sites are provided as one F(ab')2 fragment or one IgG antibody, wherein the IgG antibody comprises an antibody hinge-, CH2- and/or CH3-domain, and wherein the two first antibody binding sites are provided as two antibody scFv or scFab fragments, each of which is independently covalently connected to one of the two first antigen-binding sites, preferably via a protein linker, or (Y) the two first antigen binding sites are provided as one F(ab')2 fragment or one IgG antibody, wherein the IgG antibody comprises an antibody hinge-, CH2- and/or CH3-domain, and wherein the two first antibody binding sites are provided as two antibody scFv or scFab fragments, each of which is independently covalently connected to the two second antigen
  • C-Item 25 The isolated bispecific ABP of C-item 24, which is composed of at least one, preferably two, first antibody (heavy/light) chain sequences, and at least one, preferably two, second antibody (heavy/light) chain sequences, wherein at least one, preferably both, of the first antibody (heavy/light) chain sequences comprises in N- to C-terminal direction:
  • (1-2) optionally, one or more antibody heavy chain constant domain sequences, such as a CHI, hinge, CH2 and/or CH3 domain,
  • (1-8) optionally, an antibody heavy chain constant domain sequence; and wherein at least one, preferably both, of the second antibody (heavy/light) chain sequences comprises in N- to C- terminal direction (2-1) an antibody light chain variable domain sequence comprising at least parts of the second antigen-binding site;
  • (2-2) an antibody light chain constant domain; wherein [(1-4) to (1-5)] and [(1-7) to (1-8)] can be interchanged, and/or wherein (1-4) and (1-5); and (1-7) and (1- 8); can be (both) interchanged.
  • (1-2) optionally, one or more antibody heavy chain constant domain sequences, such as a CHI, hinge, CH2 and/or
  • (1-8) optionally, an antibody heavy chain constant domain sequence; and wherein at least one, preferably both, of the second antibody (heavy/light) chain sequences comprises in N- to C- terminal direction
  • (2-2) an antibody light chain constant domain; wherein [(1-4) to (1-5)] and [(1-7) to (1-8)] can be interchanged, and/or wherein (1-4) and (1-5); and (1-7) and (1- 8); can be (both) interchanged.
  • C-Item 28 The isolated bispecific ABP of any one of C-items 1 to l, wherein the second antigen binding site is anti PD1/PDL1 binding site.
  • C-Item 29 The isolated bispecific ABP of C-item 28, wherein the second antigen binding site comprises CDR1, CDR2 and CDR3 regions from a known inhibitory anti-PD-l/PD-Ll antibody.
  • C-Item 30 The isolated bispecific ABP of C-item 29, wherein the anti-PD-l/PD-Ll antibody is selected from nivolumab durvalumab, pembrolizumab, cemiplimab, avelumab, durvalumab, and atezolizumab, and preferably is pembrolizumab.
  • C-Item 31 An isolated bispecific ABP which competes with an ABP as recited in any one of C-items Item Bl: to 30 for binding to the ECD of the LILRB1 and/or ULRB2 protein and is able to inhibit the binding of the LILRB1 and/or ULRB2 protein or the variant thereof to a natural ligand of LILRB1 and/or ULRB2, preferably wherein the isolated ABP.
  • C-Item 32 An isolated bispecific ABP which competes with an ABP as recited in any one of C-items Item Bl: to 30 for binding to the ECD of the LILRB1 and/or ULRB2 protein and is able to inhibit the binding of the LILRB1 and/or ULRB2 protein or the variant thereof to a natural ligand of LILRB1 and/or ULRB2, preferably wherein the isolated ABP.
  • C-Item 32 An isolated bispecific ABP which competes with an ABP as recited in any one of
  • C-Item 33 The isolated ABP of any one of C-items Item Bl: to 32 that is an antibody or an antigen binding fragment thereof, wherein the antibody is a monoclonal antibody, or wherein the antigen binding fragment is a fragment of a monoclonal antibody.
  • C-Item 34 An isolated nucleic acid encoding for an ABP, or for an antigen binding fragment or a monomer of an ABP, wherein the ABP is one recited in any one of C-items Item Bl: to 33.
  • C-Item 35 The isolated nucleic acid of C-item 34, comprising a nucleic acid having a sequence encoding a heavy or light chain CDR, a combination of heavy and/or light chain CDR1, CDR2 and CDR3 or a heavy or light chain variable domain, in each case as displayed in Table 1.
  • a nucleic acid construct comprising a nucleic acid of C-item 34 or 35, optionally with one or more additional features permitting the expression of the encoded ABP or component of said ABP in a cell, such as in a host cell.
  • C-Item 37 The NAC of C-item 36, comprising two constructs, one of which includes a nucleic acid encoding the heavy antibody chain, the other of which includes a nucleic acid encoding the light antibody chain, such that expression from both constructs can generate a complete antibody molecule.
  • C-Item 38 A recombinant host cell comprising a nucleic acid recited in C-item 34 or 35, or a NAC recited in C-item 36 or 37.
  • C-Item 39 A pharmaceutical composition comprising: (i) an ABP recited in any one of C-items Item Bl: to 33, or (ii) a nucleic acid recited in C-item 34 or 35, or (iii) a NAC recited in C-item 36 or 37, or (iv) a recombinant host cell of 38, and a pharmaceutically acceptable carrier, stabiliser and/or excipient.
  • C-Item 40 A pharmaceutical composition comprising: (i) an ABP recited in any one of C-items Item Bl: to 33, or (ii) a nucleic acid recited in C-item 34 or 35, or (iii) a NAC recited in C-item 36 or 37, or (iv) a recombinant host cell of 38, and a pharmaceutically acceptable carrier, stabiliser and/or excipient.
  • a product for use in medicine wherein the product is selected from the list consisting of: (i) an ABP recited in any one of C-items Item Bl: to 33, or (ii) a nucleic acid recited in C-item 34 or 35, or (iii) a NAC recited in C-item 36 or 37, or (iv) a recombinant host cell of 38, and (iv) a pharmaceutical composition of C-item 39.
  • C-Item 41 The product for use of C-item 40, wherein the use in medicine is a treatment of a proliferative disorder, and wherein cells involved in the proliferative disorder express a natural ligand of LILRB1 and/or ULRB2, and wherein such cells induce an immune-suppressive phenotype in immune cells, such as macrophages, preferably of the subject.
  • C-Item 42 The product for use of C-item 40 or 41, wherein the product is for use in enhancing an immune response in a mammalian subject to be treated, preferably for use in promoting immune-activation of a humoral or cell-mediated immune response in the subject, such as promoting pro-inflammatory polarization of macrophages towards an Ml phenotype, for example for treating a proliferative disease, such as a cancer disease, of for treating an infectious disease.
  • a proliferative disease such as a cancer disease
  • C-Item 43 The product for use of any one of C-items 40 to 42, wherein the product has any one or any combination of the following characteristics: a. specific binding to human ULRB1 and/or (preferably and) LILRB2 (e.g. comprising the amino acid sequence of SEQ ID NO: 353 (LILRB1) and SEQ ID NO: 358 (ULRB2)), e.g., with a KD 50 nM or less, or of 20nM or less; more preferably of lOnM or less or 5nM or less b. lack of specific binding to a ULRA protein, such as preferably LILRA1 and/or ULRA3; c.
  • LILRB2 e.g. comprising the amino acid sequence of SEQ ID NO: 353 (LILRB1) and SEQ ID NO: 358 (ULRB2)
  • LILRB1 and/or ULRB2 e.g. comprising the amino acid sequence of SEQ ID NO: 353 (LILRB1) and SEQ
  • T cell activation e.g., in a mixed lymphocyte reaction (MLR) assay, as measured by increased T cell proliferation or IFN-gamma secretion, e.g.;
  • MLR mixed lymphocyte reaction
  • IFN-gamma secretion e.g.
  • d stimulates differentiation or activation of monocytes into macrophages, e.g., stimulates differentiation of monocytes into pro-inflammatory macrophages, e.g., as shown in an assay described in the Examples; e. inhibits binding of LILRB1 and/or (preferably and) ULRB2 to HLA-A and HLA-B, preferably to HLA-G;
  • f. has a binding profile as shown in Table 2;
  • g. promotes pro-inflammatory polarization of macrophages towards Ml macrophages; and h. does not induce (or trigger) basophil activation; and
  • i. induces secretion of proinflammatory cytokines in macrophages,
  • C-Item 44 The product for use of any one of C-items 40 to 43, wherein the ABP is characterized by binding to an epitope of an extracellular domain of ULRB1 and ULRB2 which is bound by the antibody clone A-045.
  • C-Item 45 A method of inducing phagocytosis in a cell, preferably a macrophage, the method comprising a step of contacting the macrophage with an ABP of any one of C-items 1 to 33.
  • a method of modulating macrophage polarization comprising a step of contacting a target cell with an ABP of any one of C-items 1 to 33, and thereby inducing or enhancing Ml like macrophage phenotype in said target cell.
  • C-Item 47 The method of C-item 46, wherein the target cell is a monocyte or macrophage.
  • C-Item 48 The method of C-item 46 or 47, wherein macrophage polarization is modulated in the presence of cells expressing LILRA1 and/or ULRA3.
  • C-Item 49 The method of any one of C-items 46 to 48, wherein the modulation is a repolarization of an M2 like phenotype into an Ml like phenotype.
  • C-Item 50 A method of treatment of a disease in a subject, the method comprising a step of administering to the subject a therapeutically effective amount of an ABP of any one of C-items 1 to 33.
  • C-Item 51 The method of C-item 50, wherein the treatment comprises a use recited in any one of C-items 40 to 44.
  • C-Item 52 The method of C-item 50 or 51, wherein the disease is a proliferative disease, preferably is a cancer.
  • C-Item 53 The method of any one of C-items 50 to 52, wherein the treatment involves antagonizing immune- suppressive macrophage polarization in the subject, such as a polarization into M2 macrophages, and thereby enhances cell-mediated immune responses against cells involved with the disease.
  • the term “comprising” is to be construed as encompassing both “including” and “consisting of”, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention.
  • “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other.
  • a and/or B is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
  • the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question.
  • the term typically indicates deviation from the indicated numerical value by ⁇ 20%, ⁇ 15%, ⁇ 10%, and for example ⁇ 5%.
  • the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect.
  • a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.
  • the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect.
  • a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.
  • Example 1 Generation and Maturation of antibodies that bind to human ULRB1 and/or LILRB2.
  • Affinity improved ULRB2 and LILRB1 cross-specific antibodies were selected by phage display from antibody gene libraries based on the parental V gene sequence with diversified CDR-H1/H2 and CDR-L3, respectively.
  • two diversified libraries were constructed: (i) keeping the light chain constant and diversifying CDR- H1 and CDR-H2, and (ii) keeping the heavy chain constant and diversifying CDR-L3.
  • CDR-H1/2 diversified libraries contained between 8e8 and 4e9 derivatives
  • CDR-L3 diversified libraries contained more than 2e7 derivatives of the respective parental sequence. The diversification of the CDR sequences was based on a rational design.
  • Antibody phage bound to recombinant ULRB1 were separated using streptavidin magnetic beads (Dynabeads M-280, ThermoFisher) and washed with DPBST including an extended washing step for 30 min in presence of 100 nM non-biotinylated target protein and 500 nM non-biotinylated recombinant LILRA1 and LILRA3.
  • the antibody phage particles were eluted using 10 ug/mL Trypsin and used to infect mid-logarithmic E. coli TGI for phage amplification.
  • Panning rounds two and three were performed equivalently to panning round one with the following modifications to increase the selection pressure for higher affinity:
  • concentration of biotinylated recombinant target protein (ULRB2 in panning round two and ULRB1 in panning round three) was further limited (1 nM in panning round two and 0.5 nM in panning round three).
  • an initial washing step with DPBST was performed.
  • the beads were suspended in 1.5 mL of PBST containing 100 nM of non-biotinylated recombinant target protein and 100 nM non-biotinylated LILRA1 and LILRA3 and incubated for up to 20 h at room temperature.
  • Antibodies A-001, A-002 and A-003 constitute the selected parental antibodies.
  • Antibodies A-004 to A-048 constitute derivative antibodies of a heavy and/or light chain sequence of one or two of the parental molecules.
  • A-004 to A-021 are heavy chain (HC) variants of parental antibody A-001.
  • A-022 to A-033 are HC variants of parental antibody A-003.
  • A-034 to A-036 are light chain (LC) variants of parental antibody A-001, A-037 is an LC variant of A-002.
  • A-038 to A-044 are LC variants of parental antibody A-003.
  • Antibody A-045 is a derivative of antibody A-001 and comprises the HC of A-010 and the LC of A-036.
  • A-046 is a derivative of antibody A-001 and comprises the HC of A-015 and the LC of A-036.
  • A-047 is combination of A-001 and A-003 derived HC and LC chains, and comprises the HC of A-025 and the LC of A-038.
  • A-048 is combination of A- 001 and A-003 derived HC and LC chains, and comprises the HC of A-025 and the LC of A-039.
  • Table 1 Amino acid sequences of CDR and variable regions of ABPs of the invention, as well as nucleic acid sequences encoding variable regions of ABPs of the invention.
  • biotinylated recombinant LILRB2, ULRBl, ULRAl, and ULRA3 were immobilized at 1 ug/mL on a streptavidin-coated 384-well Maxisorp plate.
  • the surface was blocked with 2% (w/v) bovine serum albumin (BSA) in PBST
  • BSA bovine serum albumin
  • the bacterial lysates in 2% (w/v) BSA were applied to the immobilized antigens and incubated for 1.5 h.
  • bound Fab antibodies were detected with a goat anti-human Fab antibody conjugated with horseradish peroxidase.
  • the ELISA was developed with TMB substrate.
  • the sensors were dipped into lx Kinetics Buffer supplemented with 10 ug/mL biocytin and 2% (w/v) BSA to measure the dissociation for 420 s. All sensorgrams were double referenced against lx Kinetics Buffer supplemented with 10 ug/mL biocytin and 2% (w/v) BSA, and empty streptavidin sensors to subtract sensor drifts and potential reference binding of the bacterial lysates. Dissociation rates were fitted using a 1: 1 binding model.
  • Affinities were determined by BLI on the Octet RED96e system using the ECDs of recombinant LLLRB2, LILRBl, LLLRAl, and LLLRA3. Briefly, IgGs were captured on anti-human capture (AHC) biosensors (Sartorius). The sensors were dipped into the wells containing 100 nM of recombinant LLLRB2 or LILRBl ECDs, or 900 nM of LILRA1 ECD or LLLRA3 and incubated for 300 s to measure association. Subsequently, the sensors were dipped into lx Kinetics Buffer to measure the dissociation over 420 s. All sensorgrams were double referenced against lx Kinetics Buffer and unloaded streptavidin sensors to subtract sensor drifts and potential reference binding of the analytes. The affinities were determined by applying a 1:1 binding model.
  • Binding curves of the antibody clones of the invention are provided in Figure 2. Dissociation constants (KD) are shown for the tested antibodies in Table 2 below.
  • biolayer interferometry (BLI) experiments were performed on an Octet RED96e system. Briefly, biotinylated recombinant LILRB2 ECD (extracellular domain) or LILRBl ECD was immobilized on streptavidin coated biosensors.
  • Binding curves of the antibodies of the invention are shown for LLLRB2 ( Figure 11) and LILRBl ( Figure 12).
  • A-045 showed no or only minor loss of binding potency towards LILRBl or LILRB2 in presence of LLLRAl or LILRA3, while a dramatic loss in binding potency up to no detectable binding was observed for crossspecific reference molecule (Ref062 [NGM]), due to high affinity of Ref062 towards LILA1 and LILA3. Similarly, a dramatic loss in binding potency towards LILRB1 was observed for reference molecule (Ref051 [Biond/Sanofi BND-22]) in presence of LLLRAl or LLLRA3.
  • HLA-G inhibition was tested by ELISA. 384-well Maxisorp plates were coated with 2ug/mL B2M-HLA-G fused to a mouse IgG2a-Fc and the plates were blocked with 2% (w/v) BSA in PBST. 30nM biotinylated LILRB2 ECD or LILRBl ECD were mixed with a 1: 1000 dilution of Avidin-HRP (Biolegend, 405103) in a 1:1 volumetric ratio and incubated for lh at room temperature.
  • Example 4.1 Cross-specific LILRB1/2 antibodies of the invention decrease M2- like polarization of monocyte derived macrophages
  • MO Macrophages were generated by culture of isolated monocytes in media (RPMI with 10%FBS + 1%P/S) containing 50ng/ml recombinant human M- CSF (Peprotech) and indicated concentrations of in-house cross-specific LILRB1/2 antibodies, reference cross-specific ULRB1/2 antibody (Ref062 [NGM707]), reference monospecific ULRB2 antibody (Ref047 [Merck MK-4830]), or appropriate isotype control (RefOOl) for 6 days at 37°C in 6-well tissue culture plates.
  • MO macrophages were removed from the plate with a cell scraper and polarized to M2-like macrophages in media (RPMI with 10%FBS + 1%P/S) containing 20ng/ml recombinant human IL-4 (Peprotech), 20ng/ml recombinant human IL-10 (Peprotech), and 20ng/ml recombinant human TGF-p (Peprotech) and indicated concentrations of in-house cross-specific ULRB1/2 antibodies, reference cross-specific ULRB1/2 antibody (Ref062 [NGM707]), reference monospecific LILRB2 antibody (Ref047 [Merck MK-4830]), or appropriate isotype control (RefOOl) for 2 days at 37°C in 6-well tissue culture plates. On day 8, supernatant was taken for analysis of cytokines via Luminex Assay (R8iD Systems) and M2-like macrophages were analyzed by flow cytometry for various surface markers indicative of an M2-like phenotype
  • cross-specific ULRB1/2 antibodies as well as monospecific LILRB2 antibody induced a significant decrease in M2-like macrophage phenotype marker CD163, consistent with a less suppressive M2-like polarization of the macrophages.
  • cross-specific LILRB1/2 antibodies as well as monospecific LILRB2 antibody also significantly decrease the secretion of immunosuppressive CCL13 and CCL23 cytokines of macrophages after M2-like polarization (see Figure 6B and C).
  • Example 4.2 Cross-specific LILRB1/2 antibodies of the invention decrease M2- like polarization of monocyte derived macrophages and rescue cytotoxic T cell activation
  • the myeloid suppression assay was performed as the functional myeloid assay described above. However, on day 8, after supernatant was taken for analysis of myeloid cytokines, isolated autologous T cells were added to the macrophages at a ratio of 1:5 for a co-culture for an additional 3 days. On day 11, supernatant was again taken for analysis of T cell cytokines via Luminex Assay (R8iD Systems) and T cells were analyzed by flow cytometry for surface activation markers.
  • Example 4.3 Cross-specific LJLRB1/2 antibody of the invention induce repolarization of monocyte derived Ml- and M2- like macrophages.
  • MO Macrophages were generated by culture of isolated monocytes in media (RPMI with 10%FBS + 1%P/S) containing 50ng/ml recombinant human M-CSF (Peprotech) and indicated concentrations of in-house cross-specific ULRB1/2 antibody, reference cross-specific LILRB1/2 antibody (Ref062 [NGM707]) or appropriate isotype control (RefOOl) for 6 days at 37°C in 6-well tissue culture plates.
  • MO macrophages were removed from the plate with a cell scraper and polarized to Ml- like macrophages in the presence lOOng/ml recombinant LPS or to M2-like macrophages in media (RPMI with 10%FBS + 1%P/S) containing 20ng/ml recombinant human IL-4 (Peprotech), 20ng/ml recombinant human IL-10 (Peprotech), and 20ng/ml recombinant human TGF-p (Peprotech) and indicated concentrations of in-house cross-specific ULRB1/2 antibody, reference cross-specific ULRB1/2 antibody (Ref062 [NGM707]) or appropriate isotype control (RefOOl) for 2 days at 37°C in 6-well tissue culture plates. On day 8, Ml- and M2-like macrophages were analyzed by flow cytometry for various surface markers.
  • Expi293 cells were transiently transfected to express proteins of interest ULRB1, ULRB2, ULRAl, LILRA2, and ULRA3.
  • Non-transfected Expi293 cells were used as negative control. Cells were harvested and labeled with CellTraceViolet for multiplexing.
  • mono-specific reference molecule Ref047 showed binding only to ULRB2 (1.7 nM) and none of the other targets ( Figure 7B), while ULRB1/2 cross-specific reference molecule Ref062 has high affinity for ULRB1 (9.1nM) and LILRB2 (17.3nM) but also showed binding to LILRAl (3.7nM) and, surprisingly also to LILRA3 (7.8nM) (Figure 7C).
  • A-045 showed high affinity binding to primary T cells (InM) and monocytes (0.9nM) ( Figure 7D and 7E).
  • affinity of cross-specific reference molecule Ref062 is slightly lower on both cell types with 12 nM and 4.6 nM on T cells and monocytes, respectively.
  • ULRB2 mono-specific reference antibody Ref047 only binds on monocytes (InM) due to the lack of ULRB2 expression on primary T cells, highlighting superiority of the ABP of the invention.
  • T cell suppression and tumour cell killing was analyzed by tri-culture assay, briefly describe as follows ( Figure 8a): CD14+ monocytes and CD3+ T-cells from leukapheresis donors were isolated, frozen, and processed independently. Monocytes were thawed and differentiated to macrophages in medium with M-CSF, in presence of either hIgGls_A-010 or negative control, hIgGls_Ref-001. Differentiated macrophages were scraped from flasks and polarized to M2-like macrophages with IL-4, IL-10 and TGF-B for 2 days.
  • MDA-MB- 231/Luc-2A-GFP tumour cell line (which co-expresses the luciferase gene and GFP fluorescent markers) was added for a tri-culture (tumour killing assay) in presence of either hIgGls_A-010 or negative control, hIgGls_Ref001.
  • hIgGls_A-010 or negative control hIgGls_Ref001.
  • luciferase reporter assay Figure 8C
  • % confluency mask of GFP expressing tumour cell was evaluated using Incucyte Opti-Green software (data not shown) which directly correlates with tumour cell viability.
  • T cell activation as assessed by IL-2 secretion measured in ELISA, is fully suppressed when T cells are cocultured with M2-polarized macrophages in the presence of an isotype control antibody, Ref-001.
  • This macrophage- mediated T cell suppression is significantly reverted by the addition of a functional anti-LILRB2/l antibody, A-010 ( Figure 8B).
  • Assay controls include IL-2 secretion levels of either unstimulated T cells (negative control) or stimulated T cells (positive control) in the absence of macrophages.
  • Ref-001 isotype control (with full macrophage-mediated T cell suppression) does not translate into significant tumour cell killing compared to negative assay controls (tumour cells only or tumour cells in the presence of stimulated T cells but in the absence of suppressive, M2-polarized macrophages).
  • Example 7 Displacement of LILRB1 or ULRB2 from HLA-G interaction site and inhibition of new complex formation between LILRB1 or LILRB2 with HLA-G
  • biolayer interferometry (BU) experiments were performed on an Octet RED96e system. Briefly, biotinylated recombinant HLA-G was immobilized on streptavidin coated biosensors to 1.5nm using a fixed loading threshold. To block unoccupied biotin binding sites on the streptavidin coated biosensors, biocytin was included in the assay buffer at a concentration of lOpg/mL for all further steps.
  • the sensors were dipped in wells containing the streptavidin premixed with either ULRB1 or ULRB2 to allow the tetrameric complexes of LILRB1 or LILRB2 to bind to the HLA-G immobilized on the biosensors. Subsequently, the sensors were dipped into lx kinetics buffer containing lOOnM of antigen binding fragment (Fab) to measure the displacement of the tetrameric complexes of LILRB1 or LILRB2 from the HLA-G immobilized on the biosensors over the course of 1800 seconds.
  • Fab antigen binding fragment
  • the dissociation of the tetrameric complexes of LILRB1 or LILRB2 from the HLA-G immobilized on the biosensors was analyzed by dipping the sensor in lx kinetics buffer not containing antigen binding fragment.
  • A-045, A-047, and A-048 showed stronger inhibition of complex formation between HLA-G and ULRB1 or LILRB2, respectively, than cross-specific reference molecule (Ref062 [NGM]) or antibody A-010, which both showed comparable inhibition.
  • Ref062 [NGM] cross-specific reference molecule
  • antibody A-010 which both showed comparable inhibition.
  • displacement of LILRB2 from HLA-G interaction site and inhibition of new complex formation with HLA-G was more pronounced than displacement of LILRB1.
  • MO macrophages were generated by culture of isolated monocytes in media (RPMI with 10%FBS + 1%P/S) containing 50ng/ml recombinant human M-CSF (Peprotech) for 6 days at 37°C in 6-well tissue culture plates.
  • MO macrophages were removed from the plate with a cell scraper and polarized to M2-like macrophages in media (RPMI with 10%FBS + 1%P/S) containing 20ng/ml recombinant human IL-4 (Peprotech), 20ng/ml recombinant human IL-10 (Peprotech), and 20ng/ml recombinant human TGF-p (Peprotech) or polarized to Ml-like macrophages with lOOng/ml recombinant LPS for 2 additional days at 37°C in 6-well tissue culture plates.
  • media RPMI with 10%FBS + 1%P/S
  • T cells were additionally stained for CD3 and CD8 to allow gating on CD3/ CD8 double positive cells.
  • neutrophils cells were stained for CD45 and CD66b to allow gating on CD45/CD66b double positive cells. Cells were washed again and mixed with 7-AAD viability dye before flow acquisition to test for surface binding.
  • A-045 showed high affinity binding superior to the cross-specific reference antibody Ref062 on all investigated primary immune cells, such as primary CD8 positive T cells (EC50: 15.1nM), neutrophils (EC50: 0.5nM) and monocytes (EC50: 0.9nM) ( Figure 13 A-C). Additionally, A045 showed high affinity to in vitro differentiated Ml- (EC50: 3nM) and M2-like macrophages (EC50: 2.8nM) ( Figure 13 D and E).
  • Example 9 ULRB2 antagonism of ABP of the invention was demonstrated using a specific ILT4-/HLA-G Blockade Bioassav Kit (Promega)
  • HLA-G expressing APC/CHO-K1 cells were thawed, seeded into assay plates and incubated over night at 37°C/ 5% CO2.
  • LILRB2- expressing Jurkat reporter cells were thawed and incubated with HLA-G expressing cells in the presence of A-045, a cross-specific ULRB1/2 reference antibody (Ref062 [NGM707]) or the respective isotype control (RefOOl) in a dose titration starting at 200nM. After incubation for six hours at 37°C/ 5% CO2, luciferase assay buffer was added and luminescence signal was measured.
  • A-045 showed highly efficient blocking of ULRB2 with EC50 value of 0.34nM surprisingly superior to the cross-specific reference antibody Ref062 (EC50: 0.9nM) ( Figure 15 B).
  • Example 10 Cross-specific LJLRB1/2 antibodies of the invention increasing phagocvtotic activity of macrophages
  • MO Macrophages were generated by differentiation of isolated monocytes in media (RPMI with 10%FBS + 1%P/S) containing 50ng/ml recombinant human M-CSF (Peprotech) for 6 days at 37°C in 6- well tissue culture plates. On day six, macrophages were harvested and pre-treated with A-045, a cross-specific ULRB1/2 reference antibody (Ref062 [NGM707]) and an appropriate isotype control (RefOOl) at the indicated concentrations for two hours.
  • Ref062 cross-specific ULRB1/2 reference antibody
  • RefOOl isotype control
  • A375 tumour cells were labelled with Cell Trace Far Red (CTFR) and cell necrosis was induced by heat exposure. Then macrophages were co-cultured with CFTR labelled A375 tumour cells in an effector to target ration of 1:8 in the presence of antibodies for two hours. After co-culture, cells were stained for CD45 expression to identify macrophages and phagocytosis activity was analyzed by Flow Cytometry by evaluating the sum of CD45/ CFTR double positive macrophages as percent of total CD45+ macrophages. Graphs were normalized by setting the isotype control to 100%.
  • CTR Cell Trace Far Red
  • Example 11 Sustained strong activity of A-045 despite the presence of ULRA3
  • MO Macrophages were generated by culture of isolated monocytes in media (RPMI with 10%FBS + 1%P/S) containing 50ng/ml recombinant human M-CSF (Peprotech) for 6 days at 37°C in 6-well tissue culture plates. MO macrophages were removed from the plate with a cell scraper and polarized to Ml-like macrophages with lOOng/ml recombinant LPS for 2 additional days at 37°C in 6-well tissue culture plates.
  • Example 12 [prophetic] : In-vivo anti-tumour properties of cross-specific LJLRB1/2 antibodies of the invention
  • Antibody A-045 is investigated together with the corresponding cross-specific reference molecule (Ref062 [NGM]) in a proof-of-concept efficacy in vivo study.
  • NOG-EXL mice are humanized using human hematopoietic stem cells (CD34+) isolated from human cord blood following the CROs proprietary humanization protocol or mice were purchased already humanized from the breeder. In the latter case, huNOG-EXL that have a humanization rate above 15%, will be used. Different HSC Donors are used, and the humanization rate (human CD45+ human CD33+) is tested.
  • LILRB1 and LILRB2 expression is checked post-humanization on immune cell subsets in peripheral blood of the mice.
  • mice are randomized for human HSC donors and the selected tumour cell line is inoculated unilaterally on the left flank, subcutaneously (e.g. MDA-MB-231, MSTO-211H, A-375). Treatment starts when the average tumour volume reaches ⁇ 50-100mm 3 . Treatments and corresponding schedule is provided in Table 3.
  • the humanized mice are randomized based on tumour volume, body weight, CD34 + donors and humanization rate into the following groups: A- 045, RefOOl (isotype control; vehicle), and cross-specific reference molecule (Ref062 [NGM]). Animal health is monitored daily for unexpected signs of distress. A clinical score is determined 3 times per week.
  • the tumour volume (TV) is measured 3 times per week using a caliper or by images using BioVolume.
  • percent tumour growth inhibition (%TGI) values will be calculated and reported for each treatment group (T) versus control (C)
  • T treatment group
  • C control
  • a TV exceeding 1500 mm 3 is considered as an endpoint and the mouse will be sacrificed.
  • the tumour is collected at sacrifice, weighed, and analyzed for immune cells (Flow Cytometry) and Cytokines (e.g. Luminx/ Bioplex). .
  • Flow Cytometry Flow Cytometry
  • Cytokines e.g. Luminx/ Bioplex
  • a bispecific construct targeting ULRB1/2 and PD-1 was designed and produced based on the basis of the antigen binding domain sequences of pembrolizumab and A-045 (see table 1 above).
  • the exemplary antibody was designed in a 2x2 format using the A-045 sequences as scFv fragments in the heavy chain, and the IgG frame of pembrolizumab.
  • Figure 20 shows the general format of the biABP of example 13 and its sequence construction.
  • the heavy chain sequence comprises the following mutations: L234A, L235E, G237A, A330S and P331S. Location and length of the linker sequences is shown in Figure 20 (B) and (C).
  • the bispecific antibody was produced and compared to control bispecific showed excellent monomericity.
  • Example 14.1 Expression and Purification of the bisoecific Ab of example 13
  • the bispecific antibody of example 13 (ULRB1/2 - PD-1) was expressed using suspension-adapted CHO KI cells.
  • the CHO KI cells were cultured in a chemically defined and animal-component-free medium and transfected with plasmids encoding heavy and light chains. The supernatant was harvested through centrifugation and subsequent filtration using a 0.2pm filter. The antibody was then purified employing MabSelectTM SuReTM resin.
  • the resulting bispecific antibody exhibited a yield of 160 mg/L and was formulated in PBS 100 mmol/l L- arginine.
  • Example 14.2 Size Exclusion Chromatography of the bisoecific Ab of example 13 [488] Purity was determined by analytical size exclusion chromatography with an Agilent AdvanceBio SEC column (300A, 2.7pm, 4.6 x 300mm) and 200mM sodium phosphate (pH 7.2) as running buffer at 0.35 ml/min, loading 10 pg protein.
  • HPLC-SEC was employed to evaluate the monomeric content of the target protein. Prior to preparative sizeexclusion chromatography (SEC), the monomericity was determined to be 88.2%, indicating the presence of a significant proportion of higher-order aggregates. Following preparative SEC, the monomeric fraction increased to 96.6%, demonstrating a substantial improvement in sample homogeneity and confirming the efficacy of the SEC step in enriching the monomeric form of the protein.
  • Example 14.3 SDS-PAGE Analysis of the bispecific Ab of example 13
  • Fragmentation was assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, 0.7pg of protein was dissolved in 4x Laemmli Sample Buffer (Biorad), incubated for 5 min at 90°C, and cooled down immediately on ice for 5 min. For analysis under reducing conditions, 20mM DTT (Biorad) was added. Samples were loaded on 4-20% StainFree TGX gels (BioRad) and separated at 200V for 30 min run in Tris/Glycine/SDS Buffer (Biorad). Gels were UV-activated and visualized using Biorad ChemiDoc station and ImageLab software.
  • SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis
  • SDS-PAGE analysis was performed to assess the integrity and composition of the target protein following purification. Under non-reducing conditions, a single major band was observed at approximately 250 kDa, consistent with the expected molecular weight of the intact protein complex (see Figure 21A). Densitometric analysis indicated that 84.4% of the total protein corresponded to the intact target protein after Protein A affinity purification. This proportion increased to 89.5% following preparative size-exclusion chromatography (SEC), indicating improved purity and recovery of the intact protein complex. Under reducing conditions (see Figure 21B), the SDS-PAGE profile revealed two distinct bands at approximately 75 kDa and 25 kDa, corresponding to the heavy chain (HC) and light chain (LC) subunits, respectively. These bands together accounted for greater than 99% of the total protein content, confirming the identity and high purity of the target protein subunits.
  • SEC size-exclusion chromatography
  • Example 15 Target and off-target binding data of the bisoecific Ab of example 13
  • Affinities were determined by BLI on the Octet RED96e system using the ECDs of recombinant human ULRB1, ULRB2, LILRB3, ULRAl and LILRA3 as well as cynomolgus monkey LILRB proteins. Briefly, bispecific antibody (ULRB1/2 - PD-1) was captured on Streptavidin (SA) biosensors (Sartorius) using Biotin Anti-human Fab-kappa Conjugate (ThermoFisher Scientific).
  • the loaded sensors were dipped into the wells containing a 3-fold dilution series of the respective recombinant ECD starting at the following maximum concentrations: lOOnM for human ULRB1 and ULRB2, and cyno LILRB; 300nM for human ULRB3; 900nM for human ULRAl, and ULRA3. Sensors were incubated in the analyte solutions for 300 s to measure association. Subsequently, the sensors were dipped into lx Kinetics Buffer containing 10 pg/mL Biocytin to measure dissociation. The affinities were determined by applying a 1:1 binding model.
  • Example 16 PD-1 binding data of the bisoecific Ab of example 13
  • Affinities were determined by BLI on the Octet RED96e system using the recombinant ECD of PD-1 protein. Briefly, bispecific antibody (LILRB1/2 - PD-1) was captured on Streptavidin (SA) biosensors (Sartorius) using CaptureSelectTM Biotin Anti-IgG-Fc (Human) Conjugate (ThermoFisher Scientific). The loaded sensors were dipped into the wells containing a 3-fold dilution series starting at 33.3nM of the recombinant PD-1 ECD and incubated for 300 s to measure association. Subsequently, the sensors were dipped into lx Kinetics Buffer containing lOpg/mL Biocytin to measure dissociation. The affinities were determined by applying a 1:1 binding model.
  • Dissociation constant (KD) is shown for the tested antibody in Table 5 below.
  • Table 5 Dissociation constant (KD) for the bispecific antibody (LJLRB1/2 - PD-1) binding to human PD-1 protein.
  • Example 17 Simultaneous binding of the bispecific Ab of example 13 to both targets
  • response/loading for the mixed target samples is expected to be comparable to the sum of response/loading for each of the two targets individually (proof for simultaneous binding). Accordingly, as shown in Table 6, response/loading for the target mixtures (0.56 [LJLRB2] and 0.47 [LJLRBl], respectively with PD-1) equals 97% of the sum of response/loading for PD-1 (0.17) + LLLRB2 (0.41) and 94% in case of PD-1 (0.17) + LJLRBl (0.33), matching the expectation and confirming the bispecific Ab's capability to bind both targets at the same time (see also Figure 22 A and B).
  • Table 6 Response/loading for the bispecific antibody LILRB1/2 - PD-1 in a simultaneous binding assay setup towards human ULRB1, ULRB2, PD-1, or a mixture of PD-1 with either LILRB protein.
  • Example 18 The LILRB1/2 - PD-1 bisoecific molecule of example 13 enhances T cell activation in an allogenic macrophage- T cell mixed lymphocyte reaction
  • a macrophage- mixed lymphocyte reaction with in vitro differentiated MO macrophages and allogenic T cells was performed to characterize the effects of the LILRB1/2 - PD-1 bispecific molecule on T cell stimulation in comparison to the LILRB1/2 cross-specific antibody A-045, the reference oPD-1 monospecific antibody as well as a combination of both oLILRBl/2 and oPD-1 antibodies.
  • CD14+ monocytes and CD3+ T-cells from leukapheresis donors were isolated, frozen, and processed independently.
  • Monocytes were thawed and differentiated to MO-Macrophages by culture in differentiation media (CLM medium) containing + 50 Ill/mL recombinant human M-CSF for 6 days at 37°C in 6-well tissue culture plates. On day 6, differentiated MO-macrophages were re-seeded in 96 well plates.
  • CLM medium differentiation media
  • Isolated allogenic T cells were added to the macrophages and cultured for 5 days in a co-culture setting in a cell ratio of 1: 10 in the presence of the bispecific antibody LILRB1/2 - PD-1, A-045 (both lOnM), an anti- PD-1 antibody (50nM), a combination of both A-045 (lOnM) plus anti-PD-1 (50nM) or appropriate isotype control RefOOl . After 5 days of co-culture, supernatants were harvested and analyzed for IL-2.
  • T cell activation was assessed by IL-2 secretion measured in ELISA relative to the respective isotype control.
  • IL-2 secretion was increased in the presence of cross-specific antibody A-045 or an anti-PD-1 antibody compared to the respective isotype control treatment.
  • IL-2 release is more pronounced by combining A-045 and anti-PD-1 therapy.
  • the ULRB1/2 - PD-1 bispecific molecule showed even synergistic efficacy with significantly enhanced IL-2 secretion in comparison to the isotype control or the combination therapy.
  • Assay controls included a co-culture condition of macrophages and T cells without treatment antibody (medium control) where IL-2 levels were in the same range as the isotype control condition.
  • SEQ ID NOs. 1 to 384 (Amino acid sequences of CDR and variable regions of ABPs of the invention, as well as nucleic acid sequences encoding variable regions of ABPs of the invention, see Table 1):
  • SEQ ID NO. 385 Human ULRB1 protein isoform 1; UniProt identifier Q8NHL6-1):
  • SEQ ID NO. 386 Human LJLRBl protein isoform 2; UniProt identifier Q8NHL6-2
  • SEQ ID NO. 387 Human LJLRBl protein isoform 3; UniProt identifier Q8NHL6-3
  • SEQ ID NO. 388 Human LJLRBl protein isoform 4; UniProt identifier Q8NHL6-4
  • SEQ ID NO. 389 Human LJLRBl protein isoform 5; UniProt identifier Q8NHL6-5):
  • SEQ ID NO. 390 Human LLLRB2 protein isoform 2; UniProt identifier Q8N423-2:
  • SEQ ID NO. 391 Human ULRB2 protein isoform 1; UniProt identifier Q8N423-1):
  • SEQ ID NO. 392 Human ULRB2 protein isoform 3; UniProt identifier Q8N423-3):
  • SEQ ID NO. 393 Human ULRB2 protein isoform 4; UniProt identifier Q8N423-4):
  • SEQ ID NO. 396 Pembrolizumab variable heavy chain sequence: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQG LEWMGGIN PSNGGTNFN EKFKN RVTLTTDSSTTTAYM ELKS LQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS

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Abstract

L'invention concerne des protéines bispécifiques de liaison à l'antigène (ABP), telles que des anticorps bispécifiques, qui se lient avec un premier site de liaison à l'antigène à la fois à la sous-famille B1 des récepteurs de type immunoglobuline leucocytaire (LILRB1) et LILRB2 sans se lier à, ou en se liant avec une affinité significativement moindre à la sous-famille A des récepteurs de type immunoglobuline leucocytaire (LILRA). Les ABP bispécifiques de l'invention se lient avec un second site de liaison à l'antigène à un point de contrôle immunitaire (molécules) tel que PD-1 ou PD-L1. Les ABP bispécifiques de l'invention peuvent également inhiber l'interaction entre LILRB1 et/ou LILRB2 et un ligand naturel des récepteurs ULRB (par exemple, des protéines en interaction, telles que HLA-G) sur des cellules immunitaires et l'inhibition d'une telle interaction peut réduire la suppression de cellules immunitaires et ainsi favoriser des réponses immunitaires anti-infection et antitumorales chez un sujet souffrant de telles maladies. Des molécules bispécifiques combinant l'antagonisme LILRB1/2 avec l'inhibition de points de contrôle immunitaires, telle que l'inhibition de l'axe PD-1/PD-L1, sont spécifiquement utiles dans le traitement de troubles prolifératifs. L'invention propose également des procédés de réduction de la suppression immunitaire de cellules impliquées dans une réponse immunitaire à médiation cellulaire, et/ou des procédés de traitement de maladies infectieuses et/ou prolifératives, à l'aide d'une protéine de liaison à l'antigène LILRB1 et/ou LILRB2 telle qu'un anticorps se liant à la fois à LILRB1 et/ou à LILRB2, ainsi que certains aspects associés comprenant des procédés de détection, de diagnostic et de criblage.
PCT/EP2025/068165 2024-06-26 2025-06-26 Protéines bispécifiques de liaison à l'antigène (abp) ciblant des molécules de point de contrôle immunitaire et à la fois la sous-famille b1 des récepteurs de type immunoglobuline leucocytaire (lilrb1) et lilrb2; combinaisons et utilisations associées Pending WO2026003224A2 (fr)

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