WO2024083945A1 - Protéines de liaison à un antigène - Google Patents

Protéines de liaison à un antigène Download PDF

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WO2024083945A1
WO2024083945A1 PCT/EP2023/079057 EP2023079057W WO2024083945A1 WO 2024083945 A1 WO2024083945 A1 WO 2024083945A1 EP 2023079057 W EP2023079057 W EP 2023079057W WO 2024083945 A1 WO2024083945 A1 WO 2024083945A1
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flt3lg
seq
binding protein
binding
sequence
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PCT/EP2023/079057
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English (en)
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Allison BEAL
Nikolai BELYAEV
Patrick Koenig
Geil MERANA
Ashka PATEL
Yinghui Rong
Amy Yu
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Glaxosmithkline Intellectual Property (No.3) Limited
23Andme, Inc.
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Publication of WO2024083945A1 publication Critical patent/WO2024083945A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • 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

  • the claimed subject matter of the present application was made by or on behalf of parties to a joint research agreement that was in effect on or before the date the claimed subject matter was made; the claimed subject matter was made as a result of activities undertaken within the scope of the joint research agreement; and the parties to the joint research agreement include: GlaxoSmithKline Intellectual Property (No.3) Limited and 23andMe, Inc.
  • the joint research agreement was a written contract, grant or cooperative agreement entered into by the above-mentioned parties for the performance of experimental, developmental or research work in the field of the claimed subject matter.
  • Embodiments of the present disclosure relate to FLT3LG binding proteins including anti- FLT3LG antibodies and their uses in the treatment of autoimmune diseases.
  • autoimmune diseases occur when the body's immune system abnormally attacks healthy tissues with autoantibodies or autoreactive T cells.
  • autoimmune diseases include Systemic Lupus Erythematosus (SLE), celiac disease, rheumatoid arthritis, sarcoidosis, and Sjogren's Syndrome.
  • SLE Systemic Lupus Erythematosus
  • celiac disease celiac disease
  • rheumatoid arthritis rheumatoid arthritis
  • sarcoidosis Sjogren's Syndrome
  • FLT3LG binding proteins comprising: a. (i) any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 4 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 12; or
  • FLT3LG binding proteins comprising the following 6 CDRs:
  • FLT3LG binding proteins that bind to human FLT3LG, and competes for binding to human FLT3LG with a reference FLT3LG binding protein comprising: a VH region sequence of SEQ ID NO: 8 and a VL region sequence of SEQ ID NO: 16.
  • nucleic acid sequences which encode one or both of HC and LC of a FLT3LG binding protein as defined herein.
  • expression vectors comprising one or more nucleic acid sequences as defined herein.
  • Also provided herein are methods for the production of one or more FLT3LG binding proteins comprising culturing a recombinant host cell as defined herein under conditions suitable for expression of said nucleic acid sequence(s) or expression vector(s), whereby a polypeptide comprising a FLT3LG binding protein is produced.
  • cell lines engineered to express a FLT3LG binding protein as defined herein are also provided herein.
  • compositions comprising a FLT3LG binding protein as defined herein and a pharmaceutically acceptable excipient.
  • Also provided herein are methods for treatment of an autoimmune disease in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a FLT3LG binding protein or a pharmaceutical composition as defined herein.
  • FLT3LG binding proteins are also provided herein for use in therapy.
  • FLT3LG binding proteins and pharmaceutical compositions as defined herein for use in the treatment of autoimmune disease are also provided herein. DESCRIPTION OF DRAWINGS/ FIGURES
  • FIGURE 1 illustrates ELISA binding screening of human FLT3LG (hFLT3LG) and Cynomolgus monkey FLT3LG (cyno-FLT3LG) reactive clones.
  • FIGURE 2 illustrates anti-FLT3LG antibody binding to hFLT3LG and 293T-hFLT3LG cells, as measured by ELISA and a flow cytometry (FACS) binding assay, respectively.
  • the dashed box indicates antibodies binding to both hFLT3LG and membrane-bound hFLT3LG-expressing 293T cells.
  • FIGURE 3 illustrates the blocking of hFLT3LG-hFLT3 binding by 116 hFLT3LG reactive antibodies as measured by ELISA.
  • the dashed box indicates antibodies that block more than 50% of hFLT3LG binding with hFLT3, compared to isotype IgGl.
  • FIGURE 4 illustrates the binding affinity (KD) of 55 blocking antibodies against hFLT3LG or cyno-FLT3LG as determined using SPR and calculated using a 1:1 binding model.
  • FIGURE 5 illustrates the inhibition of the binding of hFLT3LG with the hFLT3 in a competitive ELISA assay for 15 humanized anti-FLT3LG antibodies (Panel A) and a subset of 4 selected humanized anti-FLT3LG antibodies (Panel B) (h_bl.O17, h_bl.O21, h_bl.O78 and h_bl.l26).
  • FIGURE 6 illustrates hFLT3LG cell binding of the humanized anti-FLT3LG antibody panel, as determined using flow cytometry for humanized anti-FLT3LG antibodies (Panel A) and a subset of 4 selected humanized anti-FLT3LG antibodies (Panel B).
  • FIGURE 7 illustrates the inhibition curves of the humanized anti-FLT3LG antibodies blocking binding of 0.75 nM soluble FLT3LG to cell-surface FLT3 receptor.
  • R&D SYSTEM'S anti-human FLT3LG antibody (clone #40416) (R&D SYSTEMS, Cat # MAB608) was compared to four selected humanized anti-FLT3LG antibodies: h_bl.O17 (Panel A), h_bl.O21 (Panel B), h_bl.O78 (Panel C), and h_bl.l26 (Panel D).
  • FIGURE 8 illustrates blocking activity of antibody candidates in a soluble FLT3LG-induced FLT3 receptor internalization assay on RS4;11 cells in clone h_bl.O17 (Panel A), h_bl.O21 (Panel B), clone h_bl.O78 (Panel C), and h_bl.l26 (Panel D).
  • Each of the lead antibodies are shown and compared to R&D Systems' anti-human FLT3LG antibody (clone # 40416) (R&D Systems, Cat. # MAB608) and isotype controls.
  • FIGURE 9 illustrates blocking activity of anti-FLT3LG antibody candidates in a cell-cell interaction induced FLT3 internalization assay.
  • Each of the lead anti-FLT3LG antibodies are shown and compared to R&D Systems' anti-human FLT3LG antibody (clone # 40416) (R&D Systems, Cat. # MAB608) and isotype controls.
  • FIGURE 10 illustrates blocking activity of anti-FLT3LG antibody candidates against soluble FLT3LG in a phospho-AKT transduction assay for four selected humanized blocking antibodies in clone h_bl.O17 (Panel A), clone h_bl.O21 (Panel B), clone h_bl.O78 (Panel C), and clone h_bl.l26 (Panel D).
  • Each of the lead antibodies were compared to R&D Systems' anti-human FLT3LG antibody (clone # 40416) (R&D Systems, Cat. # MAB608) and isotype controls.
  • FIGURE 11 illustrates blocking activity of anti-FLT3LG antibody candidates against soluble FLT3LG in a primary DC differentiation assay.
  • Each of the lead antibodies are shown and compared to R&D Systems' anti-human FLT3LG antibody (clone # 40416) (R&D Systems, Cat. # MAB608) and isotype controls for differentiation of CDllc+ HLA-DR+ cells (Panel A), plasmacytoid DC (pDC; BDCA2+ CD123+) (Panel B), and type 2 conventional DCs (cDC2; BDCA1+) (Panel C).
  • FIGURE 12 illustrates binding of anti-FLT3LG antibody candidates to endogenous FLT3LG expressed on primary human T cells for clone h_bl.O17 (Panel A), clone h_bl.O21 (Panel B), clone h_bl.O78 (Panel C), and clone h_bl.l26 (Panel D).
  • FIGURE 13 illustrates the effect of anti-FLT3LG antibodies (h_bl.O17, h_bl.O78 and h_bl.l26) on frequency of DC populations (Panel A) and numbers of DC populations (Panel B) in the spleen of human CD34-engrafted mice dosed with hFLT3LG.
  • Frequency of DC populations are expressed as the percentage of hCD45+ cells.
  • Each datapoint represents one mouse. Circles represent mice engrafted with Donor 17 hCD34+ cells.
  • Triangles represent mice engrafted with Donor 18 hCD34+ cells.
  • Bars represent means, and error bars represent SD.
  • 17, 78, or 126 represent mice dosed with anti-FLT3LG antibodies h_bl.O17, h_bl.O78, or h_bl.l26, respectively.
  • FIGURE 14 illustrates exemplar 2D class averages of anti-FLT3LG antibody (h_bl.O78 or comparator antibody 0001) with FLT3LG by NS-EM.
  • FLT3LG means any FMS-related tyrosine kinase 3 ligand. Pseudonyms for FLT3LG include FLT3 ligand, Flt3 ligand, FL, Flt3L, FLT3L, FLG3L, and FMS-related receptor tyrosine kinase 3 ligand.
  • FLT3LG is a molecule that is encoded by the FLT3LG ene in humans and by putative homologs in other species.
  • FLT3LG specifically binds to the FMS-related receptor tyrosine kinase 3 (FLT3) receptor.
  • FLT3LG can be expressed as a cell surface membrane-bound form. FLT3LG can be a soluble molecule or shed from the cell surface.
  • hFLT3LG refers to both cell surface membrane-bound human FLT3LG and human soluble FLT3LG, unless specified otherwise.
  • FLT3 refers to the FMS-like tyrosine kinase 3 (FLT3) receptor for FLT3LG. Pseudonyms for FLT3 include CD135, FLK2, and STK1. FLT3 is encoded by the FL T3 gene in humans and by putative homologs in other species, and is a class III receptor tyrosine kinase family molecule. FLT3 consists of an extracellular region having 5 Ig-like domains, a transmembrane domain and a cytoplasmic region having a juxta-membrane domain and two tyrosine kinase domains.
  • FLT3 Upon interaction of FLT3LG with FLT3, FLT3 homodimerizes leading to phosphorylation of the tyrosine kinase domains and downstream signalling.
  • FLT3 is human FLT3 (hFLT3).
  • FLT3 can be of another organism (e.g., mouse, rat, cow, dog, cat, pig, monkey, etc.).
  • antigen binding protein refers to antibodies, antigen binding fragments thereof, and other protein constructs, such as domains, that are capable of binding to an antigen.
  • FLT3LG binding protein refers to antigen binding proteins that are capable of binding to FLT3LG.
  • a FLT3LG binding protein can be capable of binding to one or more of a human FLT3LG and a FLT3LG protein of another organism (e.g., mouse, rat, cow, dog, cat, pig, monkey, etc.).
  • a FLT3LG binding protein can be capable of binding to a fragment of, a variant of, or a mutant of FLT3LG.
  • a FLT3LG binding protein as used herein is different from the naturally occurring protein that binds FLT3LG (e.g., FLT3).
  • antibody is used herein in the broadest sense to refer to molecules with an immunoglobulin-like domain (for example, IgG, IgM, IgA, IgD, or IgE) and includes monoclonal, recombinant, polyclonal, chimeric, human, humanized, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; a single variable domain (e.g., a domain antibody (DAB)), antigen binding antibody fragments, Fab, F(abQ2, Fv, disulfide linked Fv, single chain Fv, disulfide-linked scFv, diabodies, TANDABS, etc. and modified versions of any of the foregoing.
  • DAB domain antibody
  • An antibody provided herein can comprise a heterotetra meric glycoprotein with an approximate molecular weight of 150,000 daltons.
  • An antibody comprises two heavy chains (HCs) (which are typically identical) and two light chains (LCs) (which are typically identical) linked by covalent disulfide bonds. This H2L2 structure can fold to form three functional domains: two fragment antigen binding regions (Fab regions) and a crystallisable fragment region (Fc region).
  • HCs heavy chains
  • LCs light chains
  • This H2L2 structure can fold to form three functional domains: two fragment antigen binding regions (Fab regions) and a crystallisable fragment region (Fc region).
  • a Fab region comprises a variable domain, which comprises variable heavy (VH) and variable light (VL) chains, at the amino-terminus, and a constant domain, which comprises a first domain of a constant heavy chain (CH) and a constant light chain (CL) at the carboxyl-terminus.
  • An Fc region comprises two domains formed by dimerization of paired second and third domains (CH2 and CH3) of two constant heavy chains (CH).
  • An Fc region may elicit effector functions, for example, by binding to receptors on immune cells or by binding Clq, the first component of the classical complement pathway.
  • IgM Five classes of antibodies IgM, IgA, IgG, IgE, and IgD are defined by distinct heavy chain amino acid sequences: p, a, y, £, and 6; each heavy chain can pair with either a K or A light chain.
  • IgG immunoglobulin G
  • IgGl isotypes of human IgG
  • IgG2 isotypes of human IgG
  • IgG3 isotypes of human IgG
  • Antibodies provided herein can be fully human antibodies, and can be obtained using a variety of methods, for example, using a library of human antibodies or fragments in conjunction with an antibody display system like phage or yeast display or immunizing transgenic animals (e.g., mice) that are capable of producing repertoires of human antibodies.
  • the use of human antibody libraries in some cases, transgenic animals that have been modified to express human immunoglobulin genes can be immunised with an antigen of interest and antigen-specific human antibodies can be isolated using a variety of antibody discovery techniques including B-cell cloning, hybridoma, and repertoire sequencing. Human antibodies produced using these techniques can then be screened for desired properties such as activity, affinity, developability, and selectivity.
  • Alternative antibody formats can include alternative scaffolds in which the one or more CDRs of the antigen binding protein can be arranged onto a suitable non-immunoglobulin protein scaffold or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer, or an EGF domain.
  • a suitable non-immunoglobulin protein scaffold or skeleton such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer, or an EGF domain.
  • An antibody provided herein can be a "humanized antibody,” which refers to a type of engineered antibody having CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one or more human immunoglobulin(s).
  • framework support residues may be altered to preserve binding affinity.
  • a suitable human acceptor antibody may be one selected from a conventional database (e.g., the KABAT database, Los Alamos database, and Swiss Protein database), or by homology to the nucleotide and/or amino acid sequences of the donor antibody.
  • a human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs.
  • a suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains may originate from the same acceptor antibody or different acceptor antibodies.
  • antigen binding proteins described herein may be an antibody or an antigen binding fragment thereof.
  • An antigen binding protein may be a humanized antibody or an antigen binding fragment thereof.
  • An antigen binding protein may comprise one of, a plurality of, or all of: a humanized VH region or a humanized Heavy Chain (HC) sequence; and/or a humanized VL region or a humanized Light Chain (LC) sequence.
  • the term "donor antibody” refers to an antibody that contributes the amino acid sequences of one or more of its variable regions, CDRs, or other functional fragments or analogues thereof to a first immunoglobulin partner. A donor, therefore, provides the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralising activity characteristic of a donor antibody.
  • acceptor antibody refers to an antibody that is heterologous to a donor antibody, which contributes all (or any portion) of the amino acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner.
  • a human antibody may be an acceptor antibody.
  • domain refers to a folded polypeptide structure that can retain its tertiary structure independent of the rest of the polypeptide. Generally, domains are responsible for discrete functional properties of polypeptides and in many cases may be added, removed, or transferred to other polypeptides without loss of function of the remainder of the protein and/or of the domain.
  • single variable domain refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains such as VH, VHH, and VL and/or modified antibody variable domains, for example, in which one or more loops have been replaced by sequences that are not characteristic of antibody variable domains, or antibody variable domains that have been truncated or comprise N- or C- terminal extensions, as well as folded fragments of variable domains that retain at least the binding activity and specificity of the full-length domain.
  • a single variable domain herein is capable of binding an antigen or epitope independently of a different variable region or domain.
  • a "domain antibody” or “DAB” can be a human "single variable domain”.
  • a single variable domain may be a human single variable domain, but can also be a single variable domains from a non-human species such as rodent (for example, as in WO 00/29004), a nurse shark, or a Camelid.
  • camelid VHHs are immunoglobulin single variable domain polypeptides that are derived from camelid species, such as camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain only antibodies that are naturally devoid of light chains.
  • camelid VHH domains may be humanized according to standard techniques available in the art, and such domains can be "single variable domains".
  • An antigen binding fragment may be provided by means of arrangement of one or more CDRs on one or more non-antibody protein scaffolds.
  • Protein Scaffold as used herein includes, but is not limited to, an immunoglobulin (Ig) scaffold, for example, an IgG scaffold, which may be a four chain or two chain antibody, or which may comprise only the Fc region of an antibody, or which may comprise one or more constant regions from an antibody, which constant regions may be of human or primate origin, or which may be an artificial chimera of human and primate constant regions.
  • Ig scaffold immunoglobulin
  • a protein scaffold may be an Ig scaffold, for example, an IgG or IgA scaffold.
  • An IgG scaffold may comprise some or all the domains of an antibody (i.e., CHI, CH2, CH3, VH, VL).
  • An antigen binding protein may comprise an IgG scaffold selected from IgGl, IgG2, IgG3, IgG4, or IgG4PE.
  • a scaffold may be IgGl.
  • a scaffold may consist of, or comprise, an Fc region of an antibody or a fragment thereof.
  • a protein scaffold may be a derivative of a scaffold selected from the group consisting of CTLA-4, lipocalin, Protein A derived molecules such as Z-domain of Protein A (Affibody, SpA), A- domain (Avimer/Maxibody); heat shock proteins such as GroEl and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human g- crystallin and human ubiquitin (affilins); PDZ domains; scorpion toxin kunitz type domains of human protease inhibitors; and fibronectin/adnectin which has been subjected to protein engineering in order to obtain binding to an antigen, such as FLT3, other than a natural ligand.
  • an antigen such as FLT3, other than a natural ligand.
  • multi-specific antigen binding protein refers to an antigen binding protein that comprises at least two different antigen binding sites. Each of these antigen-binding sites can be capable of binding to a different epitope than another of the antigen-binding sites; the antigen binding sites can be present on the same antigen or on different antigens.
  • a multi-specific antigen binding protein may have specificity for more than one antigen, for example, two antigens, or three antigens, or four antigens.
  • a multi-specific antigen binding protein having specificity for two antigens can be referred to as a bispecific antigen binding protein.
  • a bispecific antigen binding protein (i.e., a bispecific) can be classified as having a symmetric or asymmetric architecture.
  • a bispecific antigen binding protein can be a bispecific antibody.
  • a bispecific may have an Fc region or may be fragment-based (lacking an Fc region).
  • a fragmentbased bispecific can combine multiple antigen-binding fragments in one molecule without an Fc region or with a portion of an Fc region e.g., Fab-scFv, Fab-scFvz, orthogonal Fab-Fab, Fab-Fv, tandem scFc (e.g., BiTE and BiKE molecules), Diabody, DART, TandAb, scDiabody, tandem dAb, etc.
  • a symmetric format can combine multiple binding specificities in a single polypeptide chain or single HL pair.
  • Examples can include an Fc-fusion protein(s) of a fragment-based format or a format whereby one or more antibody fragments are fused to an antibody molecule or other antigen binding protein.
  • Examples of symmetric formats may include DVD-Ig, TVD-Ig, CODV-Ig, (scFv)4-Fc, IgG-(scFv)2, Tetravalent DART-Fc, F(ab)4CrossMab, IgG-HC-scFv, IgG-LC-scFv, mAb-dAb, etc.
  • An asymmetric format can retain as closely as possible the native architecture of a natural antibody by forcing correct HL chain pairing and/or promoting H chain heterodimerization during the co-expression of three (if common heavy or light chains are used) or four polypeptide chains, e.g., Triomab, asymmetric reengineering technology immunoglobulin (ART-Ig), CrossMab, Biclonics common light chain, ZW1 common light chain, DuoBody and knobs into holes (KiH), DuetMab, KA body, Xmab, YBODY, HET-mAb, HET-Fab, DART-Fc, SEEDbody, mouse/rat chimeric IgG.
  • Triomab Triomab
  • ART-Ig asymmetric reengineering technology immunoglobulin
  • CrossMab Biclonics common light chain
  • ZW1 common light chain ZW1 common light chain
  • DuoBody and knobs into holes KiH
  • Bispecific formats can also include an antibody fused to a non-Ig scaffold, such as Affimabs, Fynomabs, Zybodies, Anticalin-IgG fusions, or ImmTAC.
  • a non-Ig scaffold such as Affimabs, Fynomabs, Zybodies, Anticalin-IgG fusions, or ImmTAC.
  • One or more antigen binding proteins described herein may show cross-reactivity between human FLT3LG and FLT3LG from another species, such as cynomolgus FLT3LG or rhesus FLT3LG.
  • An antigen binding protein described herein may specifically bind human FLT3LG and cynomolgus FLT3LG.
  • Such cross-reactivity can be exploited during preclinical research, e.g., in one or more nonhuman primate systems such as rhesus monkey or cynomolgus monkey. Such preclinical research can be performed before the antigen binding protein is tested in humans.
  • Such cross-reactivity can be exploited to make one or more side-by-side comparisons of using an antigen binding protein herein.
  • the binding affinity of the antigen binding protein for at least cynomolgus FLT3LG and the binding affinity for human FLT3LG differ by no more than a factor of 2, 5, 10, 50, or 100.
  • Affinity also referred to as "binding affinity” is the strength of binding at a single interaction site, i.e., of one molecule, e.g., an antigen binding protein of the invention, to another molecule, e.g., FLT3LG, at a single binding site.
  • the binding affinity of an antigen binding protein to its target may be determined by equilibrium methods (e.g., using enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE analysis). For example, SPR methods as described in Example 3 may be used to measure binding affinity.
  • Avidity also referred to as functional affinity, is the cumulative strength of binding at multiple interaction sites, e.g., the sum total of the strength of binding of two molecules (or more, e.g., in the case of a bispecific or multispecific molecule) to one another at multiple sites, e.g., taking into account the valency of the interaction.
  • the equilibrium dissociation constant (KD) of an antigen binding protein-FLT3LG interaction may be 100 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. Alternatively, the KD may be between 5 and 10 nM or between 1 and 2 nM. The KD may be between 1 pM and 500 pM or between 500 pM and 1 nM. The KD may be between 100 pM and 500 pM or between 100 pM and 1 nM. The KD may be between 100 pM and 2 nM or between 100 pM and 5 nM.
  • KD equilibrium association constant
  • M -1 the equilibrium association constant
  • kd dissociation rate constant or "off-rate” describes the stability of the antigen binding protein-antigen (e.g., FLT3LG) complex, i.e., the fraction of complexes that decay per second. For example, a kd of 0.01 s 1 equates to 1% of the complexes decaying per second.
  • the dissociation rate constant (kd) of the antigen binding protein-FLT3LG interaction may be lxlO -3 s -1 or less, lxlO -4 s 1 or less, lxlO -5 s 1 or less, or lxlO -6 s 1 or less.
  • the kd may be between lxlO -5 s 1 and lxlO -4 s 1 or between lxlO -4 s 1 and lxlO -3 s -1 .
  • the kd may be between lxlO -3 s 1 and lxlO -2 s _1 .
  • the association rate constant (ka) or "on-rate” describes the rate of antigen binding proteinantigen (e.g., FLT3LG) complex formation.
  • the ka of the antigen binding protein-FLT3LG interaction may be about 1.5xl0 5 M -1 s -1 .
  • the ka may be between lxlO 6 M -1 s 1 and lxlO 5 M -1 s -1 .
  • the ka may be between lxlO 5 M -1 s 1 and 5xl0 5 M -1 s 1 or between lxlO 5 M -1 s 1 and 8xl0 5 M -1 s -1 .
  • the binding affinity of FLT3LG binding proteins for wild-type FLT3LG and the binding affinity for a variant FLT3LG having one or more mutations differ by a factor of at least 2, at least 5, at least 10, at least 50, or at least 100.
  • FLT3LG binding proteins described herein may bind to a wild-type FLT3LG and not to a variant FLT3LG having one or more amino acid mutations.
  • the variant FLT3LG can be a FLT3LG comprising an alanine at position 72.
  • the wild-type FLT3LG can be a hFLT3LG.
  • the variant FLT3LG can be a hFLT3LG.
  • FLT3LG binding proteins may bind to wild-type FLT3LG with a KD less than or equal to 2 nM and bind to a variant FLT3LG with a KD greater than or equal to 2 nM.
  • FLT3LG binding proteins may bind to wild-type FLT3LG with a KD less than or equal to 1 nM and bind to a variant FLT3LG with a KD greater than or equal to 1 nM.
  • FLT3LG binding proteins may bind to wild-type FLT3LG with a KD less than or equal to 500 pM and bind to a variant FLT3LG with a KD greater than or equal to 500 pM.
  • FLT3LG binding proteins may bind to wild-type FLT3LG with a KD less than or equal to 500 pM and bind to a variant FLT3LG with a KD greater than or equal to 1 nM.
  • FLT3LG binding proteins may bind to wild-type hFLT3LG with a KD less than or equal to 500 pM and bind to a variant hFLT3LG comprising an alanine at position 72 with a KD greater than or equal to 1 nM.
  • FLT3LG binding proteins may bind to wild-type FLT3LG with a KD greater than or equal to 2 nM and bind to a variant FLT3LG with a KD less than or equal to 2 nM.
  • FLT3LG binding proteins may bind to wild-type FLT3LG with a KD greater than or equal to 1 nM and bind to a variant FLT3LG with a KD less than or equal to 1 nM.
  • FLT3LG binding proteins may bind to wild-type FLT3LG with a KD greater than or equal to 500 pM and bind to a variant FLT3LG with a KD less than or equal to 500 pM.
  • FLT3LG binding proteins may bind to wild-type FLT3LG with a KD greater than or equal to 100 pM and bind to a variant FLT3LG with a KD less than or equal to 100 pM.
  • FLT3LG binding proteins may bind to wild-type FLT3LG with a KD greater than or equal to 500 pM and bind to a variant FLT3LG with a KD less than or equal to 100 pM.
  • isolated can be used in reference to a molecule, such as an antigen binding protein, antigen, nucleic acid, peptide, or another molecule that is removed from the environment in which it is produced, from an environment in which it may be found in nature, or from another environment.
  • An antigen binding protein described herein may be encoded by one or more isolated nucleic acid sequences.
  • Production of a FLT3LG binding protein, such as an antibody may be achieved in a cell or living organism by delivering exogenous isolated nucleic acids encoding the FLT3LG binding protein, for example, a heavy chain and a light chain of an antibody.
  • Production of a FLT3LG binding protein, such as an antibody may be achieved in a cell in vitro or in vivo by delivering exogenous isolated nucleic acids encoding the FLT3LG binding protein, for example, a heavy chain and a light chain of an antibody.
  • a subject in need may be delivered one or more nucleic acids encoding an antigen binding protein provided herein, such as a heavy chain and a light chain of an anti-FLT3LG antibody.
  • the heavy chain and the light chain of the antibody may be delivered by the same or separate nucleic acids.
  • the nucleic acids may be DNA or RIMA.
  • the nucleic acids encoding the FLT3LG binding protein may be delivered to the subject naked (i.e. without an encapsulating particle) or packaged (i.e. encapsulated in liposomes or polymer- based vehicles).
  • the nucleic acids encoding the FLT3LG binding protein may be delivered without a delivery vehicle (i.e., "naked") or delivered with a viral or non-viral delivery vehicle (i.e., as a viral vector, adsorbed to or encapsulated in liposomes or polymer-based vehicles, and the like).
  • the nucleic acid may include elements such as a poly-A tail, a 5' and/or 3' untranslated region (UTR).
  • the nucleic acids may be mRNA.
  • the mRNA may include a cap structure.
  • the mRNA may be selfreplicating RNA.
  • the nucleic acid coding for the FLT3LG binding proteins may be modified or unmodified.
  • the nucleic acids coding for the FLT3LG binding proteins may comprise at least one chemical modification.
  • Nucleic acids e.g., mRNAs
  • Such chemical modifications include, but are not limited to, a modified nucleotide, a modified sugar backbone, and the like.
  • a method of producing a FLT3LG binding protein in a cell, tissue, or organism comprising contacting said cell, tissue, or organism with a composition comprising an isolated nucleic acid comprising at least one chemical modification and which encodes the FLT3LG binding protein.
  • Also provided herein is a method of producing a FLT3LG binding protein in a cell, tissue or organism comprising contacting said cell, tissue or organism with a composition comprising a polynucleotide comprising at least one chemical modification and which encodes a FLT3LG binding protein. Also provided herein is a method of producing a FLT3LG binding protein in a cell, in vitro or in vivo, comprising contacting said cell with a composition comprising a nucleic acid comprising at least one chemical modification and which encodes a FLT3LG binding protein.
  • an expression vector can be an isolated nucleic acid which can be used to introduce a nucleic acid of interest into a cell, such as a eukaryotic cell or prokaryotic cell, or a cell free expression system where the nucleic acid sequence of interest is expressed as a peptide chain such as a protein.
  • a nucleic acid of interest can comprise a nucleic acid sequence of an antigen binding protein provide herein or a fragment thereof.
  • Such expression vectors may be, for example, cosmids, plasmids, viral sequences, transposons, and linear nucleic acids comprising a nucleic acid of interest.
  • Expression vectors within the scope of the disclosure may provide necessary elements for eukaryotic or prokaryotic expression and include viral promoter driven vectors, such as CMV promoter driven vectors, e.g., pcDNA3.1, pCEP4, and their derivatives, Baculovirus expression vectors, Drosophila expression vectors, and expression vectors that are driven by mammalian gene promoters such as human Ig gene promoters.
  • viral promoter driven vectors such as CMV promoter driven vectors, e.g., pcDNA3.1, pCEP4, and their derivatives
  • Baculovirus expression vectors Drosophila expression vectors
  • expression vectors that are driven by mammalian gene promoters such as human Ig gene promoters.
  • prokaryotic expression vectors such as T7 promoter driven vectors, e.g., pET41, lactose promoter driven vectors, and arabinose gene promoter driven vectors.
  • T7 promoter driven vectors e.g., pET41
  • lactose promoter driven vectors e.g., lactose promoter driven vectors
  • arabinose gene promoter driven vectors
  • recombinant host cell refers to a cell that comprises a nucleic acid sequence of interest that was isolated prior to its introduction into the cell.
  • the nucleic acid sequence of interest may be in an expression vector while the cell may be prokaryotic or eukaryotic.
  • exemplary eukaryotic cells are mammalian cells, such as but not limited to, COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, HepG2, 653, SP2/0, NSO, 293, HeLa, myeloma, lymphoma cells, or any derivative thereof.
  • the eukaryotic cell may be HEK293, NSO, SP2/0, or CHO cell.
  • a recombinant cell according to the disclosure may be generated by transfection, cell fusion, immortalisation, or other procedures well known in the art.
  • a nucleic acid of interest, such as an expression vector, transfected into a cell may be extrachromosomal or stably integrated into the chromosome of the cell.
  • CDR complementarity determining region amino acid sequences of antigen binding proteins. These are the hypervariable regions of antigen binding proteins herein, such as immunoglobulin heavy and light chains. Typically, there are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an antigen binding protein such as an immunoglobulin. Thus, “CDRs” as used herein can refer to three heavy chain CDRs of an antigen binding protein, three light chain CDRs of an antigen binding protein, all heavy and light chain CDRs of an antigen binding protein, or at least two CDRs of an antigen binding protein.
  • variable domain sequences and variable domain regions within full-length antigen binding sequences are numbered according to the Kabat numbering convention.
  • CDR the terms “CDR”, “CDRL1”, “CDRL2”, “CDRL3”, “CDRH1”, “CDRH2”, and “CDRH3” used in the Examples follow the Kabat numbering convention.
  • Kabat et al. Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987).
  • amino acid residues in variable domain sequences and full-length antibody sequences There are alternative numbering conventions for amino acid residues in variable domain sequences and full-length antibody sequences. Throughout this specification, amino acid residues in Fc regions, in antibody sequences or full-length antigen binding protein sequences, are numbered according to the EU index numbering convention.
  • CDR sequences There are also alternative numbering conventions for CDR sequences, for example, those set out in Chothia et al. (1989) Nature 342: 877-883.
  • the structure and protein folding of the antigen binding protein may mean that other residues are considered part of the CDR sequence and would be understood to be so by a skilled person.
  • CDR sequences available to a skilled person include “AbM” (University of Bath) and “contact” (University College London) methods.
  • the CDR regions for SEQ ID NOs: 1-4 and SEQ ID NOs: 9-12 can be defined by any numbering convention, for example, the Kabat, Chothia, AbM, and contact conventions.
  • Table 1 represents one definition using each numbering convention for CDRs, or binding unit, provided herein.
  • the Kabat numbering scheme is used in Table 1 to number the variable domain amino acid sequence. It should be noted that CDR definitions can vary depending on the individual publication used.
  • FLT3LG binding proteins comprising any one or a combination of CDRs selected from:
  • a FLT3LG binding protein described herein can comprise all 6 CDRs selected from:
  • CDRs of a FLT3LG binding protein provided herein can be modified by one or by more than one amino acid substitution, deletion, or addition, wherein the variant FLT3LG binding protein substantially retains the biological characteristics of the unmodified protein, such as inhibiting the binding of FLT3LG to FLT3.
  • each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 may be modified alone or in combination with any other CDR, in any permutation or combination.
  • a CDR may be modified by the substitution, deletion, or addition of up to 3 amino acids, for example, 1 or 2 amino acids, for example, 1 amino acid.
  • Each modification of a CDR, VH, VL, or other protein provided herein can be a conservative substitution.
  • a modification can be a conservative substitution, for example, as shown in Table 2a or in Table 2b below. Table 2a. Examples of conservative substitutions by side chain type
  • flanking residues that comprise the CDR as part of alternative definition(s), e.g., Kabat or Chothia may be substituted with a conservative amino acid residue.
  • FLT3LG binding proteins described herein may comprise: a. (i) any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 1 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 9; or
  • FLT3LG binding proteins described herein may comprise: a. (i) any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 2 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 10; or
  • FLT3LG binding proteins described herein may comprise: a. (i) any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 3 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 11; or
  • FLT3LG binding proteins described herein may comprise: a. (i) any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 4 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 12; or (ii) a CDR variant of (i), wherein the variant has 1, 2, or 3 amino acid modifications; or b. a VH region comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 8 and/or a VL region comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 16.
  • FLT3LG binding proteins described herein may comprise any one or a combination of CDRs selected from CDRH1 of SEQ ID NO: 17; CDRH2 of SEQ ID NO: 18; CDRH3 of SEQ ID NO: 19; CDRL1 of SEQ ID NO: 29; CDRL2 of SEQ ID NO: 30; and/or CDRL3 of SEQ ID NO: 31.
  • FLT3LG binding proteins described herein may comprise any one or a combination of CDRs selected from CDRH1 of SEQ ID NO: 20; CDRH2 of SEQ ID NO: 21; CDRH3 of SEQ ID NO: 22; CDRL1 of SEQ ID NO: 32; CDRL2 of SEQ ID NO: 33; and/or CDRL3 of SEQ ID NO: 34.
  • FLT3LG binding proteins described herein may comprise any one or a combination of CDRs selected from CDRH1 of SEQ ID NO: 23; CDRH2 of SEQ ID NO: 24; CDRH3 of SEQ ID NO: 25; CDRL1 of SEQ ID NO: 35; CDRL2 of SEQ ID NO: 36; and/or CDRL3 of SEQ ID NO: 37.
  • FLT3LG binding proteins described herein may comprise any one or a combination of CDRs selected from CDRH1 of SEQ ID NO: 26; CDRH2 of SEQ ID NO: 27; CDRH3 of SEQ ID NO: 28; CDRL1 of SEQ ID NO: 38; CDRL2 of SEQ ID NO: 39; and/or CDRL3 of SEQ ID NO: 40.
  • the FLT3LG binding proteins described herein may comprise a humanized sequence. Humanization may be carried out by taking any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 1 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 9 and putting on to a human framework. Humanization may be carried out by taking any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 2 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 10 and putting on to a human framework.
  • Humanization may be carried out by taking any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 3 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 11 and putting on to a human framework.
  • Humanization may be carried out by taking any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 4 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 12 and putting on to a human framework.
  • the CDRs LI, L2, L3, Hl, and H2 can structurally exhibit one of a number of main chain conformations.
  • a particular canonical structure class of a CDR can be defined by both the length of the CDR and by the loop packing, determined by residues located at key positions in both the CDRs and the framework regions (structurally determining residues or SDRs).
  • Martin and Thornton (1996; J Mol Biol 263:800-815) have generated an automatic method to define the "key residue" canonical templates.
  • Cluster analysis is used to define the canonical classes for sets of CDRs, and canonical templates are then identified by analysing buried hydrophobics, hydrogen-bonding residues, and conserved glycines and prolines.
  • the CDRs of antibody sequences can be assigned to canonical classes by comparing the sequences to the key residue templates and scoring each template using identity or similarity matrices.
  • CDR canonicals are given in Table 3 below.
  • the amino acid numbering used is Kabat.
  • CDR canonical positions per CDR there may be multiple variant CDR canonical positions per CDR, per corresponding CDR, per binding unit, per heavy or light chain variable region, per heavy or light chain, or per antigen binding protein.
  • any combination of substitutions may be present in an antigen binding protein provided herein, provided that the canonical structure of the CDR is maintained such that the antigen binding protein is capable of specifically binding FLT3LG.
  • a particular canonical structure class of a CDR can be defined by both the length of the CDR and by the loop packing, determined by residues located at key positions in both the CDRs and the framework regions.
  • epitope refers to that portion of the antigen (e.g., FLT3LG) that makes contact with a particular binding domain of an antigen binding protein provided herein (i.e., a paratope).
  • An epitope may be linear, conformational, or discontinuous.
  • a conformational or discontinuous epitope comprises amino acid residues that are separated, for example, by one or more other amino acids, i.e., not in a continuous sequence in the antigen's primary sequence when assembled by tertiary folding of the polypeptide chain.
  • residues may be from different regions of the polypeptide chain, they can be in close proximity in the three-dimensional structure of the antigen.
  • a conformational or discontinuous epitope may include residues from different peptide chains. Particular residues comprised within an epitope can be determined via a computer modelling program or via one or more three-dimensional structures obtained through a structural method such as X-ray crystallography. An epitope can be determined using epitope mapping, such as by one or more techniques, for example, peptide-based approaches such as pepscan, whereby a series of overlapping peptides can be screened for binding using one or more techniques such as ELISA or by in vitro display of large libraries of peptides or protein mutants, e.g., on phage.
  • Detailed epitope information can be determined by structural techniques, for example, X-ray crystallography, solution nuclear magnetic resonance (NMR) spectroscopy, and cryogenic-electron microscopy (cryo-EM). Mutagenesis, such as alanine scanning, can be an effective approach whereby loss of binding analysis is used for epitope mapping.
  • Another method is hydrogen/deuterium exchange (HDX) combined with proteolysis and liquid-chromatography mass spectrometry (LC-MS) analysis to characterize discontinuous or conformational epitopes.
  • HDX hydrogen/deuterium exchange
  • LC-MS liquid-chromatography mass spectrometry
  • antigen binding site refers to a site on an antigen (e.g., FLT3LG) binding protein that is capable of specifically binding to an antigen (e.g., FLT3LG).
  • An antigen binding site can be a single variable domain, or it may be paired one or more VH/VL domains, such as an antibody.
  • Single-chain Fv (ScFv) domains can also provide antigen-binding sites.
  • Competition between an antigen (e.g., FLT3LG) binding protein described herein and a reference FLT3LG binding protein, e.g., a reference antibody may be determined by one or more techniques known to the skilled person such as ELISA, FMAT, Surface Plasmon Resonance (SPR) or FORTEBIO OCTET Bio-Layer Interferometry (BLI). Such techniques can be referred to as epitope binning.
  • a competition assay may be carried out, for example, using flow cytometry-based epitope binning.
  • a FLT3LG binding protein may bind to human FLT3LG and compete for binding to human FLT3LG with a reference FLT3LG binding protein.
  • a reference FLT3LG binding protein may comprise (a) a VH region sequence of SEQ ID NO: 5 and a VL region sequence of SEQ ID NO: 13; (b) a VH region sequence of SEQ ID NO: 6 and a VL region sequence of SEQ ID NO: 14; (c) a VH region sequence of SEQ ID NO: 7 and a VL region sequence of SEQ ID NO: 15; or (d) a VH region sequence of SEQ ID NO: 8 and a VL region sequence of SEQ ID NO: 16.
  • a FLT3LG binding protein described herein may share an overlapping epitope with a reference FLT3LG binding protein comprising a VH region comprising SEQ ID NO: 5 and a VL region comprising SEQ ID NO: 13; a VH region comprising SEQ ID NO: 6 and a VL region comprising SEQ ID NO: 14; a VH region comprising SEQ ID NO: 7 and a VL region comprising SEQ ID NO: 15; or a VH region comprising SEQ ID NO: 8 and a VL region comprising SEQ ID NO: 16.
  • a FLT3LG binding protein described herein may share an overlapping epitope with a reference FLT3LG binding protein comprising an HC sequence comprising SEQ ID NO: 47 and an LC sequence comprising SEQ ID NO: 55; an HC sequence comprising SEQ ID NO: 48 and an LC sequence comprising SEQ ID NO: 56; an HC sequence comprising SEQ ID NO: 49 and an LC sequence comprising SEQ ID NO: 57; or an HC sequence comprising SEQ ID NO: 50 and an LC sequence comprising SEQ ID NO: 58.
  • a FLT3LG binding protein can be an antagonist, such as an antagonist antibody.
  • An antagonist can comprise an epitope binding protein, such as an antibody or fragment thereof, that is capable of fully or partially inhibiting the biological activity of the antigen to which it binds for example, by fully or partially blocking binding of the antigen to a receptor or by neutralising activity, such as signalling, which can be initiated by a biological activity of the antigen.
  • a FLT3LG binding protein herein can be neutralizing.
  • Negative refers to a reduction or elimination of the biological activity of the antigen (e.g., FLT3LG) in the presence of a FLT3LG binding protein as described herein, in comparison to the biological activity of the antigen in the absence of the FLT3LG binding protein, in vitro or in vivo.
  • Neutralization may be due to one or more of blocking FLT3LG binding to its receptor, preventing FLT3LG from activating its receptor, down regulating FLT3LG or its receptor, or affecting effector functionality.
  • Neutralization may be determined or measured using one or more assays, for example, as described herein. For example, the blocking assay methods described in Example 3 may be used to assess the neutralizing capability of an antigen binding protein herein.
  • a neutralizing FLT3LG binding protein may neutralize the activity of FLT3LG-FLT3 interactions (e.g., binding) by at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% relative to FLT3LG-FLT3 interactions in the absence of the FLT3LG binding protein.
  • FLT3LG binding proteins described herein may inhibit the interaction of human FLT3LG and human FLT3 by 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
  • FLT3LG binding proteins described herein may inhibit the binding of soluble FLT3LG to FLT3 by 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
  • FLT3LG binding proteins described herein may inhibit the binding of membranebound FLT3LG to FLT3 by 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
  • Percent identity or”% identity between a query nucleic acid sequence and a subject nucleic acid sequence is the "Identities" value, expressed as a percentage, that is calculated using a suitable algorithm (e.g., BLASTN, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST/KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle, or EMBOSS infoalign), over the length of the query sequence after alignment, such as a pair-wise global sequence alignment, has been performed using a suitable algorithm (e.g., Needleman- Wunsch or GenePAST/KERR) or software (e.g., DNASTAR Lasergene or GenePAST/KERR).
  • a query nucleic acid sequence may be a nucleic acid sequence disclosed herein, in particular in one or more of the claims.
  • Percent identity or”% identity between a query amino acid sequence and a subject amino acid sequence is the "Identities" value, expressed as a percentage, that is calculated using a suitable algorithm (e.g., BLASTP, FASTA, Needleman-Wunsch, Smith-Waterman, LALIGN, or GenePAST/KERR) or software (e.g., DNASTAR Lasergene, GenomeQuest, EMBOSS needle, or EMBOSS infoalign), over the length of the query sequence after alignment, such as a pair-wise global sequence alignment, has been performed using a suitable algorithm (e.g., Needleman- Wunsch or GenePAST/KERR) or software (e.g., DNASTAR Lasergene or GenePAST/KERR).
  • a query amino acid sequence may be described by an amino acid sequence disclosed herein, in particular in one or more of the claims.
  • a query sequence may be 100% identical to a subject sequence, or it may include up to an integer number of amino acid or nucleotide alterations as compared to the subject sequence, such that the % identity is less than 100%.
  • a query sequence can be at least at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a subject sequence.
  • such alterations can comprise at least one nucleotide residue deletion, substitution, or insertion, wherein said alterations may occur at the 5'- or 3'-terminal positions of the query sequence, at one or more positions between those terminal positions, interspersed either individually among the nucleotide residues in a query sequence, or in one or more contiguous groups within a query sequence.
  • such alterations can comprise at least one amino acid residue deletion, substitution (including conservative and non-conservative substitutions), or insertion, wherein said alterations may occur at the amino- or carboxy-terminal positions of a query sequence, or at one or more positions between those terminal positions, interspersed either individually among the amino acid residues in a query sequence, or in one or more contiguous groups within a query sequence.
  • a % identity may be determined across the entire length of a query sequence, including the CDRs.
  • a calculated % identity may exclude one or more or all of the CDRs.
  • all of the CDRs of an antibody may be 100% identical to a subject sequence, while a % identity in the remaining portion of the query sequence, e.g., the framework sequence, can be less than 100%, such that that the CDR sequences are fixed and intact.
  • a FLT3LG binding protein described herein may comprise a CDRH3 that is 100% identical to any one of SEQ ID NOs: 19, 22, 25, or 28.
  • a FLT3LG binding protein described herein may comprise a CDRH3 that is 100% identical to SEQ ID NO: 19.
  • a FLT3LG binding protein described herein may comprise a CDRH3 that is 100% identical to SEQ ID NO: 22.
  • a FLT3LG binding protein described herein may comprise a CDRH3 that is 100% identical to SEQ ID NO: 25.
  • a FLT3LG binding protein described herein may comprise a CDRH3 that is 100% identical to SEQ ID NO: 28.
  • a FLT3LG binding protein described herein may comprise CDRH1 that is 100% identical to SEQ ID NO: 17; CDRH2 that is 100% identical to SEQ ID NO: 18; CDRH3 that is 100% identical to
  • a FLT3LG binding protein described herein may comprise CDRH1 that is 100% identical to SEQ ID NO: 20; CDRH2 that is
  • a FLT3LG binding protein described herein may comprise CDRH1 that is 100% identical to SEQ ID NO: 23; CDRH2 that is 100% identical to SEQ ID NO: 24; CDRH3 that is 100% identical to SEQ ID NO: 25; CDRL1 that is 100% identical to SEQ ID NO: 35; CDRL2 that is 100% identical to SEQ ID NO: 36; and/or CDRL3 that is 100% identical to SEQ ID NO: 37.
  • a FLT3LG binding protein described herein may comprise CDRH1 that is 100% identical to SEQ ID NO: 26; CDRH2 that is 100% identical to SEQ ID NO: 27; CDRH3 that is 100% identical to SEQ ID NO: 28; CDRL1 that is 100% identical to SEQ ID NO: 38; CDRL2 that is 100% identical to SEQ ID NO: 39; and/or CDRL3 that is 100% identical to SEQ ID NO: 40.
  • a FLT3LG binding protein described herein may comprise CDRH1 that is 100% identical to SEQ ID NO: 17; CDRH2 that is 100% identical to SEQ ID NO: 18; CDRH3 that is 100% identical to SEQ ID NO: 19; CDRL1 that is 100% identical to SEQ ID NO: 29; CDRL2 that is 100% identical to SEQ ID NO: 30; and CDRL3 that is 100% identical to SEQ ID NO: 31.
  • a FLT3LG binding protein described herein may comprise CDRH1 that is 100% identical to SEQ ID NO: 20; CDRH2 that is 100% identical to SEQ ID NO: 21; CDRH3 that is 100% identical to SEQ ID NO: 22; CDRL1 that is 100% identical to SEQ ID NO: 32; CDRL2 that is 100% identical to SEQ ID NO: 33; and CDRL3 that is 100% identical to SEQ ID NO: 34.
  • a FLT3LG binding protein described herein may comprise CDRH1 that is 100% identical to SEQ ID NO: 23; CDRH2 that is 100% identical to SEQ ID NO: 24; CDRH3 that is 100% identical to SEQ ID NO: 25; CDRL1 that is 100% identical to SEQ ID NO: 35; CDRL2 that is 100% identical to SEQ ID NO: 36; and CDRL3 that is 100% identical to SEQ ID NO: 37.
  • a FLT3LG binding protein described herein may comprise CDRH1 that is 100% identical to SEQ ID NO: 26; CDRH2 that is 100% identical to SEQ ID NO: 27; CDRH3 that is 100% identical to SEQ ID NO: 28; CDRL1 that is 100% identical to SEQ ID NO: 38; CDRL2 that is 100% identical to SEQ ID NO: 39; and CDRL3 that is 100% identical to SEQ ID NO: 40.
  • a FLT3LG binding protein described herein may comprise a VH region that is at least 90% identical to SEQ ID NO: 5 and a VL region that is at least 90% identical to SEQ ID NO: 13.
  • a FLT3LG binding protein described herein may comprise a VH region that is at least 95% identical to SEQ ID NO: 5 and a VL region that is at least 95% identical to SEQ ID NO: 13.
  • a FLT3LG binding protein described herein may comprise a VH region that is at least 90% identical to SEQ ID NO: 6 and a VL region that is at least 90% identical to SEQ ID NO: 14.
  • a FLT3LG binding protein described herein may comprise a VH region that is at least 95% identical to SEQ ID NO: 6 and a VL region that is at least 95% identical to SEQ ID NO: 14.
  • a FLT3LG binding protein described herein may comprise a VH region that is at least 90% identical to SEQ ID NO: 7 and a VL region that is at least 90% identical to SEQ ID NO: 15.
  • a FLT3LG binding protein described herein may comprise a VH region that is at least 95% identical to SEQ ID NO: 7 and a VL region that is at least 95% identical to SEQ ID NO: 15.
  • a FLT3LG binding protein described herein may comprise a VH region that is at least 90% identical to SEQ ID NO: 8 and a VL region that is at least 90% identical to SEQ ID NO: 16.
  • a FLT3LG binding protein described herein may comprise a VH region that is at least 95% identical to SEQ ID NO: 8 and a VL region that is at least 95% identical to SEQ ID NO: 16.
  • a FLT3LG binding protein described herein may comprise a VH region that is 100% identical to SEQ ID NO: 5 and a VL region that is 100% identical to SEQ ID NO: 13; a VH region that is 100% identical to SEQ ID NO: 6 and a VL region that is 100% identical to SEQ ID NO: 14; a VH region that is 100% identical to SEQ ID NO: 7 and a VL region that is 100% identical to SEQ ID NO: 15; or a VH region that is 100% identical to SEQ ID NO: 8 and a VL region that is 100% identical to SEQ ID NO: 16.
  • a FLT3LG binding protein described herein may comprise a VH region that is 100% identical to SEQ ID NO: 5 and a VL region that is 100% identical to SEQ ID NO: 13.
  • a FLT3LG binding protein described herein may comprise a VH region that is 100% identical to SEQ ID NO: 6 and a VL region that is 100% identical to SEQ ID NO: 14.
  • a FLT3LG binding protein described herein may comprise a VH region that is 100% identical to SEQ ID NO: 7 and a VL region that is 100% identical to SEQ ID NO: 15.
  • a FLT3LG binding protein described herein may comprise a VH region that is 100% identical to SEQ ID NO: 8 and a VL region that is 100% identical to SEQ ID NO: 16.
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is at least 90% identical to SEQ ID NO: 47 and/or a Light Chain (LC) sequence that is at least 90% identical to SEQ ID NO: 55.
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is at least 95% identical to SEQ ID NO: 47 and/or a Light Chain (LC) sequence that is at least 95% identical to SEQ ID NO: 55.
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is at least 90% identical to SEQ ID NO: 48 and/or a Light Chain (LC) sequence that is at least 90% identical to SEQ ID NO: 56.
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is at least 95% identical to SEQ ID NO: 48 and/or a Light Chain (LC) sequence that is at least 95% identical to SEQ ID NO: 56.
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is at least 90% identical to SEQ ID NO: 49 and/or a Light Chain (LC) sequence that is at least 90% identical to SEQ ID NO: 57.
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is at least 95% identical to SEQ ID NO: 49 and/or a Light Chain (LC) sequence that is at least 95% identical to SEQ ID NO: 57.
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is at least 90% identical to SEQ ID NO: 50 and/or a Light Chain (LC) sequence that is at least 90% identical to SEQ ID NO: 58.
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is at least 95% identical to SEQ ID NO: 50 and/or a Light Chain (LC) sequence that is at least 95% identical to SEQ ID NO: 58.
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is 100% identical to SEQ ID NO: 47 and/or a Light Chain (LC) sequence that is 100% identical to SEQ ID NO: 55; a Heavy Chain (HC) sequence that is 100% identical to SEQ ID NO: 48 and/or a Light Chain (LC) sequence that is 100% identical to SEQ ID NO: 56; a Heavy Chain (HC) sequence that is 100% identical to SEQ ID NO: 49 and/or a Light Chain (LC) sequence that is 100% identical to SEQ ID NO: 57; or a Heavy Chain (HC) sequence that is 100% identical to SEQ ID NO: 50 and/or a Light Chain (LC) sequence that is 100% identical to SEQ ID NO: 58.
  • HC Heavy Chain
  • LC Light Chain
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is 100% identical to SEQ ID NO: 47 and a Light Chain (LC) sequence that is 100% identical to SEQ ID NO: 55.
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is 100% identical to SEQ ID NO: 48 and a Light Chain (LC) sequence that is 100% identical to SEQ ID NO: 56.
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is 100% identical to SEQ ID NO: 49 and a Light Chain (LC) sequence that is 100% identical to SEQ ID NO: 57.
  • a FLT3LG binding protein described herein may comprise a Heavy Chain (HC) sequence that is 100% identical to SEQ ID NO: 50 and a Light Chain (LC) sequence that is 100% identical to SEQ ID NO: 58.
  • a FLT3LG binding protein provided herein can comprise a sequence that is a variant amino acid sequence.
  • a nucleic acid sequence of a FLT3LG binding protein provided herein can comprise a variant nucleic acid sequence.
  • a variant nucleic acid sequence herein can be of a FLT3LG binding protein provided herein or of a variant thereof.
  • a VH or VL (or HC or LC) sequence may be a variant sequence of a VH or VL (or HC or LC) sequence provided herein with up to 10 amino acid substitutions, additions, or deletions. Such a variant sequence may have 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution(s), addition(s), or deletion(s).
  • An HC sequence may be a variant sequence of an HC sequence provided herein with up to 40 amino acid substitutions, additions, or deletions.
  • An HC variant sequence may have up to 35, up to 30, up to 25, up to 20, up to 15, or up to 10 amino acid substitutions, additions, or deletions.
  • An HC variant sequence may have 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions.
  • An LC sequence may be a variant sequence of an LC sequence provided herein with up to 20 amino acid substitutions, additions, or deletions.
  • An LC variant sequence may have up to 15, up to 10, or up to 5 amino acid substitutions, additions, or deletions.
  • An LC variant sequence may have 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions.
  • a sequence variation may exclude one or more or all of the CDRs.
  • the CDRs portion of the VH or VL (or HC or LC) sequence can be free of a sequence variation, and the variation can be present in a non-CDR portion of a VH or VL (or HC or LC) sequence, i.e., such that the CDR sequences are intact.
  • a variation can be a substitution, such as a conservative substitution, for example, as provided in Table 2a or Table 2b.
  • An antigen binding protein having a variant sequence can substantially retain the biological characteristics of an unmodified antigen binding protein, such as inhibiting binding of FLT3LG to FLT3.
  • a binding property (e.g., KD, Kd, or Ka) of a FLT3LG binding protein having a variant sequence can be substantially identical to an unmodified FLT3LG binding protein.
  • a binding property (e.g., KD, Kd, or Ka) of a variant sequence can be at least 75%, at least 90%, at least 95%, or at least 99% identical to that of an unmodified FLT3LG binding protein.
  • post- translational modifications may occur.
  • a post-translational modification can comprise the cleavage of one or more leader sequences, the addition of one or more sugar moieties such as in a glycosylation pattern, non-enzymatic glycation, deamidation, oxidation, disulfide bond scrambling and other cysteine variants such as those comprising free sulfhydryls, racemized disulfides, thioethers and trisulfide bonds, isomerisation, C-terminal lysine clipping, and N-terminal glutamine cyclisation.
  • an "antigen binding protein” or “antibody” herein can comprise an "antigen binding protein” or “antibody”, respectively, that has undergone a post-translational modification such as described herein.
  • Post translational modification can comprise glycation, which is a post-translational non- enzymatic chemical reaction between a reducing sugar, such as glucose, and a free amine group in the protein, and is typically observed at the epsilon amine of lysine side chains or at the N-Terminus of an antigen binding protein. Glycation can occur during production and storage only in the presence of reducing sugars.
  • a reducing sugar such as glucose
  • Post translational modification can comprise deamidation, which can occur during production and storage, is an enzymatic reaction primarily converting asparagine (N) to iso-aspartic acid (isoaspartate) and aspartic acid (aspartate) (D) at approximately 3:1 ratio.
  • This deamidation reaction can be therefore related to isomerization of aspartate (D) to iso-aspartate.
  • the deamidation of asparagine and the isomerisation of aspartate both involve the intermediate succinimide.
  • deamidation can occur with glutamine residues in a similar manner. Deamidation can occur, for example, in a CDR, in a Fab (non-CDR region), or in an Fc region of an antigen binding protein.
  • Post translational modification can comprise oxidation, which can occur during production and storage (i.e., in the presence of oxidizing conditions) and can result in a covalent modification of a protein, induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidation can occur primarily at methionine residues. Oxidation may occur at tryptophan and free cysteine residues. Oxidation can occur in a CDR, in a Fab (non-CDR) region, or in a Fc region of an antigen binding protein.
  • Post translational modification can comprise disulfide bond scrambling, which can occur during production and basic storage conditions.
  • One or more disulfide bonds can break or form incorrectly, which can result in one or more unpaired cysteine residues (-SH).
  • -SH free (unpaired) sulfhydryls
  • Post translational modification can comprise the formation of a thioether and racemization of a disulfide bond, which can occur under basic conditions, in production or storage, through a beta elimination of disulfide bridges back to cysteine residues via a dehydroalanine and persulfide intermediate. Subsequent crosslinking of dehydroalanine and cysteine can result in the formation of a thioether bond or the free cysteine residues can reform a disulfide bond with a mixture of D- and /.-cysteine in an antigen binding protein.
  • Trisulfides result from insertion of a sulfur atom into a disulfide bond (Cys-S-S-S-Cys) and are formed due to the presence of hydrogen sulfide in production cell culture.
  • N-terminal glutamine (Q) or glutamate (glutamic acid) (E) in the heavy chain and/or light chain may be likely to form pyroglutamate (pGlu) via cyclization.
  • pGlu pyroglutamate
  • C-terminal lysine clipping is an enzymatic reaction catalyzed by carboxypeptidases, and is commonly observed in recombinant and natural human antibodies. Variants of this process include removal of lysine from one or both heavy chains due to cellular enzymes from the recombinant host cell. Administration to a subject or human patient may be likely to result in the removal of any remaining C-terminal lysines.
  • Antigen binding proteins may be prepared by any of a number of conventional techniques.
  • an antigen binding protein may be purified from one or more cells that naturally express it (e.g., an antibody can be purified from a hybridoma that produces it) or produced in a recombinant expression system. More than one antigen binding protein can be expressed (and therefore purified) from such a natural cell or recombinant expression system.
  • host cells can be transformed with a recombinant expression vector encoding an antigen binding protein.
  • the expression vector may be maintained by the host as a separate genetic element or integrated into the host chromosome depending on the expression system.
  • a wide range of host cells can be employed, for example, Prokaryotes (including Gram negative or Gram-positive bacteria, for example, Escherichia coii, Bacilli sp., Pseudomonas sp., Corynebacterium sp.), Eukaryotes including yeast (for example, Saccharomyces cerevisiae, Pichia pastoris), fungi (for example, Aspergiius sp.), or higher Eukaryotes including insect cells and cell lines of mammalian origin (for example, CHO, NSO, PER.C6, HEK293, HeLa).
  • Prokaryotes including Gram negative or Gram-positive bacteria, for example, Escherichia coii, Bacilli sp., Pseudomonas sp., Corynebacterium sp.
  • Eukaryotes including yeast (for example, Saccharomyces cerevisiae, Pichia pastoris), fungi (for
  • a host cell may be an isolated host cell.
  • a host cell is usually not part of a multicellular organism (e.g., plant or animal).
  • a host cell can be a single celled organism, or can be an individual cell of a multicellular organism that is separate from that organism.
  • a host cell can be part of a multicellular organism, for example, a plant or animal.
  • the host cell may be a non-human host cell.
  • Appropriate cloning and expression vectors can be designed or engineered for use with bacterial, fungal, yeast, and mammalian host cells.
  • a commercial vector can be obtained and engineered to be a vector for the expression of a FLT3LG binding protein provided herein.
  • Cells of an expression system can be cultured under conditions that promote expression of the antigen binding protein using one or more of appropriate equipment, including a shake flask(s), a spinner flask(s), and a bioreactor(s).
  • the antigen binding protein can be recovered by conventional protein purification procedures or modifications thereof. Protein purification procedures can comprise of a series of unit operations comprising one or more filtration or chromatographic processes, or a combination thereof, developed to selectively isolate and/or concentrate the antigen binding protein.
  • the purified antigen binding protein may be formulated in a pharmaceutically acceptable composition.
  • Fc engineering methods can be applied to modify the functional or pharmacokinetic properties of an antigen binding protein comprising an Fc region, such as an antibody.
  • Binding to Fey can promote ADCC activity; thus, ADCC activity may be altered by making mutations in the Fc region that can increase or decrease binding to Fey receptors.
  • Binding to Clq can promote CDC activity; thus, CDC activity may be altered by making mutations in the Fc region that can increase or decrease binding to Clq receptor.
  • Modifications to the glycosylation pattern of an antigen binding protein can also be made to change the effector function.
  • the in vivo half-life of an antigen binding protein can be altered by making mutations that affect binding of the Fc region to the FcRn (Neonatal Fc Receptor).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • DCP complement-dependent cell-mediated phagocytosis
  • ADCML antibody dependent complement-mediated cell lysis
  • ADCP Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis
  • Fc region of an antigen binding protein comprising an Fc region, such as an antibody
  • FcR various Fc receptors
  • FcR including FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), FcRn, Clq, and type II Fc receptors
  • FcyRI CD64
  • FcyRII CD32
  • FcyRIII CD16
  • FcRn FcRn
  • Clq type II Fc receptors
  • FcR Fc receptors
  • Significant biological effects can be a consequence of effector functionality.
  • the ability to mediate effector function can require binding of the antigen binding protein to an antigen.
  • An antigen binding protein can mediate one of, a plurality of, or each effector function.
  • Effector function can be assessed in a number of ways including, for example, by evaluating ADCC effector function of an antibody coated to target cells mediated by Natural Killer (NK) cells via FcyRIII, or monocytes/macrophages via FcyRI, or by evaluating CDC effector function of an antigen binding protein coated to target cells mediated by complement cascade via Clq.
  • NK Natural Killer
  • an antigen binding protein described herein can be assessed for ADCC effector function in a Natural Killer cell assay.
  • mutations including mutations in the Fc region, on effector functions (including, but not limited to, FcRn binding, FcyRs and Clq binding, CDC, ADCML, ADCC, and/or ADCP) can be assessed.
  • An antigen binding protein can comprise one or more of such mutations.
  • IgG4 and IgG2 isotypes can partially or fully lack the functions of a) activation of complement by the classical pathway and b) ADCC.
  • Various modifications to the heavy chain constant region of antigen binding proteins may be carried out to alter effector function depending on the desired effector property.
  • IgGl constant regions containing specific mutations that reduce binding to Fc receptors and reduce an effector function, such as ADCC and CDC, have been described.
  • antigen binding proteins comprising a constant region such that the antigen binding protein has reduced effector function, such as reduced ADCC and/or CDC.
  • the heavy chain constant region may comprise a naturally disabled constant region of an IgG2 or IgG4 isotype or a mutated IgGl constant region.
  • suitable modifications are described in EP0307434.
  • a constant region can comprise substitution with alanine at positions 235 and 237 (EU index numbering), i.e., L235A and G237A (commonly referred to as "LAGA" mutations).
  • substitution with alanine at positions 234 and 235 can comprise substitution with alanine at positions 234 and 235 (EU index numbering), i.e., L234A and L235A (commonly referred to as "LALA” mutations).
  • Additional examples can comprise substitution with alanine at positions 234, 235 and 237 (EU index numbering), i.e., L234A, L235A, and G237A (referred to as "LALAGA” mutations).
  • Further examples, such as described in EP2691417 and US8969526, can comprise a substitution with glycine or arginine at position 329 (i.e., P329G or P329R), in combination with the LALA mutations (EU index numbering) for Fc regions including IgGl Fc regions.
  • Yet further examples can comprise a substitution with glycine or arginine at position 329 (i.e., P329G or P329R), in combination with a substitution with proline at position 228 and glutamic acid at position 235 (i.e., S228P and L235E) for Fc domains including IgG4 Fc regions (EU index numbering).
  • mutations that can be employed to decrease effector function can include (with reference to IgGl unless otherwise noted): aglycosylated N297A or N297Q or N297G; L235E; IgG4:F234A/L235A; or chimeric IgG2/IgG4.
  • IgG2 comprising H268Q/V309L/A330S/P331S or V234A/G237A/P238S/H268A/V309L/A330S/P331S substitutions can be employed to reduce FcyR and/or Clq binding.
  • L234F/L235E/P331S can include L234F/L235E/P331S; a chimeric antibody created using the CHI and hinge region from human IgG2 and the CH2 and CH3 regions from human IgG4; IgG2m4, based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S); IgG2o that contains V234A/G237A /P238S/H268A/V309L/A330S/P331S substitutions to eliminate affinity for Fey receptors and Clq complement protein; IgG4 (S228P/L234A/L235A); huIgGl L234A/L235A (AA); huIgG4 S228P/L234A/L235A; IgGlS (L234A/L235A/G237A/P238S
  • FLT3LG binding proteins described herein may comprise an IgGl constant region comprising a N297G mutation.
  • FLT3LG binding proteins described herein may comprise an Fc region comprising a N297G mutation.
  • FLT3LG binding proteins described herein may comprise a Fc region comprising SEQ ID NO: 41.
  • Half-life refers to the time required for the serum concentration of an antigen binding protein to reach half of its original value (i.e., half of a determined serum concentration achieved post administration).
  • the serum half-life of proteins can be measured by pharmacokinetic studies, for example, according to a method wherein radio-labelled protein is injected intravenously into mice and its plasma concentration is periodically measured as a function of time, for example, at about 3 minutes to about 72 hours after the injection. Other methods for pharmacokinetic analysis and determination of the half-life of a molecule can be envisioned by a skilled artisan.
  • IgG antibodies can be dependent on their binding to FcRn. Therefore, substitutions that increase the binding affinity of IgG to FcRn at pH 6.0 while maintaining the pH dependence of the interaction with target, for example, by engineering the constant region, may be employed.
  • the in vivo half-life of antigen binding proteins described herein may be altered, for example, by modification of a heavy chain constant domain or by modification of an FcRn binding domain therein.
  • FcRn also known as the neonatal Fc receptor
  • FcRn can play a key role in maintaining serum antibody levels by acting as a protective receptor that binds and salvages antibodies of the IgG isotype from degradation.
  • IgG molecules are endocytosed by endothelial cells and, if they bind to FcRn, are recycled out of the cells back into circulation.
  • FcRn may be involved in both antibody clearance and the transcytosis across tissues.
  • Human IgGl residues determined to interact directly with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435. Mutations at any of these positions may be employed in an antigen binding protein herein, for example, to enable increased serum half-life and/or altered effector properties of antigen binding proteins provided herein.
  • Antigen binding proteins described herein may have amino acid modifications that increase the affinity of the constant domain or fragment thereof for FcRn. Increasing the half-life (i.e., serum half-life) of therapeutic and diagnostic IgG antibodies and other bioactive molecules can provide benefits, which can include reducing the amount and/or frequency of dosing of these molecules.
  • An antigen binding protein may comprise all or a portion (an FcRn binding portion) of an IgG constant domain having one or more of the following amino acid modifications.
  • M252Y/S254T/T256E (commonly referred to as "YTE” mutations) and M428L/N434S (commonly referred to as "LS” mutations) can increase FcRn binding at pH 6.0.
  • a YTE or LS modification can increase FcRn binding at a pH higher than 6.0 or at a pH lower than 6.0.
  • Half-life can also be increased by T250Q/M428L, V259I/V308F/M428L, N434A, and T307A/E380A/N434A mutations (with reference to IgGl and Kabat numbering) in an antigen binding protein provided herein.
  • Half-life and FcRn binding can also be increased by introducing H433K and N434F mutations (commonly referred to as "HN” or “NHance” mutations) (with reference to IgGl) (W02006/130834) in an antigen binding protein provided herein.
  • HN H433K and N434F mutations
  • IgGl IgGl
  • An antigen binding protein provided herein can comprise a variant Fc region with altered FcRn binding affinity, which can comprise an amino acid modification at any one or more of amino acid positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439, and 447 of the Fc region (EU index numbering).
  • a modified IgG comprising an IgG constant domain of an antigen binding protein can comprise one or more amino acid modifications relative to a wild-type IgG constant domain, wherein the modified IgG can have an increased half-life compared to the half-life of an IgG having a wildtype IgG constant domain, and wherein the one or more amino acid modifications are at one or more of positions 251, 253, 255, 285-290, 308-314, 385-389, and 428-435.
  • Alanine scanning mutagenesis can be employed to alter residues in the Fc region of an antigen binding protein provided herein, for example, a human IgGl antibody, and thus alter binding to human FcRn. Positions that can effectively abrogate binding to FcRn when changed to alanine include 1253, S254, H435, and Y436. Other positions can result in a less pronounced reduction in binding when mutated, for example, as follows: E233-G236, R255, K288, L309, S415, and H433.
  • combination variants can yield a pronounced effect with respect to improved FcRn binding.
  • the E380A/N434A variant can display over 8-fold increase in binding to FcRn, relative to wild type IgGl, compared with a 2-fold increase for E380A and a 3.5-fold increase for N434A.
  • the addition of T307A to this combination (E380A/N434A/T307A) can result in a 12-fold increase in binding relative to wild type IgGl.
  • Antigen binding proteins described herein may comprise the E380A/N434A or E380A/N434A/T307A mutations and have increased binding to FcRn.
  • an improvement in IgGl-human FcRn complex stability can occur when substituting residues located in a band across the Fc-FcRn interface (e.g., M252, S254, T256, H433, N434, and Y436) or at the periphery (e.g., V308, L309, Q311, G385, Q386, P387, and N389).
  • M252Y/S254T/T256E (“YTE") and H433K/N434F/Y436H mutations can be combined to yield a high affinity to human FcRn. In some cases, such a combination can exhibit a 57-fold increase in affinity relative to the wild-type IgGl.
  • the in vivo behaviour of such a mutated human IgGl can exhibit an increase in serum half-life of up to at least 4-fold as compared to wild-type IgGl.
  • Such an increase in serum half-life can be in the serum of a human, a cynomolgus monkey, or another subject.
  • An antigen binding protein may comprise at least one amino acid modification in the Fc region of said antigen binding protein, for example, wherein said modification is at an amino acid position selected from the group consisting of 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302,
  • a FLT3LG binding protein can have 2, 3, 4, 5, 6, or more of such amino acid modifications in the Fc region of said FLT3LG binding protein.
  • a FLT3LG binding protein can have an amino acid modification in the Fc region of said FLT3LG binding protein at another amino acid position, either instead of or in addition to an amino acid modification provided herein.
  • a FLT3LG binding protein can have a modified half-life, either by introducing an FcRn- binding polypeptide into the antigen binding protein (for example, as in WO97/43316, US5869046, US5747035, WO96/32478, or WO91/14438), by fusing the antigen binding protein with antibodies whose FcRn-binding affinities are preserved, but affinities for other Fc receptors have been greatly reduced (for example, as in WO99/43713), or by fusing the antigen binding protein with FcRn binding domains of antibodies (for example, as in WO00/09560 or US4703039).
  • Antigen binding proteins herein can comprise FcRn affinity enhanced Fc variants that can improve antibody cytotoxicity and/or half-life at pH 6.0.
  • IgG variants can be produced as low fucosylated molecules. The resulting variants can result in increased serum persistence in hFcRn mice, as well as conserved enhanced ADCC.
  • Exemplary variants can include (with reference to IgGl and Kabat numbering): P230T/V303A/K322R/N389T/F404L/N434S; P228R/N434S; Q311R/K334R/Q342E/N434Y; C226G/Q386R/N434Y; T307P/N389T/N434Y; P230S/N434S;P230T/V305A/T307A/A378V/L398P/N434S; P23OT/P387S/N434S; P230Q/E269D/N434S; N276S/A378V/N434S; T307A/N315D/A330V/382V/N389T/N434Y; T256N/A378V/S383N/N434Y; N315D/A330V/N361D/A387V/N
  • substitutions in the constant region can significantly improve the functions of antigen binding proteins, such as therapeutic IgG antibodies, substitutions in the strictly conserved constant region can yield immunogenicity in humans, while substitution in the highly diverse variable region sequence can be less immunogenic.
  • the CDR residues of an antigen binding protein provided herein can be engineered to improve binding affinity of the antigen binding protein to the antigen (e.g., FLT3LG).
  • the CDR and/or framework residues can be engineered to improve stability and decrease immunogenicity risk of an antigen binding protein provided herein.
  • Improved affinity to the antigen e.g., FLT3LG
  • Improved stability of an antigen binding protein can be rationally obtained from sequence- or structure-based rational design. Decreasing the immunogenicity risk (deimmunization) of an antigen binding protein can be accomplished, for example, by one or more humanization methodologies and/or the removal of potential T-cell epitopes, which in some cases can be predicted using in silico technologies or anticipated by in vitro assays. Additionally, the variable region of an antigen binding protein can be engineered to lower the isoelectric point (pl) of the antibody. For an antigen binding protein, a longer half-life can be associated with such a reduced pl compared to wild type antigen binding proteins, in some cases despite comparable FcRn binding. A similar increase in half life can be achieved with other antigen binding proteins.
  • Engineering or selecting antigen binding proteins with pH-dependent antigen binding can be used to modify antigen binding protein and/or antigen (e.g., FLT3LG) half-life.
  • antigen e.g., FLT3LG
  • the half-life of an IgG2 antibody can be shortened if antigen -mediated clearance mechanisms can degrade the antibody when bound to the antigen.
  • an antigen :antibody complex can impact the half-life of an antigen (e.g., FLT3LG), for example, by extending half-life by protecting the antigen from the typical degradation processes, or by shortening the half-life via antibody-mediated degradation (e.g., target-mediated drug disposition).
  • FLT3LG binding proteins may have higher affinity for antigen at pH 7.4 as compared to endosomal pH (i.e., pH 5.5-6.0) such that the KD ratio at pH 5.5/pH 7.4 or at pH 6.0/pH 7.4 can be 2 or more.
  • pH-sensitive binding to the antigen binding protein can be achieved by introducing one or more histidine residues into one or more of the CDRs.
  • An antigen binding protein herein can comprise a recycling antibody engineered so that a single antibody molecule can bind to an antigen multiple times.
  • a recycling antibody can dissociate from an antigen (e.g., FLT3LG) under acidic conditions within the cell.
  • An antibody bound to a membrane-bound antigen can dissociate from the antigen in a pH-dependent manner.
  • the dissociated antibody can then be recycled by FcRn while the antigen is transferred to lysosome and degraded. This mechanism can enable the antibody to bind to other antigens repeatedly in plasma and reduces the antibody clearance.
  • An antigen binding protein can comprise a sweeping antibody, which can be engineered, for example, using a combination of variable region engineering (as described above "pH switch") to enable the antibody to bind to an antigen (e.g., FLT3LG) in plasma and dissociate from the antigen in endosome (after which the antigen undergoes lysosomal degradation), and constant region engineering to increase the cellular uptake of the antibody-antigen complex into endosome mediated (e.g., through FcRn), FcyRIIb or potentially other surface receptors.
  • a sweeping antibody can therapeutically target a soluble antigen (e.g., FLT3LG), enhancing elimination of the antigen from the circulation.
  • one or more of a panel of Fc variants with enhanced binding to FcRn including M252Y, V308P, or N434Y, with enhanced binding to FcRn that, in combination with pH-dependent binding to a target antigen (e.g., FLT3LG), can enhance clearance of a target antigen (e.g., FLT3LG) in comparison with a wild-type Fc region.
  • FcyRIIb can be used to accelerate the uptake rate of antibody-antigen complexes into cells.
  • a FLT3LG binding protein can comprise an FcyRIIb sweeping antibody, or an Fc region thereof, in which the Fc region of a pH-dependent antibody can be engineered to selectively increase human FcyRIIb binding to enhance the uptake rate of antibody-antigen complexes.
  • This inhibitory receptor can mediate the uptake of antibodyantigen complexes into liver endothelial cells (LSEC). Therefore, mediation of the uptake of an antigen binding protein (e.g., antibody):antigen complex into a cell by FcyRIIb (e.g., human FcyRIIb) can reduce antigen (e.g., FLT3LG) concentration in the circulation.
  • a FLT3LG binding protein can comprise an Fc variant (vl2) comprising the following mutations: E233D/G237D/P238D/H268D/P271G/A330R, and can have selectively increased binding affinity to human FcyRIIb.
  • vl2 variant can accelerate the clearance of antigen (e.g., FLT3LG) over that of a pH-dependent antibody with wildtype hlgGl while maintaining comparable pharmacokinetics.
  • compositions wherein a pharmaceutical composition can comprise an antigen binding protein provided herein.
  • Pharmaceutical compositions herein can be for use in the treatment of diseases, including human diseases, described herein.
  • the pharmaceutical composition may comprise an antigen binding protein, optionally in combination with one or more pharmaceutically acceptable carriers and/or excipients.
  • compositions can comprise a pharmaceutically acceptable carrier, for example, as is known by and called for by current pharmaceutical practice.
  • Pharmaceutical compositions may be administered by injection or continuous infusion via a route, that can include for example, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular, intraportal, or another route.
  • a pharmaceutical composition may be suitable for intravenous administration.
  • a pharmaceutical composition may be suitable for subcutaneous administration.
  • Pharmaceutical compositions may be suitable for topical administration (which can include, but is not limited to, epicutaneous, intranasal, or ocular administration), inhalational administration, or enteral administration (which can include, but is not limited to, oral, vaginal, or rectal administration).
  • compositions provided herein can comprise an effective amount of an antigen binding protein, such as a FLT3LG binding protein.
  • a pharmaceutical composition may comprise between 0.5 mg and 10 g of a FLT3LG binding protein, and in some cases, can comprise between 5 mg and 1 g of a FLT3LG binding protein.
  • a pharmaceutical composition may comprise between 5 mg and 500 mg of a FLT3LG binding protein, and in some cases, can comprise between 5 mg and 50 mg of a FLT3LG binding protein.
  • a pharmaceutical composition may comprise between 0.5 mg and 10 g, between 5 mg and 1 g, between 5 mg and 500 mg, or between 5 mg and 50 mg of a FLT3LG binding protein comprising an HC comprising SEQ ID NO: 47 and an LC comprising SEQ ID NO: 55.
  • a pharmaceutical composition may comprise between 0.5 mg and 10 g, between 5 mg and 1 g, between 5 mg and 500 mg, or between 5 mg and 50 mg of a FLT3LG binding protein comprising an HC comprising SEQ ID NO: 48 and an LC comprising SEQ ID NO: 56.
  • a pharmaceutical composition may comprise between 0.5 mg and 10 g, between 5 mg and 1 g, between 5 mg and 500 mg, or between 5 mg and 50 mg of a FLT3LG binding protein comprising an HC comprising SEQ ID NO: 49 and an LC comprising SEQ ID NO: 57.
  • a pharmaceutical composition may comprise between 0.5 mg and 10 g, between 5 mg and 1 g, between 5 mg and 500 mg, or between 5 mg and 50 mg of a FLT3LG binding protein comprising an HC comprising SEQ ID NO: 50 and an LC comprising SEQ ID NO: 58.
  • a pharmaceutical composition may comprise between 0.5 mg and 10 g, between 5 mg and 1 g, between 5 mg and 500 mg, or between 5 mg and 50 mg of a FLT3LG binding protein comprising an HC comprising SEQ ID NO: 63 and an LC comprising SEQ ID NO: 71.
  • a pharmaceutical composition may comprise between 0.5 mg and 10 g, between 5 mg and 1 g, between 5 mg and 500 mg, or between 5 mg and 50 mg of a FLT3LG binding protein comprising an HC comprising SEQ ID NO: 64 and an LC comprising SEQ ID NO: 72.
  • a pharmaceutical composition may comprise between 0.5 mg and 10 g, between 5 mg and 1 g, between 5 mg and 500 mg, or between 5 mg and 50 mg of a FLT3LG binding protein comprising an HC comprising SEQ ID NO: 65 and an LC comprising SEQ ID NO: 73.
  • a pharmaceutical composition may comprise between 0.5 mg and 10 g, between 5 mg and 1 g, between 5 mg and 500 mg, or between 5 mg and 50 mg of a FLT3LG binding protein comprising an HC comprising SEQ ID NO: 66 and an LC comprising SEQ ID NO: 74.
  • a pharmaceutical composition can comprise between 0.5 mg and 10 g, between 5 mg and 1 g, between 5 mg and 500 mg, or between 5 mg and 50 mg each of more than one FLT3LG binding proteins provided herein.
  • a pharmaceutical composition may be included in a kit containing the antigen binding protein together with other medicaments, and/or with instructions for use.
  • the kit may comprise the reagents in predetermined amounts with instructions for use.
  • the kit may also include one or more devices, such as a syringe, a needle, a length of tubing, or another device, that can be used for administration of the pharmaceutical composition.
  • the terms "individual”, “subject” and “patient” are used herein interchangeably.
  • the subject may be an animal.
  • the subject may be a mammal, such as a primate, for example, a marmoset or monkey.
  • the subject may be a human.
  • the antigen binding protein described herein may also be used in methods of treatment. It will be appreciated by those skilled in the art that references herein to treatment refer to the treatment of established conditions. However, antigen binding proteins disclosed herein may, depending on the condition, also be useful in the prevention of certain diseases.
  • the antigen binding protein described herein is used in an effective amount for therapeutic, prophylactic, or preventative treatment.
  • a therapeutically effective amount of the antigen binding protein described herein is an amount effective to ameliorate or reduce one or more symptoms of, or to prevent or cure, the disease.
  • kits for treating autoimmune disease in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the FLT3LG binding protein or pharmaceutical composition as defined herein.
  • the subject may be an animal or a human.
  • the subject may be a human.
  • FLT3LG binding proteins described herein are provided for use in therapy.
  • FLT3LG binding proteins are provided for use in the treatment of autoimmune disease.
  • a FLT3LG binding protein is provided for use in the treatment of autoimmune disease, wherein the FLT3LG binding protein comprises a VH region comprising SEQ ID NO: 5 and a VL region comprising SEQ ID NO: 13.
  • a FLT3LG binding protein is provided for use in the treatment of autoimmune disease, wherein the FLT3LG binding protein comprises a VH region comprising SEQ ID NO: 6 and a VL region comprising SEQ ID NO: 14.
  • a FLT3LG binding protein is provided for use in the treatment of autoimmune disease, wherein the FLT3LG binding protein comprises a VH region comprising SEQ ID NO: 7 and a VL region comprising SEQ ID NO: 15.
  • a FLT3LG binding protein is provided for use in the treatment of autoimmune disease, wherein the FLT3LG binding protein comprises a VH region comprising SEQ ID NO: 8 and a VL region comprising SEQ ID NO: 16.
  • a FLT3LG binding protein is provided for use in the treatment of autoimmune disease, wherein the FLT3LG binding protein comprises an HC sequence comprising SEQ ID NO: 47 and an LC sequence comprising SEQ ID NO: 55.
  • a FLT3LG binding protein is provided for use in the treatment of autoimmune disease, wherein the FLT3LG binding protein comprises an HC sequence comprising SEQ ID NO: 48 and an LC sequence comprising SEQ ID NO: 56.
  • a FLT3LG binding protein is provided for use in the treatment of autoimmune disease, wherein the FLT3LG binding protein comprises an HC sequence comprising SEQ ID NO: 49 and an LC sequence comprising SEQ ID NO: 57.
  • a FLT3LG binding protein is provided for use in the treatment of autoimmune disease, wherein the FLT3LG binding protein comprises an HC sequence comprising SEQ ID NO: 50 and an LC sequence comprising SEQ ID NO: 58.
  • a FLT3LG binding protein in the manufacture of a medicament for the treatment of autoimmune disease. Also provided is the use of a FLT3LG binding protein in the manufacture of a medicament for the treatment of autoimmune disease, wherein the FLT3LG binding protein comprises a VH region comprising SEQ ID NO: 5 and a VL region comprising SEQ ID NO: 13. Also provided is the use of a FLT3LG binding protein in the manufacture of a medicament for the treatment of autoimmune disease, wherein the FLT3LG binding protein comprises a VH region comprising SEQ ID NO: 6 and a VL region comprising SEQ ID NO: 14.
  • a FLT3LG binding protein in the manufacture of a medicament for the treatment of autoimmune disease, wherein the FLT3LG binding protein comprises a VH region comprising SEQ ID NO: 7 and a VL region comprising SEQ ID NO: 15. Also provided is the use of a FLT3LG binding protein in the manufacture of a medicament for the treatment of autoimmune disease, wherein the FLT3LG binding protein comprises a VH region comprising SEQ ID NO: 8 and a VL region comprising SEQ ID NO: 16.
  • a method for treatment of autoimmune disease in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a FLT3LG binding protein, wherein the FLT3LG binding protein comprises a VH region comprising SEQ ID NO: 6 and a VL region comprising SEQ ID NO: 14. Also provided is a method for treatment of autoimmune disease in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a FLT3LG binding protein, wherein the FLT3LG binding protein comprises a VH region comprising SEQ ID NO: 7 and a VL region comprising SEQ ID NO: 14.
  • a method for treatment of autoimmune disease in a subject in need thereof comprising administering to said subject a therapeutically effective amount of a FLT3LG binding protein, wherein the FLT3LG binding protein comprises a VH region comprising SEQ ID NO: 8 and a VL region comprising SEQ ID NO: 16.
  • Contemplated diseases that may be treated include acute or chronic inflammatory diseases including Type 1 and Type 2 diabetes, chronic kidney disease (CKD), including CKD caused by diabetes, diabetic nephropathy, high blood pressure, atherosclerosis, Alzheimer's disease, cancer, and associated complications of such diseases.
  • CKD chronic kidney disease
  • autoimmune diseases including, but are not limited to, NIH's autoimmune disease list which includes, but is not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes, glomerulonephritis (e.g., IgA nephropathy), granulomatosis with polyangitis, Graves' Disease, Guillain-Barre Syndrome, idiopathic thrombocytopenic purpura, juvenile idiopathic arthritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphicus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma/systemic sclerosis, Sjogren's syndrome, systemic NIH's autoimmune disease list which
  • autoimmune diseases include, but are not limited to, autoimmune neonatal thrombocytopenia, autoimmune neutropenia, autoimmunocytopenia, antiphospholipid syndrome, dermatitis, glutensensitive enteropathy, allergic encephalomyelitis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, purpura (e.g., Henoch-Schonlein purpura), Reiter's Disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation, dense deposit disease, rheumatic heart diseases, insulin dependent diabetes mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis), systemic lupus erythematosus (SLE), discoid lupus, Goodpasture's Syndrome, pemphigus, receptor autoimmunities such as, for example, (a)
  • Autoimmune diseases may be acute or chronic and may affect essentially all organs and body systems. Autoimmune diseases may include diseases with autoantibodies, tissue specific autoantigens or non-tissue specific autoantigens, collagen or connective tissue disorders and collagen vascular diseases. More extensive autoimmune disease lists can also be found at www.autoimmuneinstitute.org/resources/autoimmune-disease-list (last visited September 8, 2023) and www.autoimmuneregistry.org/autoimmune-diseases (last visited September 8, 2023).
  • the autoimmune diseases to be treated may result from increased dendritic cell (DC) differentiation and survival via FLT3LG-induced FLT3 signalling. Therefore, the FLT3LG binding proteins described herein may be effective in treating autoimmune diseases wherein DCs enhance autoimmune responses.
  • the FLT3LG binding proteins described herein may inhibit the binding of soluble FLT3LG to immobilised FLT3 receptor.
  • the half maximal inhibitory concentration (IC50) of FLT3LG binding protein inhibition of soluble FLT3LG binding to immobilised FLT3 receptor may be determined using a soluble FLT3LG blocking ELISA assay, for example, the assay described in Example 3B.
  • the FLT3LG binding proteins may inhibit the binding of soluble FLT3LG to immobilised FLT3 receptor with an IC50 of less than or equal to 1 nM, less than or equal to 0.5 nM, less than or equal to 0.25 nM, less than or equal to 0.2 nM, or less than or equal to 0.1 nM.
  • FLT3LG can exist in both a cell surface membrane-bound form and a soluble form.
  • the FLT3LG binding proteins described herein may bind to cell surface FLT3LG.
  • the half maximal effective concentration (EC50) of FLT3LG binding protein binding to cell surface FLT3LG may be determined using a flow cytometry cell surface FLT3LG binding assay, for example, the assay described in Example 3D.
  • the FLT3LG binding proteins described herein may bind to 293T-hFLT3LG cells with an EC50 of less than or equal to 10 nM, less than or equal to 5 nM, less than or equal to 1 nM, or less than or equal to 0.5 nM.
  • the FLT3LG binding proteins described herein may inhibit the binding of soluble FLT3LG binding to cell surface FLT3 receptor.
  • the IC50 of FLT3LG binding protein inhibition of soluble FLT3LG binding to cell surface FLT3 receptor may be determined using a soluble FLT3LG blocking assay, for example, the assay described in Example 3E.
  • the FLT3LG binding proteins described herein may inhibit the binding of soluble FLT3LG to cell surface FLT3 receptor with an IC50 of less than or equal to 2 nM, less than or equal to 1.5 nM, or less than or equal to 1 nM.
  • the FLT3LG binding proteins described herein may inhibit soluble FLT3LG-induced FLT3 receptor internalization.
  • the IC50 of FLT3LG binding protein inhibition of soluble FLT3LG-induced FLT3 receptor internalization may be determined using a FLT3 internalisation flow cytometry assay, for example, the assay described in Example 4A.
  • the FLT3LG binding proteins described herein may inhibit soluble FLT3LG-induced FLT3 receptor internalization with an IC50 of less than or equal to 1 nM, less than or equal to 0.5 nM, or less than or equal to 0.2 nM.
  • the FLT3LG binding proteins described herein may inhibit membrane-bound FLT3LG-induced FLT3 receptor internalization.
  • the IC50 of FLT3LG binding protein inhibition of membrane-bound FLT3LG-induced FLT3 receptor internalization may be determined using a FLT3 internalisation flow cytometry assay, for example, the assay described in Example 4B.
  • the FLT3LG binding proteins described herein may inhibit membrane-bound FLT3LG-induced FLT3 receptor internalization with an IC50 of less than or equal to 5 nM, less than or equal to 3 nM, or less than or equal to 2 nM.
  • the FLT3LG binding proteins described herein may inhibit p-AKT signalling induced by the interaction between FLT3LG and FLT3 receptor.
  • the IC50 of FLT3LG binding protein inhibition of soluble FLT3LG-induced p-AKT signalling on FLT3-expressing cells may be measured using a p-AKT signalling transduction assay, for example, the assay described in Example 5.
  • the FLT3LG binding proteins described herein may inhibit p-AKT signalling induced by the interaction between FLT3LG and FLT3 receptor with an IC50 of less than or equal to 5 nM, less than or equal to 2 nM, or less than or equal to 1.5 nM.
  • the FLT3LG binding proteins described herein may inhibit soluble FLT3LG-induced DC differentiation.
  • the IC50 of FLT3LG binding protein inhibition of soluble FLT3LG-induced DC differentiation may be measured using a DC differentiation assay, for example, the assay described in Example 6.
  • the FLT3LG binding proteins described herein may inhibit soluble FLT3 LG-induced DC differentiation with an IC50 of less than or equal to 5 nM, less than or equal to 4 nM, or less than or equal to 3 nM.
  • the FLT3LG binding proteins described herein may bind to endogenous FLT3LG expressed on human T cells.
  • the EC50 of FLT3LG binding protein binding to FLT3LG expressed on T cells may be determined using a flow cytometry assay, for example, the assay described in Example 7.
  • the FLT3LG binding proteins described herein may bind to human T cells with an EC50 or less than or equal to 2 nM, less than or equal to 1.5 nM, less than or equal to 1 nM, or less than or equal to 0.5 nM.
  • the FLT3LG binding proteins described herein may reduce the number and/or frequency of hFLT3LG-induced DCs in the spleen.
  • the DCs may be eDCs, cDCls, cDC2s, pDCs, and/or pre-pDCs.
  • the effect of FLT3LG binding proteins on the number and/or frequency of DCs may be determined using an in vivo mouse model, for example, the in vivo mouse model described in Example 8.
  • the FLT3LG binding proteins described herein may form complexes with FLT3LG dimers.
  • the FLT3LG binding proteins may form 2 FLT3LG2 : 2 mAb complexes.
  • the FLT3LG binding proteins may form complexes with 2 FLT3LG2 : 2 mAb complexes, wherein the angle between the Fab arms of the FLT3LG binding proteins is greater than 90 degrees, greater than 95 degrees, greater than 100 degrees, greater than 105 degrees, greater than 110 degrees, greater than 115 degrees, greater than 120 degrees, greater than 125 degrees, greater than 130 degrees, greater than 135 degrees, greater than 140 degrees, greater than 145 degrees, greater than 150 degrees, greater than 155 degrees, or greater than 160 degrees.
  • a FLT3LG binding protein comprising: a. (i) any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 4 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 12; any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 1 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 9; any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 2 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 10; or any one or a combination of CDRs selected from CDRH1, CDRH2, and/or CDRH3 from SEQ ID NO: 3 and/or CDRL1, CDRL2, and/or CDRL3 from SEQ ID NO: 11; or
  • a FLT3LG binding protein comprising 6 CDRs selected from the following:
  • CDRH1 of SEQ ID NO: 23 CDRH2 of SEQ ID NO: 24, CDRH3 of SEQ ID NO: 25, CDRL1 of SEQ ID NO: 35, CDRL2 of SEQ ID NO: 36, and CDRL3 of SEQ ID NO: 37.
  • the FLT3LG binding protein according to any one of the preceding clauses, wherein the binding protein comprises: a Heavy Chain (HC) sequence at least 80% identical to SEQ ID NO: 50 and/or an LC sequence at least 80% identical to SEQ ID NO: 58; an HC (HC) sequence at least 80% identical to SEQ ID NO: 47 and/or a Light Chain (LC) sequence at least 80% identical to SEQ ID NO: 55; an HC sequence at least 80% identical to SEQ ID NO: 48 and/or an LC sequence at least 80% identical to SEQ ID NO: 56; or an HC sequence at least 80% identical to SEQ ID NO: 49 and/or an LC sequence at least 80% identical to SEQ ID NO: 57.
  • HC Heavy Chain
  • LC Light Chain
  • the FLT3LG binding protein according to any one of the preceding clauses, wherein the binding protein binds to human FLT3LG with a KD less than or equal to 1 nM.
  • a FLT3LG binding protein that binds to human FLT3LG, and competes for binding to human FLT3LG with a reference FLT3LG binding protein comprising: a VH region sequence of SEQ ID NO: 8 and a VL region sequence of SEQ ID NO: 16; a VH region sequence of SEQ ID NO: 5 and a VL region sequence of SEQ ID NO: 13; a VH region sequence of SEQ ID NO: 6 and a VL region sequence of SEQ ID NO: 14; or a VH region sequence of SEQ ID NO: 7 and a VL region sequence of SEQ ID NO: 15.
  • the FLT3LG binding protein according to any one of the preceding clauses, wherein the binding protein is an antibody or an antigen binding fragment thereof.
  • nucleic acid sequence according to clause 16 wherein the sequence comprises any one of SEQ ID NOs: 63-66 encoding the HC and/or any one of SEQ ID NOs: 71-74 encoding the LC.
  • a recombinant host cell comprising the nucleic acid sequence(s) as defined in clause 16 or 17, or the expression vector(s) as defined in clause 18.
  • a method for the production of a FLT3LG binding protein comprises culturing the host cell as defined in clause 19 under conditions suitable for expression of said nucleic acid sequence(s) or expression vector(s), whereby a polypeptide comprising the FLT3LG binding protein is produced.
  • a pharmaceutical composition comprising the FLT3LG binding protein as defined in any one of clauses 1 to 15 or 21 and a pharmaceutically acceptable excipient.
  • a method for treatment of autoimmune disease in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the FLT3LG binding protein as defined in any one of clauses 1 to 15 or 21, or the pharmaceutical composition as defined in clause 23.
  • autoimmune disease is Systemic Lupus Erythematosus (SLE), rheumatoid arthritis (RA), sarcoidosis, Sjogren's syndrome, or celiac disease.
  • SLE Systemic Lupus Erythematosus
  • RA rheumatoid arthritis
  • sarcoidosis Sjogren's syndrome
  • celiac disease celiac disease
  • SLE Systemic Lupus Erythematosus
  • RA rheumatoid arthritis
  • sarcoidosis Sjogren's syndrome
  • celiac disease is Systemic Lupus Erythematosus (SLE), rheumatoid arthritis (RA), sarcoidosis, Sjogren's syndrome, or celiac disease.
  • R8iD hFLT3LG protein
  • R8iD NSO cell-derived, 308-FKN/CF
  • the R8iD hFLT3LG differs from wild-type soluble hFLT3LG (UniProt Accession # P49771.1) at position 72; R8iD hFLT3LG has an alanine mutation whilst wild-type hFLT3LG has a glycine.
  • mice were immunized with 200 pL of recombinant hFLT3LG protein mixed with a mixture of Toll-like receptor adjuvants by weekly intraperitoneal and subcutaneous injection. After 4 immunizations, serum from immunized mice was collected to test the hFLT3 LG-specific antibody titer using ELISA.
  • Spleen and lymph nodes were harvested from immunized mice 5 days after the final boost and single cell suspensions were prepared.
  • B cells were enriched then cells were stained with a fluorophore-labeled antibody panel, and hFLT3LG (R8iD, NSO cell-derived, 308-FKN/CF) labeled with Alexa647 and Alexa488 in order to isolate hFLT3 LG-specific B cells. Stained cells were then collected for cell sorting via a SONY MA900 cell sorter. Single dump channel /CD19 + /IgM /hFLT3LG duel+ stained B cells were collected and processed for lOx Genomics running and sequencing according to the manufacturer's instructions.
  • the sequencing libraries were sequenced using an Illumina NextSeq Sequencer in conjunction with the mid output sequencing kit (Illumina, Cat #: 20024904). VH and VL sequences were assembled from the sequencing data using the Cell ranger software (lOx Genomics). The cell ranger output was further analyzed to enable clone selection using a custom pipeline, which:
  • the analysis identified 3272 cells with paired VH and VL sequences belonging to 773 unique clonotypes.
  • 124 selected clones from the hFL3LG B-cell sequencing dataset were produced by DNA synthesis and transcriptionally active PCR (TAP) as chimeric human IgGl/kappa antibodies.
  • TAP transcriptionally active PCR
  • IgGl/kappa antibodies antibody variable-region DNA with 5'- and 3'-TAP universal sequences were synthesized (IDT DNA).
  • IDCT DNA antibody variable-region DNA with 5'- and 3'-TAP universal sequences
  • the variable region DNA, a promoter DNA fragment, and a heavy or light chain constant region terminal DNA fragment were assembled and amplified to produce two separate linear TAP products, one encoding the heavy chain and the other the light chain.
  • the PCR products were verified by agarose gel electrophoresis and purified using an E-Z 96 Cycle Pure kit (Omega Biotek).
  • Heavy- and light-chain TAP fragments in a 1:2 ratio were transiently co-transfected into Expi293 cells at a 3 mL scale using an ExpiFectamineTM 293 Transfection Kit (Gibco, Cat# 2401600) following the manufacturer's instructions. Cells were incubated for 6 days before supernatant was harvested by centrifugation and the expressed antibodies were then purified using protein A chromatography.
  • Example 2 Screening and characterization of anti-FLT3LG antibodies
  • This example demonstrates the methods used to screen and select anti-FLT3LG antibodies which effectively block the interaction between FLT3LG and FLT3 receptor, bind to membrane-bound hFLT3LG, and cross-react to cyno-FLT3LG.
  • Antibodies (10 pg/mL) were diluted in a blocking buffer and 100 pL per well were added and incubated at room temperature for 1 hour. Then, horseradish peroxidase (HRP)-labeled goat anti-human IgG Fc antibody (Jackson ImmunoResearch, Cat#: 109-035-098) at 1:5000 dilution in PBS-T were added at 100 pL/well as the secondary reagent. The ELISA plates were developed using a 3, 3', 5, 5'- tetramethylbenzidine (TMB) solution, and the reaction was stopped by addition of 50 pL of 2M H2SO4. Absorbance was read at 450 nm on a VersaMax microplate reader.
  • HRP horseradish peroxidase
  • TMB 3, 3', 5, 5'- tetramethylbenzidine
  • results 116 of the TAP-expressed antibodies from the 124 selected B cell clones were tested for binding to FLT3LG.
  • ELISA was employed to measure the binding of each of the 116 TAP expressed antibodies (10 pg/mL) to each of hFLT3LG (x-axis) and cyno-FLT3LG (y-axis), as shown in Figure 1. All 116 of the antibodies displayed specific binding with hFLT3LG, and 106 of 116 antibodies also showed cross-binding activity with cyno-FLT3LG (cut off at 0.5; OD450), indicated within the dashed box in Figure 1.
  • 293T cells were lentivirally transduced to stably and constitutively express hFLT3LG (sequence NCBI NMJJ01459.4; UniProt P49771).
  • hFLT3LG sequence NCBI NMJJ01459.4; UniProt P49771.
  • 0.5xl0 6 293T cells stably expressing hFLT3LG were incubated for 30 minutes at 4°C with 100 pL of anti-FLT3LG antibody (10 pg/mL) per well.
  • 100 pL of 1:100 diluted PE-conjugated goat anti-human IgG, F(ab')2 (Jackson ImmunoResearch, Cat#: 109-116-097) antibody was added to the cells and the mixture was incubated for 30 minutes at 4°C.
  • Figure 2 illustrates the binding of the antibodies to hFLT3LG as measured via ELISA in Example 2A as well as the binding of the antibodies to cell surface hFLT3LG on 293T cells.
  • 116 antibodies that showed specific binding to hFLT3LG in the ELISA assay
  • 112 of the antibodies also showed binding to membrane-bound hFLT3LG-overexpressing 293T cells.
  • Highlighted in the dashed box in Figure 2 are antibodies which bind to hFLT3LG as measured by ELISA and to membrane-bound hFLT3LG overexpressed in 293T cells as measured by flow cytometry.
  • FLT3LG binds to FLT3 receptor with a KD of 2.31E-10 M as measured by SPR.
  • a competitive ELISA assay was performed.
  • 96-vje ⁇ Maxisorp plates were coated with an anti-hFLT3 antibody (R&D, MAB812) at 1 pg/mL in PBS overnight at 4°C. The plates were blocked for 2 hours with PBS-T blocking buffer.
  • hFLT3 (1 pg/mL) R&D, 368-ST/CF
  • Bound BT-hFLT3LG was detected with a streptavidin-HRP conjugate (THERMO SCIENTIFIC, Cat#: 21140) and developed with a 3, 3', 5, 5'- tetramethylbenzidine (TMB) substrate. After quenching with 50 pL of 2M H2SO4, absorbance was read at 450 nm on a VERSAMAX microplate reader.
  • SPR Surface Plasmon Resonance
  • hFLT3LG R&D, NSO cell-derived, 308-FKN/CF
  • cyno-FLT3LG LSBIO, Cat#: G5864
  • Regeneration of the protein A surface was achieved via 30 seconds of 10 mM glycine (pH 1.5) at 50 pL/minute between each running cycle.
  • Kinetic experiments in this example were performed at 25°C.
  • the 15 anti-FLT3LG antibodies that were down-selected in Example 2 (bl.118, bl.121, bl.123, bl.126, bl.094, bl.095, bl.078, bl.015, bl.062, bl.017, bl.021, bl.039, bl.038, bl.054, and bl.004) were humanized to yield 15 humanized antibodies against FLT3LG (h_bl.H8, h_bl.l21, h_bl.l23, h_bl.l26, h_bl.O94, h_bl.O95, h_bl.O78, h_bl.O15, h_bl.O62, h_bl.O17, h_bl.O21, h_bl.O39, h_bl.O38, h_bl.O54, and h_b
  • Selected humanized antibody sequences were synthesized and cloned into mammalian expression vectors containing the IgGl (N297G) heavy chain or Kappa light chain constant region of human.
  • Expi293 cells were transfected with the vectors for antibody expression and purification.
  • the humanized antibody binding kinetics to two different sources of hFLT3LG were determined via a second format using BIACORE 8K instrument.
  • the biotinylated hFLT3LG (BT-hFLT3LG) (0.2 pg/mL) was injected and captured by a streptavidin coated Sensor CAP chip (GE Healthcare, Cat#: 2205463-AB).
  • the binding kinetics of 15 humanized antibodies to hFLT3LG at 37°C are described in Table 5, the binding kinetics of 15 humanized antibodies to hFLT3LG at 25°C are described in Table 6, the binding kinetics of 15 humanized antibodies to cyno-FLT3LG at 25°C are described in Table 7, and the binding kinetics of 15 humanized antibodies to hFLT3LG (biotinylated assay format) at 25°C are described in Table 8 (R8iD hFLT3LG) and Table 9 (Aero hFLT3LG) (select antibodies only).
  • Antibodies that have very weak binding with BT-hFLT3LG are denoted with "ND”.
  • Antibodies that have nonspecific binding with the chips are denoted with "ND*”.
  • Table 5 Binding kinetics of 15 humanized antibodies to hFL T3LG at 37°C
  • Table 9 Binding kinetics of 10 humanized antibodies to hFLT3LG (BT-hFLT3LG, Acrobiosvstems) at 25°C B. Blocking ELISA of humanized anti-FLT3LG antibodies
  • a flow cytometry-based assay (Chan et al. SLAS Discov. 2018 Aug:23 (7): 613- 623) was adopted to determine the epitope bins of the humanized antibody panel.
  • BT- hFLT3LG R&D, NSO cell-derived, 308-FKN/CF
  • LumAvidin beads Luminex, Custom Magplex-Avidin Microspheres
  • Beads were then washed 2 times with FACS buffer, pooled, and the concentration was adjusted to 1.25 x 10 6 total beads/mL.
  • the humanized antibody panel were transferred to 96-well, V-bottom plates at 20 pL/well (3.3 pg/mL final), followed by the addition of 40 pL of the pooled beads (5000 of each bead/well), and incubated for 45 minutes at room temperature. Beads were then washed 2 times with FACS buffer, and then bound antibodies were detected using a FITC conjugated goat anti-human IgG Fc antibody (Jackson ImmunoResearch, Cat#: 109-135-098) at 5 pg/mL final concentration, 50 pL/well, for 15 minutes at room temperature.
  • a FITC conjugated goat anti-human IgG Fc antibody Jackson ImmunoResearch, Cat#: 109-135-098
  • Results The competitive binding profile was compared together for the entire 15 humanized antibody panel. The correlation values across all binding profile combinations were calculated and clustered based on similarity. Based on the clustering, epitope bins were assigned. Four epitope bins A, B, C, and D for the 15 humanized antibody panel were identified as described in Table 10.
  • hFLT3LG stably transfected 293T cells were incubated at 0.5xl0 6 cells/well with 100 pL of anti-FLT3LG antibodies starting from lOOnM in a 3-fold serial dilution for 30 minutes at 4°C, followed by 100 pL of 1:100 diluted anti-human IgG Fc-APC (Jackson ImmunoResearch, Cat#: 109-135-098) antibody incubation for 30 minutes at 4°C. After washing, cells were incubated for 5 minutes in 100 pL of DAPI for viability staining. Stained cells were fixed with 4% paraformaldehyde (PFA) and fluorescence data was acquired using a CYTOFLEX flow cytometer.
  • PFA paraformaldehyde
  • Figure 6 shows the cell surface hFLT3LG binding of the humanized antibody panel (Figure 6, Panel A), and a subset of 4 selected humanized anti-FLT3LG antibodies ( Figure 6, Panel B), as determined using flow cytometry.
  • the EC50 values (along with designated epitope bins and IC50 blocking values previously determined in Example 3B/C) for the panel of 15 humanized anti- FLT3LG antibodies are detailed in Table 10. Antibodies that did not reach binding saturation are denoted with ND (not determined). All 15 humanized anti-FLT3LG antibodies showed the ability to bind to membrane-bound 293T-hFLT3LG cells. None of the antibodies showed binding to the nontransfected 293T control cells (data not shown).
  • a soluble FLT3LG binding blocking assay with RS4;11 cells was developed.
  • RS4;11 cells display detectable FLT3 surface expression.
  • biotinylated soluble FLT3LG ligand binding to the FLT3 receptor can be measured using a fluorophore-conjugated streptavidin molecule by flow cytometry.
  • Inhibition of ligand binding using anti-FLT3LG antibodies can be measured as a reduction in mean fluorescence intensity.
  • Ni-Tris-BT-hFLT3LG nickel from IM NiCI2 was first loaded onto NTA-Tris-biotin (Sigma cat. 75543) at a 1 NTA-Tris-biotin to 4.5 NiCI2 ratio.
  • the Ni-NTA-Tris- biotin was incubated with recombinant hFLT3LG-His (Aero Biosystems cat. FLL-H5223) at a 1 FLT3LG-His : 1.5 Ni-NTA-Tris-biotin ratio at room temperature; this compound is denoted herein as "NTA-Tris-BT-hFLT3LG".
  • lxlO 5 RS4;11 cells/well were first incubated with a live/dead stain (Biolegend cat. 423102) for 20 minutes. After washing once with flow cytometry buffer (Thermo cat. 00-4222- 26), cells were incubated with Fc receptor blocking solution (Biolegend cat. 422302) for 10 minutes and then washed once with FACS buffer.
  • NTA-Tris-BT-hFLT3LG was added to varying concentrations of antibody for 15 minutes before being added to cells for 30 minutes.
  • Cells were washed with the flow cytometry buffer and then incubated with streptavidin-PE (Biolegend cat. 405204) for 30 minutes. After washing cells once with the flow cytometry buffer, cells were fixed with 4% paraformaldehyde (Thermo cat. J61899.AP) for 20 minutes. All steps were performed at room temperature. After washing once and resuspending in the flow cytometry buffer, cells were analyzed using a CytoFLEX flow cytometer. Flow data was analyzed using FlowJo and MFI values were graphed and analyzed using Graphpad Prism.
  • IC50 values for the same four selected humanized anti-FLT3LG antibodies determined using the inhibition curves are provided in Table 11. Antibodies that did not display full blocking activity are denoted with ND (not determined). Without wishing to be bound by theory, it is hypothesized that the humanized anti-FLT3LG antibody h_bl.O21 did not display full blocking activity in the soluble FLT3LG blocking assay because Aero hFLT3LG (which differs at amino acid position 72 from the R8iD hFLT3LG used for antibody generation) was employed in the assay.
  • Example 4 Inhibition of FLT3 LG -induced receptor internalization by anti-FLT3LG antibodies
  • the antibodies were tested at a starting concentration of 50 nM, and at concentrations resulting from 3-fold serial dilutions for 11 points.
  • the cells were stained on ice with LIVE/DEADTM Fixable Violet Dead Cell Stain to determine cell viability (INVITROGEN, Cat. #L34955), and any human Fc receptors expressed on these cells were blocked with the TruStain FcX human Fc receptor blocking solution (BIOLEGEND, Cat. #422302).
  • the cell-surface FLT3 was determined via flow cytometry using an anti-FLT3 antibody (clone BV10A4H2) fluorescently labeled with PE (INVITROGEN, Cat.
  • the anti-FLT3LG antibodies blocked soluble hFLT3LG-induced FLT3 receptor internalization on RS4;11 cells, as indicated by the increased fluorescence (MFI) versus increased antibody concentration as shown in Figure 8 for clone h_bl.O17 (Panel A), clone h_bl.O21 (Panel B), clone h_bl.O78 (Panel C), and h_bl.l26 (Panel D). From this receptor internalization blocking data, IC50 values were calculated for each of the selected humanized blocking antibodies, as presented in Table 12. The h_bl.21 antibody did not yield a full curve and hence an IC50 value was marked as ND (not determined). The lower blocking activity observed for h_bl.O21 in the internalization assay is hypothesized to occur due to use of Aero hFLT3LG, which may not be bound strongly by antibodies specific for epitope Bin A.
  • Table 12 IC50 values of selected humanized anti-FLT3LG antibodies, as measured by a soluble FL T3LG-induced receptor internalization assay
  • FLT3LG antibodies FLT3LG stably expressed on 293T cells is able to induce FLT3 receptor internalization on RS4;11 cells when the cells are mixed.
  • the down regulation of cell-surface FLT3 was determined by flow cytometry using an anti-FLT3 antibody (clone BV10A4H2) fluorescently labeled with PE (INVITROGEN, Cat. # 12-1357-42) in comparison to mouse IgGl kappa Isotype Control (clone P3.6.2.8.1), PE (INVITROGEN, Cat. # 12-4714-82).
  • the data was acquired on a CytoFLEX flow cytometer (BECKMAN COULTER), analyzed using the FLOWJO portal, and plotted using Prism (GraphPad).
  • IC50 values of selected humanized anti-FLT3LG antibodies as measured by a membrane FLT3LG-induced receptor internalization assay
  • AKT serine/threonine kinase AKT occurs downstream of FLT3LG binding to its receptor FLT3, leading to pathways that control cell survival and proliferation in FLT3-expressing cells.
  • the human cell line RS4;11 expresses FLT3 endogenously and responds to hFLT3LG treatment, which can be detected through phosphorylation of AKT.
  • a phospho-AKT (p-AKT) transduction assay was performed to determine whether anti- FLT3LG antibodies inhibit soluble hFLT3LG-induced AKT phosphorylation by FLT3-expressing RS4;11 cells.
  • RS4;11 cells (ATCC, Cat. #: CRL-1873, Lot #: 70036117) were seeded at a density of 2.0xl0 5 cells per well in 96-well TC-treated plates (Corning, Cat. #: CLS3595) and serum-starved by washing in IX PBS (Gibco Cat. #: 14040141) and incubating in RPMI 1640 media (Gibco, Cat. #: 11875101) for 6 hours at 37°C. Cell treatments were prepared by mixing, at room temperature for 15 minutes, the recombinant human FLT3LG (R8iD, Cat.
  • 16 pL per well of the lysed cell fraction was transferred to CISBIO opaque low-volume 96- well plates (PERKIN-ELMER, Cat. No. 66PL96100).
  • 4 pL of pre-mixed phospho-AKT detection antibodies from the same HTRF kit (PERKIN-ELMER, Cat. # 64AKSPEH) were added per well. Plates were sealed and incubated at 4°C overnight and results were read via a SPECTRAMAX i3x Microplate Reader equipped with an HTRF Detection Cartridge (Molecular Devices, Cat. #: 0200-7011POS). Resulting data was analyzed in Prism to obtain best-fit curves and IC50 values.
  • results The ability of four selected blocking humanized anti-FLT3LG antibodies to block p- AKT signaling induced by the interaction between hFLT3LG and FLT3 was determined using a p-AKT signal transduction assay. Each of the four antibodies had comparable p-AKT blocking activity, as displayed in Figure 10 panel A (clone h_bl.O17), panel B (clone h_bl.O21), panel C (clone h_bl.O78), and panel D (clone h_bl.l26). R&D isotype MAB002 and human IgG isotype were used as negative controls, and the R&D tool antibody MAB608 was used as a positive control. p-AKT signaling was determined as the Homogeneous Time Resolved Fluorescence (HTRF) ratio.
  • HTRF Homogeneous Time Resolved Fluorescence
  • IC50 values were calculated for each of the selected humanized anti-FLT3LG blocking antibodies by their capacity to block p-AKT signaling induced by the interaction between hFLT3LG and FLT3, as presented in Table 14.
  • Table 14 IC50 values of selected humanized anti-FLT3LG antibodies, as measured by a p- AKT signa! transduction assay
  • the FLT3 receptor is highly expressed in hematopoietic progenitor cells and steady-state DCs and is essential for the differentiation, proliferation/expansion, and survival of major DC subsets.
  • a co-culture assay using a MS5 feeder line and human CD34+ progenitor cells was performed to determine whether anti-FLT3LG antibodies inhibit soluble FLT3LG-induced DC differentiation.
  • MS5 stromal cells (DSMZ; Cat. #: ACC 441) were grown in complete alpha- MEM (Gibco; Cat. #: 12571063) media supplemented with 10% heat-inactivated FBS, penicillin/streptomycin, 2 mM L-glutamine, and 2 mM sodium pyruvate and maintained at 37°C and 5% CO2. After incubating with a 0.05% tryspin-EDTA solution (Gibco; Cat. #: 25300054) for 5 to 10 minutes, MS5 cells were harvested, washed, and treated with 10 pg/mL mitomycin C (THERMO SCIENTIFIC; Cat.
  • Cell treatments were prepared by mixing, at room temperature for 15 minutes, the recombinant human FLT3LG (R&D, Cat. #: 308-FKN/CF) at the predetermined EC80 of 2.86 nM based on published literature and four selected humanized anti-hFLT3LG blocking antibodies titrated from a starting concentration of 28.6 nM with 3-fold serial dilutions. Pre-mixed treatments were then added to cells in duplicate on days 0, 7, and 14 post-CD34+ cell seeding before harvesting and staining of DCs for flow cytometry at day 21.
  • Dendritic cell populations were defined via flow cytometry and plotted as frequency of parent gate. Mean values across donors were plotted against titrations of blocking humanized anti-FLT3LG antibodies, and IC50 values were determined via best-fit curve. The data represent the combination of two independent experiments, representing 5 healthy CD34+ donors.
  • Each of the four anti-FLT3LG antibodies (clones h_bl.O17, h_bl.O21, h_bl.O78, and h_bl.l26) and positive control, MAB608 (R&D SYSTEMS, Catalog #MAB608) had comparable blocking activity against FLT3LG-induced DC differentiation from CD34+ human stem cell progenitors ( Figure 11, Panels A- C).
  • IC50 values were calculated for each of the selected humanized blocking antibodies by their capacity to inhibit CDllc+ HLA-DR+ DC differentiation, as shown in Table 15.
  • Table 15 IC50 values of selected humanized anti-FLT3LG antibodies, as measured by a primary DC differentiation assay
  • Example 7 Binding of anti-FLT3LG antibodies to endogenous FLT3LG on human T cells
  • a flow cytometry assay was optimized to measure binding of these antibodies to FLT3LG on human T cells.
  • an assay was optimized to upregulate FLT3LG expression on T cells and then measured binding of the anti-FLT3LG antibodies by flow cytometry as mean fluorescence intensity.
  • T cells were obtained from fresh healthy human control blood. Two human donors were included in this study. Blood was collected in EDTA tubes and mononuclear cells (MNCs), which include T cells, were obtained by density gradient centrifugation using FICOLL- PAQUE (GE, Cat# 07907) and SEPMATE tubes (STEMCELL TECHNOLOGIES, Cat# 85460). T cells were then isolated from the MNC pool by a negative selection kit (STEMCELL TECHNOLOGIES, Cat# 19661) via magnetic separation.
  • MNCs mononuclear cells
  • T cells were then plated at density of 10 6 /mL in IMMUNOCULT-XF T Cell Expansion Medium (STEMCELL TECHNOLOGIES, Cat# 10981) + 1% antibiotic-antimycotic (GIBCO, Cat# 15240062) + 10% FBS + 1% GLUTAMAX (GIBCO, Cat# 35050061) with and without lOng/mL IL-7 (R8iD, Cat# BT-007) for three days where IL-7 induces FLT3LG expression. On the third day, cells were washed with PBS and resuspended at a cell density of 0.2 x 10 6 /mL in fresh media with fresh IL-7 (see recipe above) and incubated for another three days.
  • flow cytometry buffer ("flow buffer", THERMOFISHER CAT# 00- 4222-26). 2 x 10 5 cells/well were first incubated with a live/dead stain (BIOLEGEND, Cat# 423102) for 15-25 minutes. After washing once with flow buffer, cells were incubated with Fc receptor block solution (BIOLEGEND, Cat# 422302) for 80 minutes and then washed once with flow buffer.
  • flow buffer THERMOFISHER CAT# 00- 4222-26
  • T cell surface markers CD45 APC-Cy7 (BIOLEGEND, Cat# 304014), CD3 BV650 (BIOLEGEND, Cat# 300468), CD8 PacBlue (BIOLEGEND, Cat# 344718), CD4 FITC (BIOLEGEND, Cat# 357406) for 30 minutes along with a titration of selected anti-FLT3LG antibodies starting at 250nM and titrated down 5-fold for 10 points.
  • Cells were washed once with flow buffer and then incubated with anti-human PE secondary antibody (Jackson Immunoresearch, Cat# 109- 116-097) to detect binding of anti-FLT3LG antibodies to cells.
  • Binding curves to endogenous FLT3LG of the four selected humanized anti-FLT3LG antibodies are provided in Figure 12.
  • EC50 values were calculated for each of the selected humanized anti-FLT3LG antibodies by their capacity to bind to endogenously expressed FLT3LG on T cells of two donors, as shown in Table 16.
  • Table 16 EC50 values of selected humanized anti-FLT3LG antibodies, as measured by a T cell binding assay
  • Example 8 In vivo effect of anti-FLT3LG antibodies on hFLT3LG-induced DCs
  • mice arrived at the facility and were rested for at least 3 days prior to any manipulation.
  • NOG-EXL female mice ⁇ 5-6 weeks old were irradiated at 120 centigray (cGy).
  • mice received donor human CD34+ cells by retro-orbital (r.o.) injection.
  • Two hCD34+ donors Donor 17 and Donor 18 were used in this study.
  • Body weights BW were measured daily 0-2 weeks post-engraftment (WPE) to monitor any BW loss or sign of distress after engraftment. Thereafter, BW are measured twice weekly.
  • mice were bled (survival submandibular bleeding under isoflurane anesthesia) to determine the level of humanization (% of hCD45 of all CD45 (hCD45+mCD45)). Based on %hCD45, mice were distributed into 6 different treatment groups, with 7 mice/donor assigned to each group. Mice were dosed intraperitoneally (i.p.) with 15 pg/dose of anti-FLT3LG antibodies (h_bl.O17, h_bl.O78, or h_bl.l26) on days 1 and 2 of the study.
  • anti-FLT3LG antibodies h_bl.O17, h_bl.O78, or h_bl.l26
  • hFLT3LG (R8iD, Cat# 308-FKN-250/CF, 15 pg/dose) was injected subcutaneously (s.c.) into the nape of the back of the mice neck on days 0 and 3. On day 7, mice were euthanized, blood was collected by cardiac puncture, and spleens were collected, weighed, and processed into a single cell suspension for flow cytometry analysis.
  • Results- The frequency (% of hCD45+ cells; Panel A) and number (Panel B) of hFLT3LG- induced eDC, cDCl, cDC2, pDC, and pre-pDC populations were measured in the spleen as shown in Figure 13. Compared to IgG isotype control, administration of the selected anti-FLT3LG antibodies reduced frequency and numbers of hFLT3LG-induced DCs in vivo.
  • Antigen/Antibody complexes were prepared by mixing 0.4 pM his-tagged, human FLT3LG (AcroBiosystems, Cat#FLL-H5223) with 0.1 pM anti-FLT3LG antibody h_bl.O78 or anti- FLT3LG comparator antibody 0001. Mixtures were incubated for 70-85 minutes on ice to allow for complex formation. 4 pl of sample was added to a freshly glow-discharged, continuous carbon grid (Agar Scientific Ltd, Cat#AGS160-3) and stained with 2% uranyl acetate. Grids were imaged on a THERMOFISHER SCIENTIFIC GLACIOS transmission electron microscope operated at 200 kV and a temperature of about 90 K.
  • Example 10 Amino acid and nucleic acid sequences of antibodies and antigen binding fragments thereof
  • variable heavy (VH) and variable light (VL) chains of antibodies and antigen binding fragments thereof provided herein are provided in Table 17.
  • Table 17 Variable heavy and light chain amino acid sequences of selected murine and humanized anti-FLT3LG antibodies
  • CDRs complementarity-determining regions
  • one or more humanized CDR regions can be codon optimized.
  • DNA sequences of the humanized anti-FLT3LG antibodies provided below have been codon optimized from their murine counterparts for synthesis, cloning and expression.
  • the DNA sequences of the CDRs in the humanized clones might vary from the natural murine sequences. Typically, such codon optimization does not result in amino acid sequence differences.
  • Table 18 CDR amino acid sequences of selected murine and humanized anti-FL T3L G antibodies
  • the amino acid sequence of the fragment crystallizable (Fc) region of antibodies and antigen binding fragments thereof provided herein is provided in Table 19.
  • the Fc region provided herein was utilized in both murine and humanized clones, and was the Fc disabled hlgGl N297G; however, the use of other Fc region sequences, including wild-type Fc, can be envisioned.
  • CL domains of antibodies and antigen binding fragments thereof provided herein are provided in Table 20.
  • the CL domain provided herein was utilized in both murine and humanized clones; however, the use of other CL domain sequences can be envisioned.
  • HC full-length heavy chains
  • LC light chains
  • Table 21 Full length heavy and light chain amino acid sequences of non-humanized and humanized anti-FLT3LG antibodies

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Abstract

La présente divulgation concerne des protéines de liaison à FLT3LG qui inhibent l'interaction entre FLT3LG et FLT3, et des méthodes de traitement de maladies auto-immunes avec lesdites protéines de liaison à FLT3LG.
PCT/EP2023/079057 2022-10-20 2023-10-18 Protéines de liaison à un antigène WO2024083945A1 (fr)

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