ILT7 Binding Molecules and Methods of Use Thereof

Abstract

To provide ILT7 binding molecules, e.g., anti-ILT7 antibodies and antigen-binding fragments, variants, or derivatives thereof, methods of using the antibodies and fragments, and methods for treating or preventing autoimmune diseases and conditions associated with ILT7-expressing cells.SOLUTION: The present invention provides an isolated ILT7 binding protein that can bind to the same ILT7 epitope as an antibody comprising a heavy chain variable region (VH) consisting of a specific sequence and a light chain variable region (VL) consisting of a specific sequence.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Reference to Electronically Filed Sequence Listing The content of the sequence listing filed electronically together with this application as an ASCII text file (name 2943_083STR0_SeqListing_ST25.txt, size: 143,686 bytes, creation date: March 3, 2016) is hereby incorporated by reference in its entirety into this specification.

[0002] Field of the Invention The present invention relates to ILT7 binding molecules, such as anti-ILT7 antibodies and antigen-binding fragments, variants, or derivatives thereof, methods of using antibodies and fragments, and methods of treating or preventing autoimmune diseases and conditions associated with ILT7-expressing cells.

Background Art

[0003] Plasmacytoid dendritic cells (pDC) are a distinct population of dendritic cells (DC) in peripheral blood and secondary lymphoid organs that constitute only about 0.1-0.5% of peripheral blood mononuclear cells (PBMC). However, these cells are a major source of type I interferon (IFN) and are thus particularly important regulators of the immune system. Type I IFNs promote the functions of NK cells, B cells, T cells, and myeloid dendritic cells. These IFNs are important in the initial immune response and have antiviral and antitumor activities. However, pDC and type I IFN are also thought to play a role in the development of autoimmune diseases such as systemic lupus erythematosus, chronic rheumatoid diseases, and psoriasis. Therefore, understanding methods of regulating the molecular pathways involved in IFN release is useful for controlling the immune response and treating and preventing diseases.

[0004] pDCs release IFN in response to nucleic acids sensed by Toll-like receptors (TLRs) TLR7 and TLR9, which are expressed on the surface of pDCs. The response by TLRs is regulated by receptors containing immunoreceptor tyrosine-based activation motifs (ITAMs). Immunoglobulin-like transcript 7 (ILT7), also known as LIRA4, LILRA4, or CD85g, is one such receptor.

[0005] ILT7 is a member of the immunoglobulin-like transcript (ILT) or leukocyte immunoglobulin-like receptor (LIR) gene family. ILT7 is selectively expressed on the surface of human plasmacytoid dendritic cells (pDCs), but not on myeloid dendritic cells or other peripheral blood leukocytes. Cao et al., J. Exp. Medicine 6:1399-1405 (2006). ILT7 contains four immunoglobulin-like extracellular domains and a transmembrane domain. The extracellular portion is important for interaction with bone marrow stromal cell antigen 2 (BST2), which is the ILT7 ligand, and the transmembrane domain of ILT7 contains residues with a positive charge that enable it to form a complex with FcεRIγ. The interaction between BST2 and ILT7 is hypothesized to negatively regulate the innate immune function of pDCs as a potential negative feedback mechanism. Furthermore, cross-linking of ILT7 with antibodies in vitro has been shown to negatively regulate the production of IFN-alpha and TNF-alpha by pDCs. Therefore, antibodies and other ILT7-binding molecules that are useful for neutralizing ILT7 and for regulating pDC activity and IFN release are needed, for example, to treat and prevent diseases such as autoimmune diseases. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] Provided herein are ILT7-binding molecules, such as anti-ILT7 antibodies and antigen-binding fragments thereof. MEANS FOR SOLVING THE PROBLEMS

[0007] In one example, the isolated ILT7-binding protein is an ILT7-binding protein that can bind to the same ILT7 epitope as an antibody comprising the heavy chain variable region (VH) of SEQ ID NO: 202 and the light chain variable region (VL) of SEQ ID NO: 207.

[0008] In one example, the isolated ILT7-binding protein is an ILT7-binding protein that competitively inhibits the binding of an antibody comprising the VH of SEQ ID NO: 202 and the VL of SEQ ID NO: 207 to ILT7.

[0009] In one example, the isolated ILT7-binding protein is an ILT7-binding protein comprising complementarity determining regions (CDRs) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively comprising the sequences of SEQ ID NOs: 203, 204, 205, 208, 209, and 210.

[0010] In one example, the ILT7-binding protein comprises a VH that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 202 and / or a VL that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 207.

[0011] In one example, the ILT7-binding protein comprises a VH comprising SEQ ID NO: 202 and a VL comprising SEQ ID NO: 207.

[0012] In one example, the isolated ILT7-binding protein is an ILT7-binding protein comprising a VH comprising SEQ ID NO: 202.

[0013] In one example, the isolated ILT7-binding protein is an ILT7-binding protein comprising a VH comprising SEQ ID NO: 207.

[0014] In one example, the isolated ILT7-binding protein is an ILT7-binding protein capable of binding to the same ILT7 epitope as an antibody comprising VH and VL selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 17, SEQ ID NO: 22 and SEQ ID NO: 27, SEQ ID NO: 32 and SEQ ID NO: 37, SEQ ID NO: 42 and SEQ ID NO: 47, SEQ ID NO: 52 and SEQ ID NO: 57, SEQ ID NO: 62 and SEQ ID NO: 67, SEQ ID NO: 72 and SEQ ID NO: 77, SEQ ID NO: 82 and SEQ ID NO: 87, SEQ ID NO: 92 and SEQ ID NO: 97, SEQ ID NO: 102 and SEQ ID NO: 107, SEQ ID NO: 112 and SEQ ID NO: 117, SEQ ID NO: 122 and SEQ ID NO: 127, SEQ ID NO: 132 and SEQ ID NO: 137, SEQ ID NO: 142 and SEQ ID NO: 147, SEQ ID NO: 152 and SEQ ID NO: 157, SEQ ID NO: 162 and SEQ ID NO: 167, SEQ ID NO: 172 and SEQ ID NO: 177, SEQ ID NO: 182 and SEQ ID NO: 187, SEQ ID NO: 192 and SEQ ID NO: 197, SEQ ID NO: 212 and SEQ ID NO: 217, SEQ ID NO: 222 and SEQ ID NO: 227, SEQ ID NO: 232 and SEQ ID NO: 237, and SEQ ID NO: 242 and SEQ ID NO: 247, respectively.

[0015] In one example, the isolated ILT7-binding protein is an ILT7-binding protein that competitively inhibits the binding of an antibody comprising VH and VL selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 17, SEQ ID NO: 22 and SEQ ID NO: 27, SEQ ID NO: 32 and SEQ ID NO: 37, SEQ ID NO: 42 and SEQ ID NO: 47, SEQ ID NO: 52 and SEQ ID NO: 57, SEQ ID NO: 62 and SEQ ID NO: 67, SEQ ID NO: 72 and SEQ ID NO: 77, SEQ ID NO: 82 and SEQ ID NO: 87, SEQ ID NO: 92 and SEQ ID NO: 97, SEQ ID NO: 102 and SEQ ID NO: 107, SEQ ID NO: 112 and SEQ ID NO: 117, SEQ ID NO: 122 and SEQ ID NO: 127, SEQ ID NO: 132 and SEQ ID NO: 137, SEQ ID NO: 142 and SEQ ID NO: 147, SEQ ID NO: 152 and SEQ ID NO: 157, SEQ ID NO: 162 and SEQ ID NO: 167, SEQ ID NO: 172 and SEQ ID NO: 177, SEQ ID NO: 182 and SEQ ID NO: 187, SEQ ID NO: 192 and SEQ ID NO: 197, SEQ ID NO: 212 and SEQ ID NO: 217, SEQ ID NO: 222 and SEQ ID NO: 227, SEQ ID NO: 232 and SEQ ID NO: 237, and SEQ ID NO: 242 and SEQ ID NO: 247 to ILT7.

[0016] In one example, the isolated ILT7-binding protein is an ILT7-binding protein comprising CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 selected from the group consisting of SEQ ID NO: 13, 14, 15, 18, 19, and 20, SEQ ID NO: 23, 24, 25, 28, 29, and 30, SEQ ID NO: 33, 34, 35, 38, 39, and 40, SEQ ID NO: 103, 104, 105, 108, 109, and 110, SEQ ID NO: 213, 214, 215, 218, 219, and 220, SEQ ID NO: 223, 224, 225, 228, 229, and 230, SEQ ID NO: 233, 234, 235, 238, 239, and 240, and SEQ ID NO: 243, 244, 245, 248, 249, and 250.

[0017] In one example, the ILT7-binding protein comprises VH and VL that are at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 12 and SEQ ID NO: 17, SEQ ID NO: 22 and SEQ ID NO: 27, SEQ ID NO: 32 and SEQ ID NO: 37, SEQ ID NO: 42 and SEQ ID NO: 47, SEQ ID NO: 52 and SEQ ID NO: 57, SEQ ID NO: 62 and SEQ ID NO: 67, SEQ ID NO: 72 and SEQ ID NO: 77, SEQ ID NO: 82 and SEQ ID NO: 87, SEQ ID NO: 92 and SEQ ID NO: 97, SEQ ID NO: 102 and SEQ ID NO: 107, SEQ ID NO: 112 and SEQ ID NO: 117, SEQ ID NO: 122 and SEQ ID NO: 127, SEQ ID NO: 132 and SEQ ID NO: 137, SEQ ID NO: 142 and SEQ ID NO: 147, SEQ ID NO: 152 and SEQ ID NO: 157, SEQ ID NO: 162 and SEQ ID NO: 167, SEQ ID NO: 172 and SEQ ID NO: 177, SEQ ID NO: 182 and SEQ ID NO: 187, SEQ ID NO: 192 and SEQ ID NO: 197, SEQ ID NO: 212 and SEQ ID NO: 217, SEQ ID NO: 222 and SEQ ID NO: 227, SEQ ID NO: 232 and SEQ ID NO: 237, or SEQ ID NO: 242 and SEQ ID NO: 247, respectively.

[0018] In one example, VH and VL comprise, respectively, SEQ ID NO:12 and SEQ ID NO:17, SEQ ID NO:22 and SEQ ID NO:27, SEQ ID NO:32 and SEQ ID NO:37, SEQ ID NO:42 and SEQ ID NO:47, SEQ ID NO:52 and SEQ ID NO:57, SEQ ID NO:62 and SEQ ID NO:67, SEQ ID NO:72 and SEQ ID NO:77, SEQ ID NO:82 and SEQ ID NO:87, SEQ ID NO:92 and SEQ ID NO:97, SEQ ID NO:102 and SEQ ID NO:107, SEQ ID NO:112 and SEQ ID NO:117, SEQ ID NO:122 and SEQ ID NO:127, SEQ ID NO:132 and SEQ ID NO:137, SEQ ID NO:142 and SEQ ID NO:147, SEQ ID NO:152 and SEQ ID NO:157, SEQ ID NO:162 and SEQ ID NO:167, SEQ ID NO:172 and SEQ ID NO:177, SEQ ID NO:182 and SEQ ID NO:187, SEQ ID NO:192 and SEQ ID NO:197, SEQ ID NO:212 and SEQ ID NO:217, SEQ ID NO:222 and SEQ ID NO:227, SEQ ID NO:232 and SEQ ID NO:237, or SEQ ID NO:242 and SEQ ID NO:247, respectively.

[0019] In one example, the isolated ILT7-binding protein comprises a VH comprising SEQ ID NO:12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 212, 222, 232, or 242.

[0020] In one example, the isolated ILT7-binding protein comprises a VL comprising SEQ ID NO:17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 217, 227, 237, or 247.

[0021] In one example, the ILT7-binding protein comprises an antibody or an antigen-binding fragment thereof. In one example, the antibody or the antigen-binding fragment thereof is hypofucosylated.

[0022] In one example, the ILT7-binding protein binds to the Ig1 region of ILT7. In one example, the ILT7-binding protein binds to the Ig2 region of ILT7.

[0023] In one example, the ILT7-binding protein binds to human and cynomolgus monkey ILT7.

[0024] In one example, the ILT7-binding protein suppresses interferon (IFN) alpha release from peripheral blood mononuclear cells (PBMC). In one example, the ILT7-binding protein has ADCC activity against plasmacytoid dendritic cells (pDC) in PBMC.

[0025] In one example, the ILT7-binding protein comprises a mouse, human, chimeric, humanized, or surface-reconstituted antibody or an antigen-binding fragment thereof.

[0026] In one example, the ILT7-binding protein comprises an antibody, Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-bonded Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, tribody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0027] In one example, the ILT7-binding protein comprises a monoclonal antibody or an antigen-binding fragment thereof.

[0028] In one example, the ILT7-binding protein comprises a heavy-chain immunoglobulin constant domain selected from the group consisting of (a) IgA constant domain, (b) IgD constant domain, (c) IgE constant domain, (d) IgG1 constant domain, (e) IgG2 constant domain, (f) IgG3 constant domain, (g) IgG4 constant domain, and (h) IgM constant domain.

[0029] In one example, the ILT7-binding protein comprises a light chain immunoglobulin constant domain selected from the group consisting of (a) an Ig kappa constant domain, and (b) an Ig lambda constant domain.

[0030] In one example, the ILT7-binding protein comprises a human IgG1 constant domain and a human lambda constant domain.

[0031] In one example, provided herein is a host cell that produces an ILT7-binding molecule.

[0032] In one example, provided herein is an isolated polynucleotide comprising a nucleic acid encoding a VH, wherein the VH comprises an amino acid sequence that is at least 85%, 90%, 95% identical or identical to the VH of SEQ ID NO: 202, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 212, 222, 232, or 242. In one example, the polynucleotide comprises a sequence that is at least 85%, 90%, 95% identical or identical to SEQ ID NO: 201, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 211, 221, 231, or 241.

[0033] In one example, provided herein is an isolated polynucleotide comprising a nucleic acid encoding a VL, wherein the VL comprises an amino acid sequence that is at least 85%, 90%, 95% identical or identical to a VL of 207, 17, 27, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 217, 227, 237, or 247. In one example, the polynucleotide comprises a sequence that is at least 85%, 90%, 95% identical or identical to SEQ ID NO: 206, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 216, 226, 236, or 246.

[0034] In one example, the nucleic acid is operably linked to a control sequence. In one example, an antibody or an antigen-binding fragment thereof comprising a VH or VL encoded by the nucleic acid can specifically bind to ILT7.

[0035] In one example, the polynucleotide encodes an ILT7-binding molecule provided herein.

[0036] In one example, provided herein is a vector comprising the polynucleotide.

[0037] In one example, provided herein is a polypeptide encoded by the polynucleotide.

[0038] In one example, provided herein is a host cell transformed with a polynucleotide provided herein (e.g., a polynucleotide comprising a nucleic acid encoding a VH and a polynucleotide comprising a nucleic acid encoding a VL).

[0039] In one example, provided herein is a host cell comprising a polynucleotide provided herein (e.g., a polynucleotide comprising a nucleic acid encoding VH and a polynucleotide comprising a nucleic acid encoding VL), a vector provided herein, or a polypeptide provided herein. In one example, the host cell is a mammalian host cell. In one example, the host cell is an NS0 mouse myeloma cell, a PER.C6® human cell, or a Chinese hamster ovary (CHO) cell. In one example, the host cell lacks the enzyme α-1,6-fucosyltransferase.

[0040] In one example, provided herein is a method for producing an anti-ILT7 binding molecule, the method comprising culturing a host cell provided herein and recovering the binding molecule. In one example, provided herein is an anti-ILT7 binding molecule produced by this method.

[0041] In one example, provided herein is a method for detecting ILT7 expression in a sample, the method comprising (a) contacting the sample with an ILT7 binding molecule provided herein and (b) detecting the binding of the binding molecule in the sample.

[0042] In one example, provided herein is a method for detecting plasmacytoid dendritic cells, the method comprising (a) contacting a sample containing the cells with an ILT7 binding molecule provided herein and (b) detecting the binding of the binding molecule in the sample.

[0043] In one example, provided herein is a pharmaceutical composition comprising (a) an ILT7 binding molecule provided herein, a polynucleotide provided herein, a vector provided herein, a polypeptide provided herein, or a host cell provided herein, and (b) a carrier.

[0044] In one example, provided herein is a method for reducing IFN-alpha release from plasmacytoid dendritic cells, the method comprising contacting the plasmacytoid dendritic cells with an ILT7 binding molecule provided herein, a polynucleotide provided herein, a vector provided herein, a polypeptide provided herein, a host cell provided herein, or a pharmaceutical composition provided herein.

[0045] In one example, provided herein is a method for treating a human subject having an autoimmune disease, the method comprising administering to the subject an effective amount of an ILT7 binding molecule provided herein, a polynucleotide provided herein, a vector provided herein, a polypeptide provided herein, a host cell provided herein, or a pharmaceutical composition provided herein.

[0046] In one example, provided herein is a method for preventing an autoimmune disease in a human subject, the method comprising administering to the subject an effective amount of an ILT7 binding molecule provided herein, a polynucleotide provided herein, a vector provided herein, a polypeptide provided herein, a host cell provided herein, or a pharmaceutical composition provided herein. In one example, the autoimmune disease is systemic lupus erythematosus. In one example, the autoimmune disease is rheumatoid arthritis. The present invention also relates to the following. [Item 1] An isolated ILT7-binding protein capable of binding to the same ILT7 epitope as an antibody comprising the heavy-chain variable region (VH) of SEQ ID NO: 202 and the light-chain variable region (VL) of SEQ ID NO: 207. [Item 2] An isolated ILT7-binding protein that competitively inhibits the binding of an antibody comprising the VH of SEQ ID NO: 202 and the VL of SEQ ID NO: 207 to ILT7. [Item 3] An isolated ILT7-binding protein comprising complementarity-determining regions (CDRs) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprising the sequences of SEQ ID NOs: 203, 204, 205, 208, 209, and 210. [Item 4] The isolated ILT7-binding protein according to any one of Items 1 to 3, comprising a VH that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 202 and / or a VL that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 207. [Item 5] The isolated ILT7-binding protein according to Item 4, comprising a VH comprising SEQ ID NO: 202 and a VL comprising SEQ ID NO: 207. [Item 6] An isolated ILT7-binding protein comprising a VH comprising SEQ ID NO: 202. [Item 7] An isolated ILT7-binding protein comprising a VL comprising SEQ ID NO: 207. [Item 8] SEQ ID NO: 12 and SEQ ID NO: 17, respectively, SEQ ID NO: 22 and SEQ ID NO: 27, respectively, SEQ ID NO: 32 and SEQ ID NO: 37, respectively, SEQ ID NO: 42 and SEQ ID NO: 47, respectively, SEQ ID NO: 52 and SEQ ID NO: 57, respectively, SEQ ID NO: 62 and SEQ ID NO: 67, respectively, SEQ ID NO: 72 and SEQ ID NO: 77, respectively, SEQ ID NO: 82 and SEQ ID NO: 87, respectively, SEQ ID NO: 92 and SEQ ID NO: 97, respectively, SEQ ID NO: 102 and SEQ ID NO: 107, respectively, SEQ ID NO: 112 and SEQ ID NO: 117, respectively, SEQ ID NO: 122 and SEQ ID NO: 127, respectively, SEQ ID NO: 132 and SEQ ID NO: 137, respectively, SEQ ID NO: 142 and SEQ ID NO: 147, respectively, SEQ ID NO: 152 and SEQ ID NO: 157, respectively, SEQ ID NO: 162 and SEQ ID NO: 167, respectively, SEQ ID NO: 172 and SEQ ID NO: 177, respectively, SEQ ID NO: 182 and SEQ ID NO: 187, respectively, SEQ ID NO: 192 and SEQ ID NO: 197, respectively, SEQ ID NO: 212 and SEQ ID NO: 217, respectively, SEQ ID NO: 222 and SEQ ID NO: 227, respectively, VH and VL selected from the group consisting of SEQ ID NO: 232 and SEQ ID NO: 237, respectively, and SEQ ID NO: 242 and SEQ ID NO: 247, respectively An isolated ILT7-binding protein capable of binding to the same ILT7 epitope as an antibody comprising VH and VL. [Item 9] SEQ ID NO: 12 and SEQ ID NO: 17, respectively, SEQ ID NO: 22 and SEQ ID NO: 27, respectively, SEQ ID NO: 32 and SEQ ID NO: 37, respectively, SEQ ID NO: 42 and SEQ ID NO: 47, respectively, SEQ ID NO: 52 and SEQ ID NO: 57, respectively, SEQ ID NO: 62 and SEQ ID NO: 67, respectively, SEQ ID NO: 72 and SEQ ID NO: 77, respectively, SEQ ID NO: 82 and SEQ ID NO: 87, respectively, SEQ ID NO: 92 and SEQ ID NO: 97, respectively, SEQ ID NO: 102 and SEQ ID NO: 107, respectively, SEQ ID NO: 112 and SEQ ID NO: 117, respectively, SEQ ID NO: 122 and SEQ ID NO: 127, respectively, SEQ ID NO: 132 and SEQ ID NO: 137, respectively, SEQ ID NO: 142 and SEQ ID NO: 147, respectively, SEQ ID NO: 152 and SEQ ID NO: 157, respectively, SEQ ID NO: 162 and SEQ ID NO: 167, respectively, SEQ ID NO: 172 and SEQ ID NO: 177, respectively, SEQ ID NO: 182 and SEQ ID NO: 187, respectively, SEQ ID NO: 192 and SEQ ID NO: 197, respectively, SEQ ID NO: 212 and SEQ ID NO: 217, respectively, SEQ ID NO: 222 and SEQ ID NO: 227, respectively, VH and VL selected from the group consisting of SEQ ID NO: 232 and SEQ ID NO: 237, respectively, and SEQ ID NO: 242 and SEQ ID NO: 247, respectively An isolated ILT7-binding molecule that competitively inhibits the binding of an antibody comprising VH and VL selected from the group consisting of SEQ ID NO: 232 and SEQ ID NO: 237, respectively, and SEQ ID NO: 242 and SEQ ID NO: 247, respectively, to ILT7. [Item 10] SEQ ID NO: 13, 14, 15, 18, 19, and 20, respectively, SEQ ID NO: 23, 24, 25, 28, 29, and 30, respectively, SEQ ID NO: 33, 34, 35, 38, 39, and 40, respectively, SEQ ID NO: 103, 104, 105, 108, 109, and 110, respectively, SEQ ID NO: 213, 214, 215, 218, 219, and 220, respectively, SEQ ID NO: 223, 224, 225, 228, 229, and 230, respectively, SEQ ID NO: 233, 234, 235, 238, 239, and 240, respectively, and SEQ ID NO: 243, 244, 245, 248, 249, and 250, respectively An isolated ILT7-binding molecule comprising CDR: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, selected from the group consisting of SEQ ID NO: 13, 14, 15, 18, 19, and 20, respectively, SEQ ID NO: 23, 24, 25, 28, 29, and 30, respectively, SEQ ID NO: 33, 34, 35, 38, 39, and 40, respectively, SEQ ID NO: 103, 104, 105, 108, 109, and 110, respectively, SEQ ID NO: 213, 214, 215, 218, 219, and 220, respectively, SEQ ID NO: 223, 224, 225, 228, 229, and 230, respectively, SEQ ID NO: 233, 234, 235, 238, 239, and 240, respectively, and SEQ ID NO: 243, 244, 245, 248, 249, and 250, respectively. [Item 11] SEQ ID NO: 12 and SEQ ID NO: 17, respectively, SEQ ID NO: 22 and SEQ ID NO: 27, respectively, SEQ ID NO: 32 and SEQ ID NO: 37, respectively, SEQ ID NO: 42 and SEQ ID NO: 47, respectively, SEQ ID NO: 52 and SEQ ID NO: 57, respectively, SEQ ID NO: 62 and SEQ ID NO: 67, respectively, SEQ ID NO: 72 and SEQ ID NO: 77, respectively, SEQ ID NO: 82 and SEQ ID NO: 87, respectively, SEQ ID NO: 92 and SEQ ID NO: 97, respectively, SEQ ID NO: 102 and SEQ ID NO: 107, respectively, SEQ ID NO: 112 and SEQ ID NO: 117, respectively, SEQ ID NO: 122 and SEQ ID NO: 127, respectively, SEQ ID NO: 132 and SEQ ID NO: 137, respectively, SEQ ID NO: 142 and SEQ ID NO: 147, respectively, SEQ ID NO: 152 and SEQ ID NO: 157, respectively, SEQ ID NO: 162 and SEQ ID NO: 167, respectively, SEQ ID NO: 172 and SEQ ID NO: 177, respectively, SEQ ID NO: 182 and SEQ ID NO: 187, respectively, SEQ ID NO: 192 and SEQ ID NO: 197, respectively, SEQ ID NO: 212 and SEQ ID NO: 217, respectively, SEQ ID NO: 222 and SEQ ID NO: 227, respectively, SEQ ID NO: 232 and SEQ ID NO: 237, respectively, or SEQ ID NO: 242 and SEQ ID NO: 247, respectively An isolated ILT7-binding protein according to any one of items 8 to 10, comprising VH and VL that are at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. [Item 12] Said VH and VL are SEQ ID NO: 12 and SEQ ID NO: 17, respectively, SEQ ID NO: 22 and SEQ ID NO: 27, respectively, SEQ ID NO: 32 and SEQ ID NO: 37, respectively, SEQ ID NO: 42 and SEQ ID NO: 47, respectively, SEQ ID NO: 52 and SEQ ID NO: 57, respectively, SEQ ID NO: 62 and SEQ ID NO: 67, respectively, SEQ ID NO: 72 and SEQ ID NO: 77, respectively, SEQ ID NO: 82 and SEQ ID NO: 87, respectively, SEQ ID NO: 92 and SEQ ID NO: 97, respectively, SEQ ID NO: 102 and SEQ ID NO: 107, respectively, SEQ ID NO: 112 and SEQ ID NO: 117, respectively, SEQ ID NO: 122 and SEQ ID NO: 127, respectively, SEQ ID NO: 132 and SEQ ID NO: 137, respectively, SEQ ID NO: 142 and SEQ ID NO: 147, respectively, SEQ ID NO: 152 and SEQ ID NO: 157, respectively, SEQ ID NO: 162 and SEQ ID NO: 167, respectively, SEQ ID NO: 172 and SEQ ID NO: 177, respectively, SEQ ID NO: 182 and SEQ ID NO: 187, respectively, SEQ ID NO: 192 and SEQ ID NO: 197, respectively, SEQ ID NO: 212 and SEQ ID NO: 217, respectively, SEQ ID NO: 222 and SEQ ID NO: 227, respectively, SEQ ID NO: 232 and SEQ ID NO: 237, respectively, or Each of SEQ ID NO: 242 and SEQ ID NO: 247 The ILT7 binding molecule according to item 11, comprising the same. [Item 13] An isolated ILT7 binding molecule comprising a VH comprising SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 212, 222, 232, or 242. [Item 14] An isolated ILT7 binding molecule comprising a VL comprising SEQ ID NO: 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 217, 227, 237, or 247. [Item 15] The isolated ILT7 binding molecule according to any one of items 1 to 14, comprising an antibody or an antigen-binding fragment thereof. [Item 16] The isolated ILT7 binding molecule according to item 15, wherein the antibody or the antigen-binding fragment thereof is hypofucosylated. [Item 17] The isolated ILT7 binding molecule according to any one of items 8 to 16, which binds to the Ig1 region of ILT7. [Item 18] The isolated ILT7 binding molecule according to any one of items 8 to 16, which binds to the Ig2 region of ILT7. [Item 19] The isolated ILT7 binding molecule according to any one of items 1 to 18, which binds to human and cynomolgus monkey ILT7. [Item 20] The isolated ILT7 binding molecule according to any one of items 1 to 19, which suppresses the release of interferon (IFN) alpha from peripheral blood mononuclear cells (PBMC). [Item 21] The isolated ILT7 binding molecule according to any one of items 1 to 20, which has ADCC activity against plasmacytoid dendritic cells (pDC) in PBMC. [Item 22] The isolated ILT7 binding molecule according to any one of items 1 to 21, comprising a mouse, human, chimeric, humanized, or resurfaced antibody or an antigen-binding fragment thereof. [Item 23] The isolated ILT7 binding molecule according to any one of items 1 to 22, comprising an antibody, Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-bonded Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, tribody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc. [Item 24] An isolated ILT7 binding molecule according to any one of items 1 to 23, comprising a monoclonal antibody or an antigen-binding fragment thereof. [Item 25] (a) An IgA constant domain, (b) An IgD constant domain, (c) An IgE constant domain, (d) An IgG1 constant domain, (e) An IgG2 constant domain, (f) An IgG3 constant domain, (g) An IgG4 constant domain, and (h) An IgM constant domain An isolated ILT7 binding molecule according to any one of items 1 to 24, comprising a heavy-chain immunoglobulin constant domain selected from the group consisting of. [Item 26] (a) An Ig kappa constant domain, and (b) An Ig lambda constant domain An isolated ILT7 binding molecule according to any one of items 1 to 25, comprising a light-chain immunoglobulin constant domain selected from the group consisting of. [Item 27] An isolated ILT7 binding molecule according to any one of items 1 to 26, comprising a human IgG1 constant domain and a human lambda constant domain. [Item 28] An isolated host cell producing the binding molecule according to any one of items 1 to 27. [Item 29] An isolated polynucleotide comprising a nucleic acid encoding VH, wherein VH comprises an amino acid sequence that is at least 85%, 90%, 95% identical to, or identical to, the VH of SEQ ID NO: 202, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 212, 222, 232, or 242. [Item 30] The polynucleotide according to item 29, comprising a sequence that is at least 85%, 90%, 95% identical to, or identical to, SEQ ID NO: 201, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 211, 221, 231, or 241. [Item 31] An isolated polynucleotide comprising a nucleic acid encoding VL, wherein VL comprises an amino acid sequence that is at least 85%, 90%, 95% identical to, or identical to, the VL of SEQ ID NO: 207, 17, 27, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 217, 227, 237, or 247. [Item 32] The polynucleotide according to item 31, comprising a sequence that is at least 85%, 90%, 95% identical to, or identical to, the sequence number 206, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 216, 226, 236, or 246. [Item 33] The polynucleotide according to any one of items 29 to 32, wherein the nucleic acid is operably linked to a regulatory sequence. [Item 34] The polynucleotide according to any one of items 29 to 33, wherein the antibody or antigen-binding fragment thereof comprising the VH or the VL can specifically bind to ILT7. [Item 35] A polynucleotide encoding the ILT7-binding molecule according to any one of items 1 to 27. [Item 36] A vector comprising the polynucleotide according to any one of items 29 to 35. [Item 37] A polypeptide encoded by the polynucleotide according to any one of items 29 to 35. [Item 38] A host cell transformed with the polynucleotide according to item 29 or 30 and the polynucleotide according to item 31 or 32. [Item 39] A host cell comprising the polynucleotide according to any one of items 29 to 35, the vector according to item 36, or the polypeptide according to item 37. [Item 40] The host cell according to item 38 or 39, which is a mammalian host cell. [Item 41] The mammalian host cell according to item 40, which is an NS0 mouse myeloma cell, a PER.C6® human cell, or a Chinese hamster ovary (CHO) cell. [Item 42] The host cell according to any one of items 38 to 41, lacking the enzyme α-1,6-fucosyltransferase. [Item 43] A method for producing an anti-ILT7 binding molecule, comprising culturing the host cell according to any one of items 38 to 42, and recovering the binding molecule. [Item 44] An anti-ILT7 binding molecule produced by the method according to item 43. [Item 45] A method for detecting ILT7 expression in a sample, comprising (a) contacting the sample with the ILT7 binding molecule according to any one of items 1 to 27 or 44, and (b) detecting the binding of the binding molecule in the sample. [Item 46] A method for detecting plasmacytoid dendritic cells, comprising: (a) contacting a sample containing cells with an ILT7 binding molecule according to any one of items 1 to 27 or 44; and (b) detecting the binding of the binding molecule in the sample. [Item 47] (a) An ILT7 binding molecule according to any one of items 1 to 27 or 44, a polynucleotide according to any one of items 29 to 35, a vector according to item 36, a polypeptide according to item 37, or a host cell according to any one of items 28 or 38 to 42, and (b) a carrier. A pharmaceutical composition. [Item 48] A method for reducing IFN-alpha release from plasmacytoid dendritic cells, comprising contacting plasmacytoid dendritic cells with a binding molecule according to any one of items 1 to 27 or 44, a polynucleotide according to any one of items 29 to 35, a vector according to item 36, a polypeptide according to item 37, a host cell according to any one of items 28 or 38 to 42, or a composition according to item 47. [Item 49] A method for treating a human subject having an autoimmune disease, comprising administering to the subject an effective amount of a binding molecule according to any one of items 1 to 27 or 44, a polynucleotide according to any one of items 29 to 35, a vector according to item 36, a polypeptide according to item 37, a host cell according to any one of items 28 or 38 to 42, or a composition according to item 47. [Item 50] A method for preventing an autoimmune disease in a human subject, comprising administering to the subject an effective amount of a binding molecule according to any one of items 1 to 27 or 44, a polynucleotide according to any one of items 29 to 35, a vector according to item 36, a polypeptide according to item 37, a host cell according to any one of items 28 or 38 to 42, or a composition according to item 47. [Item 51] The method according to item 49 or 50, wherein the autoimmune disease is systemic lupus erythematosus. [Item 52] The method according to item 49 or 50, wherein the autoimmune disease is rheumatoid arthritis.

Brief Description of the Drawings

[0047] [Figure 1A] It is a diagram showing the variable heavy chain (1A) and variable light chain (1B) sequence alignments of SBI28 (#28), 10D10, and 7C7 antibodies. The shaded portions indicate the CDR sequences. The squares represent the mutations introduced into 10D10 to generate 7C7. [Figure 1B] Continuation of Figure 1A. [Figure 2] It is a diagram showing the binding of an ILT7 antibody and a negative control antibody (R437) to CT-550 cells expressing human ILT7, as determined by flow cytometry. SBI33 refers to the anti-ILT7 antibody ILT7#33 provided in US Patent Application Publication No. 2009 / 0280128. [Figure 3] It is a diagram showing the binding of an ILT7 antibody and a negative control antibody (R437) to CT-125 cells expressing cynomolgus ILT7, as determined by flow cytometry. [Figure 4] It is a diagram showing the ADCC potency of an ILT7 antibody and a negative control antibody (R437) against human ILT7-expressing cells. [Figure 5] It is a diagram showing the ADCC potency of an ILT7 antibody and a negative control antibody (R437) against cynomolgus ILT7-expressing cells. [Figure 6A] It is a diagram showing the binding of an ILT7 antibody and a negative control antibody (R437) to plasmacytoid dendritic cells (pDC) in peripheral blood mononuclear cells (PBMC). [Figure 6B] Continuation of Figure 6A. [Figure 7] Figure showing the binding of afucosylated ILT7 antibody and its parental antibody to CT-550 cells expressing human (left panel) and cynomolgus monkey (right panel) ILT7, as determined by flow cytometry. [Figure 8] Figure showing the ADCC potency of afucosylated ILT7 antibody and its parental antibody against human (left panel) and cynomolgus monkey (right panel) ILT7-expressing cells. [Figure 9A] Figure showing the variable heavy chain (9A) and variable light chain (9B) sequence alignments of seven ILT70080 variants. The most closely related germline sequences (IGHV1-69*01 and IGLV3-21*01) are also shown in the alignment. [Figure 9B] Continuation of Figure 9A. [Figure 10A] Figure showing the variable heavy chain (10A) and variable light chain (10B) sequence alignments of nine ILT70083 variants. The most closely related germline sequences (IGHV3-23*01 and IGLV1-51*01) are also shown in the alignment. [Figure 10B] Continuation of Figure 10A. [Figure 11] Figure showing the binding of ILT70080 variant to cells expressing human ILT7 (CT-550, upper panel) and cells expressing cynomolgus monkey ILT7 (CT-125, lower panel). [Figure 12] Figure showing the binding of ILT70083 variant to cells expressing human ILT7 (upper panel) or cells expressing cynomolgus monkey ILT7 (lower panel). [Figure 13] Figure showing the ADCC potency of ILT70080 variant antibody against human ILT7-expressing cells. [Figure 14] Figure showing the ADCC potency of ILT70083 variant antibody against human ILT7-expressing cells. [Figure 15]Figure showing the binding of afucosylated ILT70080.6 and ILT70083 antibodies to human (left panel) and cynomolgus monkey (right panel) ILT7-expressing cells. [Figure 16] Figure showing the ADCC activity of afucosylated ILT70080.6 and ILT70083 antibodies against human (left panel) and cynomolgus monkey (right panel) ILT7-expressing cells. [Figure 17] Figure showing the cytotoxicity (left) and IFN-α secretion (right) of human PBMCs exposed to afucosylated ILT70080.6 and ILT70083 antibodies. [Figure 18] Figure showing the binding of afucosylated ILT70137 to cells expressing human ILT7 (left panel) or cynomolgus monkey ILT7 (right panel). Circles indicate afucosylated ILT70137, and triangles indicate the control. [Figure 19] Figure showing the ADCC activity of afucosylated ILT70137 against cells expressing human ILT7 (left panel) or cynomolgus monkey ILT7 (right panel). Triangles indicate afucosylated ILT70137, and circles indicate the control. [Figure 20] Figure showing the ADCC activity of afucosylated ILT70137 by measuring the inhibition of IFN alpha production as an indirect assessment of the ability of the antibody to induce ADCC of peripheral blood mononuclear cells (PBMC) in vitro. [Figure 21] Figure showing the binding of afucosylated ILT70137 to human primary plasmacytoid dendritic cells (pDC). [Figure 22] Figure showing the depletion of pDCs in cynomolgus monkeys treated with afucosylated 7C7 or afucosylated ILT70137. The arrow below the graph indicates the time point of antibody administration. [Figure 23] Figure showing IFNα production after treatment with afucosylated 7C7 or afucosylated ILT70137. The arrow below the graph indicates the time point of antibody administration.

Mode for Carrying Out the Invention

[0048] I. Definitions It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, the term "an anti-ILT7 antibody" is understood to represent one or more anti-ILT7 antibodies. Therefore, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

[0049] As used herein, the term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptides", and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of polypeptides, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting groups / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or can be produced by recombinant techniques, but does not necessarily have to be translated from a specified nucleic acid sequence. A polypeptide can be made by any method including chemical synthesis.

[0050] The polypeptide of the present invention can be a polypeptide having a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. The polypeptide can have a defined three-dimensional structure, but does not necessarily have to have such a structure. A polypeptide having a defined three-dimensional structure is called folded, and a polypeptide that does not have a defined three-dimensional structure, but rather can take on a number of different conformations, is called unfolded. As used herein, the term glycoprotein refers to a protein that is coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or nitrogen-containing side chain of an amino acid residue, such as a serine residue or an asparagine residue.

[0051] By "isolated" polypeptide or fragment, variant, or derivative thereof is meant a polypeptide that does not exist in its natural environment. A particular level of purification is not required. For example, an isolated polypeptide can be removed from its original or natural environment. Recombinant polypeptides and proteins produced by expression in a host cell are considered to be isolated for the purposes of the present invention, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0052] Similarly, fragments, derivatives, analogs, or variants of the aforementioned polypeptides, and any combination thereof, are likewise included as polypeptides of the present invention. The terms "fragment", "variant", "derivative", and "analog" when referring to the anti-ILT7 antibody or antibody polypeptide of the present invention include any polypeptide that retains at least a portion of the antigen-binding properties of the corresponding antibody or antibody polypeptide of the present invention. Fragments of the polypeptides of the present invention include proteolytic fragments as well as deletion fragments in addition to the specific antibody fragments discussed elsewhere herein. Variants of the anti-ILT7 antibodies and antibody polypeptides of the present invention include the above fragments and also include polypeptides having an amino acid sequence that has been changed by amino acid substitution, deletion, or insertion. Variants can occur naturally or may not occur naturally. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides can include conservative or non-conservative amino acid substitutions, deletions, or additions. Variant polypeptides may also be referred to herein as "polypeptide analogs". As used herein, a "derivative" of an anti-ILT7 antibody or antibody polypeptide refers to a subject polypeptide having one or more residues that have been chemically derivatized by reaction of a functional side chain group. Similarly, those peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids are also included in "derivatives". For example, 4-hydroxyproline can be used in place of proline, 5-hydroxylysine can be used in place of lysine, 3-methylhistidine can be used in place of histidine, homoserine can be used in place of serine, and ornithine can be used in place of lysine. Derivatives of the anti-ILT7 antibodies and antibody polypeptides of the present invention can include polypeptides that have been altered to exhibit additional features not found in the reference antibody or antibody polypeptide of the present invention.

[0053] The term "polynucleotide" is intended to encompass single nucleic acids as well as plural nucleic acids, and refers to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA) or plasmid DNA (pDNA). Polynucleotides can include conventional phosphodiester bonds or non-conventional bonds (such as amide bonds found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments present in a polynucleotide, such as a DNA or RNA fragment. An "isolated" nucleic acid or polynucleotide is intended to mean a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding an anti-ILT7 binding molecule, such as an antibody or an antigen-binding fragment thereof, contained in a vector is considered isolated for the purposes of the present invention. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell, or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, a polynucleotide or nucleic acid can be or can include regulatory elements such as a promoter, ribosome binding site, or transcription terminator.

[0054] As used herein, a "coding region" is a portion of a nucleic acid consisting of codons that are translated into amino acids. A "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, but can be considered part of the coding region, however any adjacent sequences such as a promoter, ribosome binding site, transcription terminator, intron, etc. are not part of the coding region. Two or more coding regions of the present invention can be present in a single polynucleotide construct, such as a single vector, or in separate polynucleotide constructs, such as separate (different) vectors. Further, any vector can contain a single coding region or can contain two or more coding regions, for example, a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. Additionally, a vector, polynucleotide, or nucleic acid of the present invention can encode a heterologous coding region, fused or unfused to a nucleic acid encoding an anti-ILT7 antibody or fragment, variant, or derivative thereof. The heterologous coding region includes, but is not limited to, specialized elements or motifs such as a secretion signal peptide or a heterologous functional domain.

[0055] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid encoding a polypeptide typically can include a promoter and / or other transcriptional or translational control elements operably associated with one or more coding regions. An operable association is the case where a coding region for a gene product, such as a polypeptide, is associated with one or more regulatory sequences such that the expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments, such as a polypeptide coding region and a promoter associated therewith, are "operably associated" when induction of promoter function results in transcription of mRNA encoding the desired gene product and the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or the ability of the DNA template to be transcribed. Thus, a promoter region will be operably associated with a nucleic acid encoding a polypeptide when the promoter is capable of effecting transcription of that nucleic acid. The promoter can be a cell-specific promoter that directs substantial transcription of DNA only in a given cell. Other transcriptional control elements besides the promoter, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein.

[0056] A variety of transcriptional control regions are known to those skilled in the art. These include transcriptional control regions that function in vertebrates, such as the promoters and enhancer segments of cytomegalovirus (immediate early promoter together with intron A), simian virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus), but are not limited thereto. Other transcriptional control regions include regions derived from vertebrate genes, such as those of actin, heat shock protein, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Further suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters inducible by interferon or interleukin).

[0057] Similarly, a variety of translational control elements are known to those skilled in the art. These include ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (in particular, internal ribosome entry sites, or IRES, also called CITE sequences), but are not limited thereto.

[0058] In other embodiments, the polynucleotide of the invention is in the form of RNA, such as messenger RNA (mRNA).

[0059] The polynucleotides and nucleic acid coding regions of the present invention can be associated with additional coding regions encoding a secretion or signal peptide that directs the secretion of the polypeptide encoded by the polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretion leader sequence that is cleaved from the mature protein when transport of the growing protein chain from the rough endoplasmic reticulum is initiated. Those skilled in the art are aware that polypeptides secreted by vertebrate cells generally have a signal sequence fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full-length" polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide, such as an immunoglobulin heavy or light chain signal peptide, is used, or a functional derivative of that sequence that retains the ability to direct the secretion of a polypeptide operably associated therewith is used. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild-type leader sequence can be replaced with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[0060] The "binding molecule" or "antigen-binding molecule" of the present invention, in its broadest sense, refers to a molecule that specifically binds to an epitope. In one embodiment, the binding molecule specifically binds to ILT7, such as full-length ILT7 or mature ILT7. In another embodiment, the binding molecule of the present invention is an antibody or an antigen-binding fragment thereof. In another embodiment, the binding molecule of the present invention comprises at least one heavy or light chain CDR of a reference antibody molecule. In another embodiment, the binding molecule of the present invention comprises at least two CDRs from one or more reference antibody molecules. In another embodiment, the binding molecule of the present invention comprises at least three CDRs from one or more reference antibody molecules. In another embodiment, the binding molecule of the present invention comprises at least four CDRs from one or more reference antibody molecules. In another embodiment, the binding molecule of the present invention comprises at least five CDRs from one or more reference antibody molecules. In another embodiment, the binding molecule of the present invention comprises at least six CDRs from one or more reference antibody molecules. In certain embodiments, the reference antibody molecule is 7C7, ILT70080, ILT70080.1 to ILT70080.7, ILT70083, ILT70083.1 to ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, or ILT70052.

[0061] The present invention relates to certain anti-ILT7 antibodies, or antigen-binding fragments, variants, or derivatives thereof. The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule as long as the antibody exhibits the desired biological activity. Antibodies can be of any of the five major classes of immunoglobulins, namely IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, which are called alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different well-known subunit structures and three-dimensional conformations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0062] The term "antibody fragment" or "fragment thereof" refers to a part of an intact antibody. An "antigen-binding fragment" or "fragment thereof" refers to a part of an intact antibody that binds to an antigen. An antigen-binding fragment can contain the antigen-determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv, and single-chain antibodies.

[0063] As used herein, "human" or "fully human" antibodies include antibodies having the amino acid sequences of human immunoglobulins, including those isolated from human immunoglobulin libraries or isolated from animals transgenic with respect to one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described below and, for example, in U.S. Patent No. 5,939,598 to Kucherlapati et al. Fully human antibodies are particularly desirable for therapeutic treatment of human patients.

[0064] Human antibodies can be made by a variety of methods known in the art, including phage display methods that utilize antibody libraries derived from the sequences of human immunoglobulins described in Vaughan et al., Nat. Biotech. 14:309-314 (1996), Sheets et al., Proc. Nat'l. Acad. Sci. 95:6157-6162 (1998), Hoogenboom and Winter, J. Mol. Biol. 227:381 (1992), and Marks et al., J. Mol. Biol. 222:581 (1991)).Further examples of phage display methods that can be used to generate and use antibodies include Rothe et al., J. Mol. Biol., 376:1182 (2008), Brinkman et al., J. Immunol. Methods 182:41-50 (1995), Ames et al., J. Immunol. Methods 184:177-186 (1995), Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994), Persic et al., Gene 187:9-18 (1997), Burton et al., Advances in Immunology 57:191-280 (1994), PCT Application No. PCT / GB91 / 01134, PCT International Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401, and U.S. Pat. Nos. 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, 6,593,081, 6,300,064, 6,653,068, 6,706,484, 7,264,963, 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108, the disclosures of each of which are incorporated herein by reference in their entireties.

[0065] In addition, as is known in the art, human antibodies can be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. For an overview of this technology, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995).

[0066] Additional techniques available in the field of antibody engineering enable the isolation of human antibodies or fragments thereof. For example, human hybridomas can be generated as described by Kontermann and Sefan, Antibody Engineering, Springer Laboratory Manuals (2001). Full-length human antibodies can likewise be produced by various display techniques, such as phage display or other viral display systems. In the phage display method, functional antibody domains are displayed on the surface of phage particles that have the polynucleotide sequences encoding them. For example, DNA sequences encoding the VH and VL regions are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of lymphoid tissue) or a synthetic cDNA library. In certain embodiments, the DNAs encoding the VH and VL regions are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., pCANTAB 6 or pComb 3 HSS). The vector is electroporated into E. coli, and the E. coli are infected with helper phage. The phage used in these methods are typically filamentous phage including fd and M13, and the VH or VL region is usually recombinantly fused to either phage gene III or gene VIII. Phage expressing an antigen-binding domain that binds to the antigen of interest (i.e., ILT7) can be selected or identified by the antigen, for example using a labeled antigen or an antigen bound or captured on a solid-phase surface or bead.

[0067] "Human" or "fully human" antibodies also include antibodies that contain at least the variable domain of the heavy chain, or at least the variable domains of the heavy and light chains, wherein the variable domain(s) have the amino acid sequence of human immunoglobulin variable domain(s).

[0068] "Human" or "fully human" antibodies also include, consist essentially of, or consist of the above "human" or "fully human" antibodies that include variants (including derivatives) of the antibody molecules (e.g., VH region and / or VL region) described herein, wherein the antibody or its antigen-binding fragment, variant, or derivative immunospecifically binds to an ILT7 polypeptide or a fragment or variant thereof. Mutations can be introduced into the nucleotide sequence encoding the human anti-ILT7 antibody using standard techniques known to those of skill in the art, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Variants (including derivatives) can encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions compared to the reference VH region, VHCDR1, VHCDR2, VHCDR3, VL region, VLCDR1, VLCDR2, or VLCDR3.

[0069] In certain embodiments, the amino acid substitutions are conservative amino acid substitutions as discussed in detail below. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants screened for biological activity to identify mutants that retain activity (e.g., the ability to bind to an ILT7 polypeptide, such as human, primate, mouse, or any combination of human, primate, and mouse ILT7). Such variants (or derivatives thereof) of "human" or "fully human" antibodies can also be referred to as "optimized" or "optimized for antigen binding" human or fully human antibodies and include antibodies having improved affinity for an antigen.

[0070] The basic immunoglobulin structure in the vertebrate system is relatively well understood. See, for example, Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press).

[0071] As will be discussed in more detail below, the term "immunoglobulin" includes various broad classes of polypeptides that can be biochemically distinguished. One of ordinary skill in the art will recognize that the heavy chains are classified as gamma, mu, alpha, delta, or epsilon, along with some of their subclasses (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody to be IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well-characterized and are known to confer functional specialization. Modified forms of each of these classes and isotypes are readily distinguishable to one of ordinary skill in the art in view of the present disclosure and are thus within the scope of the present invention. The following discussion generally targets the IgG class of immunoglobulin molecules, but all immunoglobulin classes are clearly within the scope of the present invention. With respect to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are typically joined in a "Y" shape by disulfide bonds, and the light chains surround the heavy chains, starting at the mouth of the "Y" shape and continuing through the variable regions.

[0072] The light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can bind to either a kappa or a lambda light chain. Generally, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are joined to each other by either covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by any of a hybridoma, a B cell, or a genetically modified host cell. In the heavy chains, the amino acid sequence proceeds from the N-terminus at the fork-like tip of the "Y" shape to the C-terminus at the bottom of each chain.

[0073] The base of antibody "Y" is called the Fc (fragment crystallizable) region and is composed of two heavy chains that give two or three constant domains depending on the class of the antibody. For this reason, the Fc region binds to specific classes of Fc receptors and other immune molecules such as complement proteins. Both the light and heavy chains can be classified into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be recognized that the variable domains of both the light chain (VL or VK) and heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental passage, Fc receptor binding, complement binding, etc. For convenience, the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region, and the CH3 and CL domains actually contain the carboxy termini of the heavy and light chains, respectively.

[0074] As described above, due to the variable region, an antibody can selectively recognize and specifically bind to an epitope on an antigen. That is, the VL domain and VH domain within these variable domains of the antibody, or a subset of the complementarity-determining regions (CDRs), bind to form a variable region that defines a three-dimensional antigen-binding site. This quaternary structure of the antibody forms the antigen-binding site at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs present in each of the VH and VL chains. In some examples, for instance, in certain immunoglobulin molecules derived from camelids or engineered based on camel immunoglobulins, the complete immunoglobulin molecule consists of only heavy chains and has no light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0075] In naturally occurring antibodies, the six "complementary determining regions" or "CDRs" present in each antigen-binding domain are short discontinuous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody adopts its three-dimensional structure in an aqueous environment. The remaining amino acids in the antigen-binding domain are called the "framework" regions and exhibit lower intermolecular diversity. Most of the framework regions adopt a β-sheet conformation, and the CDRs form loops that connect the β-sheet structures and, in some cases, loops that form part of them. Thus, the framework regions act to form a scaffold that provides the correct orientation of the CDRs by means of interchain non-covalent interactions. The antigen-binding domain formed by the arranged CDRs defines a surface that is complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to its cognate epitope. The amino acids that include the CDRs and framework regions, respectively, for any given heavy or light chain variable domain can be readily identified by those skilled in the art since they are precisely defined (see below).

[0076] When there are two or more definitions for terms used in and / or accepted by the art, the definitions of the terms used herein are intended to include all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementary determining region" ("CDR") to describe the discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. This particular region is described by Kabat et al. (1983) U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" and Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference, but when compared to each other, the definitions include amino acid residue overlaps or subsets. Nevertheless, regardless of which definition is applied to refer to the CDR of an antibody or its variant, it is intended to be within the scope of the terms defined and used herein. IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions including the CDR. See, for example, Lefranc, M.P. et al., Dev. Comp. Immunol. 27: 55-77(2003), which is incorporated herein by reference. The IMGT numbering system is based on the alignment of over 5,000 sequences, structural data, and the characterization of hypervariable loops, enabling easy comparison of variable and CDR regions for all species. The appropriate amino acid residues encompassing the CDR as defined by each of the references cited above are listed in Table 1 below for comparison. The exact residue numbers encompassing a particular CDR can vary depending on the sequence and size of the CDR. One of ordinary skill in the art can routinely determine which residues constitute a particular CDR given the amino acid sequence of the variable region of the antibody.

[0077] [Table 1]

[0078] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al. (1983) U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest."

[0079] The antibodies or antigen-binding fragments, variants, or derivatives of the present invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, mouse, human, humanized, primatized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), disulfide-bonded Fvs (sdFv), fragments containing either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotype (anti-Id) antibodies (e.g., anti-Id antibodies to the anti-ILT7 antibodies disclosed herein). ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. The immunoglobulin or antibody molecules of the present invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, etc.), or subclass of immunoglobulin molecule.

[0080] As used herein, the term "heavy chain moiety" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain moiety includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a binding polypeptide for use in the present invention may include a polypeptide chain comprising a CH1 domain, a polypeptide chain comprising a CH1 domain, at least a part of a hinge domain, and a CH2 domain, a polypeptide chain comprising a CH1 domain and a CH3 domain, a polypeptide chain comprising a CH1 domain, at least a part of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a part of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the present invention includes a polypeptide chain comprising a CH3 domain. Further, a binding polypeptide for use in the present invention may lack at least a part (e.g., all or part of the CH2 domain) of the CH2 domain. As described above, those skilled in the art will understand that these domains (e.g., heavy chain moieties) can be modified such that the amino acid sequence varies from that of a naturally occurring immunoglobulin molecule.

[0081] In certain anti-ILT7 antibodies, or antigen-binding fragments, variants, or derivatives thereof, disclosed herein, the heavy chain moiety of one polypeptide chain of the multimer is identical to the heavy chain moiety of the second polypeptide chain of the multimer. Alternatively, the heavy chain moiety-containing monomers of the present invention are not identical.

[0082] The heavy chain moiety of a binding molecule for use in the diagnostic and therapeutic methods disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain moiety of a polypeptide may include a C H1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain moiety may include a hinge region that is partly derived from an IgG1 molecule and partly derived from an IgG3 molecule. In another example, the heavy chain moiety may include a chimeric hinge that is partly derived from an IgG1 molecule and partly derived from an IgG4 molecule.

[0083] As used herein, the term "light chain moiety" includes an amino acid sequence derived from an immunoglobulin light chain, such as a kappa or lambda light chain. The light chain moiety may include at least one of the VL or CL domains.

[0084] The anti-ILT7 antibodies, or antigen-binding fragments, variants, or derivatives thereof, disclosed herein may be described or specified with respect to an epitope or portion of an antigen, for example, with respect to the target polypeptides (e.g., full-length or mature ILT7) disclosed herein that they recognize or specifically bind to. A portion of a target polypeptide that specifically interacts with the antigen-binding domain of an antibody is an "epitope" or "antigenic determinant". A target polypeptide may contain a single epitope, but typically contains at least two epitopes and may contain any number of epitopes depending on the size, conformation, and type of the antigen. Further, it should be noted that an "epitope" on a target polypeptide can be or can include a non-polypeptide element, for example, an epitope can include a carbohydrate side chain.

[0085] The minimum size of a peptide or polypeptide epitope of an antibody is thought to be about 4-5 amino acids. A peptide or polypeptide epitope may contain at least 7, at least 9, or at least about 15 - about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its three-dimensional form, the amino acids containing the epitope do not necessarily have to be contiguous and in some instances may not be present on the same peptide chain. The peptide or polypeptide epitope recognized by the anti-ILT7 antibody of the present invention may contain a sequence of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or about 15 - about 30 consecutive or non-consecutive amino acids of ILT7.

[0086] "Specifically bind" generally means that an antibody binds to an epitope through its antigen-binding domain and that the binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to that epitope more readily through its antigen-binding domain than to a random and unrelated epitope. The term "specificity" is used herein to determine the nature of the relative affinity of a particular antibody for binding to a particular epitope. For example, antibody "A" can be considered to have a higher specificity for a given epitope than antibody "B", or it can be said that antibody "A" binds to epitope "C" with a higher specificity than the specificity it has for a related epitope "D".

[0087] "Preferentially bind" means that an antibody specifically binds to an epitope more readily than to a related, similar, homologous, or analog epitope. Thus, an antibody that "preferentially binds" to a given epitope is likely to bind to that epitope more than to a related epitope, even if such an antibody can cross-react with the related epitope.

[0088] As a non-limiting example, an antibody can be considered to preferentially bind to a first epitope if it binds to the first epitope with a K D that is smaller than the dissociation constant (K D ) of the antibody for a second epitope. In another non-limiting example, an antibody can be considered to preferentially bind to a first antigen if it binds to the first epitope with an affinity that is at least one order of magnitude lower than the K D of the antibody for a second epitope. In another non-limiting example, an antibody can be considered to preferentially bind to a first epitope if it binds to the first epitope with an affinity that is at least two orders of magnitude lower than the K D of the antibody for a second epitope.

[0089] In another non-limiting example, an antibody can be considered to preferentially bind to a first epitope if it binds to the first epitope with an off-rate (k(off)) lower than the k(off) of the antibody for a second epitope. In another non-limiting example, an antibody can be considered to preferentially bind to a first epitope if it binds to the first epitope with an affinity at least one order of magnitude lower than the k(off) of the antibody for a second epitope. In another non-limiting example, an antibody can be considered to preferentially bind to a first epitope if it binds to the first epitope with an affinity at least two orders of magnitude lower than the k(off) of the antibody for a second epitope. The antibodies or antigen-binding fragments, variants, or derivatives thereof disclosed herein bind to the target polypeptides (e.g., ILT7, e.g., human, primate, mouse, or any other combination of human, primate, and mouse ILT7) or fragments or variants thereof disclosed herein with an off-rate (k(off)) of 5×10 -2 seconds -1 10 -2 seconds -1 5×10 -3 seconds -1 or 10 -3 seconds -1 or less can be said to bind. The antibodies of the present invention bind to the target polypeptides (e.g., ILT7, e.g., human, primate, mouse, or any other combination of human, primate, and mouse ILT7) or fragments or variants thereof disclosed herein with an off-rate (k(off)) of 5×10 -4 seconds -1 10 -4 seconds -1 5×10 -5 seconds -1 or 10 -5 seconds -1 5×10 -6 seconds -1 10 -6 seconds -1 5×10 -7 seconds -1 or 10 -7 seconds -1 or less can be said to bind.

[0090] The antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein bind to the target polypeptides disclosed herein (e.g., ILT7, e.g., human, primate, mouse, or any other combination of human, primate, and mouse ILT7) or fragments or variants thereof with an on-rate (k(on)) of 10 3 M -1 seconds -1 , 5×10 3 M -1 seconds -1 , 10 4 M -1 seconds -1 , or 5×10 4 M -1 seconds -1 or higher. The antibodies of the present invention bind to the target polypeptides disclosed herein (e.g., ILT7, e.g., human, primate, mouse, or any other combination of human, primate, and mouse ILT7) or fragments or variants thereof with an on-rate (k(on)) of 10 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , or 5×10 6 M -1 seconds -1 , or 10 7 M -1 seconds -1 or higher.

[0091] An antibody is said to competitively inhibit the binding of a reference antibody to a given epitope if it preferentially binds to the epitope or overlapping epitopes to such an extent that it blocks the binding of the reference antibody to the epitope to some degree. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. An antibody can be said to competitively inhibit the binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0092] As used herein, the term "affinity" refers to a measure of the strength of binding of an individual epitope to a CDR of an immunoglobulin molecule. See, e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.) pages 27-28. As used herein, the term "avidity" refers to the overall stability of a complex between an immunoglobulin population and an antigen, i.e., the functional binding strength of an immunoglobulin mixture with an antigen. See, e.g., pages 29-34 of Harlow. Avidity is related to both the affinity of individual immunoglobulin molecules in a population for a particular epitope and the valence of the immunoglobulin-antigen binding. For example, the interaction of a bivalent monoclonal antibody with an antigen having a highly repetitive epitope structure, such as a polymer, would be a high avidity interaction.

[0093] The anti-ILT7 antibodies or antigen-binding fragments, variants, or derivatives of the invention can also be described or specified with regard to their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody specific for one antigen to react with a second antigen, i.e., a measure of the relatedness between two different antigenic substances. Thus, an antibody is cross-reactive if it binds to an epitope other than the epitope that induced its formation. Cross-reactive epitopes generally contain many of the same complementary structural features as the inducing epitope and, in some instances, may actually fit better than the original.

[0094] For example, certain antibodies have a degree of cross-reactivity in that they bind to epitopes that are related but non-identical to the epitopes to which they are related, such as a reference epitope, having at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (when calculated using methods known in the art and described herein). An antibody can be said to have little or no cross-reactivity if it does not bind to epitopes having less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (when calculated using methods known in the art and described herein) to the reference epitope. An antibody can be considered "highly specific" for an epitope if it does not bind to any other analogs, orthologs, or homologs of that particular epitope.

[0095] The anti-ILT7 binding molecule, e.g., an antibody or an antigen-binding fragment, variant, or derivative thereof of the invention, can also be described or specified with respect to its binding affinity for the polypeptides of the invention, e.g., ILT7, e.g., human, primate, mouse, or any combination of human, primate, and mouse ILT7. Useful binding affinities include 5×10 -2 M, 10 -2 M, 5×10 -3 M, 10 -3 M, 5×10 -4 M, 10 -4 M, 5×10 -5 M, 10 -5 M, 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, 10 -8 M, 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11M, 5 × 10 -12 M, 10 -12 M, 5 × 10 -13 M, 10 -13 M, 5 × 10 -14 M, 10 -14 M, 5 × 10 -15 M, or 10 -15 Examples include an affinity having a dissociation constant or Kd of less than M.

[0096] In some embodiments, the antibody binds to human ILT7 with a dissociation constant or Kd of less than 1 nM. In some embodiments, the antibody binds to cynomolgus ILT7 with a dissociation constant or Kd of less than 5 nM. In some embodiments, the antibody binds to human ILT7 with a dissociation constant or Kd of less than 1 nM and binds to cynomolgus ILT7 with a dissociation constant or Kd of less than 5 nM.

[0097] As already shown, the subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino-terminal with respect to the hinge region of the immunoglobulin heavy chain molecule.

[0098] As used herein, the term "CH2 domain" includes the portion of the heavy chain molecule that extends from approximately residue 244 to residue 360 of the antibody (residues 244 - 360 in the Kabat numbering system, residues 231 - 340 in the EU numbering system, see Kabat EA et al.). The CH2 domain is unique in that it does not form a strict pair with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of the intact native IgG molecule. The CH3 domain extends C-terminally from the CH2 domain of the IgG molecule and is similarly well reported to contain approximately 108 residues.

[0099] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that joins the CH1 domain and the CH2 domain. This hinge region contains approximately 25 residues, is flexible, and thus the two N-terminal antigen-binding regions can move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol. 161:4083 (1998)).

[0100] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or crosslink with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to positions 239 and 242 (EU numbering system: positions 226 or 229) using the Kabat numbering system.

[0101] As used herein, the term "chimeric antibody" is considered to mean any antibody in which the immunoreactive region or site is obtained from or derived from a first species and the constant region (intact, partial, or modified according to the invention) is obtained from a second species. In certain embodiments, the target-binding region or site is of non-human origin (e.g., mouse or primate) and the constant region is human.

[0102] As used herein, the term "engineered antibody" refers to an antibody in which the variable domain of either or both the heavy and light chains has been altered by at least partial substitution of one or more CDRs from an antibody of known specificity, and optionally by partial framework region substitution and sequence changes. The CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework region is derived, but it is envisioned that the CDRs can be derived from antibodies of different classes or different species. An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity have been transplanted into a human heavy or light chain framework region is referred to herein as a "humanized" antibody. It is not necessary to exchange all of the CDRs for complete CDRs from the donor variable domain in order to transfer the antigen-binding ability of one variable domain to the other. Rather, it is only necessary to transfer those residues necessary to maintain the activity of the target binding site.

[0103] It is further recognized that the framework regions within the variable domains in the heavy chain or light chain or both of a humanized antibody can contain only residues of human origin, in which case these framework regions of the humanized antibody are referred to as "complete human framework regions". Alternatively, one or more residues of the framework region of the donor variable domain can be engineered at the corresponding positions of the human framework regions of the variable domains in the heavy chain or light chain or both of the humanized antibody, if necessary to maintain proper binding or to enhance binding to the ILT7 antigen. Thus, the human framework regions engineered in this way contain a mixture of human and donor framework residues and are referred to herein as "partially human framework regions".

[0104] For example, humanization of an anti-ILT7 antibody can be carried out essentially by replacing the rodent or mutant rodent anti-ILT7 CDR or CDR sequences with the corresponding sequences of a human antibody according to the methods of Winter and co-workers (Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-327 (1988), Verhoeyen et al., Science 239:1534-1536 (1988)). See also U.S. Pat. Nos. 5,225,539, 5,585,089, 5,693,761, 5,693,762, 5,859,205, which are incorporated herein by reference. The resulting humanized anti-ILT7 antibody will contain at least one rodent or mutant rodent CDR within the complete human framework regions of the variable domains of the heavy and / or light chains of the humanized antibody. In some examples, residues within the framework regions of one or more variable domains of the humanized anti-ILT7 antibody are replaced with the corresponding non-human (e.g., rodent) residues (see, e.g., U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370), in which case the resulting humanized anti-ILT7 antibody will contain a partially human framework region within the variable domains of the heavy and / or light chains.

[0105] Furthermore, a humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further refine the performance of the antibody (e.g., to obtain a desired affinity). Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, with all or substantially all of the CDRs corresponding to those of a non-human immunoglobulin and all or substantially all of the framework regions corresponding to those of a human immunoglobulin sequence. A humanized antibody also optionally contains at least a portion of the immunoglobulin constant region (Fc) of a human immunoglobulin. For further details, see Jones et al., Nature 331:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), which are incorporated herein by reference. Thus, such "humanized" antibodies can include antibodies in which a substantially intact but non-human variable domain has been replaced by the corresponding sequence from a non-human species. In fact, humanized antibodies are typically human antibodies in which some CDR residues and perhaps some framework residues have been replaced with residues from similar sites in a rodent antibody. See, e.g., U.S. Pat. Nos. 5,225,539, 5,585,089, 5,693,761, 5,693,762, 5,859,205. Similarly, see U.S. Pat. No. 6,180,370 and International Publication No. WO 01 / 27160, which disclose humanized antibodies with improved affinity for a given antigen and techniques for producing such humanized antibodies.

[0106] As used herein, the terms "linked", "fused" or "fusion" are used interchangeably. These terms refer to the joining of two or more elements or components to one another by any means, including chemical conjugation or recombinant means. "In-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a longer continuous ORF while maintaining the correct translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two or more segments corresponding to polypeptides encoded by the original ORFs (the segments are not normally joined as such in nature). Accordingly, the reading frame is made continuously through the fusion segments, although the segments may be physically or spatially separated, for example, by an in-frame linker sequence. For example, a polynucleotide encoding a CDR of an immunoglobulin variable region can be fused in-frame, but can be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR region, as long as the "fused" CDRs are translated simultaneously as part of a continuous polypeptide.

[0107] In the context of a polypeptide, a "linear sequence", or "sequence", is the order of amino acids in a polypeptide from the amino to the carboxyl terminal direction, where residues adjacent to one another in the sequence are contiguous in the primary structure of the polypeptide.

[0108] As used herein, the term "expression" refers to the process by which a gene produces a biochemical substance, such as a polypeptide. The process includes any expression of the functional presence of a gene within a cell, including, but not limited to, gene knockdown as well as both transient expression and stable expression. This includes, but is not limited to, transcription of the gene into messenger RNA (mRNA), and translation of such mRNA into a polypeptide. When the final desired product is a biochemical substance, expression includes the production of that biochemical substance and any precursors. The expression of a gene produces a "gene product". As used herein, a gene product can be either a nucleic acid, such as messenger RNA produced by transcription of the gene, or a polypeptide translated from the transcript. Gene products described herein further include nucleic acids having post-transcriptional modifications, such as polyadenylation, or polypeptides having post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, proteolytic cleavage, and the like.

[0109] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent (prophylaxis) or slow down (reduce) an undesired physiological change or disorder, such as the progression of an autoimmune disease. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminishment of the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or retardation of disease progression, improvement or amelioration of the disease state, and remission (partial or total), whether detectable or undetectable. "Treatment" can also mean an extension of survival as compared to expected survival if not receiving treatment. Subjects in need of treatment include those already having a condition or disorder, as well as those having a tendency to have a condition or disorder, or in which a condition or disorder is to be prevented.

[0110] "Subject" or "individual", or "animal", or "patient" or "mammal" means any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, and zoo animals, sport animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc.

[0111] As used herein, phrases such as "subjects that benefit from administration of an anti-ILT7 antibody" and "animals in need of treatment" include subjects such as mammalian subjects that benefit from administration of an anti-ILT7 antibody used, for example, for detection of an anti-ILT7 polypeptide (e.g., for a diagnostic procedure), and / or that benefit from treatment with an anti-ILT7 antibody, i.e., disease alleviation or prevention.

[0112] II. ILT7 As used herein, the terms "ILT7" and "ILT7 polypeptide" are used interchangeably. In certain embodiments, ILT7 is full-length. In another embodiment, ILT7 is mature ILT7 (amino acids 24-299). In other embodiments, ILT7 can include full-length ILT7, fragments thereof, or ILT7 variant polypeptides, and fragments of ILT7 or ILT7 variant polypeptides retain some or all of the functional properties of active ILT7.

[0113] Full-length human ILT7 is a 499 amino acid protein (accession number P59901) containing a signal peptide (amino acids 1-23), an extracellular domain (amino acids 24-446), a transmembrane domain (amino acids 447-467), and a cytoplasmic domain (amino acids 468-499). The extracellular domain contains four immunoglobulin-like C2 domains (amino acids 24-118, 123-213, 224-313, and 324-413). ILT7 is a member of the immunoglobulin-like transcript (ILT) or leukocyte immunoglobulin-like receptor (LIR) gene family. The sequence of cynomolgus monkey ILT7 is provided as SEQ ID NO: 292.

[0114] PRTHMQAENLLKPILWAEPGPVIIWKKPVTIWCQGTLEAQEYRLDKEGNSISRHMLKTLESENKAKFSIPSMMWEHAGRYHCYYQSPAGWSEPSDPLELVVTAYSRPSLSALPSPVVTSGVNVTLRCASRLGLGRFTLIEEGDHRLSWTLDSHQHNHGKFQALFPVGPLTFSNRGTFRCYGYENNTPYVWSEPSDPLQLLVSGVSRKPSLLTLQGPVVAPGDNLTLQCGSDVGYIRYALYKEGGDGLPQRPGQQSQAGLSQASFTLNPVRGSHGGQYRCYGAHNVSSKWSAPSDPLDILIAGQIPDRPSLSVQLGPTVASGEKVTLLCQSWGPMFTFLLAKEGAAHPPLRLRSTYRAQQYQAEFPMSPVTSAHAGTYRCYGSRSSDPYLLSHSSEPLELVVSEATETLNPAQNKSDSKTAPHLQDYTVENLIRMGIAGLVLVFLGILLFEAQQSQRSPTRCSQEVNSREDNAPFRVVEPWEQI (SEQ ID NO: 292.)

[0115] ILT7 is selectively expressed in a subset of peripheral blood mononuclear cells (PBMCs) called plasmacytoid dendritic cells (pDCs). pDCs are the major source of the immunomodulatory molecule interferon (IFN) alpha, and ILT7 plays a role in the regulation of IFN-alpha release from these cells.

[0116] III. Anti-ILT7 Binding Molecules In certain embodiments, the ILT7 binding molecules provided herein are antibodies or antigen-binding fragments thereof that contain the sequences and / or properties of the ILT7 binding antibodies provided herein. The SEQ ID NOs of the sequences of the ILT7 antibodies are provided in Table 2.

[0117]

Table 2

[0118] In certain embodiments, the binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof of the invention, such as antibody 7C7, ILT70080, ILT70080.1 to ILT70080.7, ILT70083, ILT70083.1 to ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, and ILT70052, binds to ILT7 and inhibits IFN-alpha release by plasmacytoid dendritic cells.

[0119] In certain embodiments, the antibody of the invention is an anti-ILT7 antibody or antigen-binding fragment, variant, or derivative thereof that binds to ILT7, such as 7C7, ILT70080, ILT70080.1 to ILT70080.7, ILT70083, ILT70083.1 to ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, and ILT70052. In certain embodiments, the anti-ILT7 antibody binds to human, primate, mouse, or any combination of human, primate, and mouse ILT7.

[0120] In one embodiment, the present invention provides an isolated binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof, that specifically binds to the same ILT7 epitope as antibody 7C7, ILT70080, ILT70080.1 to ILT70080.7, ILT70083, ILT70083.1 to ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, or ILT70052. In another embodiment, the present invention provides an isolated binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof, that specifically binds to the same ILT7 epitope as an antibody comprising the VH and VL of 7C7, ILT70080, ILT70080.1 to ILT70080.7, ILT70083, ILT70083.1 to ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, or ILT70052. In another embodiment, the present invention provides an isolated binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof, that specifically binds to the same ILT7 epitope as an antibody comprising the VH or VL of 7C7, ILT70080, ILT70080.1 to ILT70080.7, ILT70083, ILT70083.1 to ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, or ILT70052.

[0121] In another embodiment, the present invention provides an isolated binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof, that specifically binds to ILT7 and competitively inhibits the specific binding of antibody 7C7, ILT70080, ILT70080.1 to ILT70080.7, ILT70083, ILT70083.1 to ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, or ILT70052 to ILT7, such as human, primate, mouse, or any combination of human, primate, and mouse ILT7. In another embodiment, the present invention provides an isolated binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof, that specifically binds to ILT7 and competitively inhibits the specific binding of an antibody comprising the VH and VL of 7C7, ILT70080, ILT70080.1 to ILT70080.7, ILT70083, ILT70083.1 to ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, or ILT70052 to ILT7, such as human, primate, mouse, or any combination of human, primate, and mouse ILT7. In another embodiment, the present invention provides an isolated binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof, that specifically binds to ILT7 and competitively inhibits the specific binding of an antibody comprising the VH or VL of 7C7, ILT70080, ILT70080.1 to ILT70080.7, ILT70083, ILT70083.1 to ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, or ILT70052 to ILT7, such as human, primate, mouse, or any combination of human, primate, and mouse ILT7.

[0122] In certain embodiments, the binding molecule of the invention has an amino acid sequence having at least 80%, 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity with the amino acid sequence of a reference anti-ILT7 antibody molecule. In further embodiments, the binding molecule shares at least 96%, 97%, 98%, 99%, or 100% sequence identity with the reference antibody. In certain embodiments, the reference antibody is 7C7, ILT70080, ILT70080.1 to ILT70080.7, ILT70083, ILT70083.1 to ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, or ILT70052.

[0123] In another embodiment, the invention provides an isolated antibody or an antigen-binding fragment, variant, or derivative thereof, consisting essentially of or consisting of, an antibody or an antigen-binding fragment, variant, or derivative thereof, comprising a VH domain having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH amino acid sequences of SEQ ID NOs: 22, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, and 262, wherein the antibody or an antigen-binding fragment, variant, or derivative thereof comprising the VH domain specifically or preferentially binds to ILT7. In further embodiments, the antibody, or an antigen-binding fragment, variant, or derivative thereof, inhibits IFN-alpha release from plasmacytoid dendritic cells.

[0124] In a further embodiment, the invention includes, consists essentially of, or consists of an isolated antibody or an antigen-binding fragment, variant, or derivative thereof that comprises a VL domain having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL amino acid sequence of SEQ ID NO: 27, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, or 267, wherein the antibody or an antigen-binding fragment, variant, or derivative thereof that comprises the VL domain specifically or preferentially binds to ILT7. In a further embodiment, the antibody or an antigen-binding fragment, variant, or derivative thereof inhibits IFN alpha release from plasmacytoid dendritic cells.

[0125] In a further embodiment, the invention includes, consists essentially of, or consists of an isolated antibody or an antigen-binding fragment, variant, or derivative thereof that comprises a VH domain and a VL domain each having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH and VL sequences of SEQ ID NO: 22 and 27; 42 and 47; 52 and 57; 62 and 67; 72 and 77; 82 and 87; 92 and 97; 102 and 107; 112 and 117; 122 and 127; 132 and 137; 142 and 147; 152 and 157; 162 and 167; 172 and 177; 182 and 187; 192 and 197; 202 and 207; 212 and 217; 222 and 227; 232 and 237; 242 and 247; 252 and 257; or 262 and 267, wherein the antibody or an antigen-binding fragment, variant, or derivative thereof that comprises the VH and VL domains specifically or preferentially binds to ILT7. In a further embodiment, the antibody or an antigen-binding fragment, variant, or derivative thereof inhibits IFN alpha release from plasmacytoid dendritic cells.

[0126] In a further embodiment, the invention comprises, consists essentially of, or consists of an isolated antibody or an antigen-binding fragment, variant, or derivative thereof, comprising VH and VL domains having the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 sequences of SEQ ID NOs: 23, 24, 25, 28, 29, and 30; 43, 44, 45, 48, 49, and 50; 53, 54, 55, 58, 59, and 60; 63, 64, 65, 68, 69, and 70; 73, 74, 74, 78, 79, and 80; 83, 84, 85, 88, 89, and 90; 93, 94, 95, 98, 99, and 100; 103, 104, 105, 108, 109, and 110; 113, 114, 115, 118, 119, and 120; 123, 124, 125, 128, 129, and 130; 133, 134, 135, 138, 139, and 140; 143, 144, 145, 148, 149, and 150; 153, 154, 155, 158, 159, and 160; 163, 164, 165, 168, 169, and 170; 173, 174, 175, 178, 179, and 180; 183, 184, 185, 188, 189, and 190; 193, 194, 195, 198, 199, and 200; 203, 204, 205, 208, 209, and 210; 213, 214, 215, 218, 219, and 220; 223, 224, 225, 228, 229, and 230; 233, 234, 235, 238, 239, and 240; 243, 244, 245, 248, 249, and 250; 253, 254, 255, 258, 259, and 260; 263, 264, 265, 268, 269, and 270, respectively, wherein the antibody or an antigen-binding fragment, variant, or derivative thereof, comprising VH and VL domains, binds specifically or preferentially to ILT7. In a further embodiment, the antibody or an antigen-binding fragment, variant, or derivative thereof inhibits IFN-alpha release from plasmacytoid dendritic cells.

[0127] Suitable bioactive variants of the anti-ILT7 antibody of the present invention can be used in the methods of the present invention. Such variants will retain the desired binding properties of the parent anti-ILT7 antibody. Methods for making antibody variants are generally available in the art.

[0128] Methods for mutagenesis and nucleotide sequence change are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985), Kunkel et al., Methods Enzymol. 154:367-382 (1987), Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.), U.S. Patent No. 4,873,192, and references cited therein, which are hereby incorporated by reference in their entirety. Guidance regarding suitable amino acid substitutions that do not affect the biological activity of the polypeptide of interest can be found in the model of Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), pp. 345-352, which is hereby incorporated by reference in its entirety. The model of Dayhoff et al. uses the Point Accepted Mutation (PAM) amino acid similarity matrix (PAM 250 matrix) to determine suitable conservative amino acid substitutions. Conservative substitutions, such as exchanging one amino acid for another having similar properties, can be beneficial. Examples of conservative amino acid substitutions taught by the PAM 250 matrix of the model of Dayhoff et al. include, but are not limited to, Gly⇔Ala, Val⇔Ile⇔Leu, Asp⇔Glu, Lys⇔Arg, Asn⇔Gln, and Phe⇔Trp⇔Tyr.

[0129] When constructing a variant of an anti-ILT7 binding molecule, such as an antibody or an antigen-binding fragment thereof, a variant, or a derivative thereof, modifications are made so that the variant continues to retain the desired properties, such as being able to specifically bind to ILT7, and in certain embodiments, being able to inhibit IFN-alpha release. It is clear that any mutations made in the DNA encoding the variant polypeptide must not place sequences out of the reading frame. In some embodiments, any mutations made in the DNA do not create complementary regions capable of producing a secondary mRNA structure.

[0130] Methods for measuring the binding specificity of an anti-ILT7 binding molecule, such as an antibody or an antigen-binding fragment thereof, a variant, or a derivative thereof, include, but are not limited to, standard competitive binding assays, cytotoxicity assays, IFN release assays, ELISA assays, and the like.

[0131] In this specification, when considering whether any particular polypeptide comprising a constant region, CDR, VH domain, or VL domain disclosed herein is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identical to another polypeptide, % identity can be determined using methods and computer programs / software known in the art, such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482-489 to find the best segment of homology between two sequences. When using BESTFIT or any other arbitrary sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, of course, the parameters are set such that the percentage of identity is calculated over the entire length of the reference polypeptide sequence and gaps in homology up to 5% of the total number of amino acids in the reference sequence are allowed.

[0132] With respect to the object of the present invention, percent sequence identity can be determined using the Smith-Waterman homology search algorithm with a gap open penalty of 12 and a gap extension penalty of 2, using an affine gap search with the BLOSUM matrix 62. The Smith-Waterman homology search algorithm is taught by Smith and Waterman (1981) Adv. Appl. Math. 2:482-489. Variants may differ from, for example, the reference anti-ILT7 antibody (e.g., 7C7, ILT70080, ILT70080.1 to ILT70080.7, ILT70083, ILT70083.1 to ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, or ILT70052) by as few as 1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, such as 6 to 10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

[0133] The exact chemical structure of a polypeptide that can specifically bind to ILT7 and retain the desired activity depends on a number of factors. When ionizable amino and carboxyl groups are present in the molecule, certain polypeptides can be obtained as acidic or basic salts, or in the neutral form. All such preparations that retain their biological activity when placed in suitable environmental conditions are included in the definition of anti-ILT7 antibodies used herein. Furthermore, the primary amino acid sequence of a polypeptide can be enhanced by derivatization using sugar moieties (glycosylation) or other auxiliary molecules such as lipids, phosphate groups, acetyl groups, etc. It can also be enhanced by conjugation with saccharides. Certain embodiments of such enhancement are achieved through the post-translational processing system of the production host, and other such modifications can be introduced in vitro. In any case, such modifications are included in the definition of anti-ILT7 antibodies used herein as long as the desired properties of the anti-ILT7 antibody are not destroyed. Such modifications are expected to quantitatively or qualitatively affect the activity by enhancing or diminishing the activity of the polypeptide in various assays. Furthermore, individual amino acid residues in the chain can be modified by oxidation, reduction, or other derivatization, and the polypeptide can be cleaved to obtain fragments that retain the activity. Even with such changes that do not destroy the desired properties (e.g., the binding specificity of ILT7, the binding affinity, and the related activities such as the ability to inhibit cytokine release by ILT7 from mast cells and endothelial cells, and the proliferation of TF-1 cells), the polypeptide sequence is included in the definition of the anti-ILT7 antibodies for the purposes used herein.

[0134] The art provides substantial guidance regarding the preparation and use of polypeptide variants. When preparing variants of anti-ILT7 binding molecules, such as antibodies or antigen-binding fragments thereof, variants, or derivatives, one of ordinary skill in the art can readily determine which modifications to the native protein's nucleotide or amino acid sequence will result in variants suitable for use as the therapeutic active ingredient in the pharmaceutical compositions used in the methods of the invention.

[0135] The constant region of the anti-ILT7 antibody can be mutated to alter effector function in a number of ways. See, for example, U.S. Patent No. 6,737,056 B1 and U.S. Patent Application Publication No. 2004 / 0132101 A1, which disclose Fc mutations that optimize antibody binding to Fc receptors.

[0136] In certain anti-ILT7 antibodies, the Fc portion can be mutated using techniques known in the art to reduce effector function. For example, deletions or inactivations of the constant region domain (through point mutations or other means) can reduce Fc receptor binding of the modified antibody in circulation. In other examples, modifications of the constant region consistent with the present invention can mitigate complement binding and thus reduce the serum half-life and non-specific association of conjugated cytotoxins. Still other modifications of the constant region can be used to modify disulfide bonds or oligosaccharide moieties that enhance antigen specificity or enhance localization through antibody flexibility. The resulting physiological profile, biological availability, and other biochemical effects, such as biodistribution and serum half-life, can be readily measured and quantified using well-known immunological techniques without undue experimentation.

[0137] Certain ILT7 antibodies provided herein are hypofucosylated. Antibodies lacking the core fucose residue of the Fc N-glycan exhibit strong ADCC at low concentrations, show very high efficacy compared to fucosylated counterparts, and they can avoid the inhibitory effect of serum immunoglobulin G (IgG) on ADCC through their high binding to the gamma receptor IIIa (FcFcγRIIIa).

[0138] The anti-ILT7 antibodies of the present invention also include derivatives that are modified by covalently attaching any type of molecule to the antibody, such that, for example, covalent attachment does not prevent the antibody from specifically binding to its cognate epitope. For example, but not limited to, antibody derivatives include antibodies that are modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to a cell ligand or other protein, etc. A number of any chemical modifications, including but not limited to specific chemical cleavage, acetylation, formylation, etc., can be carried out by known techniques. Further, the derivative may contain one or more non-classical amino acids.

[0139] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations such as saturation mutations can be randomly introduced along all or part of the coding sequence, and the resulting mutants can be screened for biological activity to identify mutants that retain activity (e.g., the binding ability of the anti-ILT7 polypeptide).

[0140] For example, it is possible to introduce mutations only in the framework region or only in the CDR region of an antibody molecule. The introduced mutations can be silent or neutral missense mutations, i.e., they have no or little effect on the antigen-binding ability of the antibody. These types of mutations can be useful for optimizing codon usage or improving antibody production in hybridomas. Alternatively, non-neutral missense mutations can alter the antigen-binding ability of the antibody. Most of the positions of silent and neutral missense mutations probably exist in the framework region, and most of the positions of non-neutral missense mutations probably exist in the CDRs, although this is not necessarily an absolute requirement. One of ordinary skill in the art will be able to design and test mutant molecules with desired properties, such as no change in antigen-binding activity or a change in binding activity (e.g., improvement in antigen-binding activity or change in antibody specificity). After mutagenesis, the encoded protein is expressed as usual, and the functional and / or biological activity of the encoded protein (e.g., the ability to immunospecifically bind to at least one epitope of the ILT7 polypeptide) can be determined using the techniques described herein or by routinely modifying techniques known in the art.

[0141] In certain embodiments, the anti-ILT7 antibodies of the invention comprise at least one optimized complementarity determining region (CDR). An "optimized CDR" is intended to mean that the CDR has been modified and that the optimized sequence has been selected based on maintaining or improving the binding affinity and / or anti-ILT7 activity conferred on the anti-ILT7 antibody that comprises the optimized CDR. "Anti-ILT7 activity" can include, for example, the activity of modulating one or more of the following activities associated with ILT7, such as interferon release by ILT7 from plasmacytoid dendritic cells, cytotoxicity against ILT7-expressing cells, or any other activity associated with ILT7. Anti-ILT7 activity can also result from a decrease in the incidence or severity of diseases associated with ILT7 expression, including but not limited to certain types of autoimmune conditions such as systemic lupus erythematosus, rheumatoid arthritis, and psoriasis. The modification can involve substitution of amino acid residues within the CDR such that the anti-ILT7 antibody retains specificity for the ILT7 antigen and retains improved binding affinity and / or improved anti-ILT7 activity.

[0142] IV. Polynucleotides Encoding Anti-ILT7 Antibodies The invention also provides nucleic acid molecules encoding the anti-ILT7 antibodies of the invention, or antigen-binding fragments, variants, or derivatives thereof.

[0143] In a further embodiment, the present invention comprises, consists essentially of, or consists of an isolated polynucleotide comprising a nucleic acid encoding a VH domain having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference VH domain polypeptide sequence comprising SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, or 242, wherein the anti-ILT7 antibody comprising the encoded VH domain specifically or preferentially binds to ILT7. In certain embodiments, the polynucleotide encodes an antibody, or an antigen-binding fragment, variant, or derivative thereof, that inhibits IFN alpha release from plasmacytoid dendritic cells.

[0144] In a further embodiment, the present invention comprises, consists essentially of, or consists of an isolated polynucleotide comprising a nucleic acid encoding a VL domain having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a reference VL domain polypeptide sequence comprising SEQ ID NO: 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, or 247, wherein the anti-ILT7 antibody comprising the encoded VL domain specifically or preferentially binds to ILT7. In certain embodiments, the polynucleotide encodes an antibody, or an antigen-binding fragment, variant, or derivative thereof, that inhibits IFN alpha release from plasmacytoid dendritic cells.

[0145] Any of the above polynucleotides may further comprise, for example, a signal peptide for directing the secretion of the encoded polypeptide, an antibody constant region described herein, or additional nucleic acids encoding other heterologous polypeptides described herein. Similarly, as described in more detail elsewhere herein, the present invention includes compositions comprising one or more of the above polynucleotides.

[0146] In one embodiment, the present invention includes a composition comprising a first polynucleotide encoding a VH domain described herein and a second polynucleotide encoding a VL domain described herein. Specifically, the composition may consist essentially of, or consist of, a VH domain-encoding polynucleotide as set forth in SEQ ID NO: 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, or 241, and a VL domain-encoding polynucleotide as set forth in SEQ ID NO: 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, or 246. The composition may also consist essentially of, or consist of, a VH domain-encoding polynucleotide encoding the sequence set forth in SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, or 242, and a VL domain-encoding polynucleotide encoding the sequence set forth in SEQ ID NO: 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, or 267. In some embodiments, the VH domain-encoding polypeptide and the VL domain-encoding polypeptide are present in the same vector. In some embodiments, the VH domain-encoding polypeptide and the VL domain-encoding polypeptide are present in different vectors.

[0147] The present invention also includes fragments of the polynucleotides of the invention described elsewhere herein. Further, polynucleotides encoding the fusion polypeptides, Fab fragments, and other derivatives described herein are likewise contemplated by the present invention.

[0148] The polynucleotide can be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., Bio Techniques 17:242 (1994)), which briefly involves the synthesis of overlapping oligonucleotides containing a portion of the sequence encoding the antibody, the annealing and ligation of those oligonucleotides, and the amplification of the ligated oligonucleotides by PCR.

[0149] Alternatively, a polynucleotide encoding an anti-ILT7 antibody or an antigen-binding fragment, variant, or derivative thereof can be generated from a nucleic acid of a suitable origin. If a clone containing the nucleic acid encoding a particular antibody is not available but the sequence of the antibody molecule is known, the nucleic acid encoding the antibody can be chemically synthesized or obtained from a suitable origin (e.g., an antibody cDNA library or cDNA library made from any tissue or cell expressing an antibody or other anti-ILT7 antibody, such as a hybridoma cell selected to express the antibody, or a nucleic acid isolated therefrom, such as polyA+RNA) by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence, or by cloning using an oligonucleotide probe specific for a particular gene sequence, for example, cDNA clones can be identified from a cDNA library encoding an antibody or other anti-ILT7 antibody. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0150] Once the nucleotide sequence and the corresponding amino acid sequence of an anti-ILT7 antibody or its antigen-binding fragment, variant, or derivative are determined, the nucleotide sequence can be manipulated using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA technology, site-directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al. (1990) Molecular Cloning, A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel et al., eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons, NY), which are incorporated herein by reference in their entirety), to create antibodies with different amino acid sequences, for example, to make amino acid substitutions, deletions, and / or insertions.

[0151] A polynucleotide encoding an anti-ILT7 binding molecule, such as an antibody, or an antigen-binding fragment, variant, or derivative thereof, can be composed of unmodified RNA or DNA, or any polynucleotide or polydeoxynucleotide that can be modified RNA or DNA. For example, a polynucleotide encoding an anti-ILT7 antibody or an antigen-binding fragment, variant, or derivative thereof can be single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, a hybrid molecule comprising DNA and RNA that can be single-stranded, or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, a polynucleotide encoding an anti-ILT7 binding molecule, such as an antibody, or an antigen-binding fragment, variant, or derivative thereof can be composed of a triple-stranded region containing RNA or DNA, or both RNA and DNA. A polynucleotide encoding an anti-ILT7 binding molecule, such as an antibody, or an antigen-binding fragment, variant, or derivative thereof can also contain one or more modified bases, or a DNA or RNA backbone, modified for stability or for any other reason. "Modified" bases include, for example, rare bases such as tritiated bases and inosine. A variety of modifications can be made to DNA and RNA, and thus "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms.

[0152] An isolated polynucleotide encoding a non-natural variant of a polypeptide derived from an immunoglobulin (e.g., an immunoglobulin heavy chain portion or light chain portion) can be made by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions can be made at one or more non-essential amino acid residues.

[0153] V. Fusion Proteins and Antibody Conjugates As will be discussed in more detail elsewhere in this specification, an anti-ILT7 binding molecule, such as an antibody of the invention, or an antigen-binding fragment, variant, or derivative thereof, can further be recombinantly fused at the N- or C-terminus to a heterologous polypeptide or chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide or other composition. For example, an anti-ILT7 antibody can be recombinantly fused or conjugated to a molecule useful as a label in a detection assay and an effector molecule, such as a heterologous polypeptide, drug, radionuclide, or toxin. See, for example, PCT Publication Nos. WO 92 / 08495, WO 91 / 14438, WO 89 / 12624, U.S. Patent No. 5,314,995, and European Patent No. 396,387.

[0154] The anti-ILT7 antibodies of the invention, or antigen-binding fragments, variants, or derivatives thereof, can include derivatives that are modified such that the antibody is not prevented from binding to ILT7 for covalent attachment, i.e., derivatives modified by covalently attaching any type of molecule to the antibody. For example, but not limited to, antibody derivatives include antibodies modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cell ligand or other protein, etc. A number of any chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, etc. Further, the derivative can contain one or more non-classical amino acids.

[0155] An anti-ILT7 binding molecule, such as an antibody of the invention, or an antigen-binding fragment, variant, or derivative thereof, can be composed of amino acids, i.e., peptide isosteres, linked to each other by peptide bonds or modified peptide bonds, and can contain amino acids other than the 20 genetically encoded amino acids. For example, an anti-ILT7 antibody can be modified by natural processes such as post-translational processing or chemical modification techniques well known in the art. Such modifications are well described in the basic texts and more detailed monographs, as well as in the extensive research literature. Modifications can occur at any location within the anti-ILT7 binding molecule, including the peptide backbone, amino acid side chains, and amino or carboxyl termini, or moieties such as carbohydrates. It will be appreciated that the same type of modification can be present to the same or different extents at several sites within a given anti-ILT7 binding molecule. Similarly, a given anti-ILT7 binding molecule can contain many types of modifications. Anti-ILT7 binding molecules can be branched, for example, as a result of ubiquitination, and they can be cyclic, with or without branching. Cyclic, branched, and branched-cyclic anti-ILT7 binding molecules can result from natural processes after translation or can be produced by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of a covalent cross-link, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of an amino acid such as arginylation to a protein, and ubiquitination (see, for example, Proteins--Structure and Molecular Properties, T. E. Creighton, W. H. Freeman and Company, NY; 2nd ed. (1993), Johnson, ed. (1983) Posttranslational Covalent Modification of Proteins (Academic Press, NY), pgs. 1-12, Seifter et al., Meth. Enzymol. 182:626-646 (1990), Rattan et al., Ann. NY Acad. Sci. 663:48-62 (1992)).

[0156] The invention also provides a fusion protein comprising an anti-ILT7 antibody, or an antigen-binding fragment, variant, or derivative thereof, and a heterologous polypeptide. The heterologous polypeptide to which the antibody is fused can be useful for a function or can be useful for targeting anti-ILT7 polypeptide-expressing cells.

[0157] In one embodiment, the fusion protein of the present invention consists essentially of or consists of a polypeptide having any one or more amino acid sequences of the VH domain of the antibody of the present invention, or any one or more amino acid sequences of the VL domain of the antibody, fragment, variant, or derivative of the present invention, and a heterologous polypeptide sequence.

[0158] In another embodiment, the fusion protein for use in the diagnostic and treatment methods disclosed herein consists essentially of or consists of a polypeptide having any one, two, or three amino acid sequences of the CDRs of the VH domain of an anti-ILT7 antibody, or fragment, variant, or derivative thereof, or any one, two, or three amino acid sequences of the CDRs of the VL domain of an anti-ILT7 antibody, or fragment, variant, or derivative thereof, and a heterologous polypeptide sequence. In one embodiment, the fusion protein comprises the amino acid sequence of at least one VH domain of the anti-ILT7 antibody of the present invention, and the amino acid sequence of at least one VL domain of the anti-ILT7 antibody, or fragment, derivative, or variant thereof, of the present invention, and a heterologous polypeptide sequence. In some embodiments, the VH and VL domains of the fusion protein correspond to an antibody (or scFv or Fab fragment) of single origin that specifically binds to at least one epitope of ILT7. In yet another embodiment, the fusion protein for use in the diagnostic and treatment methods disclosed herein consists essentially of or consists of a polypeptide having any one, two, three, or more amino acid sequences of the CDRs of the VH domain of an anti-ILT7 antibody, and any one, two, three, or more amino acid sequences of the CDRs of the VL domain of an anti-ILT7 antibody, or fragment, or variant thereof, and a heterologous polypeptide sequence. In some embodiments, 2, 3, 4, 5, 6, or more of the CDRs of the VH domain or VL domain correspond to an antibody (or scFv or Fab fragment) of single origin of the present invention. Nucleic acid molecules encoding these fusion proteins are also encompassed by the present invention.

[0159] Exemplary fusion proteins reported in the literature include the T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84:2936-2940 (1987)), CD4 (Capon et al., Nature 337:525-531 (1989), Traunecker et al., Nature 339:68-70 (1989), Zettmeissl et al., DNA Cell Biol. USA 9:347-353 (1990), and Byrn et al., Nature 344:667-670(1990)), L-selectin (homing receptor) (Watson et al., J. Cell. Biol. 110:2221-2229 (1990), and Watson et al., Nature 349:164-167 (1991)), CD44 (Aruffo et al., Cell 61:1303-1313 (1990)), CD28 and B7 (Linsley et al., J. Exp. Med. 173:721-730 (1991)), CTLA-4 (Lisley et al., J. Exp. Med. 174:561-569 (1991)), CD22 (Stamenkovic et al., Cell 66:1133-1144 (1991)), TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991), Lesslauer et al., Eur. J. Immunol. 27:2883-2886 (1991), and Peppel et al., J. Exp. Med. 174:1483-1489 (1991)), and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. Vol. 115, Abstract No. 1448 (1991)).

[0160] As discussed elsewhere herein, an anti-ILT7 binding molecule, such as an antibody of the invention, or an antigen-binding fragment, variant, or derivative thereof, can be fused to a heterologous polypeptide to increase the in vivo half-life of the polypeptide or for use in immunoassays using methods known in the art. For example, in one embodiment, PEG can be conjugated to an anti-ILT7 antibody of the invention to increase its half-life in vivo. See Leong et al., Cytokine 16:106 (2001), Adv. in Drug Deliv. Rev. 54:531 (2002), or Weir et al., Biochem. Soc. Transactions 30:512 (2002).

[0161] Furthermore, an anti-ILT7 binding molecule, such as an antibody of the invention, or an antigen-binding fragment, variant, or derivative thereof, can be fused to a marker sequence, such as a peptide, to facilitate its purification or detection. In some embodiments, the marker amino acid sequence is a hexahistidine peptide, such as a tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), by way of example, hexahistidine provides for facile purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag corresponding to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)), and the "flag" tag.

[0162] Fusion proteins can be prepared using methods well known in the art (see, for example, U.S. Pat. Nos. 5,116,964 and 5,225,538). The exact site at which the fusion is made can be empirically selected to optimize the secretion or binding characteristics of the fusion protein. The DNA encoding the fusion protein is then transfected into a host cell for expression.

[0163] An anti-ILT7 binding molecule, such as an antibody of the invention, or an antigen-binding fragment, variant, or derivative thereof, can be used unconjugated or conjugated to at least one of a variety of molecules, for example, to improve the therapeutic properties of the molecule, to facilitate detection of the target, or for imaging or treating a patient. An anti-ILT7 binding molecule, such as an antibody of the invention, or an antigen-binding fragment, variant, or derivative thereof, can be labeled or conjugated before, after, or during purification.

[0164] In particular, the anti-ILT7 antibody of the invention, or an antigen-binding fragment, variant, or derivative thereof, can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, drug, or PEG.

[0165] One of ordinary skill in the art will recognize that the conjugate can also be assembled using a variety of techniques depending on the selected agent to which it is conjugated. For example, a conjugate with biotin can be prepared by reacting a binding polypeptide with an activated ester of biotin, such as biotin N-hydroxysuccinimide ester. Similarly, a conjugate with a fluorescent marker can be prepared in the presence of a coupling agent, such as a coupling agent described herein, or by reaction with an isothiocyanate, such as fluorescein isothiocyanate. Conjugates of the anti-ILT7 antibody of the invention, or an antigen-binding fragment, variant, or derivative thereof, are prepared in a similar manner.

[0166] The present invention further encompasses an anti-ILT7 binding molecule, such as an antibody of the present invention, or an antigen-binding fragment, variant, or derivative thereof, conjugated to a diagnostic or therapeutic agent. Anti-ILT7 antibodies, including their antigen-binding fragments, variants, and derivatives, can be used diagnostically, for example, to monitor the development or progression of a disease as part of clinical trial procedures, or to determine the effectiveness and / or prophylactic regimen of a given treatment. For example, detection can be facilitated by coupling an anti-ILT7 antibody or an antigen-binding fragment, variant, or derivative thereof to a detectable substance. Examples of detectable substances include various enzymes, avidin families, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals for use in various positron emission tomography, and non-radioactive paramagnetic metal ions. See, for example, U.S. Patent No. 4,741,900 regarding metal ions that can be conjugated to an antibody for use as a diagnostic agent according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable avidin family complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125 I, 131 I, 111 In, 90 Y, or 99 Tc.

[0167] An anti-ILT7 binding molecule, such as an antibody, or an antigen-binding fragment, variant, or derivative thereof, can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged anti-ILT7 binding molecule is then determined by detecting the presence of luminescence that occurs during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, and oxalate esters.

[0168] One method for detectably labeling an anti-ILT7 antibody, or an antigen-binding fragment, variant, or derivative thereof, is to link it to an enzyme and use the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates Quarterly Publication, Walkersville, Md.; Diagnostic Horizons 2:1-7 (1978), Voller et al., J. Clin. Pathol. 31:507-520 (1978), Butler, Meth. Enzymol. 73:482-523 (1981), Maggio, ed. (1980) Enzyme Immunoassay, CRC Press, Boca Raton, Fla., Ishikawa et al., eds. (1981) Enzyme Immunoassay (Kgaku Shoin, Tokyo). The enzyme linked to the anti-ILT7 antibody reacts with a suitable substrate, such as a chromogenic substrate, to yield a chemical moiety that can be detected by, for example, spectrophotometry, fluorometry, or visual means. Enzymes that can be used to detectably label an antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Further, detection can be achieved by colorimetric measurement using a chromogenic substrate for the enzyme. Detection can also be achieved by visual comparison of the degree of the enzyme reaction of the substrate with that of a similarly prepared standard.

[0169] Detection can also be performed using any of a variety of other immunoassays. For example, it is possible to detect a binding molecule through the use of a radioimmunoassay (RIA) by radiolabeling an anti-ILT7 binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof (see, e.g., Weintraub (March, 1986) Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques (The Endocrine Society), which is incorporated herein by reference). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.

[0170] An anti-ILT7 binding molecule, such as an antibody, or an antigen-binding fragment, variant, or derivative thereof, can also be detectably labeled using a fluorescent-emitting metal such as 152Eu, or another metal of the lanthanide series. These metals can be attached to the binding molecule using a metal chelate group such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0171] Techniques for conjugating various moieties to an antibody (e.g., an anti-ILT7 antibody) or an antigen-binding fragment, variant, or derivative thereof are well known, see, for example, Amon et al. (1985) "Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy," in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pp. 243-56, Hellstrom et al. (1987) "Antibodies for Drug Delivery," in Controlled Drug Delivery, ed. Robinson et al. (2nd ed.; Marcel Dekker, Inc.), pp. 623-53), Thorpe (1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological and Clinical Applications, ed. Pinchera et al., pp. 475-506, "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy," in Monoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin et al., Academic Press, pp. 303-16 (1985), and Thorpe et al. (1982) "The Preparation and Cytotoxic Properties of Antibody-Toxin Conjugates," Immunol. Rev. 62:119-58.

[0172] VI. Expression of Antibody Polypeptides DNA sequences encoding the light and heavy chains of the antibody can be made either simultaneously or individually using reverse transcriptase and DNA polymerase according to well-known methods. PCR can be initiated by consensus constant region primers or by more specific primers based on published heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding the antibody light and heavy chains. In this case, the library can be screened with a consensus primer or a larger homologous probe, such as a mouse constant region probe.

[0173] DNA, typically plasmid DNA, can be isolated from cells using techniques known in the art, restriction enzyme mapped, and sequenced according to standard well-known techniques described in detail in the aforementioned references related to recombinant DNA technology. Of course, the DNA can be synthetic according to the present invention at any point during the isolation method or subsequent analysis.

[0174] After manipulation of the isolated genetic material to provide an anti-ILT7 antibody, or an antigen-binding fragment, variant, or derivative of the invention, the polynucleotide encoding the anti-ILT7 antibody is typically inserted into an expression vector for introduction into a host cell that can be used to produce the desired amount of the anti-ILT7 antibody.

[0175] The recombinant expression of an antibody, its fragment, variant, or derivative, such as the heavy or light chain of an antibody that binds to a target molecule as described herein, e.g., ILT7, requires the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule or the heavy or light chain of an antibody, or a portion thereof (e.g., containing the heavy or light chain variable domain) of the present invention is obtained, a vector for producing the antibody molecule can be produced by recombinant DNA techniques using techniques well known in the art. Thus, a method for preparing a protein by expressing a polynucleotide containing an antibody-encoding nucleotide sequence is described herein. Expression vectors containing the antibody-encoding sequence and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene recombination. The present invention thus provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the present invention, or its heavy or light chain, or heavy or light chain variable domain, operably linked to a promoter. Such vectors can include a nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT International Publication No. 86 / 05807, PCT International Publication No. 89 / 01036, and U.S. Patent No. 5,122,464), and the variable domain of the antibody can be cloned in such vectors for expression of the complete heavy or light chain.

[0176] The terms "vector" or "expression vector" are used herein to mean a vector used in accordance with the present invention as a vehicle for introducing and expressing a desired gene into a host cell. As is known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors compatible with the present invention contain selectable markers and appropriate restriction sites to facilitate cloning of the desired gene and to facilitate the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0177] With respect to the object of the present invention, a number of expression vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV, or MoMLV), or SV40 virus. Other vectors involve the use of a polycistronic system along with an internal ribosome entry site. In addition, cells in which the DNA is incorporated into its chromosome can be selected by introducing one or more markers that enable the selection of transfected host cells. The markers can provide prototrophy to auxotrophic hosts, biocide (e.g., antibiotic) resistance, or resistance to heavy metals such as copper. The selectable marker gene can be ligated directly to the DNA sequence to be expressed or introduced into the same cells by co-transformation. Additional elements may also be necessary for optimal synthesis of mRNA. These elements can include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.

[0178] In some embodiments, the cloned variable region gene is inserted into an expression vector together with the heavy and light chain constant region genes (e.g., human) synthesized above. Of course, any expression vector capable of inducing expression in eukaryotic cells can be used in the present invention. Examples of suitable vectors include, but are not limited to, plasmid pcDNA3, pHCMV / Zeo, pCR3.1, pEF 1 / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, Calif.), and plasmid pCI (available from Promega, Madison, Wis.). Generally, screening a large number of transformed cells for a vector that expresses suitable high levels of immunoglobulin heavy and light chains is a routine experiment that can be performed, for example, by a robotic system.

[0179] More generally, after preparing a vector or DNA sequence encoding the monomeric subunit of an anti-ILT7 antibody, the expression vector can be introduced into a suitable host cell. Introduction of the plasmid into the host cell can be achieved by a variety of techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electroporation and electropermeabilization), protoplast fusion, calcium phosphate precipitation, cell fusion with envelope DNA, microinjection, and infection with intact virus. See Ridgway (1988) "Mammalian Expression Vectors" in Vectors, ed. Rodriguez and Denhardt (Butterworths, Boston, Mass.), Chapter 24.2, pp. 470-472. Typically, introduction of the plasmid into the host is carried out via electroporation. Host cells having the expression construct are grown under conditions appropriate for production of the light and heavy chains and assayed for heavy chain and / or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and the like.

[0180] The expression vector is transferred into the host cell by conventional techniques, and the transfected cells are cultured by conventional techniques for producing the antibody used in the methods described herein. Thus, the present invention includes a host cell containing a polynucleotide encoding an antibody of the present invention or a heavy or light chain thereof, operably linked to a heterologous promoter. In some embodiments relating to the expression of bispecific antibodies, vectors encoding both the heavy and light chains can be co-expressed in the host cell for expression of a complete immunoglobulin molecule as detailed below.

[0181] As used herein, "host cell" refers to a cell constructed using recombinant DNA technology and having a vector encoding at least one heterologous gene. In the description of methods for isolating antibodies from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to refer to the source of the antibody, unless otherwise specified. In other words, the recovery of a polypeptide from "cells" can mean either by centrifugation of the whole cells or by either a cell culture containing both the medium and the floating cells.

[0182] Using a variety of host expression vector systems, antibody molecules for use in the methods described herein can be expressed. Such host expression systems represent a medium that can produce and then purify the coding sequence of interest, but also represent cells that can express the antibody molecules of the present invention in situ when transformed or transfected with an appropriate nucleotide coding sequence. These include bacteria (e.g., Escherichia coli (E. coli), Bacillus subtilis) transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector containing an antibody coding sequence; microorganisms such as yeast (e.g., Saccharomyces, Pichia) transformed with a recombinant yeast expression vector containing an antibody coding sequence; insect cell lines infected with a recombinant virus expression vector containing an antibody coding sequence (e.g., baculovirus); plant cell lines infected with a recombinant virus expression vector containing an antibody coding sequence (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid); or mammalian cell lines (e.g., COS, CHO, BLK, 293, 3T3 cells) having a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter), but are not limited thereto. In particular, bacterial cells such as Escherichia coli or eukaryotic cells for expressing the entire recombinant antibody molecule are used for expressing the recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO) are an effective expression system for antibodies, together with a vector containing, for example, the major intermediate early gene promoter element of human cytomegalovirus (Foecking et al., Gene 45:101 (1986), Cockett et al., Bio / Technology 8:2 (1990)).

[0183] Host cell lines used for protein expression are often cells of mammalian origin, and one of ordinary skill in the art is believed to have the ability to determine the specific host cell line most suitable for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, CHO (Chinese hamster ovary), DG44 and DUXB11 (Chinese hamster ovary cell line, DHFR minus), HELA (human cervical cancer), CVI (monkey kidney cell line), COS (derivative of CVI by SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney cell line), SP2 / O (mouse myeloma), P3×63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), and 293 (human kidney). Host cell lines are typically available from the American Tissue Culture Collection, a for-profit service, or from published literature.

[0184] In addition, a host cell line can be selected that regulates the expression of the inserted sequence or modifies and processes the gene product in a particular desired way. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can be important for the function of the protein. Different host cells have mechanisms that are characteristic and specific for the post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure proper modification and processing of the foreign protein expressed. For this purpose, eukaryotic host cells that possess the cellular machinery for proper processing, glycosylation, and phosphorylation of the primary transcript of the gene product can be used.

[0185] Stable expression is useful for the long-term, high-yield production of recombinant proteins. For example, cell lines that stably express antibody molecules can be engineered. Instead of using an expression vector containing a viral origin of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, the engineered cells are grown in a rich medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection, and the cells can stably integrate the plasmid into their chromosomes, form colonies, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that stably express antibody molecules.

[0186] A number of selection systems can be used, including but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes, which can be used in tk-, hgprt-, or aprt- cells, respectively. Similarly, antimetabolite resistance can be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980), O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)), gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)), neo, which confers resistance to the aminoglycoside G-418, Clinical Pharmacy 12:488-505, Wu and Wu, Biotherapy 3:87-95 (1991), Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993), Mulligan, Science 260:926-932 (1993), and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993), TIB TECH 11(5):155-215 (May, 1993), and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods generally known in the art of recombinant DNA technology that can be used are incorporated herein by reference in their entirety, as described in Ausubel et al. (1993) Current Protocols in Molecular Biology (John Wiley & Sons, NY), Kriegler (1990) "Gene Transfer and Expression" in A Laboratory Manual (Stockton Press, NY), Dracopoli et al. (eds) (1994) Current Protocols in Human Genetics (John Wiley & Sons, NY) Chapters 12 and 13, and Colberre-Garapin et al. (1981) J. Mol. Biol. 150:1.

[0187] The expression level of the antibody molecule can be increased by amplification of the vector (see Bebbington and Hentschel (1987) "The Use of Vectors Based on Gene Amplification for the Expression of Cloned Genes in Mammalian Cells in DNA Cloning" (Academic Press, NY) Vol. 3 for a review). When the marker in the vector system expressing the antibody is amplifiable, the copy number of the marker gene increases as the level of the inhibitor present in the culture of the host cell increases. Since the amplified region is related to the antibody gene, the production of the antibody also increases (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[0188] In vitro production enables scale-up to yield large amounts of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in an airlift reactor or continuous stirred reactor, or immobilized or captured cell culture on, for example, hollow fibers, microcapsules, agarose microbeads or ceramic cartridges. If necessary and / or desirable, the solution of the polypeptide can be purified by conventional chromatography methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE cellulose, or (immuno)affinity chromatography, for example after the selective biosynthesis of a synthetic hinge region polypeptide or before or after the HIC chromatography step described herein.

[0189] The gene encoding the anti-ILT7 antibody, or antigen-binding fragment, variant, or derivative thereof of the present invention can also be expressed in non-mammalian cells such as insect, bacterial, or yeast or plant cells. Bacteria that readily take up nucleic acids include members of the Enterobacteriaceae such as Escherichia coli or Salmonella strains, the Bacillaceae, such as Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. It is further recognized that when expressed in bacteria, the heterologous polypeptide typically becomes part of inclusion bodies. The heterologous polypeptide must be assembled into a functional molecule after isolation and purification. If a tetravalent form of the antibody is desired, the subunits self-assemble into a tetravalent antibody (WO 02 / 096948 A2).

[0190] In the bacterial system, depending on the intended use of the expressed antibody molecule, a number of expression vectors can be advantageously selected. For example, when a large amount of such a protein should be produced to prepare a pharmaceutical composition of the antibody molecule, a vector that directs high-level expression of a fusion protein product that is easily purified may be a desirable vector. Such vectors include the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), pIN vectors (Inouye and Inouye, Nucleic Acids Res. 13:3101-3109 (1985), Van Heeke and Schuster, J. Biol. Chem. 24:5503-5509 (1989)), etc., where the antibody coding sequence can be individually ligated into the vector in-frame with the lacZ coding region so that a fusion protein is produced. The pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by elution in the presence of free glutathione after adsorption and binding to matrix glutathione-agarose beads. The pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0191] In addition to prokaryotic cells, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among eukaryotic microorganisms, but a number of other strains, such as Pichia pastoris, are also generally available.

[0192] For expression in Saccharomyces, for example, plasmid YRp7 (Stinchcomb et al., Nature 282:39 (1979), Kingsman et al., Gene 7:141 (1979), Tschemper et al., Gene 10:157 (1980)) is commonly used. This plasmid already contains the TRP1 gene, which provides a selectable marker for mutant strains of yeast lacking the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)). The presence of the trp1 lesion as a characteristic of the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0193] In the insect system, the Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody-encoding sequences can be individually cloned into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0194] When the binding molecule of the present invention is recombinantly expressed, it can be purified by any method known in the art for the purification of immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen after protein A, and size exclusion column chromatography), centrifugation, differences in solubility, or any other standard protein purification technique. Alternatively, a useful method for increasing the affinity of the antibodies of the present invention is disclosed in US Patent Application Publication No. 20020123057 A1.

[0195] VII. Methods of Treatment Using Therapeutic Anti-ILT7 Binding Molecules The method of the present invention relates to the use of an anti-ILT7 binding molecule, such as an antibody comprising an antigen-binding fragment, variant, and derivative thereof, for treating a patient having a disease associated with ILT7 expression or ILT7-expressing cells. "ILT7-expressing cells" are intended to mean cells that express the ILT7 antigen. Methods for detecting ILT7 expression in cells are well known in the art and include, but are not limited to, PCR techniques, immunohistochemistry, flow cytometry, Western blot, ELISA, etc.

[0196] The following considerations refer to methods of diagnosing and treating various diseases and disorders having the anti-ILT7 antibody of the present invention, but the methods disclosed herein also retain the desired properties of the anti-ILT7 antibody of the present invention, for example, can specifically bind to ILT7, and can neutralize ILT7 pathogenic activity. These are also applicable to antigen-binding fragments, variants, and derivatives of these anti-ILT7 antibodies.

[0197] In one embodiment, the treatment comprises applying or administering an anti-ILT7 binding molecule, such as an antibody or a binding fragment, variant, or derivative thereof of the present invention, to a subject or patient, or to an isolated tissue or cell line from a subject or patient having a disease, a symptom of a disease, or a predisposition to a disease. applying or administering an anti-ILT7 binding molecule. In another embodiment, the treatment also comprises applying or administering a pharmaceutical composition comprising an anti-ILT7 binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof of the present invention, to a control or patient, or to an isolated tissue or cell line from a subject or patient having a disease, a symptom of a disease, or a predisposition to a disease. applying or administering a pharmaceutical composition comprising an anti-ILT7 binding molecule.

[0198] Anti-ILT7 binding molecules, such as antibodies or antigen-binding fragments, variants, or derivatives thereof of the present invention, are useful for treating various autoimmune conditions. For example, treatment with at least one anti-ILT7 antibody elicits a beneficial physiological response, such as a reduction in interferon, for the treatment of disease states associated with ILT7-expressing cells in humans.

[0199] In one embodiment, the present invention relates to an anti-ILT7 binding molecule, such as an antibody, or an antigen-binding fragment, variant, or derivative thereof, for use in treating or preventing autoimmune conditions or diseases, in particular. Examples of autoimmune diseases include, but are not limited to, myositis, diabetes, Hashimoto's disease, autoimmune adrenal insufficiency, erythroblastosis, multiple sclerosis, rheumatic carditis, systemic lupus erythematosus, psoriasis, rheumatoid arthritis, chronic inflammation, Sjogren's syndrome, polymyositis, dermatomyositis, inclusion body myositis, juvenile myositis, and scleroderma.

[0200] According to the methods of the present invention, at least one ILT7 binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof as defined elsewhere in the present invention, is used to promote a positive therapeutic response related to an autoimmune response. As used in relation to autoimmune treatment, "positive therapeutic response" is intended to mean an improvement in the disease associated with the activity of these binding molecules, such as an antibody or an antigen-binding fragment, variant, or derivative thereof, and / or an improvement in the symptoms associated with the disease. That is, a decrease in interferon-alpha levels, a decrease in the number or activity of plasmacytoid dendritic cells, or a decrease in one or more symptoms associated with the disease can be observed. Thus, for example, improvement of the disease can be characterized as a complete remission. "Complete remission" is intended to mean the absence of a clinically detectable disease with normalization of any previous test results. Such a response must persist for at least one month after treatment according to the methods of the present invention. Alternatively, improvement of the disease can be classified as a partial remission.

[0201] Anti-ILT7 binding molecules, such as the antibodies described in the present invention, or antigen-binding fragments, variants, or derivatives thereof, may also be useful in treating autoimmune diseases and deficiencies or disorders of the immune system associated with ILT7-expressing cells. Autoimmune diseases are characterized by damage to cells, tissues, and / or organs caused by the subject's immune response against its own cells, tissues, and / or organs. In one embodiment, the autoimmune disease is systemic lupus erythematosus.

[0202] Clinical response can be evaluated using screening techniques such as blood chemistry, including but not limited to changes detectable by magnetic resonance imaging (MRI) scan, X-ray imaging, computed tomography (CT) scan, flow cytometry or fluorescence-activated cell sorting (FACS) analysis, histology, gross findings, and ELISA, RIA, chromatography, etc. In addition to these positive treatment responses, subjects being treated with an anti-ILT7 binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof, can experience a beneficial effect of improvement of symptoms associated with the disease.

[0203] A further embodiment of the invention is the use of an anti-ILT7 binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof, for diagnostic monitoring of protein levels in a tissue as part of a clinical assay procedure, for example to determine the effectiveness of a given treatment regimen. For example, detection can be facilitated by coupling an antibody to a detectable substance. Examples of detectable substances include various enzymes, avidin family molecules, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase, examples of suitable avidin family complexes include streptavidin / biotin and avidin / biotin, examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin, an example of a luminescent material includes luminol, examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 H.

[0204] VIII. Pharmaceutical Compositions and Methods of Administration Methods for preparing an anti-ILT7 binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof provided herein, and administering it to a subject in need thereof are well known to those of skill in the art and can be readily determined.

[0205] As discussed herein, an anti-ILT7 binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof provided herein, can be administered in a pharmaceutically effective amount for the in vivo treatment of ILT7-expressing cell-mediated diseases, such as certain types of autoimmune diseases. In this regard, it will be appreciated that the binding molecules of the disclosure of the present invention are formulated to facilitate administration of the active substance and promote stability. The pharmaceutical compositions according to the present invention may contain a pharmaceutically acceptable, non-toxic, sterile carrier. For the purposes of the present application, an anti-ILT7 binding molecule, such as an antibody or antigen-binding fragment, variant, or derivative thereof, conjugated or unconjugated, is considered to mean an amount sufficient to achieve effective binding to the target and to achieve a benefit, such as to improve the symptoms of a disease or condition, or to detect a substance or cell.

[0206] A pharmaceutical composition suitable for injection must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents. Suitable formulations for use in the treatment methods disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).

[0207] Consistent with the scope of the present disclosure, the anti-ILT7 antibody of the invention, or an antigen-binding fragment, variant, or derivative thereof, can be administered to a human or other animal according to the above-described treatment methods in an amount sufficient to produce a therapeutic effect. The anti-ILT7 antibody of the invention, or an antigen-binding fragment, variant, or derivative thereof, can be administered to such a human or other animal in a conventional dosage form prepared by mixing the antibody or an antigen-binding fragment, variant, or derivative of the invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by those skilled in the art that the shape and characteristics of the pharmaceutically acceptable carrier or diluent will depend on the amount of the active ingredient with which it is mixed, the route of administration, and other well-known variables. Those skilled in the art will further recognize that a cocktail comprising one or more species of anti-ILT7 binding molecules, such as an antibody or an antigen-binding fragment, variant, or derivative of the invention, can be shown to be particularly effective.

[0208] "Therapeutically effective dose or amount" or "effective amount" is intended to mean an amount of an anti-ILT7 binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof, that produces a positive therapeutic response with respect to the treatment of a patient having a disease or condition being treated when administered.

[0209] For example, the therapeutically effective amount of the composition of the invention for the treatment of ILT7-expressing cell-mediated diseases, such as certain types of autoimmune diseases including systemic lupus erythematosus, will vary depending on many different factors including the means of administration, the target site, the physiological state of the patient, whether the patient is human or an animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Usually, the patient is human, but non-human mammals including transgenic mammals can also be treated similarly. The dosage of the treatment can be titrated to optimize safety and efficacy.

[0210] The invention also provides the use of an anti-ILT7 binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof, in the manufacture of a medicament for treating an autoimmune disease, such as systemic lupus erythematosus.

[0211] IX. Diagnosis The present invention further provides a diagnostic method useful in the diagnosis of ILT7-expressing cell-mediated diseases such as certain types of autoimmune diseases including, for example, systemic lupus erythematosus. The method involves measuring the expression level of ILT7 protein or transcript in a tissue or other cells or body fluid of an individual, and comparing the measured expression level with the standard ILT7 expression level in normal tissue or body fluid, whereby an increase in the expression level compared to the standard indicates a disorder.

[0212] Using the anti-ILT7 antibodies and antigen-binding fragments, variants, and derivatives of the present invention, the ILT7 protein level in a biological sample can be assayed using classical immunohistochemical methods known to those skilled in the art (see, for example, Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985), Jalkanen et al., J. Cell Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting ILT7 protein expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable assays are described in more detail elsewhere herein.

[0213] "Assaying the expression level of an ILT7 polypeptide" is intended to measure or estimate qualitatively or quantitatively the level of an ILT7 polypeptide in a first biological sample, either directly (e.g., by determining or estimating the absolute protein level) or relatively (e.g., by comparing the disease-related polypeptide level in a second biological sample). The expression level of the ILT7 polypeptide in the first biological sample can be measured or estimated and compared to a standard ILT7 polypeptide level, where the standard is taken from a second biological sample obtained from an individual without the disorder or determined by averaging levels from a population of individuals without the disorder. As is recognized in the art, when the "standard" ILT7 polypeptide level is known, it can be repeatedly used as a standard for comparison.

[0214] "Biological sample" is intended to mean any biological sample obtained from an individual, cell line, tissue culture, or other cell source that potentially expresses ILT7. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

[0215] X. Immunassay An anti-ILT7 binding molecule, such as an antibody of the invention or an antigen-binding fragment, variant, or derivative thereof, can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include, for example, Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescence immunoassay, protein A immunoassay, and competitive and non-competitive assays using such techniques, but are not limited thereto. Such assays are conventional and well known in the art (see, for example, Ausubel et al., eds, (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1, which is hereby incorporated by reference in its entirety). Exemplary immunoassays are briefly described below (but are not intended to be limiting).

[0216] The anti-ILT7 antibody of the invention, or an antigen-binding fragment, variant, or derivative thereof, can further be used histologically in immunofluorescence, immunoelectron microscopy, or non-immunological assays for the detection of ILT7 protein or conserved variants or peptide fragments thereof in situ. In situ detection can be achieved by obtaining a histological specimen from a patient and applying thereto a labeled anti-ILT7 antibody, or an antigen-binding fragment, variant, or derivative thereof, which is applied, for example, by overlaying the labeled antibody (or fragment) onto the biological sample. Through the use of such procedures, it is possible to determine not only the presence of the ILT7 protein, or conserved variant or peptide fragment thereof, but also its distribution in the tissue being tested. Using the present invention, one of ordinary skill in the art will readily recognize that a wide variety of histological methods (such as staining procedures) can be modified to achieve such in situ detection.

[0217] Immunoassays and non-immunoassays of the ILT7 gene product or its conserved variants or peptide fragments typically incubate a sample, such as a biological fluid, tissue extract, freshly harvested cells, or cell culture, in the presence of a detectable-labeled antibody that can bind to ILT7, or its conserved variant or peptide fragment, such as a lysate of cells incubated in the presence of the antibody, and detect the bound antibody by any of a number of techniques well known in the art.

[0218] Biological samples can be immobilized by contacting them with a solid support or carrier such as nitrocellulose, or other solid supports capable of immobilizing cells, cell particles, or soluble proteins. The support can then be washed with a suitable buffer after treatment with a detectable-labeled anti-ILT7 antibody, or an antigen-binding fragment, variant, or derivative thereof. The solid support can then be washed twice with buffer to remove unbound antibody. Optionally, the antibody is then labeled. The amount of bound label on the solid support can be detected by conventional means.

[0219] The term "solid support or carrier" is intended to mean any support to which an antigen or antibody can bind. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamide, pumice, and magnetite. The nature of the carrier can be somewhat soluble or insoluble with respect to the purposes of the present invention. The support material can have substantially any possible structural configuration as long as the coupled molecules can bind to an antigen or antibody. For this reason, the shape of the support can be spherical, beads, cylindrical, the inner surface of a test tube, or the outer surface of a table. Alternatively, the surface can be flat such as a sheet, test piece, etc. An exemplary support includes polystyrene beads. Those skilled in the art are aware of many other suitable carriers for binding antibodies or antigens or will be able to identify them by using conventional experiments.

[0220] The binding activity of a given lot of anti-ILT7 antibody or antigen-binding fragment, variant, or derivative thereof can be determined according to well-known methods. Those skilled in the art will be able to determine the operating conditions and optimal assay conditions for each determination by using conventional experiments.

[0221] The binding affinity of an antibody for an antigen and the off-rate of the antibody-antigen interaction can be determined by a competitive binding assay. An example of a competitive binding assay is a radioimmunoassay that involves incubating a labeled antigen (e.g., 3 H or 125 I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen and detecting the antibody bound to the labeled antigen. The affinity and off-rate of binding of the antibody of interest for a particular antigen can be determined from the data by Scatchard plot analysis. Competition with a secondary antibody can also be determined using a radioimmunoassay. In this case, the antigen is a labeled compound (e.g., 3 H or 125Incubate with the antibody of interest conjugated to (I).

[0222] There are various methods available for measuring the affinity of antibody-antigen interactions, but relatively few methods for determining the rate constants. Most of the methods rely on either a labeled antibody or antigen, which inevitably makes the conventional measurements complex and introduces uncertainty into the measured quantities.

[0223] Surface plasmon resonance (SPR) performed on a BIACORE® instrument offers a number of advantages compared to conventional methods for measuring the affinity of antibody-antigen interactions, namely: (i) no need to label either the antibody or the antigen; (ii) no need to pre-purify the antibody and the ability to use cell culture supernatants directly; (iii) real-time measurements enabling rapid semi-quantitative comparison of different monoclonal antibody interactions, which are sufficient for many evaluation purposes; (iv) the ability to regenerate the biospecific surface so that a series of different monoclonal antibodies can be easily compared under the same conditions; (v) the analytical procedure is fully automated and a wide range of measurements can be carried out without user intervention. BIAapplications Handbook, version AB (reprinted 1998), BIACORE® code number BR-1001-86; BIAtechnology Handbook, version AB (reprinted 1998), BIACORE® code number BR-1001-84. Binding assays based on SPR require one member of the binding pair to be immobilized on the sensor surface. The immobilized binding partner is called the ligand. The binding partner in solution is called the analyte. In some examples, the ligand binds indirectly to the surface through its binding to another immobilized molecule called a capture molecule. The SPR response reflects the change in mass concentration on the detector surface when the analyte binds or dissociates.

[0224] Based on SPR, real-time BIACORE® measurements monitor interactions directly as they occur. The technique is well-suited for determining kinetic parameters. It is easy to perform comparable affinity rankings, and both kinetic and affinity constants can be derived from the sensorgram data.

[0225] When the sample is injected in individual pulses across the ligand surface, the resulting sensorgram can be classified into three essential phases: (i) association of the sample with the ligand upon injection, (ii) equilibrium or steady state upon injection if the binding rate of the sample is in equilibrium with dissociation from the complex, and (iii) dissociation of the sample from the surface during buffer flow.

[0226] The association and dissociation phases provide information on the kinetics of the analyte-ligand interaction (k a and k d , the formation and dissociation rates of the complex, k d / k a =K D ). The equilibrium phase provides information on the affinity (K D ) of the analyte-ligand interaction.

[0227] BIA evaluation software provides comprehensive convenience for curve fitting using both numerical integration and global fitting algorithms. Appropriate analysis of the data allows individual kinetic and affinity constants for the interaction to be obtained from a simple BIACORE® test. The range of affinities measurable by this technique is very wide, in the mM to pM range.

[0228] Epitope specificity is an important feature of monoclonal antibodies. Epitope mapping by BIACORE® does not require labeling or purification of the antibody, in contrast to conventional techniques using radioimmunoassay, ELISA, or other surface adsorption methods, and allows multisite specificity testing using the sequences of some monoclonal antibodies. Additionally, a large number of samples can be processed automatically.

[0229] The pairwise binding experiment tests the ability of two Mabs to bind to the same antigen simultaneously. Mabs against individual epitopes bind independently, while Mabs against identical or related epitopes interfere with each other's binding. These binding experiments by BIACORE® are easily carried out.

[0230] For example, after binding the first Mab using a capture molecule, the antigen and the second Mab can be added sequentially. The sensorgram reveals (1) how much antigen binds to the first Mab, (2) to what extent the second Mab binds to the surface-bound antigen, and (3) whether the results change if the order of the pairwise test is reversed if the second Mab does not bind.

[0231] Peptide inhibition is another technique used for epitope mapping. This method complements the pairwise antibody binding test and can relate functional epitopes to structural features when the primary sequence of the antigen is known. Peptides or antigen fragments are tested for their ability to inhibit the binding of different Mabs to a fixed antigen. A peptide that interferes with the binding of a given Mab is assumed to be structurally related to the epitope defined by that Mab.

[0232] The practice of the present invention, unless otherwise indicated, uses conventional techniques in cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the skill of the art. Such techniques are well described in the literature. For example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press), Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY), D. N. Glover ed., (1985) DNA Cloning, Volumes I and II, Gait, ed. (1984) Oligonucleotide Synthesis, Mullis et al. U.S. Pat. No. 4,683,195, Hames and Higgins, eds. (1984) Nucleic Acid Hybridization, Hames and Higgins, eds. (1984) Transcription And Translation, Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.), Immobilized Cells And Enzymes (IRL Press) (1986), Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.), Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory), Wu et al., eds., Methods In Enzymology, Vols.See 154 and 155, Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London), Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986), and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0233] The general principles of antibody engineering are described in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). The general principles of protein engineering are described in Rickwood et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). General principles regarding antibodies and antibody-hapten binding are described in Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer Associates, Sunderland, Mass.), and Steward (1984) Antibodies, Their Structure and Function (Chapman and Hall, New York, N.Y.). In addition, standard methods of immunology that are known in the art and not specifically described are generally followed as described in Current Protocols in Immunology, John Wiley & Sons, New York, Stites et al., eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods in Cellular Immunology (W.H. Freeman and Co., NY).

[0234] Standard reference studies that describe the general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York, Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY), Kennett et al., eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses (Plenum Press, NY), Campbell (1984) "Monoclonal Antibody Technology" in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden et al., (Elsevere, Amsterdam), Goldsby et al., eds. (2000) Kuby Immunology (4th ed.; H. Freemand & Co.), Roitt et al. (2001) Immunology (6th ed.; London: Mosby), Abbas et al. (2005) Cellular and Molecular Immunology (5th ed.; Elsevier Health Sciences Division), Kontermann and Dubel (2001) Antibody Engineering (Springer Verlan), Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press), Lewin (2003) Genes VIII (Prentice Hall 2003), Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press), Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

[0235] All of the references cited above, together with all references cited in this specification, are hereby incorporated by reference in their entirety into this specification.

[0236] The following examples are provided for illustration and not for limitation.

Examples

[0237] Materials and Methods Biological Samples Human peripheral blood from healthy and fit volunteers was obtained through the MedImmune blood donation program, with written informed consent and IRB approval. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood using Vacutainer CPT tubes (Becton Dickinson Biosciences, NJ, USA) supplemented with sodium citrate. The tubes were centrifuged at 17000 g for 25 minutes at 22 °C with minimal braking. After centrifugation, the serum was removed and the cell buffy coat was transferred to a 50 mL conical tube (BD Biosciences). The purified cells were washed twice with sterile phosphate-buffered saline (PBS) (Invitrogen Life Technologies, CA, USA) at 350 g for 10 minutes at 22 °C. The cells were resuspended in RPMI 1640 medium supplemented with PBS or 10% fetal bovine serum (Invitrogen) and filtered using a BD Falcon 5 mL tube equipped with a cell strainer cap (BD Biosciences). The cell density was determined using a Vi-Cell XR® cell counter (Beckman Coulter, CA, USA).

[0238] Cynomolgus monkey peripheral blood from healthy animals was obtained from Bioqual (Bioqual, Inc. MD, USA) in accordance with the National Institutes of Health guidelines for the care and use of primates. Cynomolgus monkey PBMCs were isolated using Vacutainer CPT cell preparation tubes containing sodium citrate (as described above) or by Histopaque 10771 (Sigma-Aldrich, MO, USA). Briefly, fresh whole blood was adjusted to 50 times the original blood volume with sterile PBS. Next, 25 mL of diluted blood was layered on top of 10 mL of 90% Histopaque 10771 (Sigma Aldrich), and the samples were centrifuged at 400 g for 20 minutes at room temperature with minimal braking. The buffy coat cells were harvested and transferred to a new 50 mL conical tube. The purified cells were washed twice with sterile PBS at 350 g for 10 minutes at 22 °C. The cells were resuspended in PBS or RPMI 1640 medium supplemented with 10% fetal bovine serum, filtered, and counted as described above.

[0239] Cells CT-125 and CT-550 cells were obtained from Dr. Yong-Jun Liu (University of Texas M.D. Anderson Cancer Center, Houston, TX, USA). CT-125 cells were generated by transducing the 2B4 mouse T cell hybridoma with the untagged mouse FcεR1γ and NFAT-GFP reporter genes, and CT-550 cells were generated by transducing CT-125 cells with the HA-tagged human ILT7 (Ohtsuka M. et al., PNAS 101: 8126-8131 (2004), Cao W. et al., JEM 203: pp 1399-1405 (2006)). The CT-125 Cyno ILT7 stable cell line was generated by transfecting CT-125 cells with the cynomolgus ILT7 gene cloned into the pME18X plasmid vector. CT cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1X penicillin / streptomycin (both from Invitrogen Life Technologies).

[0240] KC1333 cells were obtained from Biowa (Biowa, NJ, USA). KC1333 cells were cultured in Advance RPMI 1640 supplemented with 10% FBS, 4 mM L-glutamine, 0.2 μg / mL geneticin (all from Invitrogen), and 18.3 pg / mL recombinant human IL-2 (PeproTech, NJ, USA).

[0241] Antibodies and Reagents The anti-ILT7 humanized antibody variant, anti-ILT7 clone 7C7 (7C7), and humanized isotype control R347 were produced at MedImmune. The allophycocyanin (APC)-conjugated anti-ILT7 humanized antibody variant, 7C7, and isotype control R347 were produced using an APC monoclonal antibody labeling kit (Thermo Fisher Scientific, IL, USA). The R-phycoerythrin (PE) and FITC-labeled anti-human BDCA-2 antibody (clone AC144), R-PE anti-human BDCA-4 (clone AD5-17F6), and human FcR blocking reagent were obtained from Miltenyi Biotech, CA, USA. The anti-human CD123 (clone 7G3), Alexa Fluor 488 anti-human CD8 (clone RPA-T8), Alexa Fluor 488 anti-human CD3 (clone SP34-2), FITC anti-human CD14 (clone M5E2), FITC anti-human CD20 (clone 2H7), and PerCP-Cy 5.5 anti-human HLA-DR (clone G46-6) conjugated to either R-PE, FITC, or APC were obtained from BD Biosciences. The Pacific Blue anti-human CD56 antibody (clone MEM-188) was obtained from BioLegend, CA, USA. The DyLight 649-labeled anti-human IgG and human total IgG were obtained from Jackson Immunoresearch, PA, USA.

[0242] Staining of whole blood was performed using BD FACS lysing solution (BD Biosciences). 7-AAD was obtained from Invitrogen. AB-type human male plasma was obtained from Sigma-Aldrich. Recombinant human IL-2 was obtained from R&D Systems, MN, USA, and recombinant human interferon β (IFN-β) was obtained from PBL Biomedical, NJ, USA. CpG A ODN 2216 was obtained from InvivoGen, CA, USA.

[0243] Labeling of Human and Cynomolgus Recombinant ILT7 The protein was biotinylated via free amines using EZ-Link Sulfo-NHS-LC-Biotin (Thermo / Pierce, product: 21335). The reagent was dissolved in anhydrous dimethylformamide and the PBS-based protein solution was adjusted to approximately pH 8 with 1 M NaHCO3 in D-PBS.

[0244] The incorporation of the label was evaluated in all cases by MALDI-TOF mass spectrometry and unreacted reagent was removed by buffer exchange using disposable Sephadex G25 columns equilibrated with D-PBS. For biotinylation, the final protein concentration was determined by absorbance at 280 nm using the extinction coefficient calculated from the amino acid sequence.

[0245] ELISA binding assay Single-chain Fv fragments were displayed on phage particles and tested in binding assays to determine cross-reactivity and specificity against a panel of recombinant antigens. Phage display scFv supernatant samples were prepared in 96-well deep well plates as follows. 5 μl of culture from each well of a 96-well master plate was transferred to a Greiner deep well culture plate containing 500 μl of 2TYAG (2TY + 100 μg / ml ampicillin + 2% glucose) medium and incubated at 37 °C, 280 rpm for 5 h. K07 M13 helper phage (diluted to 1.5×10 11 pfu / ml in 2TYAG) was added at 100 μl / well and the plate was incubated at 37 °C, 150 rpm for infection. The plate was centrifuged at 3200 rpm for 10 min to remove the supernatant. The bacterial pellet was resuspended in 500 μl / well of 2TYAK (2TY + 100 μg / ml ampicillin + 50 μg / ml kanamycin) and the plate was incubated overnight at 25 °C, 280 rpm. In the morning, 500 μl of 6% (w / v) skim milk powder in 2×PBS was added to each well and the plate was incubated at room temperature for 1 h. The plate was then centrifuged at 3200 rpm for 10 min and the blocked phage display scFv supernatant was used directly in ELISA experiments.

[0246] For EC50 determination, typically purified IgG was diluted 3-fold with PBS containing 3% (w / v) dried milk powder (PBS-M) to obtain 11 concentration points. A 96-well Greiner polypropylene plate (Greiner, 650201) was used for dilution preparation. Generally, each dilution was prepared in duplicate. After blocking the IgG diluent in PBS-M at room temperature for 1 hour, it was used directly in the ELISA experiment.

[0247] The IL-T7 binding assay was an essentially plate-based ELISA performed as follows. Not all antigens were necessarily used in every experiment, but typically human, mouse, and cynomolgus monkey IL-T7 antigens were tested. The relevant control antigens (bovine insulin + IL-4RαFLAG®His, if necessary) were also used to test for non-specific binding. With the exception of bovine insulin, all antigens were biotinylated and all were produced using bacterial expression. The IL-T7 antigen was biotinylated via free sulfhydryl groups using EZ-Link Biotin-BMCC (Perbio / Pierce 21900). The method for producing IL-4RαFLAG®His used as a control antigen is described in International Publication No. WO 2010 / 070346. IL-4RαFLAG®His was biotinylated via free amines using EZ-Link Sulfo-NHS-LC-Biotin (Perbio / Pierce, 21335).

[0248] A streptavidin plate (Thermo Scientific, AB-1226) was coated with 0.5 μg / ml biotinylated antigen in PBS and incubated overnight at 4°C. The plate was washed three times with PBS and blocked for 1 hour with 300 μl / well of block buffer (PBS-M). The plate was washed once with PBS, and the blocked sample was added at 50 μl / well for 1 hour at room temperature. The plate was washed three times with PBS-T (PBS + 1% (v / v) Tween-20), and a 1:5000 dilution of the detection reagent [anti-human IgG HRP (Sigma, A0170) or anti-M13-HRP antibody (Amersham, 27-9421-01) for detecting IgG or phage display scFv, respectively] was added at 50 μl / well in PBS-M for 1 hour at room temperature. The plate was washed three times with PBS-T and developed with TMB, 50 μl / well (Sigma, T0440). After quenching the reaction with 50 μl / well of 0.1 M H2SO4, it was read at 450 nm in an EnVision™ plate reader or similar instrument.

[0249] The dose-response curve was plotted for IgG titration using Prism (Graphpad) curve-fitting software. Phage display scFv was considered to bind to the IL-T7 antigen if the absorbance at 450 nm was >0.5 and <0.1 - 0.2 for the same sample in the controls (insulin and IL-4RαFlag®His). Single-chain Fv fragments were presented on phage particles and tested as unpurified preparations in a single point ELISA screen.

[0250] Fluorescent microassay technology (FMAT) cell binding assay This homogeneous assay evaluated the binding of crude scFv supernatant samples or purified IgG to Chinese Hamster Ovary (CHO) cells expressing either human or cynomolgus ILT7 in a 384-well (Costar 3655) format. Binding of scFv or Ab to the cells was detected using either a mouse anti-His / goat anti-mouse Alexafluor®-647 labeled antibody (Molecular Probes A21236) mix or a goat anti-human Alexafluor®-647 labeled antibody (Molecular Probes A21445), respectively. Plates were read on an Applied Biosystems Cell Detection System 8200 reader. A helium neon excitation laser was focused within 100 μm of the depth of the well bottom over an area 1 mm 2 and scanned. When the cells had settled to the bottom of the well and the laser was excited at 633 nm, the beads conjugated with the phosphor (local concentration of the phosphor is relatively high compared to the unbound phosphor) emitted a signal at 650 - 685 nm, which was measured using photomultiplier tube - 1 (PMT1). The unbound phosphor in the solution was either outside the depth of excitation or at a relatively low local concentration and thus did not emit a significant signal. The presence of scFv or IgG samples bound to the cells at the bottom of the well caused an increase in the Alexafluor-labeled detection antibody within the depth of excitation. This was measured as an increase in fluorescence.

[0251] In these experiments, the assay buffer was PBS (Gibco 14190-094) containing 0.1% BSA (Sigma A9576 - 50 ml), 0.1% Tween-20 (Sigma P2287), and 0.01% sodium azide. To prepare the scFv detection mix, mouse anti-His and anti-mouse AF647 antibodies were mixed in assay buffer at 1 μg / ml and 2 μg / ml, respectively. To prepare the IgG detection mix, anti-human AF647 antibody was prepared in assay buffer at 2 μg / ml.

[0252] The cells used were CHO-K1 cells expressing either human or cynomolgus macaque ILT7 cultured using standard tissue culture techniques. The cells were grown to approximately 80% confluence in F-10 (Gibco, 22390-025) + 10% FCS (SAFC Biosciences, 13068C) + 0.5 mg / ml Zeocin (Invitrogen, R250-01), washed with PBS, detached with Accutase (PAA, L11-007), and resuspended in PBS at 1.5×10 5 cells / ml.

[0253] Crude scFv supernatant samples were prepared in 96-deep well plates. 5 μl of culture from each well of a 96-well master plate was transferred to a Greiner deep well culture plate containing 900 μl of 2TY (1.6% tryptone, 1% yeast extract, 0.5% NaCl, pH 7.0) + 100 μg / ml ampicillin + 0.1% glucose medium and incubated at 37°C and 280 rpm for 5 hours. 100 μl / well of a TY solution of 10 mM IPTG was added and the block solution was incubated overnight at 280 rpm and 30°C. In the morning, the block solution was centrifuged at 3200 rpm for 15 minutes. For high-throughput screening, the scFv supernatant from the deep well block was transferred directly to the assay plate for the required 20% dilution.

[0254] To the test wells of a 384-well clear-bottom non-binding surface black Costar plate, the following were added: 10 μl of sample (IgG or scFv), 10 μl of detection antibody or antibody mix, and 30 μl of cells. The negative controls used in these experiments typically involved the addition of an isotype (IgG) or irrelevant (ScFv) control, or assay buffer, instead of the experimental sample. The plate was sealed and incubated for 4 hours in the dark at room temperature and read on an Applied Biosystems Cell Detection System 8200 reader. Data were typically analyzed by the Velocity algorithm, and gates were set with a color ratio <0.4, size 15 - 30, and minimum count 20. Hits for crude scFv supernatant samples were defined as showing at least 50% inhibition of signal compared to total binding control wells. Dose-response curves were plotted for purified IgG titrations using Prism (Graphpad) curve-fitting software.

[0255] IC 50 For determinations, typically purified IgG was serially diluted 2-fold from 500 nM in assay buffer to obtain 11 concentration points. A 96-well Greiner polypropylene (Greiner, 650201) plate was used for dilution preparation. Generally, each dilution was prepared in duplicate. Alternatively, IgG tests were performed at a single concentration obtained in the range of 500 nM to 0.2 nM.

[0256] Evaluation of Antibody Binding in Cell Lines by Flow Cytometry The binding of anti-ILT7 variants and isotype controls to human and cynomolgus monkey ILT7 was evaluated by flow cytometry analysis using CT-550 and cynoILT7 CT125 cells, respectively. CT-125 cells were used as controls. Cells were resuspended in blocking buffer (PBS supplemented with 10% FBS) at a concentration of 5 million cells / mL and transferred to a round-bottom 96-well plate (BD FalconTM Clear Microtest Plate, BD Biosciences) at 100 μl / well. Anti-ILT7 variants and control antibodies were added to the cells at 4 °C for 30 minutes on a plate shaker. Cells were washed three times with PBS and resuspended in blocking buffer (100 μl / well). Human IgG binding on the cell surface was detected using a secondary anti-human IgG antibody conjugated to DyLight649 (1:1000 dilution). Cells were incubated on a plate shaker in the dark at 4 °C for 30 minutes. Cells were washed three times with PBS, and surface fluorescence was acquired using an LSRII flow cytometry system and FACSDiva software (both obtained from BD Biosciences).

[0257] Evaluation of antibody binding in whole blood and PBMCs by flow cytometry The binding of APC-labeled anti-ILT7 antibody and isotype control in human and cynomolgus monkey whole blood was evaluated by flow cytometry analysis. Whole blood was transferred to 50 mL conical tubes at 1 mL / tube. The APC-labeled antibody was added directly to the whole blood. Anti-BDCA-2-PE and anti-CD123-PE antibodies were used as plasmacytoid dendritic cell (pDC)-specific markers in human and cynomolgus monkey whole blood staining, respectively. Whole blood was incubated with the antibody at 4 °C in the dark for 30 minutes on a plate shaker. Blood was treated with BD FACS lysing solution according to the manufacturer's instructions. Cells were washed, and antibody binding was evaluated by flow cytometry using an LSRII flow cytometry system and FACSDiva software.

[0258] For PBMC staining, PBMC were first washed with PBS and then resuspended in PBS-based cold blocking buffer containing 50% AB-type human male plasma, 20 μg / mL human IgG, and 200 μg / mL human FcR blocking reagent at 4°C for 15 minutes on a plate shaker. After 15 minutes, APC-labeled anti-ILT7 variant or APC-labeled isotype control antibody was added directly to the blocking solution. Alternatively, anti-BDCA-2-PE and anti-BDCA-4-PE antibodies were used as pDC-specific markers for human PBMC staining. In cynomolgus monkey PBMC, pDCs were defined as HLA-DR + , Lineage - , CD11c - , and CD123 high (Malleret et al., Immunology 124: 223-233 (2008)). Therefore, anti-HLA-DR PerCP-Cy5.5, Lineage-FITC (CD3, CD8, CD20, and CD14 antibodies), and anti-CD123-PE antibodies were used as pDC-specific markers for cynomolgus monkey PBMC staining. PBMC were incubated on a plate shaker at 4°C in the dark for 30 minutes. The cells were washed and antibody binding was evaluated by flow cytometry using an LSRII flow cytometry system and FACSDiva software.

[0259] Evaluation of antibody efficacy by antibody-dependent cell-mediated cytotoxicity (ADCC) assay using cell lines The efficacy of the anti-ILT7 antibody was determined using an in vitro cell-based ADCC assay. KC1333 cells (effector) and CT cells (target) were used at a ratio of 5:1 (2.5×10 5 KC1333 cells to 0.5×10 5They were co-cultured in a round-bottom 96-well plate (in number of cells). The cells were co-cultured for 16 hours at 37 °C and 5% CO2 in RPMI 1640 medium supplemented with 10% FBS in the presence of anti-ILT7 antibody or isotype control. Next, the cells were washed and transferred to block buffer (PBS-10% FBS). KC1333 cells were detected using Pacific-Blue-anti-CD56 antibody. Dead cells were detected using 7-AAD. The viability of the target cells was evaluated by flow cytometry using an LSRII flow cytometry system and FACSDiva software. The percentage of cytotoxicity was obtained by applying the following formula: percentage of cytotoxicity = 100 - (number of surviving targets / number of surviving targets at baseline) × 100.

[0260] Evaluation of antibody efficacy by ADCC assay using human PBMC Human PBMC were washed with PBS and resuspended in RPMI medium supplemented with 10% FBS and 200 ng / mL recombinant human IL-2 at 5.0×10 6They were resuspended at a concentration of cells / mL. PBMCs were seeded at 100 μL / well in a round-bottom 96-well plate in two repeated experiments. Serial 10-fold dilutions of the anti-ILT7 antibody and a control antibody were prepared, and 100 μL of the antibody solution was added to the appropriate wells to a final concentration of 33.85 nM to 3.385 fM. The cells were incubated at 37 °C and 5% CO2 for 6 hours. After incubation, the cells were washed twice with 250 μL of cold PBS. The cells were resuspended in 100 μL of cold blocking buffer based on PBS containing 50% AB-type human male plasma, 20 μg / mL human IgG, and 200 μg / mL human FCR blocking reagent at 4 °C for 15 minutes. After the blocking step, 100 μL of cold blocking buffer containing FITC-anti-human BDCA2 and APC-anti-human CD123 antibodies was added to the appropriate wells. The plate was incubated at 4 °C with gentle shaking for 30 minutes. After incubation, the cells were washed twice with 250 μL of cold PBS and finally resuspended in 200 μL of cold PBS. 50 μL of cold 7-AAD (Invitrogen) solution was added to all wells, and 7-AAD-positive plasmacytoid dendritic cells were evaluated using an LSRII flow cytometry system and FACSDiva software.

[0261] IFNα Secretion Assay with Human PBMC Human PBMCs were washed with PBS and seeded in a round-bottom 96-well plate at a final density of 150,000 - 156,000 cells / well in RPMI medium supplemented with 10% FBS and 200 ng / mL recombinant human IL-2 in two replicate experiments. Serial 10-fold dilutions of the anti-ILT7 antibody and a control antibody were prepared, and 100 μL of the antibody solution was added to appropriate wells at a final concentration of 6.77 nM - 0.677 fM. The cells and antibodies were incubated at 37 °C, 5% CO2 for 9.5 - 10 hours. After incubation, 50 μL of ODN2216 (Invitrogen™) was added to appropriate wells at a final concentration of 0.5 μM, and the plate was incubated at 37 °C, 5% CO2 for an additional 16 hours. After incubation, the plate was centrifuged at 350 g for 10 minutes, and the supernatant was carefully collected. IFNα was quantified using a multi-subtype IFNα ELISA kit (PBL Biomedical).

[0262] IFNα Secretion Assay with Cynomolgus Monkey PBMCs Cynomolgus monkey PBMCs were washed with PBS and suspended in RPMI 1640 medium supplemented with 10% FBS, 220 ng / mL recombinant human IL-2, and 500 IU / mL recombinant human IFN-β. The maximum number of cells was added to appropriate wells at a density in the range of 314,000 - 818,000 cells / well. Serial 10-fold dilutions of the anti-ILT7 antibody and a control antibody were prepared, and 100 μL of the antibody solution was added to appropriate wells at a final concentration of 33.85 nM - 3.385 fM. The cells and antibodies were incubated at 37 °C, 5% CO2 for 9.5 - 10 hours. After incubation, 50 μL of ODN2216 (Invitrogen™) was added to appropriate wells at a final concentration of 0.5 μM, and the plate was incubated at 37 °C, 5% CO2 for an additional 16 hours. After incubation, the plate was centrifuged at 350 g for 10 minutes, and the supernatant was carefully collected. IFNα in the supernatant was quantified using a cynomolgus monkey / rhesus monkey IFNα ELISA kit (PBL Biomedical).

[0263] Statistical Analysis EC 50 and IC50 Binding curves, ADCC, and cytokine secretion assays were generated using GraphPad Prims 5 software (GraphPad Software, CA, USA).

[0264] [Example 1] Generation of a humanized ILT7 antibody from the mouse antibody SB128 Mouse mAb SBI28 (SBI28 refers to anti-ILT7 antibody ILT7#28 provided in US Patent Application Publication No. 2009 / 0280128) was humanized by framework shuffling (Dall'Acqua et al., Methods 36:43-60 (2005)). Using this method, mouse mAb SBI28 was humanized by synthesizing a combinatorial library consisting of its 6 CDRs that were in-frame fused to a pool of individual human germline frameworks. The human framework genes were selected from a pool of published antibody germline genes. These degenerate framework primer pools included 46 human germline kappa chain genes, 5 human germline Jk sequences, 44 human germline heavy chain genes, and 6 human germline JH sequences. The primer bank was designed to encode the respective frameworks of each germline gene. Antibody-specific CDR primers with degenerate ends overlapping the framework pools were also synthesized. The SBI28 framework shuffle library was constructed by pairing a variable heavy chain framework shuffle sublibrary with a variable light chain framework shuffle sublibrary. The framework shuffle sublibraries were assembled sequentially by PCR using overlap extension. A first fusion PCR was performed to synthesize each individual human germline framework fused in-frame with a portion of the corresponding CDR. Next, a second "assembly PCR" was performed using the fusion PCR products as templates to amplify full-length VH and VL sublibraries. The SBI28 framework shuffle library was cloned into an M13-based Fab expression vector using Kunkel method hybridization mutagenesis. Approximately 1300 clones from the SBI28 framework shuffle library were screened using a MesoScale Discovery (MSD) assay against CHO cells expressing recombinant ILT7 CHO cells.One humanized variant 10D10 bound to human ILT7 with 3-fold lower affinity compared to its chimeric parent (“SBI28ch”) as measured by surface plasmon resonance (SPR) on a ProteOn. SBI28ch refers to anti-ILT7 antibody ILT7#28 provided in US Patent Application Publication No. 2009 / 0280128, which is hereby incorporated by reference in its entirety.

[0265] The affinity optimization of 10D10 was initiated to improve its binding affinity to human and cynomolgus ILT7. 10D10 was first cloned into an M13-based ScFv expression vector for savings method mutagenesis. In this method, each individual amino acid of all six CDRs was randomly mutated using two different libraries (NSS and NWS) per residue position. A total of 12 independent libraries were constructed for the six CDRs using Kunkel method hybridization mutagenesis (Kunkel, T. A., et al. Methods Enzymol. 154:367 (1987)). Screening of the synthesized libraries consisted of a one-point ELISA designed to capture the limiting concentration of secreted ScFv from bacterial culture media to normalize the scFv concentration in each well. The labeled ILT7 antigen bound to the captured ScFv and the signal intensity of this interaction correlated with the relative binding affinity. Approximately 2,000 - 3,000 clones were screened. To further engineer variants with improved affinity, a small focused combinatorial library was generated in which all beneficial single amino acid changes were co-encoded. In this step, a combinatorial scFv library was constructed in which 14 individual positive hits at 9 positions in the six CDRs were co-encoded. Briefly, degenerate primers were designed to encode all beneficial amino acid changes as well as the parental residues at the same positions. This combinatorial library was screened by one-point capture ELISA as previously described. Approximately 1,200 clones were screened. The variable region of the affinity-improved variant 7C7 was cloned into a mammalian expression pOE vector and transiently expressed in HEK293 cells. Secreted soluble human IgG was purified directly from the conditioned media. The purified IgG was assayed for binding to rILT7 using ProteOn and FACS. In the ProteOn experiment, the affinity-optimized antibody 7C7 had approximately 60-fold higher K DImprovements were shown. By FAC measuring the binding to recombinant human and cynomolgus ILT7 expressed on CHO cells, 7C7 showed 2.2-fold and 14-fold better EC50 values for human and cynomolgus ILT7, respectively, compared to SBI28ch. Alignments of the VH and VL sequences of SBI28, 10D10, and 7C7 are provided in FIGS. 1A and 1B, respectively.

[0266] [Example 2] Generation of Human ILT7 Antibodies from a Human Library In addition to humanizing the mouse anti-ILT7 antibody (as described above in Example 1), human antibodies were generated using a library of human sequences. By implementing a number of strategies for generating anti-ILT7 antibodies, the opportunity to generate anti-ILT7 antibodies with distinct properties is maximized, and an ideal antibody for a particular purpose can be selected.

[0267] 2.1 Selection A large single-chain Fv (scFv) human antibody library generated using individual heavy-chain variable regions and light-chain variable regions derived from the bone marrow of adult naive donors cloned into a phagemid vector based on filamentous phage M13 was used for selection (Hutchings, C., "Generation of Naive Human Antibody Libraries" in Antibody Engineering, Dubel. Berlin, Springer Laboratory Manuals: p. 93 (2001), Lloyd et al., Protein Eng. Des. Sel. 22(3):159-68 (2009)). ILT7-specific scFv antibodies were isolated from the phage display library essentially as previously described by Vaughan et al. (Nat. Biotechnol. 14(3):309-14 (1996)) in a series of repeated selection cycles for recombinant human and / or cynomolgus monkey ILT7. Briefly, scFv-phage particles were incubated with a biotinylated recombinant ILT7 solution (biotinylated via free amines using EZ-Link Sulfo-NHS-LC-Biotin (Thermo / Pierce, product number: 21335)). Typically, scFv-phage particles were incubated with 100 nM biotinylated recombinant ILT7 for 1 hour. ScFv bound to the antigen was captured on streptavidin-coated paramagnetic beads (Dynabeads® M-280) according to the manufacturer's recommendations. Unbound phage was washed in a series of wash cycles using PBS-Tween. Phage particles retained on the antigen were eluted, infected into bacteria, and rescued for the next round of selection. Typically, 3 rounds of selection were performed in this way.

[0268] 2.2 Identification of ILT7-Specific Conjugates by Phage ELISA The scFv was displayed on phage particles and tested in binding assays to determine cross-reactivity and specificity against recombinant antigens. The detailed assay methods are provided in the Materials and Methods section. Approximately 2100 individual data points were generated from the binding assays, and the identified hits, i.e., scFv clones showing binding to recombinant ILT7, were subjected to DNA sequencing (Osbourn et al., Immunotechnology 2(3):181-96 (1996), Vaughan et al., Nat. Biotechnol. 14(3):309-14 (1996)).

[0269] 2.3 Identification of ILT7 conjugates by FMAT Unique scFv was expressed in the bacterial periplasm and its binding activity was screened in a fluorescence microassay technology (FMAT) binding assay. Binding of the scFv expressed on the cell surface to ILT7 was detected using a goat anti-mouse Alexafluor®-647 labeled antibody. The detailed assay methods are provided in the Materials and Methods section.

[0270] 2.4 Reformatting of scFv to IgG1 The most potent scFv conjugate was converted to the full immunoglobulin G1 (IgG1) antibody format essentially as described in Persic et al (Gene 187(1):9-18 (1997)), with the following modifications. To facilitate use in CHO transient cells and to enable episomal replication, the OriP fragment was included in the expression vector. The VH domain was cloned in a vector (pEU1.3) containing the human heavy chain constant domain and regulatory elements to express the entire IgG1 heavy chain in mammalian cells. Similarly, the VL domain was cloned in a vector (pEU4.4) for expressing the human light chain (lambda) constant domain and regulatory elements to express the entire IgG light chain in mammalian cells. To obtain IgG, the heavy and light chain IgG expression vectors were transfected into CHO transient mammalian cells. IgG was expressed and secreted into the medium. The harvests were pooled, filtered, and then purified. Next, IgG was purified using protein A chromatography. The culture supernatant was loaded onto a ceramic protein A (BioSepra) column of appropriate size and washed with 50 mM Tris-HCl pH 8.0, 250 mM NaCl. The bound IgG was eluted from the column using 0.1 M sodium citrate (pH 3.0) and neutralized by adding Tris-HCl (pH 9.0). The eluted material was buffer-exchanged to PBS using a Nap10 column (Amersham, #17-0854-02), and the concentration of IgG was determined spectrophotometrically using the extinction coefficient based on the amino acid sequence of IgG (Mach et al., Anal. Biochem. 200(1):74-80 (1992)).

[0271] 2.5 Binding assay of IgG The species cross-reactivity of the anti-ILT7 antibodies was determined using a FMAT binding assay. The detailed assay method is provided in the Materials and Methods section. The following 11 antibodies were identified as antibodies that successfully bound to both human and cynomolgus monkey ILT7 in the FMAT screening assay: ILT70019, ILT70028, ILT70052, ILT70076, ILT70080, ILT70083, ILT70089, ILT70100, ILT70137, ILT70142, and ILT70144.

[0272] [Example 3] The ILT7 antibodies bind to ILT7-expressing cells To determine the binding EC 50 of ILT70019, ILT70028, ILT70052, ILT70076, ILT70080, ILT70083, ILT70089, ILT70100, ILT70137, ILT70142, and ILT70144 to cells expressing human ILT7, candidates were screened by flow cytometry for binding to CT-550 cells. ILT70080 (EC 50 = 0.28 nM), ILT70083 (EC 50 = 0.37 nM), ILT70137 (EC 50 = 0.41 nM), ILT70144, ILT70142, ILT70052, and ILT70100 bound to human ILT7-expressing cells. The candidate antibodies ILT70019, ILT70028, and ILT70076 did not bind to human ILT7-expressing cells. Anti-ILT7 antibody 7C7 (7C7 is as described above in Example 1) and SBI33 (SBI33 refers to anti-ILT7 antibody ILT7#33 provided in US Patent Application Publication No. 2009 / 0280128) were used as positive controls. The isotype control R347 was used as a negative control and showed no binding to ILT7-expressing cells. The graph shown in Figure 2 represents the average value of the results of two independent experiments, and the table shown in Figure 2 shows the average value of EC 50 .

[0273] Binding EC of variants in cells expressing cynomolgus monkey ILT7 50 To determine the binding EC of the antibodies, they were screened by flow cytometry for binding to CynoILT7 CT-125 cells. ILT70052 (EC 50 = 0.35 nM), ILT70080 (EC 50 = 0.44 nM), LT70083 (EC 50 = 1.37 nM), ILT70137 (EC 50 = 1.40 nM), ILT70100 (EC 50 = 1.63 nM), and ILT70144 (EC 50 = 7.81 nM), ILT70142, and ILT70089 were positive for binding to human ILT7. ILT70019, ILT70028, and ILT70076 did not bind to cynomolgus monkey ILT7-expressing cells. The isotype control R347 showed no binding to ILT7-expressing cells. The graph in Figure 3 represents the average value of the results of two independent experiments, and the table in Figure 3 shows the average value of EC 50 .

[0274] Thus, all of ILT70052, ILT70080, ILT70083, ILT70100, ILT70137, ILT70142, and ILT70144 bind to cells expressing either cynomolgus monkey ILT7 or human ILT7. In particular, when using ILT70080, ILT70083, and ILT70137, low EC 50 values were obtained for cells expressing both cynomolgus monkey and human ILT7.

[0275] [Example 4] ADCC potency of ILT7 antibodies Anti-ILT7 antibodies were tested for ADCC potency against a human ILT7-expressing cell line using an in vitro cell-based assay. Cells expressing human ILT7 (target cells) were seeded at a 1:5 ratio with the natural killer (NK) cell line KC1333 (effector cells) in the presence of anti-ILT7 variants or isotype controls for 18 hours. During flow cytometry analysis, KC1333 cells were excluded from gating using the NK marker CD56 (Biolegend #304624), and viable cells were discriminated from dead cells using 7-AAD. Using this method, the percentage of live target cells was calculated and compared to the baseline (antibody-free control). Cytotoxicity was calculated using the following formula: Percentage cytotoxicity = 100 - (number of live targets / number of live targets in antibody-free control) × 100

[0276] ILT70080 showed the greatest ADCC potency against human ILT7-expressing cells (EC 50 = 0.022 nM), followed by ILT70137 (EC 50 = 0.044 nM) and ILT70083 (EC 50 = 0.094 nM). ILT70142, ILT70052, ILT70100, and ILT70144 also showed ADCC activity (Figure 4). Isotype controls R347 and the afucosylated form of R347 (“Afuc R347”) showed no ADCC activity against human ILT7-expressing cells.

[0277] Anti-ILT7 antibodies were tested for ADCC potency against cynomolgus ILT7-expressing cells using in vitro cell-based activity. ILT70080 showed the greatest ADCC potency against cynomolgus ILT7-expressing cells (EC 50 = 0.008 nM), followed by ILT70137 (EC 50 = 0.015 nM), ILT70142 (EC 50 = 0.058 nM), ILT70052 (EC 50 = 0.073 nM), ILT70144 (EC 50= 0.123), ILT70100 (EC 50 = 0.188 nM), and ILT70083 (EC 50 = 0.433 nM) followed. ILT70089 also showed ADCC activity. The positive control 7C7 showed ADCC, and the isotype (negative) control R347 showed no ADCC against cynomolgus ILT7-expressing cells. The graphs and tables in Figure 5 represent two independent experiments.

[0278] Thus, ILT70080 and ILT70137 showed the greatest ADCC activity in both cynomolgus and human ILT7-expressing cells.

[0279] [Example 5] Binding of ILT7 antibodies to PBMC The binding of anti-ILT7 antibodies ILT70080, ILT70083, and ILT70137 to human PBMC was evaluated by flow cytometry using an antibody concentration of 2.5 μg / ml. ITL70080, ILT70083, and ILT70137 specifically bound to pDC (BDCA-4 + cells) (Figures 6A and B). Binding was negative with the isotype control R347.

[0280] The binding of anti-ILT7 antibodies ILT70080, ILT70083, and ILT70137 to cynomolgus PBMC was also evaluated by flow cytometry. ITL70080 and ILT70083 specifically bound to pDC (HLA-DR + , Lineage - , CD123 high cells).

[0281] [Example 6] Effect of ILT7 antibodies on IFN-alpha secretion Anti-ILT7 variants were tested for ADCC potency in human and cynomolgus monkey PBMCs as described above. Secretion of IFNα in the supernatant of PBMCs cultured with anti-ILT7 antibody and CpG-A was measured by ELISA. ILT70080, ILT70083, and ILT70137 all suppressed the IFNα response to CpG-A in human and cynomolgus monkey PBMCs. ILT70080 showed the greatest inhibitory effect on the IFNα response.

[0282] [Example 7] Defucosylation of ILT70080 and ILT70083 antibodies IgG1 antibodies contain two sites for N-linked oligosaccharides in the Fc region, and these sites are highly fucosylated in human antibodies. Antibody-dependent cell cytotoxicity (ADCC) is mediated by the binding of lymphocyte receptors to the antibody Fc region, which is affected by the amount of fucosylation. An increase in ADCC has been observed with a decrease in fucosylation. Therefore, defucosylated forms of ILT7 were generated and analyzed.

[0283] 7.1 Generation of defucosylated anti-ILT7 antibodies ILT70080 and ILT70083 IgG1 were expressed in a CHO cell line lacking the enzyme α-1,6-fucosyltransferase. Expression in this cell line yielded antibodies lacking the α-1,6-fucose moiety on the N-glycan at Asn-297 of the heavy chain.

[0284] 7.2 Testing of defucosylated ILT70080 and ILT70083 anti-ILT7 antibodies Binding assays were performed with defucosylated and parental ILT70080 and ILT70083 antibodies on ILT7-expressing cells to evaluate whether defucosylation affects the binding EC 50 of the antibody. The parental and defucosylated antibodies showed similar binding to both human and cynomolgus monkey ILT7-expressing cells (Figure 7).

[0285] The ADCC potency of the afucosylated ILT70080 and ILT70083 antibodies was tested in human and cynomolgus ILT7-expressing cells using the in vitro cell-based assay described above (Example 3). Afucosylation increased the ADCC potency for all candidates tested (Figure 8). A 10-fold increase in potency was observed for the ILT70080 antibody due to afucosylation in both human and cynomolgus assays (EC 50 = 0.013 nM to EC 50 = 0.001 nM, and EC 50 = 0.006 nM to EC 50 = 0.00051 nM), while a 6- to 7-fold increase was observed for ILT70083 (EC 50 = 0.089 nM to EC 50 = 0.0105 nM, and EC 50 = 0.36 nM to EC 50 = 0.057 nM). The afucosylated isotype control R347 did not show any ADCC in ILT7-expressing cells.

[0286] The binding of the afucosylated anti-ILT7 antibodies ILT70080 and ILT70083 in human PBMC was evaluated by flow cytometry. The afucosylated variants ILT70080 and ILT70083 specifically bound to pDC (BDCA-2 + cells). Binding was negative for the isotype control R347.

[0287] The binding of the afucosylated anti-ILT7 variants ILT70080 and ILT70083 in cynomolgus PBMC was also evaluated by flow cytometry. The afucosylated variants ILT70080 and ILT70083 specifically bound to pDC (HLA-DR + Lineage - CD123 high ). Binding was negative for the isotype control R347.

[0288] [Example 8] Engineering of ILT70080 and ILT70083 Antibodies 8.1 Operation of ILT70080 The amino acid sequences of ILT70080 VH and VL were aligned with known human germline sequences in the VBASE database (Althaus H-H, Muller W and Tomlinson I: V BASE, http: / / vbase.mrc-cpe.cam.ac.uk / ), and related germline sequences were identified by sequence similarity. For the VH domain, this was VH1-69 (DP-10), and for the VL domain, this was Vlambda3-h. The VH domain (A13K, T16S, L69I * , S70T, L80M, Y84S, and D85E) and the VL domain (E3V, K20R, S22T, M46L * , M48I * , F50Y * , and I66N *Seven residues in each of the frameworks (FW) of [[ID=]] were selected to revert to the related germline sequences. The starred mutations were at positions classified as Vernier residues (Foote, J. et al. J. Mol. Biol. 224: 487 (1992)) and typically remained invariant. However, from the analysis of both the Kabat (Wu, T. T. and Kabat E. A. J. Exp. Med. 132:211-250 (1970)) and IMGT (Lefranc, M.-P. et al. Dev. Comp. Immunol. 27: 55-77 (2003)) classifications of the CDRs, these positions were thought to have a low risk of changing the binding properties of the parental antibody and to provide further opportunities to reduce immunogenicity even more. Furthermore, heavy chain N64Q mutagenesis was performed within the VH CDR2 (Kabat's definition) sequence to remove potential deamination (NG) sites at this position. Mutagenesis was performed on the ILT70080 scFv sequence in pCantab6 using standard molecular biology techniques (McCafferty et al., Appl Biochem Biotech 47:157 (1994)). Combinations of different mutagenic oligonucleotides were utilized in a number of mutagenesis reactions to generate a library of sequences containing different combinations of FW mutations. Next, a panel of ILT70080 scFv variants was tested for retention of binding to human ILT7 as crude periplasmic extracts in the FMAT cell binding assay as described above.

[0289] Seven ILT70080 variants were generated as IgG. See Figures 9A and 9B for the VH and VL sequence alignments, respectively.

[0290] 8.2 Manipulation of ILT70083 The germline sequencing of ILT70083 was similarly performed. The identified related germline sequences were VH3-23 (DP-47) and Vlambda1-b (DPL-5) for the VH and VL sequences, respectively. One FW residue was selected for mutagenesis in the VH domain (W66R), and eight FW residues were selected in the VL domain, and in this case also the selected Vernier positions (V4L * , R42T, A64G * , I66K * , S68G * , A72T, A74G, and E81G) were included. ILT70083 variants containing different combinations of mutations were generated directly on the pEU vector containing the individual VH and VL chains using standard molecular biology techniques. The ILT70083 VH and VL chains were co-transfected in different combinations to generate nine IL70083 IgG1 variants. See Figures 10A and 10B for the VH and VL alignments, respectively.

[0291] 8.3 Testing of Engineered Antibodies The resulting IgG1 was tested to confirm that the sequence changes incorporated into ILT70080 and ILT70083 did not have a detrimental effect on the binding of the parental antibody to cells expressing human ILT7 (CT-550 cells) or cynomolgus monkey ILT7 (CT-125 cells). The variants were screened for binding by flow cytometry. All of the ILT70080 variants had binding similar to the parental ILT70080 antibody to human and cynomolgus monkey ILT7 (EC 50 = 0.213 nM and 0.547 nM), respectively. See Figure 11. The binding of the ILT70083 variants was also similar to the parental antibody with respect to human ILT7 (EC 50 = 0.464 nM). See Figure 12. However, five ILT70083 variants (ILT70083.4, ILT70083.9, ILT70083.3, ILT70083.6, and ILT70083.8) had improved binding ability compared to the parental antibody with respect to cynomolgus monkey ILT7. See Figure 12.

[0292] The engineered ILT70080 and ILT70083 antibodies were tested for ADCC potency against human ILT7-expressing cell lines using in vitro cell-based assays. All ILT70080 variants had increased ADCC potency compared to the parental antibody (EC 50 <14.1 pM). See Figure 13. The two candidates with the lowest EC 50 were ILT70080.6 (EC 50 = 6.9 pM) and ILT70080.1 (EC 50 = 8.0 pM). The EC 50 values of the other ILT70080 variants were as follows: ILT70080.1 EC 50 = 10.0 pM, ILT70080.3 EC 50 = 11.0 pM, ILT70080.4 EC 50 = 11.9 pM, ILT70080.5 EC 50 = 8.6 pM, and ILT70080.7 EC 50 = 7.8 pM. All of the LT70083 variants were found to have decreased potency compared to the parental antibody (EC 50 > 89.0 pM). See Figure 14.

[0293] [Example 9] Engineering of Hypofucosylated ILT70080 and ILT70083 Antibodies A hypofucosylated form of ILT70080.6 was generated. Hypofucosylation of the ILT7080.6 antibody did not affect its binding to either human or cynomolgus monkey ILT7-expressing cells. The binding EC 50 of hypofucosylated ILT70080.6 to human and cynomolgus monkey ILT7-expressing cells was 152.3 pM and 366.2 pM, respectively. See Figure 15. The table in Figure 15 provides the mean values of the results of three independent binding experiments measuring mean fluorescence intensity (MFI).

[0294] The low-fucosylated ADCC activities of ILT70080.6 and ILT70083 (see Example 7 above) were similarly evaluated. The low-fucosylation of ILT70080.6 improved its ADCC potency by approximately 10-fold against both human and cynomolgus monkey ILT7-expressing cells. See Figure 16. The EC 50 of low-fucosylated ILT0080.6 was 1.12 pM against human ILT7-expressing cells and 0.44 pM against cynomolgus monkey ILT7-expressing cells. The table in Figure 16 provides the average values of the results of three independent ADCC assays measuring cytotoxicity.

[0295] Low-fucosylated ILT70080.6 and ILT70083 were tested for their ADCC potency in human PBMC. The cytotoxicity of the antibodies was evaluated by flow cytometry and CpG A-mediated IFNα secretion in the supernatant was measured by ELISA. The results are shown in Figure 17. In cynomolgus monkey PBMC, the EC50 values of IFNα secretion using low-fucosylated ILT70080.6 and ILT70083 antibodies were 58 pM and 5216 pM, respectively.

[0296] In human whole blood and PBMC, low-fucosylated ILT70080.6 and ILT70083 antibodies were found to specifically bind to BDCA-2 positive cells. The binding of both antibodies was restricted to human pDC at all concentrations tested (0.1 - 5.0 μg / mL).

[0297] In cynomolgus monkey whole blood, low-fucosylated ILT70080.6 and ILT70083 antibodies were found to bind to pDC (HLA-DR + Lineage - CD123 high cells) at all concentrations tested (0.5 - 2.5 μg / mL).

[0298] [Example 10] Low-fucosylation of the ILT70137 antibody The afucosylated form of the ILT70137 antibody was prepared as described above in Example 7 for the ILT70080 and ILT70083 antibodies.

[0299] 10.1 Binding to Soluble Recombinant Human ILT7 Using BIAcore (surface plasmon resonance), the kinetics (k on , k off ) constants of the binding of afucosylated IgG1 ILT70137 to human ILT7 protein were measured using an IgG capture assay format. The binding at each concentration of a two-fold serial dilution of the ILT7 protein was recorded after first capturing IgG on the sensor chip surface and then capturing either the ILT7 protein or the instrument buffer. Between each pair of injections, the IgG capture surface was regenerated. The individual association and dissociation rate constants were calculated from the binding curves obtained using a 1:1 fitting model with Biaevaluation software available through the vendor's software, which included terms for correcting for mass transport-limited binding even if detected. From the high-resolution BIAcore plot of the data, the association rate constant and dissociation rate constant of the binding of the afucosylated IgG1 ILT70137 to the ILT7 protein were determined to be 1.855×10 5 M -1 s -1 . Using the same plot similarly, the corresponding dissociation rate constant for this interaction was determined and measured to be 3.175×10 -2 s -1 . From these rate constants, K D was calculated to be 171 nM from the quotient of k off / k on . These results are summarized in Table 3 below. The individual errors in k off and k on are very low, and the overall fit to the data has a chi-squared value calculated for R maxIt was judged to be good since it was about 1% of the (maximum response). Summarizing these, this suggests that the use of a one-site binding model for fitting the data was appropriate. The evaluation shows that the binding is not at the mass transfer limit, and the measured binding rate constant is considered to be valid.

[0300]

Table 3

[0301] 10.2 Binding to ILT7-expressing cell lines The binding of afucosylated ILT70137 to ILT7 was determined using cell lines stably expressing human or cynomolgus monkey ILT7. The mean fluorescence intensity of the cell-bound antibody was evaluated by flow cytometry. The cells were incubated with a serially increasing concentration of the test antibody in the range of 0.004 - 333.3 nM at 4°C for 30 minutes. After incubation, the cells were washed with cold PBS and incubated with an anti-human Alexa Fluor 647 antibody at 4°C for 30 minutes. Next, the fluorescence intensity was determined by FACS, and the EC 50 value was calculated using a non-linear fitting equation in GraphPad Prism 6 software.

[0302] The results are shown in Fig. 18. Afucosylated ILT70137 was found to bind to recombinant human and cynomolgus monkey ILT7-expressing cells in a dose-dependent manner. No significant binding was observed with the isotype control. The average value of the half-maximal effective concentration (EC 50 ) of afucosylated ILT70137 was 0.303 nM for binding to human ILT7-expressing cells and 2.148 nM for cynomolgus monkey ILT7-expressing cells.

[0303] 10.3 ADCC activity against ILT7-expressing cell lines The potential of afucosylated ILT70137 to induce ADCC was measured by fluorescence-activated cell sorting (FACS) assay in target cells expressing human or cynomolgus ILT7. Target cells were co-cultured with the effector NK cell line KC1333 at a ratio of 1:5 in the presence of increasing concentrations (0 - 6.66×10 -9 M range) of afucosylated ILT70137 or isotype control. For assessment of target cell viability by flow cytometry, KC1333 was excluded by gating using CD56, and dead cells were excluded by gating using 7-amino-actinomycin D (7-AAD) viability staining. Viable target cells were defined as CD56-negative and 7-AAD-negative. The percentage of cytotoxicity was calculated using the following formula: % cytotoxicity = 100 - (percentage of live target cells / percentage of live targets in the antibody-free control) × 100. The half-maximal effective concentration (EC 50 ) value was calculated using a non-linear fitting formula in GraphPad Prism 6 software. X-axis: antibody concentration.

[0304] The results are shown in Figure 19. Afucosylated ILT70137 induced ADCC in a dose-dependent manner in cells expressing ILT7, with an EC 50 of 4.19 pM for cells expressing human ILT-7 and 1.89 pM for cells expressing cynomolgus ILT7.

[0305] 10.4 ADCC activity in primary plasmacytoid dendritic cells IFNα secretion in response to Toll-like receptor 9 (TLR9) agonists is mostly by plasmacytoid dendritic cells (pDCs) in peripheral blood mononuclear cell (PBMC) preparations. Therefore, the ability of defucosylated ILT70137 to induce ADCC of primary pDCs was indirectly measured by assessing its ability to block IFNα secretion in PBMCs. In these assays, purified PBMCs were seeded in 96-well round-bottom plates in medium supplemented with 10% fetal bovine serum and 200 ng / mL recombinant human IL-2. Serial dilutions of defucosylated ILT70137 and control antibody were added to appropriate wells in duplicate and incubated for 9.5 hours. After incubation, the TLR9 agonist ODN2216 was added to each well at a final concentration of 0.5 μM. IFNα in the supernatant was quantified using a multi-subtype IFNα ELISA kit and represented as pg / mL of supernatant in Figure 20. The IC 50 of ADCC was calculated using a non-linear fitting formula in GraphPad Prism v5.01 software.

[0306] Defucosylated ILT70137 dose-dependently reduced TLR9-mediated secretion of IFNα in PBMCs, and the half-maximal inhibitory concentration (IC 50 ) was 0.048 nM. These results indicate that defucosylated ILT70137 effectively depletes naturally occurring primary human pDCs in PBMCs.

[0307] 10.5 Binding to primary plasmacytoid dendritic cells The specificity of hypofucosylated ILT70137 for human primary plasmacytoid dendritic cells (pDC) was evaluated by FACS in peripheral blood mononuclear cells (PBMC). PBMC were isolated from human donors. To properly identify this dendritic cell subset, the markers CD123 (expressed on pDC and basophils) and CD304 (unique to pDC) were first utilized. pDC were CD123+CD304+ double positive, and CD304 staining was sufficient to identify pDC. See Figure 21 (upper panel). Hypofucosylated ILT70137 bound only to CD304-positive cells, indicating that it binds uniquely to pDC. See Figure 21 (lower right panel). No significant binding to this population was observed with the human IgG1 isotype hypofucosylated control antibody R3-47. See Figure 21 (lower left panel).

[0308] [Example 11] In vivo activity of ILT7 antibodies Three anti-ILT7 antibodies, namely hypofucosylated 7C7, hypofucosylated ILT70080.6, and hypofucosylated IgG1 ILT70137, were administered to male cynomolgus monkeys. All three antibodies were active in the depletion of plasmacytoid dendritic cells (pDC).

[0309] Administration of hypofucosylated ILT70080.6 was generally well tolerated. However, the following pathological findings were observed: decreased neutrophil count, angioleukocytosis, increased glomerular matrix, and vascular / perivascular inflammation. Furthermore, the appearance of antibodies against hypofucosylated ILT70080.6 (anti-drug antibodies) was associated with an increase in the clearance of hypofucosylated ILT70080.6.

[0310] In another study, the toxicokinetics of afucosylated 7C7 and afucosylated ILT70137 were investigated. In this test, five equivalent doses of the antibody were administered to cynomolgus monkeys by injection. After administration, the exposure was equivalent between afucosylated 7C7 and afucosylated ILT70137 at steady state. Furthermore, as shown in Figure 22, specific and reversible depletion of pDCs was achieved with either antibody. pDC depletion resulted in ex vivo inhibition of IFNα production. See Figure 23.

[0311] However, the pathology of animals treated with afucosylated 7C7 and afucosylated ILT70137 was different. An increase in spleen weight was observed in some animals treated with afucosylated 7C7. Microscopic findings were also observed in some animals treated with afucosylated 7C7. In particular, red pulp and macrophage hypertrophy / hyperplasia were observed in the spleen. Kupffer cell hypertrophy / hyperplasia was observed in the liver. Furthermore, immunohistochemistry showed human IgG / 7C7- and monkey IgG-containing granular deposits associated with hypertrophic / hyperplastic Kupffer cells in the liver and red pulp macrophages in the spleen. These findings are consistent with the excessive physiological clearance of immune complexes containing the drug (7C7) and anti-drug antibodies (ADA). In contrast, changes in organ weight and gross or microscopic findings were not observed with afucosylated ILT70137.

[0312] Furthermore, for two monkeys treated with the afucosylated 7C7 antibody, the neutrophil count decreased below 1 E3 / μl, but no significant change in neutrophil count was observed in monkeys treated with the control or afucosylated ILT70137.

[0313] Thus, all three antibodies depleted pDCs in vivo, but the excellent safety and lack of anti-drug antibodies after administration of afucosylated ILT70137 are surprisingly advantageous.

[0314] [Example 12] Epitope Mapping To determine the epitope to which the ILT7 antibody binds, chimeric polypeptides containing ILT7 and ILT1 polypeptides were constructed and the binding of the ILT7 antibody to these constructs was tested. ILT1 (accession number Q8N149) has the same modular structure as ILT7 and shares 65% identity with ILT7, but was not recognized by ILT7 monoclonal antibodies, and thus was selected to construct chimeric variants. The chimeric polypeptides were made by replacing the extracellular Ig domain of ILT7 with the ILT1 counterpart. All of these constructs contained an N-terminal Flag tag. The results demonstrated that ILT70080 and ILT70083 bind to the Ig1 domain of ILT7. In contrast, the 7C7 antibody binds to the Ig2 domain of ILT7.

[0315] The foregoing description of the specific embodiments fully reveals the general nature of the present invention, so that others can, by applying the knowledge of those skilled in the art, without undue experimentation and without departing from the general concept of the present invention, readily modify and / or adapt such specific embodiments. Accordingly, such adaptations and modifications are intended to be within the meaning and scope of the equivalents of the disclosed embodiments based on the teachings and guidance presented herein. The phrases or terms herein are for the purpose of description and not of limitation, and it is understood that the terms or phrases herein will be interpreted by those skilled in the art in light of the teachings and guidance.

[0316] The breadth and scope of the present invention should not be limited by any of the above exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0317] [Sequence Listing] SEQUENCE LISTING <110> VIELA BIO, INC. <120> ILT7 BINDING MOLECULES AND METHODS OF USING THE SAME <130> PA23-479 <150> US 62 / 306,125 <151> 2016-03-10 <160> 292 <170> PatentIn version 3.5 <210> 1 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> SBI28 VH <400> 1 caggttcagc tgcagcagtc tggggctgag ctggtgaagc ctggggcctc agtgaagatg 60 tcctgcaagg cttttggcta caccttcact acctatccaa tagagtggat gaagcagaat 120 catgggaaga gcctagagtg gattggaaat tttcatcctt acaatgatga tactaagtac 180 aatgaaaaat tcaagggcaa ggccaaattg actgtagaaa aatcctctag cacagtctac 240 ttggagctca gccgattaac atctgatgac tctgctgttt attactgtgc aaggggggat 300 gattacggga tggactactg gggtcaagga acctcagtca ccgtctcctc a 351 <210> 2 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> SBI28 VH <400> 2 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Phe Gly Tyr Thr Phe Thr Thr Tyr 20 25 30 Pro Ile Glu Trp Met Lys Gln Asn His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asn Phe His Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Lys Leu Thr Val Glu Lys Ser Ser Ser Thr Val Tyr 65 70 75 80 Leu Glu Leu Ser Arg Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Asp Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> SBI28 VHCDR1 <400> 3 Thr Tyr Pro Ile Glu 1 5 <210> 4 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> SBI28 VHCDR2 <400> 4 Asn Phe His Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> SBI28 VHCDR3 <400> 5 Gly Asp Asp Tyr Gly Met Asp Tyr 1 5 <210> 6 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> SBI28 VL <400> 6 gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc 60 atcacctgca aggccagtca gaatgttcgt actgctgtag cctggtatca acagaaacca 120 gggcagtctc ctaaagcact gatttacttg gcatccaacc ggcacactgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccattagcaa tgtgcaatct 240 gaagacctgg cagattattt ctgtctgcaa cattggaatt atccattcac gttcggctcg 300 gggacaaagt tggaaataaa a 321 <210> 7 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> SBI28 VL <400> 7 Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> SBI28 VLCDR1 <400> 8 Lys Ala Ser Gln Asn Val Arg Thr Ala Val Ala 1 5 10 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> SBI28 VLCDR2 <400> 9 Leu Ala Ser Asn Arg His Thr 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> SBI28 VLCDR3 <400> 10 Leu Gln His Trp Asn Tyr Pro Phe Thr 1 5 <210> 11 <211> 351 <212> DNA <213> Artificial Sequence <220> <223> 7C7 VH <400> 11 gaggtgcagc tggtggagtc tgggggaggc gtagtacagc ctgggagatc cctgagactc 60 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg tcctgtgcag cctctggatt cacctttagc atctacccca tcgagtgggt gcgacaggct 120 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr cctggacagg gcctggaatg gatcggcaac ttccacccct acaacgacga caccaagtac 180 Pro Trp Thr Gly Pro Trp Asn Asp Ser Gln Thr Pro Tyr Asn Asp Asp Thr Lys Tyr aacgagaagt tcaagggcag agtcaccatg accacagaca catccacgag cacagtgtac 240 Asn Glu Lys Phe Lys Gly Arg Ser Thr Met Thr Thr Asp His Ser Glu His Ser Tyr atggagctga gcagcctgag atctgaggac acggccgtgt attactgtac gagaggcgac 300 Met Glu Leu Ser Gln Leu Glu Asp Thr Ala Val Tyr Tyr Cys Tyr Glu Arg Asp gactacggcc tggactattg gggccagggc accctcgtga ccgtgtcctc t 351 Asp Tyr Gly Leu Asp Tyr Gly Ala Arg His Leu Val Thr Val Ser <210> 12<210> 12 <211> 117<211> 117 <212> PRT<212> PRT <213> Artificial Sequence<213> Artificial Sequence <220> <220> <223> 7C7 VH <223> 7C7 VH <400> 12 <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 20 25 30 Pro Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Phe His Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asp Asp Tyr Gly Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 7C7 VHCDR1 <400> 13 Ile Tyr Pro Ile Glu 1 5 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> 7C7 VHCDR2 <400> 14 Asn Phe His Pro Tyr Asn Asp Asp Thr Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> 7C7 VHCDR3 <400> 15 Gly Asp Asp Tyr Gly Leu Asp Tyr 1 5 <210> 16 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> 7C7 VL <400> 16 aatattcaga tgacccagag cccgagcagc ctgagcgcaa gcgttggtga tcgtgttacc 60 attacctgtg acgccagcca gaatgttcgt accgcagttg catggtatca gcagaaaccg 120 ggtaaagcac cgaaacgtct gatttatctg gcaagtaatc gtcataccgg tgttccgagc 180 cgttttagcg gtagcggttc tggcaccgat tttaccctga ccattagcag cctgcagagc 240 gaagattttg ccacctatta ttgtctgcag cattggaatt atccgtttac ctttggtccg 300 ggtacaaaac tggaaattaa a 321 <210> 17 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 7C7 VL <400> 17 Asn Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Asp Ala Ser Gln Asn Val Arg Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Trp Asn Tyr Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> 7C7 VLCDR1 <400> 18 Asp Ala Ser Gln Asn Val Arg Thr Ala Val Ala 1 5 10 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> 7C7 VLCDR2 <400> 19 Leu Ala Ser Asn Arg His Thr 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> 7C7 VLCDR3 <400> 20 Leu Gln His Trp Asn Tyr Pro Phe Thr 1 5 <210> 21 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ILT70080 VH <400> 21 caggtgcagc tggtgcaatc tggggctgag gtgaaggcgc ctgggacttc ggtgaaagtc 60 tcctgcaagg catctggaga cagcttcagg aactatgctg tcagttgggt gcgacaggcc 120 ccaggacaag gtcttgagtg gatgggagcg atgatgccta gttttggaac aagggagcac 180 tcacagcagt tcaacggcag agtcaccctt tccgcggacg aatccacgag cacagcctac 240 ttggaactga gcagtctaag atatgacgac acggccgtct attactgtgc gggtagtcgg 300 gactacaatg cttaccattt ttggactggt ccccctgact tctggggccg aggaaccctg 360 gtcaccgtct cgagt 375 <210> 22 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> ILT70080 VH <400> 22 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Ala Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Ser Phe Arg Asn Tyr 20 25 30 Ala Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe 50 55 60 Asn Gly Arg Val Thr Leu Ser Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Tyr Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro 100 105 110 Asp Phe Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 23 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ILT70080 VHCDR1 <400> 23 Asn Tyr Ala Val Ser 1 5 <210> 24 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ILT70080 VHCDR2 <400> 24 Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe Asn 1 5 10 15 Gly <210> 25 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ILT70080 VHCDR3 <400> 25 Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro Asp Phe 1 5 10 15 <210> 26 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> ILT70080 VL <400> 26 tcctatgagc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaagatt 60 tcctgtgggg gagacagcgt tggcagtaca agtgtccact ggtaccagca gaagccgggc 120 caggcccctg tgatggtcat gttttataac agtgaccggc cctcagggat ccctgagcga 180 ttctctggct ccatctctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatactagta gtgatcatcc ggagttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 27 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> ILT70080 VL <400> 27 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Lys Ile Ser Cys Gly Gly Asp Ser Val Gly Ser Thr Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Met Val Met Phe 35 40 45 Tyr Asn Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ile Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Ser Asp His 85 90 95 Pro Glu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 28 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080 VLCDR1 <400> 28 Gly Gly Asp Ser Val Gly Ser Thr Ser Val His 1 5 10 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ILT70080 VLCDR2 <400> 29 Tyr Asn Ser Asp Arg Pro Ser 1 5 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080 VLCDR3 <400> 30 Gln Val Trp Asp Thr Ser Ser Asp His Pro Glu 1 5 10 <210> 31 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.1 VH <400> 31 caggtgcagc tggtgcaatc tggggctgag gtgaagaagc ctgggtcttc ggtgaaagtc 60 tcctgcaagg catctggaga cagcttcagg aactatgctg tcagttgggt gcgacaggcc 120 ccaggacaag gtcttgagtg gatgggagcg atgatgccta gttttggaac aagggagcac 180 tcacagcagt tccagggcag agtcaccctt accgcggacg aatccacgag cacagcctac 240 atggaactga gcagtctaag atctgaggac acggccgtct attactgtgc gggtagtcgg 300 gactacaatg cttaccattt ttggactggt ccccctgact tctggggccg aggaaccctg 360 gtcaccgtct cctca 375 <210> 32 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.1 VH <400> 32 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Ser Phe Arg Asn Tyr 20 25 30 Ala Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro 100 105 110 Asp Phe Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 33 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.1 VHCDR1 <400> 33 Asn Tyr Ala Val Ser 1 5 <210> 34 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.1 VHCDR2 <400> 34 Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe Gln 1 5 10 15 Gly <210> 35 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.1 VHCDR3 <400> 35 Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro Asp Phe 1 5 10 15 <210> 36 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.1 VL <400> 36 tcctatgtgc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaggatt 60 acctgtgggg gagacagcgt tggcagtaca agtgtccact ggtaccagca gaagccgggc 120 caggcccctg tgatggtcat gttttataac agtgaccggc cctcagggat ccctgagcga 180 ttctctggct ccatctctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatactagta gtgatcatcc ggagttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 37 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.1 VL <400> 37 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asp Ser Val Gly Ser Thr Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Met Val Met Phe 35 40 45 Tyr Asn Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ile Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Ser Asp His 85 90 95 Pro Glu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 38 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.1 VLCDR1 <400> 38 Gly Gly Asp Ser Val Gly Ser Thr Ser Val His 1 5 10 <210> 39 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.1 VLCDR2 <400> 39 Tyr Asn Ser Asp Arg Pro Ser 1 5 <210> 40 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.1 VLCDR3 <400> 40 Gln Val Trp Asp Thr Ser Ser Asp His Pro Glu 1 5 10 <210> 41 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.2 VH <400> 41 caggtgcagc tggtgcaatc tggggctgag gtgaagaagc ctgggtcttc ggtgaaagtc 60 tcctgcaagg catctggaga cagcttcagg aactatgctg tcagttgggt gcgacaggcc 120 ccaggacaag gtcttgagtg gatgggagcg atgatgccta gttttggaac aagggagcac 180 tcacagcagt tccagggcag agtcaccctt tccgcggacg aatccacgag cacagcctac 240 atggaactga gcagtctaag atctgaggac acggccgtct attactgtgc gggtagtcgg 300 gactacaatg cttaccattt ttggactggt ccccctgact tctggggccg aggaaccctg 360 gtcaccgtct cctca 375 <210> 42 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.2 VH <400> 42 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Ser Phe Arg Asn Tyr 20 25 30 Ala Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe 50 55 60 Gln Gly Arg Val Thr Leu Ser Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro 100 105 110 Asp Phe Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 43 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.2 VHCDR1 <400> 43 Asn Tyr Ala Val Ser 1 5 <210> 44 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.2 VHCDR2 <400> 44 Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe Gln 1 5 10 15 Gly <210> 45 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.2 VHCDR3 <400> 45 Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro Asp Phe 1 5 10 15 <210> 46 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.2 VL <400> 46 tcctatgtgc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaggatt 60 acctgtgggg gagacagcgt tggcagtaca agtgtccact ggtaccagca gaagccgggc 120 caggcccctg tgatggtcat gttttataac agtgaccggc cctcagggat ccctgagcga 180 ttctctggct ccatctctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatactagta gtgatcatcc ggagttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 47 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.2 VL <400> 47 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asp Ser Val Gly Ser Thr Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Met Val Met Phe 35 40 45 Tyr Asn Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ile Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Ser Asp His 85 90 95 Pro Glu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 48 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.2 VLCDR1 <400> 48 Gly Gly Asp Ser Val Gly Ser Thr Ser Val His 1 5 10 <210> 49 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.2 VLCDR2 <400> 49 Tyr Asn Ser Asp Arg Pro Ser 1 5 <210> 50 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.2 VLCDR3 <400> 50 Gln Val Trp Asp Thr Ser Ser Asp His Pro Glu 1 5 10 <210> 51 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.3 VH <400> 51 caggtgcagc tggtgcaatc tggggctgag gtgaaggcgc ctgggacttc ggtgaaagtc 60 tcctgcaagg catctggaga cagcttcagg aactatgctg tcagttgggt gcgacaggcc 120 ccaggacaag gtcttgagtg gatgggagcg atgatgccta gttttggaac aagggagcac 180 tcacagcagt tccagggcag agtcaccctt tccgcggacg aatccacgag cacagcctac 240 atggaactga gcagtctaag atctgaggac acggccgtct attactgtgc gggtagtcgg 300 gactacaatg cttaccattt ttggactggt ccccctgact tctggggccg aggaaccctg 360 gtcaccgtct cctca 375 <210> 52 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.3 VH <400> 52 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Ala Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Ser Phe Arg Asn Tyr 20 25 30 Ala Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe 50 55 60 Gln Gly Arg Val Thr Leu Ser Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro 100 105 110 Asp Phe Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 53 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.3 VHCDR1 <400> 53 Asn Tyr Ala Val Ser 1 5 <210> 54 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.3 VHCDR2 <400> 54 Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe Gln 1 5 10 15 Gly <210> 55 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.3 VHCDR3 <400> 55 Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro Asp Phe 1 5 10 15 <210> 56 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.3 VL <400> 56 tcctatgtgc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaggatt 60 acctgtgggg gagacagcgt tggcagtaca agtgtccact ggtaccagca gaagccgggc 120 caggcccctg tgatggtcat gttttataac agtgaccggc cctcagggat ccctgagcga 180 ttctctggct ccatctctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatactagta gtgatcatcc ggagttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 57 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.3 VL <400> 57 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asp Ser Val Gly Ser Thr Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Met Val Met Phe 35 40 45 Tyr Asn Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ile Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Ser Asp His 85 90 95 Pro Glu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 58 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.3 VLCDR1 <400> 58 Gly Gly Asp Ser Val Gly Ser Thr Ser Val His 1 5 10 <210> 59 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.3 VLCDR2 <400> 59 Tyr Asn Ser Asp Arg Pro Ser 1 5 <210> 60 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.3 VLCDR3 <400> 60 Gln Val Trp Asp Thr Ser Ser Asp His Pro Glu 1 5 10 <210> 61 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.4 VH <400> 61 caggtgcagc tggtgcaatc tggggctgag gtgaaggcgc ctgggacttc ggtgaaagtc 60 tcctgcaagg catctggaga cagcttcagg aactatgctg tcagttgggt gcgacaggcc 120 ccaggacaag gtcttgagtg gatgggagcg atgatgccta gttttggaac aagggagcac 180 tcacagcagt tccagggcag agtcaccctt tccgcggacg aatccacgag cacagcctac 240 atggaactga gcagtctaag atctgaggac acggccgtct attactgtgc gggtagtcgg 300 gactacaatg cttaccattt ttggactggt ccccctgact tctggggccg aggaaccctg 360 gtcaccgtct cctca 375 <210> 62 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.4 VH <400> 62 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Ala Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Ser Phe Arg Asn Tyr 20 25 30 Ala Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe 50 55 60 Gln Gly Arg Val Thr Leu Ser Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro 100 105 110 Asp Phe Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 63 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.4 VHCDR1 <400> 63 Asn Tyr Ala Val Ser 1 5 <210> 64 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.4 VHCDR2 <400> 64 Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe Gln 1 5 10 15 Gly <210> 65 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.4 VHCDR3 <400> 65 Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro Asp Phe 1 5 10 15 <210> 66 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.4 VL <400> 66 tcctatgagc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaagatt 60 tcctgtgggg gagacagcgt tggcagtaca agtgtccact ggtaccagca gaagccgggc 120 caggcccctg tgatggtcat gttttataac agtgaccggc cctcagggat ccctgagcga 180 ttctctggct ccatctctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatactagta gtgatcatcc ggagttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 67 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.4 VL <400> 67 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Lys Ile Ser Cys Gly Gly Asp Ser Val Gly Ser Thr Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Met Val Met Phe 35 40 45 Tyr Asn Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ile Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Ser Asp His 85 90 95 Pro Glu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 68 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.4 VLCDR1 <400> 68 Gly Gly Asp Ser Val Gly Ser Thr Ser Val His 1 5 10 <210> 69 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.4 VLCDR2 <400> 69 Tyr Asn Ser Asp Arg Pro Ser 1 5 <210> 70 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.4 VLCDR3 <400> 70 Gln Val Trp Asp Thr Ser Ser Asp His Pro Glu 1 5 10 <210> 71 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.5 VH <400> 71 caggtgcagc tggtgcaatc tggggctgag gtgaagaagc ctgggtcttc ggtgaaagtc 60 tcctgcaagg catctggaga cagcttcagg aactatgctg tcagttgggt gcgacaggcc 120 ccaggacaag gtcttgagtg gatgggagcg atgatgccta gttttggaac aagggagcac 180 tcacagcagt tccagggcag agtcaccctt accgcggacg aatccacgag cacagcctac 240 atggaactga gcagtctaag atctgaggac acggccgtct attactgtgc gggtagtcgg 300 gactacaatg cttaccattt ttggactggt ccccctgact tctggggccg aggaaccctg 360 gtcaccgtct cctca 375 <210> 72 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.5 VH <400> 72 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Ser Phe Arg Asn Tyr 20 25 30 Ala Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro 100 105 110 Asp Phe Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 73 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.5 VHCDR1 <400> 73 Asn Tyr Ala Val Ser 1 5 <210> 74 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.5 VHCDR2 <400> 74 Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe Gln 1 5 10 15 Gly <210> 75 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.5 VHCDR3 <400> 75 Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro Asp Phe 1 5 10 15 <210> 76 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.5 VL <400> 76 tcctatgtgc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaggatt 60 acctgtgggg gagacagcgt tggcagtaca agtgtccact ggtaccagca gaagccgggc 120 caggcccctg tgatggtcat gttttataac agtgaccggc cctcagggat ccctgagcga 180 ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatactagta gtgatcatcc ggagttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 77 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.5 VL <400> 77 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asp Ser Val Gly Ser Thr Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Met Val Met Phe 35 40 45 Tyr Asn Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Ser Asp His 85 90 95 Pro Glu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 78 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.5 VLCDR1 <400> 78 Gly Gly Asp Ser Val Gly Ser Thr Ser Val His 1 5 10 <210> 79 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.5 VLCDR2 <400> 79 Tyr Asn Ser Asp Arg Pro Ser 1 5 <210> 80 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.5 VLCDR3 <400> 80 Gln Val Trp Asp Thr Ser Ser Asp His Pro Glu 1 5 10 <210> 81 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.6 VH <400> 81 caggtgcagc tggtgcaatc tggggctgag gtgaagaagc ctgggtcttc ggtgaaagtc 60 tcctgcaagg catctggaga cagcttcagg aactatgctg tcagttgggt gcgacaggcc 120 ccaggacaag gtcttgagtg gatgggagcg atgatgccta gttttggaac aagggagcac 180 tcacagcagt tccagggcag agtcaccctt accgcggacg aatccacgag cacagcctac 240 atggaactga gcagtctaag atctgaggac acggccgtct attactgtgc gggtagtcgg 300 gactacaatg cttaccattt ttggactggt ccccctgact tctggggccg aggaaccctg 360 gtcaccgtct cctca 375 <210> 82 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.6 VH <400> 82 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Ser Phe Arg Asn Tyr 20 25 30 Ala Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro 100 105 110 Asp Phe Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 83 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.6 VHCDR1 <400> 83 Asn Tyr Ala Val Ser 1 5 <210> 84 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.6 VHCDR2 <400> 84 Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe Gln 1 5 10 15 Gly <210> 85 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.6 VHCDR3 <400> 85 Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro Asp Phe 1 5 10 15 <210> 86 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.6 VL <400> 86 tcctatgtgc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaggatt 60 acctgtgggg gagacagcgt tggcagtaca agtgtccact ggtaccagca gaagccgggc 120 caggcccctg tgctggtcat ttattataac agtgaccggc cctcagggat ccctgagcga 180 ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatactagta gtgatcatcc ggagttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 87 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.6 VL <400> 87 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asp Ser Val Gly Ser Thr Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Tyr Asn Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Ser Asp His 85 90 95 Pro Glu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 88 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.6 VLCDR1 <400> 88 Gly Gly Asp Ser Val Gly Ser Thr Ser Val His 1 5 10 <210> 89 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.6 VLCDR2 <400> 89 Tyr Asn Ser Asp Arg Pro Ser 1 5 <210> 90 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.6 VLCDR3 <400> 90 Gln Val Trp Asp Thr Ser Ser Asp His Pro Glu 1 5 10 <210> 91 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.7 VH <400> 91 caggtgcagc tggtgcaatc tggggctgag gtgaagaagc ctgggtcttc ggtgaaagtc 60 tcctgcaagg catctggaga cagcttcagg aactatgctg tcagttgggt gcgacaggcc 120 ccaggacaag gtcttgagtg gatgggagcg atgatgccta gttttggaac aagggagcac 180 tcacagcagt tccagggcag agtcaccctt accgcggacg aatccacgag cacagcctac 240 atggaactga gcagtctaag atctgaggac acggccgtct attactgtgc gggtagtcgg 300 gactacaatg cttaccattt ttggactggt ccccctgact tctggggccg aggaaccctg 360 gtcaccgtct cctca 375 <210> 92 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.7 VH <400> 92 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Ser Phe Arg Asn Tyr 20 25 30 Ala Val Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro 100 105 110 Asp Phe Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 93 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.7 VHCDR1 <400> 93 Asn Tyr Ala Val Ser 1 5 <210> 94 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.7 VHCDR2 <400> 94 Ala Met Met Pro Ser Phe Gly Thr Arg Glu His Ser Gln Gln Phe Gln 1 5 10 15 Gly <210> 95 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.7 VHCDR3 <400> 95 Ser Arg Asp Tyr Asn Ala Tyr His Phe Trp Thr Gly Pro Pro Asp Phe 1 5 10 15 <210> 96 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> ILT70080.7 VL <400> 96 tcctatgtgc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaggatt 60 acctgtgggg gagacagcgt tggcagtaca agtgtccact ggtaccagca gaagccgggc 120 caggcccctg tgctggtcat gttttataac agtgaccggc cctcagggat ccctgagcga 180 ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatactagta gtgatcatcc ggagttcggc 300 ggagggacca agctgaccgt ccta 324 <210> 97 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.7 VL <400> 97 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asp Ser Val Gly Ser Thr Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Met Phe 35 40 45 Tyr Asn Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Thr Ser Ser Asp His 85 90 95 Pro Glu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 98 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.7 VLCDR1 <400> 98 Gly Gly Asp Ser Val Gly Ser Thr Ser Val His 1 5 10 <210> 99 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.7 VLCDR2 <400> 99 Tyr Asn Ser Asp Arg Pro Ser 1 5 <210> 100 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70080.7 VLCDR3 <400> 100 Gln Val Trp Asp Thr Ser Ser Asp His Pro Glu 1 5 10 <210> 101 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ILT70083 VH <400> 101 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggctg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaggcggaca 300 tattactatg atagtgatgg tcactcggat gtttttgata tttggggccg gggcaccctg 360 gtcaccgtct cgagt 375 <210> 102 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> ILT70083 VH <400> 102 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Trp Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Thr Tyr Tyr Tyr Asp Ser Asp Gly His Ser Asp Val Phe 100 105 110 Asp Ile Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 103 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ILT70083 VHCDR1 <400> 103 Ser Tyr Ala Met Ser 1 5 <210> 104 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ILT70083 VHCDR2 <400> 104 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 105 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ILT70083 VHCDR3 <400> 105 Arg Thr Tyr Tyr Tyr Asp Ser Asp Gly His Ser Asp Val Phe Asp Ile 1 5 10 15 <210> 106 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> ILT70083 VL <400> 106 cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60 tcctgttctg gaagcgcctc caatattggg agtaattttg tgtcctggta ccaacaactc 120 cccgggagag cccccaaact cctcatttat gacaatgata aacgagactt agggattcct 180 gaccgcttct ctgcctccat ctcttccacg tcagccgccc tggccatcac cggactccag 240 actgaggacg aggccgatta ttactgcgga acatgggaca ccagtctgac tgttggggtt 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> 107 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> ILT70083 VL <400> 107 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ala Ser Asn Ile Gly Ser Asn 20 25 30 Phe Val Ser Trp Tyr Gln Gln Leu Pro Gly Arg Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asp Lys Arg Asp Leu Gly Ile Pro Asp Arg Phe Ser 50 55 60 Ala Ser Ile Ser Ser Thr Ser Ala Ala Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Thr Ser Leu 85 90 95 Thr Val Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 108 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> ILT70083 VLCDR1 <400> 108 Ser Gly Ser Ala Ser Asn Ile Gly Ser Asn Phe Val Ser 1 5 10 <210> 109 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ILT70083 VLCDR2 <400> 109 Asp Asn Asp Lys Arg Asp Leu 1 5 <210> 110 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70083 VLCDR3 <400> 110 Gly Thr Trp Asp Thr Ser Leu Thr Val Gly Val 1 5 10 <210> 111 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ILT70083.1 VH <400> 111 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggctg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaggcggaca 300 tattactatg atagtgatgg tcactcggat gtttttgata tttggggccg gggcaccctg 360 gtcaccgtct cgagt 375 <210> 112 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.1 VH <400> 112 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Trp Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Thr Tyr Tyr Tyr Asp Ser Asp Gly His Ser Asp Val Phe 100 105 110 Asp Ile Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 113 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.1 VHCDR1 <400> 113 Ser Tyr Ala Met Ser 1 5 <210> 114 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.1 VHCDR2 <400> 114 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 115 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.1 VHCDR3 <400> 115 Arg Thr Tyr Tyr Tyr Asp Ser Asp Gly His Ser Asp Val Phe Asp Ile 1 5 10 15 <210> 116 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> ILT70083.1 VL <400> 116 cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60 tcctgttctg gaagcgcctc caatattggg agtaattttg tgtcctggta ccaacaactc 120 cccgggacag cccccaaact cctcatttat gacaatgata aacgagactt agggattcct 180 gaccgcttct ctgcctccat ctcttccacg tcagccaccc tgggcatcac cggactccag 240 actggggacg aggccgatta ttactgcgga acatgggaca ccagtctgac tgttggggtt 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> 117 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.1 VL <400> 117 Gln Ser Val Val Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ala Ser Asn Ile Gly Ser Asn 20 25 30 Phe Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asp Lys Arg Asp Leu Gly Ile Pro Asp Arg Phe Ser 50 55 60 Ala Ser Ile Ser Ser Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Thr Ser Leu 85 90 95 Thr Val Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 118 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.1 VLCDR1 <400> 118 Ser Gly Ser Ala Ser Asn Ile Gly Ser Asn Phe Val Ser 1 5 10 <210> 119 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.1 VLCDR2 <400> 119 Asp Asn Asp Lys Arg Asp Leu 1 5 <210> 120 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.1 VLCDR3 <400> 120 Gly Thr Trp Asp Thr Ser Leu Thr Val Gly Val 1 5 10 <210> 121 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ILT70083.2 VH <400> 121 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggctg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaggcggaca 300 tattactatg atagtgatgg tcactcggat gtttttgata tttggggccg gggcaccctg 360 gtcaccgtct cgagt 375 <210> 122 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.2 VH <400> 122 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Trp Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Thr Tyr Tyr Tyr Asp Ser Asp Gly His Ser Asp Val Phe 100 105 110 Asp Ile Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 123 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.2 VHCDR1 <400> 123 Ser Tyr Ala Met Ser 1 5 <210> 124 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.2 VHCDR2 <400> 124 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 125 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.2 VHCDR3 <400> 125 Arg Thr Tyr Tyr Tyr Asp Ser Asp Gly His Ser Asp Val Phe Asp Ile 1 5 10 15 <210> 126 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> ILT70083.2 VL <400> 126 cagtctgtcc tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60 tcctgttctg gaagcgcctc caatattggg agtaattttg tgtcctggta ccaacaactc 120 cccgggacag cccccaaact cctcatttat gacaatgata aacgagactt agggattcct 180 gaccgcttct ctgcctccat ctcttccacg tcagccaccc tgggcatcac cggactccag 240 actggggacg aggccgatta ttactgcgga acatgggaca ccagtctgac tgttggggtt 300 ttcggcggag ggaccaagct gaccgtccta 330 <210> 127 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.2 VL <400> 127 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ala Ser Asn Ile Gly Ser Asn 20 25 30 Phe Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asp Lys Arg Asp Leu Gly Ile Pro Asp Arg Phe Ser 50 55 60 Ala Ser Ile Ser Ser Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Thr Ser Leu 85 90 95 Thr Val Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 128 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.2 VLCDR1 <400> 128 Ser Gly Ser Ala Ser Asn Ile Gly Ser Asn Phe Val Ser 1 5 10 <210> 129 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.2 VLCDR2 <400> 129 Asp Asn Asp Lys Arg Asp Leu 1 5 <210> 130 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.2 VLCDR3 <400> 130 Gly Thr Trp Asp Thr Ser Leu Thr Val Gly Val 1 5 10 <210> 131 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> ILT70083.3 VH <400> 131 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180 gcagactccg tgaagggctg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaggcggaca 300 tattactatg atagtgatgg tcactcggat gtttttgata tttggggccg gggcaccctg 360 gtcaccgtct cgagt 375 <210> 132 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.3 VH <400> 132 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Trp Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Thr Tyr Tyr Tyr Asp Ser Asp Gly His Ser Asp Val Phe 100 105 110 Asp Ile Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 133 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.3 VHCDR1 <400> 133 Ser Tyr Ala Met Ser 1 5 <210> 134 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.3 VHCDR2 <400> 134 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 135 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> ILT70083.3 VHCDR3 <400> 135 Arg Thr Tyr Tyr Tyr Asp Ser Asp Gly His Ser Asp Val Phe Asp Ile 1 5 10 15 <210> 136 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> ILT70083.3 VL <400> 136 cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc 60 tcctgttctg gaagcgcctc caatattggg agtaatttt...

Claims

**Claim 1** A method for detecting ILT7 expression in a sample, comprising: detecting the binding of an anti-ILT7 antibody in a sample from a subject in need of treatment; wherein the anti-ILT7 antibody comprises complementarity determining regions (CDRs) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NOs: 203, 204, 205, 208, 209, and 210, respectively. **Claim 2** The method according to claim 1, further comprising comparing the detected ILT7 expression in the sample from the subject in need of treatment with the level of ILT7 expression in a healthy subject. **Claim 3** The method according to claim 1, wherein the sample is selected from the group consisting of cells, tissues, and blood. **Claim 4** The method according to claim 3, wherein the sample is a tissue, and further comprising determining the distribution of ILT7 expression in the tissue. **Claim 5** The method according to claim 3, wherein the sample is blood. **Claim 6** The method according to claim 1, wherein the anti-ILT7 antibody is coupled to a detectable substance. **Claim 7** The method according to claim 6, wherein the detectable substance is selected from the group consisting of an enzyme, a hapten family, a fluorescent material, a luminescent material, and a radioactive material. **Claim 8** The method according to claim 7, wherein the detectable substance is an enzyme, and the enzyme is selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase. **Claim 9** The method according to claim 7, wherein the detectable substance is a hapten family, and the hapten family is selected from the group consisting of streptavidin, biotin, and avidin. **Claim 10** The method according to claim 7, wherein the detectable substance is a fluorescent material, and the fluorescent material is selected from the group consisting of umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. **Claim 11** The method according to claim 7, wherein the detectable substance is a luminescent material, and the luminescent material is selected from the group consisting of luminol, luciferase, luciferin, and aequorin. **Claim 12** The detectable substance is a radioactive material, and the radioactive material is 125 I, 131 I, 35 S, and 3 H, and the method according to claim 7, which is selected from the group consisting of **Claim 13**: The method according to claim 1, wherein the anti-ILT7 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and VL regions each comprise an amino acid sequence that is at least 95% identical to SEQ ID NO: 202 and SEQ ID NO: 207, respectively. **Claim 14**: The method according to claim 1, wherein the subject in need of treatment has an autoimmune disease or condition. **Claim 15**: The method according to claim 14, wherein the autoimmune disease or condition is selected from the group consisting of myositis, diabetes, Hashimoto's disease, autoimmune adrenal insufficiency, erythroblastosis, multiple sclerosis, rheumatic carditis, systemic lupus erythematosus, psoriasis, rheumatoid arthritis, chronic inflammation, Sjögren's syndrome, chronic rheumatism, and scleroderma. **Claim 16**: The method according to claim 1, wherein the subject in need of treatment has a disease associated with ILT7-expressing cells. **Claim 17**: The method according to claim 15, wherein the autoimmune disease is myositis, and the myositis is selected from the group consisting of polymyositis, dermatomyositis, inclusion body myositis, and juvenile myositis. **Claim 18**: A method for detecting plasmacytoid dendritic cells in a sample, comprising: detecting the binding of an anti-ILT7 antibody in a sample from a subject in need of treatment; and the anti-ILT7 antibody comprises complementarity determining regions (CDRs) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences of SEQ ID NO: 203, 204, 205, 208, 209, and 210, respectively. **Claim 19**: The method according to claim 18, wherein the anti-ILT7 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and VL regions each comprise an amino acid sequence that is at least 95% identical to SEQ ID NO: 202 and SEQ ID NO: 207, respectively. **Claim 20**: The method according to claim 18, wherein the subject has an autoimmune disease or condition selected from the group consisting of myositis, diabetes, Hashimoto's disease, autoimmune adrenal insufficiency, erythroblastosis, multiple sclerosis, rheumatic carditis, systemic lupus erythematosus, psoriasis, rheumatoid arthritis, chronic inflammation, Sjögren's syndrome, chronic rheumatism, and scleroderma. **Claim 21**: The method according to claim 18, wherein the subject in need of treatment has a disease associated with ILT7-expressing cells. **Claim 22**: The method according to claim 20, wherein the autoimmune disease is myositis, and the myositis is selected from the group consisting of polymyositis, dermatomyositis, inclusion body myositis, and juvenile myositis.

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