Antibodies to FLT3L and their use for treatment of autoimmune and inflammatory diseases

Abstract

Provided herein are antibodies to FLT3L and their use for the treatment of autoimmune and inflammatory diseases.

Description

[0001] This application is a divisional application of an invention application with a filing date of February 13, 2019, a Chinese national application number of 201980013516.0, and an invention name of “Antibodies to McDonald’s feline sarcoma (FMS)-like tyrosine kinase 3 receptor ligand (FLT3L) and their use in the treatment of autoimmune diseases and inflammatory diseases”. Technical Field

[0002] The present application relates to the field of antibody therapy, and in particular to anti-FLT3L antibodies and methods of using these antibodies to treat autoimmune diseases and other inflammatory diseases. Background Art

[0003] Autoimmune diseases occur when the body's immune system produces its own antibodies, and are unfortunately common. For example, it is estimated that more than 23 million Americans suffer from an autoimmune disease. There are currently more than 80 recognized autoimmune diseases. Specific examples of autoimmune diseases include systemic lupus erythematosus, myositis, primary Sjögren's syndrome, multiple sclerosis, uveitis, psoriasis, and rheumatoid arthritis.

[0004] Systemic lupus erythematosus (SLE) is characterized by joint pain, lymphadenopathy and butterfly rash on the cheek. In SLE, autoantibodies for healthy tissue attack the patient's immune system, resulting in 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 [immunology and cell biology] (2002) 80: 484-488). Sjögren's syndrome is characterized by systemic chronic inflammation of exocrine organs, resulting in organ dysfunction (Holdgate N. and St.Clair E.W., F1000 Research. [F1000 research] 141210.12688 / f1000research.8352.1).

[0005] 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, itchy skin patches. Rheumatoid arthritis (RA) is an inflammatory disorder of the synovial tissue of the joints, characterized by persistent synovitis and destruction of joint cartilage and bone. The damage can progress to affect many body systems. Lupus nephritis is associated with systemic lupus erythematosus and causes inflammation of the kidneys. Once inflamed, the kidneys leak protein and can eventually fail. Uveitis is a group of inflammatory diseases that attack and can damage eye tissue, leading to vision loss.

[0006] In addition, acute and chronic proinflammatory states are associated with and may be the cause of a variety of diseases in individuals. Specific examples of diseases believed to be associated with chronic inflammation include type 1 and type 2 diabetes, chronic kidney disease (CKD, including, for example, CKD caused by diabetes, diabetic nephropathy, and hypertension), atherosclerosis, Alzheimer's disease, cancer, and the associated complications of such diseases, including heart disease, hypertension, anemia, pericarditis, renal osteodystrophy, etc. Like autoimmune diseases, in diseases associated with chronic inflammation, the body appears to mount an excessive, persistent proinflammatory response, which can lead to debilitating and often fatal complications.

[0007] The etiology of autoimmune diseases is not completely understood. Mechanistically, the underlying cause of each autoimmune disease is a persistent autoimmune response that is promoted (and / or not inhibited) by complex regulatory systems that continually replenish autoreactive immune cells. Similar mechanisms appear to operate in non-autoimmune chronic inflammatory diseases. For this reason, therapeutic interventions in autoimmune diseases and for chronic inflammation have targeted multiple regulatory systems, signaling cascades, and their components.

[0008] One class of putative therapeutic targets includes tyrosine kinase receptors (TKRs), which are transmembrane receptors that bind to different growth factors and proteins to regulate cellular homeostasis. More than fifty known human TKRs are divided into 20 different classes, which are defined by their genetic phylogeny (Robins DR et al., Oncogene. [Cancer gene] (2000) 19: 5548-5557; Lemmon MA and Schlessinger J. Cell. [Cell] (2010) 141: 1117-1134). Class III TKRs are characterized by the presence of five to seven immunoglobulin-like domains containing 70 to 100 hydrophilic residues in the extracellular portion. Among class III TKRs, McDonald feline 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 cells and B cells, monocytes and microglia. FLT3 binds FLT3 ligand (FLT3L), which is a hematopoietic cytokine expressed by various cell types (including activated T cells, activated endothelial and bone marrow stromal cells). FLT3L is expressed as both homodimers of cell surface and secretion, and signals are sent by its cognate receptor FLT3. FLT3 is expressed as a monomer on the cell surface, and is activated after being connected with FLT3L. After connecting FLT3L, FLT3 dimerization, autophosphorylation and activation signal transduction pathways, including RAS / extracellular signal-regulated kinase (ERK), phosphatidylinositol 3 kinases (PI3K) and signal transducers and transcription activators (STAT) 3 and 5. After autophosphorylation, dimerized FLT3 is internalized and degraded.

[0009] FLT3L is produced in response to inflammatory signals, particularly IL-2, IL-7 and IL-15, and its interaction with FLT3 mainly drives inflammatory processes through its role in differentiation, proliferation and survival of DC. After activation, FLT3 signaling also has a putative role in T cell and B cell survival, and it is reported that both cell types transiently upregulate receptors (Astier AL et al., J. Immunology. [Immunology] 2010v184: 685-93 and Tobon et al., Arthritis & Rheumatism. [Arthritis and rheumatism] 2010; 62 (11): 3447-56). In addition, although this observation is based on mouse data (Guimond M et al., J. Immunology [Immunology] 2010; 184: 2769-75) and has not yet been confirmed in humans, it is believed that NK cell survival is indirectly dependent on FLT3L through its demand for DC-derived IL-15.

[0010] DCs are of particular interest in inflammation because they are sentinels of the immune system, migrating from sites of inflammation to lymph nodes and initiating adaptive immune responses that are ultimately required to produce autoimmune diseases. Broadly speaking, there are two subsets of DCs: myeloid / classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). cDCs produce inflammatory cytokines (e.g., IFNI-III, IL-23, IL-12, IL-6, and IL-1□), present antigens to T cells in the context of co-stimulation, and secrete chemokines that recruit cells to sites of inflammation and ensure that they are co-localized as required for key cell-cell interactions. Through these mechanisms, cDCs stimulate neutrophils, B cells, T cells, and NK cells, leading to NETosis, autoantibody production, IL-17 production, and other inflammatory cytokine production. pDCs are the main source of type I IFNs, which are key cytokines in the innate response that enhance the activation of all arms of the immune system.

[0011] Salivary glands of patients with Sjögren's syndrome show expression of FLT3 and FLT3L on infiltrating B cells (Tobon et al. Arthritis & Rheumatism. [Arthritis and rheumatism] (2010) 62 (11): 3447-3456). In addition, patients with Sjögren's syndrome show an increase in the frequency of B cells expressing FLT3 in the circulation, and their survival rate is improved when co-cultured with human salivary cells expressing FLT3L. Individuals with MS express FLT3 protein in chronic lesions and active lesions, as well as in gray and white matter (DeBoy CA et al. Exp Mol Pathol. [Experimental Molecular Pathology] (2010); 89 (2): 109-116). In addition, FLT3 is co-localized with immature DC in the perivascular brain, indicating that FLT3-positive DC infiltrates the brain of individuals with MS (Deboy et al.). Compared with healthy individuals, in RA, synovial FLT3L levels are elevated. In addition, monocytes, NK cells and DCs from RA patients express high levels of FLT3L (Ramos M. et al. Arthritis Res Ther. (2013) 15(6): R209).

[0012] Furthermore, elevated levels of FLT3L in serum and at inflammatory sites 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).

[0013] Therefore, although FLT3-mediated proinflammatory survival (e.g., via pDC and mDC) is a favorable physiological response in healthy individuals, it may have a deleterious effect in autoimmune diseases. Therefore, the destruction or attenuation of the FLT3 / FLT3L signaling pathway may prove to be an important tool for combating autoimmune diseases and other inflammatory diseases and for reducing inflammation. Summary of the invention

[0014] Provided herein are novel FLT3L binding antibodies for use in the management of autoimmune diseases and other acute and / or chronic inflammatory diseases.

[0015] In a first aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to FLT3L, the isolated antibody or antigen-binding fragment thereof comprising 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 following amino acid sequences: (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.

[0016] 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 yet 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 yet another 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 separated antibody or antigen-binding fragment comprises a heavy chain immunoglobulin constant domain selected from the group consisting of: (a) IgA constant domain; (b) IgD constant domain; (c) IgE constant domain; (d) IgG1 constant domain; (e) IgG2 constant domain; (f) IgG3 constant domain; (g) IgG4 constant domain; and (h) IgM constant domain. In one embodiment, the separated antibody or antigen-binding fragment comprises an IgG1 constant domain. In another embodiment, the separated antibody or antigen-binding fragment comprises a light chain immunoglobulin constant domain selected from the group consisting of: (a) Igκ constant domain; and (b) 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, which are numbered according to the EU numbering index of Kabat (Edelman et al., Proc. Natl. Acad. Sci., 63:78-85 (1969)).

[0017] In a second aspect, the disclosure provides an isolated nucleic acid molecule encoding an isolated antibody or antigen-binding fragment thereof as described in the first aspect and / or embodiments thereof. In an embodiment of the second aspect, the nucleic acid molecule is operably linked to a control sequence.

[0018] In a third aspect, the present disclosure provides a vector comprising a nucleic acid molecule as described in the second aspect and / or embodiments thereof.

[0019] In a fourth aspect, the disclosure provides a host cell transformed with a nucleic acid molecule as described in the second aspect and / or an embodiment thereof or a vector as described in the third aspect. In one embodiment, the host cell is a mammalian host cell. In another embodiment, the host cell is a HEK293 cell, a NS0 mouse myeloma cell, or a Chinese hamster ovary (CHO) cell.

[0020] In a fifth aspect, the present disclosure provides a hybridoma that produces the antibody or antigen-binding fragment as described in any one of the preceding aspects or embodiments thereof.

[0021] In a sixth aspect, the present disclosure provides an isolated host cell that produces the antibody or antigen-binding fragment as described in any one of the preceding aspects or embodiments thereof.

[0022] In the seventh aspect, the present disclosure provides a method for preparing an antibody or an antigen-binding fragment thereof as described in any one of the preceding aspects or embodiments thereof, the method comprising (a) culturing a host cell expressing the antibody or antigen-binding fragment or culturing a host cell as described in the third aspect or embodiments thereof or a hybridoma as described in the fourth aspect; and (b) isolating the antibody or antigen-binding fragment thereof from the cultured host cell.

[0023] In an eighth aspect, the present disclosure provides an antibody or antigen-binding fragment thereof produced by the method of the sixth aspect.

[0024] In a ninth aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of the preceding aspects or embodiments thereof, and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition is provided for use as a medicament.

[0025] In a tenth aspect, the disclosure provides a method for treating an acute or chronic inflammatory disease, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of an antibody or fragment thereof separated as described in any one of the preceding aspects or an embodiment thereof. In one embodiment, the inflammatory disease comprises chronic kidney disease (CKD, including, for example, CKD caused by diabetes, diabetic nephropathy, and hypertension).

[0026] In an eleventh aspect, the present disclosure provides a method for treating an autoimmune disease, the method comprising: administering to a subject in need thereof a pharmaceutically effective amount of an isolated antibody or fragment thereof as described in any one of the preceding aspects or embodiments thereof. In one embodiment, the autoimmune disease comprises systemic lupus erythematosus, myositis, primary Sjögren's syndrome, multiple sclerosis, uveitis, psoriasis, or rheumatoid arthritis.

[0027] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description of the present disclosure together with the appended claims.It should be noted that the scope of the claims is limited by the description therein rather than by the specific discussion of the features and advantages set forth in this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A-1C. Selection of lead antibodies. Figure 1 A depicts the alternating panning process of huFLT3L and muFLT3L and the enrichment of the resulting BMV, CS, DP47, and Dyax phage libraries. Figure 1 B depicts the process of cloning the panning output into a vector for downstream competition HTRF. Figure 1 C depicts the competition HTRF (homogeneous time-resolved fluorescence) results of the lead candidates.

[0029] Figure 2 A-2C. Expression of FLT3L in cell lines. Figure 2 A demonstrates increased expression of FLT3 in RS4;11 cell line relative to EOL-1, MOLM13, and MV4-11 cell lines. Figure 2 B demonstrates that recombinant FLT3L binds to FLT3 on RS4;11 cells in a dose-dependent manner. Figure 2 C demonstrates that FLT3 downregulation on the surface of RS4;11 cells can be reliably detected using flow cytometric analysis with a commercially available anti-FLT3 antibody and that this downregulation occurs in a dose-dependent manner in response to ligation with FLT3L.

[0030] Figure 3 A and 3B. Derivation of EC80 and subsequent testing. Figure 3A demonstrates the titration curve of FLT3L used to derive the concentration for 80% downregulation of cell surface FLT3 on RS4;11 cells (EC80). Figure 3 B demonstrates the inhibitory properties of a commercially available anti-FLT3L antibody and a recombinant construct of the FLT3 receptor (FLT3-Fc) against 96 pM of recombinant FLT3L, which would otherwise result in 80% downregulation of cell surface FLT3 on RS4;11 cells.

[0031] Figure 4 A-4C. Inhibition of human and cynomolgus monkey sFLT3 by lead antibody candidates. Figure 4 A demonstrates the inhibitory properties of five lead candidates against 96 pM human FLT3L. Figure 4 B demonstrates the inhibitory properties of five lead candidates against 96 pM of cynomolgus monkey FLT3L. Figure 4 C Demonstration of the inhibitory properties of five lead candidates against murine FLT3L.

[0032] Figure 5 AC. Inhibition of cell surface FLT3 by lead candidates. Figure 5 A demonstrates the ability of the lead antibody to bind to human FLT3L expressed on the surface of a transduced CHO cell line. Figure 5 B demonstrates lead antibody binding to cell surface cynomolgus monkey FLT3L. Figure 5 C demonstrates lead antibody binding to cell surface mouse FLT3L.

[0033] Figure 6 AC. Lead candidates that bind to endogenous human FLT3L. Figure 6 A demonstrates the expression of FLT3L on human primary T cells after 7 days of stimulation with IL-2. Figure 6 B demonstrates the ability of all lead candidates, except clone 5D9, to bind to endogenous FLT3L on human primary T cells. Figure 6 C demonstrates that improvement in the avidity of CAT5D9 by dimerization enables dose-dependent binding of the clones to endogenous FLT3L on the surface of T cells prior to incubation with primary T cells.

[0034] Figure 7 A and 7B. Inhibition of cell surface signaling by lead candidates. Figure 7 A demonstrates the dose response curve of RS4;11 cells to CHO cells expressing FLT3L, with 1000 CHO / well determined to be the optimal number to induce 80% downregulation of FLT3 on the surface of RS4;11 cells. Figure 7 B demonstrates that all lead candidates have the ability to inhibit cell surface FLT3L on CHO cells to some extent.

[0035] Figure 8 A and 8B. Activation and neutralization of the ERK signaling pathway. Figure 8 A demonstrates a proof-of-concept study showing activation of ERK signaling in RS4;11 cells by FLT3L. Figure 8 B demonstrates inhibition of FLT3L-induced ERK activation by commercially available antibodies.

[0036] Fig. 9 A and 9B. Blockade of MEK 1 / 2 and ERK downstream signaling by lead candidates. Fig. 9 A Demonstration of functional activity of lead clone candidates against human FLT3L-induced MEK 1 / 2 phosphorylation in primary CD133+ human stem cells. Fig. 9 B Further confirmation of the functional activity of the lead clone against FLT3L-induced signaling in primary CD133+ human stem cells using ERK phosphorylation as a readout.

[0037] Fig.10 A and 10B. Target specificity and binding kinetics of lead candidates. Fig.10 A shows the Phase III pairing for the lead clonal candidate, clone 5D9, and FLT3-Fc. Fig.10 B shows the Phase III pairings of all lead clones against CAT8 and FLT3-Fc as representative of all clones except CAT5D9.

[0038] Fig.11 A and 11B. Cross-reactivity of lead candidates with huSCF and huCSF. Fig.11 A demonstrates that the lead candidate does not bind to the structural homolog huSCF. Fig.11 B demonstrates that the lead candidate does not bind to the structural homolog huCSF.

[0039] Fig.12 A and 12B. Cloning optimization. Fig.12 A demonstrates the results of the first round of clonal optimization comparing parental CAT5D9 to clone 6 (C06) using the RS4;11 FLT3 downregulation assay as readout. Fig.12 B demonstrates the results of the second round of clonal optimization comparing parental CAT5D9 with clone 6 (C06) and the final lead candidates: AM40 and SC4017.

[0040] Fig.13 A and 13B. Efficient neutralization of endogenous cell surface FLT3L. Fig.13 A shows RS4:11 FLT3 downregulation in response to serial dilutions of FLT3L-expressing CD4+ T cells. Fig.13B shows that the lead clone completely neutralizes active cell surface FLT3L on CD4+ T cells.

[0041] Fig.14 . Study outline for neutralizing FLT3L in healthy cynomolgus monkeys. As indicated, 0.03, 1, or 30 mg / kg AM40 (MEDI1116) was administered to three groups of male cynomolgus monkeys (n=4 / group) in five doses per week over a month. An eight-week follow-up period after the final dose was used to determine the persistence of the administered antibody and its effect on the circulating DC population.

[0042] Fig.15 A and 15B. Serum FLT3L protein and circulating DC frequency after administration of anti-FLT3L antibody (AM40 / MEDI1116). Fig.15 A shows target engagement of MEDI1116, measuring serum free FLT3L levels after administration at .03, 1.0 and 30 mg / kg. Daily serum measurements were performed from days 1-8 and weekly thereafter until day 85. Fig.15 B depicts the reduction and recovery of circulating CD1c+ (classical DC) frequency (left) and plasmacytoid DC frequency (right) after treatment with MEDI1116, measured as a percentage of baseline.

[0043] Fig.16 A-16D. Comparison of serum measurements of T cells and flow cytometric analysis to measure the correlation between FLT3L expression and SLEDAI scores in SLE patients. Fig.16 A depicts FLT3L levels in sera of healthy donors (HD) and SLE patients. Fig.16 B depicts the correlation between serum FLT3L SLEDAI scores. Fig.16 C depicts the frequency of circulating FLT3L+ T cells in healthy donors (HD) and SLE patients. Fig.16 D depicts the correlation between FLT3L+CD4+ T cells and SLEDAI score.

[0044] Fig.17 A-17C. FLT3L expression and SLEDAI score in CD4+ T cell subsets. Fig.17 A depicts CD4 T cells expressing FLT3L in HD and SLE patients. naive The bottom panel depicts the percentage of CD4 T cells expressing FLT3L in SLE patients. naive Correlation of cells with SLEDAI scores. Fig.17 B depicts CD4 T cells expressing FLT3L in HD and SLE patients. MEM The bottom panel depicts the percentage of CD4 T cells expressing FLT3L in SLE patients.MEM Correlation of cells with SLEDAI scores. Fig.17 C depicts CD4 T cells expressing FLT3L in HD and SLE patients. CM The bottom panel depicts the percentage of CD4 T cells expressing FLT3L in SLE patients. naive Correlation of cells with SLEDAI scores.

[0045] Fig.18 A-18C. FLT3L expression in PBMC CD4 subsets from individuals with myositis. Fig.18 A depicts FLT3L-expressing CD4 T cells in HD and myositis patients. naive The percentage of cells. Fig.18 B depicts CD4 T cells expressing FLT3L in HD and myositis patients. MEM The percentage of cells. Fig.18 C depicts FLT3L-expressing CD4 T cells in HD and myositis patients. CM The percentage of cells.

[0046] Fig.19 A-19B. Proteinuria and nephritis scores in MRL mice. Fig.19 A depicts the reduction in proteinuria at 17 weeks after anti-FLT3L administration. Fig.19 B depicts the nephritis score at 18 weeks after anti-FLT3L administration.

[0047] Fig. 20 A-20C. Splenic dendritic populations of MRL mice. Fig. 20 A depicts the changes in the frequency of CD11+siglec-H+pDCs after administration of anti-FLT3L antibody. Fig. 20 B depicts the frequency of CD11c+CD11b+mDCs after administration of anti-FLT3L antibody. Fig. 20 C depicts the frequency of CD11c+CD8+mDCs after administration of anti-FLT3L antibody.

[0048] Fig.21 A and 21B. Salivary gland pathology scoring in the NOD.H2h4 Sjögren's syndrome mouse model. Fig.21 A shows salivary gland pathology changes in the NOD.H2h4 Sjögren's syndrome mouse model following therapeutic dosing of anti-FLT3L antibody relative to isotype control. Fig.21 B shows salivary gland pathology changes in the NOD.H2h4 Sjögren's syndrome mouse model following a prophylactic dose of anti-FLT3L antibody relative to an isotype control.

[0049] Fig. 22A-22D. Changes in the presence of dendritic cells after administration of anti-FLT3L antibodies. Fig. 22 A and 22B depict changes in the frequency of plasmacytoid DCs (B220+CD11c+Siglec-H+) after administration of anti-FLT3L antibodies, visualized (A) and quantified (B) by flow cytometry. Fig. 22 C and 22D depict the classical DC frequency (B220 neg CD11c HI ) were visualized (C) and quantified (D) by flow cytometry.

[0050] Fig.23 A-23C. Anti-FLT3L antibody (MEDI1116) PK in cynomolgus monkeys correlates with functional neutralization of FLT3L, as demonstrated by inhibition and return of pDCs. Anti-FLT3L antibody (MEDI1116) was administered to cynomolgus monkeys once a week on days 1, 8, 15, 22, and 29, as indicated by arrows. Fig.23 A depicts anti-FLT3L antibody (MEDI1116) PK. Fig.23 B depicts soluble FLT3L levels at doses of 0.03 mg / kg, 1.0 mg / kg, and 30 mg / kg of anti-FLT3L antibody (MEDI1116). Fig.23 C depicts pDC frequency measured as a percentage of baseline at doses of 0.03 mg / kg, 1.0 mg / kg, and 30 mg / kg of anti-FLT3L antibody (MEDI1116).

[0051] Fig.24 A-24C. Human dosing model of anti-FLT3L antibody (MEDI1116) Q4W dosing schedule. Fig.24 A depicts anti-FLT3L antibody (MEDI1116) PK in cynomolgus monkeys. Fig.24 B depicts anti-FLT3L antibody (MEDI1116) PD in cynomolgus monkeys. Fig.24 C depicts the predicted PD of anti-FLT3L antibody (MEDI1116) in humans.

[0052] Fig.25 A-25B. Anti-FLT3L monoclonal antibody (LFC-1) effectively neutralizes FLT3L throughout treatment and leads to accumulation of circulating drug / ligand complexes. Fig.25 A depicts serum free FLT3L levels after administration of anti-FLT3L antibodies and isotype control antibodies. Free FLT3L levels were measured using FT3L-IgG as a capture reagent and sulfo-labeled anti-mouse FLT3L polyclonal antibody as a detection reagent. Fig.25B depicts serum total (free and bound) FLT3L levels after administration of anti-FLT3L antibodies and isotype control antibodies.Total FLT3L levels were measured using a polyclonal anti-mouse FLT3L antibody for capture and detection.

[0053] Fig.26 Blockade of A-26D.FLT3L inhibits NOD-H2 in the elderly h4 T cell activation in the spleen and SG-draining lymph nodes (LN) of mice. Blockade of FLT3L with an anti-FLT3L monoclonal antibody (LFC-1) resulted in antigen-experienced CD44 T cells in the spleen and salivary gland-draining LNs (24-26 weeks of age at the end of the study). HI Reduction in CD4+ and CD8+ T cell frequencies. Bar graphs are derived from flow cytometric analysis of spleens and draining LNs. Each bar represents the mean + / - standard error of the mean (SEM) of n=4-5 mice. Fig.26 A depicts CD44 in spleen after administration of anti-FLT3L antibody and isotype control antibody. HI CD4+ T cell population. Fig.26 B depicts CD44 in LNs after administration of anti-FLT3L antibody and isotype control antibody. HI CD4+ T cell population. Fig.26 C depicts CD44 in spleen after administration of anti-FLT3L antibody and isotype control antibody. HI CD8+ T cell population. Fig.26 D depicts CD44 in LNs after administration of anti-FLT3L antibody and isotype control antibody. HI CD8+ T cell population.

[0054] Fig. 27 Therapeutic anti-FLT3L blockade selectively reduces the specificity of two serum IgG autoantibodies. The corresponding serum samples were measured by the UTSW IgG autoantibody assay. DETAILED DESCRIPTION

[0055] The present invention provides an antibody or antigen binding fragment thereof that is separated and specifically binds 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 inhibit the activation of inflammatory signaling pathways. Such a method is advantageous because it attacks inflammation at the signal source, thereby allowing a more powerful anti-inflammatory therapeutic effect. Related polynucleotides, vectors, and pharmaceutical compositions comprising anti-FLT3L antibodies or antigen binding fragments thereof are also provided. It is also contemplated that methods for preparing and using the anti-FLT3L antibodies and antigen binding fragments disclosed herein, for example, methods for treating autoimmune diseases and / or chronic inflammatory diseases in subjects (used as direct therapy, adjuvant therapy, or in combination therapy).

[0056] In order to make this disclosure more easily understood, certain terms are first defined. Other definitions are set forth throughout the detailed description.

[0057] definition

[0058] Before describing the present invention in detail, it should be understood that the present invention is not limited to specific compositions or method steps, because these compositions or method steps can vary. Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by technicians in the field of the present disclosure. The following references provide general definitions of multiple terms used in the present disclosure for technicians: Singleton et al., Dictionary of Microbiology and Molecular Biology [Microbiology and Molecular Biology Dictionary] (2nd Edition, 1994); The Cambridge Dictionary of Science and Technology [Cambridge Dictionary of Science and Technology] (Walker Edited, 1988); The Glossary of Genetics [Genetics Vocabulary], 5th Edition, R. Rieger et al. (Editors), Springer Verlag (Springer Press) (1991); and Hale & Marham, The Harper Collins Dictionary of Biology [Harper Collins Biology Dictionary] (1991). As used herein, unless otherwise indicated, the following terms have the meanings assigned to them.

[0059] As used in the present disclosure, the term "antibody" (or its fragment, variant or derivative) refers to at least the smallest portion of an antibody that is capable of binding to an antigen, for example, in the case of a typical antibody produced by a B cell, at least the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL). The basic antibody structure in vertebrate systems is relatively well understood. See, for example, Harlow et al., Antibodies: A Laboratory Manual [Antibodies: Laboratory Manual], (Cold Spring Harbor Laboratory Press, 2nd edition, 1988). Antibodies or their antigen-binding fragments, variants or derivatives include, but are not limited to, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, or chimeric antibodies, epitope binding fragments such as Fab, F(ab')2, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments (Fd) comprising VL or VH domains, fragments produced by Fab expression libraries, and other antibody fragments and combinations thereof that retain antigen binding function (i.e., the ability to specifically bind to, for example, FLT3L).

[0060] A typical antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH or VL). H ) and a heavy chain constant region. The heavy chain constant region comprises three domains, namely CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL or V L ) and a light chain constant region. The light chain constant region contains one domain, namely CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FWs). Each VH and VL contains three CDRs and four FWs, arranged in the following order from the amino terminus to the carboxyl terminus: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of these antibodies can mediate the binding of immunoglobulins to host tissues or factors, including different cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Exemplary antibodies disclosed herein include anti-FLT3L antibodies (original antibodies and germlined antibodies), affinity-optimized clones, optimized antibodies lacking ADCC, conjugated antibodies (e.g., ADC), and other optimized antibodies (e.g., serum half-life optimized antibodies, including, e.g., YTE mutations, see Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006) and U.S. Pat. No. 7,083,784, which are hereby incorporated by reference in their entireties).

[0061] In certain embodiments, the CDRs (HCDR1, HCDR2, and HCDR3) of VH and the CDRs (LCDR1, LCDR2, and LCDR3) of VL consist of the following amino acid sequences: (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.

[0062] Based on the identity of the constant domain of the antibody heavy chain, which is called α, δ, ε, γ and μ, respectively, the antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or a subclass (isotype) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules (e.g., toxins, radioisotopes, etc.) to form ADCs.

[0063] "Blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it is bound, such as FLT3L. In one aspect, a blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. For example, the activation of FLT3 mediated by FLT3L can be 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 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%.

[0064] The term "FLT3L antibody", "antibody that binds to FLT3L", or "anti-FLT3L antibody" refers to an antibody or antigen-binding fragment thereof that is capable of binding to FLT3L with sufficient affinity such that the molecule can be used as a therapeutic or diagnostic agent targeting FLT3L. In a broad sense, the term "anti-FLT3L" also encompasses molecules comprising the CDRs of the antibodies disclosed herein, for example, incorporated into a scaffold.

[0065] The term "germlining" means that the amino acid at a particular position in an antibody is mutated back to the amino acid in the germline.

[0066] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chains are each composed of four FW regions connected by three CDR regions. The CDRs in each chain are held together in close proximity by the FW regions and contribute to the formation of the antigen binding site of the antibody with the CDRs from the other chain. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest [Protein sequences of interest in immunology], (5th edition, 1991, National Institutes of Health, Bethesda, Maryland)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. [Journal of Molecular Biology] 273: 927-948)). In addition, a combination of these two methods is sometimes used in the art to determine CDRs.

[0067] The Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Maryland, (1991)) is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain).

[0068] The phrases "amino acid position numbering as in Kabat" or "Kabat position" and the like refer to the numbering system of Kabat et al., 1991 for heavy chain variable domains or light chain variable domains used for the compilation of antibodies.

[0069] The terms "antigen binding domain", "antigen binding fragment" and "binding fragment" refer to a portion of an antibody molecule that contains amino acids responsible for the specific binding between the antibody and the antigen. The variable region allows the antibody or antigen binding fragment to selectively recognize and specifically bind to an epitope on the antigen. That is, a subset of the VH and VL domains or complementary determining regions (CDRs) of an antibody combines to form a variable region that defines a three-dimensional antigen binding site. More specifically, an antigen binding domain is defined by three CDRs on each VH and VL chain. As used herein, a portion of an antigen molecule that is responsible for specific interaction with an antigen binding domain is referred to as an "epitope". An antigen binding domain typically comprises an antibody light chain variable region and an antibody heavy chain variable region, however, it is not necessary to necessarily include both. For example, the so-called "Fd" antibody fragment consists only of the VH domain, but still retains some antigen binding function of the intact antibody.

[0070] By recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies, the binding fragment of the antibody is produced. Binding fragments include Fab, Fab', F(ab')2, Fv and single-chain antibodies. Digestion of antibodies with papain produces two identical antigen-binding fragments (also referred to as "Fab" fragments) and an "Fc" fragment, which have no antigen-binding activity but have the ability to crystallize. Digestion of antibodies with pepsin produces a F(ab')2 fragment, in which the two arms of the antibody molecule remain connected and contain two antigen-binding sites. The F(ab')2 fragment has the ability to cross-link antigens. When used herein, "Fv" refers to the smallest fragment of an antibody that retains both an antigen recognition site and an antigen-binding site. When used herein, "Fab" refers to a fragment of an antibody that comprises the constant domains of a light chain and the CH1 domains of a heavy chain.

[0071] As used herein, "Fc region" includes polypeptides excluding the constant region of antibodies outside the first constant region immunoglobulin domain. Therefore, 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, and the flexible hinge at the N-terminus of these domains. For IgA and IgM, Fc can include J chain. For IgG, Fc includes immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and hinges between Cgammal (Cyl) and Cgamma2 (Cy2). Although the boundaries of the Fc region can be changed, the human IgG heavy chain Fc region is generally defined as including residues C226 or P230 at its carboxyl terminus, wherein numbering is performed according to the EU index as shown in Kabat et al., 1991.

[0072] "Monoclonal antibody" refers to a homogeneous antibody population that is capable of highly specific recognition and binding to a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants.

[0073] The term "monoclonal antibody" encompasses both intact monoclonal antibodies and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain variable fragments (scFv), fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising an antigen recognition site. In addition, "monoclonal antibody" refers to such antibodies prepared in a number of ways, including but not limited to preparation by hybridoma, phage selection, recombinant expression, and transgenic animals (e.g., expression of human antibodies in transgenic mice).

[0074] The term "humanized antibody" refers to an antibody derived from a non-human (eg, murine) immunoglobulin that has been engineered to increase similarity to antibody variants produced in humans.

[0075] The term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human using any technology known in the art (e.g., recombinant expression in cultured cells or expression in transgenic animals). Therefore, the term human antibody also encompasses antibodies having an amino acid sequence corresponding to an antibody originally produced by a human (or its engineered variant or derivative) but expressed in a non-human system (e.g., produced by chemical synthesis; recombinant expression in a microorganism, mammal or insect cell; or expressed in an animal subject). Accordingly, antibodies obtained from a human subject or from a human cell (e.g., a hybridoma or cell line expressing a recombinant antibody or its fragment) and subsequently expressed in an animal (e.g., a mouse) are considered to be human antibodies. This definition of a human antibody includes complete antibodies or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy chain and / or light chain polypeptide, such as antibodies comprising a mouse light chain and a human heavy chain polypeptide.

[0076] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more animal species. Typically, the variable regions of 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, and / or affinity, and / or capacity, while the constant regions are homologous to sequences in antibodies derived from another species (usually humans) to avoid eliciting an immune response in that species.

[0077] The term "polynucleotide" is intended to encompass singular nucleic acids as well as plural 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 unconventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA or RNA fragments. An "isolated" nucleic acid or polynucleotide means a nucleic acid molecule, i.e., DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide subunit contained in a vector is considered to be isolated, as disclosed herein. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in a solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. In addition, a polynucleotide or nucleic acid may be or may include a regulatory element such as a promoter, a ribosome binding site, or a transcription terminator.

[0078] In certain embodiments, polynucleotide or nucleic acid is DNA. In the case of DNA, the polynucleotide comprising the nucleic acid encoding the polypeptide can generally include a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. Operable association or connection is to associate with one or more regulatory sequences in the following manner for the coding region of a gene product (e.g., a polypeptide), which makes the expression of the gene product under the influence or control of this or these regulatory sequences. If the induction of promoter function leads to the transcription of the mRNA encoding the desired gene product, and if the nature of the connection between the two DNA fragments does not interfere with the ability of the expression of the gene product guided by the expression of the regulatory sequence or the ability of the DNA template to be transcribed, then the two DNA fragments (such as the polypeptide coding region and the promoter associated therewith) are "operably associated" or "operably connected". Therefore, the promoter region will be operably associated with the nucleic acid encoding the polypeptide, as long as the promoter can achieve the transcription of the nucleic acid. The promoter can be a cell-specific promoter that guides the substantial transcription of DNA only in a predetermined cell. In addition to promoters, other transcription control elements such as enhancers, operators, repressors, and transcription termination signals may be operably associated with the polynucleotide in order to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

[0079] In other embodiments, the polynucleotide may be RNA, for example in the form of messenger RNA (mRNA).

[0080] A "vector" is a nucleic acid molecule introduced into a host cell to produce a transformed host cell. A vector may include a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.

[0081] A "transformed" cell or "host" cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. The transformed cell or host cell can be a bacterial cell or a eukaryotic cell.

[0082] As used herein, the term "FLT3L" refers to McDonald's feline sarcoma (FMS)-like tyrosine kinase 3 ligand, which is a polypeptide of a hematopoietic cytokine that binds to an 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) are included in the definition of FLT3L.

[0083] In the present disclosure, “comprise,” “comprising,” “containing,” and “having,” etc. may have the meanings ascribed to them in U.S. patent law and may mean “include,” “including,” etc.; “consisting essentially of or consists essentially of” likewise has the meaning assigned to it in U.S. patent law and the term is open ended, allowing for existence beyond what is recited as long as the basic or novel features recited are not altered by existence beyond what is recited, but excluding prior art embodiments.

[0084] As used herein, the terms "determining," "assessing," "assaying," "measuring," and "detecting" refer to both quantitative and qualitative determinations, and thus, the term "determining" is used interchangeably herein with "determining," "measuring," and the like. Where a quantitative determination is required, the phrase "determining the amount of" an analyte, etc. may be used. Where a qualitative and / or quantitative determination is required, the phrase "determining the level of" an analyte or "detecting" an analyte is used.

[0085] In the case of two or more nucleic acids or polypeptides, the term "identity" or "percent identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acid residues when compared and aligned (introducing gaps if necessary) for maximum correspondence and not considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of 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 by Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877) and are incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In certain embodiments, Gapped BLAST, BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, CA), or Megalign (DNASTAR) can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.

[0086] 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 an antibody that is not produced or located in its natural or natural environment. Recombinantly produced biological materials are considered isolated, as disclosed herein, and materials produced in non-natural cells (such as hybridomas) are also considered isolated. If a substance (e.g., an isolated protein such as an antibody) has been separated, fractionated, or partially or substantially purified by any suitable technique, the substance is also considered to be "isolated." For example, if an antibody is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived, the antibody is considered to be "isolated."

[0087] The term "specific binding" refers to the recognition and binding of an agent (e.g., a ligand or antibody) to a molecule (e.g., a receptor or epitope), and such binding requires some complementarity between the agent (e.g., antibody) and the molecule (e.g., ligand). By this definition, an antibody is said to "specifically bind" to a ligand when it binds to the ligand more readily than it binds to a random, unrelated molecule. The term "specificity" is used herein to characterize the relative affinity of an antibody for binding to a ligand. For example, antibody "A" may be considered to have a higher specificity for a given ligand (e.g., FLT3L) than antibody "B".

[0088] As used herein, the term "affinity" refers to a measure of the binding strength of a single epitope to the CDR of an antibody. See, e.g., Harlow et al., Antibodies: A Laboratory Manual [antibodies: Laboratory Manual], (Cold Spring Harbor Laboratory Press, 2nd edition, 1988), pp. 27-28. As used herein, the term "affinity" refers to the overall stability of the complex between an antibody population and an antigen, i.e., the functional combination strength of an antibody mixture and an antigen. See, e.g., Harlow, pp. 39-34. Avidity is related to the affinity of a single antibody in a population to a specific epitope, and is also related to the titer of the antibody and antigen.

[0089] The terms "inhibit" or "block" are used interchangeably herein and refer to any statistically significant reduction in biological activity, including complete blocking of activity. For example, "inhibit" can refer to a reduction in biological activity of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0090] The term "effector function" refers to the activity of an antibody produced by the interaction of its Fc component with an Fc receptor or complement component. These activities include, for example, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). Therefore, an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) with a changed effector function refers to an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprising a change (e.g., an amino acid substitution, a deletion or an addition, or a change in an oligosaccharide) in the Fc region, which changes the activity (e.g., ADCC, CDC, and / or ADCP) of at least one effector function. An antigen-binding protein (e.g., an antibody or its antigen-binding fragment) with an improved effector function refers to an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprising a change (e.g., an amino acid substitution, a deletion or an addition, or a change in an oligosaccharide) in the Fc region, which changes the activity (e.g., ADCC, CDC, and / or ADCP) of at least one effector function.

[0091] The term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that will be the recipient of a particular treatment. Typically, the terms "subject" and "patient" and "individual" are used interchangeably herein. Other examples of subjects include non-human mammals, such as cows, horses, dogs, sheep, or cats.

[0092] The term "pharmaceutical composition" refers to a preparation in a form such that the biological activity of the active ingredient (e.g., an anti-FLT3L antibody disclosed herein) is effective and does not include other components that are unacceptably toxic to a subject to which the composition is administered. Such compositions can be sterile.

[0093] An "effective amount" of an anti-FLT3L antibody as disclosed herein is an amount sufficient to achieve a particular purpose. An "effective amount" can be determined empirically and in a routine manner relative to the stated purpose.

[0094] The terms "therapeutically effective amount" and "pharmaceutically effective amount" refer to an amount of an anti-FLT3L antibody or other drug disclosed herein that is effective to "treat" a disease or disorder in a subject.

[0095] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to (1) therapeutic measures that cure, slow, alleviate the symptoms of a diagnosed pathological condition or disorder, and / or stop the progression of a diagnosed pathological condition or disorder; and (2) prophylactic or preventative measures that prevent and / or slow the development of a targeted pathological condition or disorder. Thus, subjects in need of treatment include: those subjects who already have the disorder; those subjects who are predisposed to have the disorder; and those subjects in whom the disorder is to be prevented. In certain aspects, a subject's autoimmune disease or inflammatory disease is successfully "treated" according to the methods of the present disclosure if the patient shows, for example, a total, partial, or temporary reduction in symptoms associated with the autoimmune disease or inflammatory disease.

[0096] The ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range of the following group, 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.

[0097] As used herein, the terms "treat, treating, treatment, etc." refer to reducing and / or ameliorating a disorder and / or symptoms associated therewith. It should be understood that, although not excluded, treating a disorder or condition does not require complete elimination of the disorder, condition, or symptoms associated therewith. For example, as contemplated herein, treating a disorder includes preventing the worsening of symptoms of the disorder.

[0098] As used herein, the term "or" should be understood as being inclusive, unless explicitly stated or obvious from the context to the contrary. As used herein, the terms "a, an," and "the" should be understood as being singular or plural, unless explicitly stated or obvious from the context to the contrary.

[0099] In addition, where used herein, "and / or" should be considered as a specific disclosure of each of two or more specified features or components with or without each other. Therefore, 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 cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; and A (alone); B (alone); and C (alone).

[0100] Unless expressly stated or apparent from the context, as used herein, the term "about" should be understood to be within the normal tolerance range in the art, for example, within 2 standard deviations of the mean. "About" can be understood to be 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% greater or less than the specified value. Unless otherwise stated, all numerical values ​​provided herein are deemed to be implicitly modified by the term "about".

[0101] FLT3L is initially expressed as a membrane-bound protein and then enzymatically cleaved into a soluble form. Both membrane-bound (mFLT3L) and secreted (sFLT3L) are functionally active. The FLT3L binding region is highly conserved between species, so that cross-species reactivity is observed between human, rodent and cynomolgus monkey ligand / receptor combinations. However, it is believed that key mutations around the binding site explain the lack of cross-species reactivity of neutralizing antibodies produced against FLT3L. Neutralizing antibodies against FLT3L can affect classical and plasmacytoid DC populations, thereby reducing the ability of the immune system to induce and maintain prolonged inflammatory responses. Secondary effects may include a decrease in circulating NK cells and a decrease in T cell and B cell activation, resulting in a decrease in the survival rate of both cell types. Overall, the downregulation of these pathways can reduce autoimmune inflammation.

[0102] In one embodiment, it is envisioned that neutralizing anti-FLT3L antibodies promote immune homeostasis by inhibiting the binding of FLT3L to FLT3. The anti-FLT3L antibody strategy targets the ligand rather than the receptor to avoid the risk of unexpected receptor dimerization or signaling. Unlike its receptor, there is no signaling domain associated with membrane-bound FLT3L.

[0103] Anti-FLT3L Antibodies

[0104] In a preferred embodiment, the present disclosure provides an isolated FLT3L binding molecule that specifically binds to FLT3L (e.g., human FLT3L), such as an antibody and an antigen-binding fragment thereof. The full-length amino acid sequence and nucleotide sequence of FLT3L are known in the art (see, for example, UniProt accession number P36888 of human FLT3L, or UniProt accession number Q00342 of mouse FLT3L). The anti-FLT3L antibodies disclosed herein inhibit the activation of FLT3 mediated by FLT3L, thereby reducing pro-inflammatory signaling in subjects and reducing inflammation.

[0105] In a preferred embodiment, the anti-FLT3L antibody does not cross-react with structurally similar TKR homologs 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. Non-specific FLT3 inhibitors that bind to other tyrosine kinase family members can cause toxicity by comprehensively inhibiting tyrosine kinase signaling. Accordingly, it is crucial that the anti-FLT3L antibody only binds to FLT3L and does not bind to structurally similar homologs. Many anti-FLT3L antibodies and inhibitors lack specificity and bind to a variety of tyrosine kinase receptors. Therefore, the preferred embodiment of the anti-FLT3L antibody must demonstrate high affinity for FLT3L and specific binding to FLT3L.

[0106] 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.

[0107] In some aspects, the FLT3L binding molecule comprises Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, IgG CH2, miniantibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb 2 , (scFv)2, or scFv-Fc. In some aspects, the anti-FLT3L antibody is of IgG type, for example, of IgG1 type (including IgG1 heavy chain immunoglobulin constant domain). In other embodiments, the anti-FLT3L antibody has IgA, IgD, IgE, IgG2, IgG3, IgG4 or IgM heavy chain immunoglobulin constant domain.

[0108] In some embodiments, the IgG constant region may comprise a light chain constant region selected from the group consisting of: an Igκ constant domain (region) and an Igλ constant domain. In a 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 such that targeted mutations in the Fc region change leucine at 243 to phenylalanine (L243F), leucine at 235 to glutamic acid (L235E), and proline at 331 to serine (P331S); amino acid numbering is performed according to the EU index. Targeted mutations reduce FcR binding and ADCC effector function (see Organesyan et al., ActaCrystallogrD BiolCrystallog r[Acta Crystallographica Sinica Series D: Biological Crystallography]. 2008 Jun 1;64(Pt 6):700-4; and WO2009100309 A2, which are incorporated by reference.

[0109] In some aspects, the anti-FLT3L antibody is a human antibody (eg, CAT5D9, SC4017, AM40, CAT8, CAT26, DTAX3, and DYAX5 antibodies).

[0110] CAT5D9 Antibody

[0111] In one embodiment, the CAT5D9 antibody refers to an antibody that specifically binds to FLT3L and includes the following complementary determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 include the amino acid sequences of SEQ ID NOs: 29, 36, 37, 32, 33, and 38, respectively.

[0112] In another embodiment, the CAT5D9 antibody refers to an antibody that specifically binds to FLT3L and comprises two VL domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:6 and two VH domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:5.

[0113] In yet another 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.

[0114] 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.

[0115] In one embodiment, the CAT5D9 antibody refers to an IgG1 antibody that specifically binds to FLT3L and includes a light chain having the amino acid sequence of SEQ ID NO:70 and a heavy chain having the amino acid sequence of SEQ ID NO:69.

[0116] In another embodiment, the CAT5D9 antibody refers to an antibody comprising a light chain encoded by the nucleic acid sequence of SEQ ID NO:72 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO:71.

[0117] SC4017 Antibody

[0118] In one embodiment, the SC4017 antibody refers to an antibody that specifically binds to FLT3L and includes the following complementary determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 include the amino acid sequences of SEQ ID NOs: 29, 30, 31, 35, 33, and 34, respectively.

[0119] In another embodiment, the SC4017 antibody refers to an antibody that specifically binds to FLT3L and comprises two VL domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:4 and two VH domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:3.

[0120] In yet another 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.

[0121] 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.

[0122] In one embodiment, the SC4017 antibody is an IgG1 antibody that specifically binds to FLT3L and includes a light chain having the amino acid sequence of SEQ ID NO:66 and a heavy chain having the amino acid sequence of SEQ ID NO:65.

[0123] In another embodiment, the SC4017 antibody refers to an antibody comprising a light chain encoded by the nucleic acid sequence of SEQ ID NO:68 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO:67.

[0124] AM40(MEDI1116) Antibody

[0125] In one embodiment, the AM40 antibody refers to an antibody that specifically binds to FLT3L and includes the following complementary determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 include the amino acid sequences of SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively.

[0126] In another embodiment, the AM40 antibody refers to an antibody that specifically binds to FLT3L and includes two VL domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:2 and two VH domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0127] In yet another 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.

[0128] 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.

[0129] In one embodiment, the AM40 antibody refers to an IgG1 antibody that specifically binds to FLT3L and includes a light chain having the amino acid sequence of SEQ ID NO:62 and a heavy chain having the amino acid sequence of SEQ ID NO:61.

[0130] In another embodiment, the AM40 antibody refers to an antibody comprising a light chain encoded by the nucleic acid sequence of SEQ ID NO:64 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO:63.

[0131] CAT8 Antibody

[0132] In one embodiment, the CAT8 antibody refers to an antibody that specifically binds to FLT3L and includes the following complementary determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 include the amino acid sequences of SEQ ID NOs: 39, 40, 41, 42, 43, and 44, respectively.

[0133] In another embodiment, the CAT8 antibody refers to an antibody that specifically binds to FLT3L and comprises two VL domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:8 and two VH domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:7.

[0134] In yet another embodiment, the 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.

[0135] In another embodiment, the 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.

[0136] In one embodiment, the CAT8 antibody refers to an IgG1 antibody that specifically binds to FLT3L and includes a light chain having the amino acid sequence of SEQ ID NO: 74 and a heavy chain having the amino acid sequence of SEQ ID NO: 73.

[0137] In another embodiment, the CAT8 antibody refers to an antibody comprising a light chain encoded by the nucleic acid sequence of SEQ ID NO:76 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO:75.

[0138] CAT26 Antibody

[0139] In one embodiment, the CAT26 antibody refers to an antibody that specifically binds to FLT3L and includes the following complementary determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 include the amino acid sequences of SEQ ID NOs: 45, 40, 46, 47, 48, and 49, respectively.

[0140] In another embodiment, the CAT26 antibody refers to an antibody that specifically binds to FLT3L and comprises two VL domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:10 and two VH domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:9.

[0141] In yet another 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.

[0142] 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.

[0143] In one embodiment, the CAT26 antibody refers to an IgG1 antibody that specifically binds to FLT3L and includes a light chain having the amino acid sequence of SEQ ID NO:78 and a heavy chain having the amino acid sequence of SEQ ID NO:77.

[0144] In another embodiment, the CAT26 antibody refers to an antibody comprising a light chain encoded by the nucleic acid sequence of SEQ ID NO:80 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO:79.

[0145] DYAX3 Antibody

[0146] In one embodiment, the Dyax3 antibody refers to an antibody that specifically binds to FLT3L and includes the following complementary determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 include the amino acid sequences of SEQ ID NOs: 50, 51, 52, 53, 54, and 55, respectively.

[0147] In another embodiment, the Dyax3 antibody refers to an antibody that specifically binds to FLT3L and comprises two VL domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:82 and two VH domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:81.

[0148] In yet another embodiment, the 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.

[0149] In another embodiment, the 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.

[0150] In one embodiment, the Dyax3 antibody refers to an IgG1 antibody that specifically binds to FLT3L and includes a light chain having the amino acid sequence of SEQ ID NO:82 and a heavy chain having the amino acid sequence of SEQ ID NO:81.

[0151] In another embodiment, the Dyax3 antibody refers to an antibody comprising a light chain encoded by the nucleic acid sequence of SEQ ID NO:84 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO:83.

[0152] DYAX5 Antibody

[0153] In one embodiment, the Dyax5 antibody refers to an antibody that specifically binds to FLT3L and includes the following complementary determining regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 include the amino acid sequences of SEQ ID NOs: 56, 57, 52, 58, 59, and 60, respectively.

[0154] In another embodiment, the Dyax5 antibody refers to an antibody that specifically binds to FLT3L and comprises two VL domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:86 and two VH domains that have at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:85.

[0155] In yet another embodiment, the 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.

[0156] In another embodiment, the 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.

[0157] In one embodiment, the Dyax5 antibody refers to an IgG1 antibody that specifically binds to FLT3L and includes a light chain having the amino acid sequence of SEQ ID NO:86 and a heavy chain having the amino acid sequence of SEQ ID NO:85.

[0158] In another embodiment, the Dyax5 antibody refers to an antibody comprising a light chain encoded by the nucleic acid sequence of SEQ ID NO:88 and a heavy chain encoded by the nucleic acid sequence of SEQ ID NO:87.

[0159] In certain embodiments, an antibody or antigen-binding fragment thereof that specifically binds to FLT3L is provided, which 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 following amino acid sequences: (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.

[0160] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein each VH and VL comprises three CDRs and four framework regions (FWs), arranged from amino terminus to carboxyl terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3 and FW4.

[0161] In certain aspects, the VH and VL regions 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.

[0162] In certain aspects, the CDRs (HCDR1, HCDR2 and HCDR3) of VH and the CDRs (LCDR1, LCDR2 and LCDR3) of VL consist of the following amino acid sequences: (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.

[0163] A summary table of anti-FLT3L antibody sequences is presented below in Table 1.

[0164] Table 1. Summary of anti-FLT3L antibody sequences.

[0165] Table 1A

[0166]

[0167]

[0168] Table 1B

[0169]

[0170]

[0171]

[0172]

[0173] Table 1C

[0174]

[0175]

[0176] Table 1D

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185] derivative

[0186] The anti-FLT3L antibodies disclosed herein may include variants of the sequences provided, which retain the ability to specifically bind to FLT3L. Such variants may be derived from the sequence of the antibody by a technician using techniques well known in the art. For example, amino acid substitutions, deletions or additions may be made in the FR region and / or CDR of the anti-FLT3L antibody that does prevent the antibody from binding to its epitope. Although the changes in FR are usually designed to improve the stability and immunogenicity of the antigen-binding domain, typically, the changes in CDR are designed to increase the affinity of the antigen-binding domain to its target. Variants of FR also include naturally occurring immunoglobulin isotypes. This change in affinity can be empirically determined by conventional techniques, which involve changing CDRs and testing the affinity of the antigen-binding domain to its target. For example, conservative amino acid substitutions may be made in any of the disclosed CDRs. Various changes may be made according to the method described in Antibody Engineering, 2nd edition, Oxford University Press, editor Borrebaeck, 1995. These changes include, but are not limited to, altering the nucleotide sequence by substituting different codons for functionally equivalent amino acid residues within the coding sequence, thereby producing a "silent" change. For example, non-polar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0187] Derivatives and analogs of the antibodies disclosed herein can be produced by a variety of techniques well 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, New York, and Bodansky et al. (1995) The Practice of Peptide Synthesis, 2nd ed., Spring Verlag, Berlin, Germany).

[0188] In one embodiment, the method for preparing a VH domain (which is an amino acid sequence variant of a VH domain disclosed herein) comprises the following steps: adding, deleting, substituting or inserting one or more amino acids in the amino acid sequence of a VH domain disclosed herein, optionally combining the VH domain thus provided with one or more VL domains, and testing the specific binding of the VH domain or one or more combinations of VH / VL to an antigen. A similar method can be used, wherein one or more sequence variants of a VL domain disclosed herein are combined with one or more VH domains.

[0189] Similar shuffling or combinatorial techniques were also disclosed by Stemmer (Nature (1994) 370:389-391), who described the technique in relation to β-lactamase genes but observed that the approach could be used to generate antibodies.

[0190] In further embodiments, random mutagenesis of one or more selected VH and / or VL genes can be used to generate novel VH or VL regions carrying one or more sequences derived from the sequences disclosed herein. Gram et al. (Proc. Nat. Acad. Sci. USA (1992) 89: 3576-3580) describe one such technique, error-prone PCR.

[0191] Another approach that can be used is to direct mutagenesis to the CDRs of the VH or VL genes. 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).

[0192] Similarly, one, two, or all three CDRs of the antigen binding domain can be grafted into a repertoire of VH or VL domains and then screened for antigen binding fragments that are specific for FLT3L.

[0193] A portion of the immunoglobulin variable domain that can be used herein may include at least one of the CDRs listed herein, and optionally an intervening framework region from a scFv fragment as listed herein. The portion may include at least about 50% of one or both of FR1 and FR4, the 50% being the C-terminal 50% of FR1 and the N-terminal 50% of FR4. The additional residues at the N-terminal or C-terminal end of the basic portion of the variable domain may be those residues that are not usually associated with the naturally occurring variable domain region. For example, constructing antibodies by recombinant DNA technology may result in the introduction of N- or C-terminal residues encoded by the introduced linker to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of a linker to connect the variable domain to other protein sequences, including immunoglobulin heavy chain constant regions, other variable domains (e.g., in the production of diabodies), or protein tags, as discussed in further detail below.

[0194] The antigen binding domains of the present disclosure described herein can be connected to another functional molecule, such as another peptide or protein (albumin, another antibody, etc.). For example, the antigen binding domain can be connected by chemical cross-linking or by recombinant methods. The antigen binding domain can also be connected to one of a variety of non-protein polymers in a manner listed in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337, such as polyethylene glycol, polypropylene glycol or polyoxyalkylene. The antigen binding domain can be chemically modified by covalent conjugation with a polymer, for example, to increase its circulation half-life. Exemplary polymers and methods of attachment thereof are also shown in U.S. Patent Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546.

[0195] The disclosed antibodies can also be changed to have a glycosylation pattern different from the natural pattern. For example, one or more carbohydrate moieties can be deleted and / or one or more glycosylation sites can be added. Adding glycosylation sites to the currently disclosed antibody fragments can be achieved by changing the amino acid sequence to include a glycosylation site consensus sequence known in the art. Another means of increasing the number of carbohydrate moieties on the 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. [CRC Biochemistry Key Review], 22: 259-306. Removal of any carbohydrate moieties from the antibody can be accomplished chemically or enzymatically, for example, as described by Hakimuddin et al. (1987) Arch. Biochem. Biophys., 259:52; and 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 label or functional marker. Detectable labels include radioactive labels, such as 131I or 99Tc, which can also be attached to the antibody fragment 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 (e.g., labeled avidin).

[0196] CDR sequences are only substantially different from those described herein in the antigen-binding domains within the scope of the present disclosure. Typically, amino acids are replaced by related amino acids with similar charge, hydrophobicity or stereochemical characteristics. Such substitutions are within the technical scope of ordinary technicians. Unlike CDR, more substantial changes can be made in FR without adversely affecting the binding properties of antibodies. Changes in FR include, but are not limited to, certain framework residues derived from or engineered by humanization non-humans (these framework residues are important for antigen contact or for stabilizing binding sites), for example, changing the class or subclass of the constant region, changing specific amino acid residues that may change effector function (such as Fc receptor binding), for example, as described in U.S. Patent Nos. 5,624,821 and 5,648,260 and Lund et al. (1991) J.Immun. [Journal of Immunology] 147: 2657-2662 and Morgan et al. (1995) Immunology [Immunology] 86: 319-324, or changing the type of derived constant region.

[0197] Those skilled in the art will appreciate that the above modifications are not exhaustive and may be applied to the protein subunits described herein, and that numerous other modifications may be made by the skilled artisan based on the teachings of the present disclosure.

[0198] Affinity and specificity of anti-FLT3L antibodies

[0199] Those skilled in the art will recognize that anti-FLT3L antibodies for use in autoimmune diseases require high affinity binding, but must lack toxicity that prevents 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 can lead to off-target toxicity. Therefore, the anti-FLT3L antibodies of the present disclosure retain binding specificity only for FLT3L, but not for structurally similar cytokines such as SCF and CSF1. Those skilled in the art will appreciate the use of, but not limited to, binding kinetics, including K on , K off and K D as a measure of binding specificity.

[0200] Serum FLT3L and circulating pDC are biomarkers that can be used as indicators of toxicity associated with anti-FLT3L antibody lead clones. When neutralizing FLT3L, the rapid decline of pDC indicates that FLT3L-mediated cell signaling that enhances immune response is suppressed. The rapid recovery of pDC frequency in the presence of free FLT3L can reflect that anti-FLT3L antibodies lack toxicity. In a preferred embodiment, when free FLT3L returns, anti-FLT3L antibodies neutralize FLT3L and allow reversible depletion of cDC and pDC. Those skilled in the art will recognize that dendritic cells decline after neutralizing FLT3L, and then return to baseline to indicate that the toxicity of the lead clone is low.

[0201] Anti-FLT3L antibody generation

[0202] The practice of the present disclosure employs, unless otherwise indicated, techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the purview of the skilled artisan. Such techniques are fully explained in the following references, such as "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" and "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction" (Mullis, 1994); "Current Protocols in Immunology (Coligan, 1991). These techniques are applicable to the production of polypeptides disclosed herein and, therefore, can be considered for use in making and practicing the present disclosure. Techniques that are particularly useful for specific embodiments are discussed in the Examples.

[0203] 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 control sequences for expression in a host cell. The isolated nucleic acid can be recombinantly incorporated into a vector and then transfected into a host cell using known techniques.

[0204] In one embodiment, the present invention contemplates a host cell transformed with an isolated nucleic acid molecule encoding an anti-FLT3L antibody or antigen-binding fragment thereof, the isolated nucleic acid molecule being operably linked to one or more control sequences. Examples of contemplated host cells include mammalian cells, such as HEK293 cells, NS0 mouse myeloma cells, or Chinese hamster ovary (CHO) cells.

[0205] In one embodiment, monoclonal anti-FLT3L antibodies (e.g., CAT5D9, SC4017, or AM40) and antigen-binding fragments thereof can be prepared using a hybridoma method, such as those described by Kohler and Milstein (1975) Nature 256: 495. Using the hybridoma method, mice, hamsters, or other appropriate host animals are immunized as described above to elicit lymphocytes that produce antibodies that will specifically bind to the immunizing antigen.

[0206] In another embodiment, lymphocytes can also be immunized in vitro.After immunization, lymphocytes are separated, and fused with suitable myeloma cell lines using, for example, polyethylene glycol to form hybridoma cells, and then these hybridoma cells can be selected from unfused lymphocytes and myeloma cells.Then, as determined by immunoprecipitation, immunoblotting or in vitro binding assays (for example, radioimmunoassay (RIA); Enzyme-linked immunosorbent assay (ELISA)), hybridomas of monoclonal antibodies specific for selected antigens can be produced using standard methods (Coding, Monoclonal Antibodies: Principles and Practice [monoclonal antibodies: Principles and Practice], Academic Press (academic press), 1986) to be cultured in vitro to breed or ascites tumors in animals in vivo.Then, monoclonal antibodies can be purified from culture medium or ascites as described above for polyclonal antibodies.

[0207] Alternatively, anti-FLT3L monoclonal antibodies (e.g., CAT5D9, SC4017, or AM40) and antigen-binding fragments thereof can also be prepared using recombinant DNA methods as described in, for example, U.S. Patent No. 4,816,567. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells, for example, by RT-PCR using oligonucleotide primers that specifically amplify genes encoding antibody heavy and light chains, and their sequences are determined using conventional procedures. The isolated polynucleotides encoding heavy and light chains are then cloned into suitable expression vectors, which, when transfected into host cells that do not otherwise produce immunoglobulins (e.g., E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells), produce monoclonal antibodies by these host cells. Similarly, 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 (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).

[0208] One or more polynucleotides encoding anti-FLT3L antibodies or antigen-binding fragments thereof can be further modified in a variety of different ways using recombinant DNA technology to produce alternative antibodies. In some aspects, for example, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be replaced by (1) such as those of a human antibody to produce a chimeric antibody, or (2) a non-immunoglobulin polypeptide to produce a fusion antibody. In some aspects, the constant region is truncated or removed to produce the desired antibody fragment of the monoclonal antibody. Site-directed mutagenesis or high-density mutagenesis of the variable region can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0209] In certain aspects, the anti-FLT3L antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment. Human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or separated from immune individuals can be produced, and these immortalized human B lymphocytes produce antibodies against target antigens (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy [monoclonal antibodies and cancer therapy], Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol. [Journal of Immunology], 147 (l): 86-95; and U.S. Pat. No. 5,750,373).

[0210] Likewise, anti-FLT3L human antibodies or antigen-binding fragments thereof can be selected from a phage library, wherein the phage library expresses human antibodies as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162, Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al., 1991, J. Mol. Biol., 222:581. Techniques for producing and using antibody phage libraries 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 in Rothe et al., 2007, J. Mol. Bio., doi: 10.1016 / j.jmb.2007.12.018 (each of which is incorporated by reference in its entirety).

[0211] Affinity maturation strategies and chain shuffling strategies (Marks et al., 1992, Bio / Technology 10:779-783, which is 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.

[0212] In some aspects, the anti-FLT3L monoclonal antibody can be a humanized antibody. Methods for engineering, humanization or resurfacing of non-human antibodies or human antibodies can also be used, and these methods are well known in the art. Humanized antibodies, resurfacing antibodies or similar engineered antibodies may have one or more amino acid residues from non-human sources (such as, but not limited to, mice, rats, rabbits, non-human primates, or other mammals). These non-human amino acid residues are replaced by residues commonly referred to as "input" residues, which are typically taken from the "input" variable domains, constant domains or other domains of known human sequences. As known in the art, such input sequences can be used to reduce immunogenicity, or reduce, enhance or modify binding, affinity, binding rate, dissociation rate, avidity, specificity, half-life or any other suitable features. In general, CDR residues directly and in most cases substantially involve affecting FLT3L binding. Accordingly, some or all of non-human or human CDR sequences are retained, and the non-human sequences of variable and constant regions can be replaced by people or other amino acids.

[0213] Antibodies can also be optionally humanized, resurfaced, engineered, or human antibodies can be engineered to retain high affinity and other favorable biological properties to the FLT3L antigen. In order to achieve this goal, humanized (or human) or engineered anti-FLT3L antibodies and resurfaced antibodies can be optionally prepared by analyzing the parental sequences and various conceptual humanization and engineering products using three-dimensional models of parental, engineered and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. These displayed inspections allow the possible effects of residues in the functional aspects of candidate immunoglobulin sequences to be analyzed, i.e., the residues that affect the ability of candidate immunoglobulins to bind their antigens (such as FLT3L) are analyzed. In this way, framework (FW) residues can be selected and combined from consensus sequences and input sequences so that desired antibody characteristics, such as increasing the affinity of one or more target antigens, are achieved.

[0214] Humanization, resurfacing or engineering of an anti-FLT3L antibody or antigen-binding fragment thereof can be performed using any known method, such as, but not limited to, those described in Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988), Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Proc. Natl. Acad. Sci. USA 89:4285 (1992); Esta 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; 5,955,358 ; 6,204,023; 6,180,370; 6,331,431; 5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,567; 5,969,108; 7,635,666; 7,723,270; 7,557,189; 7,538,195; and 7,342,110; International Application No. PCT / US 98 / 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 those in European Patent Publication No. EP 229246; each of which is incorporated herein by reference in its entirety, including the references cited therein.

[0215] Anti-FLT3L humanized antibodies and antigen-binding fragments thereof can also be prepared in transgenic mice containing human immunoglobulin loci, which are capable of producing a full repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. This method is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

[0216] In certain aspects, anti-FLT3L antibody fragments are provided. Various techniques for producing antibody fragments are known. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies (e.g., Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods [Journal of Biochemical and Biophysical Methods] 24: 107-117; Brennan et al., 1985, Science [Science], 229: 81). In certain aspects, anti-FLT3L antibody fragments are produced by recombination. Fab, Fv and scFv antibody fragments can all be expressed in Escherichia coli or other host cells and secreted therefrom, thereby allowing the production of large amounts of these fragments. Such anti-FLT3L antibody fragments can also be separated from the antibody phage library discussed above. Anti-FLT3L antibody fragments can also be linear antibodies, as described in U.S. Patent No. 5,641,870. Technical practitioners are aware of other techniques (e.g., chemical synthesis) for producing antibody fragments.

[0217] According to the present disclosure, these techniques may be suitable for producing single-chain antibodies specific for FLT3L (see, e.g., U.S. Patent No. 4,946,778). In addition, these methods may be suitable 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 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 the 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.

[0218] 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 mutating the appropriate region in the antibody or antibody fragment, or by incorporating the epitope into a peptide tag and then fusing it to one end or the middle of the antibody or antibody fragment (e.g., by DNA or peptide synthesis), or by YTE mutation. Other methods of increasing the serum half-life of an antibody or antigen-binding fragment thereof, such as conjugation to a heterologous molecule (such as PEG), are known in the art.

[0219] Pharmaceutical composition

[0220] The present invention also relates to pharmaceutical compositions comprising the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein. In certain embodiments, the present disclosure provides for the use of the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein in the manufacture of a medicament for treating a subject.

[0221] An effective amount of the pharmaceutical composition of the present disclosure should be administered, wherein an "effective amount" is defined as an amount sufficient to produce a desired preventive, therapeutic or ameliorative response in a subject. The effective amount will vary depending on the species and body weight of the subject to be administered, but can be determined using standard techniques.

[0222] In certain aspects, the disclosure provides therapeutic and prophylactic compositions for treating or preventing (reducing the likelihood of) an autoimmune disease in a subject in need thereof, including but not limited to systemic lupus erythematosus, myositis, primary Sjögren's syndrome, multiple sclerosis, uveitis, psoriasis, or rheumatoid arthritis.

[0223] In some embodiments, the pharmaceutical composition of the present disclosure comprises an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein and one or more pharmaceutically acceptable carriers, diluents or excipients. In this regard, "pharmaceutically acceptable carriers, diluents or excipients" include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier, which may or may not be approved by the United States Food and Drug Administration and are acceptable for use by humans or livestock. For example, suitable carriers are known to those skilled in the art and include stabilizers, diluents and buffers. Suitable stabilizers include carbohydrates such as sorbitol, lactose, mannitol, starch, sucrose, dextran and glucose, and proteins such as albumin or casein. Suitable diluents include saline, Hanks Balanced Salt and Ringers solution. Suitable buffering agents include alkali metal phosphates, alkali metal carbonates, or alkaline earth metal carbonates.

[0224] In certain aspects, the pharmaceutical composition of the present disclosure may further comprise one or more auxiliary substances, such as one or more lipids, phospholipids, carbohydrates and lipopolysaccharides. In some embodiments, the pharmaceutical composition of the present disclosure optionally comprises one or more other active substances.

[0225] In some cases, the pharmaceutical composition of the present disclosure can be prepared by techniques known to those skilled in the art. General considerations in the preparation and / or manufacture of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, 2005 (which is incorporated herein by reference in its entirety). Typically, the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein 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 may be subsequently added. The pharmaceutical composition of the present disclosure may be an aqueous solution, an emulsion or a suspension, or may be a dry preparation. In certain aspects, for storage or formulation purposes, the pharmaceutical composition of the present disclosure may be dried or lyophilized, for example, by freeze drying or spray drying. They may then be reconstituted into a liquid composition by adding an appropriate liquid carrier, or may be administered in the form of a dry preparation using methods known to those skilled in the art.

[0226] The pharmaceutical composition of the present disclosure can be administered to a subject via a variety of routes known in the art. Exemplary routes of administration of such pharmaceutical compositions include oral, mucosal, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal administration. Therefore, in certain embodiments, the pharmaceutical composition of the present disclosure is formulated 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, intrasternal injection or infusion techniques. In certain aspects, the pharmaceutical composition of the present disclosure is formulated to allow the anti-FLT3L antibody or its antigen-binding fragment of the present disclosure contained therein to be bioavailable after being administered to a subject.

[0227] The use of the pharmaceutical composition is selected to depend on the selected formulation. The pharmaceutical composition of the present disclosure is used in a manner compatible with the dosage formulation and in such an amount that is also effective in treatment. In certain aspects, the pharmaceutical composition of the present disclosure is formulated as a preparation in solid, semisolid, liquid or gaseous form, including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols.

[0228] In some cases, the pharmaceutical composition comprising the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein can be in solid or liquid form. In some aspects, one or more carriers are microparticles, so that these compositions are in the form of, for example, tablets or powders. In other aspects, one or more carriers are liquids, wherein the composition is, for example, an oral syrup, an injectable liquid or an aerosol, which can be used, for example, for inhalation administration. When intended for oral administration, the pharmaceutical composition comprising the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein is in solid or liquid form, wherein semi-solid, semi-liquid, suspension and gel forms are all included in the forms considered as solid or liquid herein.

[0229] In certain aspects, as a solid composition for oral administration, a pharmaceutical composition comprising an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein can be formulated in the form of a powder, granules, compressed tablets, pills, capsules, chewable tablets, wafers, etc. In some cases, such a solid composition will generally contain one or more inert diluents or edible carriers. In certain embodiments, one or more of the following may be additionally present: a binder such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum or gelatin; an excipient such as starch, lactose or dextrin, a disintegrant such as alginic acid, sodium alginate, Primogel, corn starch, etc.; a lubricant such as magnesium stearate or Sterotex; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as mint, methyl salicylate or orange flavoring; and a coloring agent.

[0230] These compositions can take the form of microspheres, solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 0.001% to 95% of the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein. Some dosage forms can contain 50 μg to 250 μg of anti-FLT3L antibodies or antigen-binding fragments thereof.

[0231] In some aspects, when the pharmaceutical composition of the present disclosure is in the form of a capsule, such as a gelatin capsule, it can also contain a liquid carrier such as polyethylene glycol or oil in addition to the materials disclosed herein. Oral formulations can also include commonly used excipients, such as, for example, pharmaceutical grade saccharin, cellulose and magnesium carbonate.

[0232] 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 can be used for oral administration or for delivery by injection. In certain embodiments, when it is intended to be used for oral administration, in addition to the anti-FLT3L antibody or its antigen-binding fragment disclosed herein, the pharmaceutical composition of the present disclosure also includes one or more of a sweetener, a preservative, a dye / colorant and a flavor enhancer. In certain aspects, in a pharmaceutical composition intended to be administered by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer and an isotonic agent may also be included.

[0233] In some cases, liquid pharmaceutical compositions comprising an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein, whether they are in the form of a solution, suspension, or other similar form, may include one or more of the following components: a sterile diluent, such as water for injection, a saline solution (e.g., physiological saline), Ringer's solution, isotonic sodium chloride, a fixed oil (e.g., a synthetic monoglyceride or diglyceride that can be used as a solvent or suspension medium), polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose. In some cases, the formulation may be packaged in an ampoule, disposable syringe, or multiple-dose vial made of glass or plastic. In some embodiments, the injectable pharmaceutical composition is preferably sterile.

[0234] In other embodiments, the pharmaceutical composition comprising the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel matrix. In certain aspects, the matrix may, for example, comprise one or more of the following: diluents such as vaseline, lanolin, polyethylene glycol, beeswax, mineral oil, water and alcohol, as well as emulsifiers and stabilizers. In other aspects, a thickener may be present in the pharmaceutical composition for topical administration. In certain embodiments, if intended for transdermal administration, the pharmaceutical composition of the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein may be included in a transdermal patch or iontophoresis device.

[0235] In yet other embodiments, the pharmaceutical composition comprising the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein is expected to be used for rectal administration in the form of, for example, a suppository. For suppositories, the binder and carrier may include, for example, a polyalkylene glycol or a triglyceride. In some cases, the composition for rectal administration includes an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, or polyethylene glycol.

[0236] In other aspects, the pharmaceutical composition comprising the anti-FLT3L antibody or its antigen-binding fragment disclosed herein comprises a dosage unit that can be administered as an aerosol. The term aerosol is used to represent various systems, ranging from those systems of colloidal nature to systems consisting of pressurized packaging. In certain embodiments, delivery is accomplished by liquefying or compressing a gas or by a suitable pump system for dispensing an active ingredient. In some embodiments, an aerosol of an anti-FLT3L antibody or its antigen-binding fragment disclosed herein can be delivered in a single-phase, two-phase or three-phase system to deliver one or more active ingredients. In other embodiments, the delivery of the aerosol includes necessary containers, activators, valves, sub-containers, etc., which together can form a kit. Those skilled in the art can easily determine specific aerosol formulations and delivery methods.

[0237] 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.

[0238] In other aspects, the pharmaceutical compositions of the present disclosure include materials that form a coating shell around the active ingredient. In some cases, the materials that form the coating shell are generally inert and can be selected from, for example, sugar, shellac and other enteric coating agents.

[0239] In yet other aspects, pharmaceutical compositions of the present disclosure in solid or liquid form include an agent that binds to an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein, thereby facilitating the delivery of the anti-FLT3L antibody or antigen-binding fragment thereof. In some cases, suitable agents that act in this capacity include proteins or liposomes.

[0240] The pharmaceutical composition to be administered to the subject takes the form of one or more dosage units, where, for example, a tablet can be a single dosage unit, while a container of an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein in aerosol form can hold multiple dosage units. Actual methods of preparing such dosage forms are known to those skilled in the art, or will be apparent; for example, see Remington: The Science and Practice of Pharmacy, 20th edition (Philadelphia College of Pharmacy, 2000). In any event, according to the teachings herein, the composition to be administered will contain a therapeutically effective amount of an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein, or a pharmaceutically acceptable salt thereof, to aid in the treatment of a disease or condition of interest.

[0241] In certain embodiments, the pharmaceutical composition of the present disclosure comprises one or more other therapeutically active substances. In other embodiments, a therapeutically effective dose of the pharmaceutical composition of the present disclosure is administered in combination with one or more other therapeutically active substances to a subject in need thereof. As used herein, "combination" refers to a combination comprising an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein and one or more other therapeutically active substances, each of which can be administered continuously (sequentially), synchronously or simultaneously.

[0242] To maintain therapeutic levels, the pharmaceutical compositions of the present disclosure are ideally administered at several intervals.The pharmaceutical compositions of the present disclosure may be used in conjunction with other bactericidal or bacteriostatic methods.

[0243] Although the description of the pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to subjects of all classifications. In certain aspects, the subject is a mammal. In certain aspects, mammals include primates, such as humans, monkeys and apes, and non-primates, such as livestock, including experimental animals and household pets and farm animals (e.g., cats, dogs, pigs, cattle, sheep, goats, horses, rabbits), and non-livestock animals, such as wild animals, birds, etc.

[0244] Autoimmune / anti-inflammatory therapy

[0245] The disclosure also features compositions and methods comprising anti-FLT3L antibodies that can be used to treat autoimmune diseases and / or other inflammatory diseases (i.e., diseases involving an overreaction and / or dysfunction of the immune system), such as those described above. In various embodiments, anti-FLT3L antibodies can be administered in combination with other immunomodulatory drugs that are designed to suppress or attenuate a subject's immune system or a specific immune response to a particular antigen or group of antigens, thereby reducing or preventing autoimmune diseases or other inflammatory diseases.

[0246] Further provided herein are methods for treating autoimmune diseases and / or other inflammatory diseases, comprising administering one or more anti-FLT3L antibodies. As shown herein, administration of anti-FLT3L antibodies can result in at least one of a reduction in immune response, expression of one or more immune signaling cascades, or a reduction in immune cell populations. In certain aspects, anti-FLT3L antibodies are administered to patients or subjects suffering from autoimmune diseases or other inflammatory diseases.

[0247] Treatment of autoimmune diseases and / or other inflammatory disease therapies including anti-FLT3L antibodies can result in, for example, a reduction in the rate of progression of an autoimmune disease or inflammatory disease, a block or stabilization of immune cell proliferation, a reduction in lesions (e.g., in MS patients), and / or disease regression. In some aspects, a measure of reduction or blockage of an autoimmune disease or inflammatory disease (e.g., reduced inflammation, the level of inflammatory cytokines, immune cell colonies, and / or related damage such as tissue damage) can be statistically significant. The measure of an autoimmune disease or inflammatory disease can be reduced by comparing the patient's measure level at baseline (before treatment) with the expected level of individual disease progression, with the expected level of disease progression based on a large number of patient populations, or with the expected level of disease progression in a control population to measure.

[0248] In one embodiment, the treatment methods as contemplated herein comprise applying or administering an anti-FLT3L binding molecule, antibody, or antigen-binding fragment, variant, or derivative thereof of the present disclosure to a subject or patient, or applying or administering an anti-FLT3L binding molecule to an isolated tissue or cell line from a subject or patient, wherein the subject or patient has a disease, has symptoms of a disease, or has a susceptibility to a disease.

[0249] Contemplated diseases include acute or chronic inflammatory diseases (including type 1 and type 2 diabetes), CKD (including, for example, CKD caused by diabetes, diabetic nephropathy and hypertension), atherosclerosis, Alzheimer's disease, cancer, and complications associated with such diseases, including heart disease, hypertension, anemia, pericarditis, renal osteodystrophy, etc. Other contemplated diseases include autoimmune diseases, including, but not limited to, systemic lupus erythematosus, myositis, primary Sjögren's syndrome, multiple sclerosis, uveitis, psoriasis, and rheumatoid arthritis.

[0250] In another embodiment, treatment is also contemplated to include applying or administering a pharmaceutical composition comprising an anti-FLT3L binding molecule of the disclosure, such as an antibody or antigen-binding fragment, variant or derivative thereof, to a subject or patient, or applying or administering a pharmaceutical composition comprising an anti-FLT3L binding molecule to an isolated tissue or cell line from a subject or patient who has a disease, has symptoms of a disease, or has a susceptibility to a disease.

[0251] According to the methods disclosed herein, at least one anti-FLT3L antibody as defined elsewhere herein is used to promote a positive therapeutic response to an autoimmune disease or inflammatory disease. The term "positive therapeutic response" refers to a reduction in symptoms associated with an autoimmune disease or inflammatory disease. Thus, for example, an improvement in a disease can be characterized as a complete response. A "complete response" refers to the absence of clinically detectable disease and any previous test results are normalized. Alternatively, an improvement in a disease can be classified as a partial response. A "positive therapeutic response" encompasses a reduction or inhibition of the progression and / or duration of an autoimmune disease or inflammatory disease, a reduction or improvement in the severity of an autoimmune disease or inflammatory disease, and / or an improvement in one or more symptoms thereof due to the administration of an anti-FLT3L binding molecule disclosed herein.

[0252] In certain embodiments, a method for treating primary Sjögren's syndrome is provided, comprising: administering to a subject in need thereof a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0253] In other embodiments, a method for treating myositis is provided, comprising: administering to a subject in need thereof a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0254] In certain embodiments, a method for treating systemic lupus erythematosus (SLE) is provided, comprising: administering a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof. In some aspects, the subject has an elevated serum level of FLT3L compared to healthy subjects, as measured by the frequency of CD4+T cells expressing FLT3L.

[0255] In some embodiments, a method for diagnosing systemic lupus erythematosus (SLE) in a subject is provided, comprising: (a) measuring the serum level of FLT3L, or (b) measuring the frequency of CD4+T cells expressing FLT3L, wherein an increase in the serum level of FLT3L in the subject or an increase in the frequency of CD4+T cells expressing FLT3L compared to a healthy donor indicates that the subject suffers from SLE. In some aspects, these CD4+T cells are effector memory cells (T EM ).

[0256] In some embodiments, a method of neutralizing membrane-bound FLT3L in a subject in need thereof is provided, the method 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.

[0257] In other embodiments, a method of neutralizing soluble FLT3L in a subject in need thereof is provided, the method comprising administering to the subject a pharmaceutically effective amount of an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein. In certain aspects, FLT3L is reversibly neutralized such that the level of soluble FLT3L can return to the level before administration.

[0258] In certain embodiments, the method of neutralizing soluble FLT3L further comprises administering an anti-FLT3L antibody or antigen-binding fragment thereof subcutaneously to a subject at a range of about 0.03 mg / kg to about 30 mg / kg once a week. In other embodiments, the method further comprises administering an anti-FLT3L antibody or antigen-binding fragment thereof to a subject at a dose of about 150 mg / kg once every four weeks.

[0259] In other embodiments, there is provided a method for reducing a population of circulating classical dendritic cells (cDC) and plasmacytoid dendritic cells (pDC) in a subject in need thereof, the method comprising administering a pharmaceutically effective amount of an antibody or its antigen-binding fragment disclosed herein to the subject. In some aspects, the population of cDC and pDC is reversibly reduced so that the population of cDC and pDC can return to the level before administration.

[0260] In certain embodiments, provided are methods of reducing FLT3L expression on CD4+ T cells, the methods comprising administering to a subject in need thereof a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0261] In other embodiments, a method of reducing the percentage of CD4+ T cells expressing FLT3L is provided, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0262] In other embodiments, a method of reducing ERK signaling in a lymphoblast is provided, the method comprising contacting the lymphoblast with an antibody or antigen-binding fragment thereof disclosed herein.

[0263] In certain embodiments, provided are methods of reducing MEK 1 / 2 phosphorylation in primary CD133+ human stem cells, the methods comprising contacting the stem cells with an antibody or antigen-binding fragment thereof disclosed herein.

[0264] The following examples are put forth so as to provide those skilled in the art with a complete disclosure and description of how to make and use the assays, screens, and therapeutic methods of the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.

[0265] Examples

[0266] The present disclosure is described with reference to the following examples. These examples are merely illustrative, and the present disclosure should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations that become apparent as a result of the teachings provided herein.

[0267] Example No. 1. Anti-FLT3L Antibody Generation

[0268] Overview:

[0269] FLT3L is a non-disulfide-linked homodimeric glycoprotein of 65 kDa. The ligand and receptor sequence homology of human, non-human primate and mouse is shown in Table 2 below.

[0270] Table 2. Identity to human FLT3 and FLT3L

[0271]

[0272] Although the homology of the complete mouse FLT3L protein is only 73%, its binding site to FLT3 is highly conserved between species. In fact, human FLT3L binds to and activates mouse FLT3 and vice versa. FLT3L has structural homology with stem cell factor (SCF or KIT ligand) and colony stimulating factor 1 (CSF1), but no obvious sequence homology with other human cytokines. The receptors for these two ligands (c-KIT and CSF1R, respectively) are also class III TKRs that commonly interact with inhibitors of FLT3, resulting in undesirable off-target toxicity in clinical applications. Taking these factors into account, FLT3L antibodies that do not cross-react with SCF or CSF1 are sought, thereby providing highly specific inhibition of the FLT3 / FLT3L pathway.

[0273] Lead antibodies are selected and tested by alternating selection on soluble human and mouse FLT3-L. Primary biochemical high-throughput screening is performed using human FLT3 / FLT3L competition to complete homogeneous time-resolved fluorescence (HTRF) assays. All single-chain variable fragment-fragment crystallization regions (scFv-Fc) hits are converted to IgG1 TM formats, and then the functional screening assay using FLT3 downregulation on the target cell surface is used to confirm the inhibitory activity of lead antibodies. Using ELISA and functional assays, mice and cynomolgus monkeys are confirmed to cross-reactivity and selectivity of FLT3L, and other family members (such as stem cell factor (scf)) are excluded.

[0274] The lead compound was further evaluated using FLT3 signaling assays in RS4;11 cell lines and primary human CD133+ stem cells. Binding to endogenous FLT3L was confirmed using primary human T cells. Specific binding sites and binding affinity were determined via Octet and BIAcore, respectively. The resulting lead antibody clones were selected for further optimization as described below.

[0275] Lead antibody candidate identification activities (C5, B10-11)

[0276] like Figure 1 As shown in, the lead antibody for anti-FLT3L binding research is produced by first screening phage library.For human FLT3L (huFLT3L) and / or mouse FLT3L (muFLT3L), panning (alternative panning or competitive panning) phage display library bone marrow Vaughan (Bone Marrow Vaughan (BMV)), merging spleen (CS), DP47 library (DP47) and Dyax human antibody library.In short, EZ- is used to connect sulfo-NHS-LC-biotin labeling kit (Thermo Scientific)) as panning antigen, and biotin is used to label the FLT3L produced internally.As described, two to three rounds of panning are carried out using internal scFv library (Xiao X et al., 2017mAbs [monoclonal antibody] 5429,996-1006 (2017)). In order to enhance the cross-reactivity of human / cynomolgus monkey, human and cynomolgus monkey recombinant FLT3L were used as panning antigens in an alternating manner in some selection processes. In order to select antibodies that inhibit FLT3L / FLT3 interactions, competitive panning was used in some experiments. For competitive panning, human FLT3L was used as the panning antigen, and an excess (>100X) of FLT3-Fc was used as a phage eluting agent instead of conventional trypsin (Xiao X et al. mAbs [monoclonal antibodies] 5429, 996-1006 (2017)).

[0277] The BMV library was enriched 100-fold, the CS library was enriched 350-fold, and the DP47 library was enriched 250-fold; the Dyax library was not enriched. Compared with the second round of panning, only the BMV and Dyax phage libraries were panned for the third time, resulting in a 100-fold enrichment for the BMV phage library and a 50-fold enrichment for the Dyax library ( Figure 1 A).

[0278] After each round of selection, monoclonal phage ELISA was performed to estimate the percentage of antigen-specific phage. Only selections that reached at least 20% positive rate were subjected to high-throughput screening. According to this set of criteria, the third output BMV panning and the second output CS and DP47 panning were cloned into pSplice V4 and pdLG or pmLG vectors and converted into scFv-Fc (Xiao X et al., PLoS One [Public Library of Science Comprehensive], October 15, 2015; 10(10): e0140691.doi:10.1371 / journal.pone.0140691) or IgG format (Xiao X et al. mAbs [monoclonal antibodies] 5429, 996-1006 (2017)) to perform functional screening by competitive HTRF.

[0279] For functional screening, first 293 freestyle cells (Thermo Fisher Scientific) were transfected with scFv-Fc or IgG constructs converted from panning output. The resulting supernatant was directly used in the FLT3L / FLT3 interaction inhibition assay based on HTRF. For HTRF assay, 10nM biotin-labeled FLT3L, 20nM streptavidin-europium cryptate (Cisbio), 10nM FLT3-mFc and 20nM anti-mFc-A647 (Cisbio) were mixed with 10 μL transfection supernatants in 384-well Greiner plates with a total volume of 20 μL / holes. The readings of 665nm and 620nm were read five minutes after mixing, and then read at intervals of one hour until the reading becomes stable. The ratio of 665nm / 620nm is then calculated. The reduction of ratio shows the inhibition of FLT3L / FLT3 interactions.

[0280] The panning outputs from direct or alternating antigen panning were converted to scFv-Fc and >4,000 colonies were selected for high throughput screening (HTS) in the HTRF FLT3L / FLT3-Fc interaction inhibition assay. Ten lead antibodies were identified and IgG TM was converted and expressed. The antibodies were retested in a second HTRF interaction inhibition assay and ten leads were identified. In parallel, more than seven hundred hits from competitive panning were converted by PmIgG and expressed in mammalian cells. The converted clones were subjected to the same HTRF FLT3L / FLT3-Fc interaction inhibition assay and two leads were identified ( Figure 1 B).

[0281] 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 downregulation, and signal transduction inhibition assays. Epitope matching and affinity assays were also performed on these leads. The screening campaign identified five lead antibodies for further study: Dyax3, Dyax5, CAT8, CAT26, and CAT5D9. Results from the competitive HTRF analysis for the lead antibodies are shown in Figure 1 C.

[0282] Example No. 2. FLT3 downregulation screening assay

[0283] After connecting FLT3L, FLT3 receptor dimerization, autophosphorylation and activation of downstream signal transduction pathways. In this process, surface FLT3 is internalized and degraded. This internalization feature is used to develop a determination for screening lead anti-FLT3L candidate clones. Cell surface FLT3 expression can be measured by flow cytometry, and the inhibition of receptor-ligand binding can be determined by quantifying the changes in cell surface FLT3 expression levels.

[0284] Cell lines RS4; 11, EOL-1, MOLM13 and MV4-11 constitutively express FLT3. Cells were cultured under normal conditions and the relative expression of FLT3 was screened after 2-24 hours of culture. FLT3 expression was measured using flow cytometry. In short, commercially available anti-CD135 (anti-FLT3) clone BV10A4H2 (BioLegend) was used for flow cytometry experiments, and mean fluorescence intensity (MFI) was reported as a measure of FLT3 expression. In all assays, commercially available mouse anti-human FLT3L monoclonal antibodies (R&D) or internal huFLT3-Fc constructs were used as positive controls for effectively neutralizing FLT3L activity.

[0285] The acute leukemia (pro-B) line RS4;11 showed consistent and high expression of FLT3 in culture compared to other commercially available lines reported to express FLT3 ( Figure 2 A). Direct binding of FLT3L to cell surface FLT3 was confirmed using serial dilutions of biotinylated recombinant huFLT3L (rhuFLT3L), which was incubated with RS4;11 cells for 30 min at 4°C. Physical binding to the cell surface was detected using BV421-streptavidin, followed by flow cytometric analysis to determine mean fluorescence intensity ( Figure 2 B).

[0286] 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. The stability of the conditions was assessed by using 2 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). After incubation with huFLT3L for 2 hours, both cell densities showed a dose-dependent downregulation of cell surface FLT3. Over the range of huFLT3L used in the assay, the allophycocyanin (APC) MFI, an indicator of FLT3 expression, was reduced by 25-fold ( Figure 2 C). These results demonstrate that the screening assay is effective in measuring a dose-dependent response of bioavailable FLT3L and is suitable for testing candidate clones for their ability to functionally neutralize FLT3L. There was no significant difference in the response whether 50,000 cells / well or 100,000 cells / well were used.

[0287] Optimization of screening assays

[0288] 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 / well were incubated with 96pM rhuFLT3L (with or without anti-FLT3L mAb candidate clones) in complete Roswell Park Memorial Institute (RPMI) medium (containing 1% bovine serum albumin (BSA)) in a humidified incubator (set at 37°C, 5% CO2) for 2 hours. Subsequent downregulation of FLT3 expression was determined by flow cytometry, as measured by raw MFI or % downregulation. 96pM rhuFLT3L (EC80) was selected because it is the point at which the exponential phase of the dose-response curve begins, thus ensuring that any functional inhibition of rhuFLT3L will be immediately reflected by changes in the level of FLT3 downregulation on the surface of RS4;11 cells. ( Figure 3 A). Assay efficacy was confirmed using a commercially available mouse anti-huFLT3L antibody control (MAB608, R&D Systems). Figure 3 B) Due to the cross-species reactivity of FLT3 with its ligand, despite being a human cell line, this assay is effective for testing clones against human, cynomolgus monkey and rodent FLT3L. For mouse FLT3L, the EC80 was 36 pM.

[0289] Example No. 3. Neutralizing activity of lead antibody candidates for soluble FLT3L After FLT3L is connected to FLT3, the receptor dimerizes, autophosphorylates and propagates signal cascades once internalized. This process measured by FLT3 downregulation can be inhibited by antibodies that bind to FLT3L. Therefore, by binding to human, mouse or cynomolgus monkey soluble FLT3L (sFLT3L), the lead candidate was tested for its ability to inhibit FLT3 downregulation on RS4;11 cells. The neutralizing ability of the lead candidate was tested using an optimized screening assay. As described, RS4;11 cells (50,000 cells / well) were incubated in complete RPMI (containing 1% BSA) for two hours in the presence of human or cynomolgus monkey FLT3L (96pM) or mouse sFLT3L (36pM). Each clone of the serial dilution was added, and the soluble FLT3-Fc construct or 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.

[0290] like Figure 4 As shown in Table 4A and 4B, all lead cloned antibodies showed the ability to inhibit human and cynomolgus monkey sFLT3L to some extent. CAT8, CAT26, Dyax3 and Dyax5 all showed similar inhibition of human sFLT3L and cynomolgus monkey sFLT3L. Although shown in Table 3 below, the IC50 values ​​of CAT8, CAT26, Dyax3 and Dyax5 still lacked effectiveness because the IC MAX .

[0291] In contrast, CAT5D9 achieves IC MAX and produced the S-shaped curve expected from FLT3L inhibition ( Figure 4 A and 4B). The IC50 values ​​of CAT5D9 presented in Table 3 indicate cross-reactivity with human and cynomolgus monkey, but not with mouse FLT3L ( Figure 4 C).

[0292] Table 3. IC50 (nM) of lead antibody candidates against 96 pM human, cynomolgus monkey or mouse sFLT3L.

[0293]

[0294] Example No. 4. Binding activity of lead antibody candidate to cell surface FLT3L

[0295] FLT3L is expressed as a cell membrane protein, and it circulates as a soluble protein after cleavage. Both membrane-bound and soluble forms are biologically active. In order to effectively block the signal transduction pathway mediated by FLT3, the lead antibody candidate should bind both soluble and membrane-bound forms of FLT3L.

[0296] In line with this, cell surface binding to human, cynomolgus monkey and mouse FLT3L was evaluated by transfecting Chinese hamster ovary (CHO) cells with the respective full-length proteins of each species. Candidate clones were incubated with FLT3L-expressing cell lines at 4 ° C for 1 hour and then washed twice to remove unbound antibodies. The cells were then incubated with PE-labeled goat anti-hu IgG secondary detection pAb to quantify bound antibodies, which were measured by flow cytometric analysis of PE signals. The huFLT3-Fc construct on the huIgG backbone was used as a positive control reagent for FLT3L expression because it cross-reacts with cynomolgus monkeys and mice in addition to human ligands.

[0297] All lead candidates bind to human FLT3L ( Figure 5 A), and all lead candidates except DYAX5 bound to cynomolgus FLT3L ( Figure 5 B). In contrast, only Dyax5 and, to a lesser extent, Dyax3 displayed cross-reactivity with mouse FLT3L ( Figure 5 C).

[0298] Thus, despite having different efficacies, all lead candidates demonstrated the ability to bind to FLT3L across species. Since there were significant differences in the degree of receptor occupancy for each clone, the EC50 and maximum occupancy (expressed as a percentage relative to FLT3-Fc) were recorded and considered in the final evaluation. Table 4 shows the results for human, cynomolgus monkey, and mouse FLT3L expressed in CHO cells.

[0299] Table 4. EC50 (nM) and maximum occupancy (% relative to FLT3-Fc)

[0300]

[0301] Example No. 5. Binding of lead antibody candidates to endogenous human FLT3L

[0302] FLT3L is expressed on the surface of primary T cells after stimulation with α-chain cytokines (i.e., IL-2, IL-7, or IL-15), regardless of TCR engagement. In Example No. 4, the lead antibody candidate exhibited the ability to bind to CHO cells transfected with human FLT3L protein. The next step was to ensure that they could bind to endogenous FLT3L from primary human cell lines. Therefore, the ability of the lead antibody candidate to bind to huFLT3L on primary T cells stimulated by IL-2 obtained from human donors was tested.

[0303] 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 (activation of T cells with anti-CD3 results in FLT3L shedding from the cell surface). After this time, expression on T cells was confirmed using the human FLT3-Fc construct ( Figure 6 A). Serial dilutions of each clone were then incubated with IL-2 stimulated T cells (100K / well) at 4°C for 30 min. 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 endogenous FLT3L on human primary T cells ( Figure 6 B). CAT5D9 binding to endogenous FLT3L was initially undetectable due to its low binding affinity. However, when CAT5D9 was dimerized with (APC-labeled) anti-IgG prior to incubation with T cells, its affinity was sufficiently enhanced to demonstrate dose-dependent binding to endogenous FLT3L ( Figure 6 C).

[0304] Example No. 6. Inhibition of cell surface FLT3L by lead antibody candidate

[0305] The ability of lead candidates to bind cell surface FLT3L provides limited insight into the functional inhibition of the ligand. Ideally, binding of lead candidates to cell surface FLT3L should reduce the signaling activity of the ligand-receptor complex.

[0306] To test this, 1,000 CHO cells / well expressing huFLT3L were plated and allowed to adhere overnight. The next day, the CHO medium was removed, the cells were gently washed with RPMI, and serially diluted antibodies were added for 30 minutes before adding FLT3+RS4;11 (100K / well). After incubation for 2 hr at 36°C, RS4;11 cells were transferred to a fresh 96-well plate on ice for staining to detect downregulation of FLT3. In addition to the staining for the standard RS4;11 FLT3 downregulation assay, anti-CD19 was included to exclude any contaminating CHO cells. Downregulation of FLT3 was measured by flow cytometry.

[0307] Lead antibody candidate blocking assays demonstrated that all lead candidates had the ability to inhibit cell surface FLT3L to some extent, although all displayed relatively low activity compared to commercially available control antibodies, reflecting low affinity ( Figure 7 ).

[0308] Example No. 7. Inhibition of FLT3 signaling induced by FLT3L by lead clones The autophosphorylation of FLT3 is mainly activated by PI3K and RAS cascades, and then activates AKT (protein kinase B, PKB), MEK and ERK. The signal cascade ultimately leads to the transcription of genes that promote cell survival and proliferation. In order to confirm that the lead antibody candidate is blocking the downstream signaling of FLT3L to FLT3 in primary human cells, the phosphorylation of ERK and MEK in CD133+ stem cells was measured with Mesoscale MSD phospho-ERK1 / 2 and phospho-MEK1 / 2 whole cell lysates.

[0309] Assay validation was established using the RS4;11 cell line, in which FLT3L-induced ERK activation was performed in a dose-dependent manner. Figure 8 A). Serial dilutions of FLT3L were incubated with 300,000 RS4;11 cells / well at 36°C for 8 minutes. Cells were then harvested, lysed and analyzed for phosphorylated ERK according to the manufacturer's instructions. As with the earlier assay, the EC80 for ERK activation of FLT3L was determined (476 pM) and used to test the inhibitory activity of candidate anti-FLT3L antibody clones. The assay utility was confirmed using a commercially available mouse anti-human FLT3L antibody as a positive control for potent neutralization ( Figure 8 B).

[0310] Using these established parameters, the lead clones were tested using in vitro expanded primary CD133+ stem cells that were verified for FLT3 expression prior to use. The lead clones were pre-incubated with 476pM FLT3L for 30 minutes and then added to the CD133+ stem cells. After incubation at 36°C for 8 minutes, the cells were harvested, lysed, and analyzed for phosphorylated ERK and MEK using the MSD assay according to the manufacturer's instructions. Commercially available mouse anti-human FLT3L was used as a positive control.

[0311] All candidate clones appeared to inhibit the action of FLT3L on MEK ( Fig. 9 A) and ERK( Fig. 9 However, only CAT5D9 reached the IC MAX and exhibited the expected sigmoidal dose response curve (Table 5). Taken together, the results presented in the Examples indicate that CAT5D9 is the best clone for the lead candidate and should continue to be optimized subject to biophysical assessment of affinity and epitope binding site.

[0312] Table 5. IC50 (nM) against 0.476 nM FLT3L

[0313]

[0314]

[0315] Example No. 8. Confirmation of target specificity

[0316] CAT5D9 appeared to be the best lead candidate through functional evaluation in bioassays. Octet epitope pairing was used to determine the binding region of CAT5D9 relative to the receptor FLT3.

[0317] Epitope matching is used to determine the lead antibody that shares the same binding region with the FLT3 receptor on FLT3L. Pairing is performed in three phases. In Phase I, biotin-FLT3L is combined with an avidin probe. In Phase II, a single antibody is combined with biotin-FLT3L. In Phase III, each test antibody is added to the Phase II antibody. Any other binding detected indicates that the two antibodies have non-competitive binding sites with the target FLT3L. Only buffer (no antibody added) and FLT3-Fc in Phase III are used as negative and positive controls, respectively. If only the buffer in Phase III is added, the resulting antibody and FLT3L dissociation kinetics reflect the inherent affinity of the clone to the target.

[0318] CAT5D9 added only at 1x and 2x concentrations inhibited the binding of FLT3-Fc, indicating that CAT5D9 hits the desired target site on FLT3L (i.e., it shares the same or overlapping epitope with FLT3-Fc) ( Fig.10 A). Importantly, in contrast, the remaining four candidate clones were not inhibited by CAT5D9, suggesting that they bind to different FLT3 epitopes ( Fig.10 A). In addition, the rapid off-rate when only buffer was added supports previous experiments indicating that CAT5D9 has a low affinity (implying a high probability of improving its performance through optimization). Consistent with this, when each of the remaining 4 clones bound in phase II (CAT8 was shown as Fig.10 B), additional binding of CAT5D9 and FLT3-Fc can be detected in phase III, reflecting that they have different binding sites. It was also observed that each of the other 4 clones was paired together and their dissociation rates were relatively slow when only buffer was added.

[0319] In summary, these data confirm that only CAT5D9 competes directly with FLT3 in the FLT3L binding region and appears to compete at low affinity binding, which suggests that it has the potential for optimization. On the other hand, based on functional data, the remaining clones all hit sites that do not directly compete with FLT3. Their slow dissociation rates indicate that they have bound to FLT3L with reasonable affinity and have little potential for optimization. The inhibition that initially existed in the initial screening assay may be due to steric hindrance or partial blocking of the receptor binding site.

[0320] Example No. 9. BIACORE Binding Kinetics (C9)

[0321] Biacore analysis was used to determine the binding kinetics of the antibody leads and confirmed the Octet data indicating that CAT5D9 had a poor affinity for FLT3L. The kinetics of anti-FLT3L fragment antigen binding and human and cynomolgus monkey FLTL3 were determined in human CAT8, CAT26, Dyax3, and Dyax5. In addition, the binding kinetics of the CAT5D9 fragment antigen for both human and cynomolgus monkey FLT3L were determined.

[0322] The results are presented in Table 6. Compared with the other lead antibodies, the equilibrium dissociation constant (K D ) was more than 50x. CAT5D9 showed low-quality fast closing kinetics (hu = 70.72, cynomolgus monkey = 70.66). As a result, steady-state binding data were obtained as the kinetic data K D The steady-state binding supported the results of kinetic binding, showing similar values ​​(Table 3). These data confirm that CAT5D9 has low affinity and that it has the possibility of improving performance through optimization.

[0323] Table 6. Binding kinetics of anti-FLT3L antibody leads.

[0324]

[0325]

[0326] Fab = fragment antigen binding; Hu = human; cyno = cynomolgus monkey; kon = binding association constant; Koff = binding dissociation constant; K D = equilibrium dissociation constant

[0327] Example No. 10. No CAT5D9 cross-reactivity

[0328] In addition to demonstrating that CAT5D9 binds to the correct FLT3L epitope, it was important to confirm that it does not bind to close structural homologs of FLT3L, stem cell factor (SCF) and colony stimulating factor (CSF1). Both factors are ligands for protein tyrosine kinase receptors (c-Kit and CSFR1, respectively), which are major off-target hits for small molecule FLT3 inhibitors currently used in the oncology setting to manage malignancies caused by constitutively activating FLT3-IT9D mutations.

[0329] To test this, ELISA plates were coated with 2 □g of recombinant human SCF or CSF1. After washing, the plates were blocked with 3% milk in tris-phosphate buffered saline (TPBS) and the lead antibody was added in serial (x2) dilutions starting at 50 □ □g / ml. After incubation, unbound antibody was removed by washing and color development of bound antibody was detected using anti-human IgG-HRP in combination with TMB substrate. Commercially available goat anti-SCF pAb and mouse anti-CSF1 mAb were used as positive binding controls.

[0330] None of these lead candidates interacted with huSCF ( Fig.11 A) or huCSF1( Fig.11 B) Cross-reactivity. Importantly, these results demonstrate the selectivity of CAT5D9 in binding only to FLT3L and not to structurally similar TKR ligand molecules.

[0331] Example No. 11. Affinity Optimization of CAT5D9

[0332] As discussed above, CAT5D9 binds FLT3L with low affinity and therefore demonstrates the possibility of improving performance through affinity optimization. The desired KD was set to 300 pM based on PK modeling. Optimization campaigns were designed to improve the KD by up to 10,000-fold.

[0333] After germlining the framework, two parallel strategies were employed: conservation mutagenesis and segmental mutagenesis. Conservation mutagenesis mutated each position in all 6 CDRs to all 20 amino acids one at a time. Clones were screened using a high-throughput approach and single beneficial mutations were grouped together. Segmental mutagenesis mutated consecutive stretches of 5 to 6 positions in the CDRs in an overlapping pattern, and the resulting library of approximately 1E6 to 1E7 clones was first enriched using phage display panning technology and then screened using a high-throughput approach.

[0334] After the first round of optimization, 30 clones from conservation mutagenesis and 24 clones from segment mutagenesis were tested in IgG format. Using molecular modeling techniques, we identified and combined the best mutations that produced clone 5D9-clone 6, the KD of these clones was 1610nM (measured by Biacore), which was 700x (see Table 7) higher than the parent 5D9 affinity. The affinity of lead clone SC4017 and AM40 exceeded the CDTP standard (<300pM). In addition, the two optimized clones all combined and neutralized endogenous cell surface FLTL, combined with the endogenous soluble FLT3L in human serum, and combined and neutralized cynomolgus monkey FLT3L.

[0335] Table 7. Summary of binding data from CAT5D9 optimized lead clones.

[0336]

[0337] This increase in affinity translated into a >1000-fold increase in functional activity as demonstrated by downregulation of FLT3 on RS4;11 cells using the method described for earlier clonal selection ( Fig.12 A) measured.

[0338] In order to achieve an affinity of 300pM, a second round of affinity optimization was performed. Clone 6 (C06) was mutated and the resulting mutants were screened in a similar manner to the first round of optimization. As part of phage panning in segment mutagenesis, clone 6 was used as a competitor in IgG form to enrich for clones with substantially higher affinity. The best clone from segment mutagenesis was clone AM40 with KD=170pM. AM40 and several combined clones of mutations from the second round of sparing mutagenesis were merged, and a superior clone SC4017 (KD=37pM) was generated. This higher affinity is again reflected in the improved functional inhibition of FLT3L, as evidenced by downregulation of FLT3 on RS4;11 cells ( Fig.12 However, further analysis attributed the superior performance of SC4017 to a single additional tryptophan incorporated near the binding region. This represented a development risk given that exposed tryptophan residues are susceptible to oxidation. For this reason, AM40 was selected as the IgG lead clone. Although AM40 had a slightly lower affinity (170pM) compared to SC4017 (37pM), AM40 still exceeded the initial target of 300pM and was determined to have a lower development risk.

[0339] Finally, we demonstrated that both clones could neutralize endogenous FLT3L on the cell surface by using an assay developed using 20 ng / ml IL-7 stimulated primary T cells co-cultured with FLT3+RS4;11 for 7 days (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 co-cultured with RS4;11 cells at a ratio of 15:1 ( Fig.13 Dose ratio responses are shown in A) and incubated overnight. FLT3 downregulation was measured by flow cytometry as described above. Both clones were shown to completely prevent FLT3 downregulation on RS4;11 cells at concentrations above 1 nM with similar efficacy ( Fig.13 B).

[0340] Example No. 12. Neutralization of FLT3L in healthy non-human primates

[0341] To determine the safety and durability of AM40 in neutralizing FLT3L, a one-month toxicity study was conducted using repeated weekly dosing, including an eight-week treatment follow-up period to track animal progress. Fig.14 The research outline is described in Fig.15 As shown in A, free soluble FLT3L levels dropped dramatically after the first administration 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) showed complete target engagement (reflected by free soluble FLT3L below BLQ) throughout Day 57, at which time soluble FLT3L levels returned to the baseline of the 1.0 mg / kg group.

[0342] Similarly, measurement of circulating DC frequencies (% of total CD45+ cells detected by flow cytometry and expressed as a percentage of pre-study baseline levels) showed a steady decrease in the frequencies of CD1c+ (classical) DC and plasmacytoid DC in the 1.0 and 30 mg / kg groups throughout day 22 ( Fig.15 B). Circulating CD1c+DC frequency remained suppressed through 50 and 85 days for the 1 mg / kg group and the 30 mg / kg group, respectively. Circulating pDC frequency remained reduced through 71 and 85 days for the 1 mg / kg group and the 30 mg / kg group, respectively. The return of the DC population in the 1.0 mg / kg group was associated with the return of serum free FLT3L, which occurred at some point between days 57 and 85. These results suggest that when AM40 neutralizes FLT3L, the DC population is reduced, but when free FLT3L is available, the DC population quickly returns to baseline.

[0343] Example No. 13. FLT3L expression is associated with the severity of systemic lupus erythematosus (SLE)

[0344] SLE is an autoimmune disease characterized by chronic inflammation and can affect almost any organ in the body and all ages. SLE commonly affects the joints, skin, kidneys, lungs, heart, and brain. Given its role in proinflammatory signaling, the expression of FLT3L in individuals with SLE has been studied to look for correlations between FLT3L levels and disease severity. To date, published studies have relied primarily on serum FLT3L levels when inferring associations with disease, and although this is the most practical measurement in the clinical setting, it has an inherent disadvantage: it reflects production minus the uptake by DCs and other activated FLT3L-consuming cells. In an inflammatory setting, the number of cells expressing FLT3 and their consumption of FLT3L will vary greatly, and this likely explains the differences in findings between studies and why there is no evidence of a direct correlation between serum FLT3L and clinical scores of disease progression. Knowing that T cells are one of the main sources of FLT3L in inflammatory settings, we developed an assay that directly measures FLT3L expression on the surface of T cells using freshly isolated peripheral blood mononuclear cells (PBMCs).

[0345] Serum and PBMC 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, and the frequency of CD4+T cells expressing FLT3L was determined using an internally developed flow cytometric analysis, where FLT3L was detected using fluorescently labeled anti-FLT3L clone MAB608 (R&D Systems). The SLE Disease Activity Index (SLEDAI) was used to determine lupus activity in the same individual. Mann Whitney and Spearman correlations were used to compare the health cohort with the disease cohort and the correlation with SLEDAI to determine significance.

[0346] Consistent with previous literature, serum FLT3L levels were elevated in SLE donors compared with HD (p < 0.05; Fig.16 A), but no significant correlation was found with disease activity (SLEDAI) (p < 0.07; Fig.16 B). In contrast, when FLT3L production was measured by the frequency of FLT3L-expressing CD4+ T cells, the increase in SLE donors compared with HD was highly significant (p<0.0001; Fig.16 C), and had a strong correlation with the SLEDAI score (r = 0.7045; p < 0.0001; Fig.16D) This data suggests that measuring the expression of FLT3L on T cells may be particularly relevant in the disease setting.

[0347] After finding 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 in SLE patients was consistent with known biology and (2) whether subset expression correlated with SLEDAI score. The CD4+ subsets studied were naive T cells (T naive ), effector memory cells (T EM ) and central memory type (T CM ) cells. The same protocol and significance levels were used to study expression and correlation as described above.

[0348] FLT3L expression across CD4+ T cell subsets is consistent with the known biology of FLT3L expression. Specifically, although there was a small but significant increase in SLE donors, it was generally found in T cells of HD. naive No FLT3L expression was observed on the cells ( Fig.17 A, top panel). T from SLE donors naive FLT3L expression on cells was significantly correlated with SLEDAI score ( Fig.17 A, bottom panel; r = 0.6629; p = 0.0004). Importantly, in HD and SLE CD4+T EM FLT3L expression was observed in both HD and SLE donors, as expected in a population that captures recently activated T cells that have been exposed to α-chain cytokines known to induce FLT3L expression. Although this response was observed in both HD and SLE donors, the elevation in the latter was more pronounced and expression in SLE donors also correlated with SLEDAI ( Fig.17 B, bottom panel; r = 0.6201; p = 0.0012). Finally, since healthy CD4+ T cells are derived from T EM Differentiation into T CM , in which FLT3L expression was decreased, but still maintained in PBMCs of SLE donors ( Fig.17 C, top panel; p < 0.0001). Likewise, there was a significant correlation between FLT3L+ T cell frequency and SLEDAI, suggesting that expression in this group reflects a chronic inflammatory state.

[0349] Overall, these studies show that FLT3L expression on CD4+T cells in SLE patients is significantly increased compared with HD. Moreover, the increase in FLT3L expression is highly correlated with the SLEDAI score. Therefore, administering anti-FLT3L antibodies to SLE patients is a reasonable therapeutic strategy for reducing the T cell population expressing FLT3L to reduce inflammation in SLE patients.

[0350] The method used above was validated using PrimeFlow in situ detection of IC FLT3L RNA and confirmed using APC-conjugated AM40.

[0351] Example No. 14. FLT3L expression in myositis

[0352] Myositis is a chronic muscle inflammation characterized by weakness, swelling and muscle pain. At the cellular level, myositis is characterized by elevated levels of type 1 interferon protein and pDC. Myositis can be associated with SLE and other proinflammatory conditions. Therefore, the expression of FLT3L in individuals with myositis was studied to find the correlation between FLT3L levels and disease severity.

[0353] Using FACS, PBMCs from individuals with myositis and HD were investigated for CD4+ T cells expressing FLT3L. naive , T EM and T CM Subsets. Mann-Whitney analysis was used to determine significance between myositis and HD samples.

[0354] like Fig.18 As shown in Figures A and 18B, the expression of FLT3L in PBMCs from individuals with myositis parallels the findings from SLE patients, as they are characterized by a significant increase in the percentage of FLT3L-positive CD4+ T cells (T naive (p<0.05; Fig.18 A), T EM (p<0.0001; Fig.18 B) and T CM (p<0.0001; Fig.18 C)). Based on these results, administration of anti-FLT3L antibodies to myositis patients is a rational therapeutic strategy for reducing the population of T cells expressing FLT3L to reduce inflammation in myositis patients.

[0355] Example No. 15. FLT3L expression in nephritis

[0356] Nephritis is an immune disorder that affects the kidney and related renal structures. The disease can be derived from SLE, certain toxins or certain infections. Nephritis can lead to permanent loss of renal function, which can be fatal. Dendritic cells have been shown to infiltrate the kidneys in lupus nephritis (Fiore et al., (2008) Mol Immunology [molecular immunology] v45: 259-265), and are believed to play a role in driving renal inflammation, therefore, it is speculated that the blocking of FLT3L can inhibit DC and prevent the progressive loss of renal function.

[0357] The MurphyRoths Large / lymphoproliferative (MRL.lpr) nephritis mouse model and mouse surrogate anti-FLT3L antibody (LFC-1) were used to investigate the effects of FLT3L blockade on proteinuria levels and nephritis scores. Isotype controls as well as anti-IFNAR antibody treatment groups were included. Mice administered anti-FLT3L antibody showed a significant reduction in proteinuria at 17 weeks post-administration ( Fig.19 In addition, mice administered anti-FLT3L antibody had reduced nephritis scores at week 18 compared to isotype controls ( Fig.19 B). Importantly, mice administered with anti-FLT3L antibodies had reduced proteinuria and reduced nephritis scores compared to mice administered with . These results support the role of FLT3L-mediated inflammation in nephritis. Based on these results, administration of anti-FLT3L antibodies to patients with nephritis is a reasonable therapeutic strategy for reducing the population of T cells expressing FLT3L to reduce inflammation in patients with nephritis.

[0358] Splenic DC populations were also examined to provide insight into FLT3L-associated changes in leukocyte populations in nephritis. Spleens were harvested from MRL mice at week 18 and examined for changes in splenic DC populations. Anti-FLT3L antibody treatment significantly reduced circulating DCs in MRL mice ( Fig. 20 AC). Specifically, Siglec-H+-pDCs were significantly reduced compared to isotype controls after administration of anti-FLT3L antibodies ( Fig. 20 A). Similarly, a significant decrease was observed in CD11b+cDC (equivalent to human CD1c+DC) and CD8+cDC (equivalent to human CD141+DC) ( Fig. 20 B and 20C). In addition, no dermatitis was observed in anti-FLT3L treated mice, compared with the usual 30%-40% incidence in untreated mice. Therefore, anti-FLT3L antibody treatment can reduce the circulating DC population in the nephritis model and improve the secondary pathology (dermatitis). According to these results, the administration of anti-FLT3L antibodies to nephritis patients can reduce inflammation and tissue damage by suppressing the DC population.

[0359] Example No. 16. FLT3L expression in Sjögren's syndrome

[0360] Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by extensive dryness of the eyes and salivary glands. In addition, the disease can lead to multi-organ dysfunction. The syndrome can occur alone or in the presence of other autoimmune diseases (such as lupus or rheumatoid arthritis). Serum FLT3L levels in individuals with pSS are elevated, and there is evidence that FLT3L and its receptors are locally expressed in inflamed salivary glands (Tobon et al., (2010) Arthritis and Rheumatism v62: 344).

[0361] The NOD.H2h4 dry mouse model was used to study the pathology of the salivary glands after prolonged anti-FLT3L antibody (LFC-1) treatment. By 16 weeks of age, these mice formed tertiary lymphoid structures (TLS) in the salivary glands (SG), which mainly contained DCs, B220+B cells and CD3+T cells, in a manner very similar to the pathological changes observed in humans. This tissue damage occurs before the development of autoantibodies and the formation of spontaneous development centers in the spleen (Mahmoud et al., 2016 Science Translational Medicine, v8361ra137). Mice were treated with a preventive regimen (starting at 5 weeks of age) or a therapeutic regimen (starting at 17 weeks of age) containing an isotype IgG control (5 mg / kg) and an anti-FLT3L antibody (5 mg / kg). For both regimens, treatment was required twice a week until the end of the study (26 weeks). Anti-FLT3L monoclonal antibody (LFC-1) effectively neutralizes FLT3L throughout the treatment course ( Fig.25 A) and leads to accumulation of circulating drug / ligand complexes ( Fig.25 B).

[0362] Lymphoid organs were collected to assess changes in peripheral immune cell populations, and salivary glands (SGs) were harvested and assessed for histopathology (TLS frequency). Although prophylactic treatment has been previously reported to prevent disease onset, there are limited reports of tissue damage that can be delayed or prevented by therapeutic intervention after disease onset. Anti-FLT3L monoclonal antibody (LFC-1) reduced antigen-experienced CD44 in spleen and salivary gland-draining LNs (24-26 weeks of age at the end of the study). HI The frequency of CD4+ and CD8+ T cells ( Fig.26 A-26D), and selective reduction of specific autoantibodies against collagen IV and platelet extracts ( Fig. 27 ).

[0363] As expected, animals treated with isotype control IgG showed increased formation of TLS in the salivary glands by 26 weeks of age (measured as frequency / mm 2 tissue), indicating salivary gland damage. Even when administered therapeutically, mice treated with anti-FLT3L had significantly reduced tissue SG damage ( Fig.21 A), and when given prophylactically, the disease is completely prevented ( Fig.21 B). Although not completely absent, the DC population as measured in the spleen was significantly suppressed ( Fig. 22 AD). Nevertheless, the reduction in inflammatory infiltration to the salivary glands was sufficient to have a significant impact on the onset and progression of the disease. These results support that inflammation in pSS is driven by a FLT3L-mediated mechanism. Based on these results, administration of anti-FLT3L antibodies is a reasonable therapeutic strategy for treating pSS in human subjects.

[0364] It will be clear from the foregoing description that changes and modifications can be made to the disclosure described herein to make it suitable for various uses and situations. Such embodiments are also within the scope of the following claims. The statement of a list of elements in any definition of a variable includes defining the variable as any single element or a combination (or sub-combination) of the listed elements herein. The statement of the embodiments herein includes the embodiments as any single embodiment or in combination with any other embodiment or part thereof. All patents and publications mentioned in this specification are incorporated herein by reference to the extent that each independent patent and publication is specifically and individually indicated to be incorporated herein by reference.

Claims

1. Use of a human monoclonal antibody in the preparation of a medicament for treating an autoimmune disease, wherein the human monoclonal antibody comprises: (1) a heavy chain comprising: (i) a variable region (VH) comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a human IgG1 heavy chain constant domain; and (2) A light chain comprising: (i) a variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2, and (ii) a human lambda light chain constant domain.

2. The use according to claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 61, and the light chain comprises the amino acid sequence of SEQ ID NO:

62.

3. The use according to claim 1, wherein the autoimmune disease is associated with an increase in the level of CD4+ T cells expressing FLT3L.

4. The use according to any one of claims 1 to 3, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

5. The use according to any one of claims 1 to 3, wherein the autoimmune disease is Sjögren's syndrome.

6. The use according to any one of claims 1 to 3, wherein the autoimmune disease is arthritis.

7. The use according to any one of claims 1 to 3, wherein the autoimmune disease is rheumatoid arthritis.

8. The use according to any one of claims 1 to 3, wherein the autoimmune disease is nephritis.

9. The use according to any one of claims 1 to 3, wherein the autoimmune disease is multiple sclerosis.

10. The use according to any one of claims 1 to 3, wherein the autoimmune disease is psoriasis.

11. The use according to claim 1, further comprising administering a second immunomodulator to the subject.

12. A human monoclonal antibody comprising: (1) a heavy chain comprising: (i) a variable region (VH) comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a human IgG1 heavy chain constant domain; and (2) A light chain comprising: (i) a variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2, and (ii) a human lambda light chain constant domain.

13. The antibody of claim 12, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 61, and the light chain comprises the amino acid sequence of SEQ ID NO:

62.

14. A pharmaceutical preparation comprising the antibody according to claim 12 or 13, further comprising a pharmaceutically acceptable excipient.

15. The pharmaceutical formulation according to claim 14, wherein the excipient comprises a surfactant, a stabilizer, an isotonicity agent, a buffer or any combination thereof.

16. A nucleic acid encoding a human monoclonal antibody, the antibody comprising: (1) a heavy chain comprising: (i) a variable region (VH) comprising the amino acid sequence of SEQ ID NO: 1, and (ii) a human IgG1 heavy chain constant domain; and (2) A light chain comprising: (i) a variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2, and (ii) a human lambda light chain constant domain.

17. The nucleic acid of claim 16, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:61, and the light chain comprises the amino acid sequence of SEQ ID NO:

62.

18. A host cell comprising the nucleic acid according to claim 16 or 17.

19. The host cell according to claim 18, wherein the host cell is a Chinese Hamster Ovary (CHO) cell or a HEK293 cell.

20. A method for preparing the antibody according to claim 11 or 12, comprising (a) culturing a host cell expressing the antibody; and (b) isolating the antibody from the cultured host cell.

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