antigen-binding proteins

Abstract

The present disclosure relates to FLT3LG-binding proteins that inhibit the interaction between FLT3LG and FLT3, and methods of treating autoimmune diseases with the FLT3LG-binding proteins.

Description

[Technical Field]

[0001] Names of participants in the joint research agreement The claimed subject matter of this application was made by or on behalf of participants in a joint research agreement that was in effect on or before the date the claimed subject matter was made, and the claimed subject matter was made as a result of activities undertaken within the scope of the joint research agreement, and the participants in the joint research agreement include GlaxoSmithKline Intellectual Property (No. 3) Limited and 23andMe, Inc.

[0002] The Joint Research Agreement was a written contract, agreement, or cooperative agreement entered into by the above-named participants for the performance of experimental, developmental, or research work in the field of the claimed subject matter.

[0003] Embodiments of the present disclosure relate to FLT3LG binding proteins, including anti-FLT3LG antibodies, and their use in the treatment of autoimmune diseases. [Background technology]

[0004] Autoimmune diseases occur when the body's immune system abnormally attacks healthy tissues with autoantibodies or autoreactive T cells. At least 80 different autoimmune diseases have been identified, including systemic lupus erythematosus (SLE), celiac disease, rheumatoid arthritis, sarcoidosis, and Sjogren's syndrome. The causes of autoimmune diseases are unknown, but genetic and environmental factors are thought to play a role. Summary of the Invention

[0005] a. (i) any one or 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 one, two, or three 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 Provided herein is a FLT3LG-binding protein comprising:

[0006] The following six CDRs: 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 CDRL3 of SEQ ID NO:40 Also provided herein is a FLT3LG binding protein comprising:

[0007] Also provided herein are FLT3LG-binding proteins that bind to human FLT3LG and compete for binding to human FLT3LG with a reference FLT3LG-binding protein comprising the VH region sequence of SEQ ID NO:8 and the VL region sequence of SEQ ID NO:16.

[0008] Also provided herein are nucleic acid sequences encoding one or both of the HC and LC of the FLT3LG binding proteins defined herein.

[0009] Also provided herein are expression vectors comprising one or more nucleic acid sequences defined herein.

[0010] Also provided herein are recombinant host cells comprising the nucleic acid sequences or expression vectors defined herein.

[0011] Also provided herein is a method for producing one or more FLT3LG-binding proteins, comprising culturing a recombinant host cell as defined herein under conditions suitable for expression of the nucleic acid sequence or expression vector, thereby producing a polypeptide comprising the FLT3LG-binding protein.

[0012] Also provided herein are cell lines engineered to express the FLT3LG binding proteins defined herein.

[0013] Also provided herein is a pharmaceutical composition comprising a FLT3LG binding protein as defined herein and a pharmaceutically acceptable excipient.

[0014] Also provided herein is a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an FLT3LG-binding protein or pharmaceutical composition defined herein.

[0015] Also provided herein are FLT3LG binding proteins and pharmaceutical compositions as defined herein for use in therapy.

[0016] Also provided herein are FLT3LG-binding proteins and pharmaceutical compositions as defined herein for use in the treatment of autoimmune diseases. [Brief explanation of the drawings]

[0017] [Figure 1] 1 illustrates ELISA binding screening of human FLT3LG (hFLT3LG) and cynomolgus monkey FLT3LG (cyno-FLT3LG) reactive clones. [Figure 2] 1 illustrates anti-FLT3LG antibody binding to hFLT3LG and 293T-hFLT3LG cells as measured by ELISA and flow cytometry (FACS) binding assays, respectively. The dashed box indicates antibody binding to both hFLT3LG and membrane-bound hFLT3LG-expressing 293T cells. [Figure 3] 1 illustrates the blocking of hFLT3LG-hFLT3 binding by 116 hFLT3LG-reactive antibodies as measured by ELISA. Dashed boxes indicate antibodies that block more than 50% of hFLT3LG binding to hFLT3 compared to isotype IgG1. [Figure 4]1 illustrates the binding affinities (KD) of 55 blocking antibodies to hFLT3LG or cyno-FLT3LG as determined using SPR and calculated using a 1:1 binding model. [Figure 5A] Illustrated is the inhibition of hFLT3LG binding to hFLT3 in a competitive ELISA assay for 15 humanized anti-FLT3LG antibodies (Panel A) and a subset of four selected humanized anti-FLT3LG antibodies (Panel B) (h_b1.017, h_b1.021, h_b1.078, and h_b1.126). [Figure 5B] Illustrated is the inhibition of hFLT3LG binding to hFLT3 in a competitive ELISA assay for 15 humanized anti-FLT3LG antibodies (Panel A) and a subset of four selected humanized anti-FLT3LG antibodies (Panel B) (h_b1.017, h_b1.021, h_b1.078, and h_b1.126). [Figure 6A] Illustrates hFLT3LG cell binding of a panel of humanized anti-FLT3LG antibodies, as determined using flow cytometry, for a humanized anti-FLT3LG antibody (Panel A) and a subset of four selected humanized anti-FLT3LG antibodies (Panel B). [Figure 6B] Illustrates hFLT3LG cell binding of a panel of humanized anti-FLT3LG antibodies, as determined using flow cytometry, for a humanized anti-FLT3LG antibody (Panel A) and a subset of four selected humanized anti-FLT3LG antibodies (Panel B). [Figure 7A] Illustrated are inhibition curves of humanized anti-FLT3LG antibodies blocking the binding of 0.75 nM soluble FLT3LG to cell surface FLT3 receptors. R&D SYSTEM's anti-human FLT3LG antibody (clone no. 40416) (R&D SYSTEMS, catalog no. MAB608) was compared with four selected humanized anti-FLT3LG antibodies: h_b1.017 (panel A), h_b1.021 (panel B), h_b1.078 (panel C), and h_b1.126 (panel D). [Figure 7B]Illustrated are inhibition curves of humanized anti-FLT3LG antibodies blocking the binding of 0.75 nM soluble FLT3LG to cell surface FLT3 receptors. R&D SYSTEM's anti-human FLT3LG antibody (clone no. 40416) (R&D SYSTEMS, catalog no. MAB608) was compared with four selected humanized anti-FLT3LG antibodies: h_b1.017 (panel A), h_b1.021 (panel B), h_b1.078 (panel C), and h_b1.126 (panel D). [Figure 7C] Illustrated are inhibition curves of humanized anti-FLT3LG antibodies blocking the binding of 0.75 nM soluble FLT3LG to cell surface FLT3 receptors. R&D SYSTEM's anti-human FLT3LG antibody (clone no. 40416) (R&D SYSTEMS, catalog no. MAB608) was compared with four selected humanized anti-FLT3LG antibodies: h_b1.017 (panel A), h_b1.021 (panel B), h_b1.078 (panel C), and h_b1.126 (panel D). [Figure 7D] Illustrated are inhibition curves of humanized anti-FLT3LG antibodies blocking the binding of 0.75 nM soluble FLT3LG to cell surface FLT3 receptors. R&D SYSTEM's anti-human FLT3LG antibody (clone no. 40416) (R&D SYSTEMS, catalog no. MAB608) was compared with four selected humanized anti-FLT3LG antibodies: h_b1.017 (panel A), h_b1.021 (panel B), h_b1.078 (panel C), and h_b1.126 (panel D). [Figure 8A] Figure 1 illustrates the blocking activity of antibody candidates in a soluble FLT3LG-induced FLT3 receptor internalization assay against RS4;11 cells for clone h_b1.017 (Panel A), h_b1.021 (Panel B), clone h_b1.078 (Panel C), and h_b1.126 (Panel D). Each lead antibody is shown and compared to R&D Systems' anti-human FLT3LG antibody (clone no. 40416) (R&D Systems, catalog no. MAB608) and an isotype control. [Figure 8B]Figure 1 illustrates the blocking activity of antibody candidates in a soluble FLT3LG-induced FLT3 receptor internalization assay against RS4;11 cells for clone h_b1.017 (Panel A), h_b1.021 (Panel B), clone h_b1.078 (Panel C), and h_b1.126 (Panel D). Each lead antibody is shown and compared to R&D Systems' anti-human FLT3LG antibody (clone no. 40416) (R&D Systems, catalog no. MAB608) and an isotype control. [Figure 8C] Figure 1 illustrates the blocking activity of antibody candidates in a soluble FLT3LG-induced FLT3 receptor internalization assay against RS4;11 cells for clone h_b1.017 (Panel A), h_b1.021 (Panel B), clone h_b1.078 (Panel C), and h_b1.126 (Panel D). Each lead antibody is shown and compared to R&D Systems' anti-human FLT3LG antibody (clone no. 40416) (R&D Systems, catalog no. MAB608) and an isotype control. [Figure 8D] Figure 1 illustrates the blocking activity of antibody candidates in a soluble FLT3LG-induced FLT3 receptor internalization assay against RS4;11 cells for clone h_b1.017 (Panel A), h_b1.021 (Panel B), clone h_b1.078 (Panel C), and h_b1.126 (Panel D). Each lead antibody is shown and compared to R&D Systems' anti-human FLT3LG antibody (clone no. 40416) (R&D Systems, catalog no. MAB608) and an isotype control. [Figure 9A] 1 illustrates the blocking activity of anti-FLT3LG antibody candidates in a cell-cell interaction-induced FLT3 internalization assay. Each of the lead anti-FLT3LG antibodies is shown and compared with R&D Systems' anti-human FLT3LG antibody (clone no. 40416) (R&D Systems, catalog no. MAB608) and an isotype control. [Figure 9B]1 illustrates the blocking activity of anti-FLT3LG antibody candidates in a cell-cell interaction-induced FLT3 internalization assay. Each of the lead anti-FLT3LG antibodies is shown and compared with R&D Systems' anti-human FLT3LG antibody (clone no. 40416) (R&D Systems, catalog no. MAB608) and an isotype control. [Figure 9C] 1 illustrates the blocking activity of anti-FLT3LG antibody candidates in a cell-cell interaction-induced FLT3 internalization assay. Each of the lead anti-FLT3LG antibodies is shown and compared with R&D Systems' anti-human FLT3LG antibody (clone no. 40416) (R&D Systems, catalog no. MAB608) and an isotype control. [Figure 9D] 1 illustrates the blocking activity of anti-FLT3LG antibody candidates in a cell-cell interaction-induced FLT3 internalization assay. Each of the lead anti-FLT3LG antibodies is shown and compared with R&D Systems' anti-human FLT3LG antibody (clone no. 40416) (R&D Systems, catalog no. MAB608) and an isotype control. [Figure 10] Illustrates the 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_b1.017 (Panel A), clone h_b1.021 (Panel B), clone h_b1.078 (Panel C), and clone h_b1.126 (Panel D). Each of the lead antibodies was compared to R&D Systems' anti-human FLT3LG antibody (clone no. 40416) (R&D Systems, catalog no. MAB608) and an isotype control. [Figure 11]Figure 1 illustrates the blocking activity of anti-FLT3LG antibody candidates against soluble FLT3LG in a primary DC differentiation assay. Each of the lead antibodies is shown and compared to R&D Systems' anti-human FLT3LG antibody (clone no. 40416) (R&D Systems, catalog no. MAB608) and an isotype control for differentiation of CD11c+HLA-DR+ cells (Panel A), plasmacytoid DCs (pDCs; BDCA2+CD123+) (Panel B), and conventional type 2 DCs (cDC2s; BDCA1+) (Panel C). [Figure 12] Binding of anti-FLT3LG antibody candidates to endogenous FLT3LG expressed on primary human T cells is depicted for clone h_b1.017 (Panel A), clone h_b1.021 (Panel B), clone h_b1.078 (Panel C), and clone h_b1.126 (Panel D). [Figure 13] Figure 1 illustrates the effect of anti-FLT3LG antibodies (h_b1.017, h_b1.078, and h_b1.126) on the frequency (Panel A) and number (Panel B) of DC populations in the spleens of human CD34-engrafted mice treated with hFLT3LG. The frequency of DC populations is expressed as a percentage of hCD45+ cells. Each data point represents one mouse. Circles represent mice engrafted with hCD34+ cells from donor 17. Triangles represent mice engrafted with hCD34+ cells from donor 18. Bars represent the mean, and error bars represent SD. 17, 78, and 126 represent mice treated with anti-FLT3LG antibodies h_b1.017, h_b1.078, or h_b1.126, respectively. [Figure 14] Illustrated are examples of 2D class averages of anti-FLT3LG antibodies (h_b1.078 or comparison antibody O001) and FLT3LG by NS-EM. Specific Description of the Invention

[0018] As used herein, "FLT3LG" refers to any FMS-related tyrosine kinase 3 ligand. Alternative names for FLT3LG include FLT3 ligand, Flt3 ligand, FL, Flt3L, FLT3L, FLG3L, and FMS-related receptor tyrosine kinase 3 ligand. FLT3LG is a molecule encoded by the FLT3LG gene 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. In general, "hFLT3LG" refers to both cell surface membrane-bound human FLT3LG and human soluble FLT3LG, unless otherwise specified.

[0019] As used herein, "FLT3" refers to the FMS-like tyrosine kinase 3 (FLT3) receptor for FLT3LG. Alternative names for FLT3 include CD135, FLK2, and STK1. FLT3 is encoded by the FLT3 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 with five Ig-like domains, a cytoplasmic region with transmembrane and juxtamembrane domains, and two tyrosine kinase domains. Upon interaction between FLT3LG and FLT3, FLT3 homodimerizes, resulting in phosphorylation of the tyrosine kinase domain and downstream signal transduction. In some instances, FLT3 is human FLT3 (hFLT3). In some instances, FLT3 can be from another organism (e.g., mouse, rat, cow, dog, cat, pig, monkey, etc.).

[0020] The term "antigen-binding protein" as used herein refers to antibodies, antigen-binding fragments thereof, and other protein constructs, such as domains, that can bind to antigens. The term "FLT3LG-binding protein" as used herein refers to an antigen-binding protein that can bind to FLT3LG. The FLT3LG-binding protein may be capable of binding to one or more of human FLT3LG and FLT3LG proteins from other organisms (e.g., mouse, rat, cow, dog, cat, pig, monkey, etc.). The FLT3LG-binding protein may be capable of binding to a fragment, variant, or mutant of FLT3LG. The FLT3LG-binding protein as used herein is different from naturally occurring proteins (e.g., FLT3) that bind to FLT3LG.

[0021] The term "antibody" is used herein in the broadest sense to refer to a molecule having an immunoglobulin-like domain (e.g., IgG, IgM, IgA, IgD, or IgE) and includes monoclonal antibodies, recombinant antibodies, polyclonal antibodies, chimeric antibodies, human antibodies, humanized antibodies, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; single variable domains (e.g., domain antibodies (DABs)), antigen-binding antibody fragments, Fab, F(ab'), Fv, disulfide-linked Fv, single-chain Fv, disulfide-linked scFv, diabodies, TANDABS, etc., as well as modified versions of any of the foregoing.

[0022] The antibodies (also known as immunoglobulins, or Igs) provided herein may comprise heterotetrameric glycoproteins with an approximate molecular weight of 150,000 daltons. Antibodies comprise two heavy chains (HC) (typically identical) and two light chains (LC) (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 fragment crystallizable region (Fc region). The Fab region comprises a variable domain at the amino terminus, comprising a variable heavy chain (VH) and a variable light chain (VL), and a constant domain at the carboxyl terminus, comprising a constant heavy chain (CH) and the first domain of a constant light chain (CL). The Fc region comprises two domains formed by the dimerization of the second and third domains (CH2 and CH3) paired with the two constant heavy chains (CH). The Fc region can elicit effector functions, for example, by binding to receptors on immune cells or by binding to C1q, the first component of the classical complement pathway. The five classes of antibodies, IgM, IgA, IgG, IgE, and IgD, are defined by distinct heavy chain amino acid sequences: μ, α, γ, ε, and δ, and each heavy chain can pair with either a K or a λ light chain. Typically, the majority of antibodies in serum belong to the IgG class, and there are four isotypes of human IgG (IgG1, IgG2, IgG3, and IgG4), whose sequences differ primarily in their hinge regions.

[0023] The antibodies provided herein may be fully human 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 such as phage or yeast display, or by immunizing transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies. Use of Human Antibody Libraries. In some cases, transgenic animals modified to express human immunoglobulin genes can be immunized 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 screening. Human antibodies produced using these techniques can then be screened for desired properties, such as activity, affinity, developability, and selectivity.

[0024] Alternative antibody formats may include alternative scaffolds in which one or more CDRs of an antigen binding protein may be arranged on a suitable non-immunoglobulin protein scaffold or framework, such as an affibody, an SpA scaffold, an LDL receptor class A domain, an avimer, or an EGF domain.

[0025] The antibodies provided herein may be "humanized antibodies," which refers to a type of engineered antibody having CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portions of the molecule derived from one or more human immunoglobulins. In addition, framework support residues may be altered to preserve binding affinity. Suitable human acceptor antibodies may be selected from conventional databases (e.g., the KABAT database, the Los Alamos database, and the Swiss Protein database) or by homology to the nucleotide and / or amino acid sequence of the donor antibody. Human antibodies characterized by homology (on an amino acid basis) to the framework regions of the donor antibody may be suitable to provide heavy chain constant regions and / or heavy chain variable framework regions for insertion of donor CDRs. Suitable acceptor antibodies 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.

[0026] One or more of the antigen-binding proteins described herein may be an antibody or an antigen-binding fragment thereof. The antigen-binding protein may be a humanized antibody or an antigen-binding fragment thereof. The antigen-binding protein may comprise one, more, or all of a humanized VH region or humanized heavy chain (HC) sequence, and / or a humanized VL region or humanized light chain (LC) sequence.

[0027] The term "donor antibody" refers to an antibody that contributes the amino acid sequence of one or more of its variable regions, CDRs, or other functional fragments or analogs to a first immunoglobulin partner. Thus, the donor provides an altered immunoglobulin coding region and the resulting expressed altered antibody with the antigen specificity and neutralizing activity characteristic of the donor antibody.

[0028] The term "acceptor antibody" refers to an antibody that is heterologous to the donor antibody and 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 the acceptor antibody.

[0029] The term "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 distinct functional properties of a polypeptide and, in many cases, can be added, removed, or transferred to other polypeptides without loss of function of the protein and / or the rest of the domain.

[0030] The term "single variable domain" refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains, e.g., VH, VHH, and VL, and / or modified antibody variable domains, e.g., those in which one or more loops have been replaced by sequences not characteristic of antibody variable domains, or antibody variable domains that are shortened or contain 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 can bind to an antigen or epitope independently of a different variable region or domain. A "domain antibody" or "DAB" may be a human "single variable domain." The single variable domain may be a human single variable domain, but may also be a single variable domain from a non-human species, e.g., a rodent (e.g., as in WO 00 / 29004), a nurse shark, or a camelid. In particular, camelid VHHs are immunoglobulin single variable domain polypeptides derived from camelid species, such as camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain-only antibodies naturally devoid of light chains. Such VHH domains may be humanized according to standard techniques available in the art, and such domains may be "single variable domains."

[0031] An antigen-binding fragment may be provided by arrangement of one or more CDRs on one or more non-antibody protein scaffolds. As used herein, "protein scaffold" includes, but is not limited to, an immunoglobulin (Ig) scaffold, such as an IgG scaffold, which may be a four-chain or two-chain antibody, or may include only the Fc region of an antibody, or may include one or more constant regions from an antibody, which may be of human or primate origin, or may be an artificial chimera of human and primate constant regions.

[0032] The protein scaffold may be an Ig scaffold, for example, an IgG or IgA scaffold. An IgG scaffold may include some or all of the domains of an antibody (i.e., CH1, CH2, CH3, VH, VL). The antigen-binding protein may include an IgG scaffold selected from IgG1, IgG2, IgG3, IgG4, or IgG4PE. For example, the scaffold may be IgG1. The scaffold may consist of or include the Fc region of an antibody or fragment thereof.

[0033] The protein scaffold may be a derivative of a scaffold selected from the group consisting of CTLA-4, lipocalin, protein A-derived molecules such as the Z-domain of protein A (affibody, SpA), A-domain (avimer / maxibody); heat shock proteins such as GroEI and GroES; transferrin (trans-body); ankyrin repeat proteins (DARPins); peptide aptamers; C-type lectin domains (tetranectin); human g-crystallin and human ubiquitin (affilin); PDZ domains; scorpion toxin Kunitz-type domains of human protease inhibitors; and antigens other than the natural ligand, such as fibronectin / adnectin, that have been subjected to protein engineering to obtain binding to FLT3.

[0034] The term "multispecific antigen-binding protein" refers to an antigen-binding protein that contains at least two different antigen-binding sites. Each of these antigen-binding sites may be capable of binding to a different epitope than another antigen-binding site, and the antigen-binding sites may be located on the same or different antigens. A multispecific antigen-binding protein may have specificity for two or more antigens, for example, two, three, or four antigens. A multispecific antigen-binding protein that has specificity for two antigens may be referred to as a bispecific antigen-binding protein.

[0035] Bispecific antigen-binding proteins (i.e., bispecifics) can be classified as having symmetric or asymmetric structures. Bispecific antigen-binding proteins can be bispecific antibodies. Bispecifics can have an Fc region or can be fragment-based (lacking an Fc region). Fragment-based bispecifics can combine multiple antigen-binding fragments in one molecule without an Fc region or with a portion of an Fc region, such as Fab-scFv, Fab-scFv2, orthogonal Fab-Fab, Fab-Fv, tandem scFc (e.g., BiTE and BiKE molecules), diabodies, DARTs, TandAbs, scDiabodies, tandem dAbs, etc.

[0036] Symmetric formats can combine multiple binding specificities in a single polypeptide chain or a single HL pair. Examples include fragment-based formats or Fc-fusion proteins in which one or more antibody fragments are fused to an antibody molecule or other antigen-binding protein. Examples of symmetric formats can include DVD-Ig, TVD-Ig, CODV-Ig, (scFv)4-Fc, IgG-(scFv)2, tetravalent DART-Fc, F(ab)4 CrossMab, IgG-HC-scFv, IgG-LC-scFv, mAb-dAb, etc.

[0037] Asymmetric formats can retain as close as possible to the native structure of natural antibodies by promoting correct H chain pairing and / or H chain heterodimerization during co-expression of three (if common heavy or light chains are used) or four polypeptide chains, and include, for example, Triomab, Asymmetric Re-Engineered Immunoglobulin (ART-Ig), CrossMab, Biclonics common light chain, ZW1 common light chain, DuoBody, and Knob-into-Hole (KiH), DuetMab, κλ body, Xmab, YBODY, HET-mAb, HET-Fab, DART-Fc, SEEDbody, and mouse / rat chimeric IgG.

[0038] Bispecific formats can also include antibodies fused to non-Ig scaffolds, for example, Affimabs, Fynomabs, Zybodies, Anticalin-IgG fusions, or ImmTACs.

[0039] One or more of the antigen-binding proteins described herein may exhibit cross-reactivity between human FLT3LG and FLT3LG from another species, such as cynomolgus monkey FLT3LG or rhesus monkey FLT3LG. The antigen-binding proteins described herein may specifically bind to human FLT3LG and cynomolgus monkey FLT3LG. Such cross-reactivity can be exploited during preclinical studies, for example, in one or more non-human primate systems, such as rhesus monkeys or cynomolgus monkeys. Such preclinical studies can be conducted before the antigen-binding proteins are tested in humans. Such cross-reactivity can be exploited to conduct one or more side-by-side comparisons using the antigen-binding proteins described herein. Cross-reactivity between other species can be used in disease models, such as dogs or other monkeys. Optionally, the binding affinity of the antigen binding protein to at least cynomolgus FLT3LG and the binding affinity to human FLT3LG differ by no more than 2-fold, no more than 5-fold, no more than 10-fold, no more than 50-fold, or no more than 100-fold.

[0040] Affinity, also referred to as "binding affinity," is the strength of binding at a single interaction site, i.e., one molecule, such as an antigen-binding protein of the present invention, to another molecule, such as 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, the SPR method described in Example 3 may be used to measure binding affinity.

[0041] Avidity, also referred to as functional affinity, is the cumulative strength of binding at multiple interaction sites, e.g., the sum of the strengths of binding of two molecules (or more, e.g., in the case of bispecific or multispecific molecules) to another at multiple sites, taking into account, e.g., the valency of the interaction.

[0042] The equilibrium dissociation constant (KD) of the 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 5 to 10 nM or 1 to 2 nM. The KD may be 1 pM to 500 pM or 500 pM to 1 nM. The KD may be 100 pM to 500 pM or 100 pM to 1 nM. The KD may be 100 pM to 2 nM or 100 pM to 5 nM. For antigen-binding proteins herein, a smaller KD value corresponds to stronger binding to an antigen, such as FLT3LG. The reciprocal of the KD (i.e., 1 / KD) is the equilibrium association constant (KA), and the M -1 For the antigen binding proteins herein, a larger KA value corresponds to stronger binding to the antigen, such as FLT3LG.

[0043] The dissociation rate constant (kd) or "off-rate" describes the stability of an antigen-binding protein-antigen (e.g., FLT3LG) complex, i.e., the rate at which the complex decays per second. For example, 0.01 s -1 The kd of the antigen-binding protein-FLT3LG interaction is 1 x 10, where kd is equal to 1% of the complex decayed per second. -3 s -1 Below, 1×10 -4 s -1 Below, 1×10 -5 s -1 or less, or 1 x 10 -6 s -1 Alternatively, kd may be 1×10 -5 s -1 ~1×10 -4 s -1 or 1×10-4 s -1 ~1×10 -3 s -1 Alternatively, kd may be 1 x 10 -3 s -1 ~1×10 -2 s -1 may be.

[0044] The association rate constant (ka) or "on-rate" describes the rate of antigen binding protein-antigen (e.g., FLT3LG) complex formation. The ka of the antigen binding protein-FLT3LG interaction is approximately 1.5×10 5 M -1 s -1 Alternatively, ka may be 1×10 6 M -1 s -1 ~1×10 5 M -1 s -1 Alternatively, ka may be 1×10 5 M -1 s -1 ~5×10 5 M -1 s -1 or 1×10 5 M -1 s -1 ~8×10 5 M -1 s -1 may be.

[0045] In some instances, the binding affinity of the FLT3LG-binding protein to wild-type FLT3LG and to variant FLT3LG with one or more mutations differs by at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold. The FLT3LG-binding protein described herein may bind to wild-type FLT3LG, but may not bind to variant FLT3LG with one or more amino acid mutations. The variant FLT3LG may be FLT3LG containing an alanine at position 72. The wild-type FLT3LG may be hFLT3LG. The variant FLT3LG may be hFLT3LG. The FLT3LG-binding protein may bind to wild-type FLT3LG with a KD of less than or equal to 2 nM, or to variant FLT3LG with a KD of more than or equal to 2 nM. The FLT3LG-binding protein may bind to wild-type FLT3LG with a KD of less than or equal to 1 nM, or to variant FLT3LG with a KD of more than or equal to 1 nM. The FLT3LG-binding protein may bind to wild-type FLT3LG with a KD of less than or equal to 500 pM, or to variant FLT3LG with a KD of more than or equal to 500 pM. The FLT3LG-binding protein may bind to wild-type FLT3LG with a KD of less than or equal to 500 pM, or to variant FLT3LG with a KD of more than or equal to 1 nM. For example, the FLT3LG-binding protein may bind to wild-type hFLT3LG with a KD of less than or equal to 500 pM, or to variant hFLT3LG containing an alanine at position 72 with a KD of more than or equal to 1 nM. The FLT3LG binding protein may bind to wild-type FLT3LG with a KD of greater than or equal to 2 nM, or to variant FLT3LG with a KD of less than or equal to 2 nM. The FLT3LG binding protein may bind to wild-type FLT3LG with a KD of greater than or equal to 1 nM, or to variant FLT3LG with a KD of less than or equal to 1 nM.The FLT3LG-binding protein may bind to wild-type FLT3LG with a KD greater than or equal to 500 pM, or to variant FLT3LG with a KD less than or equal to 500 pM. The FLT3LG-binding protein may bind to wild-type FLT3LG with a KD greater than or equal to 100 pM, or to variant FLT3LG with a KD less than or equal to 100 pM. The FLT3LG-binding protein may bind to wild-type FLT3LG with a KD greater than or equal to 500 pM, or to variant FLT3LG with a KD less than or equal to 100 pM. As used herein, "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 the environment in which it may be found in nature, or from another environment.

[0046] The antigen-binding proteins described herein, e.g., anti-FLT3LG antibodies, may be encoded by one or more isolated nucleic acid sequences. Production of FLT3LG-binding proteins, e.g., antibodies, may be achieved in cells or living organisms by delivering exogenous isolated nucleic acids encoding the heavy and light chains of the FLT3LG-binding proteins, e.g., antibodies. Production of FLT3LG-binding proteins, e.g., antibodies, may be achieved in cells in vitro or in vivo by delivering exogenous isolated nucleic acids encoding the heavy and light chains of the FLT3LG-binding proteins, e.g., antibodies. A subject in need may be delivered one or more nucleic acids encoding the heavy and light chains of the antigen-binding proteins provided herein, e.g., anti-FLT3LG antibodies. The heavy and light chains of the antibodies may be delivered by the same or separate nucleic acids. The nucleic acids may be DNA or RNA. The nucleic acid encoding the FLT3LG-binding protein may be delivered to a subject naked (i.e., without encapsulated particles) or packaged (i.e., encapsulated in a liposome or polymer-based vehicle). The nucleic acid encoding the FLT3LG-binding protein may be delivered without a delivery vehicle (i.e., "naked"), or may be delivered by a viral or non-viral delivery vehicle (i.e., as a viral vector, adsorbed to or encapsulated in a liposome or polymer-based vehicle, etc.). The nucleic acid may contain elements such as a polyA tail, a 5' and / or a 3' untranslated region (UTR). The nucleic acid may be mRNA. The mRNA may contain a cap structure. The mRNA may be self-replicating RNA.

[0047] Nucleic acids encoding FLT3LG-binding proteins may be modified or unmodified. Nucleic acids encoding FLT3LG-binding proteins may contain at least one chemical modification. Nucleic acids (e.g., mRNA) may be modified to enhance stability by including one or more chemical modifications. Such chemical modifications include, but are not limited to, modified nucleotides, modified sugar backbones, etc. Also provided herein are methods for producing FLT3LG-binding proteins in cells, tissues, or organisms, comprising contacting the cells, tissues, or organisms with a composition comprising an isolated nucleic acid encoding the FLT3LG-binding protein that contains at least one chemical modification. Also provided herein are methods for producing FLT3LG-binding proteins in cells, tissues, or organisms, comprising contacting the cells, tissues, or organisms with a composition comprising a polynucleotide encoding the FLT3LG-binding protein that contains at least one chemical modification. Also provided herein is a method for producing an FLT3LG-binding protein in a cell in vitro or in vivo, the method comprising contacting the cell with a composition that includes at least one chemical modification and that includes a nucleic acid encoding the FLT3LG-binding protein.

[0048] Also provided herein are expression vectors, which can be isolated nucleic acids that can be used to introduce a nucleic acid of interest into cells, such as eukaryotic or prokaryotic cells, or cell-free expression systems in which the nucleic acid sequence of interest is expressed as a peptide chain, such as a protein. The nucleic acid of interest can include the nucleic acid sequence of an antigen-binding protein or fragment thereof provided herein. Such expression vectors can be, for example, cosmids, plasmids, viral sequences, transposons, and linear nucleic acids containing the nucleic acid of interest. When the expression vector is introduced into a cell or a cell-free expression system (e.g., a reticulocyte lysate), the protein encoded by the nucleic acid of interest is produced by transcription / translation machinery. Expression vectors within the scope of the present disclosure can provide the 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 driven by mammalian gene promoters, such as the human Ig gene promoter. Other examples include prokaryotic expression vectors, such as T7 promoter-driven vectors, e.g., pET41, lactose promoter-driven vectors, and arabinose gene promoter-driven vectors. Those skilled in the art will recognize many other suitable expression vectors and expression systems.

[0049] Recombinant host cells are also provided herein. As used herein, the term "recombinant host cell" refers to a cell containing a nucleic acid sequence of interest that has been isolated prior to its introduction into the cell. For example, 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, including, but not limited to, COS-1 cells, COS-7 cells, HEK293 cells, BHK21 cells, CHO cells, BSC-1 cells, HepG2 cells, 653 cells, SP2 / 0 cells, NS0 cells, 293 cells, HeLa cells, myeloma cells, lymphoma cells, or any derivatives thereof. The eukaryotic cell may be a HEK293 cell, NS0 cell, SP2 / 0 cell, or CHO cell. Escherichia coli is an exemplary prokaryotic cell. Recombinant cells according to the present disclosure may be generated by transfection, cell fusion, immortalization, or other procedures well known in the art. A nucleic acid of interest, e.g., an expression vector, transfected into a cell can be extrachromosomal or can be stably integrated into a chromosome of the cell.

[0050] The complementarity determining region (CDR) amino acid sequence of the antigen-binding protein is also provided herein. The hypervariable region of the antigen-binding protein herein exists, for example, in the immunoglobulin heavy chain and light chain. Typically, there are three heavy chain and three light chain CDRs (or CDR regions) in the variable part of an antigen-binding protein, for example, an immunoglobulin. Therefore, the term "CDR" as used herein can refer to the three heavy chain CDRs of the antigen-binding protein, the three light chain CDRs of the antigen-binding protein, all the heavy and light chain CDRs of the antigen-binding protein, or at least two CDRs of the antigen-binding protein.

[0051] Throughout this specification, amino acid residues in variable domain sequences, and variable domain regions within full-length antigen-binding sequences, e.g., antibody heavy chain sequences or antibody light chain sequences, are numbered according to the Kabat numbering convention. Similarly, the terms "CDR," "CDRL1," "CDRL2," "CDRL3," "CDRH1," "CDRH2," and "CDRH3" used in the Examples follow the Kabat numbering convention. For further information, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987).

[0052] 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 the Fc region, antibody sequences or full-length antigen-binding protein sequences are numbered according to the EU index numbering convention.

[0053] Alternative numbering conventions for CDR sequences exist, such as those shown 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 will be understood as such by one of skill in the art.

[0054] Other numbering conventions for CDR sequences available to those skilled in the art include the "AbM" (University of Bath) and "contact" (University College London) conventions. The CDR regions for SEQ ID NOS: 1-4 and 9-12 can be defined by any numbering convention, such as the Kabat, Chothia, AbM, and contact conventions.

[0055] Table 1 below presents one definition using the respective numbering rules for the CDRs, or binding units, provided herein. The Kabat numbering scheme is used in Table 1 to number the amino acid sequences of the variable domains. It should be noted that CDR definitions may vary depending on the particular publication used.

[0056] [Table 1]

[0057] 1. 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 CDRL3 of SEQ ID NO: 31; 2. 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 CDRL3 of SEQ ID NO:34; 3. 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; or 4. 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 CDRL3 of SEQ ID NO:40 Provided herein are FLT3LG binding proteins comprising any one or combination of CDRs selected from:

[0058] The FLT3LG-binding proteins described herein are 1. 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 CDRL3 of SEQ ID NO: 31; 2. 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 CDRL3 of SEQ ID NO:34; 3. 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; or 4. 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 CDRL3 of SEQ ID NO:40 It may comprise all six CDRs selected from:

[0059] The CDRs of the FLT3LG binding proteins provided herein may be modified by substitution, deletion, or addition of one or more amino acids, and the variant FLT3LG binding protein substantially retains the biological characteristics of the unmodified protein, such as inhibiting binding of FLT3LG to FLT3.

[0060] It will be appreciated that each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3 may be modified alone or in any permutation or combination with any other CDR. A CDR may be modified by substituting, deleting, or adding up to three amino acids, for example, one or two amino acids, for example, one amino acid. Each modification of a CDR, VH, VL, or other protein provided herein may be a conservative substitution. The modification may be, for example, a conservative substitution as shown in Table 2a or Table 2b below.

[0061] [Table 2]

[0062] [Table 3]

[0063] For example, in a variant CDR, one or more flanking residues that comprise a CDR as part of an alternative definition, eg, Kabat or Chothia, may be substituted with a conservative amino acid residue.

[0064] Such antigen binding proteins comprising the variant CDRs described above are sometimes referred to herein as "functional CDR variants."

[0065] The FLT3LG-binding proteins described herein are a. (i) any one or 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 (ii) a CDR variant of (i), wherein the variant has one, two, or three amino acid modifications; or b. A VH region comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 5 and / or a VL region comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 13 may also include:

[0066] The FLT3LG-binding proteins described herein are a. (i) any one or 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 (ii) a CDR variant of (i), wherein the variant has one, two, or three amino acid modifications; or b. A VH region comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 6 and / or a VL region comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 14 may also include:

[0067] The FLT3LG-binding proteins described herein are a. (i) any one or 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 (ii) a CDR variant of (i), wherein the variant has one, two, or three amino acid modifications; or b. A VH region comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 7 and / or a VL region comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 15 may also include:

[0068] The FLT3LG-binding proteins described herein are a. (i) any one or 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 one, two, or three 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 may also include:

[0069] The FLT3LG-binding proteins described herein may comprise any one or 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. The FLT3LG-binding proteins described herein may comprise any one or 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. The FLT3LG-binding proteins described herein may comprise any one or 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. The FLT3LG binding proteins described herein may comprise any one or 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.

[0070] The FLT3LG-binding proteins described herein may comprise humanized sequences. Humanization may be performed by obtaining 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 placing them in a human framework. Humanization may be performed by obtaining 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 placing them in a human framework. Humanization may be performed by obtaining 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 placing them in a human framework. Humanization may be performed by taking any one or 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 placing them into a human framework.

[0071] Structurally, CDRs L1, L2, L3, H1, and H2 can exhibit one of several main-chain conformations. A particular canonical structural class of a CDR can be defined by both the length and loop packing of the CDR, determined by residues located at key positions in both the CDRs and framework regions (structurally determining residues or SDRs). Martin and Thornton (1996; J Mol Biol 263:800-815) created an automated method for defining canonical templates of "critical residues." Cluster analysis is used to define canonical classes for sets of CDRs, and canonical templates are then identified by analyzing buried hydrophobic and hydrogen-bonding residues, as well as conserved glycines and prolines. CDRs of antibody sequences can be assigned to canonical classes by comparing the sequence to critical residue templates and scoring each template using an identity or similarity matrix.

[0072] Examples of CDR canonicals are given below in Table 3. The amino acid numbering used is Kabat.

[0073] [Table 4]

[0074] 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. Thus, any combination of substitutions may be present in the antigen binding proteins provided herein, provided that the canonical structure of the CDR is maintained such that the antigen binding protein is capable of specifically binding to FLT3LG.

[0075] As discussed above, a particular canonical structural class of a CDR can be defined by both the length of the CDR and the loop packing, which is determined by residues located at key positions in both the CDR and framework regions.

[0076] As used herein, the term "epitope" refers to that portion of an antigen (e.g., FLT3LG) that contacts a specific binding domain (i.e., paratope) of an antigen-binding protein provided herein. Epitopes may be linear, conformational, or discontinuous. Conformational or discontinuous epitopes, for example, include amino acid residues that are separated by one or more other amino acids, i.e., are not a contiguous sequence in the primary sequence of the antigen when assembled by tertiary folding of the polypeptide chain. Although the residues may be from different regions of the polypeptide chain, they may be adjacent in the three-dimensional structure of the antigen. For example, for multimeric antigens, conformational or discontinuous epitopes may include residues from different peptide chains. Specific residues contained within an epitope can be determined by computer modeling programs or by one or more three-dimensional structures obtained by structural methods such as X-ray crystallography. Epitopes can be determined using epitope mapping, for example, by one or more techniques, such as peptide-based approaches like pep scans, 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 peptide or protein mutants, for example, on phage. Detailed epitope information can be determined by structural techniques, such as X-ray crystallography, solution nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM). Mutagenesis, such as alanine scanning, can be an effective approach, whereby analysis of loss of binding 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.

[0077] The term "antigen-binding site" as used herein refers to a site on an antigen (e.g., FLT3LG)-binding protein that can specifically bind to an antigen (e.g., FLT3LG). An antigen-binding site can be a single variable domain, or it can be one or more paired VH / VL domains, such as an antibody. A single-chain Fv (ScFv) domain can also provide an antigen-binding site.

[0078] 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 those skilled in the art, such as ELISA, FMAT, surface plasmon resonance (SPR), or FORTEBIO OCTET biolayer interferometry (BLI). Such techniques may be referred to as epitope binning. A competition assay may be performed, for example, using flow cytometry-based epitope binning. Competition may occur, for example, when two proteins bind to the same or overlapping epitope, when there is steric inhibition of binding, or when binding of a first protein induces a conformational change in the antigen that can prevent or reduce binding of a second protein. The FLT3LG-binding protein may bind to human FLT3LG or compete with a reference FLT3LG-binding protein for binding to human FLT3LG. The 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.

[0079] The FLT3LG-binding proteins described herein may share an overlapping epitope with reference FLT3LG-binding proteins 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. The FLT3LG-binding proteins described herein may share an overlapping epitope with reference FLT3LG-binding proteins 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.

[0080] The FLT3LG-binding protein can be an antagonist, for example, an antagonist antibody.Antagonists can include epitope-binding proteins, for example, antibodies or fragments thereof, that can completely or partially inhibit the biological activity of the antigen to which they bind, for example, by completely or partially blocking the binding of the antigen to a receptor, or by neutralizing the activity, such as signal transduction, that can be initiated by the biological activity of the antigen.

[0081] The FLT3LG-binding proteins herein can be neutralized. "Neutralizing" refers to the reduction or elimination of the biological activity of an antigen (e.g., FLT3LG) in the presence of a FLT3LG-binding protein described herein, compared to the biological activity of the antigen in the absence of the FLT3LG-binding protein, in vitro or in vivo. Neutralization can be due to one or more of the following: blocking FLT3LG binding to its receptor, preventing FLT3LG from activating its receptor, downregulating FLT3LG or its receptor, or affecting effector functionality. Neutralization can be determined or measured using one or more assays, for example, the assays described herein. For example, the blocking assay described in Example 3 can be used to evaluate the neutralizing ability of the antigen-binding proteins herein.

[0082] The effect of the FLT3LG-binding protein on the interaction between FLT3LG and FLT3 may be partial or total. A neutralizing FLT3LG-binding protein may neutralize the activity of FLT3LG-FLT3 interaction (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%, compared to the FLT3LG-FLT3 interaction in the absence of the FLT3LG-binding protein. The FLT3LG-binding proteins described herein may inhibit the interaction between 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. The 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. The FLT3LG-binding proteins described herein may inhibit the binding of membrane-bound FLT3LG to FLT3 by 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0083] "Percent identity" or "% identity" between a query nucleic acid sequence and a subject nucleic acid sequence is an "identity" value expressed as a percentage, 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, e.g., pairwise global sequence alignment, performed using a suitable algorithm (e.g., Needleman-Wunsch or GenePAST / KERR) or software (e.g., DNASTAR Lasergene or GenePAST / KERR). The query nucleic acid sequence may be a nucleic acid sequence disclosed herein, particularly one or more of the claims.

[0084] "Percent identity" or "% identity" between a query amino acid sequence and a subject amino acid sequence is an "identity" value expressed as a percentage, 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, e.g., pairwise global sequence alignment, performed using a suitable algorithm (e.g., Needleman-Wunsch or GenePAST / KERR) or software (e.g., DNASTAR Lasergene or GenePAST / KERR). The query amino acid sequence may be described by an amino acid sequence disclosed herein, particularly one or more of the claims.

[0085] The query sequence may be 100% identical to the subject sequence, or may contain up to an integer number of amino acid or nucleotide changes compared to the subject sequence such that the percent identity is less than 100%. For example, the query sequence may be 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 the subject sequence. In the case of nucleic acid sequences, such changes may include deletion, substitution, or insertion of at least one nucleotide residue, which may occur individually among the nucleotide residues in the query sequence, or interspersed in one or more contiguous groups within the query sequence, at the 5' or 3' terminal position of the query sequence, or at one or more positions between these terminal positions. In the case of amino acid sequences, such alterations can include deletion, substitution (including conservative and non-conservative substitution), or insertion of at least one amino acid residue, which can occur individually among the amino acid residues in the query sequence, or interspersed in one or more contiguous groups within the query sequence, at the amino or carboxy terminal positions of the query sequence, or at one or more positions between these terminal positions.

[0086] For antibody sequences, the percent identity may be determined over the entire length of the query sequence, including the CDRs. The calculated percent identity may exclude one or more, or all, of the CDRs. For example, all of the CDRs of an antibody may be 100% identical to the subject sequence, but the percent identity of the remainder of the query sequence, such as the framework sequences, may be less than 100%, so that the CDR sequences are fixed and intact.

[0087] The FLT3LG binding proteins described herein may comprise a CDRH3 that is 100% identical to any one of SEQ ID NOs: 19, 22, 25, or 28. The FLT3LG binding proteins described herein may comprise a CDRH3 that is 100% identical to SEQ ID NO: 19. The FLT3LG binding proteins described herein may comprise a CDRH3 that is 100% identical to SEQ ID NO: 22. The FLT3LG binding proteins described herein may comprise a CDRH3 that is 100% identical to SEQ ID NO: 25. The FLT3LG binding proteins described herein may comprise a CDRH3 that is 100% identical to SEQ ID NO: 28.

[0088] The FLT3LG-binding proteins described herein may comprise a CDRH1 that is 100% identical to SEQ ID NO: 17; a CDRH2 that is 100% identical to SEQ ID NO: 18; a CDRH3 that is 100% identical to SEQ ID NO: 19; a CDRL1 that is 100% identical to SEQ ID NO: 29; a CDRL2 that is 100% identical to SEQ ID NO: 30; and / or a CDRL3 that is 100% identical to SEQ ID NO: 31. The FLT3LG-binding proteins described herein may comprise a CDRH1 that is 100% identical to SEQ ID NO: 20; a CDRH2 that is 100% identical to SEQ ID NO: 21; a CDRH3 that is 100% identical to SEQ ID NO: 22; a CDRL1 that is 100% identical to SEQ ID NO: 32; a CDRL2 that is 100% identical to SEQ ID NO: 33; and / or a CDRL3 that is 100% identical to SEQ ID NO: 34. The FLT3LG-binding proteins described herein may comprise a CDRH1 that is 100% identical to SEQ ID NO: 23; a CDRH2 that is 100% identical to SEQ ID NO: 24; a CDRH3 that is 100% identical to SEQ ID NO: 25; a CDRL1 that is 100% identical to SEQ ID NO: 35; a CDRL2 that is 100% identical to SEQ ID NO: 36; and / or a CDRL3 that is 100% identical to SEQ ID NO: 37. The FLT3LG-binding proteins described herein may comprise a CDRH1 that is 100% identical to SEQ ID NO: 26; a CDRH2 that is 100% identical to SEQ ID NO: 27; a CDRH3 that is 100% identical to SEQ ID NO: 28; a CDRL1 that is 100% identical to SEQ ID NO: 38; a CDRL2 that is 100% identical to SEQ ID NO: 39; and / or a CDRL3 that is 100% identical to SEQ ID NO: 40.

[0089] The FLT3LG-binding proteins described herein may include a CDRH1 that is 100% identical to SEQ ID NO: 17; a CDRH2 that is 100% identical to SEQ ID NO: 18; a CDRH3 that is 100% identical to SEQ ID NO: 19; a CDRL1 that is 100% identical to SEQ ID NO: 29; a CDRL2 that is 100% identical to SEQ ID NO: 30; and a CDRL3 that is 100% identical to SEQ ID NO: 31. The FLT3LG-binding proteins described herein may include a CDRH1 that is 100% identical to SEQ ID NO: 20; a CDRH2 that is 100% identical to SEQ ID NO: 21; a CDRH3 that is 100% identical to SEQ ID NO: 22; a CDRL1 that is 100% identical to SEQ ID NO: 32; a CDRL2 that is 100% identical to SEQ ID NO: 33; and a CDRL3 that is 100% identical to SEQ ID NO: 34. The FLT3LG-binding proteins described herein may include a CDRH1 that is 100% identical to SEQ ID NO: 23; a CDRH2 that is 100% identical to SEQ ID NO: 24; a CDRH3 that is 100% identical to SEQ ID NO: 25; a CDRL1 that is 100% identical to SEQ ID NO: 35; a CDRL2 that is 100% identical to SEQ ID NO: 36; and a CDRL3 that is 100% identical to SEQ ID NO: 37. The FLT3LG-binding proteins described herein may include a CDRH1 that is 100% identical to SEQ ID NO: 26; a CDRH2 that is 100% identical to SEQ ID NO: 27; a CDRH3 that is 100% identical to SEQ ID NO: 28; a CDRL1 that is 100% identical to SEQ ID NO: 38; a CDRL2 that is 100% identical to SEQ ID NO: 39; and a CDRL3 that is 100% identical to SEQ ID NO: 40.

[0090] The FLT3LG-binding proteins described herein may comprise a VH region at least 90% identical to SEQ ID NO:5 and a VL region at least 90% identical to SEQ ID NO:13. The FLT3LG-binding proteins described herein may comprise a VH region at least 95% identical to SEQ ID NO:5 and a VL region at least 95% identical to SEQ ID NO:13. The FLT3LG-binding proteins described herein may comprise a VH region at least 90% identical to SEQ ID NO:6 and a VL region at least 90% identical to SEQ ID NO:14. The FLT3LG-binding proteins described herein may comprise a VH region at least 95% identical to SEQ ID NO:6 and a VL region at least 95% identical to SEQ ID NO:14. The FLT3LG-binding proteins described herein may comprise a VH region at least 90% identical to SEQ ID NO:7 and a VL region at least 90% identical to SEQ ID NO:15. The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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.

[0091] The FLT3LG-binding proteins described herein may comprise a VH region 100% identical to SEQ ID NO:5 and a VL region 100% identical to SEQ ID NO:13; a VH region 100% identical to SEQ ID NO:6 and a VL region 100% identical to SEQ ID NO:14; a VH region 100% identical to SEQ ID NO:7 and a VL region 100% identical to SEQ ID NO:15; or a VH region 100% identical to SEQ ID NO:8 and a VL region 100% identical to SEQ ID NO:16. The FLT3LG-binding proteins described herein may comprise a VH region 100% identical to SEQ ID NO:5 and a VL region 100% identical to SEQ ID NO:13. The FLT3LG-binding proteins described herein may comprise a VH region 100% identical to SEQ ID NO:6 and a VL region 100% identical to SEQ ID NO:14. The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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.

[0092] The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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.

[0093] The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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. The FLT3LG-binding proteins 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.

[0094] The FLT3LG-binding proteins provided herein may comprise sequences that are variant amino acid sequences. The nucleic acid sequences of the FLT3LG-binding proteins provided herein may comprise variant nucleic acid sequences. The variant nucleic acid sequences herein may be those of the FLT3LG-binding proteins provided herein or variants thereof.

[0095] The VH or VL (or HC or LC) sequence may be a variant sequence of the VH or VL (or HC or LC) sequence provided herein, having up to 10 amino acid substitutions, additions, or deletions. Such variant sequences may have 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions.

[0096] The HC sequence may be a variant sequence of the HC sequence provided herein, having up to 40 amino acid substitutions, additions, or deletions. The 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. The HC variant sequence may have 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions.

[0097] The LC sequence may be a variant sequence of the LC sequence provided herein, having up to 20 amino acid substitutions, additions, or deletions. The LC variant sequence may have up to 15, up to 10, or up to 5 amino acid substitutions, additions, or deletions. The LC variant sequence may have 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions.

[0098] The sequence mutations may eliminate one or more, or all, of the CDRs. For example, the CDR portions of the VH or VL (or HC or LC) sequence may not contain sequence mutations, or the mutations may be present in the non-CDR portions of the VH or VL (or HC or LC) sequence, i.e., such that the CDR sequences are intact.

[0099] The mutations can be substitutions, eg, conservative substitutions, eg, as provided in Table 2a or Table 2b.

[0100] An antigen-binding protein having a variant sequence can substantially retain the biological characteristics of an unmodified antigen-binding protein, such as inhibiting the binding of FLT3LG to FLT3. The binding characteristics (e.g., KD, Kd, ​​or Ka) of an FLT3LG-binding protein having a variant sequence can be substantially identical to those of an unmodified FLT3LG-binding protein. The binding characteristics (e.g., KD, Kd, ​​or Ka) of the variant sequence can be at least 75%, at least 90%, at least 95%, or at least 99% identical to those of an unmodified FLT3LG-binding protein.

[0101] Post-translational modifications may occur during the production of antigen-binding proteins, such as antibodies, in host cells. For example, post-translational modifications may include cleavage of one or more leader sequences, addition of one or more sugar moieties, such as in glycosylation patterns, non-enzymatic glycosylation, deamidation, oxidation, disulfide bond scrambling and other cysteine ​​variants, such as those containing free sulfhydryls, racemization of disulfide, thioether, and trisulfide bonds, isomerization, C-terminal lysine clipping, and N-terminal glutamine cyclization. The disclosure herein encompasses the use of antigen-binding proteins that have been subjected to or have undergone one or more post-translational modifications. Therefore, the terms "antigen-binding protein" and "antibody" herein may include "antigen-binding proteins" or "antibodies," respectively, that have undergone post-translational modifications, such as those described herein.

[0102] Post-translational modifications can include glycosylation, which is a post-translational, non-enzymatic chemical reaction between reducing sugars, such as glucose, and free amino groups in proteins, typically observed at the lysine side chains or N-terminal epsilon amine of antigen-binding proteins. Glycation can occur during production and storage only in the presence of reducing sugars.

[0103] Post-translational modifications can include deamidation, which can occur during production and storage and is an enzymatic reaction that primarily converts asparagine (N) to isoaspartic acid (isoaspartate) and aspartic acid (aspartate) (D) in an approximately 3:1 ratio. This deamidation reaction can therefore be associated with the isomerization of aspartate (D) to isoaspartate. Both the deamidation of asparagine and the isomerization of aspartate involve the intermediate succinimide. To a much lesser extent, deamidation can occur in a similar manner at glutamine residues. Deamidation can occur, for example, in the CDR, Fab (non-CDR region), or Fc region of an antigen-binding protein.

[0104] Post-translational modifications can include oxidation, which can occur during production and storage (i.e., under oxidative conditions) and can result in covalent modifications of proteins 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 can also occur at tryptophan and free cysteine ​​residues. Oxidation can occur in the CDR, Fab (non-CDR) region, or Fc region of an antigen-binding protein.

[0105] Post-translational modifications can include disulfide bond scrambling, which can occur during production and basic storage conditions. One or more disulfide bonds can be broken or formed incorrectly, resulting in one or more unpaired cysteine ​​residues (-SH). These free (unpaired) sulfhydryls (-SH) can facilitate shuffling in antigen-binding proteins.

[0106] Post-translational modifications can include thioether formation and disulfide bond racemization, which can occur under basic conditions during production or storage by beta-elimination of disulfide bridges back to cysteine ​​residues via dehydroalanine and a persulfide intermediate. Subsequent cross-linking of dehydroalanine and cysteine ​​can result in the formation of a thioether bond, or free cysteine ​​residues can rearrange disulfide bonds with a mixture of D- and L-cysteines in antigen-binding proteins.

[0107] The trisulfide results from the insertion of a sulfur atom into a disulfide bond (Cys-SSS-Cys) and is formed in the production cell culture due to the presence of hydrogen sulfide.

[0108] N-terminal glutamine (Q) or glutamate (glutamic acid) (E) in the heavy and / or light chains may potentially form pyroglutamate (pGlu) via cyclization. Most pGlu formation occurs in the production bioreactor, but it may also form non-enzymatically depending on the pH and temperature of processing and storage conditions. Cyclization of the N-terminal Q or E is commonly observed in native human antibodies.

[0109] C-terminal lysine clipping is an enzymatic reaction catalyzed by carboxypeptidase and is commonly observed in recombinant and natural human antibodies. A variant of this process involves the removal of lysines from one or both heavy chains due to cellular enzymes from recombinant host cells. Administration to a subject or human patient may result in the removal of any remaining C-terminal lysines.

[0110] Production method Antigen-binding proteins can be prepared by any of a number of conventional techniques. For example, an antigen-binding protein can 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 it can be produced in a recombinant expression system. Two or more antigen-binding proteins can be expressed (and thus purified) from such natural cells or recombinant expression systems.

[0111] Many different expression systems and purification regimes can be used to produce antigen-binding proteins. Generally, host cells are transformed with a recombinant expression vector encoding the antigen-binding protein. Depending on the expression system, the expression vector may be maintained by the host as a separate genetic element or integrated into the host chromosome. A wide range of host cells can be used, including prokaryotes (including gram-negative or gram-positive bacteria, e.g., Escherichia coli, Bacillus species, Pseudomonas species, Corynebacterium species), eukaryotes, including yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris), fungi (e.g., Aspergillus species), or higher eukaryotes, including insect cells and cell lines of mammalian origin (e.g., CHO, NS0, PER.C6, HEK293, HeLa).

[0112] The host cell may be an isolated host cell. The host cell is not usually part of a multicellular organism (e.g., a plant or an animal). For example, the host cell may be a unicellular organism or an individual cell of a multicellular organism that is separated from that organism. The host cell may be part of a multicellular organism, such as a plant or an animal. The host cell may be a non-human host cell.

[0113] Suitable cloning and expression vectors can be designed or engineered for use in bacterial, fungal, yeast, and mammalian host cells. Commercially available vectors can be obtained and engineered to become vectors for expressing the FLT3LG-binding proteins provided herein.

[0114] The cells of the expression system can be cultured under conditions that promote expression of the antigen-binding protein using one or more suitable devices, including shaker flasks, spinner flasks, and bioreactors. The antigen-binding protein can be recovered by conventional protein purification procedures or modifications thereof. Protein purification procedures can consist of a series of unit operations, including 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 can be formulated into a pharmaceutically acceptable composition.

[0115] Fc engineering methods can be applied to modify the functional or pharmacokinetic properties of antigen-binding proteins containing an Fc region, such as antibodies. Binding to Fcγ can promote ADCC activity, and therefore, ADCC activity can be altered by making mutations in the Fc region that can increase or decrease binding to the Fcγ receptor. Binding to C1q can promote CDC activity, and therefore, CDC activity can be altered by making mutations in the Fc region that can increase or decrease binding to the C1q receptor. Modifications to the glycosylation pattern of antigen-binding proteins can also be made to alter effector function. The in vivo half-life of antigen-binding proteins can be altered by making mutations that affect binding of the Fc region to FcRn (fetal Fc receptor).

[0116] The term "effector function" as used herein refers to one or more antigen-binding protein (e.g., antibody)-mediated effects, including, but not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-mediated complement activation, including complement-dependent cytotoxicity (CDC), complement-dependent cell-mediated phagocytosis (CDCP), antibody-dependent complement-mediated cytolysis (ADCML), and Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis (ADCP).

[0117] Interactions between antigen-binding proteins containing an Fc region, such as the Fc region of an antibody, and various Fc receptors (FcRs), including FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), FcRn, C1q, and type II Fc receptors, can mediate the effector functions of the antigen-binding protein. Significant biological effects can be the result of effector functionality. The ability to mediate effector functions can require binding of the antigen-binding protein to an antigen. An antigen-binding protein can mediate one, multiple, or each effector function.

[0118] Effector function can be assessed in a number of ways, including, for example, by assessing ADCC effector function of antibodies that coat cells mediated by natural killer (NK) cells via FcγRIII or by monocytes / macrophages via FcγRI, or by assessing CDC effector function of antigen binding proteins that coat cells mediated by the complement cascade via C1q. For example, the antigen binding proteins described herein can be assessed for ADCC effector function in a natural killer cell assay.

[0119] The effect of mutations, including mutations in the Fc region, on effector function (including but not limited to, FcRn binding, FcγRs and C1q binding, CDC, ADCML, ADCC, and / or ADCP) can be assessed. The antigen binding protein can contain one or more of such mutations.

[0120] Some isotypes of the human constant regions of the antigen-binding proteins provided herein, particularly IgG4 and IgG2 isotypes, may partially or completely lack a) classical pathway complement activation and b) ADCC functions. Various modifications to the heavy chain constant region of the antigen-binding protein may be made to alter effector function depending on the desired effector properties. IgG1 constant regions containing specific mutations that reduce binding to Fc receptors and reduce effector functions, such as ADCC and CDC, have been described.

[0121] Provided herein are antigen-binding proteins comprising a constant region such that the antigen-binding protein has reduced effector function, for example, reduced ADCC and / or CDC. The heavy chain constant region may comprise a non-naturally functional constant region of an IgG2 or IgG4 isotype, or a mutated IgG1 constant region. Non-limiting examples of suitable modifications are described in European Patent No. 0307434. For example, the constant region may comprise alanine substitutions at positions 235 and 237 (EU index numbering), i.e., L235A and G237A (commonly referred to as "LAGA" mutations). Another example is alanine substitutions at positions 234 and 235 (EU index numbering), i.e., L234A and L235A (commonly referred to as "LALA" mutations). Additional examples can include substitutions 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 those described in EP 2691417 and U.S. Pat. No. 8,969,526, can include a substitution with glycine or alanine at position 329 (i.e., P329G or P329R) in combination with a LALA mutation (EU index numbering) for Fc regions, including IgG1 Fc regions. Yet further examples can include a substitution with glycine or alanine at position 329 (i.e., P329G or P329R) in combination with a proline at position 228 and a glutamic acid at position 235 (i.e., S228P and L235E) for Fc domains, including IgG4 Fc regions (EU index numbering).

[0122] Other mutations that can be used to reduce effector function can include (with respect to IgG1 unless otherwise stated): aglycosylation N297A or N297Q or N297G; L235E; IgG4: F234A / L235A; or chimeric IgG2 / IgG4. IgG2 containing H268Q / V309L / A330S / P331S or V234A / G237A / P238S / H268A / V309L / A330S / P331S substitutions can be used to reduce FcγR and / or C1q binding.

[0123] Other mutations that can be used to reduce effector function include L234F / L235E / P331S; a chimeric antibody created using CH1 and hinge regions from human IgG2 and CH2 and CH3 regions from human IgG4; IgG2m4, based on the IgG2 isotype with four key amino acid residue changes from IgG4 (H268Q, V309L, A330S, and P331S); IgG2σ, which contains V234A / G237A / P238S / H268A / V309L / A330S / P331S substitutions to eliminate affinity for Fcγ receptors and the C1q complement protein; IgG4(S228P / L234A / L235A); huIgG1 L234A / L235A(AA); huIgG4 These may include S228P / L234A / L235A; IgG1s (L234A / L235A / G237A / P238S / H268A / A330S / P331S); IgG4s1 (S228P / F234A / L235A / G237A / P238S); and IgG4s2 (S228P / F234A / L235A / DG236 / G237A / P238S, where D represents a deletion) (Tam et al., Antibodies 2017, Vol. 6(3)).

[0124] The FLT3LG-binding proteins described herein may comprise an IgG1 constant region comprising an N297G mutation.The FLT3LG-binding proteins described herein may comprise an Fc region comprising an N297G mutation.The FLT3LG-binding proteins described herein may comprise an Fc region comprising SEQ ID NO:41.

[0125] Half-life (t 1 / 2 ) 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 the determined serum concentration achieved after administration). The serum half-life of a protein can be measured by pharmacokinetic studies, for example, according to a method in which a radiolabeled protein is intravenously injected into mice and its plasma concentration is measured periodically as a function of time, for example, from about 3 minutes to about 72 hours after injection. Other methods for pharmacokinetic analysis and determination of the half-life of a molecule can be envisioned by those skilled in the art.

[0126] The long half-life of IgG antibodies may depend on their binding to FcRn.Therefore, for example, by manipulating the constant region, substitutions can be used to increase the binding affinity of IgG to FcRn at pH 6.0 while maintaining the pH dependency of the interaction with the target.The in vivo half-life of the antigen-binding proteins described herein can be altered, for example, by modifying the heavy chain constant domain or by modifying their FcRn-binding domain.

[0127] In adult mammals, FcRn, also known as the fetal Fc receptor, plays an important role in maintaining serum antibody levels by acting as a protective receptor that binds to antibodies of the IgG isotype and rescues them from degradation. IgG molecules are endocytosed by endothelial cells, and upon binding to FcRn, are recycled from the extracellular space back into the circulation. In contrast, IgG molecules that enter cells and do not bind to FcRn are targeted to the lysosomal pathway where they are degraded.

[0128] FcRn can be involved in both antibody clearance and transcytosis across tissues.Human IgG1 residues determined to directly interact with human FcRn include Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435.Mutations at any of these positions can be used in the antigen-binding proteins herein to, for example, increase the serum half-life and / or alter the effector properties of the antigen-binding proteins provided herein.

[0129] The 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 that may include reducing the amount and / or frequency of dosing of these molecules. The antigen-binding proteins may comprise all or a portion of an IgG constant domain (the FcRn-binding portion) with one or more of the following amino acid modifications:

[0130] For example, for IgG1, M252Y / S254T / T256E (commonly referred to as the "YTE" mutation) and M428L / N434S (commonly referred to as the "LS" mutation) can increase FcRn binding at pH 6.0. The YTE or LS modifications can increase FcRn binding at pH above or below 6.0.

[0131] Half-life can also be increased by T250Q / M428L, V259I / V308F / M428L, N434A, and T307A / E380A / N434A mutations (with respect to IgG1 and Kabat numbering) in the antigen binding proteins provided herein.

[0132] Half-life and FcRn binding can also be increased by introducing H433K and N434F mutations (commonly referred to as "HN" or "NHance" mutations) (for IgG1) (WO 2006 / 130834) into the antigen-binding proteins provided herein.

[0133] The antigen binding proteins provided herein can comprise a variant Fc region with altered FcRn binding affinity, which can include 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 (EU index numbering) of the Fc region.

[0134] The modified IgG comprising an IgG constant domain of the 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 relative to the half-life of an IgG having a wild-type IgG constant domain, and 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.

[0135] Alanine scanning mutagenesis can be used to alter residues in the Fc region of the antigen-binding proteins provided herein, such as human IgG1 antibodies, thereby altering binding to human FcRn. Positions that can effectively abolish FcRn binding when changed to alanine include I253, S254, H435, and Y436. Other positions that can result in less significant reductions in binding when mutated include, for example, E233-G236, R255, K288, L309, S415, and H433. Some amino acid positions can show improved FcRn binding when changed to alanine, including, among others, P238, T256, E272, V305, T307, Q311, D312, K317, D376, E380, E382, S424, and N434. Other amino acid positions may show slight improvement in FcRn binding (D265, N286, V303, K360, Q362, and A378) or no change (S239, K246, K248, D249, M252, E258, T260, S267, H268, S269, D270, K274, N276, Y278, D280, V282, E283, H285, T289, K290, R292, E293, E294, Q295, Y296, N297, S298, R301, N315, E318, K320, K322, S323 ...0, K322, S323, R301, N315, E318, K 324, K326, A327, P329, P331, E333, K334, T335, S337, K338, K340, Q342, R344, E345, Q345, Q347, R356, M358, T359, K360, N361, Y373, S375, S383, N384, Q386, E388, N389, N390, K392, L398, S400, D401, K414, R416, Q418, Q419, N421, V422, E430, T437, K439, S440, S442, S444, and K447).

[0136] In some of the antigen-binding proteins provided herein, combination variants can confer significant effects in terms of improved FcRn binding. For example, at least at pH 6.0, the E380A / N434A variant can show more than an 8-fold increase in binding to FcRn compared to wild-type IgG1, compared to 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 compared to wild-type IgG1. The antigen-binding proteins described herein can contain E380A / N434A or E380A / N434A / T307A mutations and can have increased binding to FcRn.

[0137] In some of the antigen-binding proteins provided herein, improved stability of the IgG1-human FcRn complex can occur when substituting residues located in the band spanning the Fc-FcRn interference (e.g., M252, S254, T256, H433, N434, and Y436) or surrounding residues (e.g., V308, L309, Q311, G385, Q386, P387, and N389). The M252Y / S254T / T256E ("YTE") and H433K / N434F / Y436H mutations can be combined to confer high affinity for human FcRn. In some cases, such a combination can show a 57-fold increase in affinity compared to wild-type IgG1. The in vivo behavior of such mutated human IgG1 can show up to at least a four-fold increase in serum half-life compared to wild-type IgG1. Such increased serum half-life may be present in the serum of a human, a cynomolgus monkey, or another subject.

[0138] Antigen binding proteins with optimized binding to FcRn are also provided. The antigen binding protein can comprise at least one amino acid modification in the Fc region of the antigen binding protein, for example, the modification can comprise one of the following amino acids: 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, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383 , 342, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, ​​384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 4 and at an amino acid position selected from the group consisting of 00, 401, 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446, and 447. The FLT3LG-binding protein can have two, three, four, five, six, or more of such amino acid modifications in the Fc region of the FLT3LG-binding protein. The FLT3LG-binding protein can have an amino acid modification in the Fc region of the FLT3LG-binding protein at another amino acid position instead of, or in addition to, the amino acid modification provided herein.

[0139] FLT3LG-binding proteins can have an altered half-life by introducing an FcRn-binding polypeptide into the antigen-binding protein (e.g., as in WO 97 / 43316, U.S. Pat. No. 5,869,046, U.S. Pat. No. 5,747,035, WO 96 / 32478, or WO 91 / 14438), by fusing the antigen-binding protein to an antibody with preserved FcRn-binding affinity but greatly reduced affinity for other Fc receptors (e.g., as in WO 99 / 43713), or by fusing the antigen-binding protein to the FcRn-binding domain of an antibody (e.g., as in WO 00 / 09560 or U.S. Pat. No. 4,703,039).

[0140] The antigen-binding proteins herein may include Fc variants with enhanced FcRn affinity, which may improve antibody cytotoxicity and / or half-life at pH 6.0. Such IgG variants may be produced as hypofucosylated molecules. The resulting variants may result in increased serum persistence in hFcRn mice as well as preserved enhanced ADCC. Exemplary variants can include (with respect to IgG1 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 / A3 30V / 382V / N389T / N434Y;T256N / A378V / S383N / N434Y;N315D / A330V / N361D / A387 V / N434Y;V259I / N315D / M428L / N434Y;P230S / N315D / M428L / N434Y;F241L / V264E / T307P / A378V / H433R; T250A / N389K / N434Y; V305A / N315D / A330V / P395A / N434Y; V264E / Q386R / P396L / N434S / K439R; or E294del / T307P / N434Y (where "del" indicates a deletion).

[0141] Substitutions in the constant region can significantly improve the function of antigen-binding proteins, such as therapeutic IgG antibodies, but substitutions in strictly conserved constant regions can confer immunogenicity in humans, while substitutions in highly diverse variable region sequences may be less immunogenic. The CDR residues of the antigen-binding proteins provided herein can be engineered to improve the binding affinity of the antigen-binding proteins to antigens (e.g., FLT3LG). CDR and / or framework residues can be engineered to improve the stability of the antigen-binding proteins provided herein and reduce their immunogenicity risk. Improved affinity for antigens (e.g., FLT3LG) can be achieved by affinity maturation using phage or ribosome display of randomized libraries.

[0142] Improved stability of the antigen-binding proteins provided herein can be rationally obtained through sequence- or structure-based rational design. Reducing the immunogenicity risk (deimmunization) of an antigen-binding protein can be achieved, for example, by one or more humanization methodologies and / or removing potential T cell epitopes, which in some cases can be predicted using in silico techniques or anticipated by in vitro assays. Additionally, the variable region of an antigen-binding protein can be engineered to lower the isoelectric point (pI) of the antibody. A longer half-life for an antigen-binding protein can be associated with such a reduced pI compared to a wild-type antigen-binding protein, despite comparable FcRn binding in some cases. Similar increases 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 the half-life of the antigen-binding protein and / or antigen (e.g., FLT3LG). For example, the half-life of an IgG2 antibody can be shortened if antigen-mediated clearance mechanisms can degrade the antibody when bound to the antigen. Similarly, antigen:antibody complexes can affect the half-life of antigens (e.g., FLT3LG) by, for example, extending the half-life by protecting the antigen from typical degradation processes or by shortening the half-life through antibody-mediated degradation (e.g., target-mediated drug disposal). FLT3LG-binding proteins may have higher affinity for antigens at pH 7.4 compared to endosomal pH (i.e., pH 5.5-6.0), such that the KD ratio at pH 5.5 / pH 7.4 or pH 6.0 / pH 7.4 may be 2 or greater. For example, to enhance the pharmacokinetic (PK) and pharmacodynamic (PD) properties of an antigen-binding protein, pH-sensitive binding to the antigen-binding protein can be achieved by introducing one or more histidine residues into one or more CDRs.

[0143] The antigen-binding protein herein can include a recycling antibody engineered so that a single antibody molecule can bind to an antigen multiple times. Recycling antibodies can dissociate from antigens (e.g., FLT3LG) under intracellular acidic conditions. Antibodies bound to membrane-bound antigens can dissociate from the antigen in a pH-dependent manner. The dissociated antibody can then be recycled by FcRn, while the antigen is imported into lysosomes and degraded. This mechanism can allow antibodies to repeatedly bind to other antigens in plasma, reducing antibody clearance.

[0144] Antigen-binding proteins can include sweeping antibodies, which can be engineered, for example, using a combination of variable region engineering (as described above under "pH switch") to enable the antibody to bind to an antigen (e.g., FLT3LG) in plasma and dissociate from the antigen in endosomes (after the antigen undergoes lysosomal degradation), and constant region engineering to increase endosomal-mediated (e.g., FcRn-mediated) cellular uptake of the antibody-antigen complex to FcγRIIb or potentially other surface receptors. Sweeping antibodies can therapeutically target soluble antigens (e.g., FLT3LG) and enhance clearance of the antigen from circulation. In some cases, one or more of a panel of Fc variants with enhanced binding to FcRn, including M252Y, V308P, or N434Y, which have enhanced binding to FcRn in combination with pH-dependent binding to the target antigen (e.g., FLT3LG), can enhance clearance of the target antigen (e.g., FLT3LG) compared to a wild-type Fc region. FcγRIIb can be used to accelerate the uptake rate of antibody-antigen complexes into cells. The FLT3LG-binding protein can comprise an FcγRIIb-sweeping antibody or its Fc region, and the Fc region of a pH-dependent antibody can be engineered to selectively increase human FcγRIIb binding to enhance the uptake rate of antibody-antigen complexes. This inhibitory receptor can mediate the uptake of antibody-antigen complexes into liver endothelial cells (LSECs). Therefore, mediation of the uptake of antigen-binding proteins (e.g., antibodies):antigen complexes into cells by FcγRIIb (e.g., human FcγRIIb) can reduce the concentration of antigens (e.g., FLT3LG) in the circulation. The FLT3LG-binding protein can comprise an Fc variant (v12) containing the following mutations: E233D / G237D / P238D / H268D / P271G / A330R, which can have selectively increased binding affinity to human FcγRIIb. In some cases, such v12 variants can accelerate the clearance of antigens (e.g., FLT3LG) over that of pH-dependent antibodies with wild-type hIgG1 while maintaining comparable pharmacokinetics.

[0145] Pharmaceutical Composition Pharmaceutical compositions are provided herein, wherein the pharmaceutical compositions may comprise the antigen-binding proteins provided herein. The pharmaceutical compositions herein may be for use in treating diseases, including human diseases, as described herein. The pharmaceutical compositions may comprise the antigen-binding proteins, optionally in combination with one or more pharmaceutically acceptable carriers and / or excipients.

[0146] Such compositions may include, for example, a pharmaceutically acceptable carrier as known and required by current pharmaceutical practice.

[0147] The pharmaceutical composition may be administered by injection or continuous infusion via a route that may include, for example, intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, intraocular, intraportal, or another route. The pharmaceutical composition may be suitable for intravenous administration. The pharmaceutical composition may be suitable for subcutaneous administration. The pharmaceutical composition may be suitable for topical administration (which may include, but is not limited to, epicutaneous, intranasal, or ocular administration), inhalation administration, or enteral administration (which may include, but is not limited to, oral, vaginal, or rectal administration).

[0148] The pharmaceutical compositions provided herein can contain an effective amount of an antigen-binding protein, for example, a FLT3LG-binding protein. The pharmaceutical composition can contain 0.5 mg to 10 g of FLT3LG-binding protein, and in some cases, 5 mg to 1 g of FLT3LG-binding protein. The pharmaceutical composition can contain 5 mg to 500 mg of FLT3LG-binding protein, and in some cases, 5 mg to 50 mg of FLT3LG-binding protein. The pharmaceutical composition can contain 0.5 mg to 10 g, 5 mg to 1 g, 5 mg to 500 mg, or 5 mg to 50 mg of a FLT3LG-binding protein comprising an HC comprising SEQ ID NO: 47 and an LC comprising SEQ ID NO: 55. The pharmaceutical composition can contain 0.5 mg to 10 g, 5 mg to 1 g, 5 mg to 500 mg, or 5 mg to 50 mg of a FLT3LG-binding protein comprising an HC comprising SEQ ID NO: 48 and an LC comprising SEQ ID NO: 56. The pharmaceutical composition may contain 0.5 mg to 10 g, 5 mg to 1 g, 5 mg to 500 mg, or 5 mg to 50 mg of a FLT3LG-binding protein comprising a HC comprising SEQ ID NO: 49 and a LC comprising SEQ ID NO: 57. The pharmaceutical composition may contain 0.5 mg to 10 g, 5 mg to 1 g, 5 mg to 500 mg, or 5 mg to 50 mg of a FLT3LG-binding protein comprising a HC comprising SEQ ID NO: 50 and a LC comprising SEQ ID NO: 58. The pharmaceutical composition may contain 0.5 mg to 10 g, 5 mg to 1 g, 5 mg to 500 mg, or 5 mg to 50 mg of a FLT3LG-binding protein comprising a HC comprising SEQ ID NO: 63 and a LC comprising SEQ ID NO: 71. The pharmaceutical composition may contain 0.5 mg to 10 g, 5 mg to 1 g, 5 mg to 500 mg, or 5 mg to 50 mg of a FLT3LG-binding protein comprising a HC comprising SEQ ID NO: 64 and a LC comprising SEQ ID NO: 72. The pharmaceutical composition may contain 0.5 mg to 10 g, 5 mg to 1 g, 5 mg to 500 mg, or 5 mg to 50 mg of an FLT3LG binding protein comprising an HC comprising SEQ ID NO: 65 and an LC comprising SEQ ID NO: 73.The pharmaceutical composition may contain 0.5 mg to 10 g, 5 mg to 1 g, 5 mg to 500 mg, or 5 mg to 50 mg of a FLT3LG-binding protein comprising an HC comprising SEQ ID NO: 66 and an LC comprising SEQ ID NO: 74. The pharmaceutical composition may contain 0.5 mg to 10 g, 5 mg to 1 g, 5 mg to 500 mg, or 5 mg to 50 mg of each of two or more FLT3LG-binding proteins provided herein.

[0149] The pharmaceutical composition may be included in a kit containing the antigen-binding protein together with other pharmaceuticals and / or instructions for use. For convenience, the kit may contain predetermined amounts of reagents and instructions for use. The kit may also include one or more devices, such as a syringe, needle, length of tubing, or other device, that can be used to administer the pharmaceutical composition.

[0150] The terms "individual," "subject," and "patient" are used interchangeably herein. A subject may be an animal. A subject may be a mammal, e.g., a primate, e.g., a marmoset or a monkey. A subject may be a human.

[0151] The antigen-binding proteins described herein may also be used in treatment methods. Those skilled in the art will recognize that the reference herein to treatment refers to the treatment of established conditions. However, the antigen-binding proteins disclosed herein may also be useful in the prevention of certain diseases, depending on the condition. The antigen-binding proteins described herein are used in an effective amount for therapeutic, preventative, or preventive treatment. A therapeutically effective amount of the antigen-binding proteins described herein is an amount effective to ameliorate or reduce one or more symptoms of a disease, or to prevent or cure a disease.

[0152] Provided herein is a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a FLT3LG-binding protein or pharmaceutical composition as defined herein.The subject may be an animal or a human.The subject may be a human.

[0153] The FLT3LG-binding proteins described herein are provided for use in therapy.FLT3LG-binding proteins are provided for use in the treatment of autoimmune diseases.FLT3LG-binding proteins are provided for use in the treatment of autoimmune diseases, the FLT3LG-binding proteins comprising a VH region comprising SEQ ID NO:5 and a VL region comprising SEQ ID NO:13.FLT3LG-binding proteins are provided for use in the treatment of autoimmune diseases, the FLT3LG-binding proteins comprising a VH region comprising SEQ ID NO:6 and a VL region comprising SEQ ID NO:14.FLT3LG-binding proteins are provided for use in the treatment of autoimmune diseases, the FLT3LG-binding proteins comprising a VH region comprising SEQ ID NO:7 and a VL region comprising SEQ ID NO:15.FLT3LG-binding proteins are provided for use in the treatment of autoimmune diseases, the FLT3LG-binding proteins comprising 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 an 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 an 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 an 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 an 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. Use of a FLT3LG-binding protein in the manufacture of a medicament for the treatment of an autoimmune disease is also provided. Also provided is the use of an FLT3LG-binding protein in the manufacture of a medicament for the treatment of an 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 an 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. Also provided is the use of a FLT3LG-binding protein in the manufacture of a medicament for the treatment of an 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 an 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.

[0154] Also provided is a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the FLT3LG-binding protein or pharmaceutical composition described herein. Also provided is a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a FLT3LG-binding protein, 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 a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the 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 treating an autoimmune disease in a subject in need thereof, comprising administering to the 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. Also provided is a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the 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 can 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, hypertension, atherosclerosis, Alzheimer's disease, cancer, and associated complications of such diseases.Additional contemplated diseases that may be treated include autoimmune diseases, including but not limited to the NIH list of autoimmune diseases, including but not limited to alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes, glomerulonephritis (e.g., IgA nephropathy), granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, juvenile idiopathic arthritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjogren's syndrome, systemic lupus erythematosus (SLE), thyroiditis, uveitis, and vitiligo. Additional autoimmune diseases that may be treated include, but are not limited to, autoimmune neonatal thrombocytopenia, autoimmune neutropenia, autoimmune cytopenia, antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis, relapsing polychondritis, rheumatic heart disease, neuritis, uveophthalmia, polyendocrinopathy, purpura (e.g., Henoch-Schonlein purpura), Reiter's disease, stiff man syndrome, autoimmune pulmonary inflammation, dense deposit disease, rheumatic heart disease, insulin-dependent diabetes mellitus, and autoimmune ocular inflammation, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis), systemic lupus erythematosus (SLE), discoid lupus, Goodpasture's disease, and other autoimmune disorders. These include: syndromes, pemphigus, receptor autoimmunity such as (a) Graves' disease, (b) myasthenia gravis, and (c) insulin resistance, autoimmune thrombocytopenic purpura, scleroderma with anti-collagen antibodies, mixed connective tissue disease, idiopathic Addison's disease, infertility, bullous pemphigoid, diabetes mellitus, and adrenergic agonist resistance (including adrenergic agonist resistance with asthma or cystic fibrosis), chronic active hepatitis, other endocrine glandular deficiencies, vasculitis, post-MI, open-heart surgery syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, and other inflammatory, granulomatous, degenerative, and atrophic disorders, systemic sclerosis, systemic rheumatic diseases, optic neuritis, myositis, scleroderma, lupus erythematosus, and overlap syndromes. Autoimmune diseases can be acute or chronic and can affect essentially all organs and body systems.Autoimmune diseases can include autoantibodies, diseases with tissue-specific or non-tissue-specific autoantigens, collagen or connective tissue disorders, and collagen vascular diseases. A more extensive list of autoimmune diseases 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).

[0155] The treated autoimmune disease may result from increased differentiation and survival of dendritic cells (DCs) via FLT3LG-induced FLT3 signaling. Thus, the FLT3LG-binding proteins described herein may be effective in treating autoimmune diseases in which DCs enhance autoimmune responses. The FLT3LG-binding proteins described herein may inhibit the binding of soluble FLT3LG to immobilized FLT3 receptors. The half-maximal inhibitory concentration (IC50) of the FLT3LG-binding protein's inhibition of soluble FLT3LG binding to immobilized FLT3 receptors may be determined using a soluble FLT3LG blocking ELISA assay, such as the assay described in Example 3B. In some instances, the FLT3LG-binding protein may inhibit the binding of soluble FLT3LG to immobilized FLT3 receptors 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.

[0156] FLT3LG can exist in both cell surface membrane-bound form and soluble form.The FLT3LG binding protein 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 protein described herein may bind to 293T-hFLT3LG cells with an EC50 of less than or equal to 10nM, less than or equal to 5nM, less than or equal to 1nM, or less than or equal to 0.5nM.

[0157] The FLT3LG binding protein described herein may inhibit the binding of soluble FLT3LG to cell surface FLT3 receptor.The IC50 of the 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 protein described herein may inhibit the binding of soluble FLT3LG to cell surface FLT3 receptor with an IC50 of less than or equal to 2nM, less than or equal to 1.5nM, or less than or equal to 1nM.

[0158] 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 internalization flow cytometry assay, such as 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.

[0159] 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 internalization 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.

[0160] The FLT3LG-binding proteins described herein may inhibit p-AKT signaling induced by the interaction between FLT3LG and FLT3 receptor. The IC50 of FLT3LG-binding protein inhibition of soluble FLT3LG-induced p-AKT signaling in FLT3-expressing cells may be measured using a p-AKT signaling assay, for example, the assay described in Example 5. The FLT3LG-binding proteins described herein may inhibit p-AKT signaling 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.

[0161] 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, such as the assay described in Example 6. The FLT3LG-binding proteins described herein may inhibit soluble FLT3LG-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.

[0162] The FLT3LG-binding proteins described herein may bind to endogenous FLT3LG expressed on human T cells. The IC50 of the FLT3LG-binding protein binding to FLT3LG expressed on T cells may be determined using a flow cytometry assay, such as the assay described in Example 7. The FLT3LG-binding proteins described herein may bind to human T cells with an EC50 of 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.

[0163] The FLT3LG-binding proteins described herein may reduce the number and / or frequency of hFLT3LG-induced DCs in the spleen. The DCs may be cDCs, cDC1s, cDC2s, pDCs, and / or pre-pDCs. The effect of the FLT3LG-binding proteins on the number and / or frequency of DCs may be determined using an in vivo mouse model, such as the in vivo mouse model described in Example 8.

[0164] The FLT3LG-binding protein described herein may form a complex with an FLT3LG dimer. For example, the FLT3LG-binding protein may form a complex of two FLT3LG2:two mAbs. The FLT3LG-binding protein may form a complex with two FLT3LG2:two mAbs, wherein the angle between the Fab arms of the FLT3LG-binding protein 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.

[0165] term Further embodiments of the present invention are set forth below. 1.a.(i) any one or 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 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 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 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 (ii) a CDR variant of (i), wherein the variant has one, two, or three amino acid modifications; or b. a VH region comprising a sequence at least 80% identical to SEQ ID NO: 8 and / or a VL region comprising a sequence at least 80% identical to SEQ ID NO: 16; a VH region comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 5 and / or a VL region comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 13; a VH region comprising a sequence at least 80% identical to SEQ ID NO: 6 and / or a VL region comprising a sequence at least 80% identical to SEQ ID NO: 14; or a VH region comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 7 and / or a VL region comprising a sequence at least 80% identical to the sequence of SEQ ID NO: 15 FLT3LG-binding proteins comprising: The CDR of 2.a.(i) is: 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; 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; 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; or 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 Item 3. The FLT3LG-binding protein according to Item 1, 3. All six CDRs are present in the binding protein; 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 CDRL3 of SEQ ID NO:40; 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 CDRL3 of SEQ ID NO: 31; 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 CDRL3 of SEQ ID NO:34; or 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 Item 3. The FLT3LG-binding protein according to Item 1 or 2, selected from: 4. The following: 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 CDRL3 of SEQ ID NO:40; 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 CDRL3 of SEQ ID NO: 31; 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 CDRL3 of SEQ ID NO:34; or 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 A FLT3LG-binding protein comprising six CDRs selected from: 5. The binding protein is a VH region at least 80% identical to SEQ ID NO:8 and a VL region at least 80% identical to SEQ ID NO:16; a VH region at least 80% identical to SEQ ID NO:5 and a VL region at least 80% identical to SEQ ID NO:13; a VH region that is at least 80% identical to SEQ ID NO:6 and a VL region that is at least 80% identical to SEQ ID NO:14; or A VH region that is at least 80% identical to SEQ ID NO: 7 and a VL region that is at least 80% identical to SEQ ID NO: 15 Item 5. The FLT3LG-binding protein according to Item 4, comprising: 6. The binding protein is a heavy chain (HC) sequence at least 80% identical to SEQ ID NO: 50 and / or a LC sequence at least 80% identical to SEQ ID NO: 58; a heavy chain (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 Item 10. The FLT3LG-binding protein of any one of the preceding items, comprising: 7. The FLT3LG binding protein of any one of the preceding clauses, wherein the binding protein binds to human FLT3LG with a KD of less than or equal to 1 nM. 8. The FLT3LG binding protein of any one of the preceding clauses, wherein the binding protein inhibits soluble FLT3LG-induced p-AKT signaling with an IC50 of about 2 nM or less. 9. The FLT3LG binding protein of any one of the preceding clauses, wherein the binding protein inhibits soluble FLT3LG-induced FLT3 receptor internalization with an IC50 of about 1 nM or less. 10. 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. 11. The FLT3LG-binding protein of any one of the preceding clauses, wherein the binding protein is an antibody or an antigen-binding fragment thereof. 12. The FLT3LG-binding protein according to item 11, wherein the antibody or antigen-binding fragment thereof inhibits the binding of human FLT3LG to FLT3. 13. The FLT3LG-binding protein according to item 12, wherein the antibody or antigen-binding fragment thereof inhibits the binding of human soluble FLT3LG to FLT3 with an IC50 of about 2 nM or less. 14. The FLT3LG-binding protein according to any one of items 11 to 13, wherein the antibody or antigen-binding fragment thereof comprises a modified Fc region. 15. The FLT3LG binding protein of paragraph 14, wherein the modified Fc region comprises the amino acid substitution N297G (as numbered according to the EU index). 16. A nucleic acid sequence encoding one or both of the HC and LC of a FLT3LG binding protein as defined in any one of the preceding clauses. 17. The nucleic acid sequence of paragraph 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. 18. An expression vector comprising a nucleic acid sequence as defined in paragraph 16 or 17. 19. A recombinant host cell comprising a nucleic acid sequence as defined in paragraph 16 or 17, or an expression vector as defined in paragraph 18. 20. A method for producing an FLT3LG-binding protein, comprising culturing a host cell as defined in paragraph 19 under conditions suitable for expression of the nucleic acid sequence or expression vector, thereby producing a polypeptide comprising the FLT3LG-binding protein. 21. An FLT3LG-binding protein produced by the method according to item 20. 22. A cell line engineered to express the FLT3LG-binding protein according to any one of items 1 to 15 or 21. 23. A pharmaceutical composition comprising an FLT3LG-binding protein as defined in any one of items 1 to 15 or 21, and a pharmaceutically acceptable excipient. 24. A method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an FLT3LG-binding protein defined in any one of paragraphs 1 to 15 or 21, or a pharmaceutical composition defined in paragraph 23. 25. The method according to item 24, wherein the autoimmune disease is systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), sarcoidosis, Sjogren's syndrome, or celiac disease. 26. The method according to paragraph 25, wherein the autoimmune disease is SLE. 27. The method of paragraph 24 or 25, wherein the subject is a human. 28. A FLT3LG binding protein as defined in any one of clauses 1 to 15 or 21, or a pharmaceutical composition as defined in clause 23, for use in therapy. 29. An FLT3LG-binding protein as defined in any one of paragraphs 1 to 15 or 21, or a pharmaceutical composition as defined in paragraph 23, for use in the treatment of an autoimmune disease. 30. The FLT3LG-binding protein according to item 29, wherein the autoimmune disease is systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), sarcoidosis, Sjogren's syndrome, or celiac disease. [Example]

[0166] Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative, not limiting. Those skilled in the art will readily recognize that the specific examples are merely illustrative of the invention, which is more fully described in the claims that follow. It should be understood that all embodiments and features described in this application are interchangeable and combinable with all embodiments contained therein.

[0167] Example 1: Generation and selection of anti-FLT3LG antibodies This example demonstrates the generation of anti-FLT3LG antibodies using B cell cloning technology.

[0168] method A. Immunization Four 7-week-old female BALB / C and Swiss Webster mice were immunized with 50 μg of hFLT3LG protein (R&D Systems, NS0 cell-derived, 308-FKN / CF, herein referred to as R&D). R&D hFLT3LG differs from wild-type soluble hFLT3LG (UniProt accession number P49771.1) in that R&D hFLT3LG has an alanine mutation at position 72, whereas wild-type hFLT3LG has a glycine. Briefly, mice were immunized weekly intraperitoneally and subcutaneously with 200 μL of recombinant hFLT3LG protein mixed with a mixture of Toll-like receptor adjuvants. After four immunizations, serum from the immunized mice was collected and tested for hFLT3LG-specific antibody titers using ELISA.

[0169] BB cell sorting Spleens and lymph nodes were harvested from immunized mice 5 days after the final boost, and single cell suspensions were prepared. B cells were enriched, and the cells were then stained with a fluorophore-labeled antibody panel and hFLT3LG (R&D, NS0 cell-derived, 308-FKN / CF) labeled with Alexa647 and Alexa488 to isolate hFLT3LG-specific B cells. The stained cells were then collected for cell sorting using a SONY MA900 cell sorter. Single dump channel - / CD19 + / IgM - / hFLT3LG duel+ Stained B cells were collected and processed for 10x Genomics running and sequencing according to the manufacturer's instructions.

[0170] C. Single-cell sequencing and sequence analysis The sequencing library was sequenced using an Illumina NextSeq Sequencer in conjunction with a mid-output sequencing kit (Illumina, Cat. No. 20024904). VH and VL sequences were assembled from the sequencing data using Cell ranger software (10x Genomics). The cell ranger output was further analyzed to enable clonal selection using a custom pipeline, where: 1. Data was filtered for cells containing exactly one VH and one VL sequence; 2. Sequences were grouped into clonal profiles based on VH germline, CDRH3 length and CDRH3 sequence identity. 3. A clonal profile consisting of at least five clonal consensus sequences, and the Hamming distance to the consensus sequence for each clone in these clonal profiles was calculated; and 4. Potential sequence trends in CDRs were identified.

[0171] The analysis identified 3272 cells with paired VH and VL sequences belonging to 773 unique clonal populations.

[0172] From the immune repertoire sequence, 124 clones were selected for gene synthesis and small-scale expression based on the following criteria: 1. Larger clonal size, 2. A small Hamming distance to the clonal trait consensus, and 3. The minimum number of potential sequence trends in the CDR sequences.

[0173] D. Small-scale expression and purification of binders 124 selected clones from the hFL3LG B cell sequencing dataset were generated as chimeric human IgG1 / kappa antibodies by DNA synthesis and transcription-activated PCR (TAP). Briefly, antibody variable region DNA with 5'- and 3'-TAP universal sequences was synthesized (IDT DNA). Two rounds of overlapping PCR were used to assemble and amplify the variable region DNA, promoter DNA fragments, and heavy or light chain constant region terminal DNA fragments to generate two separate linear TAP products, one encoding the heavy chain and the other the light chain. PCR products were verified by agarose gel electrophoresis and purified using the EZ 96 Cycle Pure kit (Omega Biotek). Heavy and light chain TAP fragments at a 1:2 ratio were transiently co-transfected into Expi293 cells at a 3 mL scale using the ExpiFectamine™ 293 Transfection Kit (Gibco, catalog number 2401600) according to the manufacturer's instructions. Cells were incubated for 6 days, after which the supernatant was harvested by centrifugation and the expressed antibody was then purified using Protein A chromatography.

[0174] Example 2: Screening and characterization of anti-FLT3LG antibodies This example demonstrates the methods used to screen and select anti-FLT3LG antibodies that effectively block the interaction between FLT3LG and the FLT3 receptor, bind to membrane-bound hFLT3LG, and cross-react with cyno-FLT3LG.

[0175] A. Enzyme-linked immunosorbent assay (ELISA) To detect antibody clones that bind to FLT3LG, ELISA was performed on the produced TAP-expressing chimeric antibodies, which were purified as in Example 1.

[0176] Methods: 96-well microtiter plates were coated overnight with 1 μg / mL of hFLT3LG (R&D, NS0 cell-derived, 308-FKN / CF) or rhesus FLT3LG (LSBIO, catalog number: G5864; NB rhesus-FLT3LG (UniProt entry: F6ZGW9 is identical to cyno-FLT3LG (Genebank entry: XP_005589969)) and then blocked for 2 hours with 300 μL of 1% bovine serum albumin in phosphate-buffered saline with 0.05% Tween 20 (PBS-T). Antibodies (10 μg / mL) were diluted in blocking buffer, and 100 μL was added per well and incubated for 1 hour at room temperature. A 1:5000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-human IgG Fc antibody (Jackson) in PBS-T was then added. ImmunoResearch, Catalog No. 109-035-098) was added at 100 μL / well as a secondary reagent. The ELISA plate was developed using 3,3',5,5'-tetramethylbenzidine (TMB) solution, and the reaction was stopped by adding 50 μL of 2 M H2SO4. The absorbance was read at 450 nm in a VersaMax microplate reader.

[0177] Results: 116 TAP-expressed antibodies from 124 selected B cell clones were tested for binding to FLT3LG. ELISA was used to measure the binding of each of the 116 TAP-expressed antibodies (10 μg / mL) to hFLT3LG (x-axis) and cyno-FLT3LG (y-axis), as shown in Figure 1. All 116 antibodies showed specific binding to hFLT3LG, and 106 of the 116 antibodies also showed cross-binding activity with cyno-FLT3LG (cutoff at 0.5; OD450), as shown in the dashed box in Figure 1.

[0178] B. Flow cytometry (FACS) cell surface binding assay To determine whether the 116 antibodies that showed specific binding to hFLT3LG identified in the ELISA assay also bound to cell surface-expressed FLT3LG, a flow cytometry cell surface binding assay was performed.

[0179] Methods: 293T cells were transduced with lentivirus to stably and transiently express hFLT3LG (sequence NCBI NM_001459.4; UniProt P49771). 0.5 × 10 cells stably expressing hFLT3LG were used. 6 293T cells were incubated with 100 μL of anti-FLT3LG antibody (10 μg / mL) per well for 30 minutes at 4°C. After primary antibody incubation and washing, 100 μL of 1:100 diluted PE-conjugated goat anti-human IgG F(ab')2 (Jackson ImmunoResearch, catalog number: 109-116-097) antibody was added to the cells, and the mixture was incubated for 30 minutes at 4°C. After washing, the cells were incubated in 100 μL of DAPI for 5 minutes for viability staining. The stained cells were fixed with 4% paraformaldehyde (PFA), and the fluorescent staining signal was measured using a CYTOFLEX flow cytometer.

[0180] Results: Figure 2 illustrates the binding of antibodies to hFLT3LG measured by ELISA in Example 2A and to cell surface hFLT3LG on 293T cells. Of the 116 antibodies that showed specific binding to hFLT3LG in the ELISA assay, 112 antibodies also showed binding to membrane-bound hFLT3LG-overexpressing 293T cells. The dashed boxes in Figure 2 highlight antibodies that bind to hFLT3LG measured by ELISA and antibodies that bind to membrane-bound hFLT3LG overexpressed in 293T cells measured by flow cytometry.

[0181] C. Blocking assay FLT3LG binds to the FLT3 receptor with a KD of 2.31E-10 M as measured by SPR. A competitive ELISA assay was performed to detect antibodies that block the binding of soluble hFLT3LG to hFLT3.

[0182] Methods: A 96-well Maxisorp plate was coated overnight at 4°C with 1 μg / mL anti-hFLT3 antibody (R&D, MAB812) in PBS. The plate was blocked for 2 hours with PBS-T blocking buffer. hFLT3 (1 μg / mL) (R&D, 368-ST / CF) was then captured on the plate and probed with premixed biotinylated hFLT3LG (BT-hFLT3LG) (R&D, NS0 cell-derived, 308-FKN / CF) at a predetermined EC50 of 0.65 nM and anti-FLT3LG antibody (100 nM). Bound BT-hFLT3LG was detected with streptavidin-HRP conjugate (THERMO SCIENTIFIC, catalog number 21140) and developed with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. After quenching with 50 μL of 2M H 2 SO 4 , absorbance was read at 450 nm in a VERSAMAX microplate reader.

[0183] Results: 116 hFLT3LG-reactive antibodies were screened for their ability to block the binding of hFLT3LG to hFLT3 in a competitive ELISA assay as provided in Figure 3. Compared to a human IgG1 isotype control antibody, 55 of the 116 antibodies inhibited the binding of hFLT3LG to hFLT3 by more than 50% at an antibody concentration of 100 nM, as indicated by the dashed box in Figure 3.

[0184] D. Cynomolgus monkey cross-reactivity assay Surface plasmon resonance (SPR) analysis was performed to determine the cross-reactivity and binding kinetics of the 55 blocking anti-FLT3LG antibodies identified in Example 2C above to both cyno-FLT3LG and hFLT3LG.

[0185] Methods: Antibody association and dissociation rates were determined by SPR measurements using a BIACORE 8K instrument. Each antibody was captured at 0.5 μg / mL in HBS-P (0.01 M HEPES, 0.15 M NaCl, and 0.05% surfactant P20) running buffer on a Protein A chip (CYTIVA, catalog number: 29127556). To measure binding kinetics, a 3-fold dilution of 100 nM to 11 nM hFLT3LG (R&D, NS0 cell-derived, 308-FKN / CF) or cyno-FLT3LG (LSBIO, catalog number: G5864), along with blank buffer for baseline substrate, was injected at 30 μL / min for 120 seconds, followed by a 10-minute dissociation period. Regeneration of the Protein A surface was achieved with 50 μL / min of 10 mM glycine (pH 1.5) for 30 seconds between each running cycle. Kinetic experiments in this example were performed at 25°C.

[0186] Results: As shown in Figure 4, 52 of the 55 blocking antibodies showed binding to hFLT3LG, 48 antibodies showed cross-binding to cyno-FLT3LG, and showed binding affinity (K D ) ranged from 100 pm to 40 nM.

[0187] E. Down-selection of clones for humanization 124 VH and VL DNA sequence pairs were synthesized, cloned, and expressed in mammalian cells. We found that 116 of the 124 clones gave robust protein expression, with a median yield of approximately 100 μg of purified IgG in 3 mL of culture. From 48 blocking antibodies that showed binding to membrane-bound hFLT3LG protein and to hFLT3LG and cyno-FLT3LG by both ELISA and SPR, 15 clones were selected for humanization based on at least their blocking activity and hFLT3LG and cyno-FLT3LG binding affinity, as shown in Table 4.

[0188] [Table 5]

[0189] Example 3: Humanization and characterization of candidate anti-FLT3LG antibodies The 15 anti-FLT3LG antibodies (b1.118, b1.121, b1.123, b1.126, b1.094, b1.095, b1.078, b1.015, b1.062, b1.017, b1.021, b1.039, b1.038, b1.054, and b1.004) downselected in Example 2 were humanized to produce antibodies against FLT3LG. Fifteen humanized antibodies (h_b1.118, h_b1.121, h_b1.123, h_b1.126, h_b1.094, h_b1.095, h_b1.078, h_b1.015, h_b1.062, h_b1.017, h_b1.021, h_b1.039, h_b1.038, h_b1.054, and h_b1.004) were obtained.

[0190] Selected humanized antibody sequences were synthesized and cloned into mammalian expression vectors containing human IgG1(N297G) heavy chain or kappa light chain constant regions. Expi293 cells were transfected with the vectors for antibody expression and purification.

[0191] A. SPR analysis of humanized anti-FLT3LG antibody To determine the binding kinetics / affinity of the selected 15 recombinant humanized anti-FLT3LG antibody clones, SPR was performed as described below.

[0192] Methods: The association and dissociation rates of a panel of humanized antibodies were determined by SPR using a BIACORE 8K instrument as described above in Example 2D. Kinetic experiments were performed at 25°C or 37°C.

[0193] In addition to the binding formats described above, the binding kinetics of humanized antibodies against two different sources of hFLT3LG (R&D, NS0 cell-derived, 308-FKN / CF, or Acrobiosystems, FLL-H5223 (referred to herein as "Acro") (selected antibodies only)) were determined in a secondary format using a BIACORE 8K instrument. Biotinylated hFLT3LG (BT-hFLT3LG) (0.2 μg / mL) was injected and captured by a streptavidin-coated sensor CAP chip (GE Healthcare, catalog number: 2205463-AB). Each antibody (100 nM in running buffer) was serially diluted 3-fold in running buffer and then injected for 120 seconds at 50 μL / min, followed by a 10-minute dissociation period. The surface was regenerated by injection of regeneration buffer (1 M NaOH).

[0194] Results: The K on , K off , and K D values ​​for the selected 15 humanized antibody clones were measured as described above. The binding kinetics of the 15 humanized antibodies to hFLT3LG at 37°C are listed in Table 5, the binding kinetics of the 15 humanized antibodies to hFLT3LG at 25°C are listed in Table 6, the binding kinetics of the 15 humanized antibodies to cyno-FLT3LG at 25°C are listed in Table 7, and the binding kinetics of the 15 humanized antibodies to hFLT3LG (biotinylated assay format) at 25°C are listed in Table 8 (R&D hFLT3LG) and Table 9 (Acro hFLT3LG) (selected antibodies only). Antibodies with very weak binding to BT-hFLT3LG are indicated with "ND." Antibodies with nonspecific binding to the chip are indicated with "ND." * " is indicated.

[0195] [Table 6]

[0196] [Table 7]

[0197] [Table 8]

[0198] [Table 9]

[0199] [Table 10]

[0200] B. Blocking ELISA of humanized anti-FLT3LG antibody To determine and compare the blocking potency of 15 selected humanized anti-FLT3LG antibodies between soluble hFLT3LG and hFLT3, a competitive ELISA assay was performed.

[0201] Method: A 96-well Maxisorp plate was coated with 1 μg / mL of a commercially available anti-hFLT3 antibody (R&D, catalog number: MAB812) in PBS overnight at 4°C, then blocked with PBS-T blocking buffer for 2 hours. hFLT3 (1 μg / mL) (R&D, catalog number: 368-ST / CF) was then captured on the plate, followed by the addition of 100 μL of pre-incubated biotinylated hFLT3LG (BT-hFLT3LG) (R&D, derived from NS0 cells, 308-FKN / CF) (predetermined EC80 of BT-hFLT3LG: 0.55 nM). Antibodies were tested in 3-fold serial dilutions starting at 100 nM. Bound BT-hFLT3LG was detected with streptavidin poly-HRP conjugate (THERMO SCIENTIFIC, Cat. No. 21140) and developed with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. After quenching with 50 μL of 2 M H2SO4, absorbance was read at 450 nm in a VERSAMAX microplate reader.

[0202] Results: Inhibition curves of titrated humanized anti-FLT3LG antibodies against hFLT3LG binding to the hFLT3 receptor in a competitive ELISA assay are provided in Figure 5, Panel A (all 15 humanized anti-FLT3LG antibodies) and Panel B (a subset of four selected humanized anti-FLT3LG antibodies; each of the 15 antibodies was evaluated for its performance in four different assays: a BIACORE binding assay (Example 3A), a blocking ELISA assay (Example 3B), a receptor internalization assay (Example 4), and a p-AKT signaling assay (Example 5)). One antibody from each of epitope bin A (h_b1.21) and bin D (h_b1.78) and two antibodies from bin C (h_b1.17 and h_b1.126) were selected. A commercially available anti-FLT3LG antibody (R&D, MAB608) was used as a positive control in the experiment.

[0203] All 15 humanized anti-FLT3LG antibodies inhibited the binding of BT-hFLT3LG to plate-captured hFLT3 receptors. The IC50 was calculated for each antibody, and as detailed in Table 10, each of the 15 humanized anti-FLT3LG antibodies exhibited a blocking IC50 ranging from 0.03 nM to 0.28 nM. Most humanized anti-FLT3LG antibodies exhibited IC50s comparable to that of MAB608 (0.04 nM).

[0204] C. Epitope binning of humanized anti-FLT3LG antibodies To determine whether the 15 selected humanized anti-FLT3LG antibodies bind to the same or different epitopes on hFLT3LG, a flow cytometry-based epitope binning assay was performed.

[0205] Methods: A flow cytometry-based assay (Chan et al., SLAS Discov. 2018 Aug:23(7):613-623) was adapted to determine epitope bins of a humanized antibody panel. Briefly, BT-hFLT3LG (R&D, NS0 cell-derived, 308-FKN / CF) / LumAvidin beads (Luminex, Custom Magplex-Avidin Microspheres) were incubated at 5 μg / mL, 1.25 × 10 6 The beads were pre-coated with 15% humanized anti-FLT3LG antibody as a reference antibody at 1.25x10 beads / mL for 45 min at RT. The beads were then washed twice with FACS buffer, pooled, and diluted to a concentration of 1.25x10 6The concentration was adjusted to 1000 total beads / mL. The humanized antibody panel was transferred to a 96-well V-bottom plate at 20 μL / well (final 3.3 μg / mL), followed by the addition of 40 μL of pooled beads (5,000 individual beads / well) and incubation at room temperature for 45 minutes. The beads were then washed twice with FACS buffer, and bound antibodies were detected using 50 μL / well of FITC-conjugated goat anti-human IgG Fc antibody (Jackson ImmunoResearch, catalog number: 109-135-098) at a final concentration of 5 μg / mL for 15 minutes at room temperature. The beads were then washed once with FACS buffer, resuspended in 50 μL, and read on a CYTOFLEX cytometer. Only the pooled beads and reference antibody were included in the assay to allow for calculation of the secondary antibody relative to the reference antibody. The binding of each antibody relative to the reference antibody was determined by subtracting the reference fluorescent signal from the total fluorescent signal (net flow cytometry geometric mean = total geometric mean of panel antibodies (Ab) and reference Ab - geometric mean of reference Ab). This set of data points for each antibody creates its competitive binding profile. The degree of similarity between any two binding profiles was quantified by calculating the correlation coefficient calculated across all binding profile combinations. The correlation values ​​were then clustered to identify antibodies with similar competitive patterns, which were then grouped into epitope bins. Data analysis and plotting were performed using Microsoft Excel and R.

[0206] Results: Competitive binding profiles were compared together for the entire 15 humanized antibody panel. Correlation scores 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 listed in Table 10.

[0207] D. Flow cytometry cell surface binding assay To determine the binding of selected anti-FLT3LG humanized antibodies to cell surface-expressed FLT3LG, a flow cytometry cell surface binding assay was performed.

[0208] Methods: 293T cells stably transfected with hFLT3LG were incubated with 0.5 × 10 anti-FLT3LG antibodies in 100 μL of 3-fold serial dilutions starting at 100 nM. 6 Cells were incubated at 1 cell / well for 30 minutes at 4°C, followed by 100 μL of 1:100 diluted anti-human IgG Fc-APC (Jackson ImmunoResearch, Catalog No. 109-135-098) antibody incubation for 30 minutes at 4°C. After washing, cells were incubated in 100 μL of DAPI for 5 minutes for viability staining. Stained cells were fixed with 4% paraformaldehyde (PFA), and fluorescence data were acquired using a CYTOFLEX flow cytometer.

[0209] Results: Figure 6 shows cell surface hFLT3LG binding of the humanized antibody panel (Figure 6, panel A) and a subset of four selected humanized anti-FLT3LG antibodies (Figure 6, panel B), as determined using flow cytometry. EC50 values ​​for the panel of 15 humanized anti-FLT3LG antibodies (along with the designed epitope bins in Example 3B / C and previously determined IC50 blocking values) are detailed in Table 10. Antibodies that did not reach binding saturation are indicated with ND (not determined). All 15 humanized anti-FLT3LG antibodies demonstrated the ability to bind to membrane-bound 293T-hFLT3LG cells. The antibodies did not exhibit binding to non-transfected 293T control cells (data not shown).

[0210] [Table 11]

[0211] E. Soluble FLT3LG binding blocking assay To demonstrate the activity of the four selected humanized anti-FLT3LG antibodies in blocking soluble FLT3LG binding to cell surface FLT3 receptors, a soluble FLT3LG binding blocking assay was developed using RS4;11 cells. RS4;11 cells exhibit detectable FLT3 surface expression. Using biotinylated soluble FLT3LG, ligand binding to FLT3 receptors can be measured by flow cytometry using fluorophore-conjugated streptavidin molecules. Inhibition of ligand binding using anti-FLT3LG antibodies can be measured as a decrease in mean fluorescence intensity.

[0212] Methods: To generate biotinylated hFLT3LG molecules, nickel from 1 M NiCl was first loaded onto NTA-Tris-biotin (Sigma catalog 75543) at a ratio of 1 NTA-Tris-biotin to 4.5 NiCl. Ni-NTA-Tris-biotin was incubated at room temperature with recombinant hFLT3LG-His (Acro Biosystems catalog FLL-H5223) at a ratio of 1 FLT3LG-His:1.5 Ni-NTA-Tris-biotin; this compound is referred to herein as "NTA-Tris-BT-hFLT3LG."

[0213] To determine hFLT3LG binding to RS4;11 cells and its blocking by humanized anti-FLT3LG antibodies, 1 × 10 5RS4;11 cells per well were first incubated with Live / Dead stain (Biolegend catalog 423102) for 20 minutes. After washing once with flow cytometry buffer (Thermo catalog 00-4222-26), the cells were incubated with Fc receptor blocking solution (Biolegend catalog 422302) for 10 minutes and then washed once with FACS buffer. NTA-Tris-BT-hFLT3LG at 0.75 nM (the predetermined EC80 for NTA-Tris-BT-FLT3LG binding to RS4;11 cells) was then added to varying concentrations of antibody for 15 minutes, followed by addition of cells for 30 minutes. Cells were washed with flow cytometry buffer and then incubated with streptavidin-PE (Biolegend catalog 405204) for 30 minutes. After washing the cells once with flow cytometry buffer, the cells were fixed with 4% paraformaldehyde (Thermo catalog J61899.AP) for 20 minutes. All steps were performed at room temperature. After washing once with and resuspending in flow cytometry buffer, the cells were analyzed using a CytoFLEX flow cytometer. Flow data were analyzed using FlowJo, and MFI values ​​were graphed and analyzed using Graphpad Prism.

[0214] Results: Inhibition of soluble hFLT3LG binding to FLT3 by four selected blocking humanized anti-FLT3LG antibodies was observed. In the presence of the antibodies, the interaction between the receptor and ligand was blocked, as indicated by the observed decrease in fluorescence. Inhibition curves for the R&D anti-FLT3LG antibodies at 0.75 nM hFLT3LG compared with four selected humanized anti-FLT3LG antibodies (h_b1.017 (panel A), h_b1.021 (panel B), h_b1.078 (panel C), and h_b1.126 (panel D)) are provided in Figure 7. 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 exhibit complete blocking activity are indicated with ND (not determined). Without wishing to be bound by theory, it is hypothesized that the humanized anti-FLT3LG antibody h_b1.021 did not exhibit full blocking activity in the soluble FLT3LG blocking assay because Acro hFLT3LG (which differs at amino acid position 72 from the R&D hFLT3LG used to generate the antibody) was used in the assay. It is hypothesized that the antibody binding to epitope bin A containing h_b1.021 binds to an epitope containing alanine at position 72 of hFLT3LG, which is present only in R&D hFLT3LG.

[0215] [Table 12]

[0216] This example demonstrates that selected humanized anti-FLT3LG antibodies inhibit the binding of soluble FLT3LG to cell surface FLT3 receptors.

[0217] Example 4: Inhibition of FLT3LG-induced receptor internalization by anti-FLT3LG antibodies Previous examples have shown that selected humanized anti-FLT3LG antibodies effectively blocked the binding of FLT3LG to the FLT3 receptor on the cell surface. A FLT3 receptor internalization assay was performed to determine whether humanized anti-FLT3LG antibodies similarly inhibit FLT3LG-induced receptor internalization.

[0218] A. Blockade of soluble FLT3LG-induced FLT3 receptor internalization by anti-FLT3LG antibodies Methods: Four selected humanized anti-FLT3LG blocking antibodies were tested for their ability to block soluble hFLT3LG-induced FLT3 receptor internalization together with an anti-hFLT3LG antibody (clone no. 40416) (R&D, catalog no. MAB608). The anti-hFLT3LG antibodies were incubated with 218 pM (the predetermined EC80 dose for soluble hFLT3LG-induced FLT3 internalization) of hFLT3LG-His (Acrobiosystems, catalog no. FLL-H5223) at 37°C for 30 minutes. RS4;11 cells (ATCC, Catalog No. CRL-1873, Lot No. 70036117) cultured in RPMI 1640 (GIBCO, Catalog No. 11875101) + 10% heat-inactivated FBS (R&D, Catalog No. S12450H) + 1x Antibiotic-Antimycotic (GIBCO, Catalog No. 15240-062) + 1x GlutaMAX (GIBCO, Catalog No. 35050-061) were seeded at 100,000 cells per well in a 96-well round-bottom plate (CORNING, Catalog No. 3799). Cells were pelleted in each well by centrifugation, resuspended in a mixture of antibody and hFLT3LG, and incubated at 37°C for 2 hours. Antibodies were tested at a starting concentration of 50 nM and at concentrations derived from 11 three-fold serial dilutions. Cells were stained on ice with LIVE / DEAD™ Fixable Violet Dead Cell Stain (INVITROGEN, Cat. No. L34955) to determine cell viability, and any human Fc receptors expressed on these cells were blocked with TruStain FcX Human Fc Receptor Blocking Solution (BIOLEGEND, Cat. No. 422302). Cell surface FLT3 was determined by flow cytometry using an anti-FLT3 antibody (clone BV10A4H2) fluorescently labeled with PE (INVITROGEN, Cat. No. 12-1357-42) compared to a mouse IgG1 kappa isotype control (clone P3.6.2.8.1), PE (INVITROGEN, Cat. No. 12-4714-82).Data were acquired on a CytoFLEX flow cytometer (BECKMAN COULTER), analyzed using the FLOWJO portal, and plotted using Prism (GraphPad).

[0219] Results: Anti-FLT3LG antibodies blocked soluble hFLT3LG-induced FLT3 receptor internalization in RS4;11 cells, as indicated by the increase in fluorescence (MFI) with increasing antibody concentration shown in Figure 8 for clone h_b1.017 (panel A), clone h_b1.021 (panel B), clone h_b1.078 (panel C), and h_b1.126 (panel D). From this receptor internalization blocking data, IC values ​​were calculated for each of the selected humanized blocking antibodies, as presented in Table 12. The h_b1.21 antibody did not provide a complete curve; therefore, the IC value was marked as ND (not determined). The lower blocking activity observed for h_b1.021 in the internalization assay was hypothesized to result from the use of Acro hFLT3LG, which may not be bound as strongly by antibodies specific for epitope bin A.

[0220] [Table 13]

[0221] B. Blockade of membrane FLT3LG-induced FLT3 receptor internalization by anti-FLT3LG antibodies FLT3LG stably expressed on 293T cells can induce FLT3 receptor internalization on RS4;11 cells when the cells are mixed. We tested the ability of our candidate antibodies to block this membrane FLT3LG-induced FLT3 internalization.

[0222] Methods: Four selected humanized blocking antibodies were tested for their ability to block membrane-expressed hFLT3LG-induced FLT3 receptor internalization together with anti-hFLT3LG antibody (clone no. 40416) (R&D, catalog no. MAB608). 293T-hFLT3LG cells cultured in RPMI 1640 (GIBCO, Catalog No. 11875101) + 10% heat-inactivated FBS (R&D, Catalog No. S12450H) + 1x Anti-Anti (GIBCO, Catalog No. 15240-062) + 1x GlutaMAX (GIBCO, Catalog No. 35050-061) were seeded at 4,000 cells per well (EC80 previously determined from cell titration experiments) into 96-well tissue culture-treated flat-bottom plates (CORNING, Catalog No. 3596) and allowed to adhere overnight at 37°C in a CO2 incubator. The next day, spent medium was removed from the 293T-hFLT3LG cells by gentle aspiration, followed by gentle washing with culture medium. The anti-FLT3LG antibody was incubated with 293T-hFLT3LG cells for 30 minutes at 37°C. Then, 100,000 RS4;11 cells (ATCC, Catalog No. CRL-1873, Lot No. 70036117) were added to each well and then incubated for 2 hours at 37°C. After incubation, the RS4;11 cells were transferred to a 96-well round-bottom plate (CORNING, Catalog No. 3799) and stained on ice with LIVE / DEAD™ Fixable Violet Dead Cell Stain (INVITROGEN, Catalog No. L34955) to determine cell viability. The human Fc receptors expressed on these cells were blocked with the human Fc receptor blocking solution TruStain FcX (BIOLEGEND, Catalog No. 422302). RS4;11 cells were specifically gated based on their CD19 staining using an anti-human CD19 antibody (clone HIB19) fluorescently labeled with APC / Cy7 (BIOLEGEND, catalog no. 302218).Downregulation of cell surface FLT3 was determined by flow cytometry using an anti-FLT3 antibody (clone BV10A4H2) fluorescently labeled with PE (INVITROGEN, catalog number 12-1357-42) compared to a mouse IgG1 kappa isotype control (clone P3.6.2.8.1), PE (INVITROGEN, catalog number 12-4714-82). Data were acquired on a CytoFLEX flow cytometer (BECKMAN COULTER), analyzed using the FLOWJO portal, and plotted using Prism (GraphPad).

[0223] Results: Anti-FLT3LG antibodies blocked cell surface-expressed FLT3LG-induced FLT3 receptor internalization in RS4;11 cells, as indicated by the increase in fluorescence (MFI) with increasing antibody concentration shown in Figure 9 for clone h_b1.017 (panel A), clone h_b1.021 (panel B), clone h_b1.078 (panel C), and clone h_b1.126 (panel D). IC50 values ​​were calculated for each of the selected humanized blocking antibodies according to their ability to block receptor internalization, as presented in Table 13. The h_b1.21 antibody did not provide a complete curve, and therefore, the IC50 value was marked as ND (not determined).

[0224] [Table 14]

[0225] Example 5: p-AKT signaling assay Phosphorylation of the serine / threonine kinase AKT occurs downstream of FLT3LG binding to its receptor FLT3, leading to a pathway that controls cell survival and proliferation in FLT3-expressing cells. The human cell line RS4;11 endogenously expresses FLT3 and responds to hFLT3LG treatment, which can be detected by phosphorylation of AKT.

[0226] A phospho-AKT (p-AKT) transduction assay was performed to determine whether anti-FLT3LG antibodies inhibited soluble hFLT3LG-induced AKT phosphorylation by FLT3-expressing RS4;11 cells.

[0227] Methods: RS4;11 cells (ATCC, Catalog No. CRL-1873, Lot No. 70036117) were plated at 2.0 × 10 cells per well. 5 Cells were seeded at a density of 1000 cells per well into 96-well TC-treated plates (Corning, Catalog No. CLS3595), washed with 1x PBS (Gibco Catalog No. 14040141), and serum-starved by incubating in RPMI 1640 medium (Gibco, Catalog No. 11875101) for 6 hours at 37°C. Cell treatments prepared by mixing recombinant human FLT3LG (R&D, Catalog No. 308-FKN / CF) for 15 minutes at room temperature were added at a predetermined EC80 of 0.2 nM, and four selected humanized anti-hFLT3LG blocking antibodies were titrated in 3-fold serial dilutions from a starting concentration of 100 nM. The premixed cell treatments were then added in triplicate to the serum-starved cells and incubated at 37°C for 10 minutes. Cells were lysed in the same plate by rapidly adding lysis buffer from the CISBIO phospho-AKT HTRF kit (PERKIN-ELMER, catalog number 64AKSPEH) and moving to a plate shaker at 4°C for 1 hour. 16 μL of lysed cell fraction per well was transferred to a CISBIO opaque low-volume 96-well plate (PERKIN-ELMER, catalog number 66PL96100). 4 μL of premixed phospho-AKT detection antibody from the same HTRF kit (PERKIN-ELMER, catalog number 64AKSPEH) was added per well. The plate was sealed and incubated overnight at 4°C, and the results were read using a SPECTRAMAX i3x microplate reader equipped with an HTRF detection cartridge (Molecular Devices, catalog number: 0200-7011POS). The resulting data was analyzed in Prism to obtain best-fit curves and IC50 values.

[0228] 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 signaling assay. Each of the four antibodies had comparable p-AKT blocking activity, as shown in Figure 10, panels A (clone h_b1.017), B (clone h_b1.021), C (clone h_b1.078), and D (clone h_b1.126). R&D isotype MAB002 and human IgG isotype were used as negative controls, and R&D tool antibody MAB608 was used as a positive control. p-AKT signaling was determined as a homogeneous time-resolved fluorescence (HTRF) ratio.

[0229] IC50 values ​​were calculated for each of the selected humanized anti-FLT3LG blocking antibodies according to their ability to block p-AKT signaling induced by the interaction between hFLT3LG and FLT3, as presented in Table 14.

[0230] [Table 15]

[0231] Example 6: Primary DC differentiation assay The FLT3 receptor is highly expressed on hematopoietic progenitor cells and steady-state DCs and is essential for the differentiation, proliferation / expansion, and survival of major DC subsets.

[0232] A co-culture assay using the MS5 feeder line and human CD34+ progenitor cells was performed to determine whether anti-FLT3LG antibodies inhibit soluble FLT3LG-induced DC differentiation.

[0233] Methods: MS5 stromal cells (DSMZ; Catalog Number: ACC 441) were grown in complete alpha-MEM (Gibco; Catalog Number: 12571063) medium 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 5-10 minutes of incubation with 0.05% trypsin-EDTA solution (Gibco; Catalog Number: 25300054), MS5 cells were harvested, washed, and treated with 10 μg / mL mitomycin C (THERMO SCIENTIFIC; Catalog Number: AAJ67460XF) in complete alpha-MEM medium for 3-4 hours at 37°C and 5% CO2 to inhibit proliferation. The mitomycin C-treated MS5 cells were then washed with 1x PBS, resuspended in complete alpha-MEM medium, and seeded into 12-well TC-treated plates at a density of 25,000 cells per well for 24 hours at 37°C and 5% CO2. Cryopreserved human CD34+ cells (STEMCell; Catalog No. 70060) derived from peripheral blood of a healthy donor were gently overlaid on the MS5 feeder layer at a density of 50,000 cells per well, followed by the addition of the cell treatment. The cell treatment was prepared by mixing recombinant human FLT3LG (R&D, Catalog No. 308-FKN / CF) with a predetermined EC80 of 2.86 nM based on published literature for 15 minutes at room temperature, and titrated with four selected humanized anti-hFLT3LG blocking antibodies in 3-fold serial dilutions from a starting concentration of 28.6 nM. The premixed treatments were then added in duplicate to the cells on days 0, 7, and 14 after CD34+ cell seeding, after which DCs were harvested and stained for flow cytometry on day 21.

[0234] Results: Dendritic cell populations were defined by flow cytometry and plotted as the frequency of the parent gate. Mean values ​​across donors were plotted against titrations of blocking humanized anti-FLT3LG antibodies, and IC50 values ​​were determined by best-fit curves. Data represent a combination of two independent experiments representing five healthy CD34+ donors. Each of the four anti-FLT3LG antibodies (clones h_b1.017, h_b1.021, h_b1.078, and h_b1.126) and the positive control MAB608 (R&D SYSTEMS, catalog no. MAB608) had comparable blocking activity against FLT3LG-induced DC differentiation from CD34+ human stem cell precursors (Figure 11, panels A-C). IC50 values ​​were calculated for each of the selected humanized blocking antibodies according to their ability to inhibit CD11c+HLA-DR+ DC differentiation, as shown in Table 15.

[0235] [Table 16]

[0236] Example 7: Binding of anti-FLT3LG antibodies to endogenous FLT3LG in human T cells To demonstrate that the four selected humanized anti-FLT3LG antibodies can bind to endogenous FLT3LG expressed on T cells, a flow cytometry assay was optimized to measure the binding of these antibodies to FLT3LG in human T cells. Because FLT3LG is not highly expressed in healthy control human T cells, the assay was optimized to upregulate FLT3LG expression in T cells, and then the binding of the anti-FLT3LG antibodies was measured by flow cytometry as mean fluorescence intensity.

[0237] Methods: Human 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), including T cells, were obtained by density gradient centrifugation using FICOLL-PAQUE (GE, Cat. No. 07907) and SEPMATE tubes (STEMCELL TECHNOLOGIES, Cat. No. 85460). T cells were then isolated from the MNC pool by magnetic separation using a negative selection kit (STEMCELL TECHNOLOGIES, Cat. No. 19661). T cells were then cultured for 10 min in IMMUNOCULT-XF T cell expansion medium (STEMCELL TECHNOLOGIES, Cat. No. 10981) + 1% antibiotic-antimycotic (GIBCO, Cat. No. 15240062) + 10% FBS + 1% GLUTAMAX (GIBCO, Cat. No. 35050061) with and without 10 ng / mL IL-7 (R&D, Cat. No. BT-007). 6 The cells were plated at a density of 0.2 × 10 / mL for 3 days, where IL-7 induced FLT3LG expression. On day 3, the cells were washed with PBS and plated at a density of 0.2 × 10 / mL. 6 The T cells were resuspended in fresh medium (see recipe above) with fresh IL-7 at a cell density of 2 x 10 / mL and incubated for an additional 3 days. On day 6, the T cells were harvested and washed with flow cytometry buffer ("Flow Buffer", THERMOFFISHER Cat. No. 00-4222-26). 5Individual cells / well were first incubated with live / dead stain (BIOLEGEND, Cat. No. 423102) for 15-25 minutes. After washing once with flow buffer, cells were incubated with Fc receptor blocking solution (BIOLEGEND, Cat. No. 422302) for 80 minutes and then washed once with flow buffer. Cells were then stained for T cell surface markers CD45 APC-Cy7 (BIOLEGEND, Cat. No. 304014), CD3 BV650 (BIOLEGEND, Cat. No. 300468), CD8 PacBlue (BIOLEGEND, Cat. No. 344718), and CD4 FITC (BIOLEGEND, Cat. No. 357406) for 30 minutes, along with titration of selected anti-FLT3LG antibodies starting at 250 nM, titrated in 5-fold decreasing increments over 10 points. The cells were washed once with flow buffer and then incubated with anti-human PE secondary antibody (Jackson Immunoresearch, catalog number 109-116-097) to detect binding of the anti-FLT3LG antibody to the cells. The cells were washed and either left unfixed or fixed with 4% PFA (THERMOFISHER, catalog number J61899.AP) for 20 minutes. After washing once with flow cytometry buffer and resuspending, the cells were analyzed using a CYTOFLEX flow cytometer. All steps were performed at room temperature. Flow data were analyzed using FLOWJO, and MFI values ​​were graphed and analyzed using Graphpad Prism.

[0238] Results: Binding of endogenous FLT3LG in human T cells from two donors by the humanized anti-FLT3LG antibodies h_b1.017, h_b1.078, and h_b1.126 was observed. Binding curves for four selected humanized anti-FLT3LG antibodies (h_b1.017 (panel A), h_b1.021 (panel B), h_b1.078 (panel C), and h_b1.126 (panel D)) to endogenous FLT3LG are provided in Figure 12. EC50 values ​​were calculated for each of the selected humanized anti-FLT3LG antibodies according to their ability to bind to endogenously expressed FLT3LG in T cells from two donors, as shown in Table 16.

[0239] [Table 17]

[0240] Example 8: In vivo effect of anti-FLT3LG antibody on hFLT3LG-induced DCs In vivo studies were performed to determine the effect of anti-FLT3LG antibodies on the frequency and number of hFLT3LG-induced DC populations.

[0241] Methods: All mice arrived at the facility and rested for at least 3 days before any manipulation. NOG-EXL female mice (approximately 5-6 weeks old) were irradiated with 120 centigrays (cGy). After at least 4 hours of rest, the mice received donor human CD34+ cells via retroorbital (ro) injection. Two hCD34+ donors (donor 17 and donor 18) were used in this study. Body weight (BW) was measured daily from 0 to 2 weeks post-engraftment (WPE) to monitor for any BW loss or signs of distress after engraftment. Thereafter, BW was measured twice weekly.

[0242] At approximately 4 WPE, mice were bled (survival submandibular bleed under isoflurane anesthesia) to determine the level of humanization (% hCD45 of all CD45 (hCD45 + mCD45)). Based on hCD45%, mice were divided into six different treatment groups, with seven mice / donors assigned to each group. Mice were intraperitoneally (ip) dosed with 15 μg / dose of anti-FLT3LG antibody (h_b1.017, h_b1.078, or h_b1.126) on days 1 and 2 of the study. hFLT3LG (R&D, catalog number 308-FKN-250 / CF, 15 μg / dose) was injected subcutaneously (sc) into the dorsal scruff of the mouse 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 single cell suspensions for flow cytometry analysis.

[0243] Results: The frequency (% of hCD45+ cells; panel A) and number (panel B) of hFLT3LG-induced cDC, cDC1, cDC2, pDC, and pre-pDC populations were measured in the spleen as shown in Figure 13. Compared to the IgG isotype control, administration of selected anti-FLT3LG antibodies reduced the frequency and number of hFLT3LG-induced DCs in vivo.

[0244] Example 9: Evaluation of anti-FLT3LG antibody interaction with hFLT3LG by NS-EM Methods: Antigen / antibody complexes were prepared by mixing 0.4 μM his-tagged human FLT3LG (AcroBiosystems, catalog number FLL-H5223) with 0.1 μM anti-FLT3LG antibody h_b1.078 or anti-FLT3LG reference antibody O001. The mixture was incubated on ice for 70–85 minutes to allow complex formation. A 4 μl sample was added to a freshly glow-discharged continuous carbon grid (Agar Scientific Ltd, catalog number AGS160-3) and stained with 2% uranyl acetate. The grid was imaged in a THERMOFFISHER SCIENTIFIC GLACIOS transmission electron microscope operating at 200 kV and a temperature of approximately 90 K. Movies were recorded using EPU3.2 with a pixel size of 0.94 Å, a defocus of -1.4 to -2.5 μm, and 40 e - / Å 2 The images were acquired on a Falcon 4i direct electron detector at a cumulative electron fluence of 100 Hz. Ten frames were collected per movie. Data were processed using a software package that allows correction for beam-induced sample motion, image processing, particle picking, and 2D classification. The 2D class averages presented correspond to 10,017 and 2,033 particles for data associated with h_b1.078 and O001, respectively. Images shown were contrast adjusted using FIJI ImageJ v1.53t.

[0245] Results: Convergent 2D class averages showed that antibodies h_b1.078 and O001 primarily form two FLT3LG2:two mAb complexes. Comparison of representative 2D class averages from h_b1.078 and O001 suggests that the angles between fAb ​​arms are visibly different. The angle between the fAb arms in the two FLT3LG2:two h_b1.078 complex is much larger than 90 degrees, while that in the two FLT3LG2:two O001 complex is smaller than 90 degrees (as is the case in the image shown in Figure 14).

[0246] Example 10: Amino acid and nucleic acid sequences of antibodies and antigen-binding fragments thereof The amino acid sequences of the variable heavy (VH) and variable light (VL) chains of the antibodies and antigen-binding fragments thereof provided herein are provided in Table 17.

[0247] [Table 18]

[0248] The amino acid sequences of the complementarity-determining regions (CDRs) of the VH and VL chains of the antibodies and antigen-binding fragments thereof provided herein are provided in Table 18. In some embodiments, one or more humanized CDR regions may be codon-optimized. For example, the DNA sequences of the humanized anti-FLT3LG antibodies provided below have been codon-optimized from their murine counterparts for synthesis, cloning, and expression. As a result, in some embodiments, the DNA sequences of the CDRs in the humanized clones may differ from the native murine sequences. Typically, such codon optimization does not result in differences in the amino acid sequences.

[0249] [Table 19]

[0250] The amino acid sequences of the fragment crystallizable (Fc) regions of the antibodies and antigen-binding fragments thereof provided herein are provided in Table 19. The Fc regions provided herein were utilized in both murine and humanized clones, with hIgG1 N297G being a non-functional Fc; however, the use of other Fc region sequences, including wild-type Fcs, can be envisioned.

[0251] [Table 20]

[0252] The amino acid sequences of the light chain constant (CL) domains of the antibodies and antigen-binding fragments thereof provided herein are provided in Table 20. The CL domains provided herein were utilized in both murine and humanized clones; however, the use of other CL domain sequences can be envisioned.

[0253] [Table 21]

[0254] The amino acid sequences of the full-length heavy chains (HC) having the VH and Fc regions provided herein, and the light chains (LC) having the VL and CL regions provided herein, of the antibodies and antigen-binding fragments thereof provided herein are provided in Table 21, and the nucleic acid sequences of the full-length HC and LC of the antibodies and antigen-binding fragments thereof provided herein are provided in Table 22.

[0255] [Table 22] JPEG2025537495000024.jpg219170

[0256] [Table 23] JPEG2025537495000026.jpg204170JPEG2025537495000027.jpg195170JPEG20255374950 00028.jpg242170JPEG2025537495000029.jpg241170JPEG2025537495000030.jpg119170

Claims

1. (i) any one or 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 one, two, or three 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 A FLT3LG binding protein comprising:

2. a. The CDRs of (i) are 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 protein of claim 1,

3. 3. The FLT3LG binding protein of claim 1 or 2, wherein all six CDRs are present in the binding protein.

4. The following six CDRs: 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 CDRL3 of SEQ ID NO:40 A FLT3LG binding protein comprising:

5. The binding protein is A VH region that is at least 80% identical to SEQ ID NO:8 and a VL region that is at least 80% identical to SEQ ID NO:16 The FLT3LG binding protein of claim 4, comprising:

6. The binding protein is HC sequence at least 80% identical to SEQ ID NO: 50 and / or LC sequence at least 80% identical to SEQ ID NO: 58 The FLT3LG binding protein according to any one of claims 1 to 5, comprising:

7. 7. The FLT3LG binding protein of any one of claims 1 to 6, wherein the binding protein binds to human FLT3LG with a KD of less than or equal to 1 nM.

8. 8. The FLT3LG binding protein of any one of claims 1 to 7, wherein the binding protein inhibits soluble FLT3LG-induced p-AKT signaling with an IC50 of about 2 nM or less.

9. 9. The FLT3LG binding protein of any one of claims 1 to 8, wherein the binding protein inhibits soluble FLT3LG-induced FLT3 receptor internalization with an IC50 of about 1 nM or less.

10. A FLT3LG binding protein that binds to human FLT3LG and competes for binding to human FLT3LG with a reference FLT3LG binding protein comprising the VH region sequence of SEQ ID NO:8 and the VL region sequence of SEQ ID NO:

16.

11. The FLT3LG binding protein of any one of claims 1 to 10, wherein the binding protein is an antibody or an antigen-binding fragment thereof.

12. The FLT3LG binding protein of claim 11, wherein the antibody or antigen-binding fragment thereof inhibits binding of human FLT3LG to FLT3.

13. The FLT3LG binding protein of claim 12, wherein the antibody or antigen-binding fragment thereof inhibits the binding of human soluble FLT3LG to FLT3 with an IC50 of about 2 nM or less.

14. The FLT3LG binding protein of any one of claims 11 to 13, wherein the antibody or antigen-binding fragment thereof comprises a modified Fc region.

15. 15. The FLT3LG binding protein of claim 14, wherein the modified Fc region comprises the amino acid substitution N297G (as numbered according to the EU index).

16. A nucleic acid sequence encoding one or both of the HC and LC of a FLT3LG binding protein as defined in any one of claims 1 to 15.

17. 17. The nucleic acid sequence of claim 16, wherein the sequence comprises SEQ ID NO: 66 encoding the HC and / or SEQ ID NO: 74 encoding the LC.

18. 18. An expression vector comprising a nucleic acid sequence as defined in claim 16 or 17.

19. 19. A recombinant host cell comprising a nucleic acid sequence as defined in claim 16 or 17, or an expression vector as defined in claim 18.

20. A method for producing a FLT3LG binding protein, comprising culturing a host cell as defined in claim 19 under conditions suitable for expression of the nucleic acid sequence or expression vector, thereby producing a polypeptide comprising the FLT3LG binding protein.

21. 21. A FLT3LG binding protein produced by the method of claim 20.

22. A cell line engineered to express the FLT3LG binding protein of any one of claims 1 to 15 or 21.

23. A pharmaceutical composition comprising a FLT3LG binding protein as defined in any one of claims 1 to 15 or 21, and a pharmaceutically acceptable excipient.

24. 23. A method for the treatment of an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a FLT3LG binding protein as defined in any one of claims 1 to 15 or 21, or a pharmaceutical composition as defined in claim 23.

25. 25. The method of claim 24, wherein the autoimmune disease is systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), sarcoidosis, Sjogren's syndrome, or celiac disease.

26. 26. The method of claim 25, wherein the autoimmune disease is SLE.

27. 26. The method of claim 24 or 25, wherein the subject is a human.

28. A FLT3LG binding protein as defined in any one of claims 1 to 15 or 21, or a pharmaceutical composition as defined in claim 23, for use in therapy.

29. A FLT3LG binding protein as defined in any one of claims 1 to 15 or 21, or a pharmaceutical composition as defined in claim 23, for use in the treatment of an autoimmune disease.

30. 30. The FLT3LG binding protein of claim 29, wherein the autoimmune disease is systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), sarcoidosis, Sjogren's syndrome, or celiac disease.

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