Antibodies to feline mcdonough sarcoma (FMS)-like tyrosine kinase 3 receptor ligand (FLT3l) and uses thereof for treating autoimmune and inflammatory diseases

Abstract

To provide antibodies to feline McDonough sarcoma (FMS)-like tyrosine kinase 3 receptor ligand (FLT3L) and uses thereof for treating autoimmune and inflammatory diseases.SOLUTION: Provided herein are anti-FLT3L antibodies and methods of using the antibodies to treat autoimmune and other inflammatory diseases. Provided are techniques related to antibodies to feline McDonough sarcoma (FMS)-like tyrosine kinase 3 receptor ligand (FLT3L) and uses thereof for treating autoimmune and inflammatory diseases.SELECTED DRAWING: None

Description

[Technical field]

[0001] Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 receptor ligand (FLT 3L) AND THEIR USE TO TREAT AUTOIMMUNE AND INFLAMMATORY DISEASES Related techniques are provided. [Background technology]

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

[0003] Systemic lupus erythematosus (SLE) is characterized by joint pain, swollen lymph nodes, and a butterfly-shaped rash on the cheeks. In SLE, autoantibodies against healthy tissues attack the patient's immune system, causing inflammation. At the cellular level, SLE patients develop autoreactive T cells driven by dendritic cells. Cells and B cells (Palucka AK et al. Immunology and Cell Biology (2002) 80:484-488). The syndrome is characterized by systemic chronic inflammation of the exocrine organs, ultimately resulting in organ dysfunction. (Holdgate N. and St.Clair EW, F1000 Res search.1412 10.12688 / f1000 research.8352. 1).

[0004] Multiple sclerosis (MS) is a disease caused by demyelination of nerve cells in the brain and spinal cord and the central nervous system (CNS). Psoriasis is an autoimmune disease that manifests as red, sensitive skin with patches of redness. Rheumatoid arthritis (RA) is a chronic arthritis characterized by persistent synovitis and destruction of cartilage and bone within the joints. It is an inflammatory disorder of the synovial tissue. The damage can progress to impact many body systems. Lupus nephritis is associated with systemic lupus erythematosus and causes inflammation of the kidneys. When this occurs, the kidneys leak protein, which can eventually lead to kidney failure. Blindness is a group of inflammatory diseases that attack and destroy tissues in the eyes and can cause blindness.

[0005] Furthermore, acute and chronic proinflammatory states have been linked to a myriad of diseases in individuals; Examples of specific diseases that may cause them include type 1 and type 2 diabetes, e.g. diabetes mellitus, Chronic inflammation, including chronic kidney disease (CKD), including pathologic nephropathy and hypertension-induced CKD Arteriosclerosis, Alzheimer's disease, cancer and heart disease, high blood pressure, anemia, pericarditis, renal osteoporosis It is believed to be associated with the associated complications of such diseases, including dystrophies. In diseases associated with chronic inflammation, as well as immune disorders, the body faces debilitating and frequently fatal complications. This appears to promote an excessive and persistent proinflammatory response that may lead to morbid disease.

[0006] The causes of autoimmune diseases are not clearly understood. Mechanistically, the underlying Autoimmune diseases are driven by complex regulatory systems that continually energize self-reactive immune cells. An ongoing autoimmune response that is inhibited (and / or uninhibited). A similar mechanism exists for the non-autoimmune It is believed that the IL-1 receptor also plays a role in chronic inflammatory diseases. Therapeutic interventions for chronic inflammation involve the myriad regulatory systems, signaling cascades and their structures. The study targeted the components of the drug.

[0007] One class of putative therapeutic targets involves the binding of distinct growth factors and proteins to cells. These include tyrosine kinase receptors (TKRs), which are transmembrane receptors that regulate homeostasis. The more than 50 known human TKRs are classified into 20 distinct subtypes defined by their genetic lineage. Classified as 1-class (Robins DR, et al. Oncogene. (20 00)19:5548-5557; Lemmon MA and Schlessinge r J.Cell.(2010)141:1117-1134). TKR class III is 5-7 immunoglobulin-like domains in the extracellular segment containing 70-100 hydrophilic residues In class III TKR, feline McDonough sarcoma is characterized by the presence of The (FMS)-like tyrosine kinase 3 receptor (FLT3) is a transcription factor that mediates the proliferation and differentiation of human stem cells, hematopoietic cell precursors, and Membrane-bound expressed on dendritic cells, activated T and B cells, monocytes, and microglia FLT3 is a receptor for several cells, including activated T cells, activated endothelium, and bone marrow stromal cells. It binds to FLT3 ligand (FLT3L), a hematopoietic cytokine expressed by various cell types. FLT3L is expressed as both a cell surface and secreted homodimer and is expressed as a cytoplasmic homodimer that binds its cognate receptor. FLT3 signals through the cytoplasmic endothelial cell membrane (FLT3), which is expressed on the cell surface as a monomer. It is expressed and activated when ligated with FLT3L. After ligation of FLT3L FLT3 dimerizes, autophosphorylates, and binds to RAS / extracellular signal-regulated kinase (ERK). K), phosphatidylinositide 3-kinase (PI3K) and signal transduction factors, and Activates signal transduction pathways including STAT3 and STAT5. After dimerization, the dimerized FLT3 is internalized and degraded.

[0008] FLT3L mediates inflammatory signals, particularly □-chain cytokines: IL-2, IL-7, and IL It is produced in response to IFN-15 and its interaction with FLT3 is primarily involved in the differentiation, proliferation and survival of DCs. It also drives inflammatory processes through its role in the regulation of inflammatory processes by transiently upregulating the receptor. Both cell types have been reported to regulate T and B cell survival after activation. It has also been suggested that FLT3 signaling plays a role in t al., J. Immunology. 2010 v184:685-93 and Tob on et al.Arthritis&Rheumatism.2010;62(11 Furthermore, NK cell survival requires DC-derived IL-15. Although this observation is thought to be indirectly dependent on FLT3L via Guimond M et al., J.Immunology 2010;184 :2769-75) and remains unproven in humans.

[0009] DCs are essential for the immune system to function properly, as they migrate from the site of inflammation to the lymph nodes and ultimately produce autoimmune disease. These are particularly important in inflammation because they are sentinels of the immune system that mount the adaptive immune responses required for the defense against inflammation. Broadly speaking, DCs are divided into two subsets: myeloid / classical dendritic cells (cDCs) and phenotype-specific dendritic cells (PDCs). cDCs are cytoplasmic dendritic cells (pDCs). cDCs are inflammatory cytokines (e.g., IFN- III, IL-23, IL-12, IL-6 and IL-1□) and are involved in costimulation. They present antigens to T cells, secrete chemokines that recruit cells to the site of inflammation, and act as critical cell- These mechanisms ensure that they colocalize when necessary for cell interactions. Through this, cDCs stimulate neutrophils, B cells, T cells, and NK cells, resulting in NE. Tosis, autoantibody production, IL-17 production, and additional inflammatory cytokine production. pDCs are key sites in the innate response that enhance activation of all arms of the immune system. It is the major source of type I IFN, a kinase.

[0010] Salivary glands from patients with Sjögren's syndrome express FLT3 and FLT3L on infiltrating B cells. Expression is shown (Tobon et al. Arthritis & Rheumatism. 2010)62(11):3447-3456). Furthermore, patients with Sjögren's syndrome have The frequency of FLT3-expressing B cells in the ring was increased, and their survival was significantly decreased compared with that of FLT3L-expressing B cells. The expression of IL-1 in individuals with MS is enhanced when co-cultured with human salivary gland cells expressing IL-1. FLT3 protein is expressed in vascular lesions as well as in gray and white matter (DeBoy C. A.et al.Exp Mol Pathol.(2010);89(2):109- 116) Furthermore, FLT3 has been shown to increase the infiltration of FLT3-positive DCs into the brains of individuals with MS. They colocalize with immature DCs in the perivascular brain (Deboy et al.). In A, FLT3L levels in synovial fluid are increased compared to healthy individuals. Mononuclear leukocytes, NK cells, and DCs express high levels of FLT3L (Ramos M .et al.Arthritis Res Ther.(2013)15(6):R2 09).

[0011] Furthermore, increased levels of FLT3L in serum and at the site of inflammation are associated with SLE, myositis, and primary sheath inflammation. It has been reported in Anderson-Gren syndrome, MS, uveitis and RA (Andersson et al., 2013). n et al.PLoS One(2012)7:e47668;DeBoy et al. al.Exp and Mol Path(2010)89:109-16).

[0012] Thus, FLT3-mediated proinflammatory survival (e.g., via pDCs and mDCs) is a key regulator of healthy A beneficial physiological response in the individual that has deleterious effects in autoimmune diseases. Therefore, the disruption and attenuation of the FLT3 / FLT3L signaling pathway is likely to be , an important tool for combating autoimmune and other inflammatory diseases and reducing inflammation I was able to prove that it was. Summary of the Invention [Means for solving the problem]

[0013] As described herein, the present invention relates to a method for controlling autoimmune diseases and other acute and / or chronic inflammatory diseases. Novel FLT3L-binding antibodies are provided.

[0014] In a first aspect, the present disclosure provides a set of complementarity determining regions (CDRs): HCDR1, HCDR2 , which specifically binds to FLT3L, including HCDR3, LCDR1, LCDR2 and LCDR3. The present invention provides an isolated antibody or antigen-binding fragment thereof that binds to an HCDR1, HCDR2, HC DR3, LCDR1, LCDR2 and LCDR3 are (a) SEQ ID NOs: 29, 30, , 31, 32, 33 and 34; or (b) SEQ ID NOs: 29, 30, 31, 35, 3 3 and 34; or (c) the amines of SEQ ID NOs: 29, 36, 37, 32, 33 and 38, respectively. It contains the amino acid sequence.

[0015] In one embodiment of the first aspect, the isolated antibody or antigen-binding fragment thereof comprises: (a) SEQ ID NO:1 and SEQ ID NO:2; or (b) SEQ ID NO:3 and SEQ ID NO:4, respectively; or (c) SEQ ID NO: 5 and SEQ ID NO: 6 with at least 95%, 96%, 97%, 98% or It contains a heavy chain variable region (VH) and a light chain variable region (VL) with 9% sequence identity. In an embodiment, the VH and VL are (a) SEQ ID NO: 1 and SEQ ID NO: 2, respectively; or (b) (c) SEQ ID NO:5 and SEQ ID NO:6, respectively; In another embodiment, the isolated antibody or antigen-binding fragment comprises: (a) a heavy chain region comprising SEQ ID NO:61. and a light chain region comprising SEQ ID NO: 62; or (b) a heavy chain region comprising SEQ ID NO: 65 and 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 comprises the FL chain region of FLT3. In another embodiment, the isolated antibody or antigen-binding fragment thereof inhibits T3L-mediated activation. In one embodiment, the isolated antibody or The antigen-binding fragment does not cross-react with at least one of huSCF and huCSF1. In a further embodiment, the isolated antibody or antigen-binding fragment thereof is huSCF or huCSF1. In one embodiment, the isolated antibody or antigen-binding fragment thereof does not cross-react with any of the following: The antibody may be a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody or a chimeric antibody. In embodiments, the isolated antibody or antigen-binding fragment comprises: (a) an IgA constant domain; (b) an IgD constant domain; (c) IgE constant domain; (d) IgG1 constant domain; (e) IgG2 (f) an IgG3 constant domain; (g) an IgG4 constant domain; and (h) The heavy chain immunoglobulin constant domain is selected from the group consisting of IgM constant domains. In one embodiment, the isolated antibody or antigen-binding fragment comprises an IgG1 constant domain. In embodiments, the isolated antibody or antigen-binding fragment comprises: (a) an Igκ constant domain; and (b) an Igκ constant domain. g λ constant domain. 1 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 the EU numbering of Kabat L234F, L235E and P331S numbered according to the ring index (Edelman et al. ., Proc. Natl. Acad. Sci., 63:78-85 (1969)).

[0016] In a second aspect, the disclosure provides an isolated antibody or a method for the treatment of a cancer, comprising administering to said cancer patient an ... An isolated nucleic acid molecule encoding the antigen-binding fragment is provided. In this case, the nucleic acid molecule is operably linked to a regulatory sequence.

[0017] In a third aspect, the disclosure comprises a nucleic acid molecule according to the second aspect and / or embodiments thereof. A vector containing the vector is provided.

[0018] In a fourth aspect, the present disclosure relates to a nucleic acid molecule according to the second aspect and / or embodiments thereof. Alternatively, a host cell transformed with a vector according to the third aspect is provided. In one embodiment, the host cell is a mammalian host cell. In another embodiment, the host cell is a H EK293 cells, NS0 mouse myeloma cells or Chinese hamster ovary (CHO) cells It is a cell.

[0019] In a fifth aspect, the present disclosure provides an antibody or a method for the treatment of a disease comprising administering to said patient ... provides hybridomas producing antigen-binding fragments.

[0020] In a sixth aspect, the present disclosure provides an antibody or a method for the treatment of a disease comprising administering to said patient a therapeutically effective amount of said antibody or ... An isolated host cell that produces the antigen-binding fragment is provided.

[0021] In a seventh aspect, the present disclosure provides a method for producing a cellular membrane according to any one of the first to sixth aspects or embodiments thereof. 1. A method for making an antibody or antigen-binding fragment thereof, comprising: (a) contacting said antibody or antigen-binding fragment with Culturing the expressing host cell or the host cell of the third aspect or embodiment thereof. or the fourth aspect; and (b) culturing the cultured hybridoma. isolating the antibody or antigen-binding fragment thereof from the host cell.

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

[0023] In a ninth aspect, the present disclosure provides an antibody or a method for the treatment of a disease comprising administering to said patient a therapeutically effective amount of said antibody or ... provides a pharmaceutical composition comprising the antigen-binding fragment and a pharma- ceutical acceptable excipient. In an embodiment, the pharmaceutical composition is provided for use as a medicament.

[0024] In a tenth aspect, the present disclosure provides a method for treating an acute or chronic inflammatory disease, comprising: and administering to a subject in need thereof a pharma- ceutical effective amount of any of the first to ninth aspects or embodiments thereof. The present invention provides a method comprising administering an isolated antibody or fragment thereof according to an embodiment. In embodiments, the inflammatory disease is, for example, diabetes, diabetic nephropathy, and hypertension induced. This includes chronic kidney disease (CKD), including CKD that has been diagnosed as

[0025] In an eleventh aspect, the present disclosure provides a method for treating an autoimmune disease, comprising administering to said patient a therapeutically effective amount of A pharma- ceutical effective amount of any one of the first to tenth aspects or embodiments thereof may be administered to a subject in need thereof. In one embodiment, the present invention provides a method for treating a patient having a rheumatoid arthritis, comprising administering an isolated antibody or fragment thereof according to the present invention. So, autoimmune diseases include systemic lupus erythematosus, myositis, primary Sjogren's syndrome, and many others. In some cases, the conditions may include rheumatoid sclerosis, uveitis, psoriasis or rheumatoid arthritis.

[0026] These and other features and advantages of the present disclosure are set forth in the following detailed description of the present disclosure together with the appended claims. The scope of the claims will be defined by the detailed description therein. defined by the particular considerations of the features and advantages described herein. Please note that this is not the case. [Brief description of the drawings]

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

[0036] Figure 1. Outline of a study on neutralization of FLT3L in healthy cynomolgus monkeys. Three groups of male cynomolgus monkeys (n=4 per group) were administered 0.03, 1 or 30 mg / kg AM40 (MEDI1116) as indicated, 5 doses per week for one month. An 8-week follow-up period after the last dose was used to determine the persistence of the administered antibody and its effect on circulating DC populations. [Figure 15A-15B] Serum FLT3L protein and frequency of circulating DCs following administration of anti-FLT3L antibody (AM40 / MEDI1116). Figure 15A shows target engagement by MEDI1116, measuring free serum FLT3L levels following administration at 0.03, 1.0 and 30 mg / kg. Daily serum measurements were obtained from days 1-8, followed by weekly measurements up to day 85. Figure 15B shows the decrease and recovery of circulating CD1c+ (classical DCs) frequency (left) and plasmacytoid DC frequency (right), measured as a percentage of baseline, following treatment with MEDI1116. [Figures 16A-16D]Measures of the correlation between FLT3L expression and SLEDAI score in SLE patients comparing serum measurements of T cells and flow cytometry analysis. Figure 16A shows FLT3L levels in serum of healthy donors (HD) and SLE patients. Figure 16B shows the correlation between serum FLT3L and SLEDAI score. Figure 16C shows the frequency of circulating FLT3L+ T cells in healthy donors (HD) and SLE patients. Figure 16D shows the correlation between FLT3L+ CD4+ T cells and SLEDAI score. [Figures 17A-17C] FLT3L expression in CD4+ T cell subsets and SLEDAI score. Figure 17A shows the percentage of CD4 Tnaive cells expressing FLT3L in HD and SLE patients. The lower panel shows the correlation between CD4 Tnaive cells expressing FLT3L in SLE patients and SLEDAI score. Figure 17B shows the percentage of CD4 TMEM cells expressing FLT3L in HD and SLE patients. The lower panel shows the correlation between CD4 TMEM cells expressing FLT3L in SLE patients and SLEDAI score. Figure 17C shows the percentage of CD4 TCM cells expressing FLT3L in HD and SLE patients. The lower panel shows the correlation between CD4 Tnaive cells expressing FLT3L in SLE patients and SLEDAI score. [Figures 18A-18C] FLT3L expression in PBMC CD4 subsets from individuals with myositis. Figure 18A shows the percentage of CD4 Tnaive cells expressing FLT3L in HD and myositis patients. Figure 18B shows the percentage of CD4 TMEM cells expressing FLT3L in HD and myositis patients. Figure 18C shows the percentage of CD4 TCM cells expressing FLT3L in HD and myositis patients. [Figure 19A-19B] Figure 19 shows proteinuria and nephritis scores in MRL mice. Figure 19A shows the reduction in proteinuria after 17 weeks of anti-FLT3L administration. Figure 19B shows the nephritis score after 18 weeks of anti-FLT3L administration. [Figures 20A-20C]The splenic dendritic cell population in MRL mice. Figure 20A shows the change in frequency of CD11+siglec-H+pDC after administration of anti-FLT3L antibody. Figure 20B shows the frequency of CD11c+CD11b+mDC after administration of anti-FLT3L antibody. Figure 20C shows the frequency of CD11c+CD8+mDC after administration of anti-FLT3L antibody. [Figure 21A-21B] Pathology score of salivary gland in NOD.H2h4 Sjogren's syndrome mouse model. Figure 21A shows the change in salivary gland pathology in NOD.H2h4 Sjogren's syndrome mouse model after therapeutic administration of anti-FLT3L antibody against isotype control. Figure 21B shows the change in salivary gland pathology in NOD.H2h4 Sjogren's syndrome mouse model after prophylactic administration of anti-FLT3L antibody against isotype control. [Fig. 22A-22D] Changes in the presence of dendritic cells after administration of anti-FLT3L antibody. Figures 22A and 22B show changes in the frequency of plasmacytoid DCs (B220+CD11c+Siglec-H+) after administration of anti-FLT3L antibody, as demonstrated by flow cytometry (A) and quantification (B). Figures 22C and 22D show changes in the frequency of classical DCs (B220negCD11cHI) after administration of anti-FLT3L antibody, as demonstrated by flow cytometry (C) and quantification (D). [Figures 23A-23C] The PK of anti-FLT3L antibody (MEDI1116) in cynomolgus monkeys correlates with functional neutralization of FLT3L, as evidenced by suppression and restoration of pDC. Anti-FLT3L antibody (MEDI1116) was administered to cynomolgus monkeys once a week on days 1, 8, 15, 22, and 29, as indicated by the arrows. Figure 23A shows the PK of anti-FLT3L antibody (MEDI1116). Figure 23B shows the soluble FLT3L levels when anti-FLT3L antibody (MEDI1116) was administered at doses of 0.03 mg / kg, 1.0 mg / kg, and 30 mg / kg. Figure 23C shows the frequency of pDC measured as a percentage of baseline when anti-FLT3L antibody (MEDI1116) was administered at doses of 0.03 mg / kg, 1.0 mg / kg, and 30 mg / kg. [Figures 24A-24C] The human dosing model for anti-FLT3L antibody (MEDI1116) projects Q4W dosing. Figure 24A shows the PK of anti-FLT3L antibody (MEDI1116) in cynomolgus monkeys. Figure 24B shows the PD of anti-FLT3L antibody (MEDI1116) in cynomolgus monkeys. Figure 24C shows the predicted PD of anti-FLT3L antibody (MEDI1116) in humans. [Fig. 25A-25B] Anti-FLT3L monoclonal antibody (LFC-1) effectively neutralizes FLT3L throughout the course of treatment, resulting in the accumulation of circulating drug / ligand complexes. Figure 25A shows free serum FLT3L levels after administration of anti-FLT3L antibody and isotype control antibody. Free FLT3L levels were measured using FT3L-IgG as a capture reagent and sulfo-labeled anti-mouse FLT3L polyclonal antibody as a detection reagent. Figure 25B shows total (free and bound) serum FLT3L levels after administration of anti-FLT3L antibody and isotype control antibody. Total FLT3L levels were measured using polyclonal anti-mouse FLT3L antibody for capture and detection. [Figures 26A-26D]Blockade of FLT3L suppresses T cell activation in spleen and SG-draining lymph nodes (LNs) of aged NOD-H2h4 mice. Blockade of FLT3L with anti-FLT3L monoclonal antibody (LFC-1) results in a decrease in the frequency of antigen-experienced CD44HI CD4+ and CD8+ T cells in spleen and salivary gland-draining LNs (at the end of the study at 24-26 weeks of age). Bar graphs were derived from flow cytometry analysis of spleen and draining LNs. Each bar represents the mean ± standard error of the mean (SEM) of n=4-5 mice. Figure 26A shows the CD44HI CD4+ T cell population in the spleen after administration of anti-FLT3L and isotype control antibodies. Figure 26B shows the CD44HI CD4+ T cell population in the LNs after administration of anti-FLT3L and isotype control antibodies. Figure 26C shows the CD44HI CD8+ T cell population in the spleen after administration of anti-FLT3L antibody and isotype control antibody, and Figure 26D shows the CD44HI CD8+ T cell population in the LN after administration of anti-FLT3L antibody and isotype control antibody. [Figure 27] Therapeutic anti-FLT3L blockade selectively reduces two serum IgG autoantibody specificities, as measured in matched serum samples by the UTSW IgG autoantibody assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The present invention provides an isolated antibody, or antigen-binding fragment thereof, that specifically binds to FLT3L. In some aspects, such molecules include antibodies that specifically bind to FLT3L and their antigen binding sites. In one embodiment, the anti-FLT3L antibody disclosed herein is an anti-inflammatory cytokine. Inhibiting or reducing FLT3 / FLT3L binding to inhibit activation of the signal transduction pathway Such an approach could be used to attack inflammation at the source of signaling, This is beneficial for allowing for a stronger anti-inflammatory therapeutic effect. Related polynucleotides, vectors and pharmaceutical compositions comprising the binding fragments are also provided. Disclosed herein are methods for producing anti-FLT3L antibodies and antigen-binding fragments, as well as methods for the preparation of such antibodies and antigen-binding fragments. Methods of Use, E.g., Methods of Treating Autoimmune and / or Chronic Inflammatory Disease in a Subject Treatment with serotonin (direct therapy, as adjuvant therapy or in combination therapy) is also contemplated.

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

[0030] definition Before describing the present invention in detail, it is to be understood that the particular compositions or process steps may vary. Therefore, it should be understood that the present invention is not limited to any particular composition or process steps. Unless otherwise defined, all technical and scientific terms used herein are It has the same meaning as commonly understood by a person skilled in the art to which this disclosure pertains. The following references provide one of ordinary skill in the art with general definitions of many of the terms used in this disclosure: Singleton et al.,Dictionary of Microbiol ogy and Molecular Biology(2nd ed.1994);T he Cambridge Dictionary of Science and T echnology(Walker ed.,1988);The Glossary of Genetics, 5th Ed., R. Rieger et al.(eds. ), Springer Verlag (1991); and Hale & Marham, Th e Harper Collins Dictionary of Biology(1 991). As used herein, the following terms shall mean the following unless otherwise specified: have meanings assigned to them.

[0031] As used in this disclosure, the term "antibody" (or a fragment, variant, or derivative thereof) refers to, e.g., For example, in the context of a typical antibody produced by a B cell, an antigen, e.g., at least one The variable domain of one heavy chain (VH) and one light chain (VL) can be bound to The basic antibody structure in vertebrate systems is It is relatively well understood. For example, Harlow et al., Antibodies es:A Laboratory Manual,(Cold Spring Harb) or Laboratory Press, 2 nd ed., 1988). The antibody or antigen-binding fragment, variant or derivative thereof may be a polyclonal antibody, a monoclonal antibody, or a Monoclonal antibodies, human antibodies, humanized antibodies or chimeric antibodies, epitope-binding fragments (e.g. , Fab, F(ab') 2 , Fv, single chain Fv (scFv), single chain antibody, disulfide sdFv), fragments containing either the VL or VH domain (Fd), Fab The fragments and antigen-binding functions generated by the expression library, i.e., specific for FLT3L, Other antibody fragments and combinations thereof that retain the ability to bind differently are also included. The present invention is not limited to these.

[0032] A typical antibody consists of at least two heavy (H) amino acids interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (heavy or VH) and two light (L) chains. H and The heavy chain constant region is composed of three domains, C Each light chain is composed of a light chain variable region (herein referred to as VL or is V L The light chain constant region is composed of one domain, The VH and VL regions are composed of the more conserved framework regions, The complementarity determining regions (FW) are sandwiched between hypervariable regions called CDRs. Each VH and VL may be further subdivided into domains, in the following order from amino terminus to carboxy terminus: Three Cs arranged as FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4 The variable regions of the heavy and light chains are the domains that interact with antigens. The constant region of an antibody contains the binding domains of the immunoglobulin that bind to various cells of the immune system (e.g. For example, effector cells) and host tissues or factors that contain the first component of the classical complement system (Clq). Exemplary antibodies of the present disclosure include anti-FLT3L antibodies (original and and germline antibodies), affinity optimized clones, optimized antibodies lacking ADCC, conjugated Antibodies (e.g., ADCs) and other optimized antibodies (e.g., serum half-life including YTE mutations) Optimized antibodies (Dall et al., supra, which are incorporated herein by reference in their entirety). 'Acqua et al.,J.Biol.Chem.281:23514-24(2 006) and U.S. Pat. No. 7,083,784).

[0033] In certain embodiments, the CDRs (HCDR1, HCDR2 and HCDR3) of VH and The CDRs of VL (LCDR1, LCDR2 and LCDR3) are (a) SEQ ID NO: 2, 9, 30, 31, 32, 33 and 34; or (b) SEQ ID NOs: 29, 30, 31, 35, 33 and 34; or (c) SEQ ID NOs: 29, 36, 37, 32, 33 and 3, respectively. It consists of a sequence of 8 amino acids.

[0034] Antibodies are classified according to the identity of their heavy chain constant domains, which are designated α, δ, ε, γ and μ, respectively. Based on the above, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgE. gM or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3 The different classes of immunoglobulins can be either IgG1, IgG2, IgA1, or IgA2. Antibodies have different and well-known subunit structures and three-dimensional configurations. The antibodies may be naked antibodies. or can be conjugated to other molecules, such as toxins, radioisotopes, etc. to form ADCs. .

[0035] A "blocking" or "antagonist" antibody is an antibody that blocks a target protein, such as FLT3L, to which it binds. In certain embodiments, blocking antibodies or An antagonist antibody substantially or completely inhibits the biological activity of an antigen. FLT3L-mediated activation of 3 is at least 10%, at least 15%, at least 20% %, at least 25%, at least 30%, at least 35%, at least 40%, at least at least 45%, at least 50%, at least 55%, at least 60%, at least 6 5%, at least 70%, at least 75%, at least 80%, at least 85%, at least It may be reduced by at least 90%, at least 95% or even 100%.

[0036] The terms "FLT3L antibody," "antibody that binds to FLT3L," or "anti-FLT3L antibody" refer to and that the molecule is useful as a therapeutic or diagnostic reagent in targeting FLT3L. It refers to an antibody or an antigen-binding fragment thereof that can bind to FLT3L with sufficient affinity. The term "anti-FLT3L" includes, for example, any of the antibodies disclosed herein that are incorporated into a scaffold. Molecules containing the CDRs of an antibody are also broadly included.

[0037] The term "germlining" refers to the process by which amino acids at specific positions in an antibody are mutated to the antibody in the germline. This means that the value is returned by

[0038] A "variable region" of an antibody refers to the variable region of an antibody light chain or an antibody fragment thereof, either alone or in combination. The variable regions of the heavy and light chains are linked by three CDR regions. The CDRs in each chain are linked together with the CDRs from the other chain to form a FW region. CDRs are held together very closely by amino acid residues and contribute to the formation of the antigen-binding site of an antibody. There are at least two techniques for determining the sequence of Sequences of Proteins o f Immunological Interest,(5th ed.,1991,N ational Institutes of Health,Bethesda Md. .)); and (2) approaches based on crystallographic studies of antigen-antibody complexes (Al- lazikani et al. (1997) J. Molec. Biol. 273:92 7-948). Furthermore, the art has demonstrated that these two approaches to determining CDRs A combination of approaches is sometimes used.

[0039] The Kabat numbering system generally assigns residues within the variable domain (approximately 100–1500 bp in the light chain) ~107 residues of the ribosome and residues 1-113 of the heavy chain) ( For example, Kabat et al., Sequences of Immunology cal Interest,5th Ed.Public Health Service e,National Institutes of Health,Bethesda , Md. (1991).

[0040] The phrase "Kabat numbering of amino acid positions" or "Kabat position" , Kabat et al., 1991, the heavy chain variable domain or light chain variable domain of the antibody cluster Refers to the numbering system used for variable domains.

[0041] The terms "antigen-binding domain", "antigen-binding fragment" and "binding fragment" refer to the binding domain between an antibody and an antigen. The variable region refers to the portion of an antibody molecule that contains the amino acids responsible for specific binding of the antibody to the antibody. The antibody or antigen-binding fragment is capable of selectively recognizing and specifically binding to an epitope on an antigen. That is, the VH and VL domains or the complementarity determining regions (CDRs) of an antibody can be The subsets combine to form the variable regions that define a three-dimensional antigen-binding site. In the present study, an antigen-binding domain is defined by three CDRs on each of the VH and VL chains. As used herein, a portion of an antigen molecule that is responsible for specific interaction with an antigen-binding domain is The portion is referred to as an "epitope." An antigen-binding domain is typically an antibody light chain variable region. and an antibody heavy chain variable region, however, not necessarily both. For example, The so-called "Fd" antibody fragments consist of only the VH domain but still contain intact antibodies. Maintains antigen-binding function in some parts of the body.

[0042] Binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic cleavage or chemical cleavage of intact antibodies. The binding fragments are Fab, Fab', F(ab')2, Fv and single chain antibodies. Digestion of antibodies with the enzyme papain produces "Fab" fragments. and two identical antigen-binding fragments, also known as α-aminobutyric acid ... Digestion of antibodies with the enzyme pepsin produces an "Fc" fragment that has the ability to bind to the antibody. The two arms of the antibody molecule remain attached, forming an F(a The F(ab')2 fragment has the ability to cross-link antigen. As used herein, "Fv" refers to an Fv that retains both the antigen recognition and binding sites. "Fab" as used herein refers to the smallest fragment of an antibody. The term "antibody fragment" refers to a fragment of an antibody that contains the CH1 domain of the heavy chain and the CH2 domain of the heavy chain.

[0043] As used herein, an Fc region refers to an Fc region that excludes the first constant region immunoglobulin domain. Fc includes polypeptides that contain the constant region of an antibody. Thus, Fc includes IgA, IgD, and and the last two constant region immunoglobulin domains of IgG and the last two constant region immunoglobulin domains of IgE and IgM. Three constant region immunoglobulin domains with a flexible hinge N-terminal to these domains For IgA and IgM, Fc may include the J chain. For IgG, Fc is composed of the immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and The Fc region boundary contains the hinge between C gamma 1 (Cy1) and C gamma 2 (Cy2). Although the length of the Fc region of a human IgG heavy chain can vary, the human IgG heavy chain Fc region usually consists of residues C 226 or P230, where the numbering is defined as It follows the EU index as defined in (t al., 1991).

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

[0045] The term "monoclonal antibody" includes both intact and full-length monoclonal antibodies, As well as antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single chain variable fragments (sc Fv), fusion proteins containing antibody portions, as well as any other modified immunoglobulins that contain an antigen recognition site. Furthermore, "monoclonal antibodies" include those produced by hybridomas, pharmacopoeias, and the like. A number of methods, including but not limited to transgene selection, recombinant expression and transgenic animals, are also possible. The term refers to such antibodies made by methods such as human antibodies produced by recombinant human antibodies in transgenic mice. expression of anti-cancer antibodies).

[0046] The term "humanized antibody" refers to an antibody that has been humanized to increase similarity to antibody variants generated in humans. Antibodies derived from non-human (e.g., mouse) immunoglobulins that have been genetically modified for Point.

[0047] The term "human antibody" refers to an antibody produced by a human or any antibody known in the art. techniques (e.g., recombinant expression in cultured cells or expression in transgenic animals) ) having an amino acid sequence corresponding to a human-produced antibody produced using Thus, the term "human antibody" refers to an antibody that was originally produced by a human. It has an amino acid sequence but has been expressed in a non-human system (e.g., produced by chemical synthesis). recombinantly expressed in microbial, mammalian or insect cells; or expressed in an animal subject Therefore, the present invention also includes human antibodies (or genetically engineered variants or derivatives thereof) that are expressed against human Elephant or human cells (e.g., hybridomas or cell lines expressing recombinant antibodies or fragments thereof) ) and subsequently expressed in an animal, such as a mouse, is considered a human antibody. This definition of a human antibody includes intact or full-length antibodies, and / or an antibody comprising at least one human heavy and / or light chain polypeptide. For example, antibodies comprising a murine light chain and a human heavy chain polypeptide are included.

[0048] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of an immunoglobulin molecule is derived from two or more animal species. Typically, the variable regions of both the light and heavy chains are configured to provide the desired specificity and / or or a mammalian species having affinity and / or ability (e.g., mouse, rat, rabbit) The variable regions of antibodies derived from a given species correspond to the variable regions of the antibody, whereas the constant regions mediate the immune response in that species. It is necessary to have sequences that are homologous to sequences in antibodies from another species (usually human) to avoid do.

[0049] The term "polynucleotide" includes single and multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct. refers to a molecule, such as messenger RNA (mRNA) or plasmid DNA (pDNA) Polynucleotides may be formed using conventional phosphodiester bonds or non-conventional bonds (e.g., peptide bonds). The term "nucleic acid" may include amide bonds, such as those found in polar nucleic acids (PNA). Any one or more nucleic acid segments present in a polynucleotide, such as DNA or R An "isolated" nucleic acid or polynucleotide is one that has been removed from its natural environment. It is intended that the nucleic acid molecule, DNA or RNA, contained within a vector. Recombinant polynucleotides encoding the polypeptide subunits described herein are Further examples of isolated polynucleotides include heterologous polynucleotides. Recombinant polynucleotides maintained in host cells or in solution (partially or substantially ) purified polynucleotides. Isolated RNA molecules include polynucleotides. The isolated polynucleotides or nucleic acids may be in vivo or in vitro RNA transcripts. Acids further include such molecules produced synthetically. can be a regulatory element, such as a promoter, a ribosome binding site, or a transcription terminator. It may be or may include.

[0050] In certain embodiments, the polynucleotide or nucleic acid is DNA. A polynucleotide comprising a nucleic acid encoding a polypeptide typically comprises one or more coding sequences. A promoter and / or other transcriptional or translational control element operably associated with the region. An operably associated or linked gene may include a gene product, e.g., a polypeptide. The coding region is designed to place expression of the gene product under the influence or control of regulatory sequences. In this case, one or more regulatory sequences are related in such a way that two DNA fragments (polypeptides) The promoter function is determined by the transcription factor α (coding region and associated promoter). If induction results in transcription of an mRNA encoding a desired gene product and two DNA fragments are The nature of the bond between the A fragments does not interfere with their ability to direct expression of the gene product or DNA "Operably linked" or "operably associated" means that the template does not interfere with the ability of the template to be transcribed. Thus, the promoter region is a region that allows the promoter to effect transcription of the nucleic acid. A promoter would be operably linked to a nucleic acid encoding a polypeptide if it could be used to encode a polypeptide. is a cell-specific process that directs substantial transcription of DNA only in predetermined cells. Other transcription control elements besides the promoter, such as enhancers, , operators, repressors, and transcription termination signals to direct cell-specific transcription. The polynucleotide can be operably linked to a suitable promoter and other The transcriptional control regions are disclosed herein.

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

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

[0053] 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" refers to a cell into which a nucleic acid has been introduced. The gene can be introduced into such cells by transfection with a viral vector. Transformation using plasmid vectors, electroporation, lipofection This includes all techniques including the introduction of naked DNA by fusion and particle gun acceleration techniques. The cell or host cell can be a bacterial cell or a eukaryotic cell.

[0054] As used herein, the term "FLT3L" refers to FMS-like tyrosine kinase 3 receptor (FL Feline McDonald's T3 (hematopoietic cytokine) polypeptide that binds to the receptor FLT3L stands for ugh sarcoma (FMS)-like tyrosine kinase 3 receptor ligand. The protein is expressed as a membrane-bound protein and then enzymatically cleaved into a soluble form. Both membrane-bound (mFLT3L) and secreted (sFLT3L) are included within the definition of FLT3L. can be.

[0055] In this disclosure, the terms "include," "includes," "containing," "having," and the like are used in the U.S. "includes," "includes" and "comprises" may have the meanings ascribed to them in patent law. "consists essentially of" or "consists essentially of" "Subject matter" and "subject matter" likewise have the meanings ascribed to them under U.S. patent law, including without limitation and anything beyond what is referred to is fundamental or novel to what is referred to. As long as the features are not altered by the presence of more than those listed, the preceding Embodiments of the technology are excluded.

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

[0057] In the context of two or more nucleic acids or polypeptides, the terms "identical" or "percent identical" may be used. "Identity" refers to the best match without considering any conservative amino acid substitutions as part of the sequence identity. When compared and aligned (with gaps introduced if necessary) or two or more sequences having a defined percentage of nucleotides or amino acid residues. The percent identity may be determined using sequence comparison software or algorithms. The amino acid sequence or nucleotide sequence can be determined by using a sequence algorithm or by visual inspection. Various algorithms and software that can be used to obtain the alignment of The method is well known in the art (e.g., Karlin et al., 1993, Proc. .Improved in Natl. Acad. Sci., 90:5873-5877, NBL The AST and XBLAST programs (Altschul et al., 1999) 91, Nucleic Acids Res., 25:3389-3402) Karlin et al., 1990, Proc. Natl. Acad. Sci. In certain embodiments, Gapped B LAST is Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402, BLAST-2, WU-BLAST-2(Al tschul et al.,1996,Methods in Enzymology ,266:460-480),ALIGN,ALIGN-2(Genentech,So uth San Francisco,California) or Megalign( The method can be used as described in (DNASTAR).

[0058] The term "isolated" refers to a molecule that is not present in its natural environment. For example, an isolated antibody is not required to be isolated from or located in its native or natural environment. Recombinantly produced biological material includes, for example, hybridomas. Since the material is produced in a non-naturally occurring cell, it is isolated as disclosed herein. For example, a substance that is an isolated protein, such as an antibody, may also be isolated by any suitable technique. An ingredient is considered "isolated" if it has been separated, fractionated, or partially or substantially purified by For example, an antibody may be used that is capable of binding to cellular material or the cell or tissue from which it is derived. It is considered "isolated" if it is substantially free of other proteins.

[0059] The term "specifically binds" refers to the ability to recognize and bind to a molecule (e.g., a receptor or epitope). and binding occurs between an agent (e.g., an antibody) and a molecule (e.g., a ligand). This definition refers to an agent (e.g., a ligand or an antibody) that induces some kind of complementarity. By definition, an antibody will bind its ligand more readily than it would if it were to bind a random, unrelated molecule. The term "specifically binds" when the antibody binds to It can thereby be used to characterize the relative affinity of binding of a particular antibody to a particular ligand. For example, antibody "A" may bind to a given ligand (e.g., FLT3L) in a manner similar to antibody "B". " may be estimated to have a higher specificity than "

[0060] As used herein, the term "affinity" refers to the strength of binding between an individual epitope and the CDR of an antibody. For example, Harlow et al., Antibodies:A La boratory Manual,(Cold Spring Harbor Labo Ratory Press), 2 nd ed. 1988), pp. 27-28. As used herein, the term "avidity" refers to the overall affinity of a complex between a population of antibodies and antigens. This refers to the functional stability, i.e., the functional binding strength between the antibody mixture and the antigen. See pages 39-34. Avidity is the ability of individual antibodies in a population to bind to a particular epitope. It is related to both the affinity with the antibody and also the valency of the antibody and antigen.

[0061] The terms "inhibit" and "block," used interchangeably herein, refer to a complete blockade of activity. "Inhibition" refers to any statistically significant decrease in biological activity, including the abolition of Approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% of the bioactivity A reduction of 90% or 100% may be mentioned.

[0062] The term "effector function" refers to the interaction of Fc components with Fc receptors or complement as a result of These activities include, for example, antibody-dependent Cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity Thus, antigen binding with altered effector functions is The fusion protein (e.g., an antibody or an antigen-binding fragment thereof) comprises at least one effector In the Fc region, which alters the activity of the function (e.g., ADCC, CDC and / or ADCP) Contains alterations in the structure of the nucleic acid (e.g., amino acid substitutions, deletions, or additions, or oligosaccharide changes) Refers to an antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof). Antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof) that have target functions are at least Each of the antibodies has one effector function (e.g., ADCC, CDC, and / or ADCP) activity. Changes in the Fc region that increase the The term refers to an antigen-binding protein (e.g., an antibody or an antigen-binding fragment thereof) that contains a saccharide-binding variant of the saccharide. vinegar.

[0063] The term "subject" includes humans, non-human primates, rodents, etc., who will be the recipient of a particular treatment. The term refers to any animal (e.g., a mammal) including, but not limited to, an animal. Typically, the term "Subject," "patient," and "individual" are used interchangeably herein. Examples of additional animals include non-human mammals, such as cows, horses, dogs, sheep or cats. do.

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

[0065] An "effective amount" of an anti-FLT3L antibody disclosed herein is an amount that accomplishes a specifically specified purpose. "Effective amount" is an amount sufficient to achieve the intended effect. This can be determined by some routine method.

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

[0067] "Treat" or "treatment" or "to treat" or "alleviate" or The terms "to palliate" and "to alleviate" include, but are not limited to, (1) curing the progression of a diagnosed pathological condition or disorder. and therapeutic means for preventing, slowing the progression, reducing symptoms, and / or halting the progression of the disease. 2) Prophylactic or preventative, preventing and / or slowing the onset of a targeted pathological condition or disorder Therefore, subjects in need of treatment include those who already have a disorder; Subjects prone to having the disorder; and subjects in which the disorder is to be prevented. In certain embodiments, the subject is a patient who is diagnosed with a condition associated with, e.g., an autoimmune or inflammatory disease. If a complete, partial or transient reduction is observed, then the method of the present disclosure is effective in treating an autoimmune disease or is successfully "treated" for inflammatory diseases.

[0068] Ranges provided herein are understood to be shorthand for all the numerical values ​​within the range. For example, the range 1 to 50 is 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, 3 The group consisting of 9, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 It is understood that the term includes any number, combination of numbers, or subranges from.

[0069] As used herein, the terms "treat," "treating," "treatment," and the like refer to a disorder and / or or the reduction or alleviation of symptoms associated with a disease or condition, including, but not limited to, Treating a disease does not necessarily mean that the disease or its associated condition or symptoms are completely eliminated. It will be understood that the use of the compound is not required. For example, as contemplated herein, the treatment of a disorder Treatment includes preventing the symptoms of the disorder from worsening.

[0070] As used herein, the term "or" means "or" unless specifically indicated otherwise or to the contrary from the context. Unless otherwise specified, terms are to be understood as being inclusive. "A," "an," and "the" are used interchangeably unless specifically stated otherwise or the context requires. Unless the contrary is clear from the above, the terms "a," "an," and "the" are to be understood as being singular or plural.

[0071] Furthermore, as used herein, "and / or" refers to two or more stated features or to the other. should be considered a specific disclosure of each of the components with or without The term "and / or" as used in phrases such as "A and / or B" herein means "A "and B", "A or B", "A" (alone) and "B" (alone) are intended to include Similarly, the term "and / or" used in phrases such as "A, B and / or C" The following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and and C; A and B; B and C; and A (alone); B (alone); and C (alone) This is what is intended.

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

[0073] FLT3L is initially expressed as a membrane-bound protein before being enzymatically cleaved into a soluble form. Both membrane-bound (mFLT3L) and secreted (sFLT3L) are functionally active. The FLT3L binding domain is a ligand / receptor pair in humans, rodents, and cynomolgus monkeys. They are highly conserved across species in which they are observed, due to the existence of cross-species reactivity between them. While key mutations around the binding site prevented neutralizing antibodies from being generated against FLT3L, Neutralizing antibodies against FLT3L are thought to explain the lack of cross-species reactivity in and plasmacytoid DC populations, reducing the ability of the immune system to induce and sustain long-term inflammatory responses. Side effects include a reduction in circulating NK cells and reduced T and B cell activity. These include reduced cell proliferation and proliferation, which may result in reduced survival of both cell types. Downregulation of this pathway may reduce autoimmune inflammation.

[0074] In one embodiment, the neutralizing anti-FLT3L antibody inhibits FLT3L binding to FLT3. It is intended that anti-FLT3L antibodies promote immune homeostasis by inhibiting FLT3L. The strategy involves translocating the receptor to avoid the risk of unexpected receptor dimerization or signal transduction. Unlike its receptor, it targets the signaling pathway associated with membrane-bound FLT3L. There is no null transduction domain.

[0075] Anti-FLT3L antibody The present disclosure provides, in a preferred embodiment, an isolated FLT3L binding molecule, e.g., FLT3L, e.g. For example, the present invention provides an antibody and an antigen-binding fragment thereof that specifically binds to human FLT3L. The full length amino acid and nucleotide sequences for are known in the art ( For example, for human FLT3L, see UniProt accession number P36888 or For mouse FLT3L, see UniProt accession number Q00342 The anti-FLT3L antibodies of the present disclosure inhibit FLT3L-mediated activation of FLT3, thereby This reduces pro-inflammatory signaling and reduces inflammation in the subject.

[0076] In a preferred embodiment, the anti-FLT3L antibody is a structurally similar TKR cognate human stem cell factor. It does not cross-react with human colony-stimulating factor (huSCF) or human colony-stimulating factor (huCSF1). If so, SCF and CSF are ligands that also bind to tyrosine kinase receptors. It will recognize non-specific F that binds additional tyrosine kinase family members. LT3 inhibitors induce toxicity from global inhibition of tyrosine kinase signaling. Therefore, anti-FLT3L antibodies bind only to FLT3L and not to structurally similar homologues. It is extremely important to have a good understanding of the mechanism of action of FLT3L. Many anti-FLT3L antibodies and inhibitors lack specificity. Therefore, the preferred anti-FLT3L antibodies are The embodiment must demonstrate high affinity and specific binding to FLT3L. It won't happen.

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

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

[0079] In some embodiments, the IgG constant region comprises an Igκ constant domain (region) and an Igλ constant domain (region). In one particular embodiment, the light chain constant region may be selected from the group consisting of: The anti-FLT3L antibody contains a human IgG1 constant domain and a human lambda constant domain. In certain embodiments, the anti-FLT3L antibody comprises a targeted mutation in the Fc region at position 243 of the LeuR locus. 146F to phenylalanine (L243F), and 147F to glutamic acid (L23 5E) and changing the proline at position 331 to serine (P331S). -TM format. Amino acid numbering is according to the EU index. Targeted mutations reduce FcR binding and ADCC effector functions (e.g., Organesyan et al., Acta Crysta, incorporated by reference llogr D Biol Crystallogr.2008 Jun 1;64(P t 6):700-4; and WO 2009100309(A2) brochure Please refer to.

[0080] In some embodiments, the anti-FLT3L antibody is a human antibody (e.g., CAT5D9, SC401 7, AM40, CAT8, CAT26, DTAX3 and DYAX5 antibodies).

[0081] CAT5D9 antibody In one embodiment, the CAT5D9 antibody specifically binds and is complementary to FLT3L. Decision region (CDR): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and It refers to an antibody that contains HCDR1, HCDR2, HCDR3, LCDR1, L CDR2 and LCDR3 are the sequences of SEQ ID NOs: 29, 36, 37, 32, 33 and 38, respectively. Contains the amino acid sequence.

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

[0083] In a further embodiment, the CAT5D9 antibody comprises two amino acid sequences having the amino acid sequence of SEQ ID NO:6. The present invention relates to an antibody comprising a VL domain and two VH domains having the amino acid sequence of SEQ ID NO:5. vinegar.

[0084] In another embodiment, the CAT5D9 antibody is encoded by the nucleic acid sequence of SEQ ID NO:20. and two VL domains encoded by the nucleic acid sequence of SEQ ID NO: 19. This refers to an antibody that contains an antibody.

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

[0086] In another embodiment, the CAT5D9 antibody is encoded by the nucleic acid sequence of SEQ ID NO:72. and one heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 71. Point.

[0087] SC4017 antibody In one embodiment, the SC4017 antibody specifically binds and is complementary to FLT3L. Decision region (CDR): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and It refers to an antibody that contains HCDR1, HCDR2, HCDR3, LCDR1, L CDR2 and LCDR3 are represented by SEQ ID NOs: 29, 30, 31, 35, 33 and 34, respectively. Contains the amino acid sequence.

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

[0089] In a further embodiment, the SC4017 antibody comprises two amino acid sequences having the amino acid sequence of SEQ ID NO:4. The present invention relates to an antibody comprising a VL domain and two VH domains having the amino acid sequence of SEQ ID NO:3. vinegar.

[0090] In another embodiment, the SC4017 antibody is encoded by the nucleic acid sequence of SEQ ID NO:18. and two VL domains encoded by the nucleic acid sequence of SEQ ID NO: 17. This refers to an antibody that contains an antibody.

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

[0092] In another embodiment, the SC4017 antibody is encoded by the nucleic acid sequence of SEQ ID NO:68. and one heavy chain encoded by the nucleic acid sequence of SEQ ID NO:67. Point.

[0093] AM40(MEDI1116) antibody In one embodiment, the AM40 antibody specifically binds to FLT3L and has a complementarity determining Region (CDR): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and L Refers to an antibody that contains CDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCD R2 and LCDR3 are the amino acids of SEQ ID NOs: 29, 30, 31, 32, 33 and 34, respectively. It contains the amino acid sequence.

[0094] In another embodiment, the AM40 antibody specifically binds to FLT3L and has the sequence of SEQ ID NO:2. having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence and two VL domains having at least 95%, 96%, or 99% identical amino acid sequence to that of SEQ ID NO:1. It refers to an antibody comprising two VH domains that have 7%, 98% or 99% sequence identity.

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

[0096] In another embodiment, the AM40 antibody is a 2'-amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO:16. one VL domain and two VH domains encoded by the nucleic acid sequence of SEQ ID NO:15 The present invention refers to an antibody comprising the

[0097] In one embodiment, the AM40 antibody specifically binds to FLT3L and has the sequence represented by SEQ ID NO:6. 2 and one heavy chain having the amino acid sequence of SEQ ID NO: 61. Refers to an IgG1 antibody that contains IgG1 chains.

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

[0099] CAT8 antibody In one embodiment, the CAT8 antibody specifically binds to FLT3L and has a complementarity determining Region (CDR): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and L Refers to an antibody that contains CDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCD R2 and LCDR3 are the amino acids of SEQ ID NOs: 39, 40, 41, 42, 43 and 44, respectively. It contains the amino acid sequence.

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

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

[0102] In another embodiment, the CAT8 antibody is a 2A polypeptide encoded by the nucleic acid sequence of SEQ ID NO:22. one VL domain and two VH domains encoded by the nucleic acid sequence of SEQ ID NO:21 The present invention refers to an antibody comprising the

[0103] In one embodiment, the CAT8 antibody specifically binds to FLT3L and has the sequence of SEQ ID NO:7. 4 and one heavy chain having the amino acid sequence of SEQ ID NO: 73. Refers to an IgG1 antibody that contains IgG1 chains.

[0104] In another embodiment, the CAT8 antibody is a 1A polypeptide encoded by the nucleic acid sequence of SEQ ID NO:76. The present invention refers to an antibody comprising one light chain and one heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 75. .

[0105] CAT26 antibody In one embodiment, the CAT26 antibody specifically binds to FLT3L and has complementary Constant region (CDR): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and Refers to an antibody that contains LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LC DR2 and LCDR3 are represented by the sequences of SEQ ID NOs: 45, 40, 46, 47, 48 and 49, respectively. It contains the amino acid sequence.

[0106] In another embodiment, the CAT26 antibody specifically binds to FLT3L and has the sequence of SEQ ID NO:1. At least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of and two VL domains having an amino acid sequence of SEQ ID NO: 9 and at least 95%, 96% refers to an antibody comprising two VH domains having 97%, 98% or 99% sequence identity .

[0107] In a further embodiment, the CAT26 antibody comprises two amino acid sequences having the amino acid sequence of SEQ ID NO:10. The present invention relates to an antibody comprising a VL domain and two VH domains having the amino acid sequence of SEQ ID NO:9. vinegar.

[0108] In another embodiment, the CAT26 antibody is encoded by the nucleic acid sequence of SEQ ID NO:24. Two VL domains and two VH domains encoded by the nucleic acid sequence of SEQ ID NO:23. This refers to an antibody that contains

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

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

[0111] DYAX3 antibody In one embodiment, the Dyax3 antibody specifically binds to and complements FLT3L. Constant region (CDR): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and Refers to an antibody that contains LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LC DR2 and LCDR3 are represented by the sequences of SEQ ID NOs: 50, 51, 52, 53, 54 and 55, respectively. It contains the amino acid sequence.

[0112] In another embodiment, the Dyax3 antibody specifically binds to FLT3L and has the sequence of SEQ ID NO:8. 2. At least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of and two VL domains having at least 95%, 96%, or more identical to the amino acid sequence of SEQ ID NO: 81. %, 97%, 98% or 99% sequence identity to an antibody comprising two VH domains. vinegar.

[0113] In a further embodiment, the Dyax3 antibody comprises two amino acid sequences having the amino acid sequence of SEQ ID NO:12. An antibody comprising a VL domain and two VH domains having the amino acid sequence of SEQ ID NO:11. Point.

[0114] In another embodiment, the Dyax3 antibody is encoded by the nucleic acid sequence of SEQ ID NO:26. Two VL domains and two VH domains encoded by the nucleic acid sequence of SEQ ID NO:25. This refers to an antibody that contains

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

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

[0117] DYAX5 antibody In one embodiment, the Dyax5 antibody specifically binds to and complements FLT3L. Constant region (CDR): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and Refers to an antibody that contains LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LC DR2 and LCDR3 are represented by the sequences of SEQ ID NOs: 56, 57, 52, 58, 59 and 60, respectively. It contains the amino acid sequence.

[0118] In another embodiment, the Dyax5 antibody specifically binds to FLT3L and has the sequence of SEQ ID NO:8. At least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of and two VL domains having at least 95%, 96%, or more amino acid sequence of SEQ ID NO: 85. %, 97%, 98% or 99% sequence identity to an antibody comprising two VH domains. vinegar.

[0119] In a further embodiment, the Dyax5 antibody comprises two amino acid sequences having the amino acid sequence of SEQ ID NO:14. An antibody comprising a VL domain and two VH domains having the amino acid sequence of SEQ ID NO: 13. Point.

[0120] In another embodiment, the Dyax5 antibody is encoded by the nucleic acid sequence of SEQ ID NO:28. Two VL domains and two VH domains encoded by the nucleic acid sequence of SEQ ID NO:27. This refers to an antibody that contains

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

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

[0123] In certain embodiments, the set of complementarity determining regions (CDRs): HCDR1, HCDR2, HC An antibody that specifically binds to FLT3L, including LCDR3, LCDR1, LCDR2 and LCDR3. The present invention provides an antibody or an antigen-binding fragment thereof, wherein HCDR1, HCDR2, HCDR 3, LCDR1, LCDR2 and LCDR3 are represented by (a) SEQ ID NOs: 29, 30 and 3, respectively. 1, 32, 33 and 34; or (b) SEQ ID NOs: 29, 30, 31, 35, 33 and 34, respectively. and 34; or (c) the amino acids of SEQ ID NOs: 29, 36, 37, 32, 33, and 38, respectively. Contains arrays.

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

[0125] In certain aspects, the VH and VL regions are (a) SEQ ID NO:1 and SEQ ID NO:2, respectively. or (b) SEQ ID NO:3 and SEQ ID NO:4, respectively; or (c) SEQ ID NO:5 and SEQ ID NO:6, respectively. having at least 95%, 96%, 97%, 98% or 99% sequence identity with sequence number 6 It has an amino acid sequence.

[0126] In certain embodiments, the CDRs (HCDR1, HCDR2 and HCDR3) of the VH and the VL The CDRs (LCDR1, LCDR2 and LCDR3) of (a) SEQ ID NO: 29, 30, 31, 32, 33 and 34; or (b) SEQ ID NOs: 29, 30, 31, 35, respectively. , 33 and 34; or (c) SEQ ID NOs: 29, 36, 37, 32, 33 and 38, respectively. It consists of an amino acid sequence.

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

[0128] [Table 1]

[0129] [Table 2]

[0130] [Table 3]

[0131]

Table 4

[0132]

Table 5

[0133]

Table 6

[0134]

Table 7

[0135]

Table 8

[0136]

Table 9

[0137]

Table 10

[0138]

Table 11

[0139]

Table 12

[0140]

Table 13

[0141] Inducer The anti-FLT3L antibody of the present disclosure retains the ability to specifically bind to FLT3L. Such variants may be included within the meaning of the present invention as understood by those of skill in the art. The sequences of these antibodies can be derived using techniques well known in the art. For example, The amino acid substitutions, deletions, or additions of the anti-F3A antibody may interfere with the binding of the antibody to its epitope. The changes can be made within the FR regions and / or CDRs of the LT3L antibody. , which are usually designed to improve the stability and immunogenicity of the antigen-binding domain, Changes in the nucleotide sequence are typically designed to increase the affinity of the antigen-binding domain for its target. FR variants also include naturally occurring immunoglobulin allotypes. Changes that enhance affinity include altering the CDRs and the affinity of the antigen-binding domain to its target. This can be determined empirically by routine techniques, including testing for Conservative amino acid substitutions can be made within any one of the CDRs disclosed herein. The various changes are described in Antibody Engineering, 2nd ed., Oxford ord University Press, ed.Borrebaeck, 1995. These changes can be made according to the methods described. The codons encode the nucleotide residues, and thus can be altered by substitution of various codons producing "silent" changes. For example, non-polar amino acids can be used as nucleotide sequences. The acids are alanine, leucine, isoleucine, valine, proline, and phenylalanine. , tryptophan and methionine. Polar neutral amino acids include glycine, Serine, threonine, cysteine, tyrosine, asparagine and glutamine The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0142] Derivatives and analogs of the antibodies of the present disclosure can be produced using recombinant and synthetic methods (Maniatis (1999) 90)Molecular Cloning,A Laboratory Manual ,2nd ed.,Cold Spring Harbor Laboratory,C old Spring Harbor, NY and Bodansky et al. 1995)The Practice of Peptide Synthesis,2 nd ed., Spring Verlag, Berlin, Germany). They can be made by a variety of techniques well known in the art.

[0143] In one embodiment, a VH domain that is an amino acid sequence variant of a VH domain of the disclosure. The method for generating the VH domain disclosed herein includes the step of adding one or more adding, deleting, substituting or inserting amino acids of combining the VH domain thus provided with one or more VL domains; Testing one or more VH domains or VH / VL combinations for specific binding to the antigen The method comprises the steps of: A similar method can be used to combine VH domains.

[0144] Similar shuffling or combinatorial techniques have been used to engineer related β-lactamase genes. Although the present paper describes a technique for producing antibodies, it is to be noted that the approach can also be used for generating antibodies. According to Stemmer (Nature (1994) 370:389-391), It is also disclosed.

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

[0146] Another method that can be used is to direct mutagenesis to the CDRs of the VH or VL genes. Such a technique is described by Barbas et al. (Proc. Nat. Acad. Sci. 2003). Sci. USA (1994) 91: 3809-3813) and Schier (J. Mol. Biol. (1996) 263:551-567) As shown.

[0147] Similarly, one, two or all three CDRs of an antigen-binding domain may be linked to a VH or VL domain. These can then be transplanted into the repertoire of FLT3L specific The antibodies are screened for antigen-binding fragments that are targeted to the antibody.

[0148] The portions of immunoglobulin variable domains useful herein are substantially as defined herein. and optionally a scFv fragment as defined herein. The intervening framework regions may include either or both of FR1 and FR4. and wherein the 50% comprises at least about 50% of the C-terminal 50% of FR1 and the C-terminal 50% of FR2. 4. Additional residues at the N-terminus or C-terminus of a substantial part of the variable domain. The group may be a residue not normally associated with a native variable domain region. The construction of antibodies by DNA techniques allows for easy cloning and other manipulation steps. The introduced linker may result in the introduction of encoded N- or C-terminal residues. Other engineering steps include the addition of immunoglobulin heavy chain constant regions, other variable domains (e.g., In the production of anabodies) or proteinaceous labels, as discussed in more detail below. This involves the introduction of linkers to join the variable domains to the protein sequence.

[0149] The antigen binding domains of the present disclosure described herein can be coupled to other functional molecules, e.g., other peptides. It can be bound to an antigen or a protein (albumin, another antibody, etc.). The binding domains can be linked by chemical cross-linking or recombinant methods. The binding domain can be made of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene, etc. Pyrene glycol or one of the polyoxyalkylenes, U.S. Pat. No. 4,640,835 Specification; Specification No. 4,496,689; Specification No. 4,301,144; Specification No. 4, No. 670,417; No. 4,791,192; or No. 4,179,33 The antigen-binding domain can be, for example, To increase their circulation half-life, they can be chemically modified by covalent attachment to polymers. Exemplary polymers and methods for attaching them are described in U.S. Pat. Specification No. 66,106; Specification No. 4,179,337; Specification No. 4,495,285 This is also shown in the specification and in the specification of US Pat. No. 4,609,546.

[0150] The antibodies disclosed herein may be modified to have a glycosylation pattern that differs from the native pattern. For example, one or more carbohydrate sites can be deleted, and / or one or more glycosylation sites may be added. The addition of glycosylation sites to the polypeptide fragment can be accomplished by any method known in the art. This can be done by altering the amino acid sequence to contain the census sequence. Another means of increasing the number of carbohydrate sites on an antibody fragment is to attach carbohydrate moieties to amino acid residues of the antibody. By chemical or enzymatic coupling of glycosides. Such methods are described in WO 87 / 05330 pamphlet and Aplin et al. (1981) CRC Cri Rev. Biochem., 22:259-306. Removal of the desired carbohydrate moiety can be achieved, for example, by the method of Hakimuddin et al. (1987) Arch.Biochem.Biophys.,259:52;Edge et al. (1981) Anal.Biochem., 118:131 and Thotakura e. (1987) Meth. Enzymol., 138:350. The antibody fragment may be labeled with a detectable label or function. Detectable labels can also be labeled using conventional chemistries. These include radioactive labels, such as 131I or 99Tc, which may also be attached to the antibody fragment. Detectable labels include enzyme labels, horseradish peroxidase or alkaline Detectable labels also include chemical moieties, such as specific cognate detection Further detectable moieties include, for example, biotin, which can be detected via binding to labeled avidin.

[0151] Antigen-binding domains whose CDR sequences differ only non-substantially from the CDR sequences defined herein. Typically, amino acids are selected based on similar charge, hydrophobicity, or Such substitutions are made by amino acids having the stereochemical characteristics of the corresponding amino acids. Unlike CDRs, it is within the skill of the art to provide a method for modifying the binding properties of an antibody without adversely affecting the binding characteristics of the antibody. Changes to the FR can be made using non-human derived or to humanize specific fragments that are important for stabilizing antigen contact or binding sites. Manipulating framework residues, e.g., changing the class or subclass of the constant region See, for example, U.S. Pat. Nos. 5,624,821 and 5,648,260. and Lund et al. (1991) J. Immun. 147:2657-26 62 and Morgan et al. (1995) Immunology 86:319 As described in 324-34, it is possible to alter effector functions such as Fc receptor binding. By changing specific amino acid residues that may be involved in the synthesis of a constant region or by changing the species from which the constant region is derived, This includes, but is not limited to,

[0152] Those skilled in the art will appreciate that the modifications described above are not exhaustive and that the and numerous other modifications may be applicable to the protein subunits described above in light of the teachings of this disclosure. It will be appreciated that such a method would be possible for one skilled in the art.

[0153] Affinity and specificity of anti-FLT3L antibodies Those skilled in the art will appreciate that anti-FLT3L antibodies for use in autoimmune diseases have high affinity binding require a certain level of toxicity, yet be devoid of toxicity that would preclude their use in humans. FLT3L is a structurally similar homologue of stem Cellular factor (SCF, also known as KIT-ligand) and colony-stimulating factor 1 (MCF) and CSF1, also known as phage colony-stimulating factor (M-CSF). A non-specific anti-FLT3L antibody that binds to FLT3L and also to SCF and CSF1 Therefore, the anti-FLT3L antibody of the present disclosure is capable of inhibiting FLT3. L, but binds structurally similar proteins such as SCF and CSF1. Those skilled in the art will appreciate that K as a measure of binding specificity. on , K off and K D The binding kinetics include, but are not limited to, It will be.

[0154] Serum FLT3L and circulating pDCs are associated with the anti-FLT3L antibody lead clone. It is a biomarker that can be used as an indicator of the toxicity of FLT3L. Acute depletion of pDCs suppresses FLT3L-mediated cell signaling that enhances immune responses The rapid recovery of pDC frequency in the presence of FLT3L was consistent with the anti-FLT3L antibody. In a preferred embodiment, the lack of toxicity of the anti-FLT3L antibody may be a reflection of the lack of toxicity of the anti-FLT3L antibody. neutralize FLT3L and reverse the withering of cDCs and pDCs when free FLT3L is restored Those skilled in the art will appreciate that dendritic cell proliferation after neutralization of FLT3L is followed by recovery to baseline. It will be appreciated that a drop in cells is an indication of the hypotoxicity of the lead clone.

[0155] Generation of anti-FLT3L antibodies Practice of the present disclosure is clearly within the scope of those skilled in the art unless otherwise indicated. Uses techniques in molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology Such techniques are described in "Molecular Cloning: A Laboratory Manual." ory Manual”, second edition (Sambrook, 1989 ); “Oligonucleotide Synthesis” (Gait, 1984) “Animal Cell Culture” (Freshney, 1987); “M ``ethods in Enzymology'', ``Handbook of Experts'' imental Immunology” (Weir, 1996); “Gene Tra nsfer Vectors for Mammalian Cells”;(Mill er and Calos, 1987) “Current Protocols in "Molecular Biology" (Ausubel, 1987); "PCR:Th e Polymerase Chain Reaction” (Mullis, 1994 ); “Current Protocols in Immunology” (Coli These techniques are fully described in the literature, such as in the literature by Johns Hopkins et al., 1991. can be applied to the production of polypeptides and therefore should be taken into consideration when making and practicing the present disclosure. For techniques that are particularly useful for certain embodiments, see the examples below. This is discussed in Sect.

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

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

[0158] In one embodiment, an anti-FLT3L monoclonal antibody (e.g., CAT5D9, SC 4017 or AM40) and antigen-binding fragments thereof are described in Kohler and Milstein Hybrids such as those described by John F. Schmidt in (1975) Nature 256:495 The hybridoma method can be used to prepare the antibody from mice, hamsters, etc. The mouse or other suitable host animal is then cultured in lymphocytes for antibodies that will specifically bind to the immunizing antigen. To elicit production by the antibody, the subject is immunized as described above.

[0159] In another embodiment, lymphocytes may be immunized in vitro. The lymphocytes are isolated and then separated, for example using polyethylene glycol, into unfused lymphocytes and and myeloma cells. Immunoprecipitation, immunoblotting or in vitro binding assays. (e.g., radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)) Produce monoclonal antibodies specifically raised against selected antigens determined by The resulting hybridomas were then subjected to standard techniques (Coding, Monoclonal Angiography, tibodies:Principles and Practice,Academi c Press, 1986) in vitro culture or as ascites tumors in animals. The monoclonal antibodies can then be propagated either in vitro or in vivo. The polyclonal antibodies can be purified from the culture medium or ascites fluid as described above. Cut.

[0160] Alternatively, anti-FLT3L monoclonal antibodies (e.g., CAT5D9, SC4017 or AM40) and antigen-binding fragments thereof are described, for example, in U.S. Pat. No. 4,816,567. Monoclonal antibodies can be produced using recombinant DNA methods described in The polynucleotide that encodes the antibody specifically amplifies genes encoding the heavy and light chains of the antibody. Mature B cells or hamsters were detected by RT-PCR or other methods using oligonucleotide primers that Hybridoma cells are isolated and their sequences determined using conventional techniques. The isolated polynucleotides encoding the low and light chains are then cloned into a suitable expression vector. These are converted to immunoglobulin proteins that would otherwise be produced by the host cell. The antibodies were produced by Escherichia coli (E. coli) cells, monkey COS cells, and chimeric bacteria that do not produce monoclonal antibodies. Transfected into host cells such as Chinese hamster ovary (CHO) cells or myeloma cells. Furthermore, a recombinant anti-FLT3L monoclonal antibody or an antigen-binding antibody thereof of a desired species is produced. The fragment was (McCafferty et al., 1990, Nature, 348:5 52-554;Clarkson et al.,1991,Nature,352:6 24-628; and Marks et al., 1991, J. Mol. Biol., 2 22:581-597) to produce a phage display expressing the CDRs of a desired species. The antibodies may be isolated from a prey library.

[0161] Polynucleotides encoding anti-FLT3L antibodies or antigen-binding fragments thereof may be used to generate alternating antibodies. can be further modified in a number of different ways using recombinant DNA technology to produce In some aspects, for example, the constant sequences of the light and heavy chains of a mouse monoclonal antibody can be used. Domains can be (1) those regions of a human antibody, e.g., to generate chimeric antibodies; or (2) Some may be substituted with non-immunoglobulin polypeptides to generate fusion antibodies. In aspects, the constant regions can be truncated or modified to generate desired antibody fragments of a monoclonal antibody. Using site-directed or high-density mutagenesis of the variable regions, monoclonal antibodies can be isolated. The specificity, affinity, etc. of the antibody can be optimized.

[0162] In certain embodiments, the anti-FLT3L antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. Human antibodies are recombinant fragments. Human antibodies can be prepared directly using a variety of techniques known in the art. It can be produced by in vitro immunization or by generating antibodies against a target antigen. Immortalized human B lymphocytes isolated from immunized individuals can be generated (e.g., Cole et al., Monoclonal Antibodies and Ca ncer Therapy,Alan R.Liss,p.77(1985);Boem er et al., 1991, J. Immunol., 147(l):86-95; and U.S. Patent Nos. 5,750,373.

[0163] Furthermore, the anti-FLT3L antibody or antigen-binding fragment thereof is selected from a phage library. wherein the phage library can be prepared as described, for example, in Vaughan et al. l., 1996, Nat. Biotech. 14:309-314, Sheets et al. al.1998,Proc.Nat'l.Acad.Sci.95:6157-616 2, Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381 and Marks et al. 1991, J. Mol. Biol. 222 Human antibodies are expressed as described in (J. Med. Soc. 2003, 581). An antibody phage library is generated. Techniques for making and using the same are described in U.S. Pat. Nos. 5,969,108 and 6,172,733. , Specification No. 197, Specification No. 5,885,793, Specification No. 6,521,404 ; Specification No. 6,544,731; Specification No. 6,555,313; Specification No. 6,582 ,915 specification; 6,593,081 specification; 6,300,064 specification ; 6,653,068; 6,706,484 and 7,26 No. 4,963; and Rothe et al., 2007, J. Mol. Biol. .,doi:10.1016 / j.jmb.2007.12.018 (each of these (which is incorporated herein by reference in its entirety).

[0164] Affinity maturation and chain shuffling strategies (M arks et al., 1992, Bio / Technology 10:779-7 83) are known in the art and are used to produce high affinity human antibodies or antigen-binding fragments thereof. can be used to create

[0165] In some embodiments, the anti-FLT3L monoclonal antibody can be a humanized antibody. Alternatively, methods for engineering, humanizing, or resurfacing human antibodies can also be used. Humanized, resurfaced or similarly engineered antibodies are well known in the art. is a non-human, such as a mouse, rat, rabbit, non-human primate or other mammal, These non-human amino acids may have one or more amino acid residues from sources such as, but not limited to, The amino acid residues are typically selected from "import" variable domains, constant domains or They are replaced with residues often referred to as "import" residues taken from other domains. Import sequences such as off-rate, avidity, half-life or the term It may be used to reduce, enhance or modify any other suitable characteristic known in the art. In general, the CDR residues are directly and / or selectively involved in influencing FLT3L binding. Thus, part or all of the non-human or human CDR sequences are The non-human sequences in the constant regions and constant regions may be replaced with human amino acids or other amino acids. will be done.

[0166] The antibodies may optionally be humanized, resurfaced, genetically engineered, or FLT Genetically modified to retain high affinity for 3L antigens and other favorable biological properties To this end, humanized (or human) or genetically engineered antibodies may be used. The recombinant anti-FLT3L antibodies and resurfaced antibodies optionally contain the parent sequence and the gene Three-dimensional models of the recombined and humanized sequences were used to characterize the parental sequences as well as various conceptual humanized sequences. and the process of analyzing recombinant DNA products. Models are commercially available and familiar to those skilled in the art. A computer program that illustrates and presents probable three-dimensional conformational structures of globulin sequences. Tests of these representations provide insight into the function of candidate immunoglobulin sequences. Analysis of the possible roles of residues in the candidate immunoglobulins, such as FLT3L. This method allows the analysis of residues that affect the ability of a framework to bind to an antigen. (FW) residues are selected to achieve desired antibody characteristics, such as increased affinity for the target antigen. As such, the consensus sequence and the import sequence can be combined.

[0167] The humanization, resurfacement, or genetic engineering of anti-FLT3L antibodies or antigen-binding fragments thereof may be performed by Jo nes et al.,Nature 321:522(1986);Riechman et al., Nature 332:323(1988); Verhoeyen et al., Science 239:1534(1988)), Sims et a l, J. Immunol. 151:2296(1993); Chothia and L esk, J. Mol. Biol. 196:901(1987), Carter et a l, Proc. Natl. Acad. Sci. U.S.A. 89:4285(1992) ; Presta et al., J. Immunol. 151:2623(1993). U.S. Patent No. 5,639,641, No. 5,723,323; No. 5,9 76,862; No. 5,824,514; No. 5,817,483; No. 5,814,476; No. 5,763,192; No. 5,7 23,323; No. 5,766,886; No. 5,714,352; No. 5,9,55,358; No. 6,204,023; No. 6, 180,370; No. 6,331,431; No. 5,693,762 ; No. 5,530,101; No. 5,585,089; No. 5, 225,539; No. 4,816,567; No. 5,969,108 ; No. 7,635,666; No. 7,723,270; No. 7, 557,189; No. 7,538,195; and No. 7,342,11 0; International Application Publication PCT / US Patent No. S98 / 16280; PCT / US Patent No. 91 / 05939; PCT / US Patent No. 94 / 01234; P CT / UK Patent Specification No. 92 / 01755; International Patent Publication No. WO 90 / 144 Brochure No. 43; Brochure No. 90 / 14424; Brochure No. 90 / 14430 and European Patent Publication No. EP 229246, each of which is (All of which are incorporated by reference in their entirety, including the documents referenced therein) The method may be carried out by any known method, such as, but not limited to, the method described in It is possible.

[0168] Anti-FLT3L humanized antibodies and their antigen-binding fragments inhibit endogenous immunoglobulin production Human immunoglobulin genetics are capable of generating a full repertoire of human antibodies following immunization under Alternatively, the gene can be generated in a transgenic mouse containing the gene locus. See U.S. Patent Nos. 5,545,807; 5,545,806; Specification No. 5,569,825; Specification No. 5,625,126; Specification No. 5,633,42 No. 5,661,016; and US Pat. No. 5,661,016.

[0169] In certain embodiments, anti-FLT3L antibody fragments are provided. Conventionally, these fragments have been prepared by proteolytic digestion of intact antibodies. (e.g., Morimoto et al., 1993, Journal of of Biochemical and Biophysical Methods 24:107-117;Brennan et al,1985,Science,22 9:81). In certain embodiments, the anti-FLT3L antibody fragment is recombinantly produced. Fab Fv and scFv antibody fragments are all expressed in E. coli or other host cells. Such antibodies can be expressed and secreted, thus allowing the production of large amounts of these fragments. FLT3L antibody fragments can also be isolated from the antibody phage libraries discussed above. The anti-FLT3L antibody fragment can be prepared as described in U.S. Pat. No. 5,641,870. Other techniques for producing antibody fragments, such as chemical synthesis, are known. It will be clear to the trader.

[0170] According to the present disclosure, the technology is applied to generate single chain antibodies specific to FLT3L. (See, for example, U.S. Pat. No. 4,946,778). and a desired specificity for FLT3L or a derivative, fragment, analog or homolog thereof. Fab expression to allow rapid and efficient identification of monoclonal Fab fragments comprising Libraries (e.g., Huse et al., Science 246:1275- 1281 (1989)) can be adapted for constructing Antibody fragments include: (a) F(ab')2 fragments generated by pepsin digestion of an antibody molecule; (b) Fa generated by reducing the disulfide bridges of the F(ab')2 fragment (b) fragments, (c) Fab fragments produced by treatment of antibody molecules with papain and a reducing agent. and (d) Fv fragments, It is possible.

[0171] The anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein has an increased serum half-life. This can be done, for example, by modifying the Salvage receptor-binding epitopes into antibodies or antibody fragments by mutating appropriate regions By incorporation of the fragment or thereafter, the fragment or fragment of the antibody at either end or in the middle either individually (e.g., by DNA or peptide synthesis) or by YTE mutagenesis. This can be achieved by incorporating the epitope into a peptide tag that is fused to the antibody or Other methods for increasing the serum half-life of the antigen-binding fragment may be used, such as the use of heterologous moieties such as PEG. Conjugation to protons is known in the art.

[0172] Pharmaceutical Compositions The present invention relates to a pharmaceutical composition comprising the anti-FLT3L antibody or an antigen-binding fragment thereof disclosed herein. In certain embodiments, the present disclosure is directed to the manufacture of a medicament for treating a subject. The present invention provides a use of an anti-FLT3L antibody or an antigen-binding fragment thereof disclosed herein in .

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

[0174] In certain aspects, the present disclosure relates to, but is not limited to, systemic lupus erythematosus, myositis, primary sheath syndrome, and the like. Autoimmune diseases including Glenn's syndrome, multiple sclerosis, uveitis, psoriasis or rheumatoid arthritis For use in the treatment or (to reduce the likelihood of) prophylaxis in subjects in need thereof The present invention provides therapeutic and prophylactic compositions for treating

[0175] In some embodiments, the pharmaceutical compositions of the disclosure comprise an anti-FLT3L antibody or comprises an antigen-binding fragment thereof and one or more pharma- ceutically acceptable carriers, diluents or excipients. In this context, "a pharma- ceutically acceptable carrier, diluent, or excipient" includes any substance that is suitable for use in humans or domestic animals. Approved by the U.S. Food and Drug Administration as acceptable for use in Any adjuvants, carriers, excipients, glidants, sweeteners that may not be approved , diluents, preservatives, dyes / colorings, flavor enhancers, surfactants, wetting agents, dispersing agents, suspending agents, These include, but are not limited to, stabilizers, isotonicity agents, solvents or emulsifiers. For example, suitable Carriers are known to those skilled in the art and include stabilizers, diluents and buffers. Suitable stabilizers include Examples include sorbitol, lactose, mannitol, starch, sucrose, and dextran. and glucose, and proteins such as albumin or casein. Suitable buffers include saline, Hanks' balanced salt solution, and Ringer's solution. Examples of the salt include alkali metal phosphates, alkali metal carbonates, and alkaline earth metal carbonates. can be done.

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

[0177] In certain cases, the pharmaceutical compositions of the present disclosure can be prepared by techniques known to those skilled in the art. General considerations in the formulation and / or manufacture of pharmaceutical compositions are found, for example, in Remingo et al. n:The Science and Practice of Pharmacy 2 1st ed., Lippincott Williams & Wilkins, 2005 (which is incorporated herein by reference in its entirety). The anti-FLT3L antibody or antigen-binding fragment thereof may be used to form a liquid, suspension, or emulsion. One or more of the additives discussed herein may be added to the carrier or may be mixed with the carrier. The pharmaceutical composition of the present disclosure may be an aqueous solution, emulsion or suspension. In certain aspects, the pharmaceutical compositions of the present disclosure can be, for example, in a storage or dried or frozen by freeze-drying or spray-drying for formulation purposes. They can then be subsequently reconstituted into liquid compositions by the addition of a suitable liquid carrier. The composition can be reconstituted into a dry formulation using methods known to those of skill in the art. It can be administered.

[0178] The pharmaceutical compositions of the present disclosure can be administered to a subject by a variety of routes known in the art. Typical routes of administration of such pharmaceutical compositions include oral, mucosal, topical, and intravenous. These include dermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal. In embodiments, the pharmaceutical compositions of the present disclosure are administered orally, topically, transdermally, by inhalation, parenterally, sublingually, orally. The composition is adapted to be administered by a route selected from the group consisting of intranasal, intrarectal, intravaginal and intranasal routes. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, and intradermal injection. In certain aspects, the pharmaceutical compositions of the present disclosure include The anti-FLT3L antibody or antigen-binding fragment thereof of the present disclosure is bioavailable after administration to a subject. The composition is prepared to allow the composition to become

[0179] The choice of administration of the pharmaceutical composition will depend on the formulation selected. The are administered in a manner compatible with the formulation, and in such amount as is therapeutically effective. The pharmaceutical composition of the present disclosure may be in the form of a tablet, capsule, powder, granule, ointment, liquid, suppository, injection, or the like. Solids, including but not limited to liquids, inhalants, gels, microspheres and aerosols. The formulations may be prepared in solid, semi-solid, liquid or gas form.

[0180] In certain examples, a pharmaceutical comprising the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein. The composition may be in solid or liquid form. In some aspects, the carrier may be, for example, In another embodiment, the carrier is a liquid, such as in tablet or powder form. The compositions may be, for example, oral syrups, injections or aerosols that are useful for inhalation administration. When oral administration is intended, the anti-FLT3L antibody or its antigen binding site disclosed herein may be administered orally. The pharmaceutical composition comprising the combined fragment may be in either solid or liquid form, where semi-solid, semi-liquid , suspensions and gel forms are included within the forms contemplated herein as either solid or liquid. do.

[0181] In certain embodiments, a pharmaceutical composition comprising the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein is provided. The pharmaceutical compositions may be in the form of powders, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. In some cases, such solid compositions may be prepared in the form of one or more In certain embodiments, the composition may contain a binder, e.g., an inert diluent or an edible carrier such as those described above. Examples include carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, and tragacanth or gelatin; excipients, such as starch, lactose or dextrin; disintegrants, such as alginic acid, sodium alginate, primogel, cornstarch, etc.; lubricants, e.g. Magnesium stearate or Sterotex; glidants such as colloidal dioxide sweetening agents, such as sucrose or saccharin; flavoring agents, such as peppermint, salicylic acid; methyl butylate or orange flavoring; and coloring agents.

[0182] These compositions may be in the form of microspheres, solutions, suspensions, tablets, pills, capsules, sustained release The composition may take the form of a liquid preparation or powder, and may contain from about 0.001 to 95% of the antibacterial agent disclosed herein. Contains FLT3L antibody or its antigen-binding fragment. Some dosage forms are 50μg to 250μg The antibody may comprise an anti-FLT3L antibody or an antigen-binding fragment thereof.

[0183] In some embodiments, the pharmaceutical compositions of the present disclosure are in the form of a capsule, e.g., a gelatin capsule. In addition to the materials disclosed herein, liquids such as polyethylene glycol or oils may also be used. A carrier may be included. Oral formulations may contain commonly used excipients such as pharmaceutical grade saccharin, Cellulose and magnesium carbonate may also be included.

[0184] In other aspects, the pharmaceutical compositions of the present disclosure are liquids, e.g., elixirs, syrups, solutions, In certain embodiments, the liquid is for oral administration or for injection. In certain embodiments, when intended for oral administration, The pharmaceutical compositions of the present disclosure comprise an anti-FLT3L antibody or an antigen-binding fragment thereof disclosed herein. In addition, it contains one or more sweeteners, preservatives, dyes / colorants and flavor enhancers. In the pharmaceutical composition intended to be administered by injection, one or more surfactants are preferably used. Preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers and isotonic agents may be included.

[0185] In certain cases, liquid medicines comprising the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein are Pharmaceutical compositions, whether they are in solution, suspension or other similar form, contain the following components: : sterile diluents, e.g. water for injection, saline solutions, e.g. physiological saline, Ringer's solution, isotonic salts and fixed oils, such as synthetic monoglycerides or or diglyceride, polyethylene glycol, glycerin, propylene glycol or other solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as Ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid buffers, such as acetates, citrates or phosphates, and substances for adjusting isotonicity, e.g. For example, the formulation may contain one or more of the following: sodium chloride or dextrose. Samples, disposable syringes, or multi-dose bottles made of glass or plastic In some embodiments, the injectable pharmaceutical composition is preferably is sterile.

[0186] In another embodiment, the antibody comprises an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein. The pharmaceutical composition may be intended for topical administration, in which case the carrier is preferably a solution, emulsion, or the like. In certain embodiments, the base may be, for example, petrolatum, lauroyl glutamate, or ointment. Nolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, In another embodiment, the viscosity enhancing agent may comprise one or more of an emulsifier and a stabilizer for topical administration. In certain embodiments, when transdermal administration is intended, the pharmaceutical composition may be The anti-FLT3L antibody or antigen-binding fragment thereof disclosed in It can be included with the systolic device.

[0187] In yet another embodiment, the anti-FLT3L antibodies or antigen-binding fragments thereof disclosed herein are The pharmaceutical compositions comprising the compound are intended for rectal administration, for example in the form of a suppository. For the agent, binders and carriers can be, for example, polyalkalene glycols (polyalkalene glycols). In certain instances, the rectal The composition for the preparation of the present invention contains a fatty base as a suitable non-irritating excipient. Examples include, without limitation, lanolin, cocoa butter, or polyethylene glycols.

[0188] In another aspect, a pharmaceutical comprising the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein. The composition comprises a dosage unit that can be administered as an aerosol. The term aerosol refers to a colloid. Various systems range from systems of a mechanical nature to systems consisting of pressurized packages. In certain embodiments, the delivery is of a liquefied or compressed gas. This can be achieved by means of a suitable pump system that dispenses a metered amount of the active ingredient. In embodiments, the aerosol of the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein is In other embodiments, the active ingredient may be delivered in a monophasic, biphasic, or triphasic system. The delivery of the aerosol is carried out by combining the necessary containers, activators, and valves, which may be combined to form a kit. Those of skill in the art will readily appreciate that particular aerosol formulations and modes of delivery include, but are not limited to, boluses, dispensers, and the like. It can be determined.

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

[0190] In other aspects, the pharmaceutical compositions of the present disclosure form a coating shell around the active ingredient. In some cases, the materials that form the coating shell are typically inert. and may be selected from, for example, sugar, shellac and other enteric coating agents.

[0191] In yet another embodiment, the pharmaceutical compositions of the present disclosure in solid or liquid form include and binding to the anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein, thereby In particular examples, the L antibody or antigen-binding fragment thereof may be an agent that aids in the delivery of the antibody or antigen-binding fragment thereof. Suitable agents acting in this capacity include a protein or a liposome.

[0192] In certain embodiments, the pharmaceutical composition to be administered to a subject is in the form of one or more dosage units. where, for example, a tablet may be a single dose unit and may be administered using the aerosols disclosed herein. The container of the anti-FLT3L antibody or antigen-binding fragment thereof in tablet form may hold multiple dosage units. Practical methods for preparing such dosage forms are known or may be This will be apparent to those skilled in the art, and can be found, for example, in Remington: The Science and P practice of Pharmacy,20th Edition(Philade lphia College of Pharmacy and Science,20 00). In any event, the composition to be administered should be in accordance with the teachings herein. Thus, to aid in the treatment of a disease or condition in a subject, a therapeutically effective amount of the antibodies disclosed herein may be administered. Containing an FLT3L antibody or an antigen-binding fragment thereof, or a pharma- ceutical acceptable salt thereof It would be.

[0193] In certain embodiments, the pharmaceutical compositions of the present disclosure comprise one or more additional therapeutically active agents. In other embodiments, a therapeutically effective amount of the pharmaceutical composition of the present disclosure is administered to a subject in need thereof. As used herein, "combination" refers to the administration of one or more additional therapeutically active agents. The present invention relates to an anti-FLT3L antibody or antigen-binding fragment thereof and one or more additional therapeutic agents. It refers to a combination that includes active substances, each of which may be administered sequentially (sequentially), together or simultaneously. It can be administered.

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

[0195] The description of pharmaceutical compositions provided herein is primarily suitable for administration to humans. Although the present invention is directed to pharmaceutical compositions which are It will be appreciated that the subject is suitable for administration to a variety of subjects. In certain embodiments, the subject is In certain embodiments, the mammal is a primate, such as a human, monkey, or ape. and livestock and farm animals, including laboratory animals and familiar pets (e.g., cats, Non-primates such as dogs, pigs, cows, sheep, goats, horses, rabbits, as well as wild animals and birds non-domestic animals.

[0196] Autoimmune / anti-inflammatory therapy The present disclosure relates to the treatment of autoimmune and / or other inflammatory diseases (i.e., hyper-reactive and / or abnormal functions). and anti-FLT antibodies, such as those described above, that are useful for treating diseases involving the immune system. Compositions and methods comprising the 3L antibody are also featured. In various embodiments, the anti-FLT3L antibody is inhibiting a subject's immune system or a specific immune response to a particular antigen or set of antigens; designed to attenuate and thereby reduce or prevent autoimmune or other inflammatory diseases. The present invention can be administered in combination with other immunomodulatory agents that have been shown to be effective against rheumatoid arthritis.

[0197] Further provided herein are methods for treating autoimmune diseases and / or conditions comprising the administration of one or more anti-FLT3L antibodies. As provided herein, methods for treating anti-F Administration of LT3L antibodies results in a reduction in immune responses, inhibition of one or more immunological signaling cascades, In certain embodiments, the method may result in at least one of a decrease in expression or a decrease in immune cell population. An anti-FLT3L antibody is administered to a patient or subject presenting with an infectious disease or other inflammatory disease. .

[0198] Treatment of autoimmune and / or other inflammatory diseases with anti-FLT3L antibodies can be achieved, for example, by , reduction in the rate of progression of autoimmune or inflammatory diseases, slowing or stabilizing immune cell proliferation This can result in the elimination of inflammatory bowel disease, shrinkage of lesions (e.g., in MS patients) and / or regression of the disease. In some embodiments, the invention provides a method for reducing or slowing down an autoimmune or inflammatory disease (e.g., inflammation, inflammatory cycle (reduction in cytokine levels, immune cell populations and / or associated damage such as tissue lesions) The metric may be statistically significant. The reduction is based on a prediction of disease progression based on a large patient population versus the expected level of disease progression for an individual. at baseline relative to the level or relative to the expected level of disease progression in a control population This can be measured by comparison with the patient's baseline levels (before treatment).

[0199] In one embodiment, the methods of treatment contemplated herein include administering to a subject an anti-FLT3L binding molecule of the present disclosure. or administration of an antibody or antigen-binding fragment, variant, or derivative thereof to a subject or patient - Patents.com Alternatively, application or administration of the anti-FLT3L binding molecule to isolated tissue or cell lines derived from a subject or patient. wherein the subject or patient has a disease, a symptom of a disease, or a predisposition towards a disease.

[0200] Examples of contemplated diseases include acute or chronic inflammatory diseases, including type 1 and type 2 diabetes, e.g. For example, diabetes, diabetic nephropathy and CKD, including hypertension-induced CKD, atherosclerosis Alzheimer's disease, cancer and heart disease, high blood pressure, anemia, pericarditis, renal osteodystrophy Additional contemplated diseases include associated complications of such diseases, including include, but are not limited to, systemic lupus erythematosus, myositis, primary Sjogren's syndrome, multiple sclerosis autoimmune diseases including rheumatoid arthritis, uveitis, psoriasis and rheumatoid arthritis.

[0201] In another embodiment, an anti-FLT3L binding molecule, e.g., an antibody or antigen-binding fragment thereof of the disclosure. Application or administration of a pharmaceutical composition containing a fragment, variant or derivative to a subject or patient, or an anti- A pharmaceutical composition comprising an FLT3L binding molecule is administered to an isolated tissue or cell line derived from a subject or patient. Also contemplated are treatments that include the application or administration of have a predisposition towards a condition or disease.

[0202] According to the method of the present disclosure, at least one anti-FLT antibody as defined anywhere in this specification is administered. 3L antibodies can be used to promote positive therapeutic responses for autoimmune or inflammatory diseases. The term "positive therapeutic response" refers to a symptom associated with an autoimmune or inflammatory disease. Thus, for example, improvement in disease may be characterized as a complete response. A "complete response" is defined as any clinically detectable disease accompanied by normalization of any prior test results. Alternatively, improvement in disease may be considered a partial response. A "positive therapeutic response" can be classified as a positive therapeutic response to an anti-FLT3L binding agent disclosed herein. The progression and / or duration of an autoimmune or inflammatory disease resulting from administration of a molecule. reducing or inhibiting the progression of, reducing or alleviating the severity of, autoimmune or inflammatory diseases; and / or alleviation of one or more of these symptoms.

[0203] In certain embodiments, a method for treating primary Sjogren's syndrome, comprising: A pharma- tically effective amount of the antibody or antigen-binding fragment thereof disclosed herein is administered to a subject in need thereof. A method is provided comprising the step of administering

[0204] In another embodiment, a method for treating myositis includes administering to a subject in need thereof a drug The method includes administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein. A method is provided.

[0205] In certain embodiments, the method is for treating systemic lupus erythematosus (SLE). and administering to a subject in need thereof a pharma- tically effective amount of the antibody or antigen binding site disclosed herein. In some aspects, the subject is a healthy subject. FLT3, as measured by the frequency of CD4+ T cells expressing FLT3L, compared with have increased serum levels of L.

[0206] In some embodiments, the method is for diagnosing systemic lupus erythematosus (SLE) in a subject. (a) measuring a serum level of FLT3L; or (b) measuring the frequency of CD4+ T cells expressing T3L and comparing it with that of healthy donors; and elevated serum levels of FLT3L in the subject or CD4+ Methods are provided in which an increased frequency of T cells indicates that the subject has SLE. In certain embodiments, the CD4+ T cells are effector memory cells (T EM ).

[0207] In some embodiments, neutralization of membrane-bound FLT3L is performed in a subject in need thereof. The method includes administering to a subject a pharma- tically effective amount of an antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the FLT3L is a membrane-bound FL The activity of T3L is reversibly neutralized such that it can return to "pre-administration" levels.

[0208] In another embodiment, the neutralization of soluble FLT3L is achieved in a subject in need thereof. The method includes administering to a subject a pharma- tically effective amount of an anti-FLT3L antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the FLT3L is Reversibly neutralized to allow levels of soluble FLT3L to return to pre-administration levels .

[0209] In certain embodiments, the method of neutralizing soluble FLT3L comprises administering to a subject an anti-FLT3L antibody or The antigen-binding fragment is subcutaneously administered once a week in the range of about 0.03 mg / kg to about 30 mg / kg. In another embodiment, the method further comprises administering to the subject an anti-FLT3L antibody or administering the antigen-binding fragment subcutaneously once every four weeks at a dose of about 150 mg / kg. Further includes:

[0210] In another embodiment, circulating classical dendritic cells (cDCs) and plasmacytoid dendritic cells ( A method for reducing a population of erythrocytes (pDCs) in a subject in need thereof, comprising administering to said subject a pharmacologic agent effective in reducing said erythrocytes (pDCs). The method includes administering to a subject an amount of an antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, the population of cDCs and pDCs is a population of cDCs and pDCs. is reversibly reduced so that it can return to pre-administration levels.

[0211] In a particular embodiment, the method is to reduce the expression of FLT3L on CD4+ T cells. and administering to a subject in need thereof a pharma- tically effective amount of the antibody or antigen binding site disclosed herein. A method is provided comprising administering the combined fragment.

[0212] In another embodiment, the percentage of CD4+ T cells expressing FLT3L is decreased. The method comprises administering to a subject in need thereof a pharma- tically effective amount of an antibody disclosed herein. or an antigen-binding fragment thereof.

[0213] In another embodiment, the method comprises the steps of: contacting the lymphoblasts with an antibody or antigen-binding fragment thereof disclosed herein. A method is provided that includes:

[0214] In a specific embodiment, MEK 1 / 2 phosphorylation in primary CD133+ human stem cells is The method comprises contacting stem cells with an antibody or antigen-binding fragment thereof disclosed herein. A method is provided that includes the step of contacting. The present disclosure encompasses the following aspects. [Section 1] An antibody or an antigen-binding fragment thereof that specifically binds to FLT3L, CDR) set: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 is (a) SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively; or (b) SEQ ID NOs: 29, 30, 31, 35, 33, and 34, respectively; or (c) SEQ ID NOs: 29, 36, 37, 32, 33 and 38, respectively An antibody or antigen-binding fragment thereof comprising the amino acid sequence of: [Section 2] The VH and VL each comprise an amino acid sequence comprising a heavy chain variable region (VH) and a light chain variable region (VL). From the carboxy terminus, the order is: FW1, CDR1, FW2, CDR2, FW3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR20, CDR21, CDR22, CDR23, C It contains three CDRs and four framework regions (FW) arranged as R3 and FW4. wherein the VH and VL regions are (a) SEQ ID NO:1 and SEQ ID NO:2, respectively; or (b) SEQ ID NO:3 and SEQ ID NO:4, respectively; or (c) SEQ ID NO: 5 and SEQ ID NO: 6, respectively Item 1, having at least 95%, 96%, 97%, 98% or 99% sequence identity with The antibody or antigen-binding fragment thereof described herein. [Section 3] The CDRs (HCDR1, HCDR2 and HCDR3) of the VH and the VL The CDRs (LCDR1, LCDR2 and LCDR3) are (a) SEQ ID NOs: 29, 30, 31, 32, 33, and 34, respectively; or (b) SEQ ID NOs: 29, 30, 31, 35, 33, and 34, respectively; or (c) SEQ ID NOs: 29, 36, 37, 32, 33 and 38, respectively Item 3. The antibody or antigen-binding fragment thereof according to Item 2, which consists of the amino acid sequence shown below. [Section 4] The VH and VL are (a) SEQ ID NO:1 and SEQ ID NO:2, respectively; or (b) SEQ ID NO:3 and SEQ ID NO:4, respectively; or (c) SEQ ID NO: 5 and SEQ ID NO: 6, respectively The antibody or antigen-binding fragment thereof according to item 2, comprising the amino acid sequence of: [Section 5] (a) a heavy chain region having an amino acid sequence comprising SEQ ID NO:61 and an amino acid sequence comprising SEQ ID NO:62; a light chain region having the sequence (b) a heavy chain region having an amino acid sequence comprising SEQ ID NO:65 and an amino acid sequence comprising SEQ ID NO:66 a light chain region having the sequence (c) a heavy chain region having an amino acid sequence comprising SEQ ID NO: 69 and an amino acid sequence comprising SEQ ID NO: 70 Light chain region having the acid sequence The antibody or antigen-binding fragment according to item 4, comprising: [Section 6] Human stem cells, hematopoietic cell precursors, dendritic cells, activated T cells and B cells, mononuclear leukocytes or myeloid leukocytes Inhibits FLT3L-mediated activation of membrane-bound FLT3 on chromosome 1-5 The antibody or antigen-binding fragment thereof described in claim 1. [Section 7] At least one of human stem cell factor (huSCF) and human colony-stimulating factor (huCSF1) The antibody or antigen-binding fragment thereof according to item 6, which does not specifically bind to any one of the antigens. [Section 8] The antibody or its derivative according to item 6, which does not specifically bind to either huSCF or huCSF1. Fragment. [Section 9] Item 1 is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or a chimeric antibody. 9. The antibody or antigen-binding fragment thereof described in any one of 8 to 8. [Section 10] (a) IgA constant domain; (b) IgD constant domain; (c) IgE constant domain; (d) IgG1 constant domain; (e) IgG2 constant domain; (f) IgG3 constant domain; (g) an IgG4 constant domain; and (h) IgM constant domain Any of items 1 to 9, further comprising a heavy chain immunoglobulin constant domain selected from the group consisting of: The antibody or antigen-binding fragment thereof described in any one of claims 1 to 4. [Section 11] 11. The antibody or antigen-binding fragment thereof of item 10, comprising an IgG1 constant domain. [Section 12] (a) an Igκ constant domain; and (b) Igλ constant domain Any one of items 1 to 11, further comprising a light chain immunoglobulin constant domain selected from the group consisting of: The antibody or antigen-binding fragment thereof described in any one of claims 1 to 4. [Section 13] The antigen binding protein comprises a human IgG1 heavy chain constant domain and a human λ light chain constant domain. 13. The antibody or antigen-binding fragment thereof according to any one of items 1 to 12. [Section 14] The IgG1 constant domain is numbered according to the EU numbering index of Kabat. One selected from the group consisting of L234F, L235E and P331S Item 12. The antibody or antigen-binding fragment thereof according to item 11, which comprises the above amino acid substitutions. [Section 15] An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of items 1 to 14. . [Section 16] 16. The nucleic acid molecule of claim 15, operably linked to a regulatory sequence. [Section 17] Item 17. A vector comprising the nucleic acid molecule according to Item 15 or 16. [Section 18] A nucleic acid molecule according to claim 15 or 16, or a vector according to claim 17, Host cells. [Section 19] 20. The host cell of claim 18, which is a mammalian host cell. [Section 20] A hybridoma that produces the antibody or antigen-binding fragment thereof according to any one of Items 1 to 14. [Section 21] 15. An isolated host cell that produces the antibody or antigen-binding fragment thereof according to any one of items 1 to 14. [Section 22] A method for producing the antibody or antigen-binding fragment thereof according to any one of items 1 to 14, comprising: (a) culturing a host cell expressing the antibody or antigen-binding fragment thereof; and (b) and) isolating said antibody or antigen-binding fragment thereof from said cultured host cells. method. [Section 23] The antibody or antigen-binding fragment thereof according to any one of items 1 to 14 and a pharma- ceutically acceptable excipient. A pharmaceutical composition comprising an excipient. [Section 24] Item 24. The pharmaceutical composition according to item 23, for use as a medicine. [Section 25] A method for treating nephritis, comprising administering to a subject in need thereof a pharmacologic effective amount of 15. A method comprising the step of administering an antibody or antigen-binding fragment thereof described in any one of claims 1 to 14. [Section 26] 1. A method for treating primary Sjogren's syndrome, comprising administering to a subject in need thereof: A pharma- ceutical effective amount of the antibody or antigen-binding fragment thereof according to any one of items 1 to 14 is administered. A method comprising the steps of: [Section 27] A method for treating myositis, comprising administering to a subject in need thereof a pharmacologic effective amount of 15. A method comprising the step of administering an antibody or antigen-binding fragment thereof described in any one of claims 1 to 14. [Section 28] A method for treating systemic lupus erythematosus (SLE) in a patient in need thereof A pharmacologic effective amount of the antibody or antigen-binding fragment thereof according to any one of items 1 to 14 is administered to a subject. Administering [Section 29] The subject is characterized in that the frequency of CD4+ T cells expressing FLT3L is decreased compared to a healthy subject. 29. The method of claim 28, wherein the patient has an increased serum level of FLT3L as measured by [Section 30] 1. A method for diagnosing systemic lupus erythematosus (SLE) in a subject, comprising: a. Measuring serum levels of FLT3L; or b. Measuring the frequency of CD4+ T cells expressing FLT3L or an increased serum level of FLT3L in said subject compared to a healthy donor; An increased frequency of CD4+ T cells expressing FLT3L indicates that the subject has SLE. A method for indicating the above. [Section 31] The CD4+ T cells are called effector memory cells (T EM ) as described in paragraph 30 How to. [Section 32] A method for neutralizing membrane-bound FLT3L in a subject in need thereof, comprising administering Administering an effective amount of the antibody or antigen-binding fragment thereof according to any one of items 1 to 14 to the subject. The method includes the step of: [Section 33] 24. The method of claim 23, wherein the FLT3L is reversibly neutralized. [Section 34] Reduction of circulating classical dendritic cell (cDC) and plasmacytoid dendritic cell (pDC) populations A method for administering a small amount of the compound according to any one of items 1 to 14 to a subject in need thereof, comprising administering a pharmacologic effective amount of the compound according to any one of items 1 to 14. administering to said subject the antibody or antigen-binding fragment thereof of any one of claims 1 to 5. . [Section 35] The populations of cDCs and pDCs are increased by 100% CI, 0.01 to 0.25, with the populations of cDCs and pDCs returning to pre-treatment levels. Item 35. The method according to item 34, wherein the amount of the ion exchange reaction is reversibly reduced so that the amount of the ion exchange reaction is reduced. [Section 36] A method for reducing the expression of FLT3L on CD4+ T cells, comprising: A pharmacologic effective amount of the antibody or antigen-binding fragment thereof according to any one of items 1 to 14 is administered to a subject. Administering [Section 37] 1. A method for decreasing the percentage of CD4+ T cells that express FLT3L, comprising: A pharmacologic effective amount of the antibody or its derivative according to any one of items 1 to 14 is administered to a subject in need thereof. Administering an antigen-binding fragment of said [Section 38] 1. A method for reducing ERK signaling in lymphoblasts, comprising: The method includes a step of contacting the antibody or antigen-binding fragment thereof according to any one of items 1 to 14. How to do it. [Section 39] A method to reduce MEK 1 / 2 phosphorylation in primary CD133+ human stem cells. Then, the stem cells are contacted with the antibody or antigen-binding fragment thereof according to any one of items 1 to 14. The method includes the step of:

[0215] The following examples provide methods for making and using the assays, screening and treatment methods of the disclosure. This disclosure is provided to provide those of ordinary skill in the art with a complete disclosure and description of the methods. It is not intended to limit the scope of what the inventors regard as their disclosure. EXAMPLES

[0216] The present disclosure will now be described with reference to the following examples. The examples are merely illustrative and are not intended to be limiting of the present disclosure. The disclosure should in no way be construed as being limited to these examples, but rather to the specific examples provided herein. It should be construed to include any and all variations which become evident as a result of the teachings herein.

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

[0218] [Table 14]

[0219] The complete mouse FLT3L protein is only 73% identical to its FLT3 counterpart. The binding site for FLT3L is highly conserved among species. FLT3L binds to and activates stem cell factor (SCF) or It shares structural homology with KIT-ligand and colony-stimulating factor 1 (CSF1), but These two ligands have no significant sequence homology to other human cytokines. The receptors for FLT3, c-KIT and CSF1R, typically interact with FLT3 inhibitors. It is also a class III TKR that acts on undesirable off-target sites in clinical use. Taking these factors into consideration, we developed a highly specific inhibitor of the FLT3 / FLT3L pathway. Antibodies against FLT3L that will not cross-react with SCF or CSF1 was explored.

[0220] Lead antibodies were selected using alternative selection for soluble human and mouse FLT3-L The primary biochemical high-throughput screen was performed using human FLT3 / FLT3L was performed using a fully homogeneous competitive time-resolved fluorescence (HTRF) assay. The hits of single chain variable fragment-fragment crystal domain (scFv-Fc) were synthesized in IgG1 TM format. This is done by converting the IL-1 receptor agonist (IL-1 receptor) into a IL-1 receptor agonist (IL-1 receptor agonist) and then downregulating FLT3 on the target cell surface. The inhibitory activity of the lead antibodies was confirmed using a functional screening assay. Cross-reactivity and selectivity for FLT3L in humans and cynomolgus monkeys, as well as stem cell factor (S cf) Exclusion of other family members such as Confirmed.

[0221] The lead compound was shown to be effective in detecting FLT in the RS4;11 cell line and primary human CD133+ stem cells. The binding to endogenous FLT3L was further evaluated using a signal transduction assay. The specific binding sites and binding affinities were confirmed using human T cells. The resulting lead was determined as described below. A single antibody clone was selected for further optimization.

[0222] Identification of lead antibody candidates (C5, B10-11) The lead antibodies for anti-FLT3L binding studies were isolated from the phage library as shown in Figure 1. The phage display library was generated by first screening The bone marrow Vaughan (BMV), combined spleen (CS), and DP47 libraries ( DP47) and the Dyax human antibody library are targeted to human FLT3L (huFLT3L) and and / or mouse FLT3L (muFLT3L) (alternating panning or competitive Briefly, in-house generated FLT3L was used as the panning antigen via EZ-Link Sulfo-NHS-LC-Biotin Labeling Kit (Thermo Scientific) The panning was performed as described in (Xiao X et al., 201 7 mAbs 542 9,996-1006 (2017)) Two to three rounds were performed using the scFv library. To increase the response, some selection processes use human and human IgG as panning antigens in an alternating fashion. We used recombinant cyno FLT3L. We used antibodies that inhibit the FLT3L / FLT3 interaction. Competitive panning was used for some experiments to select Human FLT3L was used as the screening antigen, but phage lysis was used instead of conventional trypsin. An excess (>100-fold) of FLT3-Fc was used as the extractant (Xiao X et al. .mAbs 542 9,996-1006(2017)).

[0223] BMV library was diluted 100x, CS library was diluted 3x, 50x and DP47 live The libraries were enriched 250-fold. The Dyax libraries were not enriched. Only the Dyax phage library was panned three times, and the second round of panning and In comparison, there was a 100-fold enrichment of the BMV phage library and a 50-fold enrichment of the Dyax library. A fold enrichment occurred (Fig. 1A).

[0224] Monoclonal phage ELISA was performed to identify antigen-specific phage antigens after each selection round. For high-throughput screening, at least Only those selections that achieved a 20% positive rate were processed. Using this criteria set, the third output BMV panning of the first output together with CS and DP47 panning of the second output Cloned into ice V4 and pdLG or pmLG vectors and analyzed by competitive HTRF For functional screening, the scFv-Fc format (Xiao X, et al. .,PLoS One,2015 Oct 15;10(10):e0140691.d oi:10.1371 / journal.pone.0140691) or IgG phosphatase Mat (Xiao X, et al. mAbs 542 9, 996-1006 (201 7) was converted to one of the following:

[0225] For functional screening, 293 freestyle cells (Thermo S Scientific first generated scFv-Fc Cells were transfected with either the IgG construct or the HT Used directly in an RF-based FLT3L / FLT3 interaction inhibition assay For the HTRF assay, 10 nM biotin-labeled FLT3L, 20 nM streptavidin, Ptavidin-Europium Cryptate (Cisbio), 10 nM FLT3-mF c and 20 nM anti-mFc-A647 (Cisbio) were plated in a 384-well Greiner Mix with 10 μL of transfection supernatant in a total volume of 20 μL / well in the plate. The 665 nm and 620 nm readings were taken 5 minutes after mixing and for 1 hour until the readings stabilized. The 665nm / 620nm ratio was then calculated. The decrease in the ratio was Inhibition of FLT3L / FLT3 interaction was demonstrated.

[0226] The panning output from direct or alternate antigen panning is converted to scFv-Fc. High-throughput HTRF FLT3L / FLT3-Fc interaction inhibition assay Over 4,000 colonies were selected for high-throughput screening (HTS). 10 leads Antibodies were identified, converted to IgG TM, and expressed. These antibodies were used in the second HTRF cross-reaction. Ten leads were identified by retesting in the competitive inhibition assay. Over 700 hits from panning were converted to PmIgG and expressed in mammalian cells. The transformed clones were then subjected to the same HTRF FLT3L / FLT3-Fc interaction inhibition assay. After sequencing, two reads were identified (Figure 1B).

[0227] Ten leads from alternating antigen panning and two leads from competitive panning were expressed. The FLT3 protein was then purified in milligram quantities for further testing. Inhibition of L / FLT3-Fc interaction, receptor down-modulation and signal transduction inhibition These leads were also subjected to epitope binning and affinity determination methods. The screening activity resulted in the identification of five lead antibodies, Dyax3, Dyax5, CAT8 and CA T26 and CAT5D9 were identified and further tested. The results from the competitive HTRF analysis are shown in Figure 1C.

[0228] Example 2. Screening assay for downregulation of FLT3 After ligation of FLT3L, the FLT3 receptor dimerizes, autophosphorylates, and mediates downstream In this process, surface FLT3 is internalized and secreted into the endothelial cells, activating signaling pathways. This internalization feature can be effectively utilized to screen lead anti-FLT3L candidate clones. We developed an assay to detect cell surface FLT3 expression using flow cytometry. Inhibition of receptor-ligand binding can be measured by ELISA, and inhibition of cell surface FLT3 expression The change to the current level can be determined by quantifying it.

[0229] The cell lines RS4;11, EOL-1, MOLM13 and MV4-11 express FLT3. Cells were cultured under normal conditions, and the relative expression of FLT3 was measured after 2 to 24 hours of culture. The expression of FLT3 was measured using flow cytometry. Briefly, we used the commercially available anti-CD135 (anti-FLT3) clone BV1 0A4H2 (Biolegend) was used in flow cytometry experiments, and the mean fluorescence The median light intensity (MFI) was reported as a measure of FLT3L expression. Available mouse anti-human FLT3L monoclonal antibody (R&D) or in-house huFLT3 -Fc construct was used as a positive control for effective neutralization of FLT3L activity. .

[0230] The acute leukemia (pro-B) cell line, RS4;11, has been reported to express FLT3. showed consistent high expression of FLT3 in culture compared to other commercially available cell lines Direct binding of FLT3L to cell surface FLT3 was confirmed by biotinylated recombinant hu Serial dilutions of FLT3L (rhuFLT3L) were used to validate the antibody titer. The material was incubated with RS4;11 cells for 30 min at 4°C and then transfected with BV421-sera. Streptavidin can be used to detect physical binding to the cell surface, and then filamentation can be performed. Flow cytometry analysis was performed to determine the mean fluorescence intensity (Figure 2B).

[0231] Finally, rhuFLT3L suppressed detectable downregulation of cell surface FLT3 on RS4;11 cells. The ability of RS4;11 cells to induce upregulation of FL was determined by incubating RS4;11 cells for 2 h at 37°C. The stability of the cells was confirmed by incubation with serial dilutions of T3L. Two different concentrations of RS4;11 cells (50,000 cells (50K) and 100, 000 (100K) cells were used to evaluate the downregulation of cell surface FLT3. The expression level was determined using a fluorescently labeled anti-CD135 antibody (clone BV10A4H2). The cell density of each cell was 100% after 2 hours of incubation with huFLT3L. The alloantigen, an indicator of FLT3 expression, showed a dose-dependent downregulation. Phycocyanin (APC) MFI was measured over the range of huFLT3L used in this assay. These results indicate that the screening assay is a biomarker for Availability: Useful for measuring dose-dependent response of FLT3L and candidate clones are FL This reaction proved to be suitable for testing the ability of the antibody to functionally neutralize T3L. was significantly reduced using either 50,000 or 100,000 cells per well. There was no difference.

[0232] Optimization of screening assays To determine the ideal cell culture conditions for evaluating anti-FLT3L candidate clones, The screening assay conditions were further refined. The final conditions were 37°C, 5% CO 2 Set to The cells were incubated in a humidified incubator with 1% bovine serum albumin (BSA) for 2 hours. Anti-FLT3L mAb candidate clones were cultured in complete Roswell Park Memorial Institute (RPMI) medium containing Incubated with 96 pM rhuFLT3L with or without lawn, 1 There were 50,000 RS4;11 cells per well. Subsequent doubling of FLT3 expression Downregulation was measured either as raw MFI or as the percentage of downregulation. The EC80 of 96 pM of rhuFLT3L was determined by flow cytometry. ) was chosen because it was at the beginning of the exponential phase of the dose-response curve and therefore Functional inhibition down-regulates the level of FLT3 on the cell surface of RS4;11 cells This was because it was a point where any change in the A) Assay validation was performed using a commercially available mouse anti-huFLT3L antibody control. (MAB608, R&D Systems) (Figure 3B). Due to the cross-species reactivity with its ligand, this assay is compatible with human cell lines. It is useful for testing clones against human, cyno and rodent FLT3L. For mouse FLT3L, the EC80 was 36 pM.

[0233] Example 3. Neutralizing activity of lead antibody candidates against soluble FLT3L After ligation of FLT3L to FLT3, the receptor dimerizes and autophosphorylates. Upon internalization, it propagates the signaling cascade. This process, measured by cytotoxicity, was inhibited by antibodies that bind to FLT3L. Therefore, for lead candidates, whether they are human, mouse or cynomolgus monkey (cynomolgus monkey) ) FL on RS4;11 cells by binding to soluble FLT3L (sFLT3L) The ability to inhibit the downregulation of T3 was tested. Optimization Screening The assay was used to test the neutralizing capacity of lead candidates. As described above, RS4;11 Cells (50,000 cells per well) were incubated for 2 hours in either human or cyno In the presence of either FLT3L (96 pM) or mouse sFLT3L (36 pM), 1% Serial dilutions of each clone were added and incubated in complete RPMI containing BSA. As positive controls, soluble FLT3-Fc constructs or commercially available human anti-FLT The expression of FLT3 on RS4;11 cells was analyzed by flow cytometry. and reported as MFI.

[0234] As shown in Figures 4A and 4B, all lead clone candidates were identified as human and cyno s We demonstrated the ability to inhibit FLT3L to some degree. All of Dyax5 and Dyax3 showed similar inhibition of human sFLT3L and cyno sFLT3L. As shown in Table 3 below, CAT8, CAT26, Dyax3 and Dyax5 The IC50 value for MAX has never been reached, so it lacks validity .

[0235] In contrast, CAT5D9 uses IC MAX achieved the therapeutic effect predicted by FLT3L inhibition. The S-shaped curves for CAT5D9 presented in Table 3 were generated (Figures 4A and 4B). IC50 values ​​indicate cross-reactivity with human and cyno, but not with mouse FLT3L. No cross-reactivity was observed (Figure 4C).

[0236] [Table 15]

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

[0238] Consistent with this, cell surface binding to human, cyno, and mouse FLT3L was observed in Chinese CHO cells were transfected with each full-length protein. Candidate clones were evaluated by infecting FL Incubation with cell lines expressing T3L followed by incubation with 100 µL of ... The cells were then washed twice. To quantify the bound antibody, the cells were then stained with PE-conjugated goat anti-hu IgG secondary. Incubation with detection pAb was performed for flow cytometric analysis of PE signal. The huFLT3-Fc construct on a hu IgG backbone was added to a human ligand. In addition, it cross-reacts with cyno and mouse, making it a positive control for FLT3L expression. It was used as a solvent reagent.

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

[0240] Thus, all lead candidates have the ability to bind cross-species FLT3L, although with varying potency. The difference in receptor occupancy for each clone was evident, so the EC5 The 0 and maximal occupancy (expressed as % compared to FLT3-Fc) were recorded and used for final evaluation. Results showed that human, cyno and mouse FLT3L expressed in CHO cells The results are shown in Table 4.

[0241] [Table 16]

[0242] Example 5. Binding of lead antibody candidates to endogenous human FLT3L FLT3L is a cytokine that regulates IL-2 expression independently of TCR engagement. , is expressed on the surface of primary T cells upon stimulation with IL-7 or IL-15. In this study, the lead antibody candidate was synthesized from CH transfected with human FLT3L protein. The next step was to demonstrate their ability to bind to endogenous IgG1-specific ... The objective of the study was to confirm that the lead antibody candidates could bind to FLT3L. These results suggest that huFlt3L stimulated by IL-2 in primary T cells obtained from human donors The ability to bind was tested.

[0243] To induce FLT3L expression on the cell surface, freshly isolated human T cells were incubated for 5 days. In the absence of anti-CD3 for a long period of time (anti-CD3-induced T cell activation was observed by FL The cells were stimulated with 50 ng / mL IL-2 at 4 °C for 1 h (which would induce T3L shedding). Subsequently, expression on T cells was confirmed using a human FLT3L-Fc construct (Figure 6A). Serial dilutions of each clone were then incubated for 30 min at 4°C with IL-2 stimulated T cells (100K / well). Excess antibody was removed by washing with buffer, and the plate was then incubated with 100 μl of ... Surface-bound antibodies were detected with APC-conjugated anti-human IgG. All lead cloning except CAT5D9 The antibody bound to endogenous FLT3L on human primary T cells (Fig. 6B). Binding of CAT5D9 to IL-1 could not be detected initially due to its low binding affinity. However, before incubation with T cells, CAT5D9 was incubated with anti-IgG (APC-labeled When dimerized using the α-aminobutyric acid α-aminobutyric acid (AGA), its avidity was dose-dependent in binding to endogenous FLT3L. was sufficiently enhanced to confirm this (Figure 6C).

[0244] Example 6. Inhibition of cell surface FLT3L by lead antibody candidates The ability of lead candidates to bind cell surface FLT3L provides limitations regarding functional inhibition of the ligand. Ideally, binding of lead candidates to cell surface FLT3L would be consistent with the binding of ligand-specific FLT3L. This should decrease the signaling activity of the gand-receptor complex.

[0245] To test this, we cultured 1,000 huFLT3L-expressing CH cells per well. CHO cells were plated overnight to allow attachment. The next day, the CHO culture medium was removed and Cells were gently washed with RPMI and serial dilutions of antibodies were added for 30 min. Then, FLT3+RS4;11 (100K / well) was added. After incubation, RS4;11 was used to detect downregulation of FLT3. The cells were transferred to a fresh 96-well plate on ice for staining. In addition to the dyes used for the FLT3 downregulation assay, Anti-CD19 was included to exclude any contaminating CHO cells. Downregulation of FLT3 The activity was measured by flow cytometry.

[0246] Lead antibody candidate blocking assays demonstrated that all lead candidates blocked cell surface FLT3L to some degree. Although the candidates demonstrated the ability to inhibit IL-1, all were significantly lower than the commercially available control, reflecting their low affinity. The activity of the antibody was relatively low compared to that of the control antibody (Figure 7).

[0247] Example 7. Inhibition of FLT3L-induced FLT3 signaling in lead clones Autophosphorylation of FLT3 primarily signals through the PI3K and RAS cascades This leads to the activation of a signaling network, which in turn activates AKT (protein kinase B, PKB, MEK and ERK are activated. The signaling cascade ultimately leads to the apoptosis of the cell. Lead antibody candidates induce transcription of genes that promote survival and proliferation in primary human cells. To establish whether FLT3L-mediated downstream signaling of FLT3 was blocked, Phosphorylation of ERK and MEK in CD133+ stem cells was observed in Mesoscale MSD ho Phospho-ERK1 / 2 and phosphor-MEK1 / 2 were measured using whole cell lysates.

[0248] The validity of the assay was confirmed by measuring FLT3L-induced ERK activation in a dose-dependent manner. This was confirmed using the RS4;11 cell line (Figure 8A). Incubate with 300,000 RS4;11 cells per well for 8 min at 6°C. Cells were then harvested, lysed, and assayed for phosphorylated ERK according to the manufacturer's instructions. The assay was performed according to the manufacturer's instructions. As in the previous assay, FLT3L activation of ERK was observed. The EC80 for the antibody was determined (476 pM) and used to identify candidate anti-FLT3L antibodies. The usefulness of the assay was demonstrated by the positive control for effective neutralization. This was confirmed using a commercially available mouse anti-human FLT3L antibody as a standard antibody (Figure 8B).

[0249] Using these established parameters, lead clones were FL-tested prior to use. Using in vitro expanded primary CD133+ stem cells validated for T3 expression The lead clone was preincubated with 476 pM FLT3L for 30 min. The cells were incubated at 36°C for 8 minutes and then added to the CD133+ stem cells. After incubation, cells were harvested, lysed, and the MSD assay was performed according to the manufacturer's instructions. Phosphorylated ERK and MEK were assayed using commercially available mouse antibodies. Human FLT3L was used as a positive control.

[0250] All candidate clones showed induction of MEK (Fig. 9A) and ERK (Fig. 9B) by FLT3L. However, only CAT5D9 inhibited IC MAX Reaching the expected sigmoidal dose The reaction curves are shown in Table 5. In summary, the results presented in the examples show that CAT5D 9 was the best lead candidate clone and was characterized by affinity and biophysical properties of the epitope binding site. It is suggested that the evaluation should proceed to optimization as a condition.

[0251] [Table 17]

[0252] Example 8. Confirmation of target specificity CAT5D9 was identified as the best lead candidate based on functional evaluation in biological assays. Using the Octet epitope binning method, the C The AT5D9 binding region was determined.

[0253] Using epitope binning, we identified a region on FLT3L that shares the same binding domain as the FLT3 receptor. The binning was carried out in three stages. In the first stage, the avidin probe was used. In Phase II, individual antibodies were conjugated to biotin-FLT3L. In Phase III, each of the test antibodies was added to the Phase II antibody. Any additional binding detected indicates that the two antibodies have non-competing binding sites for the target FLT3L. In Phase III, the results showed that the buffer alone without antibody and FLT3 -Fc was used as a negative control and a positive control, respectively. When buffer alone was added, the resulting dissociation kinetics of the antibody against FLT3L was It reflects the inherent affinity of the clone for the target.

[0254] Only CAT5D9 added at 1- and 2-fold concentrations inhibited binding by FLT3L-Fc. This indicates that CAT5D9 hits the desired target site on FLT3L (i.e. These results suggest that the IL-16A- and IL-16B-specific markers share the same or overlapping epitopes with FLT3-Fc (Fig. 10A). Importantly, in contrast, the remaining four candidate clones were not amplified by CAT5D9. The inhibition of FLT3 by α- and β-lactamase was not observed in the control group, suggesting that they bound to different FLT3 epitopes. Furthermore, the steep off-rate when buffer alone was added was significantly higher than that of CAT5. Low affinity for D9 (meaning that optimizations are likely to improve performance) Consistent with this, the remaining four clones When each of the above is combined in phase II (C as shown in the representative chart presented in FIG. 10B), Additional binding of AT8), CAT5D9 and FLT3-Fc was detected in Phase III. This reflects the different binding sites of these four clonal antibodies. Each of the lines was binned together and their concentrations increased when buffer alone was added. A significantly slower dissociation rate was also observed.

[0255] Taken together, these data indicate that only CAT5D9 binds FLT3 at the FLT3L binding region. This confirms what was thought to be the case with low affinity binding, directly competing with This suggests that CAT5D9 may have potential for optimization. Some of the hits were in sites that did not directly compete with FLT3 based on functional data. Their slow dissociation rates indicate that they are already bound to FLT3L with reasonable affinity, These results suggest that the ELISA kit has little potential for optimization. Initial screening assay The inhibition initially present in the IL-1 receptor may be due to steric hindrance or partial blockage of the receptor binding site. The potential was high.

[0256] Example 9. BIACORE Binding Kinetics (C9) Biacore analysis was used to determine the binding kinetics of antibody leads, and CAT5D9 was found to be FLT This confirmed the Octet data that suggested anti-FL has poor affinity for 3L. The kinetics of T3L fragment antigen binding and human and cynomolgus monkey FLT3L were examined using human CAT8, CA Furthermore, the binding of the CAT5D9 fragment antigen was confirmed in T26, Dyax3 and Dyax5. The binding kinetics were determined for both human and cynomolgus monkey FLT3L.

[0257] The results are shown in Table 6. The equilibrium dissociation constant (K D ) compared to the remaining lead antibodies, CA T5D9 was >50-fold higher in humans and cynomolgus monkeys. CAT5D9 was a low-quality rapid oncolytic assay. The results showed a steady-state dynamics (hu=70.72, cyno=70.66). The combined data is dynamic data K D The steady-state binding showed similar values. These data support the previously reported kinetic binding results (Table 3). This confirms the feasibility of the proposed method and the possibility of improving its performance through optimization.

[0258] [Table 18]

[0259] Example 10. Absence of cross-reactivity of CAT5D9 In addition to confirming that CAT5D9 bound to the correct FLT3L epitope, , stem cell factor (SCF) and colony-stimulating factor (SCF), which are close structural homologs of FLT3L It was also important to ensure that the IL-16 receptor agonist (IL-16) would not bind to CSF1. To manage malignancies arising from constitutively activating FLT3-IT9D mutations Major off-targets for small molecule FLT3 inhibitors currently used in oncology The protein tyrosine kinase receptors c-Kit and CSFR1, which are the target genes, ) is a ligand for

[0260] To test this, ELISA plates were pre-coated with 2 μg of recombinant human SCF or CSF1. After washing, the plates were coated with 3 mL of Tris-phosphate buffered saline (TPBS). % milk and added in serial (2-fold) dilutions starting at 50 μg / mL. After incubation, unbound antibodies are removed by washing, and bound antibodies are incubated for staining. Detection was performed using anti-human IgG-HRP in combination with TMB substrate. Goat anti-SCF pAb and mouse anti-CSF1 mAb were used as positive binding controls did.

[0261] Both lead candidates cross-reacted with huSCF (Figure 11A) or huCSF1 (Figure 11B). Importantly, these results indicate that the structurally similar This demonstrates the selectivity of CAT5D9, which does not bind to TKR ligand molecules.

[0262] Example 11. Optimization of affinity of CAT5D9 As discussed above, CAT5D9 binds to FLT3L with low affinity and thus It was demonstrated that affinity optimization could improve performance. The desired 300 pM The KD of was set based on PK modeling. Optimization activity was achieved with a KD of up to 10,000-fold. It was designed to achieve an improvement in D.

[0263] After germlining the framework, two parallel strategies, parsimony ( ous mutagenesis and block mutagenesis were used. Parsimonious mutagenesis was used for all 6 All 20 amino acids are mutated at each position in the CDRs, one at a time. The lines are screened using high-throughput methods, and individual beneficial mutations are identified. Block mutagenesis is performed by combining 5-6 CDRs in an overlapping pattern. Mutate a continuous stretch of positions, resulting in approximately 1E6-1E7 clones The library was first enriched using a phage display panning method and then The antibodies are screened using high-throughput methods.

[0264] After the first optimization round, 30 clones from parsimonious mutagenesis and block mutations were identified. 24 clones from the mutations were tested in IgG format. We found a KD of 1610 nM (measured by Biacore), close to the parent 5D9. The best clone yielded clone 5D9-clone 6, which achieved a 700-fold improvement in affinity compared to the The following mutations were identified and combined (see Figure 7). The affinities of AM40 and AM41 exceeded the CDTP criteria (<300 pM). Lone binds to and neutralizes endogenous cell surface FLTL and neutralizes endogenous soluble FLT in human serum. It binds to cyno FLT3L and neutralizes it.

[0265] [Table 19]

[0266] This increase in affinity was confirmed by RS clone selection using the methods described for the initial clonal selection. The function was measured by downregulation of FLT3 on 4;11 cells (Figure 12A). This translated into a more than 1000-fold improvement in potent activity.

[0267] A second round of affinity optimization was performed to achieve an affinity of 300 pM. The clone 6 (C06) was mutated, and the resulting mutant was The screening was carried out in the same manner as above. As such, clone 6 is a clone with substantially higher affinity in the IgG format. The best clones from block mutations were used as competitors to enrich the loans. The clone used was AM40 with KD = 170 pM. Combine the mutations from the analytical clones and the second round of parsimony mutagenesis to obtain the KD One excellent clone, SC4017, was generated with a β-binding affinity of 0.01 pM. The effect was confirmed by downregulation of FLT3 in RS4;11 cells. Again reflecting improved functional inhibition of FLT3L (Fig. 12B). Subsequent analysis demonstrated that the superior performance of SC4017 was due to the incorporation of a single They found that the extra tryptophan in the tryptophan residues exposed to oxidation was responsible. Given the vulnerability of the base, this represented a development risk. For this reason, AM40 was the lead It was selected as an IgG clone. Its affinity was slightly lower than that of SC4017 (37 pM). Despite being low (170pM), the AM40 still beats the initial target of 300pM. Because it exceeded its benchmark, it was determined to have a lower development risk.

[0268] Finally, we demonstrated that in co-culture with FLT3+RS4;11 over a 7-day period Use primary T cells stimulated with 20 ng / mL IL-7 (cells on primary T cells) This was found to be the most effective protocol for inducing surface FLT3L expression. We developed an assay to confirm that both clones neutralize endogenous FLT3L on the cell surface. Briefly, IL-7-stimulated CD4+ T cells were co-cultured with RS4;11 cells. Both were administered overnight at a ratio of 15:1 (dose ratio response shown in Figure 13A) either alone or in combination with the candidate clones. The cells were incubated in the presence of serial dilutions of the FL Downregulation of T3 was confirmed by flow cytometry as previously described. Both clones inhibited RS4;1 at concentrations above 1 nM (Figure 13B). 1 completely prevented the downregulation of FLT3 in cells with similar efficacy. It was proven that:

[0269] Example 12. Neutralization of FLT3L in healthy non-human primates To determine the safety and durability of AM40 in neutralizing FLT3L, Toxicity studies were conducted over a one-month period using one repeated dose. Animal growth was tracked. To assess the efficacy and safety of schizophrenia, an 8-week treatment follow-up period was included. As shown in FIG. 15A, free soluble FLT3L levels increased with increasing initial dose of AM40. The effect of 0.3 mg / kg was significantly reduced after the first dose, but the effect was not significantly reduced after the first dose. The high dose groups (1 mg / kg and 30 mg / kg ) in the 1.0 mg / kg group, soluble FLT3L levels were at baseline at that time point. Consistently full target engagement (below BLQ) through day 57 of return The results showed that FLT3L levels in the IL-16 / IL-2 cells were significantly higher than in the control cells (reflected as free soluble FLT3L).

[0270] Similarly, the frequency of circulating DCs (as detected by flow cytometry and measured at baseline) was Measurements of the percent of total CD45+ cells expressed as a percent of in-level were performed at 1.0 and 30 mg / mL. The frequency of CD1c+ (classical) DCs and plasmacytoid DCs by day 22 in the 1 kg group was The results showed a steady decrease in the 1 mg / kg and 30 mg / kg groups (Fig. 15B). For each, the frequency of circulating CD1c+DC was suppressed by days 50 and 85. In the 1 mg / kg and 30 mg / kg groups, circulating pDCs The incidence of bronchitis remained decreased until days 71 and 85. The recovery of DC populations was related to the return of free serum FLT3L, which occurred sometime between days 57 and 85. These results suggest that DC populations are more susceptible to FLT3L neutralization by AM40 than DCs. However, it rapidly recovers to baseline when free FLT3L becomes available. This shows that:

[0271] Example 13. Expression of FLT3L correlates with the severity of systemic lupus erythematosus (SLE) R SLE is an autoimmune disease characterized by chronic inflammation that affects almost all organs and tissues in the body. SLE commonly affects the joints, skin, kidneys, lungs, heart, and brain. Given its role in proinflammatory signaling, FLT3L To explore the correlation between FL levels and disease severity, we The expression of T3L was investigated. Studies published to date have not shown any correlation with disease. Most of these are dependent on serum FLT3L levels, and serum FLT3L levels are clinically In this situation, it is the most practical measurement, but it is utilized by DCs and other activated FLT3L-consuming cells. It has the inherent disadvantage of reflecting the negative effects of the production of FL in the setting of inflammation. This is because the number of T3-expressing cells and their consumption of FLT3L are highly variable. This is due to the variability in observational findings between studies and the association of serum FLT3L with clinical scores for disease progression. This likely explains why T cells play a key role in inflammatory conditions, and therefore show no direct correlation with the immune system. Therefore, the inventors have developed a new single Direct expression of FLT3L on the T cell surface using isolated peripheral blood mononuclear cells (PBMCs). We developed an assay to quantitatively measure

[0272] Serum and PBMCs were collected from individuals with SLE (n=24) and healthy donors (HD; n=1 5) Serum FLT3L was measured using ELISA (R&D Systems). CD4+ T cells expressing FLT3L were measured using a 100-mL IgG antibody according to the manufacturer's instructions. FLT3L was determined using a flow cytometry analysis developed in-house and was assayed with fluorescently labeled anti-F Detection was performed using the LT3L clone MAB608 (R&D Systems). To determine the activity of lupus in an individual, the SLE Disease Activity Index (SLEDAI) Significance was assessed for comparisons of healthy vs. diseased cohorts and for the SLEDAI and Correlations were determined using Mann-Whitney and Spearman correlations.

[0273] Consistent with previous literature, serum FLT3L levels were higher in SLE donors compared with HD. (p<0.05; Fig. 16A), but no significant correlation with disease activity (SLEDAI) was observed. In contrast, FLT3L production was not suppressed by FLT3L expression (p<0.07; Fig. 16B). As measured by the frequency of CD4+ T cells expressing 3L, SLE donors were more likely to develop pulmonary fibrosis compared with HD donors. There was a highly significant increase in the sham-induced pulmonary function (p<0.0001; Fig. 16C) and SLEDAI scores. A strong correlation between the two (r = 0.7045; p < 0.0001; Figure 16D) was observed. The data suggest that measuring FLT3L expression on T cells may be particularly important in disease settings. This suggests that

[0274] We found a correlation between SLEDAI scores and CD4+ T cells expressing FLT3L. Therefore, (1) expression of FLT3L across CD4+ T cell subsets from SLE patients was (2) whether the expression of the subset was consistent with the SLEDAI score Subsets of CD4+ cells were examined to determine whether they correlated with the The D4+ subset is composed of naive T cells (T naive ), effector memory cells (T EM ) and Central Memory (T CM ) cells. To assess the significance of the variances in the mean mean, we used the same protocol and significance levels as described above.

[0275] Expression of FLT3L across CD4+ T cell subsets was consistent with known In particular, FLT3L expression was generally associated with T cell death in HD. naiv e A small but significant increase was observed in SLE donors, but not in cells ( (Figure 17A, top). T cells from SLE donors naive Expression of FLT3L on SL cells It was significantly correlated with the EDAI score (Figure 17A, lower; r = 0.6629; p = 0.0 004). Importantly, expression of FLT3L is known to induce expression of FLT3L. capture recently activated T cells that may have been exposed to β-chain cytokines As expected in this population, HD and SLE CD4+T EM Observed in both This response was seen in both HD and SLE donors, but was significantly less in the latter. Furthermore, expression in SLE donors correlated with SLEDAI (Figure 17B , downward; r = 0.6201; p = 0.0012). Finally, FLT3L expression was significantly associated with increased risk of developing glaucoma in healthy subjects. D4+ T cells are EM From T CM However, in SLE donors, The expression of FLT3 was maintained in PBMCs of mice (Fig. 17C, upper panel; p<0.0001). There was a significant correlation between the frequency of L+ T cells and SLEDAI, which may explain the association between the frequency of L+ T cells and SLEDAI in this group. These results suggest that its expression reflects a chronic inflammatory state.

[0276] Taken together, these studies demonstrated that FLT3L expression on CD4+ T cells in SLE patients We have demonstrated that FLT3L expression is significantly increased in HD patients compared with that in HD patients. Increased expression of 3L is highly correlated with SLEDAI score. Administration of anti-FLT3L antibodies inhibits FLT3L to reduce inflammation in SLE patients. It is a rational therapeutic strategy to reduce expressing T cell populations.

[0277] The method used above was the PrimeFlow in silico analysis of IC FLT3L RNA. Validated using tu detection and confirmed using APC-conjugated AM40 Ta.

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

[0279] PBMCs from individuals with myositis and HD were analyzed using FACS to determine whether they expressed FLT3L. We tested CD4+ T cells that express T naive Subset, T EM Subset and T CM The significance between myositis and HD samples was Determined using Mann-Whitney analysis.

[0280] As can be seen from Figures 18A and 18B, FLT in PBMCs from individuals with myositis The expression of FLT3L is naive (p<0.05; Figure 18A), T EM (p<0.0001; Fig. 18B) and T CM (p<0.0001; Figure 18C) The study was characterized by a significant increase in the percentage of positive CD4+ T cells, and thus SL In light of these results, the use of anti-FLT in patients with myositis is Administration of FLT3L antibody targets FLT3L-expressing T cells to reduce inflammation in patients with myositis. It is a rational therapeutic strategy to reduce the cell population.

[0281] Example 15. Expression of FLT3L in nephritis Nephritis is an immune disorder that affects the kidneys and is related to the structure of the kidney. This condition is Nephritis can be fatal. Dendritic cells have been shown to infiltrate the kidney in lupus nephritis, which can result in permanent loss of kidney function. It has been reported (Fiore et al., (2008) Mol Immunology v45:259-265), which is thought to play a role in driving inflammation in the kidney. Therefore, blockade of FLT3L can suppress DC and prevent the progressive loss of renal function. It was hypothesized that this could be prevented.

[0282] Murphy Roths Large / lymphoproliferative (MRL.lpr) nephritis Using a mouse model and a mouse surrogate anti-FLT3L antibody (LFC-1), The effect of FLT3L blockade on urine levels and nephritis scores was examined. The control and anti-IFNAR antibody treatment groups were included. Mice administered anti-FLT3L antibody showed a significant reduction in proteinuria after 17 weeks of treatment (Figure 19A). The nephritis scores of mice treated with anti-FLT3L antibody were higher than those of isotype control. (Figure 19B). Importantly, the anti-FLT3L antibody-administered mice The proteinuria and nephritis scores in the treated mice were reduced compared to those in the untreated mice. These results support a role for FLT3L-mediated inflammation in nephritis. In light of these results, administration of anti-FLT3L antibodies to patients with nephritis may reduce inflammation in these patients. Rational therapeutic strategies to reduce FLT3L-expressing T cell populations It is.

[0283] To provide insight into FLT3L-related changes in leukocyte populations in nephritis, spleen DC populations were also examined. Spleens were harvested from MRL mice at 18 weeks of age, and splenic DC populations were We examined the changes in circulating DC populations in MRL mice. Treatment with anti-FLT3L antibodies increased the number of DC populations in MRL mice. Specifically, Siglec-H+-pDCs significantly reduced the expression of anti-F After administration of LT3L antibody, it was significantly decreased compared to the isotype control (Figure 20A Similarly, CD11b+cDC (equivalent to human CD1c+DC) and CD8+cDC (equivalent to human A significant decrease was also observed in the CD141+ DCs (Fig. 20B and 20C). Furthermore, the incidence of dermatitis in anti-FLT3L-treated mice was significantly higher than that in untreated mice. This was not observed compared with the usual incidence of 30-40% in patients with anti-FLT. Treatment with 3L antibody reduced circulating DC populations and reduced secondary pathology in a nephritis model. In light of these results, the administration of anti-FLT3L antibodies to patients with nephritis is , which may reduce inflammation and tissue damage by suppressing DC populations.

[0284] Example 16. Expression of FLT3L in Sjogren's syndrome Primary Sjögren's syndrome (pSS) is an autosomal recessive disease characterized by widespread dryness of the eyes and salivary glands. This is an immune condition. In addition, this condition can induce multiple organ failure. This syndrome can occur alone or in combination with can occur in the presence of an additional autoimmune disease, such as lupus or rheumatoid arthritis. Serum levels of FLT3L in individuals with pSS are increased and inflamed saliva There is evidence that both FLT3L and its receptors are expressed locally in the gland. (Tobon et al.,(2010)Arthritis and Rheu matism v62:344).

[0285] Using the NOD.H2h4 Sjögren's syndrome mouse model, we investigated the efficacy and safety of long-term anti-FLT3L antibodies. The mice were examined for salivary gland pathology after treatment with LFC-1. By 16 weeks of age, In the salivary glands (SG), mainly in a manner closely resembling the pathological changes seen in humans. and tertiary lymphoid structures (TLS) containing DCs, B220+ B cells, and CD3+ T cells. This tissue damage is preceded by the development of autoantibodies and the formation of idiopathic germinal centers in the spleen. (Mahmoud et al.,2016 Science Translati Onal Medicine, v8 361ra137). Mice were treated with isotype Ig G control (5 mg / kg), prophylactic treatment with anti-FLT3L antibody (5 mg / kg) Mice were treated with either a standard (starting at 5 weeks of age) or therapeutic (starting at 17 weeks of age) protocol. For both protocols, treatment continued with twice-weekly dosing until the end of the study (26 weeks). Anti-FLT3L monoclonal antibody (LFC-1) inhibited FLT3L throughout the course of treatment. (Figure 25A), resulting in the accumulation of the drug / ligand complex in the circulation (Figure 2 5B).

[0286] Collect lymphoid organs to assess changes to peripheral immune cell populations, including salivary glands (SGs) Histopathology (incidence of TLS) was evaluated. Although it has been reported that tissue damage can be delayed or prevented by therapeutic intervention after disease initiation, Previous reports on the prevention of FLT3L-associated leukemia have been limited. The antibody (LFC-1) inhibited CD44 expression in the spleen and salivary gland-draining LNs. HI CD4+ and and reduced the frequency of CD8+ T cells in mice (aged 24–26 weeks at the end of the study) 26A-26D), and specific autoantibodies against collagen IV and platelet extract were selected. Selectively reduced the levels of α-amyloides (Figure 27).

[0287] As expected, animals treated with isotype control IgG showed a 26 By the age of 1 week, TLS formation, an indicator of salivary gland damage, was increased in the salivary glands (1 mm tissue 2 Anti-FLT3L treated mice showed a higher IFN-γ-γ expression level than did therapeutically administered FLT3L. showed a significant reduction in tissue SG damage even when administered prophylactically (Figure 21A). The disease was completely prevented (FIG. 21B). The DC population measured in the spleen was significantly suppressed. However, the salivary gland was not completely ablated (Fig. 22A-D). It has been shown to significantly impact disease initiation and progression by reducing inflammatory infiltration into the These results suggest that inflammation in pSS is mediated by FLT3L. In light of these results, the administration of anti-FLT3L antibodies Administration of rifampicin is a rational therapeutic strategy for treating pSS in human subjects.

[0288] From the above description, it is understood that the disclosure described herein can be adapted to a variety of uses and conditions. It will be apparent that variations and modifications may be made to the embodiments described herein for purposes of illustration only. Any such embodiment is within the scope of the appended claims. The enumeration of the list may be used as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes any single embodiment. The present invention includes the embodiments as a single embodiment or in combination with any other embodiment or portion thereof. All patents and publications referred to herein are the property of their respective owners. This document is incorporated by reference to the same extent as if it were specifically and individually indicated to be incorporated by reference. INCORPORATED INTO THE SPECIFICATION.

Claims

[Claim 1] The invention described in the specification.

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