Antibodies against feline McDonough sarcoma (FMS)-like tyrosine kinase 3 receptor ligand (FLT3L) and their use to treat autoimmune and inflammatory diseases

Abstract

To provide anti-FLT3L antibodies and methods of using the antibodies to treat autoimmune and other inflammatory diseases.SOLUTION: The invention provides an antibody or antigen-binding fragment thereof that binds to FLT3L, where the antibody or antigen-binding fragment thereof comprises a set of Complementarity-Determining Regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, where the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise respective specific amino acid sequences.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Provided are technologies relating to antibodies against feline McDonough sarcoma (FMS)-like tyrosine kinase 3 receptor ligand (FLT3L) and their use to treat autoimmune and inflammatory diseases. [Background technology]

[0002] Autoimmune diseases, which occur when the body's immune system produces autoantibodies, are unfortunately common. For example, it is estimated that more than 23 million Americans suffer from autoimmune diseases. Currently, more than 80 types of autoimmune diseases are recognized. Specific examples of autoimmune diseases include systemic lupus erythematosus, myositis, primary Sjogren's syndrome, multiple sclerosis, uveitis, psoriasis, and rheumatoid arthritis.

[0003] 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 attack the patient's immune system, causing inflammation. At the cellular level, SLE patients have autoreactive T cells and B cells driven by dendritic cells (Palucka AK et al., Immunology and Cell Biology (2002) 80:484-488). Sjögren's syndrome is characterized by systemic chronic inflammation of the exocrine organs, ultimately resulting in organ dysfunction (Holdgate N. and St. Clair EW, F1000 Research.1412 10.12688 / f1000 research.8352.1).

[0004] 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 as red patches and sensitive skin. Rheumatoid arthritis (RA) is an inflammatory disorder of the synovial tissue of joints, characterized by persistent synovitis and destruction of cartilage and bone within the joints. The damage can progress and impact many body systems. Lupus nephritis is related to systemic lupus erythematosus and causes inflammation of the kidneys. When inflammation occurs, the kidneys leak proteins, which can eventually lead to kidney failure. Uveitis is a group of inflammatory diseases that attack and destroy tissue in the eye, potentially causing blindness.

[0005] Furthermore, acute and chronic pro-inflammatory states are associated with and may contribute to a myriad of diseases in individuals. Examples of certain diseases are thought to be associated with chronic inflammation, including type 1 and type 2 diabetes, chronic kidney disease (CKD), including diabetes, diabetic nephropathy, and hypertension-induced CKD; arteriosclerosis, Alzheimer's disease, cancer, and related complications of such diseases, including heart disease, hypertension, anemia, pericarditis, renal osteodystrophy, etc. As with autoimmune diseases, in diseases associated with chronic inflammation, the body appears to mount an excessive and ongoing pro-inflammatory response that can lead to debilitating and frequently fatal comorbidities.

[0006] The causes of autoimmune diseases are not clearly understood. Mechanistically, underlying each autoimmune disease is an ongoing autoimmune response that is driven (and / or uninhibited) by complex regulatory systems that continually energize autoreactive immune cells. Similar mechanisms appear to operate in non-autoimmune chronic inflammatory diseases. Thus, therapeutic interventions in autoimmune diseases and for chronic inflammation have targeted the myriad regulatory systems, signaling cascades, and their components.

[0007] One class of putative therapeutic targets includes tyrosine kinase receptors (TKRs), which are transmembrane receptors that bind distinct growth factors and proteins to regulate cellular homeostasis. More than 50 known human TKRs are classified into 20 distinct classes defined by their genetic lineage (Robins DR, et al. Oncogene. (2000) 19:5548-5557; Lemmon MA and Schlessinger J. Cell. (2010) 141:1117-1134). TKR class III is characterized by the presence of five to seven immunoglobulin-like domains within the extracellular segment, containing 70 to 100 hydrophilic residues. Among class III TKRs, feline McDonough sarcoma (FMS)-like tyrosine kinase 3 receptor (FLT3) is a membrane-bound receptor expressed on human stem cells, hematopoietic cell precursors, dendritic cells, activated T and B cells, monocytes, and microglia. FLT3 binds to FLT3 ligand (FLT3L), a hematopoietic cytokine expressed by multiple cell types, including activated T cells, activated endothelium, and bone marrow stromal cells. FLT3L is expressed both as a cell surface and secreted homodimer and signals through its cognate receptor, FLT3. FLT3 is expressed on the cell surface as a monomer and is activated upon ligation with FLT3L. After ligation with FLT3L, FLT3 dimerizes, autophosphorylates, and activates signaling pathways including RAS / extracellular signal-regulated kinase (ERK), phosphatidylinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT) 3 and 5. After autophosphorylation, dimerized FLT3 is internalized and degraded.

[0008] FLT3L is produced in response to inflammatory signals, particularly the □-chain cytokines IL-2, IL-7, and IL-15, and its interaction with FLT3 drives the inflammatory process primarily through its role in DC differentiation, proliferation, and survival. Furthermore, FLT3 signaling has also been suggested to play a role in T cell and B cell survival after activation, with both cell types reported to transiently upregulate the receptor (Astier AL et al., J. Immunology. 2010 v184:685-93 and Tobon et al. Arthritis & Rheumatism. 2010;62(11):3447-56). Furthermore, NK cell survival is thought to indirectly depend on FLT3L through a 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 remains unproven in humans.

[0009] DCs are particularly important in inflammation because they are the sentinels of the immune system, migrating from inflammatory sites to lymph nodes and ultimately initiating the adaptive immune responses required to produce autoimmune disease. Broadly, there are two subsets of DCs: myeloid / classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). cDCs produce proinflammatory cytokines (e.g., IFN-III, IL-23, IL-12, IL-6, and IL-1), present antigens to T cells in the context of costimulation, secrete chemokines that recruit cells to the inflammatory site, and ensure their colocalization when necessary for critical cell-cell interactions. Through these mechanisms, cDCs stimulate neutrophils, B cells, T cells, and NK cells, resulting in NETosis, autoantibody production, IL-17 production, and the production of additional inflammatory cytokines. pDCs are the primary source of type I IFN, a key cytokine in the innate response that enhances activation of all arms of the immune system.

[0010] Salivary glands from patients with Sjögren's syndrome show expression of FLT3 and FLT3L on infiltrating B cells (Tobon et al. Arthritis & Rheumatism. (2010) 62(11):3447-3456). Furthermore, patients with Sjögren's syndrome show elevated frequencies of circulating FLT3-expressing B cells, and their survival is enhanced when co-cultured with human salivary gland cells expressing FLT3L. Individuals with MS express FLT3 protein in chronic and active lesions as well as in gray and white matter (DeBoy C. A. et al. Exp Mol Pathol. (2010); 89(2):109-116). Furthermore, FLT3 colocalizes with immature DCs in the perivascular brain, indicating infiltration of FLT3-positive DCs into the brains of individuals with MS (Deboy et al.). In RA, FLT3L levels in synovial fluid are increased compared to healthy individuals. Furthermore, mononuclear leukocytes, NK cells, and DCs from RA patients express high levels of FLT3L (Ramos M. et al. Arthritis Res Ther. (2013) 15(6):R209).

[0011] Furthermore, increased levels of FLT3L in serum and at sites of inflammation have been reported in SLE, myositis, primary Sjögren'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).

[0012] Thus, although FLT3-mediated proinflammatory survival (e.g., via pDCs and mDCs) is a beneficial physiological response in healthy individuals, it is likely to have detrimental effects in autoimmune diseases. Thus, disruption and attenuation of the FLT3 / FLT3L signaling pathway could prove to be an important tool for combating autoimmune and other inflammatory diseases and reducing inflammation. Summary of the Invention [Means for solving the problem]

[0013] Provided herein are novel FLT3L-binding antibodies for controlling autoimmune diseases and other acute and / or chronic inflammatory diseases.

[0014] In a first aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to FLT3L, comprising a set of complementarity-determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of (a) SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively; or (b) SEQ ID NOs: 29, 30, 31, 35, 33, and 34, respectively; or (c) SEQ ID NOs: 29, 36, 37, 32, 33, and 38, respectively.

[0015] In one embodiment of the first aspect, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL) having at least 95%, 96%, 97%, 98%, or 99% sequence identity to (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, the VH and VL comprise (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, 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 activation of FLT3. 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 thereof has one or more of the following: (a) an IgA constant domain; (b) an IgD constant domain; (c) an IgE constant domain; (d) an IgG1 constant domain; (e) an IgG2 constant domain. (f) an IgG3 constant domain; (g) an IgG4 constant domain; and (h) an IgM constant domain. In one embodiment, the isolated antibody or antigen-binding fragment comprises an IgG1 constant domain. In another embodiment, the isolated antibody or antigen-binding fragment comprises a light chain immunoglobulin constant domain selected from the group consisting of: (a) an Igκ constant domain; and (b) an Igλ constant domain. In one embodiment, the antigen-binding protein comprises a human IgG1 constant domain and a human λ 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 EU numbering index of Kabat (Edelman et al., Proc. Natl. Acad. Sci., 63:78-85 (1969)).

[0016] In a second aspect, the present disclosure 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 regulatory sequence.

[0017] In a third aspect, the present disclosure provides a vector comprising a nucleic acid molecule according to the second aspect and / or embodiments thereof.

[0018] In a fourth aspect, the present disclosure 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.

[0019] In a fifth aspect, the present disclosure provides a hybridoma producing the antibody or antigen-binding fragment of any of the first to fourth aspects or embodiments thereof.

[0020] In a sixth aspect, the present disclosure provides an isolated host cell that produces the antibody or antigen-binding fragment of the first to fifth aspects and / or embodiments thereof.

[0021] In a seventh aspect, the present disclosure provides a method of making an antibody or antigen-binding fragment thereof according to any of the first to sixth aspects or embodiments thereof, the method comprising: (a) culturing a host cell expressing the antibody or antigen-binding fragment, or culturing a host cell of the third aspect or an embodiment thereof, or a hybridoma according to the fourth aspect; and (b) isolating the antibody or antigen-binding fragment thereof from the cultured host cell.

[0022] In an eighth aspect, the present disclosure provides an antibody or antigen-binding fragment thereof produced according to the method of the sixth aspect.

[0023] In a ninth aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any of the first to eighth aspects or embodiments thereof and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition is provided for use as a medicament.

[0024] In a tenth aspect, the present disclosure provides a method for treating acute or chronic inflammatory disease, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of an isolated antibody or fragment thereof according to any of the first to ninth aspects or embodiments thereof. In one embodiment, the inflammatory disease comprises chronic kidney disease (CKD), including, for example, diabetes, diabetic nephropathy, and hypertension-induced CKD.

[0025] In an eleventh aspect, the present disclosure provides a method for treating an autoimmune disease, comprising administering to a subject a therapeutically effective amount of a compound selected from the group consisting of ribonucleotides, ribonucleotides, nucleotides, and nucleotides required for the treatment of an autoimmune disease. The present invention provides a method comprising administering to a subject in need thereof a pharmaceutically effective amount of an isolated antibody or fragment thereof according to any of the first to tenth aspects or embodiments thereof. In one embodiment, the autoimmune disease comprises systemic lupus erythematosus, myositis, primary Sjogren's syndrome, multiple sclerosis, uveitis, psoriasis, or rheumatoid arthritis.

[0026] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description of the disclosure together with the appended claims, with the understanding that the scope of the claims is defined by the recitations therein, and not by any specific discussion of the features and advantages set forth herein. [Brief explanation of the drawings]

[0027] [Figures 1A-1C] Selection of lead antibodies. Figure 1A shows the alternating panning process using huFLT3L and muFLT3L and the resulting enrichment of BMV, CS, DP47, and Dyax phage libraries. Figure 1B shows the process by which panning outputs were cloned into vectors for downstream competitive HTRF. Figure 1C shows the results of competitive HTRF (homogeneous time-resolved fluorescence) for lead candidates. [Figures 2A-2C] Expression of FLT3L in cell lines. Figure 2A shows increased expression of FLT3 in the RS4;11 cell line compared to EOL-1, MOLM13, and MV4-11 cell lines. Figure 2B confirms that recombinant FLT3L binds to FLT3 on RS4;11 cells in a dose-dependent manner. Figure 2C shows that downregulation of FLT3 on the surface of RS4;11 cells can be reliably detected using flow cytometry analysis with a commercially available anti-FLT3 antibody, and that this downregulation occurs in a dose-dependent manner in response to ligation with FLT3L. [Figure 3A-3B] Induction of EC80 and subsequent testing. Figure 3A shows the FLT3L titration curve used to induce the concentration at which 80% of cell surface FLT3 is downregulated (EC80) on RS4;11 cells. Figure 3B shows the inhibition profiles of a commercially available anti-FLT3L antibody and a recombinant construct of the FLT3 receptor (FLT3-Fc) against 96 pM recombinant FLT3L, which would otherwise result in 80% downregulation of cell surface FLT3 on RS4;11 cells. [Figures 4A-4C]Inhibition of human and cyno sFLT3 lead antibody candidates. Figure 4A shows the inhibition profiles of five lead candidates against human FLT3L at 96 pM. Figure 4B shows the inhibition profiles of five lead candidates against cynomolgus monkey FLT3L at 96 pM. Figure 4C shows the inhibition profiles of five lead candidates against mouse FLT3L. [Figures 5A-5C] Inhibition of cell surface FLT3 by lead candidates. Figure 5A shows the ability of lead antibodies to bind to human FLT3L expressed on the surface of a transduced CHO cell line. Figure 5B shows lead antibodies binding to cell surface cyno (cynomolgus monkey) FLT3L. Figure 5C shows lead antibodies binding to cell surface mouse FLT3L. [Figures 6A-6C] These lead candidates bind to endogenous human FLT3L. Figure 6A shows FLT3L expression on human primary T cells after 7 days of stimulation with IL-2. Figure 6B shows the ability of all lead candidates, except for clone 5D9, to bind to endogenous FLT3L on human primary T cells. Figure 6C shows that improving the avidity of CAT5D9 by dimerization before incubation with primary T cells allows for detection of dose-dependent binding of the clone to endogenous FLT3L on the T cell surface. [Figures 7A-7B] Inhibition of cell surface signal transduction by lead candidates. Figure 7A shows the dose-response curve of RS4;11 cells to CHO cells expressing FLT3L, determining 1000 CHO cells per well as the optimal number to induce 80% downregulation of FLT3 on the surface of RS4;11 cells. Figure 7B shows that all lead candidates have the ability to inhibit cell surface FLT3L on CHO cells to some extent. [Figure 8A-8B] Activation and neutralization of the ERK signaling pathway. Figure 8A shows a proof-of-concept study demonstrating ERK signaling activation in RS4;11 cells by FLT3L. Figure 8B shows the inhibition of FLT3L-induced ERK activation by commercially available antibodies. [Figure 9A-9B]Blockade of MEK 1 / 2 and ERK downstream signaling by lead candidates. Figure 9A shows the functional activity of lead clone candidates on human FLT3L-induced MEK 1 / 2 phosphorylation in primary CD133+ human stem cells. Figure 9B provides further confirmation of the functional activity of lead clones on FLT3L-induced signaling in primary CD133+ human stem cells using ERK phosphorylation as a readout. [Figures 10A-10B] Figure 10A shows phase III binning of lead clone candidates and FLT3-Fc against clone 5D9. Figure 10B shows phase III binning of all lead clones and FLT3-Fc against CAT8, as a representative of all clones other than CAT5D9. [Figures 11A-11B] Cross-reactivity of lead candidates to huSCF and huCSF. Figure 11A shows that lead candidates do not bind to the structural homolog huSCF. Figure 11B shows that lead candidates do not bind to the structural homolog huCSF. [Figures 12A-12B] Figure 12A shows the results of a first round of clone optimization comparing parental CAT5D9 with clone 6 (C06) using the RS4;11 FLT3 downregulation assay as a readout. Figure 12B shows the results of a second round of clone optimization comparing parental CAT5D9 with clone 6 (C06) and the final lead candidates AM40 and SC4017. [Figures 13A-13B] Effective neutralization of endogenous cell-surface FLT3L. Figure 13A shows downregulation of RS4:11 FLT3 in response to serial dilutions of CD4+ T cells expressing FLT3L. Figure 13B shows that the lead clone completely neutralizes the activity of cell-surface FLT3L on CD4+ T cells. [Figure 14]

[0023] Figure 1 is a schematic of a study on neutralization of FLT3L in healthy cynomolgus monkeys. Three groups of male cynomolgus monkeys (n = 4 per group) were administered 0.03, 1, or 30 mg / kg of AM40 (MEDI1116) as indicated, five times weekly for one month. An 8-week follow-up period after the final dose was used to determine the persistence of the administered antibody and its effect on circulating DC populations. [Figures 15A-15B] Serum FLT3L protein and circulating DC frequencies after administration of anti-FLT3L antibody (AM40 / MEDI1116). Figure 15A shows target engagement by MEDI1116 by measuring free serum FLT3L levels after administration at 0.03, 1.0, and 30 mg / kg. Daily serum measurements were obtained from days 1-8, followed by weekly measurements until day 85. Figure 15B shows the decrease and recovery of circulating CD1c+ (classical DC) frequencies (left) and plasmacytoid DC frequencies (right), measured as a percentage of baseline, after treatment with MEDI1116. [Figures 16A-16D] Figure 16 shows the correlation between FLT3L expression and SLEDAI scores in SLE patients, comparing serum measurements of T cells and flow cytometry analysis. Figure 16A shows FLT3L levels in the serum of healthy donors (HDs) and SLE patients. Figure 16B shows the correlation between serum FLT3L and SLEDAI scores. Figure 16C shows the frequency of circulating FLT3L+ T cells in healthy donors (HDs) and SLE patients. Figure 16D shows the correlation between FLT3L+ CD4+ T cells and SLEDAI scores. [Figures 17A-17C]FLT3L expression in CD4+ T cell subsets and SLEDAI scores. Figure 17A shows the percentage of CD4 T naive cells expressing FLT3L in HD and SLE patients. The lower panel shows the correlation of CD4 T naive cells expressing FLT3L in SLE patients with SLEDAI scores. Figure 17B shows the percentage of CD4 TMEM cells expressing FLT3L in HD and SLE patients. The lower panel shows the correlation of CD4 TMEM cells expressing FLT3L in SLE patients with SLEDAI scores. Figure 17C shows the percentage of CD4 TMEM cells expressing FLT3L in HD and SLE patients. The lower panel shows the correlation between CD4 T naive cells expressing FLT3L in SLE patients with SLEDAI scores. [Figures 18A-18C] Figure 18 shows FLT3L expression in PBMC CD4 subsets from individuals with myositis. Figure 18A shows the percentage of CD4 Tnaive cells expressing FLT3L in HD and myositis patients. Figure 18B shows the percentage of CD4 TMEM cells expressing FLT3L in HD and myositis patients. Figure 18C shows the percentage of CD4 TCM cells expressing FLT3L in HD and myositis patients. [Figures 19A-19B] 19A and 19B show proteinuria and nephritis scores in MRL mice. Fig. 19A shows a reduction in proteinuria after 17 weeks of anti-FLT3L administration. Fig. 19B shows nephritis scores after 18 weeks of anti-FLT3L administration. [Figures 20A-20C] Figure 20A shows the change in the frequency of CD11+siglec-H+pDC after administration of anti-FLT3L antibody. Figure 20B shows the frequency of CD11c+CD11b+mDC after administration of anti-FLT3L antibody. Figure 20C shows the frequency of CD11c+CD8+mDC after administration of anti-FLT3L antibody. [Figures 21A-21B]Pathology scores of salivary glands in a NOD.H2h4 mouse model of Sjogren's syndrome. Figure 21A shows changes in salivary gland pathology in a NOD.H2h4 mouse model of Sjogren's syndrome after therapeutic administration of an anti-FLT3L antibody relative to an isotype control. Figure 21B shows changes in salivary gland pathology in a NOD.H2h4 mouse model of Sjogren's syndrome after prophylactic administration of an anti-FLT3L antibody relative to an isotype control. [Figures 22A-22D] Changes in the presence of dendritic cells after administration of anti-FLT3L antibody. Figures 22A and 22B show changes in the frequency of plasmacytoid DCs (B220+CD11c+Siglec-H+) after administration of anti-FLT3L antibody, as evidenced by flow cytometry (A) and quantification (B). Figures 22C and 22D show changes in the frequency of classical DCs (B220negCD11cHI) after administration of anti-FLT3L antibody, as evidenced by flow cytometry (C) and quantification (D). [Figures 23A-23C] The PK of anti-FLT3L antibody (MEDI1116) in cynomolgus monkeys correlates with functional neutralization of FLT3L, as evidenced by the suppression and recovery of pDCs. As indicated by the arrows, anti-FLT3L antibody (MEDI1116) was administered to cynomolgus monkeys once weekly on days 1, 8, 15, 22, and 29. Figure 23A shows the PK of anti-FLT3L antibody (MEDI1116). Figure 23B shows soluble FLT3L levels when anti-FLT3L antibody (MEDI1116) was administered at doses of 0.03 mg / kg, 1.0 mg / kg, and 30 mg / kg. Figure 23C shows the frequency of pDCs, measured as a percentage of baseline, when anti-FLT3L antibody (MEDI1116) was administered at doses of 0.03 mg / kg, 1.0 mg / kg, and 30 mg / kg. [Figures 24A-24C] The human dosing model for anti-FLT3L antibody (MEDI1116) projects Q4W dosing. Figure 24A shows the PK of anti-FLT3L antibody (MEDI1116) in cynomolgus monkeys. Figure 24B shows the PD of anti-FLT3L antibody (MEDI1116) in cynomolgus monkeys. Figure 24C shows the predicted PD of anti-FLT3L antibody (MEDI1116) in humans. [Figures 25A-25B] Anti-FLT3L monoclonal antibody (LFC-1) effectively neutralizes FLT3L throughout the course of treatment, resulting in the accumulation of circulating drug / ligand complexes. Figure 25A shows free serum FLT3L levels after administration of anti-FLT3L antibody and isotype control antibody. Free FLT3L levels were measured using FLT3L-IgG as the capture reagent and sulfo-labeled anti-mouse FLT3L polyclonal antibody as the detection reagent. Figure 25B shows total (free and bound) serum FLT3L levels after administration of anti-FLT3L antibody and isotype control antibody. Total FLT3L levels were measured using polyclonal anti-mouse FLT3L antibody for capture and detection. [Figures 26A-26D] Blockade of FLT3L suppresses T cell activation in the spleen and salivary gland-draining lymph nodes (LNs) of aged NOD-H2h4 mice. Blockade of FLT3L using an anti-FLT3L monoclonal antibody (LFC-1) resulted in a reduction in the frequency of antigen-experienced CD44HI 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). Bar graphs were derived from flow cytometry analysis of the spleen and draining LNs. Each bar represents the mean ± standard error of the mean (SEM) for n = 4–5 mice. Figure 26A shows the CD44HI CD4+ T cell population in the spleen after administration of anti-FLT3L and isotype control antibodies. Figure 26B shows the CD44HI CD4+ T cell population in the LNs after administration of anti-FLT3L and isotype control antibodies. Figure 26C shows the CD44HI CD8+ T cell population in the spleen after administration of anti-FLT3L antibody and isotype control antibody, and Figure 26D shows the CD44HI CD8+ T cell population in the LN after administration of anti-FLT3L antibody and isotype control antibody. [Figure 27] Therapeutic anti-FLT3L blockade selectively reduces two serum IgG autoantibody specificities, as measured in matched serum samples by the UTSW IgG autoantibody assay. DETAILED DESCRIPTION OF THE INVENTION

[0028] 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, the anti-FLT3L antibodies disclosed herein can be used to inhibit or reduce FLT3 / FLT3L binding to block activation of inflammatory signaling pathways. Such an approach is beneficial for attacking inflammation at the source of signaling, allowing for more robust anti-inflammatory therapeutic effects. Related polynucleotides, vectors, and pharmaceutical compositions comprising anti-FLT3L antibodies or antigen-binding fragments thereof are also provided. Additionally, methods of producing and using the anti-FLT3L antibodies and antigen-binding fragments disclosed herein, for example, to treat autoimmune diseases and / or chronic inflammatory diseases in subjects (as direct therapy, adjuvant therapy, or in combination therapy), are also contemplated.

[0029] In order that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are provided throughout the detailed description.

[0030] definition 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 defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this disclosure: 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 given to them below, unless otherwise specified.

[0031] As used in this disclosure, the term "antibody" (or fragment, variant, or derivative thereof) refers to an antibody that is capable of binding to an antigen, e.g., at least one variable domain of a heavy chain (VH) and one variable domain of a light chain (VL), e.g., in the context of a typical antibody produced by a B cell. The term "antibody" refers to at least the smallest portion of an antibody that can be produced. Basic antibody structure in vertebrate systems is relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2002). nded., 1988). Antibodies or antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, or chimeric antibodies, epitope-binding fragments (e.g., Fab, F(ab'), Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments containing either the VL or VH domain (Fd), fragments produced by a Fab expression library, and other antibody fragments that retain antigen-binding function, i.e., the ability to specifically bind, for example, to FLT3L, and combinations thereof.

[0032] A typical antibody comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as VH or V H Each light chain is composed of a light chain variable region (herein abbreviated as VL or VL) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. LThe heavy and light chain variable regions contain a binding domain that interacts with an antigen. The antibody constant region can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), separated by more conserved regions called framework regions (FWs). Each VH and VL region is composed of three CDRs and four FWs, arranged in the following order from the amino terminus to the carboxy terminus: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. Exemplary antibodies of the present disclosure include anti-FLT3L antibodies (original and germline antibodies), affinity-optimized clones, optimized antibodies lacking ADCC, conjugated antibodies (e.g., ADCs), and other optimized antibodies (e.g., serum half-life optimized antibodies containing YTE mutations) (see Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006) and U.S. Patent No. 7,083,784, which are incorporated by reference in their entireties).

[0033] In certain embodiments, the CDRs of VH (HCDR1, HCDR2, and HCDR3) and the CDRs of VL (LCDR1, LCDR2, and LCDR3) consist of the amino acid sequences of (a) SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively; or (b) SEQ ID NOs: 29, 30, 31, 35, 33, and 34, respectively; or (c) SEQ ID NOs: 29, 36, 37, 32, 33, and 38, respectively.

[0034] Antibodies can be any of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, called α, δ, ε, γ, and μ, respectively. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or can be conjugated to other molecules, such as toxins, radioisotopes, etc., to form ADCs.

[0035] A "blocking" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of an antigen to which it binds, such as FLT3L. In certain embodiments, a blocking or antagonist antibody substantially or completely inhibits the biological activity of the antigen. For example, FLT3L-mediated activation of FLT3 is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 510%, at least 520%, at least 530%, at least 540%, at least 550%, at least 560%, at least 570%, at least 580%, at least 590%, at least 610%, at least 620%, at least 630%, at least 64 It can be reduced by at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or even 100%.

[0036] The terms "FLT3L antibody," "antibody that binds to FLT3L," or "anti-FLT3L antibody" refer to an antibody or antigen-binding fragment thereof that can bind to FLT3L with sufficient affinity such that the molecule is useful as a therapeutic or diagnostic reagent in targeting FLT3L. The term "anti-FLT3L" also broadly encompasses molecules comprising, for example, the CDRs of the antibodies disclosed herein incorporated within a scaffold.

[0037] The term "germlining" means that an amino acid at a particular position in an antibody is mutated back to the antibody in the germline.

[0038] 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, either alone or in combination. Each heavy and light chain variable region consists of four FW regions connected by three CDR regions. The CDRs within each chain, along with the CDRs from the other chain, are held closely together by the FW regions and contribute to the formation of the antibody's antigen-binding site. There are at least two techniques 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). Furthermore, a combination of these two approaches is sometimes used in the art to determine CDRs.

[0039] The Kabat numbering system is commonly used when referring to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0040] The phrase "Kabat amino acid position numbering" or "Kabat position" and the like refer to the numbering system used for the heavy or light chain variable domains of a collection of antibodies in Kabat et al., 1991.

[0041] The terms "antigen-binding domain," "antigen-binding fragment," and "binding fragment" refer to a portion of an antibody molecule containing amino acids responsible for specific binding between the antibody and an antigen. The variable region enables the antibody or antigen-binding fragment to selectively recognize and specifically bind to an epitope on an antigen. That is, the VH domain and VL domain or a subset of complementarity-determining regions (CDRs) of an antibody combine to form the 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 chain and VL chain. As used herein, the portion of an antigen molecule responsible for specific interaction with the antigen-binding domain 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, but does not necessarily contain both. For example, a so-called "Fd" antibody fragment consists only of a VH domain but still retains some of the antigen-binding function of an intact antibody.

[0042] Binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Binding fragments are produced by cleavage. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain antibodies. Digestion of antibodies with the enzyme papain produces two identical antigen-binding fragments, also known as "Fab" fragments, and an "Fc" fragment, which has no antigen-binding activity but has the ability to crystallize. Digestion of antibodies with the enzyme pepsin produces an F(ab')2 fragment, in which the two arms of the antibody molecule remain linked and contain two antigen-binding sites. The F(ab')2 fragment has the ability to cross-link antigen. As used herein, "Fv" refers to the minimum fragment of an antibody that retains both the antigen-recognition and antigen-binding sites. As used herein, "Fab" refers to a fragment of an antibody that contains the constant domain of the light chain and the CH1 domain of the heavy chain.

[0043] As used herein, the term "Fc region" refers to the polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, plus the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes the immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and the hinge between Cgamma1 (Cy1) and Cgamma2 (Cy2). Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 at its carboxyl terminus, where numbering is according to the EU index as defined by Kabat et al., 1991.

[0044] A "monoclonal antibody" refers to a homogeneous antibody population that is involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants.

[0045] The term "monoclonal antibody" includes both intact and full-length monoclonal antibodies, as well as antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain variable fragments (scFv), fusion proteins containing an antibody portion, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies made in a number of ways, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals (e.g., expression of human antibodies in transgenic mice).

[0046] The term "humanized antibody" refers to antibodies derived from non-human (e.g., murine) immunoglobulins that have been genetically engineered to increase the similarity of the antibody variants produced in humans.

[0047] The term "human antibody" refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human or created using any technique known in the art (e.g., recombinant expression in cultured cells or expression in a transgenic animal). Thus, the term "human antibody" also includes antibodies (or genetically engineered variants or derivatives thereof) that have an amino acid sequence corresponding to an antibody originally produced by a human, but that are expressed in a non-human system (e.g., produced by chemical synthesis; recombinantly expressed in a microbial, mammalian, or insect cell; or expressed in an animal subject). Thus, an antibody obtained from a human subject or human cell (e.g., a hybridoma or cell line expressing a recombinant antibody or fragment thereof) and subsequently expressed in an animal, such as a mouse, is considered a human antibody. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide, e.g., an antibody comprising a murine light chain and a human heavy chain polypeptide.

[0048] The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecules are derived from two or more animal species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and / or capacity, while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

[0049] The term "polynucleotide" includes single and multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds as found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present within a polynucleotide. An "isolated" nucleic acid or polynucleotide is intended to be a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide subunit contained within a vector is considered isolated as disclosed herein. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides that have been purified (partially or substantially) in solution. Isolated RNA molecules include in vivo or in vitro RNA transcription products of polynucleotides. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. Furthermore, the polynucleotide or nucleic acid may be or include a regulatory element, such as a promoter, a ribosome binding site, or a transcription terminator.

[0050] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide will typically include a promoter and / or other transcriptional or translational control elements operably associated with one or more coding regions. Operable association or linkage occurs when one or more regulatory sequences are associated with a coding region for a gene product, e.g., a polypeptide, in such a way as to place expression of that gene product under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and its associated promoter) are "operably associated" or "operably linked" if induction of promoter function results in transcription of mRNA encoding the desired gene product and if the nature of the binding between the two DNA fragments does not interfere with their ability to direct expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter is capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements besides a promoter, for example, enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

[0051] In other embodiments, the polynucleotide may be RNA, for example, in the form of messenger RNA (mRNA).

[0052] A "vector" is a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may contain nucleic acid sequences that allow it to replicate in the host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements known in the art.

[0053] A "transformed" or "host" cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. A transformed cell is a cell into which a nucleic acid molecule has been introduced. As used herein, the term "transformation" includes all techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with a viral vector, transformation with a plasmid vector, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration methods. Transformed or host cells can be bacterial or eukaryotic cells.

[0054] As used herein, the term "FLT3L" refers to feline McDonough sarcoma (FMS)-like tyrosine kinase 3 receptor ligand, a polypeptide that is a hematopoietic cytokine that binds to the FMS-like tyrosine kinase 3 receptor (FLT3) receptor. FLT3L is initially expressed as a membrane-bound protein and then enzymatically cleaved into a soluble form. Both membrane-bound (mFLT3L) and secreted (sFLT3L) forms are included within the definition of FLT3L.

[0055] In this disclosure, "comprises," "including," "containing," "having," and the like can have the meaning ascribed to them in U.S. patent law and can mean "comprises," "including," etc. "Consisting essentially of" or "consisting essentially of" likewise have the meaning ascribed to them in U.S. patent law and are open-ended, allowing for the presence of more than what is recited so long as the basic or novel characteristics of what is recited are not changed by the presence of more than what is recited, but excluding prior art embodiments.

[0056] As used herein, the terms "determine," "assess," "assay," "measure," and "detect" refer to both quantitative and qualitative determinations, and thus the term "determine" is used synonymously with "assay," "measure," etc. Where a quantitative determination is intended, the phrase "determine the amount" of an analyte, etc. may be used. Where a qualitative and / or quantitative determination is intended, the phrase "determine the level" of an analyte or "detect" an analyte is used.

[0057] The term "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 identical or have a specified percentage of nucleotide or amino acid residues when compared and aligned (with gaps, if necessary) for best correspondence without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be determined using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to align amino acid or nucleotide sequences are known in the art (see, e.g., Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402)). In certain embodiments, Gapped BLAST is performed using the algorithms described in Altschul et al., 1997, Nucleic Acids Res. 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, California) or Megalign (DNASTAR) can be used as described.

[0058] The term "isolated" refers to a molecule that is not in its natural environment. No particular level of purification is required. For example, an isolated antibody is one that is not produced or located in its native or natural environment. "Isolated" refers to an antibody that is not isolated from the host. Recombinantly produced biological material is considered isolated as disclosed herein, as is material produced in a non-native cell, such as a hybridoma. A material that is 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 cell or tissue source from which it is derived.

[0059] The term "specifically binds" refers to an agent (e.g., a ligand or antibody) that recognizes and binds to a molecule (e.g., a receptor or epitope), and where the binding induces some complementarity between the agent (e.g., antibody) and the molecule (e.g., ligand). By this definition, an antibody is said to "specifically bind" if it binds to its ligand more readily than it would bind to a random, unrelated molecule. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular ligand. For example, antibody "A" can be predicted to have higher specificity for a given ligand (e.g., FLT3L) than antibody "B."

[0060] As used herein, the term "affinity" refers to a measure of the strength of binding between an individual epitope and the CDR of an antibody. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press), 2 nd See, e.g., Harlow, ed. 1988, pp. 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of antibodies and antigens, i.e., the functional binding strength between the antibody mixture and the antigen. See, e.g., Harlow, pp. 39-34. Avidity relates to both the affinity of individual antibodies within a population for a particular epitope and also the valency of the antibody and antigen.

[0061] The terms "inhibit" or "block," used interchangeably herein, refer to any statistically significant decrease in biological activity, including complete blocking of the activity. For example, "inhibition" can refer to about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% decrease in biological activity.

[0062] The term "effector function" refers to an activity of an antibody that results in the interaction of their Fc component with an Fc receptor or a component of complement. These activities include, for example, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). Accordingly, an antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) with altered effector function refers to an antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) that contains alterations in the Fc region (e.g., amino acid substitutions, deletions, or additions, or oligosaccharide alterations) that alter the activity of at least one effector function (e.g., ADCC, CDC, and / or ADCP). An antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) with improved effector function refers to an antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) that contains alterations in the Fc region (e.g., amino acid substitutions, deletions, or additions, or oligosaccharide alterations) that increase the activity of at least one effector function (e.g., ADCC, CDC, and / or ADCP).

[0063] The term "subject" refers to any animal (e.g., mammal), including, but not limited to, a human, a non-human primate, a rodent, etc., that will be the recipient of a particular treatment. Typically, the terms "subject," "patient," and "individual" are used interchangeably herein. Additional examples of subjects include non-human mammals, such as cows, horses, dogs, sheep, or cats.

[0064] The term "pharmaceutical composition" refers to a formulation in which the biological activity of an active ingredient (e.g., an anti-FLT3L antibody disclosed herein) is in an acceptable form to be effective and does not contain additional ingredients that are unacceptably harmful to a subject to which the composition will be administered. Such compositions may be sterile.

[0065] An "effective amount" of an anti-FLT3L antibody disclosed herein is an amount sufficient to carry out the purpose specifically specified. An "effective amount" can be determined empirically and by routine methods relevant to the purpose described herein.

[0066] The terms "therapeutically effective amount" and "pharmacologically effective amount" refer to an amount of an anti-FLT3L antibody disclosed herein or other drug effective to "treat" a disease or disorder in a subject.

[0067] The terms "treat," or "treatment," or "to treat," or "alleviate," or "to alleviate," and the like, refer to both (1) therapeutic measures that cure, slow the progression of, reduce symptoms of, and / or halt the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent and / or slow the onset of the targeted pathological condition or disorder. Thus, subjects in need of treatment include those already with the disorder; those prone to having the disorder; and those in whom the disorder is to be prevented. In certain embodiments, a subject is successfully "treated" for an autoimmune or inflammatory disease according to the methods of the present disclosure when the patient exhibits, for example, a complete, partial, or transient reduction in symptoms associated with the autoimmune or inflammatory disease.

[0068] Ranges provided herein are understood to be shorthand for all numbers within the range. For example, a range of 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.

[0069] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or alleviating a disorder and / or its associated symptoms. It will be understood, although not excluded, that treating a disease or condition does not require that the disease, its associated condition, or symptoms be completely eliminated. For example, as contemplated herein, treating a disorder includes preventing the symptoms of the disorder from worsening.

[0070] As used herein, the term "or" should be understood to be inclusive unless specifically stated otherwise or clear from the context. As used herein, the terms "a," "an," and "the" should be understood to be singular or plural unless specifically stated otherwise or clear from the context.

[0071] Furthermore, "and / or" as used herein should be considered a specific disclosure of each of two or more of the specified features or components, with or without the other. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to include 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; and A (alone); B (alone); and C (alone).

[0072] Unless specifically stated otherwise or clear from the context, the term "about" as used herein means , within a range of normal acceptance in the art, e.g., within two standard deviations of the mean. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% more or less of the stated value. Unless otherwise indicated, all numerical values ​​provided herein are considered to be implicitly modified by the term "about."

[0073] FLT3L is initially expressed as a membrane-bound protein before being enzymatically cleaved into a soluble form. Both membrane-bound (mFLT3L) and secreted (sFLT3L) forms are functionally active. The FLT3L binding region is highly conserved across observed species, with cross-species reactivity observed between human, rodent, and cynomolgus monkey ligand / receptor combinations. However, key mutations surrounding the binding site may explain the lack of cross-species reactivity of neutralizing antibodies generated against FLT3L. Neutralizing antibodies against FLT3L may affect classical and plasmacytoid DC populations by reducing the immune system's ability to induce and sustain long-term inflammatory responses. Side effects include a reduction in circulating NK cells and reduced T and B cell activation, which may result in reduced survival of both cell types. Taken together, downregulation of these pathways may reduce autoimmune inflammation.

[0074] In one embodiment, a neutralizing anti-FLT3L antibody is intended to promote immune homeostasis by inhibiting FLT3L from binding to FLT3. The anti-FLT3L antibody strategy targets the ligand beyond the receptor to avoid the risk of unexpected receptor dimerization or signal transduction. Unlike its receptor, there is no signal transduction domain associated with membrane-bound FLT3L.

[0075] Anti-FLT3L antibody In a preferred embodiment, the present disclosure provides isolated FLT3L-binding molecules, e.g., antibodies and antigen-binding fragments thereof, that specifically bind to FLT3L, e.g., human FLT3L. The full-length amino acid sequence and nucleotide sequence for FLT3L are known in the art (see, e.g., UniProt Accession No. P36888 for human FLT3L or UniProt Accession No. Q00342 for mouse FLT3L). The anti-FLT3L antibodies of the present disclosure inhibit FLT3L-mediated activation of FLT3, thereby reducing pro-inflammatory signaling and reducing inflammation in a subject.

[0076] In preferred embodiments, the anti-FLT3L antibody does not cross-react with the structurally similar TKR cognate human stem cell factor (huSCF) or human colony-stimulating factor (huCSF1). Those skilled in the art will recognize that SCF and CSF are ligands that also bind to tyrosine kinase receptors. Nonspecific FLT3 inhibitors that bind to additional tyrosine kinase family members induce toxicity from global inhibition of tyrosine kinase signaling. Therefore, it is crucial that the anti-FLT3L antibody binds only to FLT3L and not to structurally similar homologs. Many anti-FLT3L antibodies and inhibitors lack specificity and bind to a wide range of tyrosine kinase receptors. Therefore, preferred embodiments of the anti-FLT3L antibody must demonstrate high affinity and specific binding to FLT3L.

[0077] In one embodiment, the anti-FLT3L antibody of the present disclosure is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and / or a chimeric antibody.

[0078] In some embodiments, the FLT3L binding molecule is a Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgG CH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb 2 , (s In some embodiments, the anti-FLT3L antibody is an IgG type antibody, e.g., an IgG1 type antibody (containing an IgG1 heavy chain immunoglobulin constant domain). In other embodiments, the anti-FLT3L antibody has an IgA, IgD, IgE, IgG2, IgG3, IgG4, or IgM heavy chain immunoglobulin constant domain.

[0079] In some embodiments, the IgG constant region may comprise a light chain constant region selected from the group consisting of an Igκ constant domain and an Igλ constant domain. In one specific embodiment, the anti-FLT3L antibody comprises a human IgG1 constant domain and a human λ constant domain. In another specific embodiment, the anti-FLT3L antibody has an IgG1-TM format, in which targeted mutations in the Fc region change leucine at position 243 to phenylalanine (L243F), leucine at position 235 to glutamic acid (L235E), and proline at position 331 to serine (P331S). Amino acid numbering is according to the EU index. Targeted mutations reduce FcR binding and ADCC effector function (see, e.g., Organesyan et al., Acta Crystallogr D Biol Crystallogr. 2008 Jun 1;64(Pt 6):700-4; and WO 2009100309(A2), which are incorporated by reference).

[0080] In some aspects, the anti-FLT3L antibody is a human antibody (e.g., CAT5D9, SC4017, AM40, CAT8, CAT26, DTAX3, and DYAX5 antibodies).

[0081] CAT5D9 antibody In one embodiment, the CAT5D9 antibody refers to an antibody that specifically binds to FLT3L and comprises the complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 29, 36, 37, 32, 33, and 38, respectively.

[0082] In another embodiment, the CAT5D9 antibody refers to an antibody that specifically binds to FLT3L and comprises two VL domains having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 6 and two VH domains having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 5.

[0083] In a further embodiment, the CAT5D9 antibody refers to an antibody comprising two VL domains having the amino acid sequence of SEQ ID NO:6 and two VH domains having the amino acid sequence of SEQ ID NO:5.

[0084] In another embodiment, the CAT5D9 antibody refers to an antibody comprising 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.

[0085] In one embodiment, the CAT5D9 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises one light chain having the amino acid sequence of SEQ ID NO: 70 and one heavy chain having the amino acid sequence of SEQ ID NO: 69.

[0086] In another embodiment, the CAT5D9 antibody refers to an antibody comprising one light chain encoded by the nucleic acid sequence of SEQ ID NO:72 and one heavy chain encoded by the nucleic acid sequence of SEQ ID NO:71.

[0087] SC4017 antibody In one embodiment, the SC4017 antibody refers to an antibody that specifically binds to FLT3L and comprises the complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 29, 30, 31, 35, 33, and 34, respectively.

[0088] In another embodiment, the SC4017 antibody refers to an antibody that specifically binds to FLT3L and comprises two VL domains having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 4 and two VH domains having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 3.

[0089] In a further embodiment, the SC4017 antibody refers to an antibody comprising two VL domains having the amino acid sequence of SEQ ID NO:4 and two VH domains having the amino acid sequence of SEQ ID NO:3.

[0090] In another embodiment, the SC4017 antibody refers to an antibody comprising 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.

[0091] In one embodiment, the SC4017 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises one light chain having the amino acid sequence of SEQ ID NO: 66 and one heavy chain having the amino acid sequence of SEQ ID NO: 65.

[0092] In another embodiment, the SC4017 antibody refers to an antibody comprising one light chain encoded by the nucleic acid sequence of SEQ ID NO:68 and one heavy chain encoded by the nucleic acid sequence of SEQ ID NO:67.

[0093] AM40(MEDI1116) antibody In one embodiment, the AM40 antibody refers to an antibody that specifically binds to FLT3L and comprises the complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively.

[0094] In another embodiment, the AM40 antibody refers to an antibody that specifically binds to FLT3L and comprises two VL domains having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO:2 and two VH domains having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO:1.

[0095] In a further embodiment, the AM40 antibody refers to an antibody comprising two VL domains having the amino acid sequence of SEQ ID NO:2 and two VH domains having the amino acid sequence of SEQ ID NO:1.

[0096] In another embodiment, the AM40 antibody refers to an antibody comprising 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.

[0097] In one embodiment, the AM40 antibody specifically binds to FLT3L and has one light chain having the amino acid sequence of SEQ ID NO: 62 and one heavy chain having the amino acid sequence of SEQ ID NO: 61. Refers to IgG1 antibodies containing the IgG1 chain.

[0098] In another embodiment, the AM40 antibody refers to an antibody comprising one light chain encoded by the nucleic acid sequence of SEQ ID NO:64 and one heavy chain encoded by the nucleic acid sequence of SEQ ID NO:63.

[0099] CAT8 antibody In one embodiment, CAT8 antibody refers to an antibody that specifically binds to FLT3L and comprises the complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 39, 40, 41, 42, 43, and 44, respectively.

[0100] In another embodiment, a CAT8 antibody refers to an antibody that specifically binds to FLT3L and comprises two VL domains having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 8 and two VH domains having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 7.

[0101] In a further embodiment, a CAT8 antibody refers to an antibody comprising two VL domains having the amino acid sequence of SEQ ID NO:8 and two VH domains having the amino acid sequence of SEQ ID NO:7.

[0102] In another embodiment, a CAT8 antibody refers to an antibody comprising 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.

[0103] In one embodiment, a CAT8 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises one light chain having the amino acid sequence of SEQ ID NO: 74 and one heavy chain having the amino acid sequence of SEQ ID NO: 73.

[0104] In another embodiment, a CAT8 antibody refers to an antibody comprising one light chain encoded by the nucleic acid sequence of SEQ ID NO:76 and one heavy chain encoded by the nucleic acid sequence of SEQ ID NO:75.

[0105] CAT26 antibody In one embodiment, the CAT26 antibody refers to an antibody that specifically binds to FLT3L and comprises the complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 45, 40, 46, 47, 48, and 49, respectively.

[0106] In another embodiment, the CAT26 antibody refers to an antibody that specifically binds to FLT3L and comprises two VL domains having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 10 and two VH domains having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 9.

[0107] In a further embodiment, the CAT26 antibody refers to an antibody comprising two VL domains having the amino acid sequence of SEQ ID NO: 10 and two VH domains having the amino acid sequence of SEQ ID NO: 9.

[0108] In another embodiment, the CAT26 antibody refers to an antibody comprising 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.

[0109] In one embodiment, the CAT26 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises one light chain having the amino acid sequence of SEQ ID NO: 78 and one heavy chain having the amino acid sequence of SEQ ID NO: 77.

[0110] In another embodiment, the CAT26 antibody refers to an antibody comprising one light chain encoded by the nucleic acid sequence of SEQ ID NO:80 and one heavy chain encoded by the nucleic acid sequence of SEQ ID NO:79.

[0111] DYAX3 antibody In one embodiment, a Dyax3 antibody refers to an antibody that specifically binds to FLT3L and comprises the complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 50, 51, 52, 53, 54, and 55, respectively.

[0112] In another embodiment, a Dyax3 antibody refers to an antibody that specifically binds to FLT3L and comprises two VL domains having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of 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.

[0113] In a further embodiment, Dyax3 antibody refers to an antibody comprising two VL domains having the amino acid sequence of SEQ ID NO:12 and two VH domains having the amino acid sequence of SEQ ID NO:11.

[0114] In another embodiment, a Dyax3 antibody refers to an antibody comprising 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.

[0115] In one embodiment, Dyax3 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises one light chain having the amino acid sequence of SEQ ID NO: 82 and one heavy chain having the amino acid sequence of SEQ ID NO: 81.

[0116] In another embodiment, a Dyax3 antibody refers to an antibody comprising one light chain encoded by the nucleic acid sequence of SEQ ID NO:84 and one heavy chain encoded by the nucleic acid sequence of SEQ ID NO:83.

[0117] DYAX5 antibody In one embodiment, a Dyax5 antibody refers to an antibody that specifically binds to FLT3L and comprises the complementarity determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 56, 57, 52, 58, 59, and 60, respectively.

[0118] In another embodiment, the Dyax5 antibody specifically binds to FLT3L and has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:86. and two VH domains having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 85.

[0119] In a further embodiment, Dyax5 antibody refers to an antibody comprising two VL domains having the amino acid sequence of SEQ ID NO:14 and two VH domains having the amino acid sequence of SEQ ID NO:13.

[0120] In another embodiment, a Dyax5 antibody refers to an antibody comprising 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.

[0121] In one embodiment, Dyax5 antibody refers to an IgG1 antibody that specifically binds to FLT3L and comprises one light chain having the amino acid sequence of SEQ ID NO: 86 and one heavy chain having the amino acid sequence of SEQ ID NO: 85.

[0122] In another embodiment, a Dyax5 antibody refers to an antibody comprising one light chain encoded by the nucleic acid sequence of SEQ ID NO:88 and one heavy chain encoded by the nucleic acid sequence of SEQ ID NO:87.

[0123] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to FLT3L is provided, comprising a set of complementarity-determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of (a) SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively; or (b) SEQ ID NOs: 29, 30, 31, 35, 33, and 34, respectively; or (c) SEQ ID NOs: 29, 36, 37, 32, 33, and 38, respectively.

[0124] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), each of which comprises three CDRs and four framework regions (FW) arranged from amino-terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, and FW4.

[0125] In certain aspects, the VH region and VL region have amino acid sequences that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to (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.

[0126] In certain aspects, the CDRs of VH (HCDR1, HCDR2 and HCDR3) and the CDRs of VL (LCDR1, LCDR2 and LCDR3) consist of the amino acid sequences of (a) SEQ ID NOs: 29, 30, 31, 32, 33 and 34, respectively; or (b) SEQ ID NOs: 29, 30, 31, 35, 33 and 34, respectively; or (c) SEQ ID NOs: 29, 36, 37, 32, 33 and 38, respectively.

[0127] A summary of the anti-FLT3L antibody sequences is provided in Table 1 below.

[0128] [Table 1]

[0129] [Table 2]

[0130] [Table 3]

[0131] [Table 4]

[0132] [Table 5]

[0133] [Table 6]

[0134]

Table 7

[0135]

Table 8

[0136]

Table 9

[0137]

Table 10

[0138]

Table 11

[0139]

Table 12

[0140]

Table 13

[0141] inducer The anti-FLT3L antibodies of the present disclosure may include sequence variants, provided that they retain the ability to specifically bind to FLT3L. Such variants can be derived from the sequences of these antibodies by those skilled in the art using techniques well known in the art. For example, amino acid substitutions, deletions, or additions can be made within the FR regions and / or CDRs of anti-FLT3L antibodies that disrupt binding of the antibody to its epitope. While changes within the FRs are typically designed to improve the stability and immunogenicity of the antigen-binding domain, changes within the CDRs 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 empirically determined by routine techniques involving altering the CDRs and testing the affinity of the antigen-binding domain for its target. For example, conservative amino acid substitutions can be made within any one of the CDRs disclosed herein. Various changes can be made according to the methods described in Antibody Engineering, 2nd ed., Oxford University Press, ed. Borrebaeck, 1995. These changes include, but are not limited to, nucleotide sequences altered by substituting various codons that encode functionally equivalent amino acid residues within the sequence, thus producing "silent" changes. For example, nonpolar 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.

[0142] Derivatives and analogs of antibodies of the present disclosure can be made by a variety of techniques known in the art, including recombinant and synthetic methods (Maniatis (1990) Molecular Cloning, A Laboratory Manual, 2nd 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).

[0143] In one embodiment, a method for generating a VH domain that is an amino acid sequence variant of a VH domain of the present disclosure comprises the steps of adding, deleting, substituting or inserting one or more amino acids into the amino acid sequence of a VH domain disclosed herein, optionally combining the VH domain so provided with one or more VL domains, and testing one or more VH domains or VH / VL combinations for specific binding to an antigen. Similar methods can be used to combine VH domains.

[0144] Similar shuffling or combinatorial techniques have also been described by Stemmer (Nature (1994) 370:389-391), who describes techniques related to β-lactamase genes but observes that the approach can be used for antibody generation.

[0145] In a further embodiment, random mutagenesis of one or more selected VH and / or VL genes can be used to generate novel VH or VL regions having one or more sequences derived from the sequences disclosed herein. One such technique, error-prone PCR, is described by Gram et al. (Proc. Nat. Acad. Sci. USA (1992) 89:3576-3580).

[0146] Another method that can be used is to direct mutagenesis to the CDRs of the VH or VL gene. Such techniques are disclosed by Barbas et al. (Proc. Nat. Acad. Sci. USA (1994) 91:3809-3813) and Schier et al. (J. Mol. Biol. (1996) 263:551-567).

[0147] Similarly, one, two or all three CDRs of an antigen-binding domain can be grafted into a repertoire of VH or VL domains, which are then screened for antigen-binding fragments that are specific for FLT3L.

[0148] A portion of an immunoglobulin variable domain useful herein may comprise at least one CDR substantially as defined herein and, optionally, an intervening framework region derived from an scFv fragment as defined herein. The portion may comprise at least about 50% of either or both of FR1 and FR4, the 50% being the C-terminal 50% of FR1 and the N-terminal 50% of FR4. Additional residues at the N- or C-terminus of a substantial portion of the variable domain may be residues not normally associated with naturally occurring variable domain regions. For example, 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 engineering steps. Other engineering steps include the introduction of linkers to join the variable domain to additional protein sequences, including immunoglobulin heavy chain constant regions, other variable domains (e.g., in the generation of diabodies), or proteinaceous tags, as discussed in more detail below.

[0149] The antigen-binding domains of the present disclosure described herein can be conjugated to other functional molecules, such as another peptide or protein (such as albumin or another antibody). For example, the antigen-binding domains can be conjugated by chemical cross-linking or recombinant methods. The antigen-binding domains can also be conjugated to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, using methods described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. The antigen-binding domains can be chemically modified by covalent conjugation to a polymer, for example, to increase their circulatory half-life. Typical polymers and methods for attaching them are also shown in US Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546.

[0150] The antibodies disclosed herein can also be altered to have a glycosylation pattern that differs from the native pattern. For example, one or more carbohydrate sites can be deleted, and / or one or more glycosylation sites can be added. Addition of glycosylation sites to the antibody fragments disclosed herein can be accomplished by altering the amino acid sequence so that it contains a glycosylation site consensus sequence known in the art. Another means of increasing the number of carbohydrate sites on an antibody fragment is by chemically or enzymatically coupling 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 moieties from the antibody can be accomplished chemically or enzymatically, as described, for example, in Hakimuddin et al. (1987) Arch. Biochem. Biophys., 259:52; Edge et al. (1981) Anal. Biochem., 118:131; and 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 conventional chemistry. Detectable labels also include enzyme labels, such as horseradish peroxidase or alkaline phosphatase. Detectable labels further include chemical moieties, such as biotin, which can be detected via binding to a specific cognate detectable moiety, such as labeled avidin.

[0151] Antigen-binding domains in which the CDR sequences differ only insubstantially from those defined herein are included within the scope of the present disclosure. Typically, amino acids are substituted with related amino acids having similar charge, hydrophobicity, or stereochemical characteristics. Such substitutions would be within the skill of one of ordinary skill in the art. Unlike CDRs, more substantial changes can be made within FRs without adversely affecting the binding properties of the antibody. Changes to the FRs include, but are not limited to, humanizing a non-human origin or manipulating specific framework residues important for stabilizing antigen contact or binding sites, e.g., changing the class or subclass of the constant region, changing specific amino acid residues that may alter effector function such as Fc receptor binding, or changing the species from which the constant region is derived, as described, e.g., in U.S. Pat. Nos. 5,624,821 and 5,648,260 and Lund et al. (1991) J. Immun. 147:2657-2662 and Morgan et al. (1995) Immunology 86:319-324.

[0152] Those skilled in the art will appreciate that the modifications described above are not entirely exclusive and are applicable to the protein subunits described herein, and that numerous other modifications will be possible to those skilled in the art in light of the teachings of the present disclosure.

[0153] Affinity and specificity of anti-FLT3L antibodies Those skilled in the art will recognize that anti-FLT3L antibodies for use in autoimmune diseases require high-affinity binding, yet must lack toxicity that would prevent their use in humans. Structurally similar homologs of FLT3L 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"). Non-specific anti-FLT3L antibodies that bind to FLT3L and also bind to SCF and CSF1 may cause non-specific toxicity. Thus, the anti-FLT3L antibodies of the present disclosure retain binding specificity only for FLT3L, but do not specifically bind to structurally similar cytokines such as SCF and CSF1. Those skilled in the art will recognize that K as a measure of binding specificity. on , K. off and K. D It will be known to use binding kinetics including, but not limited to:

[0154] Serum FLT3L and circulating pDCs are associated with the anti-FLT3L antibody lead clone. This is a biomarker that can be used as an indicator of the cytotoxicity of anti-FLT3L antibodies. The rapid decline in pDCs when FLT3L is neutralized indicates the suppression of FLT3L-mediated cell signaling, which enhances immune responses. The rapid recovery of pDC frequency in the presence of FLT3L may reflect the lack of cytotoxicity of anti-FLT3L antibodies. In a preferred embodiment, the anti-FLT3L antibody neutralizes FLT3L and allows reversible depletion of cDCs and pDCs when free FLT3L is restored. Those skilled in the art will recognize that a decline in dendritic cells after neutralization of FLT3L, followed by a recovery to baseline, is an indicator of the low cytotoxicity of the lead clone.

[0155] Generation of anti-FLT3L antibodies The practice of the present disclosure will employ, unless otherwise indicated, techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology that are well within the knowledge of those skilled in the art. Such techniques are explained fully in such references 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); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polypeptides of the present disclosure and therefore can be taken into consideration when making and practicing the present disclosure. Techniques that are particularly useful for certain embodiments are discussed in the Examples section below.

[0156] In one embodiment, the isolated nucleic acid molecule encoding the anti-FLT3L antibody or antigen-binding fragment thereof is operably linked to one or more regulatory sequences for expression in a host cell. The isolated nucleic acid can be recombinantly incorporated into a vector, which is then transfected into the host cell using known techniques.

[0157] In one embodiment, host cells transformed with an isolated nucleic acid molecule encoding an anti-FLT3L antibody or antigen-binding fragment thereof operably linked to one or more regulatory sequences are contemplated herein. Examples of contemplated host cells include mammalian cells such as HEK293 cells, NS0 mouse myeloma cells, or Chinese hamster ovary (CHO) cells.

[0158] In one embodiment, anti-FLT3L monoclonal antibodies (e.g., CAT5D9, SC4017, or AM40) and antigen-binding fragments thereof can be prepared using hybridoma methods, such as those described by 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 elicit the production by lymphocytes of antibodies that will specifically bind to the immunizing antigen.

[0159] In another embodiment, lymphocytes can be immunized in vitro. After immunization, lymphocytes are isolated and fused, e.g., using polyethylene glycol, with a suitable myeloma cell line to form hybridoma cells, which can then be selected and removed from unfused lymphocytes and myeloma cells. Immunoprecipitation, immunoblotting, or in vitro binding assays (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)) can be used. Hybridomas producing monoclonal antibodies specifically directed against a selected antigen as determined by

[0049] can then be grown either in in vitro culture or in vivo as ascites tumors in animals using standard methods (Coding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986). Monoclonal antibodies can then be purified from the culture medium or ascites fluid as described above for polyclonal antibodies.

[0160] Alternatively, anti-FLT3L monoclonal antibodies (e.g., CAT5D9, SC4017, or AM40) and antigen-binding fragments thereof can be produced using recombinant DNA methods, for example, as described in U.S. Pat. No. 4,816,567. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells by, for example, RT-PCR using oligonucleotide primers that specifically amplify genes encoding the antibody heavy and light chains, and their sequences are determined using conventional techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which are transfected into host cells, such as Escherichia coli (E. coli) cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, that do not otherwise produce immunoglobulin proteins or monoclonal antibodies produced by the host cells. Furthermore, recombinant anti-FLT3L monoclonal antibodies or antigen-binding fragments thereof of the desired species can be isolated from phage display libraries expressing the CDRs of the desired species as described in (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).

[0161] Polynucleotides encoding anti-FLT3L antibodies or antigen-binding fragments thereof can be further modified in a number of different ways using recombinant DNA technology to generate alternative antibodies. In some embodiments, for example, the constant domains of the light and heavy chains of a murine monoclonal antibody can be replaced with (1) those regions of a human antibody, e.g., to generate chimeric antibodies, or (2) with non-immunoglobulin polypeptides to generate fusion antibodies. In some embodiments, the constant regions are truncated or removed to generate desired antibody fragments of the monoclonal antibody. Site-directed or high-density mutagenesis of the variable regions can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0162] In certain embodiments, the anti-FLT3L antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. Human antibodies can be prepared directly using various techniques known in the art. Immortalized human B lymphocytes isolated from in vitro immunized individuals that produce antibodies against target antigens can be generated (see, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol., 147(1):86-95; and U.S. Patent No. 5,750,373).

[0163] Furthermore, anti-FLT3L antibodies or antigen-binding fragments thereof can be selected from phage libraries, where the phage libraries are described, for example, in Vaughan et al., 1996, Nat. Biotech. 14:309-314, Sheets et al. Human antibodies are expressed as described in (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; and Marks et al., 1991, J. Mol. Biol. 222:581). An antibody phage library is generated. Techniques for making and using them are also described in U.S. Pat. Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484 and 7,264,963; and Rothe et al., 2007, J. Mol. Bio., doi:10.1016 / j.jmb.2007.12.018 (each of which is incorporated by reference herein in its entirety).

[0164] Affinity maturation and chain 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 antibodies or antigen-binding fragments thereof.

[0165] In some embodiments, the anti-FLT3L monoclonal antibody may be a humanized antibody. Methods for genetically engineering, humanizing, or resurfacing non-human or human antibodies can also be used and are well known in the art. Humanized, resurfaced, or similarly engineered antibodies may contain one or more amino acid residues derived from non-human sources, such as, but not limited to, mouse, rat, rabbit, non-human primate, or other mammalian sources. These non-human amino acid residues are typically replaced with residues often referred to as "import" residues taken from an "import" variable, constant, or other domain of a known human sequence. Such import sequences can be used to reduce immunogenicity or to reduce, enhance, or modify binding, affinity, on-rate, off-rate, avidity, half-life, or any other suitable characteristic known in the art. Generally, the CDR residues are directly and most substantially involved in influencing FLT3L binding. Thus, some or all of the non-human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions can be substituted with human or other amino acids.

[0166] Antibodies can optionally be humanized, resurfaced, genetically engineered, or even engineered human antibodies while retaining high affinity for the FLT3L antigen and other favorable biological properties. To achieve this goal, humanized (or human) or genetically engineered anti-FLT3L antibodies and resurfaced antibodies can optionally be prepared by a process of analyzing the parental sequences and various conceptual humanized and genetically engineered products using three-dimensional models of the parental and genetically engineered and humanized sequences. Three-dimensional immunoglobulin models are commercially available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the potential role of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of a candidate immunoglobulin, such as FLT3L, to bind to its antigen. In this manner, framework (FW) residues can be selected and combined from consensus and import sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen.

[0167] Humanization, resurfacing, or genetic engineering of anti-FLT3L antibodies or antigen-binding fragments thereof can be performed using techniques such as 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), and Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992). Presta et al., J. Immunol. 151:2623 (1993), U.S. Patent Nos. 5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5 , 714,352; 5,9,55,358; 6,204,023; 6,180,370; 6,331,431; 5,693,762 Specification; Specification No. 5,530,101; Specification No. 5,585,089; Specification No. 5,225,539; Specification No. 4,816,567; Specification No. 5,969,108; Specification No. 7,63 and 7,342,110; International Patent Application Publication Nos. PCT / US S98 / 16280; PCT / US 91 / 05939; PCT / US 94 / 01234; PCT / GB 92 / 01755; International Patent Application Publication Nos. WO 90 / 14443; WO 90 / 14424; WO 90 / 14430; and European Patent Publication No. EP 229246 (each of which is incorporated herein by reference in its entirety, including the documents cited therein).

[0168] Humanized anti-FLT3L antibodies and antigen-binding fragments thereof can also be produced in transgenic mice containing human immunoglobulin loci, which are capable of producing a full repertoire of human antibodies after immunization in the absence of endogenous immunoglobulin production. This approach is described in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

[0169] In certain embodiments, anti-FLT3L antibody fragments are provided. Various techniques are known for producing antibody fragments. Traditionally, these fragments are derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1993, Journal of of Biochemical and Biophysical Methods 24:107-117; Brennan et al., 1985, Science, 229:81). In certain embodiments, anti-FLT3L antibody fragments are recombinantly produced. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, thus enabling the production of large amounts of these fragments. 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 U.S. Pat. No. 5,641,870. Other techniques for producing antibody fragments, such as chemical synthesis, will be apparent to those skilled in the art.

[0170] According to the present disclosure, techniques for producing single-chain antibodies specific to FLT3L can be applied (see, e.g., U.S. Pat. No. 4,946,778). Furthermore, methods can be adapted for constructing Fab expression libraries (see, e.g., Huse et al., Science 246:1275-1281 (1989)) to allow rapid and efficient identification of monoclonal Fab fragments with the desired specificity for FLT3L or its derivatives, fragments, analogs, or homologs. Antibody fragments can be produced by techniques in the art, including, but not limited to, (a) F(ab')2 fragments produced by pepsin digestion of antibody molecules; (b) Fab fragments produced by reducing disulfide bridges of F(ab')2 fragments; (c) Fab fragments produced by treating antibody molecules with papain and a reducing agent; and (d) Fv fragments. It is possible.

[0171] The anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein can be modified to increase their serum half-life. This can be achieved, for example, by incorporating a salvage receptor-binding epitope into the antibody or antibody fragment by mutation of the appropriate region in the antibody or antibody fragment, or by incorporating the epitope into a peptide tag that is then fused to the antibody or antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis), or by YTE mutation. Other methods for increasing the serum half-life of antibodies or antigen-binding fragments thereof, such as conjugation to heterologous molecules such as PEG, are known in the art.

[0172] Pharmaceutical Composition The present invention is also directed to pharmaceutical compositions comprising the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein. In certain embodiments, the present disclosure provides use of the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein in the manufacture of a medicament for treating a subject.

[0173] An effective amount of the pharmaceutical composition of the present disclosure must be administered, where "effective amount" is defined as an amount sufficient to produce the desired prophylactic, therapeutic, or palliative response in the subject. The effective amount will vary depending on the species and weight of the subject to which it is administered, but can be determined using standard techniques.

[0174] In certain aspects, the present disclosure provides therapeutic and prophylactic compositions for use in the treatment or prevention (to reduce the likelihood of) autoimmune diseases, including, without limitation, systemic lupus erythematosus, myositis, primary Sjogren's syndrome, multiple sclerosis, uveitis, psoriasis, or rheumatoid arthritis, in a subject in need thereof.

[0175] In some embodiments, the pharmaceutical compositions of the present disclosure comprise an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. In this context, "pharmaceutically acceptable carriers, diluents, or excipients" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier, which may or may not be approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine. For example, suitable carriers are known to those skilled in the art and include stabilizers, diluents, and buffers. Suitable stabilizers include 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 buffering agents include alkali metal phosphates, alkali metal carbonates or alkaline earth metal carbonates.

[0176] In certain aspects, the pharmaceutical compositions of the present disclosure can further contain one or more auxiliary substances, such as one or more lipids, phospholipids, carbohydrates, and lipopolysaccharides. In some embodiments, the pharmaceutical compositions of the present disclosure optionally include one or more additional active agents.

[0177] In certain cases, pharmaceutical compositions of the present disclosure can be prepared by techniques known to those skilled in the art. General considerations in the formulation and / or manufacture of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety). In general, the present disclosure The disclosed anti-FLT3L antibody or antigen-binding fragment thereof is mixed with a carrier to form a solution, suspension, or emulsion. One or more additives discussed herein may be added to the carrier or subsequently added. The pharmaceutical compositions of the present disclosure may be an aqueous solution, emulsion, or suspension, or may be a dried formulation. In certain embodiments, the pharmaceutical compositions of the present disclosure may be dried, for example, by freeze-drying or spray-drying, or lyophilized for storage or formulation purposes. They may subsequently be reconstituted into a liquid composition by the addition of a suitable liquid carrier, or may be administered in a dried formulation using methods known to those skilled in the art.

[0178] The pharmaceutical compositions of the present disclosure can be administered to a subject by various routes known in the art. Typical routes of administration of such pharmaceutical compositions include oral, mucosal, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. Thus, in certain embodiments, the pharmaceutical compositions of the present disclosure are prepared to be administered by a route selected from the group consisting of oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal routes. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, and intradermal injection or infusion techniques. In certain aspects, the pharmaceutical compositions of the present disclosure are prepared to allow the anti-FLT3L antibody or antigen-binding fragment thereof contained therein to become bioavailable after administration to a subject.

[0179] The choice of administration of the pharmaceutical composition will depend on the formulation selected. The pharmaceutical compositions of the present disclosure are administered in a manner compatible with the formulation and in an amount that is therapeutically effective. In certain embodiments, the pharmaceutical compositions of the present disclosure are prepared into solid, semi-solid, liquid, or gaseous forms, including, but not limited to, tablets, capsules, powders, granules, ointments, liquids, suppositories, injections, inhalants, gels, microspheres, and aerosols.

[0180] In certain examples, pharmaceutical compositions comprising the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein can be in solid or liquid form. In some embodiments, the carrier is a particulate material, e.g., such that the composition is in tablet or powder form. In other embodiments, the carrier is a liquid, and the composition is, for example, an oral syrup, an injectable solution, or an aerosol that is useful for inhalation administration. When intended for oral administration, pharmaceutical compositions comprising the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein can be in either solid or liquid form, where semi-solid, semi-liquid, suspension, and gel forms are included in the forms discussed herein as either solid or liquid.

[0181] In certain aspects, pharmaceutical compositions comprising the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein can be prepared in the form of powders, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. In some cases, such solid compositions will typically contain one or more inert diluents or edible carriers. In certain embodiments, one or more of the following may additionally be present: a binder such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; an excipient such as starch, lactose, or dextrin; a disintegrant such as alginic acid, sodium alginate, Primogel, cornstarch, or the like; a lubricant such as magnesium stearate or Sterotex; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; a flavoring such as peppermint, methyl salicylate, or orange flavoring; and a coloring agent.

[0182] These compositions may take the form of microspheres, liquids, suspensions, tablets, pills, capsules, sustained-release formulations, or powders and contain about 0.001-95% of the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein. Some dosage forms may contain 50 μg-250 μg of the anti-FLT3L antibody or antigen-binding fragment thereof.

[0183] In some embodiments, when the pharmaceutical composition of the present disclosure is in the form of a capsule, e.g., 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 contain commonly used excipients such as pharmaceutical grades of saccharin, cellulose, and magnesium carbonate.

[0184] In other aspects, the pharmaceutical composition of the present disclosure 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 intended for oral administration, the pharmaceutical composition of the present disclosure contains, in addition to the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein, one or more sweeteners, preservatives, dyes / coloring agents, and flavor enhancers. In certain aspects, pharmaceutical compositions intended to be administered by injection may contain one or more surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonic agents.

[0185] In certain cases, liquid pharmaceutical compositions comprising the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein, whether in solution, suspension, or other similar form, may contain one or more of the following components: a sterile diluent, such as water for injection, saline, e.g., physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic monoglycerides or diglycerides, polyethylene glycol, glycerin, propylene glycol, or other solvents that can function as solvents or suspending media; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate, and an agent for adjusting isotonicity, such as sodium chloride or dextrose. In some cases, the formulations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic. In some embodiments, the injectable pharmaceutical compositions are preferably sterile.

[0186] In other embodiments, pharmaceutical compositions comprising the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment, or gel base. In certain aspects, the base may comprise, for example, one or more of petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. In other aspects, a thickening agent may be present in a pharmaceutical composition for topical administration. In certain embodiments, when intended for transdermal administration, the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein may be included with a transdermal patch or iontophoresis device.

[0187] In yet other embodiments, pharmaceutical compositions comprising the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein are intended for rectal administration, for example, in the form of suppositories. For suppositories, binders and carriers may include, for example, polyalkalene glycols or triglycerides. In certain examples, compositions for rectal administration contain a fatty base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, or polyethylene glycol.

[0188] In another aspect, pharmaceutical compositions comprising the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein comprise 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 colloidal nature to systems consisting of pressurized packages. In certain embodiments, delivery is achieved by liquefied or compressed gas or by a suitable pump system that dispenses a metered amount of the active ingredient. In some embodiments, aerosols of the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein can be delivered in a monophasic, biphasic, or triphasic system to deliver the active ingredient. In other embodiments, delivery of the aerosol can be achieved by adding the necessary containers, activators, valves, and other components that can together form a kit. These include tubes, dispensers, etc. Those skilled in the art can readily determine the particular aerosol formulation and mode of delivery.

[0189] The pharmaceutical compositions of the present disclosure may be administered in a suitable non-toxic pharmaceutical carrier, may be contained in microcapsules, microbeads, and / or may be contained in a sustained release implant.

[0190] In other aspects, the pharmaceutical compositions of the present disclosure include 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.

[0191] In yet other embodiments, the pharmaceutical compositions of the present disclosure, in solid or liquid form, include an agent that binds to the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein, thereby assisting in the delivery of the anti-FLT3L antibodies or antigen-binding fragments thereof. In particular examples, suitable agents that act in this capacity include proteins or liposomes.

[0192] In certain embodiments, the pharmaceutical composition to be administered to a subject takes the form of one or more dosage units, where, for example, a tablet can be a single dosage unit, and a container of an anti-FLT3L antibody or antigen-binding fragment thereof in aerosol form disclosed herein can hold multiple dosage units. Practical methods for preparing such dosage forms are known or apparent to those skilled in the art; see, for example, 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, contain a therapeutically effective amount of the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutically acceptable salt thereof, to aid in treating the subject's disease or condition in accordance with the teachings herein.

[0193] In certain embodiments, the pharmaceutical compositions of the present disclosure comprise one or more additional therapeutically active substances. In other embodiments, a therapeutically effective amount of the pharmaceutical composition of the present disclosure is administered to a subject in need thereof in combination with one or more additional therapeutically active substances. As used herein, "combination" 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 may be administered sequentially (sequentially), together, or simultaneously.

[0194] The pharmaceutical compositions of the present disclosure may be administered at intervals to maintain therapeutic levels, if desired. The pharmaceutical compositions of the present disclosure may be used in conjunction with other bactericidal or bacteriostatic methods.

[0195] While the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to all types of subjects. In certain embodiments, the subject is a mammal. In certain embodiments, mammals include humans, primates such as monkeys and apes, and non-primates such as domestic and farm animals (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), including laboratory animals and familiar pets, as well as non-domestic animals such as wild animals and birds.

[0196] Autoimmune / anti-inflammatory therapy The disclosure also features compositions and methods comprising anti-FLT3L antibodies, such as those described above, that are useful for treating autoimmune diseases and / or other inflammatory diseases (i.e., diseases involving an over-reactive and / or dysfunctional immune system). In various embodiments, the anti-FLT3L antibodies , can be administered in combination with other immunomodulatory agents designed to inhibit or attenuate a subject's immune system or a specific immune response to a particular antigen or set of antigens, thereby reducing or preventing autoimmune or other inflammatory diseases.

[0197] Further provided herein are methods for treating autoimmune diseases and / or other inflammatory diseases, comprising administering one or more anti-FLT3L antibodies. As demonstrated herein, administration of an anti-FLT3L antibody can result in at least one of a reduction in immune response, expression of one or more immunological signaling cascades, or a reduction in immune cell populations. In certain embodiments, an anti-FLT3L antibody is administered to a patient or subject presenting with an autoimmune disease or other inflammatory disease.

[0198] Treatment of autoimmune and / or other inflammatory disease therapies with anti-FLT3L antibodies may, for example, reduce the rate of progression of the autoimmune or inflammatory disease, slow or stabilize immune cell proliferation, shrink lesions (e.g., in MS patients), and / or cause disease regression. In some embodiments, a metric measuring the reduction or slowing of an autoimmune or inflammatory disease (e.g., reduction in inflammation, inflammatory cytokine levels, immune cell populations, and / or associated damage such as tissue lesions) may be statistically significant. A reduction in an autoimmune or inflammatory disease metric can be measured by comparing the patient's level of the metric at baseline (pre-treatment) to an expected level of disease progression for the individual, to an expected level of disease progression based on a larger patient population, or to an expected level of disease progression for a control population.

[0199] In one embodiment, the therapeutic methods contemplated herein involve application or administration of an anti-FLT3L binding molecule, antibody, or antigen-binding fragment, variant, or derivative thereof of the present disclosure to a subject or patient, or application or administration of an anti-FLT3L binding molecule to an isolated tissue or cell line derived from a subject or patient, wherein the subject or patient has a disease, symptoms of a disease, or a predisposition toward a disease.

[0200] Examples of contemplated diseases include acute or chronic inflammatory diseases, including type 1 and type 2 diabetes, e.g., diabetes, CKD, including diabetic nephropathy and hypertension-induced CKD, arteriosclerosis, Alzheimer's disease, cancer, and associated complications of such diseases, including heart disease, hypertension, anemia, pericarditis, renal osteodystrophy, etc. Additional examples of contemplated diseases include autoimmune diseases, including, without limitation, systemic lupus erythematosus, myositis, primary Sjogren's syndrome, multiple sclerosis, uveitis, psoriasis, and rheumatoid arthritis.

[0201] In another embodiment, treatments are also contemplated that involve application or administration of a pharmaceutical composition comprising an anti-FLT3L binding molecule, e.g., an antibody of the present disclosure or an antigen-binding fragment, variant, or derivative thereof, to a subject or patient, or application or administration of a pharmaceutical composition comprising an anti-FLT3L binding molecule to an isolated tissue or cell line derived from a subject or patient, wherein the subject or patient has a disease, symptoms of a disease, or a predisposition toward a disease.

[0202] According to the methods of the present disclosure, at least one anti-FLT3L antibody, as defined anywhere herein, is used to promote a positive therapeutic response for an autoimmune or inflammatory disease. The term "positive therapeutic response" refers to a reduction in symptoms associated with an autoimmune or inflammatory disease. Thus, for example, an improvement in disease can be characterized as a complete response. A "complete response" is intended to be the absence of clinically detectable disease accompanied by normalization of any previous test results. Alternatively, an improvement in disease can be classified as a partial response. A "positive therapeutic response" includes a reduction or inhibition of the progression and / or duration of an autoimmune or inflammatory disease, a reduction or alleviation of the severity of an autoimmune or inflammatory disease, and / or alleviation of one or more symptoms thereof, resulting from administration of an anti-FLT3L binding molecule disclosed herein.

[0203] In certain embodiments, there is provided a method for treating primary Sjogren's syndrome, comprising administering to a subject in need thereof a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0204] In other embodiments, there is provided a method for treating myositis, comprising administering to a subject in need thereof a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0205] In certain embodiments, methods are provided for treating systemic lupus erythematosus (SLE), 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 has an increased serum level of FLT3L compared to a healthy subject, as measured by the frequency of CD4+ T cells expressing FLT3L.

[0206] In some embodiments, a method for diagnosing systemic lupus erythematosus (SLE) in a subject comprises: (a) measuring serum levels of FLT3L; or (b) measuring the frequency of CD4+ T cells that express FLT3L; and detecting increased serum levels of FLT3L or CD4+ T cells that express FLT3L in the subject compared to healthy donors. Methods are provided wherein an increased frequency of T cells indicates that the subject has SLE. In certain embodiments, the CD4+ T cells are effector memory cells (T EM )

[0207] In some embodiments, there is provided a method of neutralizing membrane-bound FLT3L in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein. In certain aspects, FLT3L is reversibly neutralized such that the activity of membrane-bound FLT3L can return to "pre-administration" levels.

[0208] In another embodiment, a method of neutralizing soluble FLT3L in a subject in need thereof is provided, 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 such that the level of soluble FLT3L can return to pre-administration levels.

[0209] In certain embodiments, the method for neutralizing soluble FLT3L further comprises subcutaneously administering to the subject an anti-FLT3L antibody or antigen-binding fragment thereof once weekly at a dose ranging from about 0.03 mg / kg to about 30 mg / kg. In other embodiments, the method further comprises subcutaneously administering to the subject an anti-FLT3L antibody or antigen-binding fragment thereof once every four weeks at a dose of about 150 mg / kg.

[0210] In other embodiments, provided are methods of reducing the population of circulating classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein. In certain aspects, the population of cDCs and pDCs is reversibly reduced, allowing the population of cDCs and pDCs to return to pre-administration levels.

[0211] In certain embodiments, provided is a method for reducing expression of FLT3L on CD4+ T cells, comprising administering to a subject in need thereof a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0212] In another embodiment, a method for decreasing the percentage of CD4+ T cells that express FLT3L comprises administering to a subject in need thereof a pharmaceutically effective amount of an antibody disclosed herein. or an antigen-binding fragment thereof.

[0213] In another embodiment, a method of reducing ERK signaling in lymphoblasts is provided, comprising contacting lymphoblasts with an antibody or antigen-binding fragment thereof disclosed herein.

[0214] In certain embodiments, a method of reducing MEK 1 / 2 phosphorylation in primary CD133+ human stem cells is provided, comprising contacting the stem cells with an antibody or antigen-binding fragment thereof disclosed herein. The present disclosure encompasses the following aspects. [Section 1] An antibody or antigen-binding fragment thereof that specifically binds to FLT3L, comprising a set of complementarity-determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are: (a) SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively; or (b) SEQ ID NOs: 29, 30, 31, 35, 33 and 34, respectively; or (c) SEQ ID NOs: 29, 36, 37, 32, 33 and 38, respectively An antibody or antigen-binding fragment thereof comprising the amino acid sequence: [Section 2] Each VH and VL comprises a heavy chain variable region (VH) and a light chain variable region (VL), each VH and VL comprising three CDRs and four framework regions (FW), arranged from amino-terminus to carboxy-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, and FW4, wherein the VH and VL regions comprise: (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 The antibody or antigen-binding fragment thereof according to item 1, having at least 95%, 96%, 97%, 98%, or 99% sequence identity with [Section 3] The CDRs (HCDR1, HCDR2, and HCDR3) of the VH and the CDRs (LCDR1, LCDR2, and LCDR3) of the VL are (a) SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively; or (b) SEQ ID NOs: 29, 30, 31, 35, 33 and 34, respectively; or (c) SEQ ID NOs: 29, 36, 37, 32, 33 and 38, respectively Item 3. The antibody or antigen-binding fragment thereof according to Item 2, which consists of the amino acid sequence: [Section 4] The VH and VL are (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 Item 3. The antibody or antigen-binding fragment thereof according to Item 2, comprising the amino acid sequence: [Section 5] (a) a heavy chain region having an amino acid sequence comprising SEQ ID NO: 61 and a light chain region having an amino acid sequence comprising SEQ ID NO: 62; or (b) a heavy chain region having an amino acid sequence comprising SEQ ID NO: 65 and a light chain region having an amino acid sequence comprising SEQ ID NO: 66; or (c) a heavy chain region having an amino acid sequence comprising SEQ ID NO: 69 and a light chain region having an amino acid sequence comprising SEQ ID NO: 70 The antibody or antigen-binding fragment according to Item 4, comprising: [Section 6] Item 6. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 5, which inhibits FLT3L-mediated activation of membrane-bound FLT3 on human stem cells, hematopoietic cell precursors, dendritic cells, activated T cells and B cells, monocytes, or microglia. [Section 7] Item 7. The antibody or antigen-binding fragment thereof according to Item 6, which does not specifically bind to at least one of human stem cell factor (huSCF) and human colony-stimulating factor (huCSF1). [Section 8] Item 7. The antibody or fragment thereof according to Item 6, which does not specifically bind to either huSCF or huCSF1. [Section 9] Item 9. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 8, which is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or a chimeric antibody. [Section 10] (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) an IgG4 constant domain; and (h) IgM constant domain Item 10. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, further comprising a heavy chain immunoglobulin constant domain selected from the group consisting of: [Section 11] Item 11. The antibody or antigen-binding fragment thereof of Item 10, comprising an IgG1 constant domain. [Section 12] (a) an Igκ constant domain; and (b) Igλ constant domain Item 12. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 11, further comprising a light chain immunoglobulin constant domain selected from the group consisting of: [Section 13] Item 13. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 12, wherein the antigen-binding protein comprises a human IgG1 heavy chain constant domain and a human λ light chain constant domain. [Section 14] Item 12. The antibody or antigen-binding fragment thereof according to Item 11, wherein 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 EU numbering index of Kabat. [Section 15] 15. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of Items 1 to 14. [Section 16] 16. The nucleic acid molecule of paragraph 15, operably linked to a regulatory sequence. [Section 17] Item 17. A vector comprising the nucleic acid molecule of Item 15 or 16. [Section 18] A host cell transformed with the nucleic acid molecule of Item 15 or 16 or the vector of Item 17. [Section 19] 19. The host cell of paragraph 18, which is a mammalian host cell. [Section 20] A hybridoma that produces the antibody or antigen-binding fragment according to any one of Items 1 to 14. [Section 21] Item 15. An isolated host cell that produces the antibody or antigen-binding fragment thereof according to any one of Items 1 to 14. [Section 22] A method for producing the antibody or antigen-binding fragment thereof according to any one of Items 1 to 14, comprising: (a) culturing host cells that express the antibody or antigen-binding fragment thereof; and (b) isolating the antibody or antigen-binding fragment thereof from the cultured host cells. [Section 23] Item 15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of Items 1 to 14 and a pharmaceutically acceptable excipient. [Section 24] Item 24. The pharmaceutical composition according to Item 23, for use as a pharmaceutical. [Section 25] A method for treating nephritis, comprising the step of administering to a subject in need thereof a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof according to any one of Items 1 to 14. [Section 26] A method for treating primary Sjögren's syndrome, comprising the step of administering to a subject in need thereof a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof according to any one of Items 1 to 14. [Section 27] A method for treating myositis, comprising the step of administering to a subject in need thereof a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof according to any one of items 1 to 14. [Section 28] A method for treating systemic lupus erythematosus (SLE), comprising the step of administering to a subject in need thereof a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof according to any one of Items 1 to 14. [Section 29] 29. The method of paragraph 28, wherein the subject has an increased serum level of FLT3L compared to a healthy subject as measured by the frequency of CD4+ T cells expressing FLT3L. [Section 30] 1. A method for diagnosing systemic lupus erythematosus (SLE) in a subject, comprising: a. Measuring serum levels of FLT3L; or b. Measuring the frequency of CD4+ T cells expressing FLT3L wherein an increased serum level of FLT3L or an increased frequency of CD4+ T cells expressing FLT3L in the subject compared to a healthy donor indicates that the subject has SLE. [Section 31] The CD4+ T cells are called effector memory cells (T EM Item 31. The method according to Item 30, wherein [Section 32] A method for neutralizing membrane-bound FLT3L in a subject in need thereof, comprising the step of administering to the subject a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof according to any one of Items 1 to 14. [Section 33] Item 24. The method of Item 23, wherein the FLT3L is reversibly neutralized. [Section 34] A method for reducing the population of circulating classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) in a subject in need thereof, comprising the step of administering to the subject a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof according to any one of Items 1 to 14. [Section 35] 35. The method of paragraph 34, wherein the population of cDCs and pDCs is reversibly reduced such that the population of cDCs and pDCs can return to pre-administration levels. [Section 36] A method for reducing FLT3L expression on CD4+ T cells, comprising the step of administering to a subject in need thereof a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof of any one of Items 1 to 14. [Section 37] A method for reducing the percentage of CD4+ T cells that express FLT3L, comprising the step of administering to a subject in need thereof a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof of any one of Items 1 to 14. [Section 38] A method for reducing ERK signaling in lymphoblasts, the method comprising the step of contacting the lymphoblasts with the antibody or antigen-binding fragment thereof according to any one of Items 1 to 14. [Section 39] A method for reducing MEK 1 / 2 phosphorylation in primary CD133+ human stem cells, comprising contacting the stem cells with the antibody or antigen-binding fragment thereof described in any one of items 1 to 14.

[0215] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening and treatment methods of the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. [Example]

[0216] The present disclosure will now be described with reference to the following examples, which are illustrative only and the present disclosure should in no way be considered limited to these examples, but rather should be considered to include any and all variations that become evident as a result of the teachings provided herein.

[0217] Example 1. Generation of anti-FLT3L antibodies Overview: FLT3L is a 65 kDa non-disulfide-linked homodimeric glycoprotein. The sequence homologies of the ligand and receptor for humans, non-human primates, and mice are shown in Table 2 below.

[0218] [Table 14]

[0219] Although the homology of the complete mouse FLT3L protein is only 73%, its binding site with FLT3 is highly conserved across species. Indeed, human FLT3L binds to and activates mouse FLT3, and vice versa. FLT3L shares structural homology with stem cell factor (SCF or KIT-ligand) and colony-stimulating factor (CSF1), but lacks significant sequence homology with other human cytokines. The receptors for these two ligands, c-KIT and CSF1R, are also class III TKRs that typically interact with FLT3 inhibitors, resulting in undesirable off-target toxicity in clinical use. Given these considerations, a search was made for an antibody against FLT3L that would provide highly specific inhibition of the FLT3 / FLT3L pathway and would not cross-react with SCF or CSF1.

[0220] Lead antibodies were selected and tested using a binary selection against soluble human and mouse FLT3-L. Primary biochemical high-throughput screening was performed using a human FLT3 / FLT3L competitive fully homogeneous time-resolved fluorescence (HTRF) assay. All single-chain variable fragment-fragment crystal domain (scFv-Fc) hits were converted to IgG1™ format, and then the inhibitory activity of the lead antibodies was confirmed using a functional screening assay using downregulation of FLT3 on the surface of target cells. Cross-reactivity and selectivity for mouse and cynomolgus monkey FLT3L, as well as exclusion of other family members such as stem cell factor (scf), were confirmed using ELISA and functional assays.

[0221] The lead compounds were further evaluated using FLT3 signaling assays in the RS4;11 cell line and primary human CD133+ stem cells. Binding to endogenous FLT3L was confirmed using primary human T cells. Specific binding sites and binding affinities were determined by Octet and BIAcore, respectively. The resulting lead antibody clones were selected for further optimization, as described below.

[0222] Identification of lead antibody candidates (C5, B10-11) Lead antibodies for anti-FLT3L binding studies were generated by first screening phage libraries, as shown in Figure 1. Phage display libraries, including bone marrow Vaughan (BMV), combined spleen (CS), DP47 library (DP47), and Dyax human antibody library, were panned (alternate or competitive) against human FLT3L (huFLT3L) and / or mouse FLT3L (muFLT3L). Briefly, in-house generated FLT3L was labeled with biotin using the EZ-Link Sulfo-NHS-LC-Biotin Labeling Kit (Thermo Scientific) as the panning antigen. Panning was performed over two to three rounds using an in-house scFv library, as described (Xiao X et al., 2017 mAbs 542 9, 996-1006 (2017)). To enhance human / cyno cross-reactivity, some selection processes used human and cyno recombinant FLT3L as panning antigens in an alternating fashion. To select antibodies that inhibit the FLT3L / FLT3 interaction, competitive panning was used for some experiments. For competitive panning, human FLT3L was used as the panning antigen, but excess (>100-fold) FLT3-Fc was used as the phage elution agent instead of conventional trypsin (Xiao X et al. mAbs 542 9, 996-1006 (2017)).

[0223] The BMV library was enriched 100-fold, the CS library 3.50-fold, and the DP47 library 250-fold. The Dyax library was not enriched. Three rounds of panning of the BMV and Dyax phage libraries alone resulted 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 (Figure 1A).

[0224] Monoclonal phage ELISA was performed to estimate the percentage of antigen-specific phage after each selection round. Only selections that achieved a positive rate of at least 20% were processed for high-throughput screening. Using this criteria set, the BMV panning from the third output, along with the CS and DP47 panning from the second output, were cloned into pSplice V4 and pdLG or pmLG vectors and converted into either scFv-Fc format (Xiao X, et al., PLoS One, 2015 Oct 15;10(10):e0140691.doi:10.1371 / journal.pone.0140691) or IgG format (Xiao X, et al., mAbs 542 9,996-1006(2017)) for functional screening by competitive HTRF.

[0225] For functional screening, 293 freestyle cells (Thermo Scientific) were first transfected with either the scFv-Fc construct or the IgG construct converted from the panning output. The resulting supernatant was used directly in an HTRF-based FLT3L / FLT3 interaction inhibition assay. For the HTRF assay, 10 nM biotin-labeled FLT3L, 20 nM streptavidin-europium cryptate (Cisbio), 10 nM FLT3-mFc, and 20 nM anti-mFc-A647 (Cisbio) were mixed with 10 μL of transfection supernatant in a total volume of 20 μL per well in a 384-well Greiner plate. Readings at 665 nm and 620 nm were obtained 5 min after mixing and at 1-h intervals thereafter until the readings stabilized. The 665 nm / 620 nm ratio was then calculated. A decrease in the ratio indicated an inhibition of the FLT3L / FLT3 interaction.

[0226] Panning outputs from direct or alternating antigen panning were converted to scFv-Fc, and over 4,000 colonies were selected for high-throughput screening (HTS) in an HTRF FLT3L / FLT3-Fc interaction inhibition assay. Ten lead antibodies were identified, converted to IgG TM, and expressed. These antibodies were retested in a second round of HTRF interaction inhibition assays, and 10 leads were identified. In parallel, over 700 hits from competitive panning were converted to PmIgG and expressed in mammalian cells. When the converted clones were subjected to the same HTRF FLT3L / FLT3-Fc interaction inhibition assay, two leads were identified (Figure 1B).

[0227] Ten leads from the alternating antigen panning and two leads from the competitive panning were expressed and purified in milligram quantities for further testing. Additional testing included HTRF FLT3L / FLT3-Fc interaction inhibition, receptor downmodulation, and signal transduction inhibition assays. These leads also underwent epitope binning and affinity determination. The screening campaign identified five lead antibodies, Dyax3, Dyax5, CAT8, CAT26, and CAT5D9, which were further tested. Results from the competitive HTRF analysis of the lead antibodies are shown in Figure 1C.

[0228] Example 2. Screening assay for downregulation of FLT3 After ligation of FLT3L, the FLT3 receptor dimerizes, autophosphorylates, and activates downstream signaling pathways. During this process, surface FLT3 is internalized and degraded. Taking advantage of this internalization feature, we developed an assay to screen lead anti-FLT3L candidate clones. Cell surface FLT3 expression can be measured by flow cytometry, and inhibition of receptor-ligand binding can be determined by quantifying changes in cell surface FLT3 expression levels.

[0229] The cell lines RS4;11, EOL-1, MOLM13, and MV4-11 constitutively express FLT3. Cells were cultured under normal conditions and screened for relative FLT3 expression after 2 to 24 hours of culture. FLT3 expression was measured using flow cytometry. Briefly, a commercially available anti-CD135 (anti-FLT3) clone, BV10A4H2 (Biolegend), was used in flow cytometry experiments, and mean fluorescence intensity (MFI) was reported as a measure of FLT3L expression. In all assays, a commercially available mouse anti-human FLT3L monoclonal antibody (R&D) or an in-house huFLT3-Fc construct was used as a positive control for effective neutralization of FLT3L activity.

[0230] The acute leukemia (pro-B) cell line, RS4;11, exhibits consistently high expression of FLT3 in culture compared with other commercially available cell lines reported to express FLT3. Direct binding of FLT3L to cell surface FLT3 was confirmed using serial dilutions of biotinylated recombinant huFLT3L (rhuFLT3L), which could be detected for physical binding to the cell surface using BV421-streptavidin after 30 min of incubation with RS4;11 cells at 4°C, followed by flow cytometry analysis to determine mean fluorescence intensity (Fig. 2B).

[0231] Finally, the ability of rhuFLT3L to induce detectable downregulation of cell surface FLT3 on RS4;11 cells was confirmed by incubating RS4;11 cells with serial dilutions of FLT3L for 2 hours at 37°C. Stability was assessed using two different concentrations of RS4;11 cells: 50,000 (50K) and 100,000 (100K) cells. Downregulation of cell surface FLT3 was determined using a fluorescently labeled anti-CD135 antibody (clone BV10A4H2). Both cell densities showed dose-dependent downregulation of cell surface FLT3 after 2 hours of incubation with huFLT3L. Allophycocyanin (APC) MFI, an indicator of FLT3 expression, decreased 25-fold across the range of huFLT3L used in the assay (Figure 2C). These results demonstrated that the screening assay was effective in measuring the dose-dependent response of bioavailable FLT3L and would be suitable for testing candidate clones for their ability to functionally neutralize FLT3L. This response did not differ significantly whether 50,000 or 100,000 cells per well were used.

[0232] Optimization of screening assays The screening assay conditions were further refined to determine the ideal cell culture conditions for evaluating anti-FLT3L candidate clones. The final conditions were 50,000 RS4;11 cells per well incubated with 96 pM rhuFLT3L, with or without anti-FLT3L mAb candidate clones, in complete Roswell Park Memorial Institute (RPMI) medium containing 1% bovine serum albumin (BSA) for 2 hours in a humidified incubator set at 37°C and 5% CO2. Subsequent downregulation of FLT3 expression was determined by flow cytometry, measured either as raw MFI or percent downregulation. The EC80 of 96 pM rhuFLT3L was chosen because it marks the beginning of the exponential phase of the dose-response curve, ensuring that any functional inhibition of rhuFLT3L would be immediately reflected by a change in the level of downregulated FLT3 on the RS4;11 cell surface (Figure 3A). The validity of the assay was confirmed using a commercially available mouse anti-huFLT3L antibody control (MAB608, R&D Systems) (Figure 3B). Due to the cross-species reactivity of FLT3 with its ligand, this assay was valid for testing clones against human, cyno, and rodent FLT3L, despite being a human cell line. For mouse FLT3L, the EC80 was 36 pM.

[0233] Example 3. Neutralizing activity of lead antibody candidates against soluble FLT3L After ligation of FLT3L to FLT3, the receptor dimerizes, autophosphorylates, and internalizes, propagating a signaling cascade. This process, measured by downregulation of FLT3, can be inhibited by antibodies that bind to FLT3L. Therefore, lead candidates were tested for their ability to inhibit FLT3 downregulation on RS4;11 cells by binding to human, mouse, or cynomolgus monkey (cyno) soluble FLT3L (sFLT3L). An optimized screening assay was used to test the neutralizing ability of lead candidates. As described above, RS4;11 cells (50,000 cells per well) were incubated for 2 hours in complete RPMI containing 1% BSA in the presence of either human or cyno FLT3L (96 pM) or mouse sFLT3L (36 pM). Serial dilutions of each clone were added, and the neutralizing ability was assessed. A soluble FLT3-Fc construct or a commercially available human anti-FLT3L antibody was used as a positive control. FLT3 expression on RS4;11 cells was determined using flow cytometry and reported as MFI.

[0234] As shown in Figures 4A and 4B, all lead clone candidates demonstrated the ability to inhibit both human and cyno sFLT3L to some extent. CAT8, CAT26, Dyax3, and Dyax5 all demonstrated similar inhibition of human and cyno sFLT3L. As shown in Table 3 below, the IC50 values ​​for CAT8, CAT26, Dyax3, and Dyax5 were IC MAX was never reached, and therefore lacks validity.

[0235] In contrast, CAT5D9 uses IC MAX The IC50 values ​​for CAT5D9 presented in Table 3 demonstrate cross-reactivity with human and cyno, but not mouse, FLT3L (Figure 4C).

[0236] [Table 15]

[0237] Example 4. Binding activity of lead antibody candidates to cell surface FLT3L FLT3L is expressed as a plasma membrane protein and circulates as a soluble protein upon cleavage. Both the membrane-bound and soluble forms are biologically active. To effectively block the FLT3-mediated signaling pathway, lead antibody candidates must bind to both the soluble and membrane-bound forms of FLT3L.

[0238] Consistent with this, cell surface binding to human, cyno, and mouse FLT3L was assessed by transfecting Chinese hamster ovary (CHO) cells with each full-length protein. Candidate clones were incubated with FLT3L-expressing cell lines for 1 hour at 4°C and then washed twice to remove unbound antibody. Cells were then incubated with a PE-labeled goat anti-hu IgG secondary detection pAb to quantify bound antibody, which was measured by flow cytometric analysis of the PE signal. The huFLT3-Fc construct on the hu IgG backbone was used as a positive control for FLT3L expression because it cross-reacts with cyno and mouse, as well as human, ligands.

[0239] All lead candidates bound to human FLT3L (Fig. 5A), and all except DYAX5 bound to cyno FLT3L (Fig. 5B). In contrast, only Dyax5, and to a lesser extent Dyax3, showed cross-reactivity with mouse FLT3L (Fig. 5C).

[0240] Therefore, all lead candidates demonstrated the ability to bind to cross-species FLT3L, albeit with varying potencies. Because differences in receptor occupancy for each clone were evident, the EC50 and maximum occupancy (expressed as % compared to FLT3-Fc) were recorded and incorporated into the final evaluation. The results are shown in Table 4 for human, cyno, and mouse FLT3L expressed in CHO cells.

[0241] [Table 16]

[0242] Example 5. Binding of lead antibody candidates to endogenous human FLT3L FLT3L is expressed on the surface of primary T cells upon stimulation with IL-2, IL-7, or IL-15, independent of TCR engagement and other cytokines. In Example 4, lead antibody candidates demonstrated the ability to bind to CHO cells transfected with human FLT3L protein. The next step was to confirm that they could bind to endogenous FLT3L from primary human cell lines. Therefore, lead antibody candidates were tested for their ability to bind to huFlt3L on IL-2 stimulated primary T cells obtained from human donors.

[0243] To induce FLT3L expression on the cell surface, freshly isolated human T cells were stimulated with 50 ng / mL IL-2 for 5 days in the absence of anti-CD3 (anti-CD3 activation of T cells would cause FLT3L to be shed from the cell surface). Expression on T cells was then confirmed using a human FLT3L-Fc construct (Figure 6A). Serial dilutions of each clone were then incubated with IL-2-stimulated T cells (100K / well) for 30 minutes at 4°C. Excess antibody was removed by washing with buffer, and surface-bound antibody was detected with APC-labeled anti-human IgG. All lead clones except CAT5D9 bound to endogenous FLT3L on human primary T cells (Figure 6B). Binding of CAT5D9 to endogenous FLT3L was initially undetectable due to its low binding affinity. However, when CAT5D9 was dimerized with anti-IgG (APC-labeled) before incubation with T cells, its avidity was sufficiently enhanced to confirm dose-dependent binding to endogenous FLT3L (Fig. 6C).

[0244] Example 6. Inhibition of cell surface FLT3L by lead antibody candidates The ability of the lead candidate to bind to cell surface FLT3L provided limited insight into the functional inhibition of the ligand. Ideally, binding of the lead candidate to cell surface FLT3L should reduce the signaling activity of the ligand-receptor complex.

[0245] To test this, 1,000 huFLT3L-expressing CHO cells were plated per well overnight to allow attachment. The next day, the CHO culture medium was removed, the cells were gently washed with RPMI, and serial dilutions of antibodies were added for 30 minutes, followed by FLT3 + RS4;11 (100K / well). After 2 hours of incubation at 36°C, the RS4;11 cells were transferred to a fresh 96-well plate on ice for staining to detect FLT3 downregulation. In addition to the dye used for our standard RS4;11 FLT3 downregulation assay, anti-CD19 was included to exclude any contaminating CHO cells. FLT3 downregulation was measured by flow cytometry.

[0246] Lead antibody candidate blocking assays demonstrated that all lead candidates had the ability to inhibit cell surface FLT3L to some extent, but all candidates exhibited relatively low activity compared to commercially available control antibodies, reflecting low affinity (Figure 7).

[0247] Example 7. Inhibition of FLT3L-induced FLT3 signaling in lead clones Autophosphorylation of FLT3 leads to activation of signaling networks, primarily through the PI3K and RAS cascades, which in turn activate AKT (protein kinase B, PKB), MEK, and ERK. The signaling cascade ultimately results in the transcription of genes that promote cell survival and proliferation. To confirm whether the lead antibody candidate blocked downstream signaling of FLT3 by FLT3L in primary human cells, phosphorylation of ERK and MEK in CD133+ stem cells was measured using Mesoscale MSD phospho-ERK1 / 2 and phosphor-MEK1 / 2 whole cell lysates.

[0248] The validity of the assay was confirmed using the RS4;11 cell line for FLT3L-induced ERK activation in a dose-dependent manner (Figure 8A). Serial dilutions of FLT3L were incubated with 300,000 RS4;11 cells per well for 8 minutes at 36°C. Cells were then harvested, lysed, and assayed for phosphorylated ERK according to the manufacturer's instructions. As with the previous assay, the EC80 for FLT3L activation of ERK was determined (476 pM) and used to test the inhibitory activity of candidate anti-FLT3L antibody clones. The usefulness of the assay was confirmed using a commercially available mouse anti-human FLT3L antibody as a positive control for effective neutralization (Figure 8B).

[0249] Using these established parameters, the lead clone was tested using in vitro expanded primary CD133+ stem cells that had been validated for FLT3 expression prior to use. The lead clone was preincubated with 476 pM FLT3L for 30 minutes and then added to the CD133+ stem cells. After an 8-minute incubation at 36°C, the cells were harvested, lysed, and assayed for phosphorylated ERK and MEK using the MSD assay according to the manufacturer's instructions. A commercially available mouse anti-human FLT3L was used as a positive control.

[0250] All candidate clones appeared to inhibit the induction of MEK (Fig. 9A) and ERK (Fig. 9B) by FLT3L. However, only CAT5D9 inhibited the IC MAX The antibody reached a concentration of 0.01g / mL and showed the expected sigmoidal dose-response curve (Table 5). Taken together, the results presented in the Examples suggest that CAT5D9 is the best lead candidate clone and should be further optimized for affinity and biophysical evaluation of the epitope binding site.

[0251] [Table 17]

[0252] Example 8. Confirmation of target specificity CAT5D9 appeared to be the best lead candidate based on functional evaluation in biological assays. Octet epitope binning method was used to determine the CAT5D9 binding region relative to the receptor FLT3.

[0253] An epitope binning method was used to determine lead antibodies that share the same binding region with the FLT3 receptor on FLT3L. Binning was performed in three stages. In Stage I, biotin-FLT3L binding to an avidin probe was performed. In Stage II, individual antibodies were bound to biotin-FLT3L. In Stage III, each test antibody was added to the Stage II antibody. Any additional binding detected indicated that the two antibodies had non-competing binding sites for the target FLT3L. In Stage III, buffer alone and FLT3-Fc, without any antibody added, were used as negative and positive controls, respectively. When buffer alone was added in Stage III, the resulting dissociation kinetics of the antibody against FLT3L reflected the intrinsic affinity of the clone for the target.

[0254] Only CAT5D9 added at 1x and 2x concentrations inhibited binding by FLT3L-Fc, suggesting that CAT5D9 hits the desired target site on FLT3L (i.e., shares the same or overlapping epitope with FLT3-Fc) (Figure 10A). Importantly, in contrast, the remaining four candidate clones were not inhibited by CAT5D9, suggesting that they bound to different FLT3 epitopes (Figure 10A). Furthermore, the steep off-rate when buffer alone was added supports previous experiments suggesting that CAT5D9 has low affinity (implying that its performance through optimization is likely to improve). Consistent with this, when each of the remaining four clones bound in Phase II (CAT8, as shown in the representative chart presented in Figure 10B), additional binding of CAT5D9 and FLT3-Fc could be detected in Phase III, reflecting their distinct binding sites. Furthermore, it was also observed that each of the other four clones were binned together and that their dissociation rates were significantly slower when buffer alone was added.

[0255] Taken together, these data confirm that only CAT5D9 directly competes with FLT3 at the FLT3L binding domain and appears to do so with low affinity binding, which is , suggesting that CAT5D9 has potential for optimization. On the other hand, all of the remaining clones hit sites that, based on functional data, do not directly compete with FLT3. Their slow dissociation rates suggested that they already bound FLT3L with reasonable affinity and had little potential for optimization. The initial inhibition in the initial screening assays was likely due to steric hindrance or partial blockage of the receptor binding site.

[0256] Example 9. BIACORE Binding Kinetics (C9) Biacore analysis was used to determine the binding kinetics of the antibody leads, confirming Octet data suggesting that CAT5D9 has poor affinity for FLT3L. Anti-FLT3L fragment antigen binding and kinetics for human and cynomolgus monkey FLT3L were determined for human CAT8, CAT26, Dyax3, and Dyax5. Furthermore, binding kinetics of CAT5D9 fragment antigen was determined for both human and cynomolgus monkey FLT3L.

[0257] The results are shown in Table 6. The equilibrium dissociation constant (K D ) was over 50-fold higher in CAT5D9 humans and cynomolgus monkeys compared to the remaining lead antibodies. CAT5D9 exhibited poor, rapid off-kinetics (hu=70.72, cyno=70.66). As a result, steady-state binding data were significantly lower than the kinetic data K D The steady-state binding was obtained as a check on the above. The steady-state binding supported the kinetic binding results, showing similar values ​​(Table 3). These data confirm the low affinity of CAT5D9 and the potential for improved performance through optimization.

[0258] [Table 18]

[0259] Example 10. Absence of cross-reactivity of CAT5D9 In addition to confirming that CAT5D9 bound to the correct FLT3L epitope, it was also important to ensure that it would not bind to stem cell factor (SCF) and colony-stimulating factor (CSF1), which are close structural homologs of FLT3L. Both factors are major off-targets for small molecule FLT3 inhibitors currently used in oncology to manage malignancies arising from constitutively activating FLT3-IT9D mutations. They are ligands for the protein tyrosine kinase receptors c-Kit and CSFR1, respectively.

[0260] To test this, ELISA plates were coated with 2 μg of recombinant human SCF or CSF1. After washing, plates were blocked with 3% milk in Tris-phosphate-buffered saline (TPBS), and serial (2-fold) dilutions starting at 50 μg / mL were added. After incubation, unbound antibody was removed by washing, and bound antibody was detected using anti-human IgG-HRP 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.

[0261] None of the lead candidates cross-reacted with huSCF (Figure 11A) or huCSF1 (Figure 11B). Importantly, these results demonstrate the selectivity of CAT5D9, which binds only to FLT3L and not to structurally similar TKR ligand molecules.

[0262] Example 11. Affinity optimization of CAT5D9 As discussed above, CAT5D9 binds to FLT3L with low affinity, demonstrating the potential for improved performance through affinity optimization. A desired KD of 300 pM was established based on PK modeling. Optimization efforts were designed to achieve up to a 10,000-fold improvement in KD.

[0263] After germlining the framework, two parallel strategies were used: parsimonious mutagenesis and block mutagenesis. Parsimonious mutagenesis mutates all 20 amino acids at each position in all six CDRs, one at a time. Clones are screened using high-throughput methods, and individual beneficial mutations are combined together. Block mutagenesis mutates consecutive stretches of 5-6 positions in the CDRs in an overlapping pattern, and the resulting library of approximately 1E6-1E7 clones is first enriched using phage display panning and then screened using high-throughput methods.

[0264] After the first optimization round, 30 clones from parsimony mutagenesis and 24 clones from block mutations were tested in an IgG format. Using molecular modeling techniques, we identified and combined the best mutations that resulted in clone 5D9-clone 6, which achieved a KD of 1610 nM (measured by Biacore), a 700-fold improvement in affinity over parental 5D9 (see Figure 7). The affinities of lead clones SC4017 and AM40 exceeded the CDTP criterion (<300 pM). Furthermore, both optimized clones bind and neutralize endogenous cell-surface FLT3L, endogenous soluble FLT3L in human serum, and cyno FLT3L.

[0265] [Table 19]

[0266] This increase in affinity translated into a more than 1000-fold improvement in functional activity as measured by downregulation of FLT3 on RS4;11 cells (Figure 12A) using the methods described for the initial clonal selection.

[0267] 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 a similar manner to the first optimization round. As part of the phage panning for block mutagenesis, clone 6 was used as a competitor to enrich for clones with substantially higher affinity in the IgG format. The best clone from the block mutagenesis was clone AM40, with a KD of 170 pM. Combining several combinatorial clones of AM40 and mutations from the second round of parsimony mutagenesis generated one superior clone, SC4017, with a KD of 37 pM. This higher affinity again reflected improved functional inhibition of FLT3L, as evidenced by downregulation of FLT3 on RS4;11 cells (Figure 12B). However, subsequent analysis revealed that the superior performance of SC4017 was due to the incorporation of a single additional tryptophan adjacent to the binding region. This represented a development risk given the vulnerability of tryptophan residues exposed to oxidation. For this reason, AM40 was selected as the lead IgG clone. Despite having a slightly lower affinity (170 pM) compared to SC4017 (37 pM), AM40 still exceeded the initial target of 300 pM and was therefore determined to have a lower development risk.

[0268] Finally, we confirmed that both clones could neutralize endogenous FLT3L on the cell surface by developing an assay using primary T cells stimulated with 20 ng / mL IL-7 for 7 days in coculture with FLT3+RS4;11 (which was found to be the most effective protocol for inducing cell surface FLT3L expression on primary T cells). Briefly, IL-7-stimulated CD4+ T cells were incubated overnight with RS4;11 cells at a 15:1 ratio (dose ratio response shown in Figure 13A), either alone or in the presence of serial dilutions of candidate clones SC4017 and AM40. FL Downregulation of FLT3 was measured by flow cytometry as previously described. Both clones were shown to completely prevent downregulation of FLT3 on RS4;11 cells with similar efficacy at concentrations above 1 nM (Figure 13B).

[0269] Example 12. Neutralization of FLT3L in healthy non-human primates To determine the safety and durability of AM40 in neutralizing FLT3L, a toxicity study was conducted over a one-month period using repeated weekly dosing. An 8-week treatment follow-up period was included to track animal growth. A summary of the study is illustrated in Figure 14. As shown in Figure 15A, free soluble FLT3L levels fell sharply after the first dose of AM40 at all doses, but 0.3 mg / kg was insufficient to maintain target engagement for a full week. The higher dose groups (1 mg / kg and 30 mg / kg) consistently demonstrated complete target engagement (reflected as free soluble FLT3L below BLQ) through day 57, at which point soluble FLT3L levels returned to baseline in the 1.0 mg / kg group.

[0270] Similarly, measurement of circulating DC frequencies (% of total CD45+ cells detected by flow cytometry and expressed as a % of pre-study baseline levels) revealed a steady decline in the frequencies of CD1c+ (classical) DCs and plasmacytoid DCs by day 22 in the 1.0 and 30 mg / kg groups (Figure 15B). For the 1 mg / kg and 30 mg / kg groups, the frequencies of circulating CD1c+ DCs remained suppressed until days 50 and 85. For the 1 mg / kg and 30 mg / kg groups, the frequencies of circulating pDCs remained reduced until days 71 and 85. Recovery of the DC population in the 1.0 mg / kg group correlated with the recovery of free serum FLT3L, which occurred sometime between days 57 and 85. These results indicate that the DC population declines when FLT3L is neutralized by AM40 but rapidly recovers to baseline once free FLT3L becomes available.

[0271] Example 13. FLT3L expression correlates with the severity of systemic lupus erythematosus (SLE) SLE is an autoimmune disease characterized by chronic inflammation, affecting nearly every organ in the body and all age groups. SLE commonly affects the joints, skin, kidneys, lungs, heart, and brain. Given its role in proinflammatory signaling, we investigated FLT3L expression in individuals with SLE to explore correlations between FLT3L levels and disease severity. Most published studies to date have relied on serum FLT3L levels to draw correlations with disease. While serum FLT3L levels are the most realistic measure in clinical settings, they have the inherent drawback of reflecting the production minus that is utilized by DCs and other activated FLT3L-consuming cells. In the context of inflammation, the number of FLT3-expressing cells and their consumption of FLT3L vary greatly, which likely explains the variability in observations between studies and the lack of a direct correlation between serum FLT3L levels and clinical scores of disease progression. Since T cells were found to be one of the most important receptors for FLT3L in inflammatory situations, we developed an assay to directly measure the expression of FLT3L on the surface of T cells using freshly isolated peripheral blood mononuclear cells (PBMCs).

[0272] Serum and PBMCs were isolated from individuals with SLE (n = 24) and healthy donors (HD; n = 15). Serum FLT3L was measured using ELISA (R&D Systems) according to the manufacturer's instructions. FLT3L-expressing CD4+ T cells were determined using an in-house developed flow cytometry assay. FLT3L was detected using a fluorescently labeled anti-FLT3L clone, MAB608 (R&D Systems). The SLE Disease Activity Index (SLEDAI) was used to determine lupus activity in an individual. Significance was determined using Mann-Whitney and Spearman correlations for comparisons of healthy vs. diseased cohorts and correlations with SLEDAI, respectively.

[0273] Consistent with previous literature, serum FLT3L levels were elevated in SLE donors compared with HD donors (p<0.05; Fig. 16A), but no significant correlation with disease activity (SLEDAI) was found (p<0.07; Fig. 16B). In contrast, when FLT3L production was measured by the frequency of FLT3L-expressing CD4+ T cells, it was highly significantly increased in SLE donors compared with HD donors (p<0.0001; Fig. 16C) and strongly correlated with SLEDAI scores (r=0.7045; p<0.0001; Fig. 16D). These data suggest that measuring FLT3L expression on T cells may be particularly important in the setting of disease.

[0274] Having found a correlation between SLEDAI score and CD4+ T cells expressing FLT3L, we examined subsets of CD4+ cells to determine (1) whether FLT3L expression across CD4+ T cell subsets from SLE patients was consistent with known biology, and (2) whether subset expression correlated with SLEDAI score. The CD4+ subsets examined included naive T cells (T naive ), effector memory cells (T EM ) and Central Memory (T CM ) cells. The same protocols and significance levels as described above were used to test for expression and correlation.

[0275] The expression of FLT3L across CD4+ T cell subsets was consistent with the known biology of FLT3L expression. In particular, FLT3L expression is generally associated with T cell proliferation in HD. naive A small but significant increase was observed in SLE donors, but not in T cells (Fig. 17A, top). naiveExpression of FLT3L on cells was significantly correlated with SLEDAI score (Figure 17A, bottom; r = 0.6629; p = 0.0004). Importantly, FLT3L expression was significantly associated with HD and SLE CD4+ T cell proliferation, as expected in this population capturing recently activated T cells that would have been exposed to □-chain cytokines known to induce FLT3L expression. EM This response was observed in both HD and SLE donors, but was significantly elevated in the latter. Furthermore, expression in SLE donors correlated with SLEDAI (Fig. 17B, bottom; r = 0.6201; p = 0.0012). Finally, FLT3L expression was significantly elevated in healthy CD4+ T cells, suggesting that they were T EM From T CM Although FLT3L+ T cell expression declines with differentiation into leukemia-associated leukemia (LE)-associated leukemia (LE), it is maintained in PBMCs of SLE donors (Fig. 17C, upper panel; p<0.0001). Furthermore, there is a significant correlation between the frequency of FLT3L+ T cells and SLEDAI, suggesting that expression in this population reflects a chronic inflammatory state.

[0276] Collectively, these studies demonstrate that FLT3L expression on CD4+ T cells in SLE patients is significantly increased compared with that in HD patients. Furthermore, increased FLT3L expression is highly correlated with SLEDAI scores. Therefore, administration of anti-FLT3L antibodies to SLE patients is a rational therapeutic strategy to reduce the FLT3L-expressing T cell population and reduce inflammation in SLE patients.

[0277] The method used above was validated using PrimeFlow in situ detection of IC FLT3L RNA and confirmed using APC-conjugated AM40.

[0278] Example 14. Expression of FLT3L in myositis Myositis is a chronic muscle inflammation characterized by weakness, swelling, and muscle pain. At the cellular level, myositis is characterized by elevated levels of interferon type 1 proteins and pDCs. may be associated with SLE and other pro-inflammatory conditions. Therefore, we investigated the expression of FLT3L in individuals with myositis to explore correlations between FLT3L levels and disease severity.

[0279] PBMCs from individuals with myositis and HD were tested for CD4+ T cells expressing FLT3L using FACS. naive Subset, T EM Subset and T CM Significance between myositis and HD samples was determined using Mann-Whitney analysis.

[0280] As can be seen from Figures 18A and 18B, the expression of FLT3L in PBMCs from individuals with myositis was significantly higher than that of FLT3L (T naive (p<0.05; Figure 18A), T EM (p<0.0001; Figure 18B) and T CM These patients were characterized by a significant increase in the percentage of CD4+ T cells positive for FLT3L (p<0.0001; Figure 18C), which is comparable to observations from SLE patients. In light of these results, administration of anti-FLT3L antibodies to myositis patients is a rational therapeutic strategy to reduce the T cell population expressing FLT3L to reduce inflammation in myositis patients.

[0281] Example 15. Expression of FLT3L in nephritis Nephritis is an immune disorder that affects the kidney and is related to the structure of the kidney. This condition can result from SLE, certain toxins, or certain infections. Nephritis can result in 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), which is thought to play a role in driving inflammation in the kidney, it was hypothesized that blockade of FLT3L could suppress DCs and prevent the progressive loss of kidney function.

[0282] We examined the effects of FLT3L blockade on proteinuria levels and nephritis scores using a mouse model of Murphy Roths Large / lymphoproliferative (MRL.lpr) nephritis and a mouse surrogate anti-FLT3L antibody (LFC-1). Isotype control and anti-IFNAR antibody treatment groups were included. Mice treated with anti-FLT3L antibody showed a significant reduction in proteinuria after 17 weeks of treatment (Figure 19A). Furthermore, nephritis scores after 18 weeks were reduced in mice treated with anti-FLT3L antibody compared with isotype controls (Figure 19B). Importantly, proteinuria and nephritis scores were reduced in mice treated with anti-FLT3L antibody compared with untreated mice. These results support the role of FLT3L-mediated inflammation in nephritis. In light of these results, administration of anti-FLT3L antibodies to patients with nephritis is a rational therapeutic strategy to reduce the FLT3L-expressing T cell population to reduce inflammation in patients with nephritis.

[0283] To provide insight into FLT3L-related changes in leukocyte populations during nephritis, we also examined splenic DC populations. Spleens were harvested from MRL mice at 18 weeks of age and examined for changes in splenic DC populations. Treatment with anti-FLT3L antibody significantly reduced circulating DCs in MRL mice (Figures 20A-C). Specifically, Siglec-H+-pDCs were significantly reduced after anti-FLT3L antibody administration compared to isotype control (Figure 20A). Similarly, significant reductions were observed in CD11b+ cDCs (equivalent to human CD1c+ DCs) and CD8+ cDCs (equivalent to human CD141+ DCs) (Figures 20B and 20C). Furthermore, the incidence of dermatitis in anti-FLT3L-treated mice was low compared to the usual 30-40% incidence in untreated mice. Thus, treatment with anti-FLT3L antibody reduced circulating DC populations and ameliorated secondary pathology (dermatitis) in this 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.

[0284] Example 16. Expression of FLT3L in Sjögren's syndrome Primary Sjögren's syndrome (pSS) is an autoimmune condition characterized by widespread dryness of the eyes and salivary glands. Furthermore, this condition can lead to multiple organ failure. This syndrome can occur alone or in the presence of additional autoimmune diseases such as lupus or rheumatoid arthritis. Serum levels of FLT3L are increased in individuals with pSS, and there is evidence that both FLT3L and its receptor are expressed locally in inflamed salivary glands (Tobon et al., (2010) Arthritis and Rheumatism v62:344).

[0285] We used the NOD.H2h4 Sjögren's syndrome mouse model to examine salivary gland pathology after long-term anti-FLT3L antibody (LFC-1) treatment. By 16 weeks of age, these mice develop tertiary lymphoid structures (TLS) in the salivary glands (SGs), primarily composed of DCs, B220+ B cells, and CD3+ T cells, in a manner closely resembling the pathological changes seen in humans. This tissue damage is preceded by the development of autoantibodies and the formation of spontaneous germinal centers in the spleen (Mahmoud et al., 2016 Science Translational Medicine, v8 361ra137). Mice were treated with either an isotype IgG control (5 mg / kg) or an anti-FLT3L antibody (5 mg / kg) in either a prophylactic (starting at 5 weeks of age) or therapeutic (starting at 17 weeks of age) protocol. For both protocols, treatment continued with twice-weekly administration until the end of the study (26 weeks). Anti-FLT3L monoclonal antibody (LFC-1) effectively neutralized FLT3L throughout the course of treatment (FIG. 25A), resulting in the accumulation of the drug / ligand complex in the circulation (FIG. 25B).

[0286] Lymphoid organs were collected to assess changes in peripheral immune cell populations, and salivary glands (SGs) were harvested and evaluated for histopathology (frequency of TLS). While it has previously been reported that prophylactic treatment can prevent disease onset, there have been limited previous reports on whether tissue damage can be delayed or prevented by therapeutic intervention after disease onset. Anti-FLT3L monoclonal antibody (LFC-1) inhibited CD44 expression in the spleen and salivary gland-draining LNs. HI It reduced antigen-associated CD4+ and CD8+ T cell frequencies (at 24-26 weeks of age at the end of the study) (Figures 26A-26D), and selectively reduced specific autoantibodies against collagen IV and platelet extract (Figure 27).

[0287] As expected, animals treated with isotype control IgG had increased TLS formation, an indicator of salivary gland damage, in the salivary glands by 26 weeks of age (1 mm of tissue). 2 Anti-FLT3L-treated mice showed a significant reduction in tissue SG damage, even when administered therapeutically (Figure 21A), and when administered prophylactically, disease was completely prevented (Figure 21B). DC populations measured in the spleen were significantly suppressed but not completely deleted (Figures 22A-D). Nevertheless, this was sufficient to significantly affect disease initiation and progression by reducing inflammatory infiltration into the salivary glands. These results support that inflammation in pSS is driven by a FLT3L-mediated mechanism. In light of these results, administration of anti-FLT3L antibodies is a reasonable therapeutic strategy for treating pSS in human subjects.

[0288] From the above description, it will be apparent that variations and modifications may be made to the disclosure described herein to adapt it to various uses and conditions. Such embodiments are also within the scope of the appended claims. The recitation of a list of elements in any definition of a variable herein includes a definition of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein does not limit the scope of any single embodiment. This includes embodiments in their entirety or in combination with any other embodiment or portion thereof. All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

1. A composition for reducing binding of feline McDonough sarcoma (FMS)-like tyrosine kinase 3 receptor ligand (FLT3L) by FLT3, comprising an antibody or antigen-binding fragment thereof that binds to FLT3L, wherein the antibody or antigen-binding fragment thereof comprises a set of complementarity-determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 29, 30, 31, 32, 33 and 34, respectively.

2. The composition of claim 1 , wherein the FLT3L is expressed on the cell surface.

3. The composition of claim 1 , wherein the FLT3L is expressed as a secreted homodimer.

4. The composition of claim 1 , wherein the reduction is in vitro.

5. 2. The composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise amino acid sequences having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

6. The composition of claim 5, wherein the VH and VL comprise the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

7. The composition of claim 1 , comprising the antibody, wherein the antibody is a monoclonal antibody or a recombinant antibody.

8. The antigen-binding fragment of the antibody includes Fab, F(ab') ２ , Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments produced by an Fab expression library, and combinations thereof.

9. The composition of claim 7 , wherein the antibody is a monoclonal antibody.

10. The composition of claim 1 , wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 61 and a light chain comprising the amino acid sequence of SEQ ID NO:

62.

11. A composition for treating a disease, comprising an antibody or antigen-binding fragment thereof specific to feline McDonough sarcoma (FMS)-like tyrosine kinase 3 receptor ligand (FLT3L), wherein the antibody or antigen-binding fragment thereof comprises a set of complementarity-determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively; the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprise amino acid sequences having at least 95% sequence identity to SEQ ID NO: 1 and SEQ ID NO: 2, respectively; and the disease is an autoimmune disease or an inflammatory disease.

12. The composition of claim 11, wherein the VH and VL comprise the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

13. The composition of claim 11 , wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 61 and a light chain comprising the amino acid sequence of SEQ ID NO:

62.

14. The composition of claim 11 , wherein the disease is an autoimmune disease.

15. The composition of claim 11 , wherein the disease is an inflammatory disease.

16. 15. The composition of claim 14, wherein the autoimmune disease is selected from the group consisting of Sjogren's syndrome, nephritis, myositis, systemic lupus erythematosus, multiple sclerosis, uveitis, psoriasis, and rheumatoid arthritis.

17. 16. The composition of claim 15, wherein the inflammatory disease is selected from the group consisting of type 1 diabetes, type 2 diabetes, chronic kidney disease, nephropathy, arteriosclerosis, Alzheimer's disease, cancer, heart disease, hypertension, anemia, pericarditis, renal osteodystrophy, and any complications thereof.

18. 17. The composition of claim 16, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

19. 17. The composition of claim 16, wherein the autoimmune disease is Sjogren's syndrome.

20. 17. The composition of claim 16, wherein the autoimmune disease is rheumatoid arthritis.

21. The composition of claim 16, wherein the autoimmune disease is nephritis.

22. 17. The composition of claim 16, wherein the autoimmune disease is multiple sclerosis.

23. The composition of claim 16, wherein the autoimmune disease is psoriasis.

24. The composition of claim 11 , wherein the treatment further comprises administration of a second immunomodulatory agent.

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