RU2788128C2 - Antibodies to ligand of receptor tyrosine kinase 3 similar to product of protooncogene of feline sarcoma virus (fms) of mcdonough (flt3l) strain and options of their use for treatment of autoimmune and inflammatory diseases - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: antibody or its antigen-binding fragment are proposed, which specifically bind to ligand of receptor tyrosine kinase 3 similar to a product of protooncogene of a feline sarcoma virus (hereinafter – FMS) of McDonough (FLT3L) strain. Polynucleotide encoding the specified antibody, an expression vector and hybridoma containing the specified polynucleotide, and a pharmaceutical composition containing the specified antibody are also proposed. A method for the production of an antibody and the use of an antibody for the treatment or prevention of an autoimmune disease are also proposed.

EFFECT: invention provides reduction in inflammation in autoimmune diseases.

26 cl, 27 dwg, 7 tbl, 16 ex

Description

BACKGROUND OF THE INVENTION

[0001] Autoimmune diseases, which occur when the body's immune system produces autoantibodies, are unfortunately widespread. For example, it is estimated that over 23 million Americans suffer from autoimmune diseases. Currently, more than 80 autoimmune diseases are known. Specific examples of autoimmune diseases include systemic lupus erythematosus, myositis, primary Sjögren's syndrome, multiple sclerosis, uveitis, psoriasis, and rheumatoid arthritis.

[0002] Systemic lupus erythematosus (SLE) is characterized by joint pain, swollen lymph nodes, and a butterfly-shaped rash on the cheeks. In SLE, autoantibodies against healthy tissues produced by the patient's immune system attack them, leading to inflammation. At the cellular level, SLE patients have autoreactive T and B cells driven by dendritic cells (Palucka A.K. et al. Immunology and Cell Biology (2002) 80: 484-488). Sjögren's syndrome is characterized by systemic chronic inflammation of the exocrine organs resulting in organ dysfunction (Holdgate N. and St. Clair E.W., F1000 Research. 1412 10.12688/f1000research.8352.1).

[0003] Multiple sclerosis (MS) is characterized by demyelination of nerve cells in the brain and spinal cord and inflammation of the central nervous system (CNS). Psoriasis is an autoimmune disease that manifests itself as red, itchy patches of skin. Rheumatoid arthritis (RA) is an inflammatory disorder of the synovial tissue of the joint, characterized by persistent synovitis and destruction of the cartilage and bone tissue of the joint. Damage can progress, affecting multiple body systems. Lupus nephritis is associated with systemic lupus erythematosus and leads to the development of kidney inflammation. When the kidneys become inflamed, they leak protein and eventually fail. Uveitis is a group of inflammatory diseases in which damage and possibly destruction of eye tissue occurs, resulting in loss of vision.

[0004] In addition, acute and chronic pro-inflammatory conditions have been associated with a variety of diseases in humans and may be the cause of their development. Specific examples of diseases that are believed to be associated with chronic inflammation include type 1 and type 2 diabetes, chronic kidney disease (CKD), including, for example, CKD that has developed on the background of diabetes, diabetic nephropathy, and high blood pressure. blood pressure; atherosclerosis, Alzheimer's disease, cancer, and associated complications of such diseases, including heart disease, hypertension, anemia, pericarditis, renal osteodystrophy, and others. Similar to autoimmune diseases, in diseases associated with chronic inflammation, the body appears to over-prolong the pro-inflammatory response, which can lead to the development of debilitating and often fatal comorbidities.

[0005] The causes of autoimmune diseases are not well understood. In terms of mechanism, at the heart of every autoimmune disease is an ongoing autoimmune response that is stimulated (and/or not suppressed) by complex regulatory systems that constantly replenish the number of autoreactive immune cells. A similar mechanism appears to occur in non-autoimmune chronic inflammatory diseases. For this reason, therapeutic interventions for autoimmune diseases and chronic inflammation target multiple regulatory systems, signaling cascades, and their constituent components.

[0006] One class of putative therapeutic targets includes receptor tyrosine kinases (TKRs), which are transmembrane receptors that bind various growth factors and proteins to regulate cellular homeostasis. More than fifty known human TKRs are classified into 20 different classes defined by their genetic phylogeny (Robins D.R., et al. Oncogene. (2000) 19: 5548-5557; Lemmon M.A., and Schlessinger J. Cell. (2010) 141: 1117-1134 ). Class III TKRs are characterized by the presence of five to seven immunoglobulin-like domains in an extracellular region containing 70 to 100 hydrophilic residues. Among class III TKRs, McDonough feline sarcoma virus (FMS) proto-oncogene product-like receptor tyrosine kinase 3 (FLT3) is a membrane-bound receptor expressed on human stem cells, hematopoietic progenitor cells, dendritic cells, activated T and B cells, monocytes, and microglial cells. FLT3 binds the FLT3 ligand (FLT3L), which is a hematopoietic cytokine expressed by a variety of cell types, including activated T cells, activated endothelium, and bone marrow stromal cells. FLT3L is expressed both on the cell surface and as a secreted homodimer and signals through its cognate receptor, FLT3. FLT3 is expressed on the cell surface as a monomer and is activated when ligated to FLT3L. After ligation with FLT3L, FLT3 undergoes dimerization, autophosphorylation, and activates signaling pathways, including RAS/extracellular signal-regulated kinase (ERK), phosphatidylinositide 3-kinase (PI3K), and signal transducers and transcription activators (STAT) 3 and 5. After autophosphorylation dimerized FLT3 is internalized and degraded.

[0007] FLT3L is produced in response to inflammatory signals, in particular the following γ-chain cytokines: IL-2, IL-7 and IL-15, and its interaction with FLT3 triggers the inflammatory process mainly through its role in differentiation, proliferation and survival of DC. A role for FLT3 signaling in the survival of T and B cells after activation has also been suggested, with both cell types reported to transiently upregulate the expression of this receptor (Astier AL et al., J. Immunology. 2010 v184: 685-93 and Tobon et al Arthritis & Rheumatism 2010;62(11):3447-56). In addition, NK cell survival is thought to be indirectly dependent on FLT3L due to its requirement for DC-derived IL-15, although this observation is based on mouse data (Guimond M et al., J. Immunology 2010; 184: 2769-75) and has not yet been demonstrated in humans.

[0008] DCs are of particular interest in inflammation because they are immune system sentinels migrating from the site of inflammation to the lymph node and initiating the adaptive immune response that is ultimately required for the development of an autoimmune disease. Broadly speaking, there are two subpopulations of DC: myeloid/classic dendritic cells (cDC) and plasmacytoid dendritic cells (pDC). cDCs produce inflammatory cytokines (eg, IFNI-III, IL-23, IL-12, IL-6, and IL-1β), present antigen to T cells in the context of costimulation, and secrete chemokines that recruit cells to the site of inflammation and promote their cooperative localization necessary for important intercellular interactions. Through these mechanisms, cDC cells stimulate neutrophils, B cells, T cells, and NK cells, resulting in netosis, autoantibody production, IL-17 production, and additional production of inflammatory cytokines. pDCs are the main source of type I IFN, which is a key cytokine in the development of an innate response that enhances the activation of all branches of the immune system.

[0009] The salivary glands of patients with Sjögren's syndrome are characterized by the expression of FLT3 and FLT3L on infiltrating B cells (Tobon et al. Arthritis & Rheumatism. (2010) 62(11): 3447-3456). In addition, patients with Sjögren's syndrome are characterized by an increased frequency of FLT3-expressing B cells in the circulation, and their survival is increased when co-cultured with FLT3L-expressing human salivary gland cells. In individuals with MS, the FLT3 protein is expressed in chronic and active lesions, as well as in the gray and white matter (DeBoy C.A. et al. Exp Mol Pathol. (2010); 89(2): 109-116). In addition, FLT3 colocalizes with immature DCs in the perivascular space of the brain, suggesting infiltration of FLT3-positive DCs into the brains of individuals with MS (Deboy et al.). In RA, synovial fluid FLT3L levels are elevated compared to healthy individuals. In addition, monocytes, NK cells and DCs from RA patients are characterized by high levels of FLT3L expression (Ramos M. et al. Arthritis Res Ther. (2013) 15(6): R209).

[0010] Moreover, elevated serum and inflammatory FLT3L levels have been reported in SLE, myositis, primary Sjogren's syndrome, MS, uveitis, and RA (Andersson et al. PLoS One (2012) 7: e47668; DeBoy et al. Exp and Mol Path (2010) 89: 109-16).

[0011] Therefore, although FLT3-mediated pro-inflammatory survival (eg, involving pDCs and mDCs) is an effective physiological response in healthy individuals, it is likely to have negative effects in autoimmune diseases. Thus, disrupting or attenuating the functioning of the FLT3/FLT3L signaling pathway may be an important tool to combat autoimmune diseases and other inflammatory diseases, as well as to reduce inflammation.

BRIEF DESCRIPTION OF THE INVENTION

[0012] Provided herein are new FLT3L binding antibodies for controlling autoimmune diseases and other acute and/or chronic inflammatory diseases.

[0013] In a first aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to FLT3L, comprising a group of complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3 , LCDR1, LCDR2 and LCDR3 contain the amino acid sequences: (a) under SEQ ID NO: 29, 30, 31, 32, 33 and 34, respectively; or (b) under SEQ ID NO: 29, 30, 31, 35, 33 and 34, respectively; or (c) under SEQ ID NO: 29, 36, 37, 32, 33 and 38, respectively.

[0014] In one embodiment of the first aspect, the isolated antibody, or antigen-binding fragment thereof, has a heavy chain variable region (VH) and a light chain variable region (VL) characterized by at least 95%, 96%, 97%, 98%, or 99% sequence identity with: (a) SEQ ID NO: 1 and SEQ ID NO: 2, respectively; or (b) SEQ ID NO: 3 and SEQ ID NO: 4, respectively; or (c) SEQ ID NO: 5 and SEQ ID NO: 6, respectively. In another embodiment, VH and VL provide (a) SEQ ID NO: 1 and SEQ ID NO: 2, respectively; or (b) SEQ ID NO: 3 and SEQ ID NO: 4, respectively; or (c) SEQ ID NO: 5 and SEQ ID NO: 6, respectively. In a further embodiment, the isolated antibody or antigen-binding fragment comprises (a) a heavy chain region comprising SEQ ID NO: 61 and a light chain region comprising SEQ ID NO: 62; or (b) a heavy chain region comprising SEQ ID NO: 65 and a light chain region comprising SEQ ID NO: 66; or (c) a heavy chain region comprising SEQ ID NO: 69 and a light chain region comprising SEQ ID NO: 70. In one embodiment, the isolated antibody or antigen-binding fragment thereof inhibits FLT3L-mediated FLT3 activation. In another embodiment, the isolated antibody or antigen-binding fragment thereof does not cross-react with structurally similar TKR ligand molecules. In one embodiment, the isolated antibody, or antigen-binding fragment thereof, does not cross-react with at least one of huSCF and huCSF1. In a further embodiment, the isolated antibody or antigen-binding fragment thereof does not cross-react with either huSCF or huCSF1. In one embodiment, the isolated antibody, or antigen-binding fragment thereof, is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or a chimeric antibody. In one embodiment, the isolated antibody or antigen-binding fragment comprises an immunoglobulin heavy chain constant domain selected from the group consisting of: (a) an IgA constant domain; (b) IgD constant domain; (c) IgE constant domain; (d) IgG1 constant domain; (e) IgG2 constant domain; (f) IgG3 constant domain; (g) IgG4 constant domain; and (h) IgM constant domain. In one embodiment, the isolated antibody or antigen-binding fragment contains an IgG1 constant domain. In another embodiment, the isolated antibody or antigen binding fragment comprises an immunoglobulin light chain constant domain selected from the group consisting of: (a) an Ig kappa chain constant domain and (b) an Ig lambda chain constant domain. In one embodiment, the antigen binding protein comprises a human IgG1 constant domain and a human lambda chain constant domain. In one embodiment, the IgG1 constant domain comprises one or more amino acid substitutions selected from the group consisting of L234F, L235E, and P331S, numbered according to the Kabat EU numbering system (Edelman et al., Proc. Natl. Acad. Sci., 63:78-85 (1969)).

[0015] In a second aspect, the present invention provides an isolated nucleic acid molecule encoding an isolated antibody or antigen-binding fragment thereof according to the first aspect and/or embodiments thereof. In one embodiment of the second aspect, the nucleic acid molecule is operably linked to a control sequence.

[0016] In a third aspect, the present invention provides a vector containing a nucleic acid molecule according to the second aspect and/or embodiments thereof.

[0017] In a fourth aspect, the present invention provides a host cell transformed with a nucleic acid molecule according to the second aspect and/or embodiments thereof or a vector according to the third aspect. In one embodiment, the host cell is a mammalian host cell. In another embodiment, the host cell is a HEK293 cell, an NS0 mouse myeloma cell, or a Chinese hamster ovary (CHO) cell.

[0018] In a fifth aspect, the present invention provides a hybridoma producing an antibody or antigen-binding fragment according to any of the previous aspects or their embodiments.

[0019] In a sixth aspect, the present invention provides an isolated host cell producing an antibody or antigen-binding fragment according to any of the previous aspects or embodiments thereof.

[0020] In a seventh aspect, the present invention provides a method for producing an antibody or antigen-binding fragment according to any of the previous aspects or their embodiments, comprising (a) culturing a host cell expressing said antibody or antigen-binding fragment, or culturing the host cell according to the third aspect or its variant implementation or hybridoma according to the fourth aspect; and (b) isolating said antibody or antigen-binding fragment thereof from said cultured host cell.

[0021] In an eighth aspect, the present invention provides an antibody, or an antigen-binding fragment thereof, obtained according to the method of the sixth aspect.

[0022] In a ninth aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any of the previous aspects or embodiments thereof and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition is for use as a drug.

[0023] In a tenth aspect, the present invention provides a method for treating an acute or chronic inflammatory disease, comprising administering to a subject in need thereof a pharmaceutically effective amount of an isolated antibody or fragment thereof, in accordance with any of the preceding aspects or embodiments thereof. In one embodiment, the inflammatory disease includes chronic kidney disease (CKD), including, for example, CKD secondary to diabetes, diabetic nephropathy, and high blood pressure.

[0024] In an eleventh aspect, the present invention provides a method for treating an autoimmune disease, comprising administering to a subject in need thereof a pharmaceutically effective amount of an isolated antibody or fragment thereof, in accordance with any of the preceding aspects or embodiments thereof. In one embodiment, the autoimmune disease includes systemic lupus erythematosus, myositis, primary Sjögren's syndrome, multiple sclerosis, uveitis, psoriasis, or rheumatoid arthritis.

[0025] These and other features and advantages of the present invention will be better understood from the following detailed description of the present invention in conjunction with the appended claims. It should be noted that the scope of the claims is determined by the wording given therein, and not by a specific consideration of the features and advantages set forth in the present description.

BRIEF DESCRIPTION OF GRAPHICS

[0026] FIG. 1A-1C. Selection of leading antibodies. In FIG. 1A shows the alternating panning method using huFLT3L and muFLT3L and the resulting enrichment of BMV, CS, DP47 and Dyax phage libraries. In FIG. 1B depicts the manner in which panning products were inserted into a vector for subsequent competitive HTRF analysis. In FIG. 1C shows the results of a competitive HTRF (Time-Resolved Homogeneous Fluorescence) analysis for the lead candidates.

[0027] FIG. 2A-2C. Expression of FLT3L in cell lines. In FIG. 2A shows increased expression of FLT3 in the RS4;11 cell line compared to the EOL-1, MOLM13 and MV4-11 cell lines. In FIG. 2B shows results confirming that recombinant FLT3L binds to FLT3 on RS4;11 cells in a dose dependent manner. In FIG. 2C demonstrates that downregulation of FLT3 on the surface of RS4;11 cells can be reliably detected with a commercially available anti-FLT3 antibody using flow cytometric analysis, and such downregulation occurs in response to ligation to FLT3L in a dose-dependent manner.

[0028] FIG. 3A and 3B. Obtaining EC80 and subsequent testing. In FIG. 3A shows the FLT3L titration curve used to obtain the concentration at which 80% of FLT3 on the cell surface of RS4;11 (EC80) cells is downregulated. In FIG. 3B shows the inhibition profile for a commercially available anti-FLT3L antibody and recombinant FLT3 receptor construct (FLT3-Fc) relative to 96 pM recombinant FLT3L, which would otherwise result in 80% downregulation of FLT3 on the cell surface of RS4 cells;11.

[0029] FIG. 4A - 4C. Inhibition of human and cynomolgus monkey sFLT3 by candidate leader antibodies. In FIG. 4A shows the inhibition profiles for the five lead candidates against 96 pM human FLT3L. In FIG. 4B shows inhibition profiles for five lead candidates against 96 pM FLT3L cynomolgus monkey. In FIG. 4C shows the inhibition profiles for five mouse FLT3L lead candidates.

[0030] FIG. 5A-C. Inhibition of FLT3 on the cell surface by leading candidates. In FIG. 5A demonstrates the ability of leader antibodies to bind human FLT3L expressed on the surface of transduced CHO cell lines. In FIG. 5B shows binding of leader antibodies to cynomolgus FLT3L on the cell surface. In FIG. 5C shows binding of leader antibodies to mouse FLT3L on the cell surface.

[0031] FIG. 6A-C. Binding of candidate leaders to endogenous human FLT3L. In FIG. 6A shows FLT3L expression on primary human T cells 7 days after stimulation with IL-2. In FIG. 6B demonstrates the ability of all lead candidates to bind endogenous FLT3L on primary human T cells, with the exception of clone 5D9. In FIG. 6C demonstrates that increasing the avidity of CAT5D9 by dimerization prior to incubation with primary T cells allowed detection of dose-dependent clone binding to endogenous FLT3L on the surface of T cells.

[0032] FIG. 7A and 7B. Suppression of cell surface signaling by candidate leaders. In FIG. 7A shows a dose-response curve for RS4;11 cells versus FLT3L-expressing CHO cells, indicating 1000 CHO cells per well as the optimal amount to induce 80% FLT3 downregulation on the surface of RS4;11 cells. In FIG. 7B demonstrates that all lead candidates have the ability to inhibit FLT3L to some extent on the cell surface of CHO cells.

[0033] FIG. 8A and 8B. Activation and neutralization of ERK signaling pathways. In FIG. 8A shows the results of a proof-of-concept study showing FLT3L activation of ERK signaling in RS4;11 cells. In FIG. 8B demonstrates the suppression of commercially available FLT3L antibody-induced ERK activation.

[0034] FIG. 9A and 9B. Blocking by candidate leaders of signaling further along the signaling pathway involving MEK 1/2 and ERK. In FIG. 9A shows the functional activity of candidate leader clones on FLT3L-induced MEK 1/2 phosphorylation in primary human CD133+ stem cells. In FIG. 9B shows further confirmation of the functional activity of leader clones in relation to FLT3L-induced signaling in primary human CD133+ stem cells using ERK phosphorylation as a readout.

[0035] FIG. 10A and 10B. Target specificity and binding kinetics of candidate leaders. In FIG. 10A shows phase III sorting of candidate leader clones and FLT3-Fc relative to clone 5D9. In FIG. 10B shows phase III sorting of all leader clones and FLT3-Fc against CAT8 as representative of all clones other than CAT5D9.

[0036] FIG. 11A and 11B. Cross-reactivity of leading candidates with huSCF and huCSF. In FIG. 11A demonstrates that the leader candidates do not bind to the huSCF structural homologue. In FIG. 11B demonstrates that the leader candidates do not bind to the huCSF structural homologue.

[0037] FIG. 12A and 12B. Clone optimization. In FIG. 12A shows the results of the first round of clone optimization comparing the original CAT5D9 with clone 6 (C06) using the FLT3 downregulation assay of RS4;11 as the readout. In FIG. 12B shows the results of the second round of clone optimization comparing the original CAT5D9 with clone 6 (C06) and the final lead candidates AM40 and SC4017.

[0038] FIG. 13A and 13B. Effective neutralization of endogenous FLT3L on the cell surface. In FIG. 13A shows the downregulation of FLT3 in RS4:11 in response to serial dilution of FLT3L-expressing CD4+ T cells. In FIG. 13B shows that the leader clones completely neutralize FLT3L activity on the cell surface of CD4+ T cells.

[0039] FIG. 14. Study design for FLT3L neutralization in healthy cynomolgus monkeys. Three groups of male cynomolgus monkeys (n=4 per group) were administered 0.03, 1 or 30 mg/kg AM40 (MEDI1116) in five weekly doses for a month as indicated. An eight-week follow-up period after the last dose was used to determine the persistence of the administered antibody and its effect on the circulating DC population.

[0040] FIG. 15A and 15B. Serum FLT3L protein concentration and circulating DC frequency after administration of anti-FLT3L antibody (AM40/MEDI1116). In FIG. 15A shows the interaction of MEDI1116 with a target when measuring free FLT3L serum levels after administration at 0.03, 1.0 and 30 mg/kg. Serum was measured daily on days 1-8, followed by weekly measurements until day 85. In FIG. 15B depicts the decline and recovery in circulating CD1c+ (classic DCs) (left) and plasmacytoid DCs (right), measured as a percentage of baseline, after treatment with MEDI1116.

[0041] FIG. 16A-16D. FLT3L expression measurements and correlation of SLEDAI score in SLE patients when comparing serum measurements and T cell flow cytometry results. In FIG. 16A depicts serum levels of FLT3L in healthy (HD) donors and SLE patients. In FIG. 16B depicts the correlation between serum FLT3L levels and SLEDAI scores. In FIG. 16C depicts the circulating frequency of FLT3L+T cells in healthy donors (HD) and in SLE patients. In FIG. 16D shows the correlation between FLT3L+ CD4+ T cells and SLEDAI scores.

[0042] FIG. 17A-17C. FLT3L expression in CD4+ T cell subpopulations and SLEDAI scores. In FIG. 17A depicts the percentage of CD4 T _naive cells expressing FLT3L in HD and SLE patients. The lower panel shows the correlation of FLT3L-expressing CD4 T _naive cells in SLE patients compared to the SLEDAI score. In FIG. 17B shows the percentage of CD4 T _MEM cells expressing FLT3L in HD and SLE patients. The lower panel shows the correlation of FLT3L-expressing CD4 T _MEM cells in SLE patients compared with the SLEDAI score. In FIG. 17C depicts the percentage of CD4 T _CM cells expressing FLT3L in HD and SLE patients. The lower panel shows the correlation of FLT3L-expressing CD4 T _naive cells in SLE patients compared to the SLEDAI score.

[0043] FIG. 18A-18C. FLT3L expression in CD4 PBMC subpopulations from individuals with myositis. In FIG. 18A depicts the percentage of CD4 T _naive cells expressing FLT3L in HD and myositis patients. In FIG. 18B depicts the percentage of CD4 T _MEM cells expressing FLT3L in HD and myositis patients. In FIG. 18C shows the percentage of CD4 T _CM cells expressing FLT3L in HD and myositis patients.

[0044] FIG. 19A-19B. Rates of proteinuria and nephritis in MRL mice. In FIG. 19A shows the reduction in proteinuria 17 weeks after administration of anti-FLT3L antibody. In FIG. 19B depicts nephrite grade scores 18 weeks after anti-FLT3L antibody administration.

[0045] FIG. 20A-20C. Splenic dendritic cell populations in MRL mice. In FIG. 20A shows changes in CD11+ siglec-H+pDC frequency after administration of anti-FLT3L antibody. In FIG. 20B depicts the frequency of CD11c+CD11b+mDC following administration of an anti-FLT3L antibody. In FIG. 20C depicts the frequency of CD11c+CD8+mDC after administration of anti-FLT3L antibody.

[0046] FIG. 21A and 21B. Salivary gland pathology score in a mouse model of Sjögren's syndrome NOD.H2h4. In FIG. 21A shows changes in salivary gland pathology in the mouse model of Sjögren's syndrome NOD.H2h4 after administration of a therapeutic dose of anti-FLT3L antibody compared to isotype control. In FIG. 21B shows changes in salivary gland pathology in the mouse model of Sjögren's syndrome NOD.H2h4 after administration of a prophylactic dose of anti-FLT3L antibody compared to isotype control.

[0047] FIG. 22A-22D. Changes in the presence of dendritic cells after the introduction of antibodies to FLT3L. In FIG. 22A and 22B depict changes in the incidence of plasmacytoid DCs (B220+CD11c+Siglec-H+) after anti-FLT3L antibody administration as shown by flow cytometry (A) and quantification (B). In FIG. 22C and 22D depict changes in the incidence of classic DC (B220 ^neg CD11c ^HI ) after administration of anti-FLT3L antibody as shown by flow cytometry (C) and quantification (D).

[0048] FIG. 23A-23C. PK data for an anti-FLT3L antibody (MEDI1116) in cynomolgus monkeys correlated with functional neutralization of FLT3L, as evidenced by suppression and return of pDC. As shown by arrows, anti-FLT3L antibody (MEDI1116) was administered to cynomolgus monkeys once a week on days 1, 8, 15, 22, 29. FIG. 23A shows PK data for an anti-FLT3L antibody (MEDI1116). In FIG. 23B depicts soluble FLT3L levels at doses of anti-FLT3L antibody (MEDI1116) of 0.03 mg/kg, 1.0 mg/kg, and 30 mg/kg. In FIG. 23C depicts the incidence of pDC, measured as a percentage of baseline, at doses of anti-FLT3L antibody (MEDI1116) of 0.03 mg/kg, 1.0 mg/kg, and 30 mg/kg.

[0049] FIG. 24A-24C. The anti-FLT3L antibody (MEDI1116) human dosing model predicts Q4W dosing. In FIG. 24A depicts PK data for an anti-FLT3L antibody (MEDI1116) in cynomolgus monkeys. In FIG. 24B shows PD data for an anti-FLT3L antibody (MEDI1116) in cynomolgus monkeys. In FIG. 24C depicts predicted PD data for an anti-FLT3L antibody (MEDI1116) in humans.

[0050] FIG. 25A-25B. Monoclonal antibody to FLT3L (LFC-1) effectively neutralizes FLT3L throughout the course of treatment and leads to the accumulation of the drug/ligand complex in the bloodstream. In FIG. 25A depicts serum levels of free FLT3L following administration of anti-FLT3L antibody and isotype control antibody. Free FLT3L levels were measured using FT3L-IgG as capture and sulfo-labelled polyclonal anti-mouse FLT3L antibody as detection reagent. In FIG. 25B depicts serum levels of total (free and bound) FLT3L following administration of anti-FLT3L antibody and isotype control antibody. Total FLT3L levels were measured using a polyclonal anti-mouse FLT3L antibody for capture and detection.

[0051] FIG. 26A-26D. Blocking FLT3L suppresses T cell activation in the spleen and SG draining lymph node (LN) in elderly NOD-H2 ^h4 mice. Blocking FLT3L with the anti-FLT3L monoclonal antibody (LFC-1) resulted in a reduced incidence of CD44 ^HI antigen-exposed CD4+ and CD8+ T cells in the spleen and salivary draining LN (end of study at 24-26 weeks of age). Histograms were constructed based on the results of the analysis of flow cytometry of the spleen and draining LN. Each bar represents the mean +/- standard error of the mean (SEM) for n=4-5 mice. In FIG. 26A depicts CD44 ^HI CD4+ T cell populations in the spleen following administration of anti-FLT3L antibody and isotype control antibody. In FIG. 26B depicts CD44 ^HI CD4+ T cell populations in LN after administration of anti-FLT3L antibody and isotype control antibody. In FIG. 26C depicts CD44 ^HI CD8+ T cell populations in the spleen following administration of anti-FLT3L antibody and isotype control antibody. In FIG. 26D depicts CD44 ^HI CD8+ T cell populations in LN after administration of anti-FLT3L antibody and isotype control antibody.

[0052] FIG. 27. Therapeutic blocking with anti-FLT3L antibodies selectively reduces the specificities of two serum IgG autoantibodies. Corresponding serum samples were measured using the UTSW IgG autoantibody assay.

DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention provides isolated antibodies, or antigen-binding fragments thereof, that specifically bind to FLT3L. In some aspects, such molecules are antibodies and antigen-binding fragments thereof that specifically bind to FLT3L. In one embodiment, anti-FLT3L antibodies disclosed herein can be used to suppress or reduce FLT3/FLT3L binding to suppress the activation of inflammatory signaling pathways. This approach is advantageous because it targets the inflammation at the source of the signal, providing a stronger anti-inflammatory effect of the treatment. Related polynucleotides, vectors, pharmaceutical compositions containing anti-FLT3L antibodies or antigen-binding fragments thereof are also provided. Also provided are methods of making, as well as methods of using the anti-FLT3L antibodies and antigen-binding fragments disclosed herein, for example, methods of treating autoimmune and/or chronic inflammatory diseases in a subject (as direct therapy, adjuvant therapy, or in combination therapy).

[0054] For easier understanding of the present invention, some terms are defined first. Additional definitions are set forth in the detailed description.

[0055] Definitions

[0056] Before describing the present invention in detail, it should be understood that the present invention is not limited to specific compositions or process steps, as these may vary. Unless otherwise indicated, all technical and scientific terms used herein have the meanings commonly understood by a person skilled in the art to which the present invention pertains. The following references provide general definitions for many of the terms used in the present invention for those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ^ed . 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th ^Ed ., R. Rieger et al. (eds.), Springer Verlag (1991) and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings prescribed below, unless otherwise indicated.

[0057] The term "antibody" (or fragment, variant or derivative thereof) as used herein refers to at least the minimum portion of an antibody that is capable of binding to an antigen, e.g., at least the heavy chain variable domain (VH) and a light chain variable domain (VL) in the context of a typical antibody produced by a B cell. The basic structures of antibodies in vertebrate systems are relatively well understood. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ^ed ., 1988). Antibodies or antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, epitope-binding fragments (e.g., Fab, F(ab′) ₂ , Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments containing either a VL or VH domain (Fd), fragments generated by a Fab expression sequence library, and other antibody fragments and combinations thereof that retain antigen-binding function, i.e., the ability to specifically bind , for example, FLT3L.

[0058] A typical antibody contains at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH or V _H ) and a heavy chain constant region. The heavy chain constant region consists of three domains: CHI, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL or V _L ) and a light chain constant region. The light chain constant region consists of a single domain, which is CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with regions that are more conserved called framework regions (FWs). Each VH and VL consists of three CDRs and four FWs arranged from amino to carboxy in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains constitute the binding domain that interacts with the antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system. Exemplary antibodies of the present invention include anti-FLT3L antibodies (original and germline modified), affinity optimized clones, optimized antibodies without ADCC, conjugated antibodies (e.g., ADC), and other optimized antibodies (e.g., antibodies with optimized half-life). in serum, including for example YTE mutations, see Dall'Acqua et al., J Biol Chem 281:23514-24 (2006) and US Pat. No. 7,083,784, which are hereby incorporated by reference in their entirety) .

[0059] In certain embodiments, the VH CDRs (HCDR1, HCDR2, and HCDR3) and VL CDRs (LCDR1, LCDR2, and LCDR3) are composed of the amino acid sequences: (a) under SEQ ID NOs: 29, 30, 31, 32, 33, and 34 respectively; or (b) under SEQ ID NO: 29, 30, 31, 35, 33 and 34, respectively; or (c) under SEQ ID NO: 29, 36, 37, 32, 33 and 38, respectively.

[0060] An antibody can belong to any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their constant heavy chain domains called alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies may be "naked" or conjugated to other molecules, such as toxins, radioisotopes, etc., to form an ADC.

[0061] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen it binds, such as FLT3L. In a certain aspect, blocking antibodies or antagonistic antibodies significantly or completely inhibit the biological activity of the antigen. For example, FLT3L-mediated FLT3 activation can be reduced by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35% , at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, by at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or even 100%.

[0062] The terms "anti-FLT3L antibody", "antibody that binds to FLT3L", or "anti-FLT3L antibody" refer to an antibody or antigen-binding fragment thereof that is capable of binding FLT3L with sufficient affinity such that the molecule is useful as a therapeutic agent or diagnostic reagent with a targeted effect on FLT3L. The term "anti-FLT3L antibody" also broadly encompasses molecules containing, for example, the CDRs of the antibodies disclosed herein, included in a framework.

[0063] The term "germline modification" means that amino acids at certain positions in the antibody are mutated back to amino acids that occur at the germline level.

[0064] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. The variable region of each of the heavy and light chains consists of four FW regions connected by three CDR regions. The CDRs in each chain are held together in close proximity by the FW regions and, together with the CDRs from the other chain, contribute to the formation of an antibody antigen-binding site. There are at least two methods for determining CDRs: (1) an approach based on interspecies sequence variability (i.e. Kabat et al. Sequences of Proteins of Immunological Interest, (5th ^ed ., 1991, National Institutes of Health, Bethesda Md .)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948)). In addition, combinations of the two approaches are sometimes used in the art to determine CDR.

[0065] The Kabat numbering system is generally used to denote a residue in a variable domain (about light chain residues 1-107 and heavy chain residues 1-113) (e.g., Kabat et al., Sequences of Immunological Interest, 5th ^Ed . Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0066] The phrases "numbering amino acid positions according to Kabat" or "position according to Kabat" and the like. refer to the numbering system used for heavy chain variable domains or light chain variable domains of an antibody compilation in Kabat et al., 1991).

[0067] The terms "antigen-binding domain", "antigen-binding fragment", and "binding fragment" refer to the portion of an antibody molecule that contains the amino acids responsible for the specific binding of an antibody to an antigen. The variable region allows an antibody or antigen-binding fragment to selectively recognize and specifically bind epitopes on antigens. That is, the VH and VL domain or a subset of complementarity determining regions (CDRs) of an antibody are combined to form a variable region that defines a three-dimensional antigen binding site. More specifically, the antigen binding domain is defined by three CDRs on each of the VH and VL chains. The portion of an antigen molecule that is responsible for specific interactions with an antigen-binding domain, as used herein, is referred to as an "epitope". An antigen binding domain typically contains an antibody light chain variable region and an antibody heavy chain variable region, however, it does not always include both. For example, the so-called "Fd" antibody fragment consists only of the domain VH, but still retains some of the antigen-binding function of the intact antibody.

[0068] Binding antibody fragments are obtained using recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv and single chain antibodies. Cleavage of antibodies with an enzyme called papain results in two identical antigen-binding fragments, also known as "Fab" fragments, and a "Fc" fragment, which has no antigen-binding activity but is capable of crystallization. Cleavage of antibodies with an enzyme called pepsin results in an F(ab')2 fragment in which the two arms of the antibody molecule remain linked and contain two antigen-binding sites. F(ab')2 fragment has the ability to crosslink the antigen. "Fv" as used herein refers to the minimum antibody fragment that retains both antigen recognition and antigen binding sites. "Fab" as used herein refers to an antibody fragment that contains a light chain constant domain and a heavy chain CH1 domain.

[0069] The Fc region, as used herein, includes polypeptides containing the constant region of an antibody, with the exception of the first immunoglobulin domain of the constant region. Thus, Fc refers to the last two domains of the constant region of immunoglobulins IgA, IgD and IgG and to the last three domains of the constant region of immunoglobulins IgE and IgM, as well as to the flexible hinge, which is located at the N-terminus of these domains. In the case of IgA and IgM, the Fc may include a J chain. In the case of IgG, Fc contains the immunoglobulin domains C-gamma-2 and C-gamma-3 (Cy2 and Cy3) and a hinge between C-gamma-1 (Cy1) and C-gamma-2 (Cy2). Although the boundaries of the Fc region may vary, the Fc region of the human IgG heavy chain is usually defined as containing C226 or P230 residues towards its carboxyl terminus, with the numbering following the EU system as given in Kabat et al., 1991.

[0070] "Monoclonal antibody" refers to a homogeneous population of antibodies involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This distinguishes them from polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants.

[0071] The term "monoclonal antibody" encompasses both intact and full length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain variable fragments (scFv), fusion proteins, containing part of the antibody and any other modified immunoglobulin molecule containing antigen recognition site. Moreover, "monoclonal antibody" refers to those antibodies that are produced by any of a number of methods, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals (eg, expression of a human antibody in a transgenic mouse).

[0072] The term "humanized antibody" refers to an antibody derived from a non-human (eg, mouse) immunoglobulin that has been engineered to increase similarity to antibody variants produced in humans.

[0073] The term "human antibody" means an antibody produced by a human body, or an antibody characterized by an amino acid sequence corresponding to a human-produced antibody, obtained using a technique known in the art (for example, recombinant expression in cell cultures or expression in transgenic animals ). Thus, the term human antibody also encompasses an antibody having an amino acid sequence corresponding to an antibody that was originally produced by the human body (or an engineered variant or derivative thereof) but that is expressed in a system other than humans (e.g., produced by chemical synthesis; recombinantly expressed microbial, mammalian, or insect cells; or expressed in the body of an animal subject). Accordingly, a human antibody is defined as an antibody derived from a human subject or from human cells (eg, a hybridoma or a cell line expressing a recombinant antibody or fragment thereof) and subsequently expressed in an animal body, eg, mice. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies containing at least one human heavy and/or light chain polypeptide, such as, for example, an antibody containing mouse light chain and human heavy chain polypeptides.

[0074] The term "chimeric antibodies" refers to antibodies where the amino acid sequence of the immunoglobulin molecule is derived from two or more animal species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from a single mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity and/or affinity and/or ability , while the constant regions are homologous to sequences in antibodies derived from another species (usually human) in order to avoid the development of an immune response in that species.

[0075] The term "polynucleotide" is intended to encompass a single nucleic acid as well as a collection of nucleic acids and refers to an isolated nucleic acid molecule or a construct based on it, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The polynucleotide may comprise a conventional phosphodiester bond or an unconventional bond (eg, an amide bond such as that found in peptido nucleic acids (PNAs)). The term "nucleic acid" refers to one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is meant a nucleic acid molecule, i. DNA or RNA that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide subunit contained in a vector is considered isolated as disclosed herein. Additional examples of an isolated polynucleotide include recombinant polynucleotides contained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides. Isolated polynucleotides or nucleic acids further include those molecules that are obtained by synthesis. In addition, the polynucleotide or nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or transcription terminator.

[0076] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide containing a nucleic acid that encodes a polypeptide may normally include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. A functional association or relationship occurs when the coding region of a gene product, such as a polypeptide, is associated with one or more regulatory sequences in such a manner as to place the expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide-coding region and its associated promoter) are "operably associated" or "operably linked" if induction of the promoter function results in transcription of the mRNA encoding the desired gene product, and if the nature of the link between the two DNA fragments does not negative effect on the ability of the expression control sequences to direct the expression of the gene product, or no negative effect on the ability to transcribe the template DNA. Thus, a promoter region will be operably associated with a nucleic acid encoding a polypeptide if the promoter is capable of transcribing that nucleic acid. The promoter may be a cell type-specific promoter that drives essential transcription of DNA only in the desired cells. In addition to the promoter, other transcriptional control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably linked to the polynucleotide to drive cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

[0077] In other embodiments, the implementation of the polynucleotide may be RNA, for example, in the form of messenger RNA (mRNA).

[0078] A "vector" is a nucleic acid molecule introduced into a host cell, thereby producing a transformed host cell. The vector may include nucleic acid sequences that allow it to replicate in the host cell, such as an origin of replication. The vector may also include one or more selectable marker genes and other genetic elements known in the art.

[0079] A "transformed" cell or "host cell" is a cell into which a nucleic acid molecule has been introduced using molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of deproteinized DNA by electroporation, lipofection, and particle insertion accelerated by a gene gun. . The transformed cell or host cell may be a bacterial cell or a eukaryotic cell.

[0080] As used herein, the term "FLT3L" refers to the McDonough feline sarcoma virus (FMS) tyrosine kinase 3 proto-oncogene product-like ligand, which is a polypeptide that is a hematopoietic cytokine that binds to the FMS-like receptor tyrosine kinase 3 (FLT3) receptor. ). FLT3L is initially expressed as a membrane-bound protein before being cleaved by enzymes into a soluble form. The definition of FLT3L includes both membrane bound (mFLT3L) and secreted (sFLT3L).

[0081] As used herein, "provides", "providing", "comprising", and "having", etc., may have the meanings assigned to them in the US Patent Law and may mean "includes", "including", etc. P.; "substantially consisting of" or "substantially consisting of" similarly has the meaning prescribed in the US Patent Law, and the term is open-ended, allowing for more elements than those specifically listed, provided that the essential or novel characteristics of what is what is listed does not change when there are more items than listed, but excludes prior art embodiments.

[0082] As used herein, the terms "definition", "assessment", "analysis", "measurement" and "detection" refer to both quantitative and qualitative determinations, and therefore the term "determination" in this document can be used interchangeably with the terms "analysis", "measurement", etc. If quantification is meant, the phrase "quantitation" of the analyte and the like can be used. If qualitative and/or quantitative determination is implied, the phrase "determining the level" of the analyte or "detection" of the analyte is used.

[0083] The terms "identical" or "percent identity" in the context of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that are the same or are characterized by a certain percentage of nucleotides or amino acid residues that are the same when compared and aligned ( with the introduction of gaps, if necessary) to achieve maximum match without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual assessment. Various algorithms and software are known in the art that can be used to obtain amino acid or nucleotide sequence alignment results (see, for example, Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, which modified in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and included in the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402) In certain embodiments, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology , 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, CA) or Megalign (DNASTAR).

[0084] The term "isolated" refers to a molecule that is not in its natural environment. No specific level of purification is required. For example, an isolated antibody is one that is not produced or found in its natural or natural environment. Biological materials produced by a recombinant process are considered isolated as disclosed herein, as are materials that are produced in a non-naturally occurring cell, such as a hybridoma. A substance, such as an isolated protein such as an antibody, is also considered "isolated" if it has been separated, fractionated, or partially or substantially purified by any suitable technique. For example, an antibody is considered "isolated" if it is substantially free of cellular material or other proteins from the cellular or tissue source from which it originates.

[0085] The term "specifically binds" refers to an agent (for example, a ligand or antibody) that recognizes and binds a molecule (for example, a receptor or epitope), and such binding is determined by some complementarity between the agent (for example, an antibody) and the molecule ( like a ligand). Under this definition, an antibody is said to "specifically bind" to a ligand if it binds to that ligand more easily than it would to a random unrelated molecule. The term "specificity" in this document is used for a qualitative assessment of the relative affinity with which a particular antibody binds to a particular ligand. For example, antibody "A" may be considered to have higher specificity for a given ligand (eg, FLT3L) than antibody "B".

[0086] As used herein, the term "affinity" refers to a measure of the binding strength of an individual epitope to the CDR of an antibody. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press), 2nd ^ed . 1988) on pages 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of antibodies and an antigen, that is, the functional strength of association of a mixture of antibodies with an antigen. See, for example, Harlow on pages 39-34. Avidity is related both to the affinity of individual antibodies in a population with particular epitopes, and to the valence of antibodies and antigen.

[0087] The terms "inhibit" or "block" are used interchangeably herein and refer to any statistically significant decrease in biological activity, including complete blockage of activity. For example, "inhibition" may mean a reduction in biological activity of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0088] The term "effector function" refers to the activities of antibodies that result from the interaction of their Fc components with Fc receptors or components of the complement system. Such activities include, for example, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). Thus, an antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) with an altered effector function means an antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) that contains a change in the Fc region (e.g., an amino acid substitution, deletion, or addition or change in oligosaccharide) that alters the activity of at least one effector function (eg ADCC, CDC and/or ADCP). Antigen-binding protein (for example, an antibody or antigen-binding fragment thereof) with improved effector function means an antigen-binding protein (for example, an antibody or antigen-binding fragment thereof) that contains a change in the Fc region (for example, an amino acid substitution, deletion, or addition or change in an oligosaccharide), which increases the activity of at least one effector function (eg ADCC, CDC and/or ADCP).

[0089] The term "subject" refers to any animal (eg, mammal), including without limitation humans, non-human primates, rodents, and the like, that is to be the recipient of a particular treatment. Generally, the terms "subject" and "patient" and "individual" are used interchangeably herein. Additional examples of subjects include non-human mammals such as those of cattle, horses, dogs, sheep, or cats.

[0090] The term "pharmaceutical composition" refers to a preparation that is presented in a form that provides the effectiveness of the biological activity of the active ingredient (for example, disclosed in this document antibodies to FLT3L) and which does not contain additional components that are unacceptably toxic to the subject who will be enter this song. Such a composition may be sterile.

[0091] An "effective amount" of an anti-FLT3L antibody disclosed herein is an amount sufficient to achieve the stated purpose. An "effective amount" can be determined empirically and by a standard method, depending on the claimed purpose.

[0092] The terms "therapeutically effective amount" and "pharmaceutically effective amount" refer to the amount of an anti-FLT3L antibody or other drug disclosed herein that is effective to "treat" a disease or disorder in a subject.

[0093] Terms such as "administering treatment" or "treatment" or "treat" or "reducing" or "reducing" refers to both (1) therapeutic measures that provide cure, slow down, reduce symptoms and/or stopping the progression of the diagnosed condition or disorder, and (2) prophylactic or preventive measures that prevent and/or slow the development of the target condition or disorder. Thus those in need of treatment include those who already have the disorder; those who are prone to developing the disorder; and those who need to prevent the development of the disorder. In certain aspects, a subject is successfully "treated" for an autoimmune or inflammatory disease in accordance with the methods of the present invention if the patient experiences, for example, a complete, partial, or temporary reduction in symptoms associated with the autoimmune or inflammatory disease.

[0094] The ranges presented herein are understood as shorthand for all values within the range. For example, the range from 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50.

[0095] As used herein, the terms "treat," "treat," "treat," and the like. refer to the reduction and/or alleviation of the disorder and/or associated symptoms. It will be understood, although not excluded, that the treatment of a disorder or condition does not need to completely eliminate the disorder, condition, or associated symptoms. For example, as provided herein, treating a disorder includes preventing the symptoms of the disorder from exacerbating.

[0096] As used herein, the term "or" is understood to be inclusive unless otherwise specifically indicated or evident from the context. As used herein, the singular forms are understood to mean the singular or plural forms, unless otherwise specifically indicated or evident from the context.

[0097] Moreover, "and/or" when used herein should be considered as a specific disclosure of each of the two or more specified features or components, with or without the other component. Thus, the term "and/or" used in a phrase such as "A and/or B" is herein meant to include "A and B", "A or B", "A" (individually), and "B" (individually). Likewise, the term "and/or" used in a phrase such as "A, B and/or C" is meant to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; as well as A (separately); B (individually); and also C (separately).

[0098] Unless otherwise noted or otherwise apparent from the context, the term "about" as used herein is understood to be within the range of normal tolerances in the art, such as within 2 standard deviations of the mean. "Approximately" can be understood as deviating up or down within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0 .1%, 0.05%, or 0.01% of the specified value. Unless otherwise indicated, all numerical values given in this document are considered to be modified by the term "approximately" by implication.

[0099] FLT3L is initially expressed as a membrane-bound protein, before being cleaved by enzymes into a soluble form. Both the membrane bound (mFLT3L) and secreted (sFLT3L) forms are functionally active. The FLT3L binding region is so highly conserved across species that there is cross-species reactivity observed among ligand/receptor combinations in humans, rodents, and cynomolgus monkeys. However, the lack of cross-species reactivity in neutralizing antibodies produced against FLT3L is believed to be due to key mutations around the binding site. Neutralizing antibodies to FLT3L can affect classical and plasmacytoid DC populations by reducing the ability of the immune system to induce and maintain a prolonged inflammatory response. Secondary effects may include a decrease in NK cells in the circulation and reduced activation of T and B cells, resulting in decreased survival of both cell types. Collectively, downregulating these signaling pathways can reduce autoimmune inflammation.

[00100] In one embodiment, it is provided that a neutralizing antibody to FLT3L promotes immune homeostasis by suppressing the binding of FLT3L to FLT3. The strategy of using anti-FLT3L antibodies is directed to the ligand and not to the receptor in order to avoid the risk of unexpected receptor dimerization or signaling from it. Unlike its receptor, membrane-bound FLT3L lacks a signaling domain associated with it.

[00101] Antibodies to FLT3L

[00102] In a preferred embodiment, the present invention provides isolated FLT3L binding molecules, eg antibodies and antigen binding fragments thereof, that specifically bind FLT3L, eg human FLT3L. The full length amino acid and nucleotide sequences for FLT3L are known in the art (see, for example, UniProt Access # P36888 for human FLT3L or UniProt Access # Q00342 for mouse FLT3L). Anti-FLT3L antibodies of the present invention inhibit FLT3L-mediated FLT3 activation and thus reduce pro-inflammatory signaling and reduce inflammation in a subject.

[00103] In a preferred embodiment, anti-FLT3L antibodies do not cross-react with structurally similar TKR homologs of human stem cell factor (huSCF) or human colony stimulating factor (huCSF1). One skilled in the art will appreciate that SCF and CSF are ligands that also bind receptor tyrosine kinases. Non-specific inhibitors of FLT3 that bind additional members of the tyrosine kinase family cause toxicity due to global downregulation of tyrosine kinase signaling. Accordingly, it is critical that anti-FLT3L antibodies bind only FLT3L and not structurally similar homologues. Many antibodies to FLT3L and its inhibitors are not specific and bind a wide range of receptor tyrosine kinases. Thus, in a preferred embodiment, the anti-FLT3L antibody should show high affinity for FLT3L and specific binding thereof.

[00104] In one embodiment, the FLT3L antibodies of the present invention are monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and/or chimeric antibodies.

[00105] In some aspects, FLT3L binding molecules include Fab, Fab', F(ab') ₂ , Fd, single chain Fv or scFv, Fv disulfide-linked, V-NAR domain, IgNar, intraantibody, CH2 IgG, minibody, F (ab') ₃ , tetrabody, tribody, diabody, single domain antibody, DVD-Ig, Fcab, mAb ² , (scFv) ₂ or scFv-Fc. In some aspects, the anti-FLT3L antibodies are of an IgG type, such as an IgG1 type (comprises the IgG1 immunoglobulin heavy chain constant domain). In other embodiments, the anti-FLT3L antibodies have an immunoglobulin heavy chain constant domain of IgA, IgD, IgE, IgG2, IgG3, IgG4, or IgM.

[00106] In some embodiments, the IgG constant region may comprise a light chain constant region selected from the group consisting of an Ig kappa chain constant domain (region) and an Ig lambda chain constant domain. In one specific embodiment, the anti-FLT3L antibodies comprise a human IgG1 constant domain and a human lambda chain constant domain. In another specific embodiment, anti-FLT3L antibodies are in the IgG1-TM format such that targeted mutations in the Fc region result in the replacement of leucine at position 243 with phenylalanine (L243F), leucine at position 235 with glutamic acid (L235E) and proline at position 331 for serine (P331S); while the numbering of amino acids is given in accordance with the EU system. Such targeted mutations reduce FcR binding and ADCC effector function (see Organesyan et al., Acta Crystallogr D Biol Crystallogr. 2008 Jun 1; 64(Pt 6): 700-4 and WO 2009100309 A2, which is incorporated by reference).

[00107] In some aspects, anti-FLT3L antibodies are human antibodies (eg, CAT5D9, SC4017, AM40, CAT8, CAT26, DTAX3, and DYAX5 antibodies).

[00108] CAT5D9 antibody

[00109] In one embodiment, a CAT5D9 antibody refers to an antibody that specifically binds to FLT3L and provides complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 provide for the amino acid sequences of SEQ ID NOs: 29, 36, 37, 32, 33 and 38, respectively.

[00110] In another embodiment, a CAT5D9 antibody refers to an antibody that specifically binds to FLT3L and provides two VL domains characterized by at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence under SEQ ID NO: 6, and two VH domains characterized by at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence under SEQ ID NO: 5.

[00111] In a further embodiment, a CAT5D9 antibody refers to an antibody that provides two VL domains characterized by the amino acid sequence of SEQ ID NO: 6 and two VH domains characterized by the amino acid sequence of SEQ ID NO: 5.

[00112] In another embodiment, a CAT5D9 antibody refers to an antibody that provides two VL domains encoded by the nucleic acid sequence of SEQ ID NO: 20 and two VH domains encoded by the nucleic acid sequence of SEQ ID NO: 19.

[00113] In one embodiment, a CAT5D9 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises a light chain characterized by the amino acid sequence of SEQ ID NO: 70 and a heavy chain characterized by the amino acid sequence of SEQ ID NO: 69.

[00114] In another embodiment, a CAT5D9 antibody refers to an antibody that provides a light chain encoded by the nucleic acid sequence of SEQ ID NO: 72 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 71.

[00115] SC4017 Antibody

[00116] In one embodiment, the SC4017 antibody refers to an antibody that specifically binds to FLT3L and provides complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 provide for the amino acid sequences of SEQ ID NOs: 29, 30, 31, 35, 33 and 34, respectively.

[00117] In another embodiment, the SC4017 antibody refers to an antibody that specifically binds to FLT3L and provides two VL domains characterized by at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence under SEQ ID NO: 4, and two VH domains characterized by at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence under SEQ ID NO: 3.

[00118] In a further embodiment, the SC4017 antibody refers to an antibody that provides two VL domains characterized by the amino acid sequence of SEQ ID NO: 4 and two VH domains characterized by the amino acid sequence of SEQ ID NO: 3.

[00119] In another embodiment, the SC4017 antibody refers to an antibody that provides two VL domains encoded by the nucleic acid sequence of SEQ ID NO: 18 and two VH domains encoded by the nucleic acid sequence of SEQ ID NO: 17.

[00120] In one embodiment, the SC4017 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises a light chain characterized by the amino acid sequence of SEQ ID NO: 66 and a heavy chain characterized by the amino acid sequence of SEQ ID NO: 65.

[00121] In another embodiment, the SC4017 antibody refers to an antibody that provides a light chain encoded by the nucleic acid sequence of SEQ ID NO: 68 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 67.

[00122] AM40 antibody (MEDI1116)

[00123] In one embodiment, an AM40 antibody refers to an antibody that specifically binds to FLT3L and provides complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 provide for the amino acid sequences of SEQ ID NOs: 29, 30, 31, 32, 33 and 34, respectively.

[00124] In another embodiment, an AM40 antibody refers to an antibody that specifically binds to FLT3L and provides two VL domains characterized by at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence under SEQ ID NO: 2, and two VH domains characterized by at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence under SEQ ID NO: 1.

[00125] In a further embodiment, an AM40 antibody refers to an antibody that provides two VL domains characterized by the amino acid sequence of SEQ ID NO: 2 and two VH domains characterized by the amino acid sequence of SEQ ID NO: 1.

[00126] In another embodiment, an AM40 antibody refers to an antibody that provides two VL domains encoded by the nucleic acid sequence of SEQ ID NO: 16 and two VH domains encoded by the nucleic acid sequence of SEQ ID NO: 15.

[00127] In one embodiment, the AM40 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises a light chain characterized by the amino acid sequence of SEQ ID NO: 62 and a heavy chain characterized by the amino acid sequence of SEQ ID NO: 61.

[00128] In another embodiment, an AM40 antibody refers to an antibody that provides a light chain encoded by the nucleic acid sequence of SEQ ID NO: 64 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 63.

[00129] CAT8 antibody

[00130] In one embodiment, a CAT8 antibody refers to an antibody that specifically binds to FLT3L and provides complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 provide for the amino acid sequences of SEQ ID NOs: 39, 40, 41, 42, 43 and 44, respectively.

[00131] In another embodiment, a CAT8 antibody refers to an antibody that specifically binds to FLT3L and provides two VL domains characterized by at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence under SEQ ID NO: 8, and two VH domains characterized by at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence under SEQ ID NO: 7.

[00132] In a further embodiment, a CAT8 antibody refers to an antibody that provides two VL domains characterized by the amino acid sequence of SEQ ID NO: 8 and two VH domains characterized by the amino acid sequence of SEQ ID NO: 7.

[00133] In another embodiment, a CAT8 antibody refers to an antibody that provides two VL domains encoded by the nucleic acid sequence of SEQ ID NO: 22 and two VH domains encoded by the nucleic acid sequence of SEQ ID NO: 21.

[00134] In one embodiment, a CAT8 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises a light chain characterized by the amino acid sequence of SEQ ID NO: 74 and a heavy chain characterized by the amino acid sequence of SEQ ID NO: 73.

[00135] In another embodiment, a CAT8 antibody refers to an antibody that provides a light chain encoded by the nucleic acid sequence of SEQ ID NO: 76 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 75.

[00136] CAT26 antibody

[00137] In one embodiment, a CAT26 antibody refers to an antibody that specifically binds to FLT3L and provides complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 provide for the amino acid sequences of SEQ ID NOs: 45, 40, 46, 47, 48 and 49, respectively.

[00138] In another embodiment, a CAT26 antibody refers to an antibody that specifically binds to FLT3L and provides two VL domains characterized by at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence under SEQ ID NO: 10, and two VH domains characterized by at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence under SEQ ID NO: 9.

[00139] In a further embodiment, a CAT26 antibody refers to an antibody that provides two VL domains characterized by the amino acid sequence of SEQ ID NO: 10 and two VH domains characterized by the amino acid sequence of SEQ ID NO: 9.

[00140] In another embodiment, a CAT26 antibody refers to an antibody that provides two VL domains encoded by the nucleic acid sequence of SEQ ID NO: 24 and two VH domains encoded by the nucleic acid sequence of SEQ ID NO: 23.

[00141] In one embodiment, a CAT26 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises a light chain characterized by the amino acid sequence of SEQ ID NO: 78 and a heavy chain characterized by the amino acid sequence of SEQ ID NO: 77.

[00142] In another embodiment, a CAT26 antibody refers to an antibody that provides a light chain encoded by the nucleic acid sequence of SEQ ID NO: 80 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 79.

[00143] DYAX3 antibody

[00144] In one embodiment, a Dyax3 antibody refers to an antibody that specifically binds to FLT3L and provides complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 provide for the amino acid sequences of SEQ ID NOs: 50, 51, 52, 53, 54 and 55, respectively.

[00145] In another embodiment, a Dyax3 antibody refers to an antibody that specifically binds to FLT3L and provides two VL domains characterized by at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence under SEQ ID NO: 82, and two VH domains having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 81.

[00146] In a further embodiment, a Dyax3 antibody refers to an antibody that provides two VL domains characterized by the amino acid sequence of SEQ ID NO: 12 and two VH domains characterized by the amino acid sequence of SEQ ID NO: 11.

[00147] In another embodiment, a Dyax3 antibody refers to an antibody that provides two VL domains encoded by the nucleic acid sequence of SEQ ID NO: 26 and two VH domains encoded by the nucleic acid sequence of SEQ ID NO: 25.

[00148] In one embodiment, the Dyax3 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises a light chain characterized by the amino acid sequence of SEQ ID NO: 82 and a heavy chain characterized by the amino acid sequence of SEQ ID NO: 81.

[00149] In another embodiment, a Dyax3 antibody refers to an antibody that provides a light chain encoded by the nucleic acid sequence of SEQ ID NO: 84 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 83.

[00150] DYAX5 antibody

[00151] In one embodiment, a Dyax5 antibody refers to an antibody that specifically binds to FLT3L and provides complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 provide for the amino acid sequences of SEQ ID NOs: 56, 57, 52, 58, 59 and 60, respectively.

[00152] In another embodiment, a Dyax5 antibody refers to an antibody that specifically binds to FLT3L and provides two VL domains characterized by at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence under SEQ ID NO: 86, and two VH domains having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 85.

[00153] In a further embodiment, a Dyax5 antibody refers to an antibody that provides two VL domains characterized by the amino acid sequence of SEQ ID NO: 14 and two VH domains characterized by the amino acid sequence of SEQ ID NO: 13.

[00154] In another embodiment, a Dyax5 antibody refers to an antibody that provides two VL domains encoded by the nucleic acid sequence of SEQ ID NO: 28 and two VH domains encoded by the nucleic acid sequence of SEQ ID NO: 27.

[00155] In one embodiment, the Dyax5 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises a light chain characterized by the amino acid sequence of SEQ ID NO: 86 and a heavy chain characterized by the amino acid sequence of SEQ ID NO: 85.

[00156] In another embodiment, a Dyax5 antibody refers to an antibody that provides a light chain encoded by the nucleic acid sequence of SEQ ID NO: 88 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 87.

[00157] In certain embodiments, an antibody or antigen-binding fragment thereof is provided that specifically binds to FLT3L, comprising a group of complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contain the amino acid sequences: (a) under SEQ ID NOs: 29, 30, 31, 32, 33 and 34, respectively; or (b) under SEQ ID NO: 29, 30, 31, 35, 33 and 34, respectively; or (c) under SEQ ID NO: 29, 36, 37, 32, 33 and 38, respectively.

[00158] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), where each of the VH and VL contains three CDRs and four framework regions (FW) located from the amino terminus to carboxy terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3 and FW4.

[00159] In certain aspects, the VH and VL regions are characterized by an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with: (a) SEQ ID NO: 1 and SEQ ID NO: 2 respectively; or (b) SEQ ID NO: 3 and SEQ ID NO: 4, respectively; or (c) SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[00160] In certain aspects, the VH CDRs (HCDR1, HCDR2, and HCDR3) and VL CDRs (LCDR1, LCDR2, and LCDR3) are composed of the amino acid sequences: (a) under SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively ; or (b) under SEQ ID NO: 29, 30, 31, 35, 33 and 34, respectively; or (c) under SEQ ID NO: 29, 36, 37, 32, 33 and 38, respectively.

[00161] Table 1 provides a summary table of anti-FLT3L antibody sequences.

[00162] Table 1. Summary Table of Anti-FLT3L Antibody Sequences

Table 1A

Name of the antibody VH (amino acid) VL (amino acid) AM40 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYALSWVRQAPGQGLEWMGTRPPTSRTASYAQKFQGRVTITVDESTSTGYMELSSLRSED TAVYYCASNDFVYGSYRFWGQGTTVTVSSA
(SEQ ID NO: 1) NFMLTQPHSVSESPGKTVTISCTRTSGNIAGYFVQWYQQRPGSSPTTVIYEDYQRPSGVPDRFSGSIDSSSSNSASLTISGLKTEDEADYYCQSYDDYRRAAFGGGTKLTVL
(SEQ ID NO: 2) SC4017 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYALSWVRQAPGQGLEWMGTRPPTSRTASYAQKFQGRVTITVDESTSTGYMELSSLRSEDTAVYYCASNDFVYGSYRFWGQGTTVTVSS
(SEQ ID NO: 3) NFMLTQPHSVSESPGKTVTISCTRTSGWIAGYFVQWYQQRPGSSPTTVIYEDYQRPSGVPDRFSGSIDSSSSNSASLTISGLKTEDEADYYCQSYDDYRRAAFGGGTKLTVL
(SEQ ID NO: 4) CAT5D9 QVQLVQSGAEVKKPGSSVKVSCKISGGTFSSYALSWVRQAPGQGLEWMGGIIPVFRTASYAQKFQGRVTITVDESASTGYIELSSLKSEDTATYYCASNNYVWGSYRFWGQGTTVTVSS
(SEQ ID NO: 5) NFMLTQPHSVSESPGKTVTISCTRTSGNIAGYFVQWYQQRPGSSPTTVIYEDYQRPSGVPDRFSGSIDRSSSNSASLTISGLKPDDEADYYCQSYDDTSQGVFGAGTKVTVL
(SEQ ID NO: 6) CAT8 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSGYYGANFDFWGQGTTVTVSS
(SEQ ID NO: 7) QSVLTQPPSASGTPGQRVAISCSGSSSNIGSGYVYWYQQVPGTAPTLLIHRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGYVFGTGTKVTV
(SEQ ID NO: 8) CAT26 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAVSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDAYGSSWYFYYFDYWGQGTMVTVSS
(SEQ ID NO: 9) QSVLTQPPSASGTPGQRVTISCSGSSSNIGINPVNWYQQLPGTAPKVLIYSDKYRPSGVADRFSGSKSGTSASLAISGLQSEDEADYFCAAWDDSLNGRVFGTGTKLTVL
(SEQ ID NO: 10) Dyax3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSMYEMRWVRQAPGKGLEWVSVIPSGGKTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYSRWFGQLGFYSHYAMDVWSQGTTVTVSS
(SEQ ID NO: 11) DIQMTQSPSSLSASVGDRVAITCRASQSIDTYLNWYQQKPGKAPKLLIYAASKLEDGVPSRFSGSGTGTDFTLTIRSLQPEDFASYFCQQSYSSPGITFGPGTKVEIK
(SEQ ID NO: 12) Dyax5 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMVWVRQAPGKGLEWVSSIYSSGGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRYSRWFGQLGFYSHYAMDVWSQGTTVTVSS
(SEQ ID NO: 13) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPWTFGQGTKVEIK
(SEQ ID NO: 14)

TABLE 1B

Name of the antibody VH (DNA) VL (DNA) AM40 caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttcagcagttatgctcttagctgggtgcgacaggcccctggacaagggcttgagtggatgggaacgcggccgccgacctcccggacagcaagctacgcacagaaatttcagggcagagtcacgattaccgtggacgaatccacgagcacaggctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgtcaaacgacttcgtgtacgggagttatcgtttctggggccaagggaccacggtcaccgtctcctcagcg
(SEQ ID NO: 15) aattttatgctgactcagccccactctgtgtcggagtctccggggaagacggtaaccatctcctgcacccgcaccagtgggaacattgccggctactttgtgcagtggtaccagcagcgcccgggcagttcccccaccactgtgatctatgaggattaccaacgaccctctggggtccctgatcggttctctggctccatcgacagctcctccaactctgcctccctcaccatctctggactgaagactgaggacgaggctgactactattgtcagtcttatgatgactaccggcgggcggcgttcggcggagggaccaagctgaccgtccta
(SEQ ID NO: 16) SC4017 Caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttcagcagttatgctcttagctgggtgcgacaggcccctggacaagggcttgagtggatgggaacgcggccgccgacctcccggacagcaagctacgcacagaaatttcagggcagagtcacgattaccgtggacgaatccacgagcacaggctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgtcaaacgacttcgtgtacgggagttatcgtttctggggccaagggaccacggtcaccgtctcctca
(SEQ ID NO: 17) Aattttatgctgactcagccccactctgtgtcggagtctccggggaagacggtaaccatctcctgcacccgcaccagtgggtggattgccggctactttgtgcagtggtaccagcagcgcccgggcagttcccccaccactgtgatctatgaggattaccaacgaccctctggggtccctgatcggttctctggctccatcgacagctcctccaactctgcctccctcaccatctctggactgaagactgaggacgaggctgactactattgtcagtcttatgatgactaccggcgggcggcgttcggcggagggaccaagctgaccgtccta
(SEQ ID NO: 18) CAT5D9 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGATTTCTGGAGGCACCTTCAGCAGTTATGCTCTTAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTGTCTTTCGGACAGCAAGCTACGCACAGAAATTTCAGGGCAGAGTCACGATTACCGTGGACGAATCCGCGAGCACAGGCTACATAGAACTGAGCAGCCTGAAATCTGAGGACACGGCCACATATTACTGTGCGTCAAATAATTACGTTTGGGGGAGTTATCGTTTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCA
(SEQ ID NO: 19) AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTCACCATCTCCTGCACCCGCACCAGTGGGAACATTGCCGGCTACTTTGTGCAGTGGTACCAGCAGCGCCCGGGCAGTTCCCCCACCACTGTGATCTATGAGGATTACCAACGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGGTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGCCTGACGACGAGGCTGACTACTATTGTCAGTCTTATGATGACACCTCTCAAGGTGTGTTCGGCGCAGGGACCAAGGTCACCGTCCTA
(SEQ ID NO: 20) CAT8 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAAGCAGCGGCTACTACGGGGCCAATTTTGACTTCTGGGGGCAGGGGACCACGGTCACCGTCTCGAGT
(SEQ ID NO: 21) CAGTCTGTGCTGACGCAGCCGCCCTCAGCGTCCGGGACCCCCGGGCAGAGGGTCGCCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTGGTTATGTATACTGGTATCAGCAGGTCCCAGGAACGGCCCCCACACTCCTCATCCATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCGTGGGATGACAGCCTGAGTGGTTATGTCTTCGGAACTGGGACCAAGGTCACCGTC
(SEQ ID NO: 22) CAT26 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCGTGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGTGAAAGACGCATATGGCAGCAGCTGGTACTTTTACTACTTTGACTACTGGGGCCAAGGGACAATGGTCACCGTCTCGAGT
(SEQ ID NO: 23) CAGTCTGTGTTGACGCAGCCGCCTTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAATCAATCCTGTGAACTGGTACCAACAACTCCCCGGAACGGCCCCCAAAGTCCTCATTTATAGTGATAAATACCGGCCCTCAGGGGTCGCTGACCGCTTCTCTGGCTCCAAGTCTGGAACCTCAGCCTCCCTGGCCATCAGTGGCCTCCAGTCTGAGGATGAGGCTGATTACTTCTGTGCAGCATGGGATGACAGCCTGAATGGTCGCGTCTTCGGAACTGGGACCAAGCTGACCGTCCTA
(SEQ ID NO: 24) Dyax3 GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTATGTACGAGATGCGTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTGTTATCCCTTCTGGTGGCAAGACTTTTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACGGCCGTGTATTACTGTGCGAGATACAGCAGATGGTTCGGGCAGCTAGGGTTTTACTCCCACTACGCTATGGACGTCTGGAGCCAAGGGACCACGGTCACCGTCTCAAGC
(SEQ ID NO: 25) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCGCCATCACTTGCCGCGCAAGTCAGAGCATCGACACCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTATGCTGCATCCAAGTTGGAAGACGGGGTCCCATCAAGATTCAGTGGCAGTGGAACTGGGACAGATTTCACTCTCACCATCAGAAGTCTGCAACCTGAAGATTTTGCAAGTTATTTCTGTCAACAGAGCTACTCTAGTCCAGGGATCACTTTCGGCCCTGGGACCAAGGTGGAGATCAAA
(SEQ ID NO: 26) Dyax5 GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTTCTTACATTATGGTTTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTCTATCTATTCTTCTGGTGGCTCTACTTCTTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACAGCCGTGTATTACTGTACGAGATACAGCAGATGGTTCGGGCAGCTAGGGTTTTACTCCCACTACGCTATGGACGTCTGGAGCCAAGGGACCACGGTCACCGTCTCAAGC
(SEQ ID NO: 27) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
(SEQ ID NO: 28)

TABLE 1C

Name of the antibody HCDR1 HCDR2 HDDR3 LCDR1 LCDR2 LCDR3 AM40 SYALS
(SEQ ID NO: 29) TRPPTSRTASYAQKFQG
(SEQ ID NO: 30) NDFVYGSYRF
(SEQ ID NO: 31) TRTSGNIAGYFVQ
(SEQ ID NO: 32) EDYQRPS
(SEQ ID NO: 33) QSYDDYRRAA
(SEQ ID NO: 34) SC4017 SYALS
(SEQ ID NO: 29) TRPPTSRTASYAQKFQG
(SEQ ID NO: 30) NDFVYGSYRF
(SEQ ID NO: 31) TRTSGWIAGYFVQ
(SEQ ID NO: 35) EDYQRPS
(SEQ ID NO: 33) QSYDDYRRAA
(SEQ ID NO: 34) CAT5D9 SYALS
(SEQ ID NO: 29) GIIPVFRTASYAQKFQG
(SEQ ID NO: 36) NNYVWGSYRF
(SEQ ID NO: 37) TRTSGNIAGYFVQ
(SEQ ID NO: 32) EDYQRPS
(SEQ ID NO: 33) QSYDDTSQGV
(SEQ ID NO: 38) CAT8 SYAMS
(SEQ ID NO: 39) AISGSGGSTYYADSVKG
(SEQ ID NO: 40) SSGYYGANFDF
(SEQ ID NO: 41) SGSSSNIGSGYVY
(SEQ ID NO: 42) RNNQRPS
(SEQ ID NO: 43) AAWDDSLSGYV
(SEQ ID NO: 44) CAT26 SYAVS
(SEQ ID NO: 45) AISGSGGSTYYADSVKG
(SEQ ID NO: 40) DAYGSSWYFYYFDY
(SEQ ID NO: 46) SGSSSNIGINPVN
(SEQ ID NO: 47) SDKYRPS
(SEQ ID NO: 48) AAWDDSLNGRV
(SEQ ID NO: 49) DYAX3 MYEMR
(SEQ ID NO: 50) VIPSGGKTFYADSVKG
(SEQ ID NO: 51) YSRWFGQLGFYSHYAMDV
(SEQ ID NO: 52) RASQSIDTYLN
(SEQ ID NO: 53) AASKLED
(SEQ ID NO: 54) QQSYSSPGIT
(SEQ ID NO: 55) DYAX5 SYIMV
(SEQ ID NO: 56) SIYSSGGSTSYADSVKG
(SEQ ID NO: 57) YSRWFGQLGFYSHYAMDV
(SEQ ID NO: 52) RASQSISSYLN
(SEQ ID NO: 58) AASSLQS
(SEQ ID NO: 59) QQSYSTPPWT
(SEQ ID NO: 60)

TABLE 1D

Name of the antibody HC LC AM40
(amino acid) Qvqlvqsgaevkkpgssvkvsckasggtfssyalswvrqapgqglewmgtrpptsrtasyaqkfqgrvtitvdeststgymelsslrsedtavyycasndfvygsyrfwgqgttvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdkthtcppcpapefeggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk
(SEQ ID NO: 61) nfmltqphsvsespgktvtisctrtsgniagyfvqwyqqrpgsspttviyedyqrpsgvpdrfsgsidsssnsasltisglktedeadyycqsyddyrraafgggtkltvlgqpkaapsvtlfppsseelqankatlvclisdfypgavtvawkadsspvkagvetttpskqsnnnkyaassylsltpeqwks
(SEQ ID NO: 62) AM40 (DNA)
(SEQ ID NO: 63)
(SEQ ID NO: 64) SC4017 (amino acid) qvqlvqsgaevkkpgssvkvsckasggtfssyalswvrqapgqglewmgtrpptsrtasyaqkfqgrvtitvdeststgymelsslrsedtavyycasndfvygsyrfwgqgttvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdkthtcppcpapefeggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk
(SEQ ID NO: 65) nfmltqphsvsespgktvtisctrtsgWiagyfvqwyqqrpgsspttviyedyqrpsgvpdrfsgsidsssnsasltisglktedeadyycqsyddyrraafgggtkltvlgqpkaapsvtlfppsseelqankatlvclisdfypgavtvawkadsspvkagvetttpskqsnnkyaassylscltpeqwkshrtecshr
(SEQ ID NO: 66) SC4017 (DNA) atgggatggagctgtatcatcctcttcttggtagcaacagctacaggtaaggggctcacagtagcaggcttgaggtctagacatatatatgggtgacaatgacatccactttgcctttctctccacaggtgtacactcccaggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttcagcagttatgctcttagctgggtgcgacaggcccctggacaagggcttgagtggatgggaacgcggccgccgacctcccggacagcaagctacgcacagaaatttcagggcagagtcacgattaccgtggacgaatccacgagcacaggctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgtcaaacgacttcgtgtacgggagttatcgtttctggggccaagggaccacggtcaccgtctcctcagcgtcgaccaagggcccatccgtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcctggaactcaggcgctctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaattcgaggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcat aatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcctccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtctacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcttaagcctgtctccgggtaaa
(SEQ ID NO: 67)
(SEQ ID NO: 68) CAT5D9 (amino acid) QVQLVQSGAEVKKPGSSVKVSCKISGGTFSSYALSWVRQAPGQGLEWMGGIIPVFRTASYAQKFQGRVTITVDESASTGYIELSSLKSEDTATYYCASNNYVWGSYRFWGQGTTVTVSSAStkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdkthtcppcpapefeggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk
(SEQ ID NO: 69) NFMLTQPHSVSESPGKTVTISCTRTSGNIAGYFVQWYQQRPGSSPTTVIYEDYQRPSGVPDRFSGSIDRSSSNSASLTISGLKPDDEADYYCQSYDDTSQGVFGAGTKVTVL
(SEQ ID NO: 70) CAT5D9 (DNA)
(SEQ ID NO: 71)
(SEQ ID NO: 72)

[00163] Derivatives

[00164] Anti-FLT3L antibodies of the present invention may include variants of the present sequences that retain the ability to specifically bind FLT3L. Such variants can be generated by one of skill in the art from antibody sequences using techniques well known in the art. For example, amino acid substitutions, deletions, or additions can be made in the FR region and/or CDRs of anti-FLT3L antibodies that prevent the antibodies from binding to their epitopes. Whereas changes in the FR are typically designed to improve the stability and immunogenicity of the antigen-binding domain, changes in the CDR are typically designed to increase the affinity of the antigen-binding domain for its target. FR variants also include naturally occurring immunoglobulin allotypes. Such affinity-enhancing changes can be determined empirically using standard techniques that involve changing the CDR and testing the affinity of the antigen-binding domain for its target. For example, conservative amino acid substitutions can be made to any of the disclosed CDRs. Various changes can be made in accordance with the methods described in Antibody Engineering, 2nd ^ed ., Oxford University Press, ed. Borrebaeck, 1995. Such changes include, but are not limited to, nucleotide sequences that are changed by changing different codons that code for a functionally equivalent amino acid residue within the sequence, thereby producing a "silent" change. For example, non-polar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[00165] Derivatives and analogs of antibodies of the present invention can be obtained using various methods well known in the art, including using recombinant methods and methods of synthesis (Maniatis (1990) Molecular Cloning, A Laboratory Manual, 2 ^nd ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Bodansky et al (1995) The Practice of Peptide Synthesis, 2nd ed., Spring Verlag, Berlin, Germany).

[00166] In one embodiment, a method for obtaining a VH domain, which is a variant VH domain amino acid sequence of the present invention, includes the step of adding, deleting, replacing, or inserting one or more amino acids in the amino acid sequence of a VH domain of the present invention, optionally combining the resulting a VH domain pattern with one or more VL domains; and testing the VH domain or the VH/VL combination or combinations for specific antigen binding. A similar method can be used in which one or more VL domain sequence variants disclosed herein are combined with one or more VH domains.

[00167] Similar shuffling or combining techniques are also described in Stemmer (Nature (1994) 370: 389-391), which describes a technique for the β-lactamase gene, but notes that this approach can be used to generate antibodies.

[00168] In additional embodiments, new VH or VL regions can be generated carrying one or more sequences derived from the sequences disclosed herein using random mutagenesis of one or more selected VH and/or VL genes. One such technique, which is error-prone PCR (PCR), is described by Gram et al. (Proc. Nat. Acad. Sci. U.S.A. (1992) 89: 3576-3580).

[00169] Another method that can be used is site-directed mutagenesis of CDRs in the VH or VL genes. Such techniques are disclosed in Barbas et al. (Proc. Nat. Acad. Sci. U.S.A. (1994) 91: 3809-3813) and Schier et al. (J. Mol. Biol. (1996) 263: 551-567).

[00170] Similarly, one, two, or all three CDRs of an antigen-binding domain can be grafted into a repertoire of VH or VL domains, which is then screened for an antigen-binding fragment specific for FLT3L.

[00171] An immunoglobulin variable domain portion suitable for use according to this document may comprise at least one of the CDRs that is as set forth herein, and optionally intermediate framework regions from scFv fragments as set forth herein . This portion may include at least about 50% of any one or both of FR1 and FR4, with 50% being C-terminal 50% FR1 and N-terminal 50% FR4. Additional residues at the N-terminus or C-terminus of a significant portion of the variable domain may be residues that are not normally associated with naturally occurring regions of the variable domain. For example, the construction of antibodies by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other operational steps. Other operational steps include the introduction of linkers to attach variable domains to additional protein sequences, including immunoglobulin heavy chain constant regions, other variable domains (eg, in the preparation of diabodies), or protein tags, which are discussed in more detail below.

[00172] The antigen-binding domains of the present invention described herein can be coupled to another functional molecule, such as another peptide or protein (albumin, another antibody, etc.). For example, antigen binding domains can be linked by chemical crosslinking or by recombinant methods. Antigen binding domains can also be linked to one of a number of non-protein polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, by the methods set forth in US Pat. , for example, to increase their half-life in the bloodstream. Illustrative polymers and methods for attaching them are also shown in US Pat.

[00173] The disclosed antibodies can also be modified to have a glycosylation pattern that differs from the natural pattern. For example, one or more carbohydrate moieties may be removed and/or one or more glycosylation sites may be added. The addition of glycosylation sites to antibody fragments disclosed herein can be accomplished by altering the amino acid sequence to include glycosylation site consensus sequences known in the art. Other means of increasing the amount of carbohydrate moieties on antibody fragments is to chemically or enzymatically link the glycosides to the amino acid residues of the antibody. Such methods are described in WO 87/05330 and Aplin et al. (1981) CRC Crit. Rev. Biochem., 22: 259-306. Removal of any carbohydrate fragments from antibodies can be accomplished by chemical or enzymatic methods, for example, as described in Hakimuddin et al. (1987) Arch. Biochem. Biophys., 259:52 and Edge et al. (1981) Anal. Biochem., 118: 131, as well as Thotakura et al. (1987) Meth. Enzymol., 138: 350. Antibody fragments can also be labeled with a detectable or functional label. Detectable labels include radioactive labels such as 131I or 99Tc, which can also be attached to antibody fragments using traditional chemical methods. Detectable labels also include enzymatic labels such as horseradish peroxidase or alkaline phosphatase. Detectable labels further include chemical moieties, such as biotin, which can be detected by binding to a specific cognate detectable moiety, such as labeled avidin.

[00174] The scope of the present invention encompasses antigen-binding domains in which the CDR sequences differ only marginally from those set forth herein. Typically, an amino acid is replaced with a related amino acid having similar charge, hydrophobic, or stereochemical characteristics. Such substitutions will be within the skill of the art. Unlike CDRs, more significant changes can be made to FRs without adversely affecting the binding properties of the antibody. Alterations in FR include, without limitation, the humanization of certain non-human or engineered scaffold residues that are important for antigen contact or binding site stabilization, e.g. as binding to the Fc receptor, for example, as described in US patent No. 5624821 and 5648260, as well as in Lund et al. (1991) J. Immun. 147: 2657-2662 and in Morgan et al. (1995) Immunology 86: 319-324, or changing the species from which the constant region is derived.

[00175] One skilled in the art will appreciate that the modifications described above that may be applied to the protein subunits described herein are not all inclusive, and that many other modifications are possible for one skilled in the art in light of the teachings of the present invention. .

[00176] Anti-FLT3L antibody affinity and specificity

[00177] Those skilled in the art will appreciate that anti-FLT3L antibodies for use in an autoimmune disease must have high affinity binding, but must not have toxicity that precludes their use in humans. Structurally similar FLT3L homologues include stem cell factor (SCF, also known as KIT ligand) and colony stimulating factor 1 (CSF1, also known as macrophage colony stimulating factor "M-CSF"). A non-specific anti-FLT3L antibody that binds FLT3L and also binds SCF and CSF1 can lead to off-target toxicity. Therefore, the anti-FLT3L antibodies of the present invention retain binding specificity only for FLT3L, but not for structurally similar cytokines such as SCF and CSF1. One skilled in the art will know how to use, without limitation, binding kinetics characteristics, including K _on , K _off , and K _D , as indicators of binding specificity.

[00178] The level of FLT3L in blood serum and the amount of pDC in the bloodstream are biomarkers that can be used as an indicator of toxicity associated with clone leaders of anti-FLT3L antibodies. A rapid decrease in pDC upon FLT3L neutralization indicates suppression of FLT3L-mediated signaling that enhances the immune response. The rapid recovery of pDC frequency in the presence of free FLT3L may reflect the lack of toxicity of the anti-FLT3L antibody. In a preferred embodiment, the anti-FLT3L antibody neutralizes FLT3L and provides a reversible reduction in cDC and pDC while returning free FLT3L. One of skill in the art will appreciate that a drop in dendritic cell count upon FLT3L neutralization followed by a return to baseline is indicative of low toxicity of the leader clone.

[00179] Obtaining antibodies to FLT3L

[00180] The practice of the present invention involves the use, unless otherwise indicated, of the techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the competence of a person skilled in the art. Such techniques are fully covered in the literature such as "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to obtain polypeptides of the present invention and, therefore, may be considered in the creation and implementation of the present invention. Particularly applicable techniques for specific embodiments will be discussed in the examples.

[00181] In one embodiment, an isolated nucleic acid molecule encoding an anti-FLT3L antibody, or antigen-binding fragment thereof, is operably linked to one or more control sequences to allow expression in a host cell. The isolated nucleic acid can be recombinantly incorporated into a vector, which in turn is transfected into a host cell using known techniques.

[00182] In one embodiment, provided herein are host cells transformed with an isolated nucleic acid molecule encoding an anti-FLT3L antibody, or antigen-binding fragment thereof, that is operably linked to one or more control sequences. Examples of envisaged host cells include mammalian cells such as HEK293 cells, mouse NS0 myeloma cells, or Chinese hamster ovary (CHO) cells.

[00183] In one embodiment, anti-FLT3L monoclonal antibodies (eg, CAT5D9, SC4017, or AM40) and antigen-binding fragments thereof can be generated using hybridoma methods such as those described in Kohler and Milstein (1975) Nature 256:495. Using the hybridoma method, a mouse, hamster, or other suitable host animal is immunized as described above to induce lymphocytes to produce antibodies that will specifically bind to the immunizing antigen.

[00184] In another embodiment, lymphocytes can also be immunized in vitro. After immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol to produce hybridoma cells, which can then be separated from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies specifically directed to a selected antigen, as determined by immunoprecipitation, immunoblotting, or in vitro binding assays (e.g., radioimmunoassay (RIA); ELISA), can then be propagated either in in vitro culture using standard methods (Coding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986), or in vivo as ascitic tumors in an animal. The monoclonal antibodies can then be purified from the culture medium or ascitic fluid as described above for polyclonal antibodies.

[00185] Alternatively, anti-FLT3L monoclonal antibodies (e.g., CAT5D9, SC4017, or AM40) and antigen-binding fragments thereof can also be generated using recombinant DNA techniques, as described, for example, in US Pat. No. 4,816,567. Polynucleotides encoding a monoclonal antibody are isolated from mature B cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify genes encoding antibody heavy and light chains and sequence them using standard procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, whereby after transfection with host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which otherwise do not produce immunoglobulin protein, monoclonal antibodies are produced by host cells. Also, recombinant anti-FLT3L monoclonal antibodies or antigen-binding fragments of the desired species can be isolated from phage display libraries expressing CDRs of the desired species, as described (McCafferty et al., 1990, Nature, 348:552-554; Clarkson et al., 1991, Nature , 352:624-628 and Marks et al., 1991, J. Mol. Biol., 222:581-597).

[00186] The polynucleotide(s) encoding an anti-FLT3L antibody or antigen-binding fragment thereof can be further modified in a number of different ways using recombinant DNA technology to generate alternative antibodies. In some aspects, the light and heavy chain constant domains of, for example, a murine monoclonal antibody can be replaced with (1) the corresponding regions of, for example, a human antibody to form a chimeric antibody, or (2) to a non-immunoglobulin polypeptide to form a fusion antibody. In some aspects, the constant regions are truncated or deleted to obtain the desired monoclonal antibody fragment. To optimize specificity, affinity, etc. monoclonal antibody in relation to the variable region, site-directed mutagenesis or high density mutagenesis can be used.

[00187] In certain aspects, an anti-FLT3L antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. Human antibodies can be directly obtained using various techniques known in the art. It is possible to obtain immortalized human B lymphocytes, immunized in vitro or isolated from an immunized individual, which produce an antibody directed to the target antigen (see, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 ( 1985); Boemer et al., 1991, J. Immunol., 147(l):86-95 and US Pat. No. 5,750,373).

[00188] Also, a human anti-FLT3L antibody, or antigen-binding fragment thereof, can be selected from a phage library, such phage library expressing human antibodies, as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162, Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381 and Marks et al., 1991, J. Mol. Biol., 222:581). Techniques for creating and using antibody phage libraries are also described in US Pat. Nos. 5,969,108; 6,172,197; 6544731; 6555313; 6582915; 6593081; 6300064; 6653068; 6706484 and 7264963 and in Rothe et al., 2007, J. Mol. Bio., doi:10.1016/j.jmb.2007.12.018 (each incorporated by reference in its entirety).

[00189] Affinity maturation and strand shuffling strategies (Marks et al., 1992, Bio/Technology 10:779-783, incorporated by reference in its entirety) are known in the art and can be used to generate high affinity human or their antigen-binding fragments.

[00190] In some aspects, the anti-FLT3L monoclonal antibody may be a humanized antibody. Methods for constructing, humanizing, or resurfacing non-human and human antibodies can also be used and are well known in the art. A humanized, resurfaced, or similarly engineered antibody may have one or more amino acid residues derived from a non-human source, such as, but not limited to, a mouse, rat, rabbit, non-human primate, or other mammal. These non-human amino acid residues are replaced with what are often referred to as "import" residues, which are usually taken from an "import" variable, constant or other domain of a known human sequence. Such imported sequences can be used to reduce immunogenicity, or reduce, enhance or modify binding, affinity, association rate, dissociation rate, avidity, specificity, half-life, or any other suitable characteristic as known in the art. In general, the residues in the CDR are directly and most significantly involved in influencing FLT3L binding. Therefore, some or all of the non-human or human CDR sequences are retained, while the non-human variable and constant region sequences can be replaced with human or other amino acids.

[00191] Antibodies can also optionally be humanized, resurfaced, engineered, or human antibodies engineered to retain high affinity for the FLT3L antigen and other beneficial biological properties. To achieve this goal, humanized (or human) or engineered anti-FLT3L antibodies and resurfaced antibodies can optionally be obtained during the analysis of the original sequences and various conceptual humanized and engineered products using three-dimensional models of the original, engineered and humanized sequences. Three-dimensional immunoglobulin models are publicly available and known to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. The evaluation of these mappings makes it possible to analyze the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e. analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen, such as FLT3L. In this manner, framework (FW) region residues can be selected and combined with residues from the consensus and import sequences such that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.

[00192] Humanization, resurfacing, or construction of anti-FLT3L antibodies or antigen-binding fragments thereof can be carried out using any known method, such as, without limitation, those described in Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239: 1534 (1988)), Sims et al., J. Immunol. 151 : 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), US Pat. Nos. 5,639,641, 5,723,323; 5976862; 5824514; 5817483; 5814476; 5763192; 5723323; 5766886; 5714352; 5955358; 6204023; 6180370; 6331431; 5693762; 5530101; 5585089; 5225539; 4816567; 5969108; 7635666; 7723270; 7557189; 7538195 and 7342110; International Patent Applications No. PCT/US98/16280; PCT/US91/05939; PCT/US94/01234; PCT/GB92/01755; publications of international patent applications No. WO90/14443; WO90/14424; WO90/14430 and European Patent Application Publication No. EP 229246; each of which is incorporated herein by reference in its entirety, including the references cited therein.

[00193] Humanized anti-FLT3L antibodies and antigen-binding fragments thereof can also be produced in transgenic mice containing human immunoglobulin loci that are capable of producing the full repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. This approach is described in US Pat. Nos. 5,545,807; 5545806; 5569825; 5625126; 5633425 and 5661016.

[00194] In certain aspects, an anti-FLT3L antibody fragment is provided. There are various methods for obtaining fragments of antibodies. Traditionally, these fragments are obtained by proteolytic cleavage of intact antibodies (eg, Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods 24: 107-117; Brennan et al., 1985, Science, 229:81). In certain aspects, anti-FLT3L antibody fragments are generated recombinantly. All of the Fab, Fv and scFv can be expressed in and secreted from E. coli or other host cells, allowing large amounts of these fragments to be obtained. Such anti-FLT3L antibody fragments can also be isolated from the antibody phage libraries discussed above. Anti-FLT3L antibody fragments can also be linear antibodies, as described in US Pat. No. 5,641,870. Other techniques for producing antibody fragments, such as chemical synthesis, will be apparent to those skilled in the art.

[00195] According to the present invention, the methods can be adapted to obtain single-chain antibodies specific for FLT3L (see, for example, US patent No. 4946778). In addition, the methods can be adapted to construct Fab expression libraries (see, for example, Huse et al., Science 246: 1275-1281 (1989)) to enable rapid and efficient identification of monoclonal Fab fragments with the desired specificity for FLT3L or their derivatives, fragments, analogues or homologues. Using techniques known in the art, antibody fragments can be generated, including, without limitation, (a) an F(ab')2 fragment obtained by pepsin digestion of an antibody molecule; (b) Fab fragment obtained by reducing the disulphide bridges of the F(ab')2 fragment, (c) Fab fragment obtained by treating the antibody molecule with papain and a reducing agent, and (d) Fv fragments.

[00196] An anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein can be modified to increase its serum half-life. This can be done, for example, by incorporating a reutilization receptor binding epitope into the antibody or antibody fragment, by mutating the appropriate region in the antibody or antibody fragment, or by incorporating the epitope into a peptide tag, which is then fused to the antibody or antibody fragment either at the end or in the middle ( for example, via DNA synthesis or peptide synthesis), or via YTE mutation. Other methods are known in the art to increase the serum half-life of an antibody or antigen-binding fragment thereof, such as conjugation to a heterologous molecule such as PEG.

[00197] Pharmaceutical compositions

[00198] The present invention is also directed to pharmaceutical compositions containing anti-FLT3L antibodies or antigen-binding fragments disclosed herein. In certain embodiments, the present invention contemplates the use of an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein in the manufacture of a medicament for the treatment of a subject.

[00199] An effective amount of the pharmaceutical composition of the present invention should be administered, with an "effective amount" being defined as an amount that is sufficient to produce the desired prophylactic, therapeutic, or ameliorating response in a subject. The effective amount will vary with the species and weight of the subject being administered, but can be determined using standard techniques.

[00200] In certain aspects, the present invention provides therapeutic and prophylactic compositions for use in subjects in need thereof in the treatment or prevention (reducing the likelihood) of the development of autoimmune diseases, including, without limitation, systemic lupus erythematosus, myositis, primary Sjögren's syndrome, multiple sclerosis , uveitis, psoriasis or rheumatoid arthritis.

[00201] In some embodiments, a pharmaceutical composition of the present invention comprises an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. In this regard, "pharmaceutically acceptable carriers, diluents, or adjuvants" include, without limitation, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, coloring/coloring agent, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, diluent or emulsifier, which may or may not have been approved by the US Food and Drug Administration as acceptable for use in humans or pets. For example, suitable carriers are known to those skilled in the art and include stabilizers, diluents, and buffers. Suitable stabilizers include carbohydrates such as sorbitol, lactose, mannitol, starch, sucrose, dextran and glucose, as well as proteins such as albumin or casein. Suitable diluents include saline, Hank's balanced salt solution and Ringer's solution. Suitable buffers include alkali metal phosphate, alkali metal carbonate or alkaline earth metal carbonate.

[00202] In certain aspects, the pharmaceutical compositions of the present invention may additionally contain one or more auxiliary materials, such as one or more lipids, phospholipids, carbohydrates, and lipopolysaccharides. In some embodiments, the implementation of the pharmaceutical compositions of the present invention optionally contain one or more additional active substances.

[00203] In certain cases, the pharmaceutical compositions of the present invention can be obtained by methods known to specialists in this field of technology. General approaches to the formulation and/or manufacture of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy 21 ^st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety). Typically, an anti-FLT3L antibody or antigen-binding fragment thereof of the present invention is mixed with a carrier to form a solution, suspension or emulsion. One or more of the additives discussed herein may be added to the carrier or may be added subsequently. The pharmaceutical compositions of the present invention may be in the form of an aqueous solution, emulsion or suspension, or may be presented as a dried preparation. In certain aspects, the pharmaceutical compositions of the present invention may be desiccated or lyophilized for storage or formulation purposes, such as by freeze drying or spray drying. They can then be dissolved into liquid compositions by the addition of a suitable liquid carrier, or administered in a dry formulation using methods known to those skilled in the art.

[00204] The pharmaceutical compositions of the present invention can be administered to a subject by various methods known in the art. Exemplary routes of administration for such pharmaceutical compositions include oral, transmucosal, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. Thus, in certain embodiments, the pharmaceutical composition of the present invention is formulated for administration by routes selected from the group consisting of oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal routes. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. In certain aspects, the pharmaceutical compositions of the present invention are formulated such that the anti-FLT3L antibody or antigen-binding fragment thereof of the present invention contained therein is bioavailable when administered to a subject.

[00205] The choice of method of administration of the pharmaceutical composition will depend on the selected composition. The pharmaceutical compositions of the present invention are administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically effective. In certain aspects, the pharmaceutical composition of the present invention is formulated in solid, semi-solid, liquid or gaseous form, including, without limitation, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols.

[00206] In certain instances, a pharmaceutical composition comprising an anti-FLT3L antibody or antigen-binding fragment disclosed herein may be in the form of a solid or liquid. In some aspects, the carrier(s) are(are) in particulate form, such that the compositions are, for example, in the form of a tablet or powder. In other aspects, the carrier(s) are liquid, and the composition is, for example, an oral syrup, injectable liquid or aerosol, which is applicable, for example, for inhalation administration. When intended for oral administration, a pharmaceutical composition comprising an anti-FLT3L antibody or antigen-binding fragment disclosed herein is presented in either solid or liquid form, with semi-solid forms considered herein as either solid or liquid forms. , semi-liquid, suspension and gel forms.

[00207] In certain aspects, as a solid composition for oral administration, a pharmaceutical composition comprising an anti-FLT3L antibody or antigen-binding fragment disclosed herein may be formulated as a powder, granule, compressed tablet, pill, capsule, chewing gum, cachet, or the like. forms. In some cases, such a solid composition will typically contain one or more inert diluents or food carriers. In certain embodiments, one or more of the following may additionally be present: binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch, lactose or dextrins; disintegrants such as alginic acid, sodium alginate, primogel, corn starch and the like; lubricants such as magnesium stearate or stereotex; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring such as peppermint, methyl salicylate or orange flavoring; as well as a coloring agent.

[00208] Such compositions may be in the form of microspheres, solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders and contain from about 0.001 to 95% of the anti-FLT3L antibody or antigen-binding fragment disclosed herein. Some formulations may contain 50 μg to 250 μg of the anti-FLT3L antibody or antigen-binding fragment thereof.

[00209] In some aspects, if the pharmaceutical composition of the present invention is in the form of a capsule, such as a gelatin capsule, it may contain, in addition to the materials disclosed herein, a liquid carrier such as polyethylene glycol or an oil. Oral formulations may also include commonly used excipients such as, for example, pharmaceutical grades of saccharin, cellulose, and magnesium carbonate.

[00210] In other aspects, the pharmaceutical composition of the present invention is in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. In certain embodiments, the liquid may be for oral administration or for delivery by injection. In certain embodiments, when the pharmaceutical compositions of the present invention are intended for oral administration, they contain, in addition to the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein, one or more of a sweetener, preservatives, a pigment/colorant, and a flavor enhancer. . In certain aspects, a pharmaceutical composition to be administered by injection may include one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent.

[00211] In certain instances, liquid pharmaceutical compositions containing an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein, whether in solutions, suspensions, or the like, may include one or more of the following: sterile diluents , such as water for injection, saline solution such as saline, Ringer's solution, isotonic sodium chloride solution, fixed oils such as synthetic mono- or diglycerides which may serve as a solvent or suspending medium, polyethylene glycols, glycerol, propylene glycol or others solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity adjusters such as sodium chloride or dextrose. In some cases, the drug may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In some embodiments, the injectable pharmaceutical composition is preferably sterile.

[00212] In other embodiments, a pharmaceutical composition comprising an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein may be for topical application, in which case the carrier may preferably be a solution, emulsion, ointment, or gel base. In certain aspects, the base, for example, may contain one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. In other aspects, thickeners may be present in the topical pharmaceutical composition. In certain embodiments, when the pharmaceutical composition is for transdermal administration, the pharmaceutical composition based on the anti-FLT3L antibody or antigen-binding fragment disclosed herein can be incorporated into a transdermal patch or iontophoresis device.

[00213] In yet other embodiments, a pharmaceutical composition comprising an anti-FLT3L antibody or antigen-binding fragment disclosed herein is for rectal administration in the form of, for example, a suppository. In the case of suppositories, binders and carriers may include, for example, polyalkylene glycols or triglycerides. In certain cases, the composition for rectal administration contains an oily base as a suitable non-irritating excipient. Such bases include, without limitation, lanolin, cocoa butter, or polyethylene glycol.

[00214] In other aspects, a pharmaceutical composition comprising an anti-FLT3L antibody or antigen-binding fragment disclosed herein contains dosage units that can be administered as an aerosol. The term aerosol is used to refer to a variety of systems ranging from systems of a colloidal nature to systems consisting of pressurized packages. In certain embodiments, delivery is by liquefied or compressed gas or a suitable pumping system that dispenses the active ingredients. In some embodiments, to deliver the active ingredient(s), aerosols of an anti-FLT3L antibody disclosed herein, or an antigen-binding fragment thereof, can be delivered in single-phase, two-phase, or three-phase systems. In other embodiments, delivery of the aerosol includes the necessary containers, activators, valves, lifts, and the like, which together may form a kit. One of ordinary skill in the art will readily be able to determine specific aerosol formulations and methods for their delivery.

[00215] The pharmaceutical compositions of the present invention may be administered in a suitable non-toxic pharmaceutical carrier, they may be contained in microcapsules, microgranules and/or may be contained in a sustained release implant.

[00216] In other aspects, the pharmaceutical composition of the present invention includes materials that form a coating shell around the active ingredients. In some cases, the materials that form the coating shell are typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents.

[00217] In still other aspects, the pharmaceutical compositions of the present invention in solid or liquid form include an agent that binds to the anti-FLT3L antibody or antigen-binding fragment disclosed herein and thereby facilitates the delivery of the anti-FLT3L antibody or antigen-binding fragment. In certain instances, suitable agents which act in this way include a protein or a liposome.

[00218] In certain aspects, the pharmaceutical compositions to be administered to a subject are in the form of one or more dosage units, whereby, for example, one dosage unit may be a tablet and the container containing the anti-FLT3L antibody or antigen-binding fragment disclosed herein in the form of an aerosol may contain a plurality of dosage units. Actual methods for preparing such dosage forms are known or will be apparent to those skilled in the art; for example, see Remington: The Science and Practice of Pharmacy, 20th ^Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will in any event comprise a therapeutically effective amount of an anti-FLT3L antibody or antigen-binding fragment thereof or a pharmaceutically acceptable salt thereof disclosed herein to assist in the treatment of the disease or condition of interest in accordance with the teachings disclosed herein.

[00219] In certain embodiments, the implementation of the pharmaceutical compositions of the present invention contain one or more additional therapeutically active substances. In other embodiments, a therapeutically effective dose of the pharmaceutical compositions of the present invention is administered to a subject in need thereof in combination with one or more additional therapeutically active agents. The term "combination" as used herein refers to a combination comprising an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein and one or more additional therapeutically active substances, each of which can be administered intermittently (sequentially), in parallel or simultaneously.

[00220] To maintain therapeutic levels, it may be necessary to perform the administration of the pharmaceutical compositions of the present invention at several intervals. The pharmaceutical compositions of the present invention may be used in combination with other bactericidal or bacteriostatic methods.

[00221] Although the descriptions of pharmaceutical compositions presented herein generally relate to pharmaceutical compositions that are suitable for administration to humans, one of skill in the art will appreciate that such compositions are generally suitable for administration to any other subject. In certain aspects, the subject is a mammal. In certain aspects, a mammal includes primates, such as humans, non-human and great apes, and non-primate animals, such as domesticated animals, including laboratory and companion animals, and farm animals (e.g., cats, dogs, pigs). , cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wild animals, birds or the like.

[00222] Anti-autoimmune/anti-inflammatory therapy

[00223] The present invention also provides compositions and methods that are useful in the treatment of autoimmune and/or other inflammatory diseases (i.e., diseases associated with an overreactive and/or improperly functioning immune system) comprising anti-FLT3L antibodies, such as described above. In various embodiments, anti-FLT3L antibodies may be administered in combination with other immunoregulatory drugs designed to suppress or attenuate a subject's immune system activity or specific immune response to a particular antigen or group of antigens and thereby reduce or prevent the development of an autoimmune or other inflammatory disease. .

[00224] Additionally, provided herein are methods for treating autoimmune and/or other inflammatory diseases, comprising administering one or more anti-FLT3L antibodies. As shown herein, administration of anti-FLT3L antibodies can result in at least one of a reduction in the immune response, expression of participants in one or more immunological signaling cascades, or a decrease in immune cell populations. In certain aspects, an anti-FLT3L antibody is administered to a patient or subject suffering from an autoimmune disease or other inflammatory disease.

[00225] Treatment of an autoimmune and/or other inflammatory disease with an anti-FLT3L antibody results in, for example, a reduction in the rate of progression of the autoimmune disease or inflammatory disease, a slowing or stabilization of immune cell proliferation, a reduction in the size of a lesion (e.g., as in case of patients with MS) and/or regression of the disease. In some aspects, indicators that measure the reduction or slowing of the development of an autoimmune disease or inflammatory disease (for example, reduced inflammation, reduced levels of inflammatory cytokines, immune cell populations and/or associated damage, such as tissue lesions), can be statistically meaningful. Reduction in autoimmune disease or inflammatory disease scores can be measured by comparison with a patient's score at baseline (before treatment) versus an individual's expected rate of disease progression, versus an expected rate of disease progression based on a large patient group, or versus an expected rate of disease progression in a control. groups.

[00226] In one embodiment, the method of treatment provided herein comprises administering or administering a FLT3L binding molecule, antibody or antigen-binding fragment, variant or derivative thereof of the present invention to a subject or patient, or administering or administering an FLT3L binding molecule performed on an isolated tissue or cell line from a subject or patient, wherein the subject or patient has a disease, a symptom of a disease, or a predisposition to develop a disease.

[00227] Contemplated diseases include acute or chronic inflammatory diseases, including type 1 and type 2 diabetes, CKD, including, for example, CKD secondary to diabetes, diabetic nephropathy, and high blood pressure; atherosclerosis, Alzheimer's disease, cancer, and associated complications of such diseases, including heart disease, hypertension, anemia, pericarditis, renal osteodystrophy, and others. Additional contemplated diseases include autoimmune diseases including, but not limited to, systemic lupus erythematosus, myositis, primary Sjögren's syndrome, multiple sclerosis, uveitis, psoriasis, and rheumatoid arthritis.

[00228] In another embodiment, the treatment also involves the use or administration of a pharmaceutical composition containing a FLT3L-binding molecule, for example, an antibody or antigen-binding fragment thereof, a variant or derivative of the present invention, performed on a subject or patient, or the use or administration of a pharmaceutical composition, containing an FLT3L-binding molecule, performed on an isolated tissue or cell line from a subject or patient who has a disease, a symptom of a disease, or a predisposition to developing a disease.

[00229] In accordance with the methods of the present invention, at least one antibody to FLT3L, as defined elsewhere in this document, is used to stimulate a positive therapeutic response to an autoimmune or inflammatory disease. The term "positive therapeutic response" means a reduction in symptoms associated with an autoimmune or inflammatory disease. For example, an improvement in the course of a disease may be characterized by a complete response. By "complete response" is meant the absence of clinically detectable disease with normalization of all previously obtained test results. Alternatively, improvement in the course of the disease can be categorized as a partial response. "A positive therapeutic response" encompasses the reduction or suppression of the progression and/or duration of an autoimmune or inflammatory disease, the reduction or alleviation of the severity of an autoimmune or inflammatory disease, and/or the alleviation of one or more symptoms thereof, resulting from the administration of the FLT3L binding molecule disclosed herein. .

[00230] In certain embodiments, a method for treating primary Sjögren's syndrome is provided, comprising administering to a subject in need thereof a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[00231] In other embodiments, a method for treating myositis is provided, comprising administering to a subject in need thereof a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[00232] In certain embodiments, a method of treating systemic lupus erythematosus (SLE) is provided, comprising administering to a subject in need thereof a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein. In some aspects, the subject is characterized by elevated serum levels of FLT3L, as measured by the frequency of FLT3L-expressing CD4+ T cells, compared to a healthy subject.

[00233] In some embodiments, a method is provided for diagnosing systemic lupus erythematosus (SLE) in a subject, comprising (a) measuring serum levels of FLT3L, or (b) measuring the frequency of FLT3L-expressing CD4+ T cells, wherein elevated serum FLT3L levels blood or an increased frequency of FLT3L-expressing CD4+ T cells in a subject compared to a healthy donor indicates that the subject has SLE. In certain aspects, CD4+ T cells are effector memory cells (T _EM ).

[00234] In some embodiments, a method is provided for neutralizing membrane-bound FLT3L in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of an antibody or antigen-binding fragment disclosed herein. In certain aspects, FLT3L is reversibly neutralized so that membrane-bound FLT3L activity can return to "pre-administration" levels.

[00235] In other embodiments, a method is provided for neutralizing soluble FLT3L in a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein. In certain aspects, FLT3L is reversibly neutralized so that soluble FLT3L levels can return to "pre-administration" levels.

[00236] In specific embodiments, the method for neutralizing soluble FLT3L further comprises subcutaneously administering an anti-FLT3L antibody, or antigen-binding fragment thereof, to the subject once a week in the range of about 0.03 mg/kg to about 30 mg/kg. In other embodiments, the method further comprises subcutaneously administering the anti-FLT3L antibody, or antigen-binding fragment thereof, to the subject once every four weeks at a dose of approximately 150 mg/kg.

[00237] In other embodiments, a method is provided for reducing populations of classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) in the bloodstream of a subject in need thereof, the method comprising administering to the subject a pharmaceutically effective amount of an antibody or antigen-binding fragment disclosed herein . In certain aspects, cDC and pDC populations are reversibly reduced so that cDC and pDC populations are able to return to pre-administration levels.

[00238] In certain embodiments, a method is provided for reducing FLT3L expression on the surface of CD4+ T cells, comprising administering to a subject in need thereof a pharmaceutically effective amount of an antibody or antigen-binding fragment disclosed herein.

[00239] In other embodiments, a method is provided for reducing the percentage of CD4+ T cells expressing FLT3L, comprising administering to a subject in need thereof a pharmaceutically effective amount of an antibody or antigen-binding fragment disclosed herein.

[00240] In other embodiments, a method for reducing ERK signaling in a lymphoblast is provided, comprising contacting the lymphoblast with an antibody or antigen-binding fragment disclosed herein.

[00241] In certain embodiments, a method for reducing the level of MEK 1/2 phosphorylation in primary human CD133+ stem cells is provided, comprising contacting the stem cells with an antibody or antigen-binding fragment disclosed herein.

[00242] The following examples are provided for the purpose of providing those skilled in the art with a complete disclosure and description of how to make and use the assay, screening, and treatment methods of the present invention, and are not intended to limit the scope of what the present inventors consider their invention. .

EXAMPLES

[00243] The present invention is described with reference to the following examples. The examples are illustrative only, and the present invention should in no way be construed as being limited to these examples, but rather should be construed as covering any and all variations that become apparent as a result of the ideas presented herein.

[00244] EXAMPLE #1 GENERATION OF ANTIBODY TO FLT3L

[00245] Overview

[00246] FLT3L is a 65 kDa non-disulfide homodimeric glycoprotein. Table 2 below shows the homology of the ligand and receptor sequences in humans, non-human primates and mice.

Table 2 Identity with human FLT3 and FLT3L

FLT3 ECD FLT3-L Javanese macaque 96% 95% rhesus monkey 97% 96% Mouse 85% 73%

[00247] Although the homology of the full-length mouse FLT3L protein is only 73%, its FLT3 binding site is highly conserved across species. Indeed, human FLT3L binds and activates mouse FLT3 and vice versa. FLT3L shares structural homology with stem cell factor (SCF or KIT ligand) and colony stimulating factor 1 (CSF1), but does not share significant sequence homology with other human cytokines. The receptors for these two ligands, c-KIT and CSF1R, respectively, are also class III TKRs that commonly interact with FLT3 inhibitors, resulting in unwanted off-target toxicity in clinical use. With these considerations in mind, a search was made for anti-FLT3L antibodies that would not cross-react with SCF or CSF1, providing highly specific suppression of the FLT3/FLT3L signaling pathway.

[00248] Lead antibodies were selected and tested in alternating selection procedures using soluble human and mouse FLT3-L. Primary biochemical high-throughput screens were performed using human FLT3/FLT3L competitive assays with time-resolved homogeneous fluorescence (HTRF). All matches for a single chain variable fragment with a crystallizing fragment region (scFv-Fc) were converted to IgG1 TM format, and then a functional screening assay using downregulation of FLT3 on the surface of the target cell was applied to confirm the inhibitory activity of leader antibodies. Mouse and cynomolgus cross-reactivity and selectivity for FLT3L, as well as exclusion of other members of the family such as stem cell factor (scf), were confirmed by ELISA and functional assays.

[00249] Lead compounds were further evaluated using FLT3 signaling assays in RS4;11 cell lines and primary human CD133+ stem cells. Binding to endogenous FLT3L was confirmed using primary human T cells. Site specific binding and binding affinity values were determined using Octet and BIAcore, respectively. The resulting leader antibody clone was selected for further optimization as described below.

[00250] LEADER ANTIBODY CAMPAIGN (C5, B10-11)

[00251] Lead antibodies for FLT3L binding assays were generated primarily by screening phage libraries as shown in FIG. 1. Phage display libraries comprising the Vaughan bone marrow (BMV) cell library, pooled spleen (CS) cell library, DP47 (DP47) library, and Dyax human antibody library were panned (alternative panning or competitive panning) against human FLT3L ( huFLT3L) and/or mouse FLT3L (muFLT3L). Briefly, self-produced FLT3L was labeled with biotin using the EZ-Link Sulfo-NHS-LC-Biotin labeling kit (Thermo Scientific) as the panning antigen. Panning was performed for two to three rounds using self-produced scFv libraries as described (Xiao X et al., 2017 mAbs 542 9, 996-1006 (2017)). To enhance cross-reactivity between human and cynomolgus monkey, human and cynomolgus recombinant FLT3L were alternately used as panning antigen in some selection procedures. Competitive panning was used in some experiments to select for antibodies suppressing FLT3L/FLT3 interactions. In the case of competitive panning, human FLT3L was used as the antigen for panning, while an excess (>100X) of FLT3-Fc (>100X) of FLT3-Fc (Xiao X et al. mAbs 542 9, 996-1006 ( 2017)).

[00252] The BMV library was enriched 100-fold, the CS library was enriched 350-fold, and the DP47 library was enriched 250-fold; the Dyax library was not enriched. The third time only the BMV and Dyax phage libraries were panned, resulting in a 100-fold enrichment of the BMV phage library and a 50-fold enrichment of the Dyax library compared to the second round of panning (FIG. 1A).

[00253] To assess the percentage of antigen-specific phages after each round of selection, ELISA of monoclonal phages was performed. For high throughput screening, only screening results that achieved at least a 20% positive score were processed. With this set of criteria, the third round BMV panning products, along with the second round CS and DP47 panning products, were inserted into pSplice V4 and pdLG or pmLG vectors and converted to either scFv-Fc format (Xiao X, et al., PLoS One, 2015 Oct 15; 10(10):e0140691. doi: 10.1371/journal.pone.0140691), or to IgG format (Xiao X, et al. mAbs 542 9, 996-1006 (2017)) for functional screens by competitive HTRF analysis.

[00254] For functional screening, FreeStyle 293 cells (Thermo Scientific) were first transfected with either scFv-Fc constructs or IgG constructs converted from panning products. The resulting supernatant was used directly in HTRF-based FLT3L/FLT3 interaction suppression assays. For the HTRF assay, 10 nM biotin-labeled FLT3L, 20 nM europium cryptate with streptavidin (Cisbio), 10 nM FLT3-mFc, and 20 nM anti-mFc A647 (Cisbio) were mixed with 10 µl of transfection-derived supernatant in a total volume of 20 µl/well in a Greiner 384 well plate. Readings at 665 nM and 620 nM were recorded five minutes after mixing and then one hour apart until the readings stabilized. The ratios 665 nM/620 nM were then calculated. A decrease in the ratio indicated suppression of the FLT3L/FLT3 interaction.

[00255] Panning products from direct or alternating antigen panning were converted to scFv-Fc and more than 4,000 colonies were selected for high throughput screening (HTS) in the FLT3L/FLT3-Fc interaction suppression HTRF assay. Ten lead antibodies were identified, converted to IgG-TM and expressed. The antibodies were retested in a second HTRF interaction suppression assay and ten leaders were identified. In parallel, over seven hundred competitive panning matches were converted to PmIgG and expressed in mammalian cells. Transformed clones were subjected to the same FLT3L/FLT3-Fc interaction suppression HTRF assay and two leaders were identified (FIG. 1B).

[00256] Ten leaders from alternating panning of antigens and two leaders from competitive panning were expressed and purified in milligram amounts for further testing. Additional testing included an HTRF FLT3L/FLT3-Fc interaction suppression assay, receptor down-modulation assays, and signaling suppression assays. The leaders were also subjected to epitope-specific sorting and affinity determination. During the screening campaign, five leading antibodies were identified, which were subjected to additional study: Dyax3, Dyax5, CAT8, CAT26 and CAT5D9. The results of the competitive HTRF assay for the lead antibodies are shown in FIG. 1C.

[00257] EXAMPLE #2 SCREENING ASSAY FOR FLT3 DOWN REGULATION

[00258] After ligation with FLT3L, the FLT3 receptor undergoes dimerization, autophosphorylation, and activates signaling downstream signaling pathways. In the course of this, surface FLT3 is internalized and destroyed. This internalization characteristic was used to develop an assay for screening candidate anti-FLT3L leader antibody clones. Cell surface FLT3 expression can be measured by flow cytometry, and inhibition of receptor binding to ligand can be determined by quantifying changes in cell surface FLT3 expression levels.

[00259] The RS4;11, EOL-1, MOLM13, and MV4-11 cell lines constitutively express FLT3. These cells were cultured under normal conditions and after 2-24 hours of culture were subjected to screening in relation to the relative level of expression of FLT3. Flow cytometry was used to measure FLT3 expression. Briefly, the commercial anti-CD135 (anti-FLT3) clone BV10A4H2 (Biolegend) was used in flow cytometry experiments, and the mean fluorescence intensity (MFI) was recorded as an indicator of FLT3 expression. In all assays, a commercially available mouse anti-human FLT3L monoclonal antibody (R&D) or a self-produced huFLT3-Fc construct was used as a positive control to effectively neutralize FLT3L activity.

[00260] RS4;11, an acute leukemia (pro-B) cell line, had consistent and high expression of FLT3 in culture compared to other commercially available lines reported to express FLT3 (FIG. 2A). Direct binding of FLT3L to FLT3 on the cell surface was confirmed by serial dilution of biotinylated recombinant huFLT3L (rhuFLT3L), which, after 30 min incubation with RS4;11 cells at 4°C, can be detected in a state physically bound to the cell surface using BV421-streptavidin followed by flow cytometry analysis to determine the mean fluorescence intensity (Figure 2B).

[00261] Finally, the ability of rhuFLT3L to induce detectable downregulation of FLT3 on the cell surface of RS4;11 cells was confirmed by incubating RS4;11 cells with serial dilutions of FLT3L at 37°C for 2 hours. Condition stability was assessed using 2 different concentrations of RS4;11 cells (50,000 (50,000) and 100,000 (100,000) cells). Downregulation of FLT3 on the cell surface was determined using a fluorescently labeled anti-CD135 antibody (clone BV10A4H2). At both cell densities, a dose-dependent downregulation of FLT3 was observed on the cell surface 2 hours after incubation with huFLT3L. Allophycocyanin (APC) MFI, serving as an indicator of FLT3 expression, was subject to a 25-fold decrease compared to the huFLT3L range used in this assay (Fig. 2C). These results indicated that the screening assay was effective in measuring the dose-response response of bioavailable FLT3L and would be useful for testing the ability of candidate clones to functionally neutralize FLT3L. The response was not significantly different regardless of whether 50 or 100 thousand cells per well were used.

[00262] OPTIMIZING THE SCREENING ANALYSIS

[00263] The screening assay conditions were further refined to determine ideal cell culture conditions for evaluating candidate anti-FLT3L antibody clones. End conditions were as follows: 50,000 RS4;11 cells per well for 2 hours in a humidified incubator set at 37°C, 5% CO ₂ , incubated with 96 pM rhuFLT3L, with or without candidate anti-FLT3L mAb clones, in complete medium Roswell Park Memorial Institute (RPMI) with one percent bovine serum albumin (BSA). Subsequent downregulation of FLT3 expression was determined by flow cytometry, the measurements of which were presented as either baseline MFI or % downregulation. A concentration of 96 pM rhuFLT3L (EC80) was chosen as this is the point at which the exponential phase of the dose-response curve begins and therefore provides an immediate reflection of any functional inhibition of rhuFLT3L by changes in the level of FLT3 downregulation at the cell surface. RS4 cells;11. (Fig. 3A). The performance of the assay was confirmed with a control, which was a commercially available mouse anti-huFLT3L antibody (MAB608, R&D Systems) (FIG. 3B). Due to the cross-species reactivity of FLT3 with its ligand, this assay, despite being performed on a human cell line, was effective for testing clones against human, cynomolgus and rodent FLT3L. In the case of mouse FLT3L, EC80 was 36 pM.

[00264] EXAMPLE 3. NEUTRALIZING ACTIVITY OF CANDIDATE LEADER ANTIBODIES DIRECTED TO SOLUBLE FLT3L

[00265] After FLT3L is ligated to FLT3, the receptor undergoes dimerization, autophosphorylation, and propagates the signaling cascade after internalization. This process, as measured by the downregulation of FLT3, can be suppressed by antibodies that bind to FLT3L. Therefore, lead candidates were tested for their ability to downregulate FLT3 on RS4;11 cells by binding human, mouse or cynomolgus monkey (sFLT3L) soluble FLT3L (sFLT3L). An optimized screening assay was used to test the neutralizing ability of the leading candidates. As described, RS4;11 cells (50,000 cells per well) were incubated for two hours in complete RPMI with one percent BSA in the presence of either human FLT3L (96 pM) or macaque or mouse sFLT3L (36 pM). Serial dilutions of each clone were made and either a soluble FLT3-Fc construct or a commercially available human anti-FLT3L antibody was used as positive controls. FLT3 expression on RS4;11 cells was determined by flow cytometry and recorded as MFI.

[00266] As seen in FIG. 4A and 4B, all leader antibody clones showed the ability to inhibit both human and macaque sFLT3L to some extent. All of CAT8, CAT26, Dyax3 and Dyax5 showed similar levels of inhibition of human sFLT3L and macaque sFLT3L. Although shown in Table 3 below, the IC ₅₀ values for CAT8, CAT26, Dyax3 and Dyax5 are not adequate because the IC _MAX value was not reached in any of the cases.

[00267] In contrast, CAT5D9 achieved IC _MAX and provided the S-curve expected for FLT3L inhibition (FIGS. 4A and 4B). The IC ₅₀ values for CAT5D9 presented in Table 3 indicated cross-reactivity with human and macaque FLT3L but not with mouse FLT3L (FIG. 4C).

TABLE 3. IC ₅₀ (nM) of candidate leader antibodies directed against 96 pM human, macaque, or mouse sFLT3L.

Person macaque Mouse R&D 0.04 0.10 0.12 CAT8 43.5 3001 n.a. CAT26 618 2584 n.a. DYAX3 54.8 3066 1489 DYAX5 245 6015 354 CAT5D9 385 564 n.a.

[00268] EXAMPLE 4. BINDING ACTIVITY OF CANDIDATE LEADER ANTIBODIES DIRECTED AGAINST FLT3L ON THE CELL SURFACE

[00269] FLT3L is expressed as a protein on the cell membrane and, after cleavage, circulates in the bloodstream as a soluble protein. Both membrane-bound and soluble forms are biologically active. To effectively block FLT3-mediated signaling pathways, candidate leader antibodies must bind both soluble and membrane-bound forms of FLT3L.

[00270] Accordingly, cell surface binding to human, macaque, and mouse FLT3L was assessed by transfection of Chinese Hamster Ovary (CHO) cells with the appropriate full-length protein for each species. Lead clones were incubated with FLT3L expressing cell lines for 1 hour at 4° C. before washing twice to remove unbound antibody. Cells were then incubated with PE labeled goat anti-human IgG secondary detection pAb to quantify bound antibody, which was measured by flow cytometry analysis of the signal from PE. The huFLT3-Fc construct on a human IgG backbone was used as a positive control reagent for FLT3L expression because, in addition to reacting with human ligand, it cross-reacts with macaque and mouse ligand.

[00271] All lead candidates bound human FLT3L (FIG. 5A), and all except DYAX5 bound macaque FLT3L (FIG. 5B). In contrast, only Dyax5 and to a lesser extent Dyax3 showed cross-reactivity with mouse FLT3L (FIG. 5C).

[00272] Thus, the ability of cross-species binding of FLT3L was observed in all candidate leaders, although with varying efficiency. Since differences in the degree of receptor occupancy for each clone were evident, both EC ₅₀ and maximum occupancy (expressed as a percentage of FLT3-Fc) were noted and taken into account in the final assessment. Table 4 shows the results for human, macaque and mouse FLT3L expressed in CHO cells.

Table 4. EC ₅₀ (nM) and maximum occupancy (% relative to FLT3-Fc)

Person macaque Mouse _EC50 % busy. _EC50 % busy. _EC50 % busy. FLT3-Fc 0.09 one hundred 0.6 one hundred 0.14 one hundred CAT8 0.08 60 9.0 32 n.a. n.a. CAT26 0.19 46 1.1 96 n.a. n.a. DYAX 3 0.27 33 1.8 35 0.39 sixteen DYAX 5 0.66 28 n.a. n.a. 0.11 51 CAT 5D9 0.157 40 1.1 one hundred n.a. n.a.

[00273] EXAMPLE 5. BINDING OF CANDIDATE LEADER ANTIBODIES TO HUMAN ENDOGENOUS FLT3L

[00274] FLT3L is expressed on the surface of primary T cells when stimulated by γ-chain cytokines, namely IL-2, IL-7, or IL-15, regardless of TCR involvement. In Example 4, candidate leader antibodies demonstrated the ability to bind to CHO cells transfected with human FLT3L protein. The next step was to ensure that they could bind endogenous FLT3L from a primary human cell line. Therefore, candidate leader antibodies were tested for their ability to bind huFLT3L on IL-2 stimulated primary T cells derived from human donors.

[00275] To induce cell surface expression of FLT3L, freshly isolated human T cells were stimulated with 50 ng/ml IL-2 in the absence of anti-CD3 antibody (activation of T cells with anti-CD3 antibody would shed FLT3L from the cell surface) for 5 days . After this time period, expression on T cells was confirmed using the human FLT3-Fc construct (FIG. 6A). Serial dilutions of each clone were then incubated with IL-2 stimulated T cells (100k/well) for 30 minutes at 4°C. Excess antibodies were removed by washing with buffer, and surface-bound antibody was detected with an APC-labeled anti-human IgG antibody. All clone leaders except CAT5D9 bound endogenous FLT3L on primary human T cells (FIG. 6B). Binding of CAT5D9 to endogenous FLT3L was not initially detectable due to its low binding affinity. However, when CAT5D9 was dimerized with an anti-IgG antibody (labeled with APC) prior to incubation with T cells, its avidity increased sufficiently to confirm dose-dependent binding to endogenous FLT3L (FIG. 6C).

[00276] EXAMPLE 6. INHIBITION BY CANDIDATE LEADER ANTIBODIES FLT3L ON THE CELL SURFACE

[00277] The ability of lead candidates to bind FLT3L on the cell surface provided limited insight into functional ligand inhibition. Ideally, binding of candidate leaders to FLT3L on the cell surface should reduce the signaling activity of the ligand-receptor complex.

[00278] In order to test this, 1000 huFLT3L expressing CHO cells/well were seeded overnight for adherence. The next day, CHO culture medium was removed, cells were gently washed with RPMI, and serial antibody dilutions were added for 30 minutes prior to addition of FLT3+ RS4;11 (100 k/well). After 2 hours of incubation at 36° C., RS4;11 cells were transferred to a new 96-well plate on ice for staining to detect downregulation of FLT3. In addition to the dye used for our standard FLT3 downregulation assays for RS4;11, an anti-CD19 antibody was included to exclude any contaminating CHO cells. Downregulation of FLT3 was measured by flow cytometry.

[00279] The results of the blocking assay with candidate leader antibodies indicated that all candidate leader had the ability to inhibit FLT3L at the cell surface to some extent, although all candidates showed relatively low activity compared to the commercial control antibody, reflecting low affinity (Fig. 7 ).

[00280] EXAMPLE #7 SUPPRESSION BY LEADER CLONES OF FLT3L-INDUCED FLT3 SIGNALING

[00281] FLT3 autophosphorylation leads to activation of signaling networks primarily through PI3K and the RAS cascade, which in turn leads to activation of AKT (protein kinase B, PKB), MEK and ERK. The signaling cascade ultimately leads to the transcription of genes that promote cell survival and proliferation. To confirm that candidate leader antibodies block subsequent FLT3 signaling induced by FLT3L in primary human cells, ERK and MEK phosphorylation in CD133+ stem cells was measured using the Mesoscale MSD Phospho-ERK1/2 and Phosphor MEK1/2 Whole Cell Lysate kits.

[00282] The assay was validated using the RS4;11 cell line with FLT3L-induced ERK activation in a dose-dependent manner (FIG. 8A). Serial dilutions of FLT3L were incubated with 300,000 RS4;11 cells per well for 8 minutes at 36°C. The cells were then harvested, lysed and analyzed for phosphorylated ERK according to the manufacturer's instructions. As in earlier assays, EC80 (476 pM) for ERK activation by FLT3L was determined and used to test the inhibitory activity of candidate anti-FLT3L antibody clones. The suitability of the assay was confirmed using a commercially available mouse anti-human FLT3L antibody as a positive control for effective neutralization (FIG. 8B).

[00283] Using these set parameters, the leader clones were tested using in vitro expanded primary CD133+ stem cells that were validated for FLT3 expression prior to use. Clones-leaders were pre-incubated with 476 pM FLT3L for 30 minutes, then they were added to CD133+ stem cells. After incubation for 8 minutes at 36° C., cells were harvested, lysed and analyzed for phosphorylated ERK and MEK using MSD assays according to the manufacturer's instructions. A commercially available mouse anti-human FLT3L antibody was used as a positive control.

[00284] All candidate clones appear to suppress MEK (FIG. 9A) and ERK (FIG. 9B) induction by FLT3L. However, only CAT5D9 achieved IC _MAX and provided the expected S-shaped dose-response curve (Table 5). Taken together, the results presented in the examples suggested that CAT5D9 was the best candidate leader clone and should be used for further optimization based on biophysical assessment of affinity and epitope binding site.

Table 5. IC ₅₀ (nM) for binding to 0.476 nM FLT3L

MEK ERK R&D 0.19 0.19 CAT8 ^7.4e6 0 CAT26 2.1e ¹⁷ 2.4e ³⁸ DYAX3 93 2.4 DYAX5 ^2.4e7 25 CAT5D9 181 187

[00285] EXAMPLE #8 CONFIRMATION OF TARGET SPECIFICITY

[00286] According to the results of functional evaluation in biological assays, CAT5D9 appeared to be the best lead candidate. Epitope-specific sorting using the Octet system was used to determine the CAT5D9 binding region for the FLT3 receptor.

[00287] Epitope-specific sorting was used to identify leader antibodies that share the same FLT3L binding region as the FLT3 receptor. Sorting was carried out in three phases. In phase I, the binding of the biotin-FLT3L complex to the avidin probe was performed. In phase II, individual antibodies were bound to the biotin-FLT3L complex. In phase III, each of the tested antibodies was added to the phase II antibody. Any additional binding detected was indicative of the presence of non-competitive binding sites for the FLT3L target in the two antibodies. Buffer alone was used as negative and positive controls without the addition of phase III antibodies and FLT3-Fc, respectively. If only buffer was added in phase III, the resulting kinetics of dissociation of the antibody from FLT3L reflected the clone's characteristic affinity for the target.

[00288] Only CAT5D9 added at 1x and 2x concentrations suppressed binding to FLT3-Fc, indicating that CAT5D9 acted on the required target site on FLT3L (i.e., it had the same or overlapping epitope as FLT3- Fc) (FIG. 10A). Importantly, in contrast, the remaining four candidate clones were not inhibited by CAT5D9, indicating that they bound to a different FLT3 epitope (FIG. 10A). In addition, the fast dissociation rate with the addition of buffer alone confirmed the results obtained in the previous experiment, indicating that the affinity of CAT5D9 is low (meaning that its potential to improve efficiency through optimization was high). Accordingly, if each of the 4 remaining clones bound in phase II (CAT8 is shown as a representative graph in Figure 10B), additional binding of CAT5D9 and FLT3-Fc can be detected in phase III, reflecting the presence of different sites in them. binding. It was also observed that each of the 4 remaining clones sorted together and that with the addition of buffer alone, their dissociation rate was relatively low.

[00289] Together, these data confirm that only CAT5D9 competed directly with FLT3 for the FLT3L binding region and appears to do so with low affinity binding, suggesting its potential for optimization. On the other hand, all remaining clones interacted with a site that, based on functional data, did not directly compete with FLT3. Their slow dissociation rate indicated that they already bound with sufficient affinity for FLT3L and would have little potential for optimization. The inhibition initially present in the initial screening assays was likely due to steric hindrance or partial blockage of the binding site in the receptor.

[00290] EXAMPLE #9 BIACORE (C9) BINDING KINETICS

[00291] The Biacore assay was used to determine the binding kinetics of the leader antibodies and confirm the Octet assay data, which indicated that CAT5D9 had low affinity for FLT3L. The binding kinetics of the antigen-binding fragment of the antibody to human FLT3L and FLTL3 and cynomolgus monkey was determined on human CAT8, CAT26, Dyax3 and Dyax5. In addition, the kinetics of antigen binding to the CAT5D9 fragment was determined for both human FLT3L and cynomolgus FLT3L.

[00292] The results are shown in Table 6. The equilibrium dissociation constant (K _D ) was more than 50 times higher in human and cynomolgus CAT5D9 compared to the rest of the lead antibodies. CAT5D9 was characterized by low efficient fast dissociation kinetics (Human = 70.72, Monkey = 70.66). As a result, equilibrium binding data were obtained to check the K _D kinetic data. Equilibrium binding confirmed the results for kinetic binding, showing similar values (table 3). These data supported the low affinity of CAT5D9 and its potential to improve performance through optimization.

Table 6. Binding kinetics of leader antibodies to FLT3L

Fab to FLT3L Species affiliation FLT3L kon
(xE+5/Ms) koff
(xE-3/s) K _D (kin.: koff/kon)
(nM) K _D (equilibrium)
(nM) CAT8 people 6.28 5.56 8.85 n. about. CAT26 people 1.91 0.84 4.37 n. about. Dyax3 people 0.66 1.23 18.69 n. about. Dyax5 people 2.28 5.01 21.96 n. about. CAT5D9 people 0.61 70.72 1157 1595 CAT5D9 macaques 0.61 70.66 1157 1607

Fab=antigen binding fragment; people = person; macaque = Javanese macaque; kon=association constant during binding; Koff=dissociation constant during binding; K _D= equilibrium dissociation constant.

[00293] EXAMPLE #10 NO CROSS REACTIVITY IN CAT5D9

[00294] In addition to confirming that CAT5D9 binds to the correct FLT3L epitope, it was also important to confirm that it would not bind to structurally related FLT3L homologs of stem cell factor (SCF) and colony stimulating factor (CSF1). Both factors are ligands for receptor protein tyrosine kinases (c-Kit and CSFR1, respectively), which are the major off-target molecules for small molecule FLT3 inhibitors currently used in oncology to control cancer resulting from a constitutively activated mutation of FLT3- IT9D.

[00295] In order to test this, the ELISA plate was coated with 2 μg of recombinant human SCF or CSF1. After washing, the plate was blocked with 3% milk in Tris-Phosphate Buffered Saline (TPBS) and serial (x2) dilutions of leader antibodies were added, starting at 50 μg/ml. After incubation, unbound antibody was removed by washing, and bound antibody was detected using an HRP-conjugated anti-human IgG antibody in combination with TMB substrate for color development. Commercially available goat anti-SCF pAb and mouse anti-CSF1 mAb were used as positive binding controls.

[00296] None of the lead candidates cross-reacted with huSCF (FIG. 11A) or huCSF1 (FIG. 11B). Importantly, these results show that CAT5D9 is selective in binding only FLT3L, but not structurally similar TKR ligand molecules.

[00297] EXAMPLE #11 CAT5D9 AFFINITY OPTIMIZATION

[00298] As discussed above, CAT5D9 bound to FLT3L with low affinity and therefore showed the potential to improve performance through affinity optimization. The desired KD of 300 pM was set from the results of PK modeling. The optimization campaign was designed to achieve a 10,000x improvement in KD.

[00299] After modifying the framework regions at the germline gene level, two parallel strategies were used - sparing mutagenesis and block mutagenesis. Lean mutagenesis mutates every position in all 6 CDRs for all 20 amino acids, one at a time. Clones were screened using high-throughput methods, and individual beneficial mutations were combined. Block mutagenesis mutates contiguous regions of 5 to 6 positions in the CDR in an overlapping pattern, and the resulting libraries of approximately 1E6-1E7 clones are first enriched by phage display panning techniques and then screened by high throughput methods.

[00300] After the first round of optimization, 30 sparing mutagenesis clones and 24 block mutagenesis clones were tested in IgG format. Using molecular modeling techniques, the present inventors identified and combined the best mutations, resulting in clone 5D9-clone 6, which reached a KD of 1610 nM (as measured by Biacore), corresponding to a 700-fold improvement in affinity compared to the original 5D9 (see table 7). The affinity of the leading clones SC4017 and AM40 exceeded the CDTP criteria (<300 pM). In addition, both optimized clones bound and neutralized endogenous cell surface FLTL, bound endogenous human serum soluble FLT3L and bound and neutralized macaque FLT3L.

Table 7 Summary of binding data for leader clones from CAT5D9 optimization

kon
(1E+5/Ms) koff
(1E-4/s) K _D
(pM) hFLT3/hFLT3L receptor 3.6 81.2 22.340 Original 5D9/hFLT3L original 0.6 707.2 1,157,000 5D9-C06/hFLT3L Round 1 5.5 8.8 1.610 AM40/hFLT3L Round 2 10.9 1.8 170 AM40/cFLT3L Round 2 26.6 1.7 63 SC4017/hFLT3L Round 2 14.5 0.5 37 SC4017/cFLT3L Round 2 58.4 0.5 8.8

[00301] This increase in affinity equated to a >1000-fold improvement in functional activity as measured by FLT3 downregulation performed on RS4;11 cells (FIG. 12A) using the method described for earlier clone selection procedures.

[00302] A second round of affinity optimization was performed to achieve an affinity of 300 pM. Clone 6 (C06) was mutated and the resulting mutants were screened in the same manner as in the first optimization round. As part of phage panning during block mutagenesis, clone 6 was used in IgG format as a competitor to enrich clones with significantly higher affinity. The best clone obtained by block mutagenesis was clone AM40 with KD=170 pM. Combined several combinatorial clones of AM40 and mutations resulting from the second round of sparing mutagenesis, and received one superior clone, which is SC4017 with KD=37 pM. This higher affinity was again reflected in improved functional inhibition of FLT3L as demonstrated by the downregulation of FLT3 on RS4;11 cells (FIG. 12B). However, in additional analysis, the superior performance of SC4017 was attributed to one additional tryptophan added adjacent to the binding region. This posed a design risk given the vulnerability to oxidation of environmentally exposed tryptophan residues. For this reason, AM40 was chosen as the clone leader of IgG. Despite a slightly lower affinity (170 pM) compared to SC4017 (37 pM), AM40 still exceeded the original target of 300 pM and was determined to be at lower risk for development.

[00303] Finally, the present inventors confirmed that both clones could neutralize endogenous FLT3L at the cell surface by designing an assay using primary T cells stimulated with 20 ng/ml IL-7 for 7 days (this was found to be most efficient protocol to induce cell surface FLT3L expression in primary T cells) in co-culture with FLT3+ RS4;11. Briefly, IL-7 stimulated CD4+ T cells were incubated with RS4;11 cells overnight at a ratio of 15:1 (dose ratio response shown in FIG. 13A) either alone or in the presence of serial dilution of candidate SC4017 clones. and AM40. Downregulation of FLT3 was measured by flow cytometry as described previously. Both clones with similar efficacy were shown to completely prevent FLT3 downregulation on RS4;11 cells at concentrations above 1 nM (FIG. 13B).

[00304] EXAMPLE #12 FLT3L NEUTRALIZATION IN HEALTHY NON-HUMAN PRIMATES

[00305] To determine the safety and stability of AM40 in FLT3L neutralization, a toxicity study was performed over a period of one month with multiple weekly doses. An eight-week post-treatment follow-up period was included in the study to track progress in animals. The study scheme is shown in Fig. 14. As shown in FIG. 15A, free soluble FLT3L levels dropped dramatically after the first AM40 administration at all doses, but the 0.3 mg/kg dose was insufficient to maintain the target interaction throughout the week. The higher dose groups (1mg/kg and 30mg/kg) had complete interaction with target molecules (expressed as free soluble FLT3L below BLQ) consistently until day 57, after which soluble FLT3L levels returned to baseline in group 1, 0 mg/kg.

[00306] Similarly, measuring the incidence of DC in the circulation (% of total CD45+ cells detected by flow cytometry and expressed as a percentage of baseline levels before the study) revealed a steady decrease in the incidence of CD1c+ (classic) DC and plasmacytoid DC until the day 22 in the 1.0 and 30 mg/kg groups (FIG. 15B). The incidence rate of CD1c+ DC in the circulation remained reduced until day 50 and day 85 for the 1 mg/kg group and the 30 mg/kg group, respectively. The incidence of pDC in the circulation remained reduced until day 71 and day 85 for the 1 mg/kg group and the 30 mg/kg group, respectively. Recovery of DC population levels in the 1.0 mg/kg group correlated with recovery of free FLT3L serum levels, which occurred at some point in time between day 57 and day 85. These results indicate that DC population levels decreased when neutralizing FLT3L with AM40, but quickly returned to baseline when free FLT3L became available.

[00307] EXAMPLE #13 FLT3L EXPRESSION CORRELATES WITH Severity of Systemic Lupus Erythematosus (SLE)

[00308] SLE is an autoimmune disease characterized by chronic inflammation and can affect almost every organ in the body and in all age groups. SLE usually affects the joints, skin, kidneys, lungs, heart, and brain. Given its role in pro-inflammatory signaling, FLT3L expression in individuals with SLE was examined in search of a correlation between FLT3L levels and disease severity. The studies published to date have largely been based on serum FLT3L levels when correlated with disease, and although this is the most practical measure in clinical settings, it has the inherent disadvantage of being a reflection of production minus that taken up by DC cells and other activated FLT3L-consuming cells. Under conditions of inflammation, the number of FLT3-expressing cells and their consumption of FLT3L will vary greatly, and this probably explains the differences in the results obtained in various studies, and why none of them showed a direct correlation between serum FLT3L and clinical indicators. disease progression. Given that T cells are one of the predominant sources of FLT3L under inflammatory conditions, the present inventors developed an assay to measure FLT3L expression directly on the surface of T cells using freshly isolated peripheral blood mononuclear cells (PBMCs).

[00309] Serum and PBMC were isolated from individuals with SLE (n=24) and healthy donors (HD; n=15). Serum FLT3L was measured by ELISA (R&D Systems) according to the manufacturer's instructions, and the frequency of FLT3L-expressing CD4+ T cells was determined using a self-developed flow cytometry assay, with FLT3L detected using a fluorescently labeled anti-FLT3L antibody clone, MAB608 (R&D Systems). The SLE disease activity index (SLEDAI) was used to determine lupus activity in the same individuals. Significance was determined using the Mann-Whitney and Spearman correlation to compare healthy and diseased cohorts and correlation scores with SLEDAI, respectively.

[00310] Consistent with previous data from the literature, serum FLT3L levels were assessed in SLE donors versus HD (p<0.05; FIG. 16A), but no significant correlation with disease activity was found (SLEDAI) (p< 0.07, Fig. 16B). In contrast, when FLT3L production was measured by the frequency of FLT3L-expressing CD4+ T cells, there was a very significant increase in SLE donors compared to HD (p<0.0001; Fig. 16C) and a strong correlation with SLEDAI scores (r=0, 7045; p<0.0001; Fig. 16D). These data suggest that measuring FLT3L expression on T cells may be particularly relevant in disease settings.

[00311] After finding a correlation between SLEDAI scores and FLT3L-expressing CD4+ T cells, subpopulations of CD4+ cells were examined to determine if (1) FLT3L expression in subpopulations of CD4+ T cells from SLE-suffering patients with known biology, and (2) whether subpopulation expression correlates with SLEDAI scores. The studied subpopulations of CD4+ cells were naive T cells (T _naive ), effector memory cells (T _EM ) and central memory cells (T _CM ). The same protocols and significance levels as described above were used to study expression and correlations.

[00312] FLT3L expression in CD4+ T cell subpopulations was comparable to the known biology of FLT3L expression. In particular, FLT3L expression was not generally observed on HD T _naive cells, although there was a slight but significant increase in SLE donors (FIG. 17A, upper panel). FLT3L expression on T _naive cells from SLE donors was significantly correlated with SLEDAI scores (Fig. 17A, lower panel; r=0.6629; p=0.0004). Importantly, FLT3L expression was observed on CD4+ T _EM cells in both HD and SLE subjects, as would be expected for this population, which captures newly activated T cells that will be exposed to cytokines with γ- chain known to induce FLT3L expression. Although this response was observed in both HD and SLE donors, it was significantly increased in the latter, and again, expression in SLE donors correlated with SLEDAI (Fig. 17B, lower panel; r=0.6201 ; p=0.0012). Finally, FLT3L expression decreased in healthy CD4+ T cells as they differentiated from T _EM to T _CM but was retained in PBMC from SLE donors (Fig. 17C, top; p<0.0001). Again, there is a significant correlation between the frequency of FLT3L+ T cells and SLEDAI scores, suggesting that expression in this group is a reflection of a chronic inflammatory condition.

[00313] Taken together, these studies show that FLT3L expression on the surface of CD4+ T cells from SLE patients is significantly increased compared to expression in HD patients. Moreover, increased FLT3L expression strongly correlated with SLEDAI scores. Thus, administration of anti-FLT3L antibodies to SLE patients is a reasonable therapeutic strategy to reduce FLT3L-expressing T cell populations in order to reduce inflammation in SLE patients.

[00314] The above method was validated using in situ detection according to the PrimeFlow IC method of FLT3L RNA and confirmed using APC-conjugated AM40.

[00315] EXAMPLE #14 FLT3L EXPRESSION IN MYOSITIS

[00316] Myositis is a chronic muscle inflammation that is characterized by weakness, swelling, and muscle pain. At the cellular level, myositis is characterized by elevated levels of type 1 interferon proteins and pDC. Myositis may be associated with SLE and other pro-inflammatory conditions. Therefore, the expression of FLT3L in individuals with myositis was investigated in search of a correlation between FLT3L levels and the severity of the disease.

[00317] Using FACS, PBMCs from myositis and HD individuals were examined for FLT3L-expressing CD4+ T cells. CD4+ T cells were divided into subpopulations T _naive , T _EM and T _CM . Significance between myositis and HD samples was determined using Mann-Whitney analysis.

[00318] As seen in FIG. 18A and 18B, FLT3L expression in PBMCs from individuals with myositis was consistent with the results obtained from SLE patients, as they were characterized by a significant increase in the percentage of CD4+ T cells positive for FLT3L (T _naive (p<0.05; Fig. 18A ), T _EM (p<0.0001; Fig. 18B) and T _CM (p<0.0001; Fig. 18C)). In light of these results, administration of anti-FLT3L antibodies to patients with myositis is a reasonable therapeutic strategy to reduce populations of FLT3L-expressing T cells in order to reduce inflammation in patients with myositis.

[00319] EXAMPLE #15 FLT3L EXPRESSION IN NEPHRITE

[00320] Nephritis is an immune disorder that affects the kidneys and associated kidney structures. This pathological condition may occur due to SLE, certain toxins, or certain infections. Nephritis can lead to permanent loss of kidney function, which can be fatal. Dendritic cells have been shown to infiltrate the kidney in lupus nephritis (Fiore et al., (2008) Mol Immunology v45: 259-265) and are thought to play a role in triggering inflammation in the kidney, so it has been suggested that the blockage FLT3L can suppress DC and prevent progressive loss of kidney function.

[00321] Murphy Roths Large/lymphoproliferative mouse model of nephritis (MRL.lpr) and mouse anti-FLT3L surrogate antibody (LFC-1) were used to study the effect of FLT3L blocking on proteinuria levels and nephritis score. An isotype control was included as well as an anti-IFNAR antibody treatment group. Mice injected with anti-FLT3L antibody showed a significant reduction in proteinuria scores 17 weeks after administration (FIG. 19A). In addition, anti-FLT3L treated mice had reduced nephritis scores at 18 weeks compared to isotype controls (FIG. 19B). Importantly, the levels of proteinuria and nephritis in anti-FLT3L treated mice were lower than in control treated mice. These results supported the role of FLT3L-mediated inflammation in nephritis. In light of these results, administration of anti-FLT3L antibodies to patients with nephritis is a reasonable therapeutic strategy to reduce populations of FLT3L-expressing T cells in order to reduce inflammation in patients with nephritis.

[00322] Spleen DC populations were also examined to gain insight into FLT3L-associated changes in leukocyte populations in nephritis. Spleens were removed from MRL mice at 18 weeks and changes in spleen DC populations were examined. Anti-FLT3L antibody treatment significantly reduced circulating DCs in MRL mice (Fig. 20A-C). In particular, after administration of the anti-FLT3L antibody, the amount of Siglec-H+-pDC significantly decreased compared to the isotype controls (FIG. 20A). Similarly, significant reductions in numbers were observed in CD11b+ cDC (equivalent to human CD1c+ DC) and CD8+ cDC (equivalent to human CD141+ DC) (FIGS. 20B and 20C). In addition, no cases of dermatitis were observed in mice treated with anti-FLT3L antibody compared to the usual 30-40% incidence in mice without treatment. Therefore, anti-FLT3L antibody treatment reduced circulating DC populations and improved secondary pathology (dermatitis) in the nephritis model. In light of these results, administration of anti-FLT3L antibodies to patients with nephritis may reduce inflammation and tissue damage by suppressing DC populations.

[00323] EXAMPLE #16 FLT3L EXPRESSION IN SJOGREN'S SYNDROME

[00324] Primary Sjögren's syndrome (pSS) is an autoimmune condition characterized by extensive dryness of the eyes and salivary glands. In addition, this pathological condition can cause multiple organ dysfunction. This syndrome occurs on its own or in the presence of additional autoimmune diseases such as lupus or rheumatoid arthritis. Individuals with pSS have elevated serum levels of FLT3L, and there is evidence of local expression of both FLT3L and its receptor in the inflamed salivary gland (Tobon et al., (2010) Arthritis and Rheumatism v62: 344).

[00325] To study the pathology of the salivary glands after prolonged treatment with an antibody to FLT3L (LFC-1), a mouse model of Sjögren's syndrome NOD.H2h4 was used. At 16 weeks of age, these mice develop tertiary lymphoid structures (TLS) in the salivary gland (SG) that predominantly contain DC, B220+ B cells, and CD3+ T cells, thus highly similar to the pathological changes seen in humans. . This tissue damage is preceded by the production of autoantibodies and the formation of spontaneous germinal centers in the spleen (Mahmoud et al., 2016 Science Translational Medicine, v8 361ra137). Mice were treated using either prophylactic (starting at 5 weeks of age) or therapeutic (starting at 17 weeks of age) protocol with IgG isotype controls (5 mg/kg), anti-FLT3L antibody (5 mg/kg). For both protocols, treatment was continued by dosing twice a week until the end of the study (26 weeks). The anti-FLT3L monoclonal antibody (LFC-1) effectively neutralized FLT3L throughout the course of treatment (FIG. 25A) and resulted in circulating drug/ligand complex accumulation (FIG. 25B).

[00326] To assess changes in peripheral immune cell populations, lymphoid organs were harvested, and salivary glands (SG) were harvested and evaluated for tissue pathology (TLS frequency). While it has been previously reported that prophylactic treatments can prevent the onset of disease, there are currently limited publications that therapeutic intervention can slow or prevent tissue damage after disease onset. The anti-FLT3L monoclonal antibody (LFC-1) reduced the incidence of CD44 ^HI antigen-exposed CD4+ and CD8+ T cells in the spleen and salivary gland-draining LNs (at the end of the study at 24-26 weeks of age) (Fig. 26A - 26D), and also selectively reduced the levels of specific autoantibodies to collagen IV and platelet extract (Fig. 27).

[00327] As expected, isotype IgG control animals had increased TLS production in the salivary gland (measured as frequency per mm ^{2 of} tissue) by 26 weeks of age, indicative of salivary gland damage. In mice treated with anti-FLT3L antibody, even at therapeutic doses, there was a significant reduction in SG tissue damage (FIG. 21A) and disease progression was completely prevented at prophylactic doses (FIG. 21B). The DC cell populations measured in the spleen were significantly suppressed, although not completely eliminated (Fig. 22A-D). Yet this was enough to have a significant impact on the onset and progression of the disease by reducing inflammatory infiltration into the salivary gland. These results confirmed that inflammation in pSS is driven by FLT3L-mediated mechanisms. In light of these results, administration of anti-FLT3L antibodies is a reasonable therapeutic strategy to treat pSS in human subjects.

It will be apparent from the above description that the present invention described herein may be subject to changes and modifications to adapt it to various applications and conditions. Such embodiments are also within the scope of the following claims. Reference to a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The designation of an embodiment herein includes such embodiment as any single embodiment or in combination with any other embodiments or parts thereof. All patents and publications mentioned in this specification are incorporated herein by reference to the same extent as if each independent patent and each independent publication were specifically and individually identified as being incorporated by reference.

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SEQUENCE LIST

<110> Viela Bio, Inc.

<120> ANTIBODIES TO RECEPTOR TYROSINE KINASE 3 LIGAND LIKE PRODUCT

PROTOONCOGENE OF CAT SARCOMA VIRUS (FMS) OF MCDONOUGH STRAIN (FLT3L), AND

VARIANTS OF THEIR APPLICATION FOR THE TREATMENT OF AUTOIMMUNE AND INFLAMMATORY

DISEASES

<130> 054493-502001WO

<150> US 62/630,571

<151> 2018-02-14

<160> 72

<170> PatentIn version 3.5

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<223> "AM40_VL"

<400> 2

Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys

1.5 10 15

Thr Val Thr Ile Ser Cys Thr Arg Thr Ser Gly Asn Ile Ala Gly Tyr

20 25 30

Phe Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val

35 40 45

Ile Tyr Glu Asp Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly

65 70 75 80

Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp

85 90 95

Tyr Arg Arg Ala Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 3

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "SC4017_VH"

<400> 3

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Thr Arg Pro Pro Thr Ser Arg Thr Ala Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Val Asp Glu Ser Thr Ser Thr Gly Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Asn Asp Phe Val Tyr Gly Ser Tyr Arg Phe Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 4

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "SC4017_VL"

<400> 4

Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys

1.5 10 15

Thr Val Thr Ile Ser Cys Thr Arg Thr Ser Gly Trp Ile Ala Gly Tyr

20 25 30

Phe Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val

35 40 45

Ile Tyr Glu Asp Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly

65 70 75 80

Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp

85 90 95

Tyr Arg Arg Ala Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 5

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT5D9_VH"

<400> 5

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ile Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Arg Thr Ala Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Val Asp Glu Ser Ala Ser Thr Gly Tyr

65 70 75 80

Ile Glu Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Asn Asn Tyr Val Trp Gly Ser Tyr Arg Phe Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 6

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT5D9_VL"

<400> 6

Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys

1.5 10 15

Thr Val Thr Ile Ser Cys Thr Arg Thr Ser Gly Asn Ile Ala Gly Tyr

20 25 30

Phe Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val

35 40 45

Ile Tyr Glu Asp Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly

65 70 75 80

Leu Lys Pro Asp Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp

85 90 95

Thr Ser Gln Gly Val Phe Gly Ala Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 7

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT8_VH"

<400> 7

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ser Gly Tyr Tyr Gly Ala Asn Phe Asp Phe Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 8

<211> 109

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT8_VL"

<400> 8

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1.5 10 15

Arg Val Ala Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Gly

20 25 30

Tyr Val Tyr Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro Thr Leu Leu

35 40 45

Ile His Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Gly Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val

100 105

<210> 9

<211> 123

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT26_VH"

<400> 9

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Asp Ala Tyr Gly Ser Ser Trp Tyr Phe Tyr Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 10

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT26_VL"

<400> 10

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1.5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ile Asn

20 25 30

Pro Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Val Leu

35 40 45

Ile Tyr Ser Asp Lys Tyr Arg Pro Ser Gly Val Ala Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Arg Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 11

<211> 126

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX3_VH"

<400> 11

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr

20 25 30

Glu Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Val Ile Pro Ser Gly Gly Lys Thr Phe Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Tyr Ser Arg Trp Phe Gly Gln Leu Gly Phe Tyr Ser His Tyr Ala

100 105 110

Met Asp Val Trp Ser Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 12

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX3_VL"

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Ala Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Thr Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Lys Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Thr Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ser Tyr Phe Cys Gln Gln Ser Tyr Ser Ser Pro Gly

85 90 95

Ile Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 13

<211> 127

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX5_VH"

<400> 13

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1.5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Ser Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Tyr Ser Arg Trp Phe Gly Gln Leu Gly Phe Tyr Ser His Tyr

100 105 110

Ala Met Asp Val Trp Ser Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 14

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX5_VL"

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1.5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> "AM40_VH"

<400> 15

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agttatgctc ttagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaacg cggccgccga cctcccggac agcaagctac 180

gcacagaaat ttcagggcag agtcacgatt accgtggacg aatccacgag cacaggctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gtcaaacgac 300

360

<210> 16

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> "AM40_VL"

<400> 16

aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60

tcctgcaccc gcaccagtgg gaacattgcc ggctactttg tgcagtggta ccagcagcgc 120

ccgggcagtt cccccaccac tgtgatctat gaggattacc aacgaccctc tggggtccct 180

gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240

ctgaagactg aggacgaggc tgactactat tgtcagtctt atgatgacta ccggcgggcg 300

gcgttcggcg gagggaccaa gctgaccgtc cta 333

<210> 17

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> "SC4017_VH"

<400> 17

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agttatgctc ttagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaacg cggccgccga cctcccggac agcaagctac 180

gcacagaaat ttcagggcag agtcacgatt accgtggacg aatccacgag cacaggctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gtcaaacgac 300

ttcgtgtacg ggagttatcg tttctggggc caagggacca cggtcaccgt ctcctca 357

<210> 18

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> "SC4017_VL"

<400> 18

aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60

tcctgcaccc gcaccagtgg gtggattgcc ggctactttg tgcagtggta ccagcagcgc 120

ccgggcagtt cccccaccac tgtgatctat gaggattacc aacgaccctc tggggtccct 180

gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240

ctgaagactg aggacgaggc tgactactat tgtcagtctt atgatgacta ccggcgggcg 300

gcgttcggcg gagggaccaa gctgaccgtc cta 333

<210> 19

<211> 357

<212> DNA

<213> Artificial sequence

<220>

<223> "CAT5D9_VH"

<400> 19

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaaga tttctggagg caccttcagc agttatgctc ttagctgggt gcgacaggcc 120

ccctggacaag ggcttgagtg gatgggaggg atcatccctg tctttcggac agcaagctac 180

gcacagaaat ttcagggcag agtcacgatt accgtggacg aatccgcgag cacaggctac 240

atagaactga gcagcctgaa atctgaggac acggccacat attactgtgc gtcaaataat 300

tacgtttggg ggagttatcg tttctggggc caggggacca cggtcaccgt ctcctca 357

<210> 20

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> "CAT5D9_VL"

<400> 20

aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtcaccatc 60

tcctgcaccc gcaccagtgg gaacattgcc ggctactttg tgcagtggta ccagcagcgc 120

ccgggcagtt cccccaccac tgtgatctat gaggattacc aacgaccctc tggggtccct 180

gatcggttct ctggctccat cgacaggtcc tccaactctg cctccctcac catctctgga 240

ctgaagcctg acgacgaggc tgactactat tgtcagtctt atgatgacac ctctcaaggt 300

gtgttcggcg cagggaccaa ggtcaccgtc cta 333

<210> 21

<211> 360

<212> DNA

<213> Artificial sequence

<220>

<223> "CAT8_VH"

<400> 21

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaagcagc 300

ggctactacg gggccaattt tgacttctgg gggcagggga ccacggtcac cgtctcgagt 360

<210> 22

<211> 327

<212> DNA

<213> Artificial sequence

<220>

<223> "CAT8_VL"

<400> 22

cagtctgtgc tgacgcagcc gccctcagcg tccgggaccc ccgggcagag ggtcgccatc 60

tcttgttctg gaagcagctc caacatcggga agtggttatg tatactggta tcagcaggtc 120

cgggaacgg cccccacact cctcatccat aggaataatc agcggccctc aggggtccct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240

tccgaggatg aggctgatta ttactgtgca gcgtgggatg acagcctgag tggttatgtc 300

ttcggaactg ggaccaaggt caccgtc 327

<210> 23

<211> 369

<212> DNA

<213> Artificial sequence

<220>

<223> "CAT26_VH"

<400> 23

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctctggatt cacctttagc agctatgccg tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgt gaaagacgca 300

tatggcagca gctggtactt ttactacttt gactactggg gccaagggac aatggtcacc 360

gtctcgagt 369

<210> 24

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<223> "CAT26_VL"

<400> 24

cagtctgtgt tgacgcagcc gccttcagcg tctgggaccc ccgggcagag ggtcaccatc 60

tcttgttctg gaagcagctc caacatcgga atcaatcctg tgaactggta ccaacaactc 120

ccgggaacgg cccccaaagt cctcatttat agtgataaat accggccctc aggggtcgct 180

gaccgcttct ctggctccaa gtctggaacc tcagcctccc tggccatcag tggcctccag 240

tctgaggatg aggctgatta cttctgtgca gcatgggatg acagcctgaa tggtcgcgtc 300

ttcggaactg ggaccaagct gaccgtccta 330

<210> 25

<211> 378

<212> DNA

<213> Artificial sequence

<220>

<223> "DYAX3_VH"

<400> 25

gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60

tcttgcgctg cttccggatt cactttctct atgtacgaga tgcgttggggt tcgccaagct 120

ccctggtaaag gtttggagtg ggtttctgtt atcccttctg gtggcaagac tttttatgct 180

gactccgtta aaggtcgctt cactatctct agagacaact ctaagaatac tctctacttg 240

cagatgaaca gcttaagggc tgaggacacg gccgtgtatt actgtgcgag atacagcaga 300

360

acggtcaccg tctcaagc 378

<210> 26

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> "DYAX3_VL"

<400> 26

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga cagagtcgcc 60

atcacttgcc gcgcaagtca gagcatcgac acctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaaactcct gatctatgct gcatccaagt tggaagacgg ggtcccatca 180

agattcagtg gcagtggaac tgggacagat ttcactctca ccatcagaag tctgcaacct 240

gaagattttg caagttattt ctgtcaacag agctactcta gtccagggat cactttcggc 300

cctgggacca aggtggagat caaa 324

<210> 27

<211> 381

<212> DNA

<213> Artificial sequence

<220>

<223> "DYAX5_VH"

<400> 27

gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60

tcttgcgctg cttccggatt cactttctct tcttacatta tggtttgggt tcgccaagct 120

cctggtaaag gtttggagtg ggtttcttct atctattctt ctggtggctc tacttcttat 180

gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa tactctctac 240

ttgcagatga acagcttaag ggctgaggac acagccgtgt attactgtac gagatacagc 300

agatggttcg ggcagctagg gttttactcc cactacgcta tggacgtctg gagccaaggg 360

accacggtca ccgtctcaag c 381

<210> 28

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> "DYAX5_VL"

<400> 28

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctccgtg gacgttcggc 300

caagggacca aggtggaaat caaa 324

<210> 29

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "AM40 and SC4017 and CAT5D9_HCDR1"

<400> 29

Ser Tyr Ala Leu Ser

15

<210> 30

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "AM40 and SC4017_HCDR2"

<400> 30

Thr Arg Pro Pro Thr Ser Arg Thr Ala Ser Tyr Ala Gln Lys Phe Gln

1.5 10 15

gly

<210> 31

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "AM40 and SC4017_HCDR3"

<400> 31

Asn Asp Phe Val Tyr Gly Ser Tyr Arg Phe

1.5 10

<210> 32

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "AM40 and CAT5D9_LCDR1"

<400> 32

Thr Arg Thr Ser Gly Asn Ile Ala Gly Tyr Phe Val Gln

1.5 10

<210> 33

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "AM40 and SC4017 and CAT5D9_LCDR2"

<400> 33

Glu Asp Tyr Gln Arg Pro Ser

15

<210> 34

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "AM40 and SC4017_LCDR3"

<400> 34

Gln Ser Tyr Asp Asp Tyr Arg Arg Ala Ala

1.5 10

<210> 35

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "SC4017_LCDR1"

<400> 35

Thr Arg Thr Ser Gly Trp Ile Ala Gly Tyr Phe Val Gln

1.5 10

<210> 36

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT5D9_HCDR2"

<400> 36

Gly Ile Ile Pro Val Phe Arg Thr Ala Ser Tyr Ala Gln Lys Phe Gln

1.5 10 15

gly

<210> 37

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT5D9_HCDR3"

<400> 37

Asn Asn Tyr Val Trp Gly Ser Tyr Arg Phe

1.5 10

<210> 38

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT5D9_LCDR3"

<400> 38

Gln Ser Tyr Asp Asp Thr Ser Gln Gly Val

1.5 10

<210> 39

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT8_HCDR1"

<400> 39

Ser Tyr Ala Met Ser

15

<210> 40

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT8 and CAT26_HCDR2"

<400> 40

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1.5 10 15

gly

<210> 41

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT8_HCDR3"

<400> 41

Ser Ser Gly Tyr Tyr Gly Ala Asn Phe Asp Phe

1.5 10

<210> 42

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT8_LCDR1"

<400> 42

Ser Gly Ser Ser Ser Asn Ile Gly Ser Gly Tyr Val Tyr

1.5 10

<210> 43

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT8_LCDR2"

<400> 43

Arg Asn Asn Gln Arg Pro Ser

15

<210> 44

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT8_LCDR3"

<400> 44

Ala Ala Trp Asp Asp Ser Leu Ser Gly Tyr Val

1.5 10

<210> 45

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT26_HCDR1"

<400> 45

Ser Tyr Ala Val Ser

15

<210> 46

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT26_HCDR3"

<400> 46

Asp Ala Tyr Gly Ser Ser Trp Tyr Phe Tyr Tyr Phe Asp Tyr

1.5 10

<210> 47

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT26_LCDR1"

<400> 47

Ser Gly Ser Ser Ser Asn Ile Gly Ile Asn Pro Val Asn

1.5 10

<210> 48

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT26_LCDR2"

<400> 48

Ser Asp Lys Tyr Arg Pro Ser

15

<210> 49

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT26_LCDR3"

<400> 49

Ala Ala Trp Asp Asp Ser Leu Asn Gly Arg Val

1.5 10

<210> 50

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX3_HCDR1"

<400> 50

Met Tyr Glu Met Arg

15

<210> 51

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX3_HCDR2"

<400> 51

Val Ile Pro Ser Gly Gly Lys Thr Phe Tyr Ala Asp Ser Val Lys Gly

1.5 10 15

<210> 52

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX3 and DYAX5_HCDR3"

<400> 52

Tyr Ser Arg Trp Phe Gly Gln Leu Gly Phe Tyr Ser His Tyr Ala Met

1.5 10 15

Asp Val

<210> 53

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX3_LCDR1"

<400> 53

Arg Ala Ser Gln Ser Ile Asp Thr Tyr Leu Asn

1.5 10

<210> 54

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX3_LCDR2"

<400> 54

Ala Ala Ser Lys Leu Glu Asp

15

<210> 55

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX3_LCDR3"

<400> 55

Gln Gln Ser Tyr Ser Ser Pro Gly Ile Thr

1.5 10

<210> 56

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX5_HCDR1"

<400> 56

Ser Tyr Ile Met Val

15

<210> 57

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX5_HCDR2"

<400> 57

Ser Ile Tyr Ser Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys

1.5 10 15

gly

<210> 58

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX5_LCDR1"

<400> 58

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1.5 10

<210> 59

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX_LCDR2"

<400> 59

Ala Ala Ser Ser Leu Gln Ser

15

<210> 60

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "DYAX_LCDR3"

<400> 60

Gln Gln Ser Tyr Ser Thr Pro Pro Trp Thr

1.5 10

<210> 61

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "AM40_HC"

<400> 61

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Thr Arg Pro Pro Thr Ser Arg Thr Ala Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Val Asp Glu Ser Thr Ser Thr Gly Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Asn Asp Phe Val Tyr Gly Ser Tyr Arg Phe Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 62

<211> 217

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "AM40_LC"

<400> 62

Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys

1.5 10 15

Thr Val Thr Ile Ser Cys Thr Arg Thr Ser Gly Asn Ile Ala Gly Tyr

20 25 30

Phe Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val

35 40 45

Ile Tyr Glu Asp Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly

65 70 75 80

Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp

85 90 95

Tyr Arg Arg Ala Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 63

<211> 1347

<212> DNA

<213> Artificial sequence

<220>

<223> "AM40_HC"

<400> 63

caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60

tcctgcaagg cttctggagg caccttcagc agttatgctc ttagctgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaacg cggccgccga cctcccggac agcaagctac 180

gcacagaaat ttcagggcag agtcacgatt accgtggacg aatccacgag cacaggctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gtcaaacgac 300

360

tcgaccaagg gcccatccgt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcctgg 480

aactcaggcg ctctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagagagt tgagcccaaa 660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaattcga ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagcctcca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtc tacaccctgc ccccatcccg ggaggagatg 1080

accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1260

cagggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcttaa gcctgtctcc gggtaaa 1347

<210> 64

<211> 651

<212> DNA

<213> Artificial sequence

<220>

<223> "AM40_LC"

<400> 64

aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60

tcctgcaccc gcaccagtgg gaacattgcc ggctactttg tgcagtggta ccagcagcgc 120

ccgggcagtt cccccaccac tgtgatctat gaggattacc aacgaccctc tggggtccct 180

gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240

ctgaagactg aggacgaggc tgactactat tgtcagtctt atgatgacta ccggcgggcg 300

gcgttcggcg gagggaccaa gctgaccgtc ctaggtcagc ccaaggcggc gccctcggtc 360

actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540

agctacctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc a 651

<210> 65

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "SC4017_HC"

<400> 65

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Thr Arg Pro Pro Thr Ser Arg Thr Ala Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Val Asp Glu Ser Thr Ser Thr Gly Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Asn Asp Phe Val Tyr Gly Ser Tyr Arg Phe Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 66

<211> 217

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "SC4017_LC"

<400> 66

Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys

1.5 10 15

Thr Val Thr Ile Ser Cys Thr Arg Thr Ser Gly Trp Ile Ala Gly Tyr

20 25 30

Phe Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val

35 40 45

Ile Tyr Glu Asp Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly

65 70 75 80

Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp

85 90 95

Tyr Arg Arg Ala Ala Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 67

<211> 1486

<212> DNA

<213> Artificial sequence

<220>

<223> "SC4017_HC"

<400> 67

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtaa ggggctcaca 60

gtagcaggct tgaggtctag acatatatat gggtgacaat gacatccact ttgcctttct 120

ctccacaggt gtacactccc aggtgcagct ggtgcagtct ggggctgagg tgaagaagcc 180

tgggtcctcg gtgaaggtct cctgcaaggc ttctggaggc accttcagca gttatgctct 240

tagctgggtg cgacaggccc ctggacaagg gcttgagtgg atgggaacgc ggccgccgac 300

ctcccggaca gcaagctacg cacagaaatt tcagggcaga gtcacgatta ccgtggacga 360

atccacgagc acaggctaca tggagctgag cagcctgaga tctgaggaca cggccgtgta 420

ttactgtgcg tcaaacgact tcgtgtacgg gagttatcgt ttctggggcc aagggaccac 480

ggtcaccgtc tcctcagcgt cgaccaaggg cccatccgtc ttccccctgg caccctcctc 540

caagagcacc tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga 600

accggtgacg gtgtcctgga actcaggcgc tctgaccagc ggcgtgcaca ccttcccggc 660

tgtcctacag tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag 720

cttgggcacc cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga 780

caagagagtt gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc 840

tgaattcgag gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat 900

gatctcccgg acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga 960

ggtcaagttc aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg 1020

ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga 1080

ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcctccat 1140

cgagaaaacc atctccaaag ccaaagggca gccccgagaa ccacaggtct acaccctgcc 1200

cccatcccgg gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt 1260

ctatcccagc gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa 1320

gaccacgcct cccgtgctgg actccgacgg ctccttcttc ctctatagca agctcaccgt 1380

ggacaagagc aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct 1440

gcacaaccac tacacgcaga agagcttaag cctgtctccg ggtaaa 1486

<210> 68

<211> 651

<212> DNA

<213> Artificial sequence

<220>

<223> "SC4017_LC"

<400> 68

aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60

tcctgcaccc gcaccagtgg gtggattgcc ggctactttg tgcagtggta ccagcagcgc 120

ccgggcagtt cccccaccac tgtgatctat gaggattacc aacgaccctc tggggtccct 180

gatcggttct ctggctccat cgacagctcc tccaactctg cctccctcac catctctgga 240

ctgaagactg aggacgaggc tgactactat tgtcagtctt atgatgacta ccggcgggcg 300

gcgttcggcg gagggaccaa gctgaccgtc ctaggtcagc ccaaggcggc gccctcggtc 360

actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540

agctacctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc a 651

<210> 69

<211> 449

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT5D9_HC"

<400> 69

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1.5 10 15

Ser Val Lys Val Ser Cys Lys Ile Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Val Phe Arg Thr Ala Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Val Asp Glu Ser Ala Ser Thr Gly Tyr

65 70 75 80

Ile Glu Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Asn Asn Tyr Val Trp Gly Ser Tyr Arg Phe Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 70

<211> 217

<212> PROTEIN

<213> Artificial sequence

<220>

<223> "CAT5D9_LC"

<400> 70

Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys

1.5 10 15

Thr Val Thr Ile Ser Cys Thr Arg Thr Ser Gly Asn Ile Ala Gly Tyr

20 25 30

Phe Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser Pro Thr Thr Val

35 40 45

Ile Tyr Glu Asp Tyr Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly

65 70 75 80

Leu Lys Pro Asp Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Asp

85 90 95

Thr Ser Gln Gly Val Phe Gly Ala Gly Thr Lys Val Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu

115 120 125

Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 71

<211> 1346

<212> DNA

<213> Artificial sequence

<220>

<223> "CAT5D9_HC"

<400> 71

aggtgcagct ggtgcagtct ggggctgagg tgaagaagcc tgggtcctcg gtgaaggtct 60

cctgcaaggc ttctggaggc accttcagca gttatgctct tagctgggtg cgacaggccc 120

ctggacaagg gcttgagtgg atgggaacgc ggccgccgac ctcccggaca gcaagctacg 180

cacagaaatt tcagggcaga gtcacgatta ccgtggacga atccacgagc acaggctaca 240

tggagctgag cagcctgaga tctgaggaca cggccgtgta ttactgtgcg tcaaacgact 300

tcgtgtacgg gagttatcgt ttctggggcc aagggaccac ggtcaccgtc tcctcagcgt 360

cgaccaaggg cccatccgtc ttccccctgg caccctcctc caagagcacc tctgggggca 420

cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg gtgtcctgga 480

actcaggcgc tctgaccagc ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac 540

tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc cagacctaca 600

tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagagagtt gagcccaaat 660

cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaattcgag gggggaccgt 720

cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg acccctgagg 780

tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc aactggtacg 840

tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag tacaacagca 900

cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt 960

acaagtgcaa ggtctccaac aaagccctcc cagcctccat cgagaaaacc atctccaaag 1020

ccaaagggca gccccgagaa ccacaggtct acaccctgcc cccatcccgg gaggagatga 1080

ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc gacatcgccg 1140

tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct cccgtgctgg 1200

actccgacgg ctccttcttc ctctatagca agctcaccgt ggacaagagc aggtggcagc 1260

aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac tacacgcaga 1320

aggcttaag cctgtctccg ggtaaa 1346

<210> 72

<211> 651

<212> DNA

<213> Artificial sequence

<220>

<223> "CAT5D9_LC"

<400> 72

aattttatgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtcaccatc 60

tcctgcaccc gcaccagtgg gaacattgcc ggctactttg tgcagtggta ccagcagcgc 120

ccgggcagtt cccccaccac tgtgatctat gaggattacc aacgaccctc tggggtccct 180

gatcggttct ctggctccat cgacaggtcc tccaactctg cctccctcac catctctgga 240

ctgaagcctg acgacgaggc tgactactat tgtcagtctt atgatgacac ctctcaaggt 300

gtgttcggcg cagggaccaa ggtcaccgtc ctaggtcagc ccaaggcggc gccctcggtc 360

actctgttcc cgccctcctc tgaggagctt caagccaaca aggccacact ggtgtgtctc 420

ataagtgact tctacccggg agccgtgaca gtggcctgga aggcagatag cagccccgtc 480

aaggcgggag tggagaccac cacaccctcc aaacaaagca acaacaagta cgcggccagc 540

agctacctga gcctgacgcc tgagcagtgg aagtcccaca gaagctacag ctgccaggtc 600

acgcatgaag ggagcaccgt ggagaagaca gtggccccta cagaatgttc a 651

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Claims (46)

1. An antibody or antigen-binding fragment thereof that specifically binds to a receptor tyrosine kinase 3 ligand similar to the McDonough strain feline sarcoma virus (FMS) proto-oncogene product (FLT3L) containing complementarity-determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOS: 29, 30, 31, 32, 33 and 34, respectively. 2. An antibody or antigen-binding fragment thereof according to claim 1, containing a heavy chain variable region (VH) and a light chain variable region (VL), where the VH and VL regions contain an amino acid sequence that is at least 95%, 96%, 97 %, 98% or 99% identical to the sequence of SEQ ID NO:1 and SEQ ID NO:2, respectively. 3. An antibody or antigen-binding fragment thereof according to claim 2, wherein VH and VL consist of SEQ ID NO:1 and SEQ ID NO:2, respectively. 4. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a recombinant antibody selected from the group consisting of a monoclonal antibody, a human antibody, a humanized antibody, and a chimeric antibody. 5. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain constant domain selected from the group consisting of: (a) IgA constant domain; (b) IgD constant domain; (c) IgE constant domain; (d) IgG1 constant domain; (e) IgG2 constant domain; (f) IgG3 constant domain; (g) IgG4 constant domain and (h) IgM constant domain. 6. An antibody or antigen-binding fragment thereof according to claim 5, wherein the immunoglobulin heavy chain constant domain is IgG1, wherein the immunoglobulin heavy chain constant domain IgG1 comprises one or more amino acid substitutions selected from the group consisting of L234F, L235E, and P331S, numbered in according to the EU numbering system according to Kabat. 7. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment comprises an immunoglobulin light chain constant domain selected from the group consisting of: (a) an Ig kappa chain constant domain and (b) Ig lambda chain constant domain. 8. An antibody or antigen-binding fragment thereof according to claim 7, wherein the immunoglobulin light chain constant domain is a kappa lambda IgG constant domain. 9. A polynucleotide encoding an antibody or antigen-binding fragment thereof according to any one of claims 1-8. 10. An expression vector containing a polynucleotide according to claim 9. 11. A hybridoma that produces an antibody or antigen-binding fragment according to any one of claims 1 to 8, wherein the hybridoma contains a polynucleotide according to claim 9. 12. A pharmaceutical composition for the treatment or prevention of an autoimmune disease, wherein the composition comprises an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8 and a carrier. 13. A method for producing an antibody or antigen-binding fragment according to any one of claims 1 to 8, including (a) culturing a host cell expressing the antibody or antigen-binding fragment thereof; and (b) isolating the antibody or antigen-binding fragment thereof from the cultured host cell. 14. The use of an antibody or antigen-binding fragment specific for FLT3L for the treatment or prevention of an autoimmune disease, where the antibody or antigen-binding fragment contains: complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contain the amino acid sequences of SEQ ID NOS: 29, 30, 31, 32, 33 and 34, respectively. 15. Use according to claim 14, wherein the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, myositis, Sjögren's syndrome, multiple sclerosis, uveitis, psoriasis, nephritis, and rheumatoid arthritis. 16. Use according to claim 14 wherein the disease is selected from the group consisting of type 1 diabetes, type 2 diabetes and chronic kidney disease. 17. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in a method for neutralizing membrane-bound FLT3L. 18. The use of an antibody or antigen-binding fragment according to any one of claims 1 to 8 in a method for reducing populations of classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) in the bloodstream. 19. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in a method for reducing FLT3L expression on the surface of CD4+ T cells. 20. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in a method for reducing the percentage of CD4+ T cells expressing FLT3L. 21. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in a method for reducing ERK signaling in a lymphoblast. 22. The use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in a method for reducing the level of MEK 1/2 phosphorylation in primary human CD133+ stem cells. 23. Monoclonal antibody that specifically binds to FLT3L, where the antibody contains: i. complementarity determining regions (CDR) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 contain the amino acid sequence of SEQ ID NO: 29, 30, 31, 32, 33 and 34, respectively; ii. human IgG1 heavy chain constant domain; and iii. human lambda Ig light chain constant domain. 24. A monoclonal antibody according to claim 23, containing a heavy chain variable region (VH) and a light chain variable region (VL), where the VH and VL regions contain an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:1 and SEQ ID NO:2, respectively. 25. A monoclonal antibody according to claim 24, comprising a heavy chain containing the amino acid sequence of SEQ ID NO:61 and a light chain containing the amino acid sequence of SEQ ID NO:62. 26. A host cell for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the cell contains a polynucleotide according to claim 9 or a vector according to claim 10.

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