ILT7 binding molecules and methods of using the same

Abstract

To provide ILT7 binding molecules, e.g., anti-ILT7 antibodies, and methods for treating or preventing conditions and diseases associated with ILT7-expressing cells such as autoimmune diseases.SOLUTION: There are provided an isolated ILT7 binding protein that can bind to the same ILT7 epitope as an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL) having specific sequences, an isolated host cell producing the isolated ILT7 binding molecule, a polynucleotide encoding the ILT7 binding molecule, and a vector.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] References to electronically submitted sequence listings The contents of the Sequence Listing filed with this application and submitted electronically as an ASCII text file (Name: 2943_083STR0_SeqListing_ST25.txt, Size: 143,686 bytes, Created: March 3, 2016) are incorporated herein by reference in their entirety.

[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 the antibodies and fragments, and methods of treating or preventing autoimmune diseases and conditions associated with ILT7-expressing cells. [Background technology]

[0003] Plasmacytoid dendritic cells (pDCs) are a distinct population of dendritic cells (DCs) in peripheral blood and secondary lymphoid organs, constituting only approximately 0.1–0.5% of peripheral blood mononuclear cells (PBMCs). However, these cells are particularly important regulators of the immune system because they are a major source of type I interferons (IFNs). Type I IFNs promote the function 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 activity. However, pDCs and type I IFNs are also thought to play a role in the development of autoimmune diseases such as systemic lupus erythematosus, chronic rheumatoid disease, and psoriasis. Therefore, understanding how to modulate molecular pathways involved in IFN release is useful for controlling immune responses and treating and preventing disease.

[0004] pDCs release IFN in response to nucleic acids sensed by the Toll-like receptors (TLRs), TLR7 and TLR9, which are expressed on their surface. TLR responses are 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 the ILT7 ligand, bone marrow stromal cell antigen 2 (BST2), and the transmembrane domain of ILT7 contains positively charged residues that enable it to form a complex with FcεRIγ. The interaction between BST2 and ILT7 has been hypothesized to negatively regulate the innate immune function of pDCs, potentially as a negative feedback mechanism. Furthermore, in vitro cross-linking of ILT7 with antibodies 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 modulating pDC activity and IFN release are needed to treat and prevent diseases such as, for example, autoimmune diseases. Summary of the Invention [Problem 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 problem]

[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 a heavy chain variable region (VH) of SEQ ID NO: 202 and a 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 a VH of SEQ ID NO: 202 and a 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, HDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the sequences of SEQ ID NOs: 203, 204, 205, 208, 209, and 210, respectively.

[0010] In one example, an 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 selected from the group consisting of 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:13 137, 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, 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, and SEQ ID NO:242 and SEQ ID NO:247, respectively.

[0015] In one example, the isolated ILT7 binding protein is selected from the group consisting of 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: 149, respectively, SEQ ID NO: 150 and SEQ ID NO: 151, respectively, SEQ ID NO: 152 and SEQ ID NO: 153, respectively, SEQ ID NO: 154 and SEQ ID NO: 155, respectively, SEQ ID NO: 156 and SEQ ID NO: 157, respectively, SEQ ID NO: 157 and SEQ ID NO: 158, respectively, SEQ ID NO: 159 and SEQ ID NO: 160, respectively, SEQ ID NO: 161 and SEQ ID NO: 162, respectively, SEQ ID NO: 163 and SEQ ID NO: 164, respectively, SEQ ID NO: 165 and SEQ ID NO: and SEQ ID NO:137, 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, and SEQ ID NO:242 and SEQ ID NO:247, respectively.

[0016] In one example, the isolated ILT7 binding protein comprises a CDR selected from the group consisting of SEQ ID NOs: 13, 14, 15, 18, 19, and 20, respectively; SEQ ID NOs: 23, 24, 25, 28, 29, and 30, respectively; SEQ ID NOs: 33, 34, 35, 38, 39, and 40, respectively; SEQ ID NOs: 103, 104, 105, 108, 109, and 110, respectively; SEQ ID NOs: 213, 214, 215, 218, 219, and 220, respectively; SEQ ID NOs: 223, 224, 225, 228, 229, and 230, respectively; SEQ ID NOs: 233, 234, 235, 238, 239, and 240, respectively; and SEQ ID NOs: 243, 244, 245, 248, 249, and 250, respectively. It is an ILT7 binding protein that contains HCDR1, HDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0017] In one example, the ILT7 binding protein is selected from the group consisting of 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: 133, respectively and VH and VL sequences that are at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:137, 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.

[0018] In one example, the VH and VL are selected from the group consisting of 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:123, respectively, SEQ ID NO:124 and SEQ ID NO:125, respectively, SEQ ID NO:126 and SEQ ID NO:127, respectively, SEQ ID NO:128 and SEQ ID NO:129, respectively, SEQ ID NO:130 and SEQ ID NO:131, respectively, SEQ ID NO:132 and SEQ ID NO:133, respectively, SEQ ID NO:134 and SEQ ID NO:135, respectively, SEQ ID NO:136 and SEQ ID NO:137, respectively, SEQ ID NO:138 and SEQ ID NO:139, respectively, SEQ ID NO:140 and SEQ ID NO:141, respectively, SEQ ID NO:142 and SEQ ID NO:143, respectively, SEQ ID NO:144 and SEQ ID NO:145, respectively, SEQ ID NO:146 and SEQ ID NO:147, respectively, SEQ ID NO:148 and SEQ ID NO:149, respectively, SEQ ID NO:150 and SEQ ID Sequence number 127 includes 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.

[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 antigen-binding fragment thereof. In one example, the antibody or 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 ILT7.

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

[0025] In one example, the ILT7 binding protein comprises a murine, human, chimeric, humanized, or resurfaced antibody or 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-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, 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) 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.

[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 a 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 regulatory sequence. In one example, an antibody or antigen-binding fragment thereof comprising the 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 a 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 a VH and a polynucleotide comprising a nucleic acid encoding a 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 of 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 of detecting ILT7 expression in a sample, the method comprising: (a) contacting the sample with an ILT7 binding molecule provided herein; and (b) detecting binding of the binding molecule in the sample.

[0042] In one example, provided herein is a method of detecting plasmacytoid dendritic cells, the method comprising: (a) contacting a sample containing the cells with an ILT7-binding molecule provided herein; and (b) detecting 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 of reducing IFNalpha release from plasmacytoid dendritic cells, the method comprising contacting 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 of 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 (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 chronic 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 comprising the sequences of SEQ ID NOs: 203, 204, 205, 208, 209, and 210, respectively. [Item 4] 4. The isolated ILT7 binding protein of 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] 5. The isolated ILT7-binding protein of item 4, comprising a VH comprising SEQ ID NO: 202 and a VL comprising SEQ ID NO: 207. [Item 6] 1. An isolated ILT7 binding protein comprising a VH comprising SEQ ID NO: 202. [Item 7] 1. 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; 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 a VH and VL selected from the group consisting of: [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; 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 to ILT7 of an antibody comprising a VH and a VL selected from the group consisting of: [Item 10] SEQ ID NOs: 13, 14, 15, 18, 19, and 20, respectively; SEQ ID NOs: 23, 24, 25, 28, 29, and 30, respectively; SEQ ID NOs: 33, 34, 35, 38, 39, and 40, respectively; SEQ ID NOs: 103, 104, 105, 108, 109, and 110, respectively; SEQ ID NOs: 213, 214, 215, 218, 219, and 220, respectively; SEQ ID NOs: 223, 224, 225, 228, 229, and 230, respectively; SEQ ID NOs: 233, 234, 235, 238, 239, and 240, respectively; and SEQ ID NOs: 243, 244, 245, 248, 249, and 250, respectively 1. An isolated ILT7 binding molecule comprising CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 selected from the group consisting of: [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 11. The isolated ILT7 binding protein of any one of items 8 to 10, comprising a VH and VL that are at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to [Item 12] The 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 SEQ ID NO: 242 and SEQ ID NO: 247, respectively 12. The ILT7-binding molecule of item 11, comprising: [Item 13] 14. 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. 15. 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. 15. The isolated ILT7-binding molecule of any one of items 1 to 14, comprising an antibody or antigen-binding fragment thereof. [Item 16] 16. The isolated ILT7-binding molecule of paragraph 15, wherein the antibody or antigen-binding fragment thereof is hypofucosylated. [Item 17] 17. The isolated ILT7-binding molecule of any one of items 8 to 16, which binds to the Ig1 region of ILT7. [Item 18] 17. The isolated ILT7-binding molecule of any one of paragraphs 8 to 16, which binds to the Ig2 region of ILT7. [Item 19] 19. The isolated ILT7-binding molecule of any one of items 1 to 18, which binds to human and cynomolgus ILT7. [Item 20] 20. The isolated ILT7-binding molecule of any one of paragraphs 1 to 19, which inhibits interferon (IFN) alpha release from peripheral blood mononuclear cells (PBMC). [Item 21] 21. The isolated ILT7-binding molecule of any one of paragraphs 1 to 20, which has ADCC activity against plasmacytoid dendritic cells (pDCs) in PBMCs. [Item 22] 22. The isolated ILT7-binding molecule of any one of paragraphs 1 to 21, comprising a murine, human, chimeric, humanized, or resurfaced antibody or antigen-binding fragment thereof. [Item 23] 23. The isolated ILT7-binding molecule of any one of paragraphs 1 to 22, comprising an antibody, Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc. [Item 24] 24. The isolated ILT7-binding molecule of any one of items 1 to 23, comprising a monoclonal antibody or an antigen-binding fragment thereof. [Item 25] (a) IgA constant domain, (b) IgD constant domain, (c) IgE constant domain, (d) IgG1 constant domain; (e) IgG2 constant domain, (f) IgG3 constant domain; (g) an IgG4 constant domain, and (h) IgM constant domain 25. The isolated ILT7-binding molecule of any one of paragraphs 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) Ig lambda constant domain 26. The isolated ILT7-binding molecule of any one of paragraphs 1 to 25, comprising a light chain immunoglobulin constant domain selected from the group consisting of: [Item 27] 27. The isolated ILT7-binding molecule of any one of paragraphs 1 to 26, comprising a human IgG1 constant domain and a human lambda constant domain. [Item 28] 28. An isolated host cell producing the binding molecule of any one of items 1 to 27. [Item 29] 1. 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 a 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] 30. The polynucleotide of item 29, comprising a sequence 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. [Item 31] 1. 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 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] 32. The polynucleotide of item 31, comprising a sequence 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. [Item 33] 33. The polynucleotide of any one of items 29 to 32, wherein the nucleic acid is operably linked to a regulatory sequence. [Item 34] 34. The polynucleotide of any one of items 29 to 33, wherein an antibody or antigen-binding fragment thereof comprising the VH or VL can specifically bind to ILT7. [Item 35] 28. A polynucleotide encoding the ILT7-binding molecule of 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] 40. The host cell of item 38 or 39, which is a mammalian host cell. [Item 41] 41. The mammalian host cell of paragraph 40, which is an NS0 mouse myeloma cell, a PER.C6® human cell, or a Chinese hamster ovary (CHO) cell. [Item 42] 42. The host cell of any one of items 38 to 41, which lacks the enzyme alpha-1,6-fucosyltransferase. [Item 43] 43. A method for producing an anti-ILT7 binding molecule, the method comprising culturing the host cell according to any one of items 38 to 42, and recovering the binding molecule. [Item 44] 44. An anti-ILT7 binding molecule produced by the method of item 43. [Item 45] 10. A method for detecting ILT7 expression in a sample, the method comprising (a) contacting the sample with an ILT7-binding molecule according to any one of items 1 to 27 or 44, and (b) detecting binding of the binding molecule in the sample. [Item 46] 10. A method for detecting plasmacytoid dendritic cells, the method comprising (a) contacting a sample containing the cells with an ILT7-binding molecule according to any one of items 1 to 27 or 44, and (b) detecting binding of the binding molecule in the sample. [Item 47] 1. A pharmaceutical composition comprising (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. [Item 48] 1. A method for reducing IFN-alpha release from plasmacytoid dendritic cells, the method comprising contacting plasmacytoid dendritic cells with the binding molecule of any one of items 1 to 27 or 44, the polynucleotide of any one of items 29 to 35, the vector of item 36, the polypeptide of item 37, the host cell of any one of items 28 or 38 to 42, or the composition of item 47. [Item 49] 1. A method for treating a human subject having an autoimmune disease, comprising administering to the subject an effective amount of the binding molecule of any one of items 1 to 27 or 44, the polynucleotide of any one of items 29 to 35, the vector of item 36, the polypeptide of item 37, the host cell of any one of items 28 or 38 to 42, or the composition of item 47. [Item 50] 1. A method for preventing an autoimmune disease in a human subject, comprising administering to a subject an effective amount of the binding molecule of any one of items 1 to 27 or 44, the polynucleotide of any one of items 29 to 35, the vector of item 36, the polypeptide of item 37, the host cell of item 28 or any one of items 38 to 42, or the composition of item 47. [Item 51] 51. The method of item 49 or 50, wherein the autoimmune disease is systemic lupus erythematosus. [Item 52] 51. The method of item 49 or 50, wherein the autoimmune disease is chronic rheumatoid arthritis. [Brief explanation of the drawings]

[0047] [Figure 1A]

[0023] Figure 1A shows the variable heavy chain (1A) and variable light chain (1B) sequence alignment of SBI28 (#28), 10D10, and 7C7 antibodies. Shaded areas indicate CDR sequences. Boxes represent mutations introduced into 10D10 to generate 7C7. [Figure 1B] This is a continuation of Figure 1A. [Figure 2] Figure 1 shows binding of ILT7 antibodies 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] FIG. 1 shows binding of ILT7 antibodies and a negative control antibody (R437) to CT-125 cells expressing cynomolgus ILT7 as determined by flow cytometry. [Figure 4] FIG. 1 shows the ADCC efficacy of ILT7 antibody and negative control antibody (R437) against human ILT7-expressing cells. [Figure 5] FIG. 1 shows the ADCC efficacy of ILT7 antibody and negative control antibody (R437) against cynomolgus monkey ILT7-expressing cells. [Figure 6A] FIG. 1 shows binding of ILT7 antibody and negative control antibody (R437) to plasmacytoid dendritic cells (pDC) in peripheral blood mononuclear cells (PBMC). [Figure 6B] This is a continuation of Figure 6A. [Figure 7] Figure 1 shows binding of underfucosylated ILT7 antibodies and their parental antibodies to CT-550 cells expressing human (left panel) and cynomolgus monkey (right panel) ILT7 as determined by flow cytometry. [Figure 8] FIG. 1 shows the ADCC efficacy of underfucosylated ILT7 antibodies and their parental antibodies against human (left panel) and cynomolgus monkey (right panel) ILT7-expressing cells. [Figure 9A] 9A and 9B show variable heavy (9A) and variable light (9B) chain sequence alignments of seven ILT70080 variants, with the closest germline sequences (IGHV1-69*01 and IGLV3-21*01) also shown in the alignment. [Figure 9B] This is a continuation of Figure 9A. [Figure 10A] Figure 10A shows the variable heavy (10A) and variable light (10B) chain sequence alignment of nine ILT70083 variants, with the closest germline sequences (IGHV3-23*01 and IGLV1-51*01) also shown in the alignment. [Figure 10B] This is a continuation of Figure 10A. [Figure 11] Figure 1 shows binding of ILT70080 variants to cells expressing human ILT7 (CT-550, upper panel) and cells expressing cynomolgus ILT7 (CT-125, lower panel). [Figure 12] Figure 1 shows binding of ILT70083 variants to cells expressing human ILT7 (top panel) or cynomolgus ILT7 (bottom panel). [Figure 13] FIG. 1 shows the ADCC efficacy of ILT70080 variant antibodies against human ILT7-expressing cells. [Figure 14] FIG. 1 shows the ADCC efficacy of ILT70083 variant antibodies against human ILT7-expressing cells. [Figure 15]Figure 1 shows binding of underfucosylated ILT70080.6 and ILT70083 antibodies to human (left panel) and cynomolgus monkey (right panel) ILT7-expressing cells. [Figure 16] FIG. 1 shows the ADCC activity of underfucosylated ILT70080.6 and ILT70083 antibodies against human (left panel) and cynomolgus monkey (right panel) ILT7-expressing cells. [Figure 17] Figure 1 shows cytotoxicity (left) and IFN-α secretion (right) of human PBMCs exposed to underfucosylated ILT70080.6 and ILT70083 antibodies. [Figure 18] Figure 1 shows binding of underfucosylated ILT70137 to cells expressing human ILT7 (left panel) or cynomolgus ILT7 (right panel). Circles indicate underfucosylated ILT70137 and triangles indicate control. [Figure 19] Figure 1 shows the ADCC activity of underfucosylated ILT70137 against cells expressing human ILT7 (left panel) or cynomolgus ILT7 (right panel). Triangles indicate underfucosylated ILT70137, and circles indicate the control. [Figure 20] FIG. 1 shows the ADCC activity of underfucosylated ILT70137 by measuring the inhibition of IFN-alpha production as an indirect assessment of the ability of an antibody to induce ADCC of peripheral blood mononuclear cells (PBMC) in vitro. [Figure 21] Figure 1 shows binding of underfucosylated ILT70137 to human primary plasmacytoid dendritic cells (pDCs). [Figure 22] Figure 1 shows pDC depletion in cynomolgus monkeys treated with de-fucosylated 7C7 or de-fucosylated ILT70137. The arrows below the graph indicate the time points of antibody administration. [Figure 23] Figure 1 shows IFNα production following treatment with underfucosylated 7C7 or underfucosylated ILT70137. The arrows below the graph indicate the time of antibody administration. DETAILED DESCRIPTION OF THE INVENTION

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

[0049] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as 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, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids is 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 products of post-expression modifications of polypeptides, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant technology, but need not necessarily be translated from a designated nucleic acid sequence. Polypeptides can be made by any method, including chemical synthesis.

[0050] Polypeptides of the present invention can be polypeptides 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 in size. Polypeptides can have a defined three-dimensional structure, but do not necessarily have such a structure. Polypeptides that have a defined three-dimensional structure are referred to as folded, while polypeptides that do not have a defined three-dimensional structure, but rather can adopt a number of different conformations, are referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety attached to the protein via an oxygen- or nitrogen-containing side chain of an amino acid residue, such as a serine or asparagine residue.

[0051] By "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural environment. A particular level of purification is not required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for 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 also included as polypeptides of the present invention. The terms "fragment," "variant," "derivative," and "analog," when referring to anti-ILT7 antibodies or antibody polypeptides of the present invention, include any polypeptide that retains at least some 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 and 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-described fragments, as well as polypeptides having altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be generated using mutagenesis techniques known in the art. Variant polypeptides can contain 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 chemically derivatized by reaction of a functional side group. Similarly, those peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids are also included as "derivatives." For example, 4-hydroxyproline can be substituted for proline, 5-hydroxylysine can be substituted for lysine, 3-methylhistidine can be substituted for histidine, homoserine can be substituted for serine, and ornithine can be substituted for lysine. Derivatives of the anti-ILT7 antibodies and antibody polypeptides of the invention can include polypeptides that have been altered to exhibit additional features not found in the reference antibody or antibody polypeptide of the invention.

[0053] The term "polynucleotide" is intended to encompass a singular nucleic acid as well as multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). Polynucleotides can contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. An "isolated" nucleic acid or polynucleotide is intended to refer to 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, e.g., an antibody or 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 heterologous host cells 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, the polynucleotide or nucleic acid may be or 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 translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered part of the coding region; however, any adjacent sequences, such as promoters, ribosome binding sites, transcription terminators, introns, etc., are not part of the coding region. Two or more coding regions of the invention can be present in a single polynucleotide construct, e.g., a single vector, or in separate polynucleotide constructs, e.g., separate (different) vectors. Furthermore, any vector can contain a single coding region or can include 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, vectors, polynucleotides, or nucleic acids of the invention can encode heterologous coding regions, fused or unfused to the nucleic acid encoding an anti-ILT7 antibody or fragment, variant, or derivative thereof. Heterologous coding regions include specialized elements or motifs, such as, but not limited to, secretory signal peptides or heterologous functional domains.

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

[0056] A variety of transcription control regions are known to those skilled in the art. These include, but are not limited to, transcription control regions that function in vertebrates, such as promoter and enhancer segments from cytomegalovirus (immediate-early promoter with intron A), simian virus 40 (early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include regions derived from vertebrate genes such as 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 transcription control regions include tissue-specific promoters and enhancers, and 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, including, but not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly IRES, also called internal ribosome entry sites, or CITE sequences).

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

[0059] Polynucleotide and nucleic acid coding regions of the present invention can be associated with additional coding regions encoding secretory or signal peptides that direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once export of the growing protein chain from the rough endoplasmic reticulum has begun. 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 that 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. 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 term "binding molecule" or "antigen-binding molecule" of the present invention refers, in its broadest sense, to a molecule that specifically binds to an antigenic determinant. In one embodiment, a binding molecule specifically binds to ILT7, e.g., full-length ILT7 or mature ILT7. In another embodiment, a binding molecule of the present invention is an antibody or an antigen-binding fragment thereof. In another embodiment, a binding molecule of the present invention comprises at least one heavy or light chain CDR of a reference antibody molecule. In another embodiment, a binding molecule of the present invention comprises at least two CDRs from one or more reference antibody molecules. In another embodiment, a binding molecule of the present invention comprises at least three CDRs from one or more reference antibody molecules. In another embodiment, a binding molecule of the present invention comprises at least four CDRs from one or more reference antibody molecules. In another embodiment, a binding molecule of the present invention comprises at least five CDRs from one or more reference antibody molecules. In another embodiment, a 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" refers to an immunoglobulin molecule that recognizes and specifically binds to 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 containing an antibody, and any other modified immunoglobulin molecule so long as the antibody exhibits the desired biological activity. An antibody may belong to any of the five major immunoglobulin classes, namely, IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or can be conjugated to other molecules such as toxins, radioisotopes, etc.

[0062] The term "antibody fragment" or "antibody fragment thereof" refers to a portion of an intact antibody. An "antigen-binding fragment" or "antigen-binding fragment thereof" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment may contain the antigenic determining variable regions of the 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, a "human" or "fully human" antibody includes antibodies having the amino acid sequence of a human immunoglobulin, including antibodies isolated from human immunoglobulin libraries, as described below and, for example, in U.S. Patent No. 5,939,598 to Kucherlapati et al., or isolated from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins. 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 using antibody libraries derived from human immunoglobulin sequences as 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 make and use antibodies include those described in 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), and Burton et al., Advances in Immunology 57:191-280, each of which is incorporated by reference herein in its entirety. (1994), PCT Application No. PCT / GB91 / 01134, PCT Application Publication No. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401, and U.S. Patent Nos. 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081; Included are methods disclosed in Nos. 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.

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

[0066] Additional technologies available in the field of antibody engineering make it possible to isolate human antibodies or fragments thereof. For example, human hybridomas can be generated as described by Kontermann and Sefan. Antibody Engineering, Springer Laboratory Manuals (2001). Fully human antibodies can also be produced by various display technologies, such as phage display or other viral display systems. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. For example, DNA sequences encoding VH and VL regions are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of lymphoid tissues) or a synthetic cDNA library. In certain embodiments, the DNA encoding the VH and VL regions is linked 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 is 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 antigen, for example, using labeled antigen or antigen bound or captured to a solid surface or bead.

[0067] A "human" or "fully human" antibody also includes antibodies comprising at least the variable domain of a heavy chain, or at least the variable domains of a heavy and light chain, wherein the variable domain(s) have the amino acid sequence of a human immunoglobulin variable domain(s).

[0068] "Human" or "fully human" antibodies also include such "human" or "fully human" antibodies that comprise, consist essentially of, or consist of variants (including derivatives) of the antibody molecules (e.g., VH and / or VL regions) described herein, where the antibodies or antigen-binding fragments, variants, or derivatives thereof immunospecifically bind to an ILT7 polypeptide or a fragment or variant thereof. Mutations can be introduced into the nucleotide sequence encoding a human anti-ILT7 antibody using standard techniques known to those skilled in the art, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. A variant (including a derivative) may encode fewer than 50 amino acid substitutions, fewer than 40 amino acid substitutions, fewer than 30 amino acid substitutions, fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to a 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 more 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 can be 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 of "human" or "fully human" antibodies (or derivatives thereof) can also be referred to as "optimized" or "antigen-binding optimized" human or fully human antibodies, and include antibodies with improved affinity for the antigen.

[0070] Basic immunoglobulin structure in vertebrate systems is relatively well understood, see, e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press).

[0071] As discussed in more detail below, the term "immunoglobulin" encompasses a wide variety of biochemically distinguishable polypeptide classes. Those skilled in the art will recognize that heavy chains, along with some subclasses thereof (e.g., γ1-γ4), are classified as gamma, mu, alpha, delta, or epsilon. It is the nature of this chain that determines the "class" of an antibody as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc., are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to those of skill in the art in light of the present disclosure and, therefore, are within the scope of the present invention. While the following discussion generally focuses on the IgG class of immunoglobulin molecules, all immunoglobulin classes are expressly within the scope of the present invention. For IgG, a typical immunoglobulin molecule contains two identical light polypeptide chains with a molecular weight of approximately 23,000 daltons and two identical heavy polypeptide chains with a molecular weight of 53,000-70,000. The four chains are typically joined by disulfide bonds in a "Y" configuration, with the light chains surrounding the heavy chains, which begin at the mouth of the "Y" and continue through the variable region.

[0072] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can bind to either kappa or lambda light chains. Generally, light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. In the heavy chain, the amino acid sequence runs from the N-terminus at the forked tips of the Y to the C-terminus at the bottom of each chain.

[0073] The base of the antibody "Y" is called the Fc (fragment crystallizable) region and is composed of two heavy chains that contribute two or three constant domains depending on the class of antibody. The Fc region thus binds to a specific class of Fc receptor and other immune molecules, such as complement proteins. Both light and heavy chains can be divided 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 (VL or VK) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light (CL) and heavy (CH1, CH2, or CH3) chains confer important biological properties, such as secretion, placental transport, Fc receptor binding, complement binding, etc. For convenience, the numbering of 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, the C-terminal portion is the constant region, and the CH3 and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0074] As described above, the variable regions enable an antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL and VH domains, or a subset of complementarity-determining regions (CDRs), within these variable domains of an antibody combine to form the variable regions that define a three-dimensional antigen-binding site. This quaternary structure of an antibody forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs present on each of the VH and VL chains. In some cases, for example, in certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, the complete immunoglobulin molecule consists only of heavy chains and does not have light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

[0075] In naturally occurring antibodies, the six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are short, noncontiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional structure in an aqueous environment. The remaining amino acids in the antigen-binding domain, called "framework" regions, exhibit less inter-molecular variability. The framework regions mostly adopt a β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, the framework regions act as a scaffold that provides proper orientation of the CDRs through interchain non-covalent interactions. The antigen-binding domain formed by the arranged CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody to its cognate epitope. The amino acids that comprise 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 have been precisely defined (see below).

[0076] When there are more than one definition for a term used and / or accepted in the art, the definition of the term used herein is intended to encompass all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe the non-contiguous 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) US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest," and Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), both of which are incorporated herein by reference; however, when compared with each other, the definitions include overlapping or subsets of amino acid residues. Nevertheless, the application of either definition to refer to the CDR of an antibody or variant thereof is intended to be within the scope of the term defined and used herein. IMGT (ImMunoGeneTics) also provides a numbering system for immunoglobulin variable regions, including CDRs. See, for example, Lefranc, MP 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 characterization of hypervariable loops, allowing for easy comparison of variable and CDR regions across all species. The appropriate amino acid residues encompassing the CDRs defined by each of the above-cited references are set forth in Table 1 below for comparison. The exact residue numbers encompassing a particular CDR may vary depending on the sequence and size of the CDR. One skilled in the art can routinely determine which residues constitute a particular CDR given the amino acid sequence of the variable region of an 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 reliance 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] Antibodies or antigen-binding fragments, variants, or derivatives thereof of the present invention include, but are not limited to, polyclonal antibodies, monoclonal antibodies, murine, 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-linked Fvs (sdFv), fragments comprising either the VL or VH domains, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (e.g., anti-Id antibodies directed against 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. Immunoglobulin or antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.), or subclass of immunoglobulin molecule.

[0080] As used herein, the term "heavy chain portion" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion comprises 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 can comprise a polypeptide chain comprising a CH1 domain, a polypeptide chain comprising a CH1 domain, at least a portion 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 portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present invention comprises a polypeptide chain comprising a CH3 domain. Furthermore, a binding polypeptide for use in the present invention can lack at least a portion of a CH2 domain (e.g., all or a portion of the CH2 domain). As noted above, it will be understood by those skilled in the art that these domains (eg, heavy chain portions) can be modified to vary in amino acid sequence from naturally occurring immunoglobulin molecules.

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

[0082] The heavy chain portions of the binding molecules for use in the diagnostic and treatment methods disclosed herein can be derived from different immunoglobulin molecules. For example, the heavy chain portion of the polypeptide can be derived from a C IgG1 molecule. H1 In another example, a heavy chain portion may comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion may comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.

[0083] As used herein, the term "light chain portion" includes amino acid sequences derived from an immunoglobulin light chain, such as a kappa or lambda light chain. A light chain portion can include at least one of a VL or CL domain.

[0084] Anti-ILT7 antibodies disclosed herein, or antigen-binding fragments, variants, or derivatives thereof, may be described or specified in terms of the epitope or portion of the antigen, e.g., the target polypeptide (e.g., full-length or mature ILT7) disclosed herein that they recognize or specifically bind. The portion of the target polypeptide that specifically interacts with the antigen-binding domain of the 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 antigen. Furthermore, it should be noted that an "epitope" on a target polypeptide may be or include non-polypeptide elements; for example, an epitope may include a carbohydrate side chain.

[0085] The minimum size of a peptide or polypeptide epitope of an antibody is believed to be about 4 to 5 amino acids. A peptide or polypeptide epitope may contain at least 7, at least 9, or at least about 15 to about 30 amino acids. Because the CDRs can recognize an antigenic peptide or polypeptide in its three-dimensional form, the amino acids comprising the epitope do not necessarily have to be contiguous and, in some cases, may not even be present on the same peptide chain. A peptide or polypeptide epitope recognized by an 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 to about 30 contiguous or non-contiguous amino acids of ILT7.

[0086] "Specifically binds" generally means that an antibody binds to an epitope via 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 via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to determine the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" can be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" can be said to bind to epitope "C" with higher specificity than it has for the related epitope "D."

[0087] By "preferentially binds" is meant that an antibody specifically binds to an epitope more readily than it binds to a related, similar, homologous, or analogous epitope. Thus, an antibody that "preferentially binds" to a given epitope will more likely bind to that epitope than to a related epitope, even if such an antibody may cross-react with the related epitope.

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

[0089] In another non-limiting example, an antibody can be considered to bind preferentially to a first epitope if it binds to the first epitope with an off-rate (k(off)) that is lower than the antibody's k(off) for the second epitope. In another non-limiting example, an antibody can be considered to bind preferentially to a first epitope if it binds to the first epitope with an affinity that is at least one order of magnitude lower than the antibody's k(off) for the second epitope. In another non-limiting example, an antibody can be considered to bind preferentially 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 antibody's k(off) for the second epitope. An antibody or antigen-binding fragment, variant, or derivative thereof disclosed herein can be used to bind a target polypeptide disclosed herein (e.g., ILT7, e.g., human, primate, mouse, or any other combination of human, primate, and mouse ILT7), or a fragment or variant thereof, at a concentration of 5x10 -2 seconds -1 , 10 -2 seconds -1 , 5×10 -3 seconds -1 , or 10 -3 seconds -1 The antibodies of the invention can be said to bind to a target polypeptide disclosed herein (e.g., ILT7, e.g., human, primate, mouse, or any other combination of human, primate, and mouse ILT7), or a fragment or variant thereof, 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 It can be said that they bind with the following off-rate (k(off)):

[0090] The antibodies disclosed herein, or antigen-binding fragments, variants, or derivatives thereof, may bind to a target polypeptide disclosed herein (e.g., ILT7, e.g., human, primate, mouse, or any other combination of human, primate, and mouse ILT7), or a fragment or variant thereof, in an amount of 10 or more. 3 M -1 seconds -1 , 5×10 3 M -1 seconds -1 , 10 4 M -1 seconds -1 , or 5 x 10 4 M -1 seconds -1 The antibodies of the present invention can be said to bind to a target polypeptide disclosed herein (e.g., ILT7, e.g., human, primate, mouse, or any other combination of human, primate, and mouse ILT7), or a fragment or variant thereof, with an on rate (k(on)) of 10 or greater. 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , or 5 x 10 6 M -1 seconds -1 , or 10 7 M -1 seconds -1 It can bind with an on-rate (k(on)) of 1 or more.

[0091] An antibody is said to competitively inhibit the binding of a reference antibody to a given epitope if it preferentially binds to that epitope or an overlapping epitope to the extent that it blocks, to some extent, the binding of the reference antibody to the epitope. 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 the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0092] The term "affinity" as used herein refers to a measure of the strength of binding of an individual epitope to the 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. The term "avidity" as used herein refers to the overall stability of the complex between an immunoglobulin population and an antigen, i.e., the functional binding strength of an immunoglobulin mixture with an antigen. See, e.g., Harlow, pages 29-34. Avidity relates to both the affinity of individual immunoglobulin molecules in a population with a particular epitope and the binding valency of the immunoglobulin with the antigen. For example, the interaction of a bivalent monoclonal antibody with an antigen with a highly repetitive epitope structure, such as a polymer, would be a high avidity interaction.

[0093] Anti-ILT7 antibodies of the present invention, or antigen-binding fragments, variants, or derivatives thereof, can also be described or specified in terms of 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 relationship 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 induced epitope and, in some instances, may actually be a better fit than the original.

[0094] For example, certain antibodies have some degree of cross-reactivity in that they bind to epitopes that have 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 (as calculated using methods known in the art and described herein) to related but non-identical epitopes, e.g., a reference epitope. An antibody can be said to have little or no cross-reactivity if it does not bind to epitopes that have 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 (as calculated using methods known in the art and described herein) to the reference epitope. An antibody can be considered "highly specific" for a particular epitope if it does not bind to any other analogs, orthologs, or homologs of that epitope.

[0095] Anti-ILT7 binding molecules, e.g., antibodies of the invention or antigen-binding fragments, variants, or derivatives thereof, can also be described or specified in terms of their binding affinity to a polypeptide of the invention, e.g., ILT7, e.g., human, primate, mouse, or any combination of human, primate, and mouse ILT7. Useful binding affinities include those of 5×10 -2 M, 10 -2 M, 5 x 10 -3 M, 10 -3 M, 5 x 10 -4 M, 10 -4 M, 5 x 10 -5 M, 10 -5 M, 5 x 10 -6 M, 10 -6 M, 5 x 10 -7 M, 10 -7 M, 5 x 10 -8 M, 10 -8 M, 5 x 10 -9 M, 10 -9 M, 5 x 10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11M, 5 x 10 -12 M, 10 -12 M, 5 x 10 -13 M, 10 -13 M, 5 x 10 -14 M, 10 -14 M, 5 x 10 -15 M or 10 -15 An affinity having a dissociation constant or Kd less than M is included.

[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 to cynomolgus ILT7 with a dissociation constant or Kd of less than 5 nM.

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

[0098] As used herein, the term "CH2 domain" includes the portion of an antibody heavy chain molecule extending from about residue 244 to residue 360 ​​using the conventional numbering scheme (residues 244-360 in the Kabat numbering system, or residues 231-340 in the EU numbering system; see Kabat EA et al.). The CH2 domain is unique in that it is not strictly paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains in intact native IgG molecules. The CH3 domain, which extends C-terminally from the CH2 domains of IgG molecules and comprises approximately 108 residues, is also well documented.

[0099] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 and CH2 domains. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and bottom 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 using the Kabat numbering system (positions 226 or 229 in the EU numbering system).

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

[0102] As used herein, the term "engineered antibody" refers to an antibody in which either or both heavy or light chain variable domains have been altered by at least partial replacement of one or more CDRs from an antibody of known specificity, and, if necessary, partial framework region replacement and sequence changes. The CDRs may be derived from antibodies of the same class or even subclass as the antibody from which the framework regions are derived, although it is envisioned that the CDRs may be derived from antibodies of a different class or species. Engineered antibodies in which one or more "donor" CDRs from a non-human antibody of known specificity have been grafted onto human heavy or light chain framework regions are referred to herein as "humanized" antibodies. Transferring the antigen-binding capacity of one variable domain to another does not necessarily require the replacement of all CDRs with the complete CDRs from the donor variable domain. 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 of the heavy or light chain, or both, of a humanized antibody may comprise only residues of human origin, in which case these framework regions of the humanized antibody are referred to as "fully human framework regions." Alternatively, one or more residues of the framework regions of the donor variable domain can be engineered into the corresponding positions of the human framework regions of the variable domains of the heavy or light chain, or both, of the humanized antibody, if necessary to maintain proper binding or to enhance binding to the ILT7 antigen. Thus, human framework regions that have been engineered in this manner comprise a mixture of human and donor framework residues and are referred to herein as "partially human framework regions."

[0104] For example, humanization of anti-ILT7 antibodies can be performed essentially according to the method of Winter and coworkers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)) by substituting rodent or mutant rodent anti-ILT7 CDRs or CDR sequences for the corresponding sequences of a human antibody. See also U.S. Patent Nos. 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205, which are incorporated herein by reference. The resulting humanized anti-ILT7 antibody will comprise at least one rodent or mutant rodent CDR within fully human framework regions of the heavy and / or light chain variable domains of the humanized antibody. In some instances, residues within the framework regions of one or more variable domains of the humanized anti-ILT7 antibody are substituted with corresponding non-human (e.g., rodent) residues (see, e.g., U.S. Patent Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370), in which case the resulting humanized anti-ILT7 antibody will comprise partially human framework regions within the heavy and / or light chain variable domains.

[0105] Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain a desired affinity). Generally, humanized antibodies will comprise substantially all of at least one, and 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. The humanized antibody also optionally will comprise at least a portion of an immunoglobulin constant region (Fc), typically 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. Accordingly, such "humanized" antibodies can include antibodies in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies. See, e.g., U.S. Pat. Nos. 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205. See also U.S. Pat. No. 6,180,370 and International Application WO 01 / 27160, which disclose humanized antibodies and techniques for producing humanized antibodies with improved affinity for a given antigen.

[0106] As used herein, the terms "linked," "fused," and "fusion" are used interchangeably. These terms refer to the joining of two or more elements or components together by any means, including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a longer, contiguous 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 usually so joined in nature). Thus, the reading frame is created contiguous throughout the fused segments, but the segments can be physically or spatially separated, for example, by in-frame linker sequences. For example, polynucleotides encoding CDRs of immunoglobulin variable regions can be fused in-frame but separated by polynucleotides encoding at least one immunoglobulin framework region or additional CDR regions, so long as the "fused" CDRs are simultaneously translated as part of a contiguous polypeptide.

[0107] In the context of a polypeptide, a "linear sequence" or "sequence" is the order of amino acids in the polypeptide from amino to carboxyl terminus, in which residues adjacent to each other 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, e.g., a polypeptide. The process includes any expression of the functional presence of a gene in a cell, including, but not limited to, gene knockdown and both transient and stable expression. This includes, but is not limited to, transcription of a gene into messenger RNA (mRNA) and translation of such mRNA into a polypeptide. Where the final desired product is a biochemical, expression includes the production of that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids with post-transcriptional modifications, e.g., polyadenylation, or polypeptides with post-translational modifications, e.g., methylation, glycosylation, lipid addition, association with other protein subunits, proteolytic cleavage, etc.

[0109] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the goal is to prevent (prevent) or slow (reduce) an undesired physiological change or disorder, such as the progression of an autoimmune disease. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Subjects in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder, or those in whom the 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, livestock, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc.

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

[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, a fragment thereof, or an ILT7 variant polypeptide, where a fragment of ILT7 or an ILT7 variant polypeptide retains 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] PRTHMQAENLLKPILWAEPGPVIIWKKPVTIWCQGTLEAQEYRLDKEGNSISRHMLKTLESENKAKFSIPSMMWEHAGRYHCYYQSPAGWSEPSDPLELVVTAYSRPSLSALPSPVVTSGVNV TLRCASRLGLGRFTLIEEGDHRLSWTLDSHQHNHGKFQALFPVGPLTFSNRGTFRCYGYENNTPYVWSEPSDPLQLLVSGVSRKPSLLTLQGPVVAPGDNLTLQCGSDVGYIRYALYKEGGDG LPQRPGQQSQAGLSQASFTLNPVRGSHGGQYRCYGAHNVSSKWSAPSDPLDILIAGQIPDRPSLSVQLGPTVASGEKVTLLCQSWGPMFTFLLAKEGAAHPPLRLRSTYRAQQYQAEFPMSPVTSAHAGTYRCYGSRSSDPYLLSHSSEPLELVVSEATETLNPAQNKSDSKTAPHLQDYTVENLIRMGIAGLVLVFLGILLFEAQQSQRSPTRCSQEVNSREDNAPFRVVEPWEQI (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 a major source of the immunoregulatory molecule interferon (IFN)-alpha, and ILT7 plays a role in regulating the release of IFN-alpha from these cells.

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

[0117] [Table 2]

[0118] In certain embodiments, binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof of the invention, such as antibodies 7C7, ILT70080, ILT70080.1 to ILT70080.7, ILT70083, ILT70083.1 to ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, and ILT70052, bind to ILT7 and inhibit IFN-alpha release by plasmacytoid dendritic cells.

[0119] In certain embodiments, antibodies of the invention include anti-ILT7 antibodies or antigen-binding fragments, variants, or derivatives thereof that bind 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 antibodies bind to human, primate, mouse, or any combination of human, primate, and mouse ILT7.

[0120] In one embodiment, the invention provides an isolated binding molecule, e.g., an antibody or 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 invention provides an isolated binding molecule, such as an antibody or 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 invention provides an isolated binding molecule, such as an antibody or 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 invention provides an isolated binding molecule, e.g., an antibody or 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, e.g., human, primate, mouse, or any combination of human, primate and mouse ILT7. In another embodiment, the invention provides an isolated binding molecule, e.g., an antibody or 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, e.g., human, primate, mouse, or any combination of human, primate and mouse ILT7. In another embodiment, the invention provides an isolated binding molecule, e.g., an antibody or 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, e.g., human, primate, mouse, or any combination of human, primate and mouse ILT7.

[0122] In certain embodiments, a binding molecule of the invention has an amino acid sequence that shares 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 antigen-binding fragment, variant, or derivative thereof comprising, consisting essentially of, or consisting of 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 sequence 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 antigen-binding fragment, variant, or derivative thereof comprising the VH domain specifically or preferentially binds to ILT7. In a further embodiment, the antibody, or antigen-binding fragment, variant, or derivative thereof inhibits IFN-alpha release from plasmacytoid dendritic cells.

[0124] In a further embodiment, the invention comprises an isolated antibody or antigen-binding fragment, variant, or derivative thereof comprising, consisting essentially of, or consisting of 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 antigen-binding fragment, variant, or derivative thereof comprising the VL domain specifically or preferentially binds to ILT7. In a further embodiment, the antibody, or antigen-binding fragment, variant, or derivative thereof inhibits IFNalpha release from plasmacytoid dendritic cells.

[0125] In a further embodiment, the present invention relates to SEQ ID NOs: 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 263 and 267, wherein the antibody or antigen-binding fragment, variant, or derivative thereof comprising the VH and VL domains specifically or preferentially binds to ILT7. In a further embodiment, the antibody or antigen-binding fragment, variant, or derivative thereof inhibits IFN-alpha release from plasmacytoid dendritic cells.

[0126] In a further embodiment, the present invention provides 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, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, and 160; 15, 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 antigen-binding fragment, variant, or derivative thereof comprising the VH and VL domains specifically or preferentially binds to ILT7. In a further embodiment, the antibody or antigen-binding fragment, variant, or derivative thereof inhibits IFN-alpha release from plasmacytoid dendritic cells.

[0127] Suitable biologically active variants of the anti-ILT7 antibodies of the invention can be used in the methods of the 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 alterations are well known in the art. See, e.g., 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, NY), U.S. Patent No. 4,873,192, and references cited therein, which are incorporated herein by reference. Guidance regarding suitable amino acid substitutions that do not affect the biological activity of a 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, DC), pp. 345-352, which is incorporated herein by reference in its entirety. The Dayhoff et al. model uses a Point Accepted Mutation (PAM) amino acid similarity matrix (PAM 250 matrix) to determine suitable conservative amino acid substitutions. Conservative substitutions, such as replacing one amino acid with another amino acid with similar properties, can be beneficial. Examples of conservative amino acid substitutions taught by the PAM 250 matrix of the Dayhoff et al. model include, but are not limited to, Gly⇔Ala, Val⇔Ile⇔Leu, Asp⇔Glu, Lys⇔Arg, Asn⇔Gln, and Phe⇔Trp⇔Tyr.

[0129] When constructing variants of anti-ILT7 binding molecules, e.g., antibodies, or binding fragments, variants, or derivatives thereof, modifications are made so that the variants continue to retain the desired properties, e.g., the ability to specifically bind to ILT7 and, in certain embodiments, the ability to inhibit IFN-alpha release. It is clear that any mutations made in the DNA encoding the variant polypeptide must not place the sequence out of reading frame. In some embodiments, any mutations made in the DNA do not create complementary regions that could produce secondary mRNA structures.

[0130] Methods for determining the binding specificity of an anti-ILT7 binding molecule, e.g., an antibody, or antigen-binding fragment, variant, or derivative thereof, include, but are not limited to, standard competitive binding assays, cytotoxicity assays, IFN release assays, ELISA assays, etc.

[0131] As used herein, 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, percent 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 sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence in accordance with the present invention, the parameters are of course set so that the percentage of identity is calculated over the entire length of the reference polypeptide sequence and so that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

[0132] For purposes of the present invention, percent sequence identity can be determined using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, and a BLOSUM matrix of 62. The Smith-Waterman homology search algorithm is taught by Smith and Waterman (1981) Adv. Appl. Math. 2:482-489. A variant may, for example, differ from a 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 precise chemical structure of a polypeptide capable of specifically binding to ILT7 and retaining the desired activity depends on many factors. The presence of ionizable amino and carboxyl groups on a molecule allows a particular polypeptide to be obtained as an acidic or basic salt, or as a neutral form. All such preparations that retain their biological activity when placed under appropriate environmental conditions are included in the definition of an anti-ILT7 antibody as used herein. Furthermore, the primary amino acid sequence of a polypeptide can be enhanced by derivatization with sugar moieties (glycosylation) or other accessory molecules, such as lipids, phosphate groups, acetyl groups, etc. Enhancement can also be achieved by conjugation with sugars. Certain aspects of such enhancement are achieved through the post-translational processing systems of the production host, while other such modifications can be introduced in vitro. Regardless, such modifications are included in the definition of an anti-ILT7 antibody as used herein, so long as they do not destroy the desired properties of the anti-ILT7 antibody. It is expected that such modifications may affect activity, either quantitatively or qualitatively, by enhancing or impairing 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 activity. Such alterations that do not destroy the desired properties (e.g., ILT7 binding specificity, binding affinity, and associated activities, such as the ability to inhibit cytokine release by ILT7 from mast cells, endothelial cells, and proliferation of TF-1 cells) still fall within the definition of anti-ILT7 antibody of interest as 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, variants, or derivatives thereof, one of skill in the art can readily determine what modifications to the nucleotide or amino acid sequence of the native protein result in variants that are suitable for use as therapeutically active ingredients in pharmaceutical compositions used in the methods of the invention.

[0135] The constant region of an 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 to reduce effector function using techniques known in the art. For example, deletion or inactivation of constant region domains (through point mutations or other means) can reduce Fc receptor binding of the modified antibody in circulation. In another example, constant region modifications consistent with the present invention can attenuate complement binding, thus reducing the serum half-life and nonspecific association of a conjugated cytotoxin. Still other modifications of the constant region can be used to modify disulfide bonds or oligosaccharide moieties that allow for enhanced antigen specificity or enhanced localization due to antibody flexibility. The physiological profile, bioavailability, and other biochemical effects of the resulting modifications, 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, exhibit significantly higher efficacy compared to their fucosylated counterparts, and are able to circumvent the inhibitory effects of serum immunoglobulin G (IgG) on ADCC through their enhanced binding to gamma receptor IIIa (FcFcγRIIIa).

[0138] The anti-ILT7 antibodies of the present invention also include derivatives that have been modified, e.g., by the covalent attachment of any type of molecule to the antibody such that the 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 have been modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to a cellular ligand or other protein, etc. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, etc. Additionally, derivatives may contain one or more non-classical amino acids.

[0139] A "conservative amino acid substitution" is one 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 with similarly charged side chains have been defined in the art. These families include amino acids with 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations, such as saturation mutagenesis, can be introduced randomly along all or part of the coding sequence and the resulting mutants screened for biological activity to identify mutants that retain activity (e.g., the ability to bind an anti-ILT7 polypeptide).

[0140] For example, it is possible to introduce mutations only into the framework regions or only into the CDR regions of an antibody molecule. The introduced mutations may 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 may be useful for optimizing codon usage or improving antibody production in hybridomas. Alternatively, non-neutral missense mutations may alter the antigen-binding ability of the antibody. Most silent and neutral missense mutations are likely to be located in framework regions, and most non-neutral missense mutations are likely to be located in CDRs, although this is not necessarily an absolute requirement. Those skilled in the art will be able to design and test mutant molecules with desired properties, such as no change in antigen-binding activity or changes in binding activity (e.g., improved antigen-binding activity or changed antibody specificity). Following mutagenesis, the encoded protein can be routinely expressed and the functional and / or biological activity of the encoded protein (e.g., the ability to immunospecifically bind to at least one epitope of an ILT7 polypeptide) can be determined using techniques described herein or by routinely modifying techniques known in the art.

[0141] In certain embodiments, the anti-ILT7 antibodies of the present invention comprise at least one optimized complementarity-determining region (CDR). By "optimized CDR," we mean that the CDR has been modified and the optimized sequence has been selected based on maintaining or improving the binding affinity and / or anti-ILT7 activity conferred to the anti-ILT7 antibody comprising the optimized CDR. "Anti-ILT7 activity" can include, for example, the activity of modulating one or more of the following ILT7-associated activities: ILT7-mediated interferon release from plasmacytoid dendritic cells, cytotoxicity against ILT7-expressing cells, or any other ILT7-associated activity. Anti-ILT7 activity can also result in a reduction in the incidence or severity of certain types of autoimmune conditions, such as systemic lupus erythematosus, chronic rheumatoid arthritis, and psoriasis, associated with ILT7 expression. The modification can involve the substitution of amino acid residues within the CDR so 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 present 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 invention comprises an isolated polynucleotide comprising, consisting essentially of, or consisting of 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 an anti-ILT7 antibody comprising the encoded VH domain specifically or preferentially binds ILT7. In certain embodiments, the polynucleotide encodes an antibody, or antigen-binding fragment, variant, or derivative thereof, that inhibits IFN-alpha release from plasmacytoid dendritic cells.

[0144] In a further embodiment, the invention comprises an isolated polynucleotide comprising, consisting essentially of, or consisting of 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 an anti-ILT7 antibody comprising the encoded VL domain specifically or preferentially binds ILT7. In certain embodiments, the polynucleotide encodes an antibody, or antigen-binding fragment, variant, or derivative thereof, that inhibits IFN-alpha release from plasmacytoid dendritic cells.

[0145] Any of the above polynucleotides can further comprise additional nucleic acid encoding, for example, a signal peptide to direct secretion of the encoded polypeptide, an antibody constant region as described herein, or other heterologous polypeptide as 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 invention includes a composition comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a VH domain described herein and the second polynucleotide encodes a VL domain described herein. In particular, the composition may comprise, consist essentially of, or consist of a VH domain-encoding polynucleotide 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 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 comprise, 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 on the same vector. In some embodiments, the VH domain-encoding polypeptide and the VL domain-encoding polypeptide are on different vectors.

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

[0148] Polynucleotides can be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of an antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)); briefly, this method involves synthesizing overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligating those oligonucleotides, and amplifying the ligated oligonucleotides by PCR.

[0149] Alternatively, polynucleotides encoding anti-ILT7 antibodies, or antigen-binding fragments, variants, or derivatives thereof, can be generated from nucleic acid of a suitable source. If a clone containing nucleic acid encoding a particular antibody is not available but the sequence of the antibody molecule is known, nucleic acid encoding the antibody can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library made from any tissue or cell that expresses the antibody or other anti-ILT7 antibody, such as hybridoma cells selected to express the antibody, or nucleic acid isolated therefrom, e.g., polyA+ RNA) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence to identify cDNA clones from a cDNA library encoding the antibody or other anti-ILT7 antibody, for example. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0150] Once the nucleotide sequence and corresponding amino acid sequence of an anti-ILT7 antibody, or antigen-binding fragment, variant, or derivative thereof, has been 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, NY) and Ausubel et al., eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons, NY), which are incorporated herein by reference in their entireties), to generate antibodies with different amino acid sequences, for example to make amino acid substitutions, deletions, and / or insertions.

[0151] Polynucleotides encoding anti-ILT7 binding molecules, e.g., antibodies, or antigen-binding fragments, variants, or derivatives thereof, can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA, or modified RNA or DNA. For example, polynucleotides encoding anti-ILT7 antibodies or antigen-binding fragments, variants, or derivatives thereof can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, or hybrid molecules comprising DNA and RNA, which can be single-stranded, or more typically double-stranded, or a mixture of single- and double-stranded regions. In addition, polynucleotides encoding anti-ILT7 binding molecules, e.g., antibodies, or antigen-binding fragments, variants, or derivatives thereof, can be composed of RNA or DNA, or triple-stranded regions comprising both RNA and DNA. Polynucleotides encoding anti-ILT7 binding molecules, e.g., antibodies, or antigen-binding fragments, variants, or derivatives thereof, can also contain one or more modified bases or DNA or RNA backbones modified for stability or for any other reason. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA, and thus "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

[0152] Isolated polynucleotides encoding non-naturally occurring variants of polypeptides derived from immunoglobulins (e.g., immunoglobulin heavy or light chain portions) 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 discussed in more detail elsewhere herein, anti-ILT7 binding molecules, e.g., antibodies of the invention, or antigen-binding fragments, variants, or derivatives thereof, can further be recombinantly fused at the N- or C-terminus to heterologous polypeptides or chemically conjugated (including covalent and non-covalent conjugation) to polypeptides or other compositions. For example, anti-ILT7 antibodies can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules, such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT Publication Nos. WO 92 / 08495, WO 91 / 14438, WO 89 / 12624, U.S. Pat. No. 5,314,995, and EP 396,387.

[0154] Anti-ILT7 antibodies, or antigen-binding fragments, variants, or derivatives thereof, of the present invention may include derivatives that have been modified such that the covalent attachment does not prevent the antibody from binding to ILT7, i.e., by covalently attaching any type of molecule to the antibody. For example, but not limited to, antibody derivatives include antibodies that have been modified by, e.g., glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, etc. Additionally, derivatives may contain one or more non-classical amino acids.

[0155] Anti-ILT7 binding molecules, such as antibodies of the invention, or antigen-binding fragments, variants, or derivatives thereof, can be composed of amino acids linked together by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and can contain amino acids other than the 20 genetically encoded amino acids. For example, anti-ILT7 antibodies can be modified by natural processes, such as post-translational processing, or by chemical modification techniques well known in the art. Such modifications are well described in basic texts and more detailed monographs, as well as in the voluminous research literature. Modifications can occur anywhere in the anti-ILT7 binding molecule, including the peptide backbone, the amino acid side-chains, and the amino- or carboxyl-terminus, or moieties such as carbohydrates. It will be recognized that the same type of modification can be present in the same or varying degrees at several sites in 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 may result from post-translation natural processes or may be made 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 covalent cross-links, 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 amino acids to proteins such as arginylation, and ubiquitination (see, e.g., 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), pp. 1-12; Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. NY Acad. Sci. 663:48-62 (1992).

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

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

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

[0159] Exemplary fusion proteins reported in the literature include T-cell receptors (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 (a homing receptor) (Watson et al., J. Cell. Biol. 110:2221-2229 (1990), and Watson et al., Nature 349:164-167 (1990)). (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, anti-ILT7 binding molecules, such as antibodies of the invention, or antigen-binding fragments, variants, or derivatives thereof, can be fused to heterologous polypeptides 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, anti-ILT7 binding molecules, e.g., antibodies of the present invention, or antigen-binding fragments, variants, or derivatives thereof, can be fused to a marker sequence, such as a peptide, to facilitate their purification or detection. In some embodiments, the marker amino acid sequence is a hexahistidine peptide, such as the tag provided in pQE vectors (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. For example, hexahistidine provides for convenient purification of the fusion protein, as described in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989). Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds 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, e.g., U.S. Patent Nos. 5,116,964 and 5,225,538). The precise site at which the fusion is made can be selected empirically to optimize the secretion or binding characteristics of the fusion protein. DNA encoding the fusion protein is then transfected into a host cell for expression.

[0163] The anti-ILT7 binding molecule, e.g., an antibody of the invention, or antigen-binding fragment, variant, or derivative thereof, can be used in unconjugated form or can be conjugated to at least one of a variety of molecules, e.g., to improve the therapeutic properties of the molecule, to facilitate detection of a target, or for imaging or treatment of a patient. The anti-ILT7 binding molecule, e.g., an antibody of the invention, or antigen-binding fragment, variant, or derivative thereof, can be labeled or conjugated before or after purification, or as the purification is performed.

[0164] In particular, the anti-ILT7 antibodies of the invention, or antigen-binding fragments, variants, or derivatives thereof, can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, drug, or PEG.

[0165] Those skilled in the art will recognize that conjugates can also be assembled using a variety of techniques, depending on the agent selected to be conjugated. For example, conjugates with biotin are prepared by reacting a binding polypeptide with an activated ester of biotin, such as the biotin N-hydroxysuccinimide ester. Similarly, conjugates with fluorescent markers can be prepared in the presence of a coupling agent, such as those described herein, or by reaction with an isothiocyanate, such as fluorescein isothiocyanate. Conjugates of the anti-ILT7 antibodies of the invention, or antigen-binding fragments, variants, or derivatives thereof, are prepared in a similar manner.

[0166] The present invention further encompasses anti-ILT7 binding molecules, e.g., antibodies of the present invention, or antigen-binding fragments, variants, or derivatives thereof, conjugated to diagnostic or therapeutic agents. Anti-ILT7 antibodies, including their antigen-binding fragments, variants, and derivatives, can be used diagnostically, e.g., to monitor disease development or progression as part of a clinical trial procedure, e.g., to determine the efficacy of a given treatment and / or prevention regimen. For example, detection can be facilitated by coupling an anti-ILT7 antibody or its antigen-binding fragment, variant, or derivative to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, various positron-emitting metals using positron emission tomography, and non-radioactive paramagnetic metal ions. See, e.g., U.S. Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostic agents in accordance with the present invention. examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group 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, 111 In, 90 Y, or 99 Examples include Tc.

[0167] An anti-ILT7 binding molecule, e.g., an antibody, or 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 arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0168] One way in which an anti-ILT7 antibody, or antigen-binding fragment, variant, or derivative thereof, can be detectably labeled is by linking it to an enzyme and using 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 (Kugaku Shoin, (Tokyo). The enzyme conjugated to the anti-ILT7 antibody reacts with an appropriate substrate, e.g., a chromogenic substrate, to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric, or visual means. Enzymes that can be used to detectably label antibodies 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, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Furthermore, detection can be achieved by colorimetric methods using a chromogenic substrate for the enzyme. Detection can also be achieved by visual comparison of the extent of enzymatic reaction of the substrate in comparison with similarly prepared standards.

[0169] Detection can also be carried out using any of a variety of other immunoassays. For example, by radioactively labeling an anti-ILT7 binding molecule, such as an antibody or an antigen-binding fragment, variant, or derivative thereof, it is possible to detect the binding molecule through the use of a radioimmunoassay (RIA) (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). Radioactive isotopes can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.

[0170] Anti-ILT7 binding molecules, e.g., antibodies, or antigen-binding fragments, variants, or derivatives thereof, can also be detectably labeled using fluorescence-emitting metals such as 152Eu, or other metals of the lanthanide series, which can be attached to the binding molecule using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0171] Techniques for conjugating various moieties to antibodies (e.g., anti-ILT7 antibodies) or antigen-binding fragments, variants, or derivatives thereof are well known and are described, for example, in 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 Antibodies 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 an antibody can be generated either simultaneously or separately using reverse transcriptase and DNA polymerase according to well-known methods. PCR can be initiated with consensus constant region primers or with 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 antibody light and heavy chains. In this case, libraries can be screened with consensus primers or larger homologous probes, such as mouse constant region probes.

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

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

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

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

[0177] For the purposes of the present invention, numerous 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, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Other vectors involve the use of polycistronic systems with internal ribosome entry sites. In addition, cells into which the DNA has integrated into their chromosomes can be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers can provide prototrophy to auxotrophic hosts, resistance to biocides (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed or can be introduced into the same cell by cotransformation. Additional elements may also be necessary for optimal synthesis of mRNA. These elements can include signal sequences, splice signals, and transcriptional promoters, enhancers, and termination signals.

[0178] In some embodiments, the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (e.g., human) synthesized above. Of course, any expression vector capable of eliciting expression in eukaryotic cells can be used in the present invention. Examples of suitable vectors include, but are not limited to, the plasmids pcDNA3, pHCMV / Zeo, pCR3.1, pEF1 / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, Calif.), and the plasmid pCI (available from Promega, Madison, Wis.). Generally, screening large numbers of transformed cells for vectors expressing suitably high levels of immunoglobulin heavy and light chains is routine experimentation that can be performed, for example, by robotic systems.

[0179] More generally, after preparing a vector or DNA sequence encoding a 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 electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped 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 accomplished via electroporation. Host cells harboring the expression construct are grown under conditions appropriate for the production of light and heavy chains and assayed for heavy 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 a host cell by conventional techniques, and the transfected cells are cultured by conventional techniques to produce the antibody for use in the methods described herein. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter. In some embodiments for expression of double-chain antibodies, vectors encoding both the heavy and light chains can be co-expressed in a host cell for expression of a complete immunoglobulin molecule, as described in more detail below.

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

[0182] A variety of host-expression vector systems can be utilized to express antibody molecules for use in the methods described herein. Such host-expression systems represent vehicles in which coding sequences of interest can be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing an antibody molecule of the invention in situ. These include, but are not limited to, bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; microorganisms such as yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter). Bacterial cells such as Escherichia coli or eukaryotic cells, particularly for the expression of whole recombinant antibody molecules, are used to express recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) are effective expression systems for antibodies, with vectors containing, for example, the major intermediate-early gene promoter element of the 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 those skilled in the art are credited with the ability to determine the particular 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 lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / O (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). Host cell lines are typically available from commercial services such as the American Tissue Culture Collection, or from published literature.

[0184] In addition, a host cell strain can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.

[0185] Stable expression is useful for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express antibody molecules can be engineered. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the foreign DNA, engineered cells are grown in enrichment medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection, and cells can stably integrate the plasmid into their chromosomes and form foci, 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, 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 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)), and 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 for recombinant DNA technology that can be used are 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, which are incorporated herein by reference in their entireties.

[0187] The expression level of an antibody molecule can be increased by amplifying the vector (for a review, 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). If the marker in the antibody expression vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, antibody production will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[0188] In vitro production allows for scale-up to generate large quantities 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 airlift reactors or continuous stirred reactors, or immobilized or entrapped cell culture on, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE cellulose, or (immuno)affinity chromatography, for example, after selective biosynthesis of the synthetic hinge region polypeptide, or before or after the HIC chromatography step described herein.

[0189] Genes encoding the anti-ILT7 antibodies, or antigen-binding fragments, variants, or derivatives 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 enterobacteria, such as Escherichia coli or Salmonella strains, Bacillaceae, e.g., Bacillus subtilis, Pneumococcus, Streptococcus, and Haemophilus influenzae. It is further recognized that when expressed in bacteria, heterologous polypeptides typically become part of inclusion bodies. After isolation and purification, heterologous polypeptides must be assembled into functional molecules. If a tetravalent form of the antibody is desired, subunits self-assemble into a tetravalent antibody (WO 02 / 096948 A2).

[0190] In bacterial systems, numerous expression vectors can be advantageously selected depending on the intended use of the expressed antibody molecule. For example, if large quantities of such protein are to be produced to prepare pharmaceutical compositions of the antibody molecule, vectors directing high-level expression of easily purified fusion protein products may be desirable. Such vectors include, but are not limited to, 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)), and the like, in which antibody coding sequences can be individually ligated in frame with the lacZ coding region to produce a fusion protein. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. 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 prokaryotes, eukaryotic microorganisms can be used as well. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, although a number of other strains, such as Pichia pastoris, are commonly available.

[0192] For expression in Saccharomyces, for example, the 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 selection 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 insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda cells. Antibody coding sequences can be cloned individually into non-essential regions (e.g., the polyhedrosis gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrosis promoter).

[0194] When a binding molecule of the invention is recombinantly expressed, it can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity, particularly for specific antigens following Protein A, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Alternatively, useful methods for increasing the affinity of antibodies of the invention are disclosed in U.S. Patent Application Publication No. 20020123057 A1.

[0195] VII. Methods of Treatment Using Therapeutic Anti-ILT7 Binding Molecules The methods of the present invention relate to the use of anti-ILT7 binding molecules, e.g., antibodies, including antigen-binding fragments, variants, and derivatives thereof, to treat patients with diseases associated with ILT7 expression or ILT7-expressing cells. By "ILT7-expressing cells" is intended a cell that expresses 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] Although the following discussion refers to methods of diagnosing and treating various diseases and disorders with the anti-ILT7 antibodies of the invention, the methods disclosed herein are also applicable to antigen-binding fragments, variants, and derivatives of these anti-ILT7 antibodies that retain the desired properties of the anti-ILT7 antibodies of the invention, e.g., are capable of specifically binding to ILT7 and neutralizing ILT7 pathogenic activity.

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

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

[0199] In one embodiment, the invention relates to an anti-ILT7 binding molecule, such as an antibody, or antigen-binding fragment, variant, or derivative thereof, particularly for use in the treatment or prevention of an autoimmune condition or disease, including, but not limited to, myositis, diabetes, Hashimoto's disease, autoimmune adrenal insufficiency, pure red cell aplasia, 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, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof as defined elsewhere in the present invention, is used to promote a positive therapeutic response regarding an autoimmune response. A "positive therapeutic response" in the context of autoimmune treatment refers to an improvement in the disease and / or symptoms associated with the activity of these binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof. 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, an improvement in the disease can be characterized as a complete response. A "complete response" is intended to refer to the absence of clinically detectable disease with any standardized test results to date. Such a response must persist for at least one month after treatment according to the methods of the present invention. Alternatively, the improvement in the disease can be classified as a partial response.

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

[0202] Clinical responses can be assessed using screening techniques such as magnetic resonance imaging (MRI) scans, X-radiography, computed tomography (CT) scans, flow cytometry or fluorescence activated cell sorting (FACS) analysis, histology, macroscopic findings, and blood chemistries, including, but not limited to, changes detectable by ELISA, RIA, chromatography, etc. In addition to these positive therapeutic responses, subjects receiving treatment with an anti-ILT7 binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, can experience the beneficial effect of amelioration of symptoms associated with the disease.

[0203] A further embodiment of the invention is the use of an anti-ILT7 binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, for diagnostic monitoring of protein levels in tissues as part of a clinical testing procedure, e.g., to determine the effectiveness of a given treatment regimen. Detection can be facilitated, for example, by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, 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 prosthetic group 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 is one example.

[0204] VIII. Pharmaceutical Compositions and Methods of Administration Methods for preparing and administering anti-ILT7 binding molecules, such as antibodies provided herein, or antigen-binding fragments, variants, or derivatives thereof, to a subject in need thereof are well known and readily determined by those of skill in the art.

[0205] As discussed herein, anti-ILT7 binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives 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 present disclosure will be formulated to facilitate administration and promote stability of the active agent. Pharmaceutical compositions in accordance with the present invention may include a pharmaceutically acceptable, non-toxic, sterile carrier. For purposes of this application, a conjugated or unconjugated anti-ILT7 binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, is considered to mean an amount sufficient to achieve effective binding to the target and to achieve a benefit, e.g., to ameliorate the symptoms of a disease or condition, or to detect a substance or cell.

[0206] Pharmaceutical compositions suitable for injection must be sterile and fluid to the extent that easy syringability exists. They must be stable under the conditions of manufacture and storage and be beneficially preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of microbial action can be achieved by various antibacterial and antifungal agents. Suitable formulations for use in the therapeutic 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, anti-ILT7 antibodies of the present invention, or antigen-binding fragments, variants, or derivatives thereof, can be administered to humans or other animals according to the above-described treatment methods in an amount sufficient to produce a therapeutic effect. Anti-ILT7 antibodies of the present invention, or antigen-binding fragments, variants, or derivatives thereof, can be administered to such humans or other animals in conventional dosage forms prepared by mixing the antibodies of the present invention, or antigen-binding fragments, variants, or derivatives thereof, with conventional pharmaceutically acceptable carriers or diluents in accordance with known techniques. Those skilled in the art will recognize that the form and characteristics of the pharmaceutically acceptable carrier or diluent will depend on the amount of 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 cocktails comprising one or more species of anti-ILT7 binding molecules, such as antibodies of the present invention, or antigen-binding fragments, variants, or derivatives thereof, may prove particularly effective.

[0208] By "therapeutically effective dose or amount" or "effective amount" is intended the amount of an anti-ILT7 binding molecule, e.g., an antibody or antigen-binding fragment, variant, or derivative thereof, that, when administered, results in a positive therapeutic response for the treatment of a patient having the disease or condition being treated.

[0209] The therapeutically effective amount of the compositions of the present invention for treating 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 animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, can be treated as well. Treatment dosages can be titrated to optimize safety and efficacy.

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

[0211] IX. Diagnosis The present invention further provides diagnostic methods that are useful in diagnosing ILT7-expressing cell-mediated diseases, such as certain types of autoimmune diseases, including systemic lupus erythematosus, the methods involving measuring the expression level of ILT7 protein or transcript in tissue or other cells or body fluids of an individual and comparing the measured expression level with a standard ILT7 expression level in a normal tissue or body fluid, whereby an increased expression level compared to the standard is indicative of a disorder.

[0212] The anti-ILT7 antibodies and antigen-binding fragments, variants, and derivatives thereof of the present invention can be used to assay ILT7 protein levels in biological samples using classical immunohistochemical methods known to those skilled in the art (see, e.g., 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] By "assaying the expression level of an ILT7 polypeptide" is intended to qualitatively or quantitatively measure or estimate the level of an ILT7 polypeptide in a first biological sample directly (e.g., by determining or estimating absolute protein levels) or relatively (e.g., by comparing disease-associated polypeptide levels in a second biological sample). The ILT7 polypeptide expression level in a 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 is determined by averaging levels from a population of individuals without the disorder. As recognized in the art, if a "standard" ILT7 polypeptide level is known, it can be used repeatedly as a standard for comparison.

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

[0215] X. Immunoassay Anti-ILT7 binding molecules, such as antibodies of the present invention or antigen-binding fragments, variants, or derivatives thereof, can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assays using techniques such as Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitin reaction, gel diffusion precipitin reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescence immunoassay, and protein A immunoassay. Such assays are conventional and well known in the art (see, e.g., Ausubel et al., eds. (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1, incorporated herein by reference in its entirety). Exemplary immunoassays are briefly described below (but are not intended to be limiting):

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

[0217] Immunoassays and non-immunoassays for ILT7 gene products or conserved variants or peptide fragments thereof typically involve incubating a sample, such as a biological fluid, tissue extract, freshly harvested cells, or a lysate of cells that have been incubated in cell culture, in the presence of a detectably labeled antibody that can bind to ILT7, or a conserved variant or peptide fragment thereof, and detecting the bound antibody by any of a number of techniques well known in the art.

[0218] The biological sample can be contacted and immobilized on a solid support or carrier such as nitrocellulose, or other solid support capable of immobilizing cells, cell particles, or soluble proteins. The support can then be washed with a buffer appropriate for treatment with a detectably labeled anti-ILT7 antibody, or its antigen-binding fragment, variant, or derivative. The solid support can then be washed twice with a 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] By "solid phase support or carrier" is intended any support capable of binding an antigen or antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified celluloses, polyacrylamide, gabbro, and magnetite. The nature of the support can be soluble to some extent or insoluble for purposes of the present invention. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the shape of the support can be a sphere, a bead, a cylinder, the interior surface of a test tube, or the exterior surface of a rod. Alternatively, the surface can be flat, such as a sheet, test strip, etc. Exemplary supports include polystyrene beads. Those of skill in the art will know many other suitable carriers for binding antibodies or antigens, or will be able to ascertain such by use of routine experimentation.

[0220] The binding activity of a given lot of an 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 routine experimentation.

[0221] The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. An example of a competitive binding assay is a method in which a labeled antigen (e.g., 3 H or 125 A radioimmunoassay involves incubating a labeled compound (e.g., a IgG1A antibody) with an antibody of interest in the presence of increasing amounts of unlabeled antigen, and detecting antibody binding to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rate 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 incubated with a labeled compound (e.g., a IgG1A antibody) in the presence of increasing amounts of an unlabeled secondary antibody. 3 H or 125I) and incubate with the antibody of interest conjugated to

[0222] Although there are a variety of methods available for measuring the affinity of antibody-antigen interactions, relatively few methods determine rate constants. Most of the methods rely on either labeled antibody or antigen, which necessarily complicates routine measurements and introduces uncertainty into the measured quantities.

[0223] Surface plasmon resonance (SPR) performed in BIACORE® offers numerous advantages over conventional methods for measuring the affinity of antibody-antigen interactions: (i) neither the antibody nor the antigen needs to be labeled; (ii) the antibody does not need to be pre-purified, and cell culture supernatants can be used directly; (iii) real-time measurements are possible, allowing rapid, semi-quantitative comparisons of different monoclonal antibody interactions, which is sufficient for many evaluation purposes; (iv) the biospecific surface can be regenerated, allowing a series of different monoclonal antibodies to be easily compared under identical conditions; and (v) the analytical procedure is fully automated, allowing a wide range of measurements to be performed without user intervention. (BIA Applications Handbook, version AB (1998 reprint), BIACORE® code number BR-1001-86; BIA Technology Handbook, version AB (1998 reprint), BIACORE® code number BR-1001-84). SPR-based binding tests require one member of a 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 cases, the ligand binds indirectly to the surface through binding to another immobilized molecule called the capture molecule. The SPR response reflects the change in mass concentration on the detector surface as 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. Comparative affinity rankings are easy to perform, and both kinetic and affinity constants can be derived from sensorgram data.

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

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

[0227] The BIA evaluation software provides comprehensive facilities for Karp fitting using both numerical integration and global fitting algorithms. With appropriate analysis of the data, individual rate and affinity constants for interactions can be obtained from simple BIACORE® experiments. The range of affinities measurable by this technique is very broad, ranging from mM to pM.

[0228] Epitope specificity is a key feature of monoclonal antibodies. In contrast to conventional techniques using radioimmunoassays, ELISAs, or other surface adsorption methods, epitope mapping with BIACORE® does not require antibody labeling or purification, and allows multi-site specificity testing using the sequence of a portion of a monoclonal antibody. Furthermore, large numbers of samples can be processed automatically.

[0229] Pairwise binding experiments test the ability of two MAbs to simultaneously bind to the same antigen. MAbs to distinct epitopes bind independently, while MAbs to the same or closely related epitopes block each other's binding. These binding experiments are easily performed by BIACORE®.

[0230] For example, a capture molecule can be used to bind a first Mab, followed by sequential addition of an antigen and a second Mab. The sensorgram will reveal (1) how much antigen binds to the first Mab, (2) how much the second Mab binds to the surface-bound antigen, and (3) whether reversing the order of the pairwise tests would change the results if the second Mab did not bind.

[0231] Peptide inhibition is another technique used for epitope mapping. This method complements pairwise antibody binding studies and can relate functional epitopes to structural features when the primary sequence of the antigen is known. Peptides or antigen fragments are tested for inhibition of binding of different MAbs to the immobilized antigen. Peptides that interfere with binding of a given MAb are assumed to be structurally related to the epitope defined by that MAb.

[0232] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology that are within the skill of the art and are fully explained 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), DN Glover ed., (1985) DNA Cloning, Volumes I and II, Gait, ed. (1984) Oligonucleotide Synthesis, Mullis et al. US 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., NY), 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] General principles of antibody engineering are described in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford University Press). General principles of protein engineering are described in Rickwood et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford University Press, Oxford, Eng.). General principles of 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, NY). Additionally, standard immunological methods known in the art and not specifically described will generally be 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 works describing 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 Hall2003), Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press), Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press). .

[0235] All references cited above, along with all references cited herein, are incorporated herein by reference in their entirety.

[0236] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0237] Materials and Methods biological samples Human peripheral blood from healthy 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 cell preparation tubes (Becton Dickinson Biosciences, NJ, USA) supplemented with sodium citrate. The tubes were spun down at 17,000 xg for 25 minutes at 22°C with minimal braking. After centrifugation, serum was removed, and the cell buffy coat was transferred to a 50-mL conical tube (BD Biosciences). Purified cells were washed twice with sterile phosphate-buffered saline (PBS) (Invitrogen Life Technologies, CA, USA) at 350 xg for 10 minutes at 22°C. 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). 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 50x the initial 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 sample was centrifuged at 400 g for 20 minutes at room temperature with minimal braking. Disks of cells were harvested and transferred to a new 50 mL conical tube. Purified cells were washed twice with sterile PBS at 350 g for 10 minutes at 22°C. Cells were resuspended in PBS or RPMI 1640 medium supplemented with 10% fetal bovine serum, filtered, and counted as above.

[0239] cell CT-125 and CT-550 cells were obtained from Dr. Yong-Jun Liu (University of Texas MD Anderson Cancer Center, Houston, TX, USA). CT-125 cells were generated by transducing the 2B4 murine T cell hybridoma with untagged mouse FcεR1γ and the NFAT-GFP reporter gene, and CT-550 cells were generated by transducing CT-125 cells with 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 monkey ILT7 gene cloned into the pME18X plasmid vector. CT cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1× penicillin / streptomycin (all from Invitrogen Life Technologies).

[0240] KC1333 cells were obtained from Biowa (Biowa, NJ, USA) and 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 the APC Monoclonal Antibody Labeling Kit (Thermo Fisher Scientific, IL, USA). 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. Anti-human CD123 (clone 7G3) conjugated to either R-PE, FITC, or APC, 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) were obtained from BD Biosciences. Pacific Blue anti-human CD56 antibody (clone MEM-188) was obtained from BioLegend, CA, USA. DyLight 649-labeled anti-human IgG and human total IgG were obtained from Jackson Immunoresearch, PA, USA.

[0242] Whole blood staining was performed using BD FACS lysing solution (BD Biosciences). 7-AAD was obtained from Invitrogen. AB 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 monkey recombinant ILT7 Proteins were 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 pH ∼8 with 1 M NaHCO in D-PBS.

[0244] Label incorporation was assessed in all cases by MALDI-TOF mass spectrometry, and unreacted reagent was removed by buffer exchange using a disposable Sephadex G25 column 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-displayed scFv supernatant samples were generated in 96-well deep-well plates as follows: 5 μl of culture from each well of the 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 and 280 rpm for 5 hours. K07 M13 helper phage (1.5 × 10 in 2TYAG) was added. 11 100 μl / well of 2TYAK (diluted to 100 μg / ml ampicillin and 50 μg / ml kanamycin) was added, and the plates were incubated at 37°C and 150 rpm for infection. The plates were spun down at 3200 rpm for 10 minutes, and the supernatant was removed. The bacterial pellet was resuspended in 500 μl / well of 2TYAK (2TY + 100 μg / ml ampicillin + 50 μg / ml kanamycin), and the plates were incubated overnight at 25°C and 280 rpm. In the morning, 500 μl of 6% (w / v) skim milk powder in 2x PBS was added to each well, and the plates were incubated at room temperature for 1 hour. The plates were then centrifuged at 3200 rpm for 10 minutes, and the blocked phage-displayed scFv supernatant was used directly for ELISA experiments.

[0246] For EC50 determination, purified IgG was typically diluted 3-fold with 3% (w / v) dry milk powder in PBS (PBS-M) to obtain 11 concentration points. 96-well Greiner polypropylene plates (Greiner, 650201) were used for dilution preparation. Typically, each dilution was prepared in duplicate. IgG dilutions were blocked in PBS-M at room temperature for 1 hour before being used directly in ELISA experiments.

[0247] The IL-T7 binding assay was a plate-based ELISA performed essentially as follows. While not all antigens were used in every experiment, human, mouse, and cynomolgus IL-T7 antigens were typically tested. Relevant control antigens (bovine insulin + IL-4RαFLAG® His, as needed) were also used to test for nonspecific binding. With the exception of bovine insulin, all antigens were biotinylated and all were produced using bacterial expression. 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 WO 2010 / 070346. IL-4RαFLAG® His was biotinylated via the free amine using EZ-link Sulfo-NHS-LC-Biotin (Perbio / Pierce, 21335).

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

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

[0250] Fluorescence Microvolume Assay Technology (FMAT) cell binding assay This homogeneous assay assessed 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 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 a 100 μm depth into the bottom of the wells to capture an area of ​​1 mm. 2 The cells were scanned. When the cells settled to the bottom of the well and the laser was excited at 633 nm, the fluorophore-conjugated beads (where the local concentration of fluorophore was relatively high compared to unbound fluorophore) emitted a signal at 650–685 nm, which was measured using a photomultiplier tube (PMT1). Unbound fluorophore in solution was either outside the excitation depth or at a relatively low local concentration, and therefore did not emit a significant signal. The presence of scFv or IgG samples binding to cells at the bottom of the well caused an increase in Alexafluor-labeled detection antibody within the excitation depth, which 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 make the ScFv detection mix, mouse anti-His and anti-mouse AF647 antibodies were mixed at 1 μg / ml and 2 μg / ml, respectively, in assay buffer. To make the IgG detection mix, anti-human AF647 antibody was prepared at 2 μg / ml in assay buffer.

[0252] The cells used were CHO-K1 cells expressing either human or cynomolgus ILT7, cultured using standard tissue culture techniques. 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 plated at 1.5 × 10 cells / ml in PBS. 5 The cells were resuspended at 100 cells / ml.

[0253] Crude scFv supernatant samples were prepared in 96-deep-well plates. Five μl of culture from each well of the 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 10 mM IPTG in TY solution 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, scFv supernatant from the deep-well block was transferred directly to an assay plate for the required dilution of 20%.

[0254] Test wells of a 384-well, clear-bottom, non-binding, black Costar plate were filled with the following: 10 μl of sample (IgG or scFv), 10 μl of detection antibody or antibody mix, and 30 μl of cells. 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 samples. Plates were sealed and incubated in the dark at room temperature for 4 hours before being read on an Applied Biosystems Cell Detection System 8200 reader. Data were typically analyzed using the Velocity algorithm, with gates set at a color ratio of <0.4, a size of 15–30, and a minimum count of 20. Crude scFv supernatant sample hits were defined as those showing 50% or greater inhibition of signal compared to the total binding control wells. Dose-response curves were plotted for purified IgG titrations using Prism (Graphpad) curve-fitting software.

[0255] I C 50 For determination, purified IgG was typically diluted 2-fold in assay buffer starting from 500 nM to obtain 11 concentration points. 96-well Greiner polypropylene (Greiner, 650201) plates were used for dilution preparation. Typically, each dilution was prepared in duplicate. Alternatively, IgG testing was performed at a single concentration ranging from 500 nM to 0.2 nM.

[0256] Assessment of antibody binding in cell lines by flow cytometry Binding of anti-ILT7 variants and isotype controls to human and cynomolgus monkey ILT7 was assessed by flow cytometry analysis using CT-550 and cynoILT7 CT125 cells, respectively. CT-125 cells were used as a control. 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 Falcon™ Clear Microtest Plate, BD Biosciences) at 100 μl / well. Anti-ILT7 variant and control antibodies were added to the cells for 30 minutes at 4°C 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 for 30 minutes on a plate shaker in the dark at 4°C. Cells were washed three times with PBS and surface fluorescence was acquired using an LSRII flow cytometry system and FACSDiva software (both from BD Biosciences).

[0257] Assessment of antibody binding in whole blood and PBMCs by flow cytometry The binding of APC-labeled anti-ILT7 antibodies and isotype controls in human and cynomolgus monkey whole blood was assessed by flow cytometry analysis. Whole blood was transferred to 50 mL conical tubes at 1 mL per tube. APC-labeled antibodies were 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 antibodies on a plate shaker at 4°C in the dark for 30 minutes. Blood was treated with BD FACS lysing solution according to the manufacturer's instructions. Cells were washed, and antibody binding was assessed by flow cytometry using an LSRII flow cytometry system and FACSDiva software.

[0258] For PBMC staining, PBMCs were first washed with PBS and resuspended in cold PBS-based blocking buffer containing 50% AB human male plasma, 20 μg / mL human IgG, and 200 μg / mL human FcR blocking reagent for 15 minutes at 4°C 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 PBMCs, pDCs were identified by HLA-DR1. + , 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 staining cynomolgus monkey PBMCs. PBMCs were incubated on a plate shaker at 4°C in the dark for 30 minutes. Cells were washed, and antibody binding was assessed by flow cytometry using an LSRII flow cytometry system and FACSDiva software.

[0259] Assessment of antibody potency by cell line-based antibody-dependent cellular cytotoxicity (ADCC) assays The potency of anti-ILT7 antibodies was determined using an ADCC in vitro cell-based assay. KC1333 cells (effector) and CT cells (target) were cultured at a 5:1 ratio (2.5 × 10 KC1333 cells). 5 0.5 × 10 CT cells per 5Cells were co-cultured in round-bottom 96-well plates with 1000 cells / mL of ILT7 antibody (number of viable targets) and 1000 cells / mL of isotype control (number of viable targets at baseline) in RPMI 1640 medium supplemented with 10% FBS at 37°C and 5% CO2 for 16 hours. Cells were then washed and transferred to blocking buffer (PBS-10% FBS). KC1333 cells were detected using Pacific-Blue-anti-CD56 antibody. Dead cells were detected using 7-AAD. Target cell viability was assessed by flow cytometry using an LSRII flow cytometry system and FACSDiva software. The percentage of cytotoxicity was obtained by applying the following formula: percentage cytotoxicity = 100 - (number of viable targets / number of viable targets at baseline) × 100.

[0260] Assessment of antibody potency by ADCC assay using human PBMCs Human PBMCs were washed with PBS and then cultured at 5.0 × 10 in RPMI medium supplemented with 10% FBS and 200 ng / mL recombinant human IL-2. 6The PBMCs were resuspended at a concentration of 100 cells / mL in a round-bottom 96-well plate in duplicate at 100 μL per well. Ten-fold serial dilutions of the anti-ILT7 antibody and control antibody were prepared, and 100 μL of the antibody solution was added to the appropriate wells to achieve final concentrations ranging from 33.85 nM to 3.385 fM. The cells were incubated for 6 hours at 37°C and 5% CO2. After incubation, the cells were washed twice with 250 μL of cold PBS. The cells were resuspended in 100 μL of cold PBS-based block buffer containing 50% AB human male plasma, 20 μg / mL human IgG, and 200 μg / mL human FCR blocking reagent for 15 minutes at 4°C. After the blocking step, 100 μL of cold block buffer containing FITC-anti-human BDCA2 and APC-anti-human CD123 antibodies was added to the appropriate wells. The plate was incubated for 30 minutes at 4°C with gentle shaking. 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 by human PBMCs Human PBMCs were washed with PBS and seeded in duplicate in round-bottom 96-well plates 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. Ten-fold serial dilutions of anti-ILT7 antibodies and control antibodies were prepared, and 100 μL of antibody solution was added to the appropriate wells at final concentrations of 6.77 nM–0.677 fM. The cells and antibodies were incubated for 9.5–10 hours at 37°C and 5% CO2. After incubation, 50 μL of ODN2216 (Invitrogen™) was added to the appropriate wells at a final concentration of 0.5 μM, and the plates were incubated for an additional 16 hours at 37°C and 5% CO2. After incubation, plates were centrifuged at 350 g for 10 min, the supernatants were carefully collected, and IFNα was quantified using a multisubtype IFNα ELISA kit (PBL Biomedical).

[0262] IFNα secretion assay using 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 the appropriate wells at densities ranging from 314,000 to 818,000 cells / well. Ten-fold serial dilutions of anti-ILT7 antibodies and control antibodies were prepared, and 100 μL of antibody solution was added to the appropriate wells at final concentrations of 33.85 nM to 3.385 fM. The cells and antibodies were incubated for 9.5 to 10 hours at 37°C and 5% CO2. After incubation, 50 μL of ODN2216 (Invitrogen™) was added to the appropriate wells at a final concentration of 0.5 μM, and the plate was incubated for an additional 16 hours at 37°C and 5% CO2. After incubation, plates were centrifuged at 350 g for 10 min, the supernatants were carefully collected, and IFNα in the supernatants was quantified using a rhesus / cynomolgus 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 murine antibody SB128 Murine mAb SBI28 (SBI28 refers to the anti-ILT7 antibody ILT7#28 provided in U.S. Patent Application Publication No. 2009 / 0280128) was humanized by framework shuffling (Dall'Acqua et al., Methods 36:43-60 (2005)). Using this method, murine mAb SBI28 was humanized by synthesizing a combinatorial library consisting of its six CDRs fused in-frame to a pool of individual human germline frameworks. The human framework genes were selected from a pool of published antibody germline genes. These universal framework primer pools included 46 human germline kappa chain genes, 5 human germline J sequences, 44 human germline heavy chain genes, and 6 human germline J sequences. Primer banks were designed to encode each framework of each germline gene. Antibody-specific CDR primers with degenerate ends that overlapped with the framework pool were also synthesized. The SBI28 framework shuffle library was constructed by pairing a variable heavy chain framework shuffle sub-library with a variable light chain framework shuffle sub-library. The framework shuffle sub-libraries were sequentially assembled using PCR by 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. A second "assembly PCR" was then performed using the fusion PCR products as templates to amplify the full-length VH and VL sub-libraries. The SBI28 framework shuffle library was cloned into an M13-based Fab expression vector using Kunkel hybridization mutagenesis. Approximately 1,300 clones from the SBI28 framework shuffle library were screened on recombinant ILT7 CHO cells expressing ILT7 CHO cells using the MesoScale Discovery (MSD) assay.One humanized variant, 10D10, bound with 3-fold lower affinity to human ILT7 compared to its chimeric parent ("SBI28ch") as measured by surface plasmon resonance (SPR) in ProteOn. SBI28ch refers to the anti-ILT7 antibody ILT7#28 provided in U.S. Patent Application Publication No. 2009 / 0280128, which is incorporated herein by reference in its entirety.

[0265] Affinity optimization of 10D10 was initiated to improve its binding affinity to human and cynomolgus monkey ILT7. 10D10 was first cloned into an M13-based ScFv expression vector for parsimony mutagenesis. In this method, individual amino acids in each of all six CDRs were 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 hybridization mutagenesis (Kunkel, TA, et al. Methods Enzymol. 154:367 (1987)). Screening of the synthesized libraries consisted of a single-point ELISA designed to capture a limiting concentration of secreted ScFv from bacterial culture medium to normalize the scFv concentration in each well. Labeled ILT7 antigen bound to the captured ScFv and the signal intensity of this interaction were correlated with 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 nine positions in six CDRs were co-encoded. Briefly, degenerate primers were designed to encode all beneficial amino acid changes as well as the parent residue at the same position. This combinatorial library was screened by single-point capture ELISA as previously described. Approximately 1,200 clones were screened. The variable regions of the affinity-improved variant 7C7 were cloned into a mammalian expression pOE vector and transiently expressed in HEK293 cells. Secreted soluble human IgG was purified directly from the conditioned medium. The purified IgG was assayed for binding to rILT7 using ProteOn and FACS. In ProteOn experiments, the affinity-optimized antibody 7C7 exhibited a K of approximately 60-fold higher than that of SBI28ch. DBy FAC, which measures binding to recombinant human and cynomolgus ILT7 expressed on CHO cells, 7C7 demonstrated 2.2-fold and 14-fold better EC50s for human and cynomolgus ILT7, respectively, compared to SBI28ch. Alignments of the VH and VL sequences of SBI28, 10D10, and 7C7 are provided in Figures 1A and 1B, respectively.

[0266] [Example 2] Generation of human ILT7 antibodies from a human library In addition to humanizing murine anti-ILT7 antibodies (as described above in Example 1), libraries of human sequences were used to generate human antibodies. By pursuing multiple strategies to generate anti-ILT7 antibodies, the chances of generating anti-ILT7 antibodies with distinct traits are maximized, allowing for the selection of ideal antibodies for specific purposes.

[0267] 2.1 Selection A large single-chain Fv (scFv) human antibody library, generated using individual heavy and light chain variable regions derived from the bone marrow of naive adult donors cloned into a phagemid vector based on the 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 in a series of repeated selection cycles on recombinant human and / or cynomolgus ILT7, essentially as previously described by Vaughan et al. (Nat. Biotechnol. 14(3):309-14 (1996)). Briefly, scFv-phage particles were incubated with a biotinylated recombinant ILT7 solution (biotinylated via the free amino acid 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. Antigen-bound ScFvs were captured on streptavidin-coated paramagnetic beads (Dynabeads® M-280) according to the manufacturer's recommendations. Unbound phages were washed in a series of washing cycles using PBS-Tween. Phage particles retained on the antigen were eluted, infected into bacteria, and rescued for the next selection round. Typically, three rounds of selection were performed in this manner.

[0268] 2.2 Identification of ILT7-specific binders by phage ELISA The scFvs were displayed on phage particles and tested in binding assays to determine cross-reactivity and specificity to the recombinant antigen. Detailed assay procedures are provided in the Materials and Methods section. Approximately 2,100 individual data points were generated from the binding assays, and identified hits, i.e., scFv clones that showed 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 binders by FMAT The unique scFvs were expressed in bacterial periplasm and their binding activity was screened in a fluorescence microvolume assay (FMAT) binding assay. Binding of the cell surface-expressed scFvs to ILT7 was detected using a goat anti-mouse Alexafluor®-647-labeled antibody. Detailed assay procedures are provided in the Materials and Methods section.

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

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

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

[0273] EC binding of variants in cells expressing cynomolgus ILT7 50 To determine the binding potential of ILT70052 (EC 50 =0.35nM), ILT70080(EC 50 =0.44nM), LT70083(EC 50 =1.37nM), ILT70137(EC 50 =1.40nM), 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 did not show any binding to ILT7-expressing cells. The graph in Figure 3 represents the average results of two independent experiments, and the table in Figure 3 shows the EC 50 The average value of

[0274] Thus, ILT70052, ILT70080, ILT70083, ILT70100, ILT70137, ILT70142, and ILT70144 all bind to cells expressing either cynomolgus monkey ILT7 or human ILT7. In particular, ILT70080, ILT70083, and ILT70137 show low EC20 binding activity on cells expressing both cynomolgus monkey and human ILT7. 50 values ​​were obtained.

[0275] [Example 4] ADCC potency of ILT7 antibodies Anti-ILT7 antibodies were tested for ADCC potency against human ILT7-expressing cell lines 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 gated out using the NK marker CD56 (Biolegend #304624), and 7-AAD was used to distinguish live from dead cells. Using this method, the percentage of live target cells was calculated and compared to the baseline (no antibody control). Cytotoxicity was calculated using the following formula: Percent cytotoxicity = 100 - (number of viable targets / number of viable targets in no-antibody control) x 100

[0276] ILT70080 showed the greatest ADCC potency against human ILT7-expressing cells (EC 50 =0.022nM), and ILT70137 (EC 50 =0.044 nM) and ILT70083 (EC 50 =0.094 nM). ILT70142, ILT70052, ILT70100, and ILT70144 also demonstrated ADCC activity (Figure 4). The isotype controls R347 and the low-fucose version of R347 ("Afuc R347") did not demonstrate any ADCC activity against human ILT7-expressing cells.

[0277] Anti-ILT7 antibodies were tested for ADCC potency against cynomolgus monkey ILT7-expressing cells using in vitro cell-based activity. ILT70080 showed the greatest ADCC potency against cynomolgus monkey ILT7-expressing cells (EC 50 =0.008nM), and ILT70137 (EC 50 =0.015nM), ILT70142(EC 50 =0.058nM), ILT70052(EC 50 =0.073nM), ILT70144(EC 50=0.123), ILT70100(EC 50 =0.188nM), and ILT70083 (EC 50 =0.433 nM). ILT70089 also demonstrated ADCC activity. The positive control 7C7 demonstrated ADCC, and the isotype (negative) control R347 did not demonstrate any ADCC against cynomolgus ILT7-expressing cells. The graph and table in Figure 5 are representative of two independent experiments.

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

[0279] [Example 5] Binding of ILT7 antibody to PBMCs Binding of anti-ILT7 antibodies ILT70080, ILT70083, and ILT70137 in human PBMCs was assessed by flow cytometry using an antibody concentration of 2.5 μg / ml. ILT70080, ILT70083, and ILT70137 bind to pDCs (BDCA-4 + The antibody specifically bound to the IgG1-dependent IgG1 receptor agonist (IgG1-dependent agonist) and IgG2-dependent agonist (IgG1-dependent agonist) (Figures 6A and 6B). The isotype control R347 showed negative binding.

[0280] Binding of anti-ILT7 antibodies ILT70080, ILT70083, and ILT70137 to cynomolgus monkey PBMCs was also assessed by flow cytometry. ILT70080 and ILT70083 bind to pDCs (HLA-DR + , Lineage - , CD123 high It specifically bound to the IgG4-dependent IgG4 receptor agonist (IgG4-dependent agonist) and IgG4-dependent ...).

[0281] [Example 6] Effect of ILT7 antibody on IFN-alpha secretion Anti-ILT7 variants were tested for ADCC efficacy in human and cynomolgus monkey PBMCs as described above. IFNα secretion in the supernatants of PBMCs cultured with anti-ILT7 antibodies 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] Low fucose content 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 cellular cytotoxicity (ADCC) is mediated by lymphocyte receptor binding to the antibody Fc region and is affected by the amount of fucosylation. Increased ADCC has been observed with decreased fucosylation. Therefore, a low-fucose form of ILT7 was generated and analyzed.

[0283] 7.1 Generation of low-fucose forms of anti-ILT7 antibodies ILT70080 and ILT70083 IgG1s were expressed in a CHO cell line lacking the enzyme α-1,6-fucosyltransferase, resulting in antibodies lacking an α-1,6-fucose moiety on the N-glycan at Asn-297 of the heavy chain.

[0284] 7.2 Testing of hypofucosylated ILT70080 and ILT70083 anti-ILT7 antibodies Binding assays with low fucosylated and parental ILT70080 and ILT70083 antibodies in ILT7-expressing cells were performed to demonstrate that low fucosylation reduces the binding EC of the antibodies. 50 The parental and underfucosylated antibodies showed similar binding to both human and cynomolgus ILT7-expressing cells (Figure 7).

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

[0286] The binding of the low-fucosylated anti-ILT7 antibodies ILT70080 and ILT70083 in human PBMCs was assessed by flow cytometry. The low-fucosylated variants ILT70080 and ILT70083 inhibited the binding of pDCs (BDCA-2 + The antibody specifically bound to the IgG1-specific antibody (IgG1-specific antibody) and the isotype control R347 showed negative binding.

[0287] Binding of the low-fucosylated anti-ILT7 variants ILT70080 and ILT70083 in cynomolgus monkey PBMCs was also assessed by flow cytometry. The low-fucosylated variants ILT70080 and ILT70083 bind to pDCs (HLA-DR + Lineage - CD123 high ) specifically bound to the isotype control R347. Binding was negative for the isotype control R347.

[0288] [Example 8] Engineering of ILT70080 and ILT70083 antibodies 8.1 Operating the ILT70080 The amino acid sequences of ILT70080 VH and VL were aligned with known human germline sequences in the VBASE database (Althaus HH, Muller W and Tomlinson I: V BASE, http: / / vbase.mrc-cpe.cam.ac.uk / ), and closely 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 VL domain (E3V, K20R, S22T, M46L * , M48I * , F50Y * , and I66N *Seven residues in each framework (FW) of VH CDR2 were selected to revert to the close germline sequence. Mutations marked with an asterisk are positions classified as Vernier residues (Foote, J. et al. J. Mol. Biol. 224: 487 (1992)) and typically remain unchanged. However, analysis of both the Kabat (Wu, TT and Kabat EAJ Exp. Med. 132:211-250 (1970)) and IMGT (Lefranc, M.-P. et al. Dev. Comp. Immunol. 27: 55-77 (2003)) classifications of CDRs indicated that these positions pose a low risk of altering the binding properties of the parent antibody and offer additional opportunities to further reduce immunogenicity. Additionally, heavy chain N64Q mutagenesis was performed within the VH CDR2 (Kabat definition) sequence to remove a potential deamination (NG) site 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)). Different combinations of mutagenic oligonucleotides were utilized in multiple mutagenesis reactions to generate a library of sequences containing different combinations of FW mutations. The panel of ILT70080 scFv variants was then 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 IgGs, see Figures 9A and 9B for VH and VL sequence alignments, respectively.

[0290] 8.2 Operating the ILT70083 Germlining of ILT70083 was also performed. The closely related germline sequences identified 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, again at selected Vernier positions (V4L). * , R42T, A64G * , I66K * , S68G * , A72T, A74G, and E81G). ILT70083 variants containing different combinations of mutations were generated directly on pEU vectors containing individual VH and VL chains using standard molecular biology techniques. ILT70083 VH and VL chains were co-transfected in different combinations to generate nine IL70083 IgG1 variants. See Figures 10A and 10B for VH and VL alignments, respectively.

[0291] 8.3 Testing of Engineered Antibodies The resulting IgG1s were tested to confirm that the sequence changes incorporated into ILT70080 and ILT70083 did not adversely affect the binding of the parental antibodies to cells expressing human ILT7 (CT-550 cells) or cynomolgus ILT7 (CT-125 cells). The variants were screened for binding by flow cytometry. All ILT70080 variants had similar binding to the parent ILT70080 antibody to human and cynomolgus ILT7 (EC 50 =0.213 nM and 0.547 nM). See Figure 11. Binding of the ILT70083 variants was also similar to the parent antibody for 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 for cynomolgus ILT7 compared to the parent antibody. See Figure 12.

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

[0293] [Example 9] Low fucose content of engineered ILT70080 and ILT70083 antibodies A de-fucosylated version of ILT70080.6 was generated. De-fucosylation of the ILT7080.6 antibody did not affect its binding to either human or cynomolgus ILT7-expressing cells. Binding EC of de-fucosylated ILT70080.6 to human and cynomolgus ILT7-expressing cells 50 were 152.3 pM and 366.2 pM, respectively. See Figure 15. The table in Figure 15 provides the average results of three independent binding experiments measuring mean fluorescence intensity (MFI).

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

[0295] De-fucosylated ILT70080.6 and ILT70083 were tested for ADCC efficacy in human PBMCs. Antibody cytotoxicity was assessed by flow cytometry, and CpGA-mediated IFNα secretion in the supernatant was measured by ELISA. The results are shown in Figure 17. In cynomolgus monkey PBMCs, the EC50 values ​​for IFNα secretion using de-fucosylated ILT70080.6 and ILT70083 antibodies were 58 pM and 5216 pM, respectively.

[0296] In human whole blood and PBMCs, the defucosylated ILT70080.6 and ILT70083 antibodies were found to bind specifically to BDCA-2-positive cells. Binding of both antibodies was restricted to human pDCs at all concentrations tested (0.1–5.0 μg / mL).

[0297] In cynomolgus monkey whole blood, the low-fucosylated ILT70080.6 and ILT70083 antibodies inhibited pDC (HLA-DR) at all concentrations tested (0.5-2.5 μg / mL). + Lineage - CD123 high It was found to bind to IgG4-dependent phospholipase A (IgG4-dependent phospholipase A) and IgG4-dependent phospholipase A (IgG4-dependent phospholipase A) receptors.

[0298] [Example 10] Low fucosylation of ILT70137 antibody A de-fucosylated version of the ILT70137 antibody was generated as described above in Example 7 for the ILT70080 and ILT70083 antibodies.

[0299] 10.1 Binding to soluble recombinant human ILT7 BIAcore (surface plasmon resonance) was used to analyze the binding kinetics (k) of the de-fucosylated IgG1 ILT70137 to human ILT7 protein using an IgG capture assay format. on , k off ) constants were measured. Binding of each concentration of two-fold serial dilutions of ILT7 protein was recorded by first capturing IgG on the sensor chip surface, followed by capture of either ILT7 protein or instrument buffer. Between each pair of injections, the IgG capture surface was regenerated. Individual association and dissociation rate constants were calculated from the resulting binding curves using a 1:1 fitting model using Biaevaluation software available through the vendor's software, which included a term to correct for mass-transfer-limited binding, if detected. From a high-resolution BIAcore plot of the data, the association and dissociation rate constants for binding of ILT7 protein to the underfucosylated IgG1ILT70137 were calculated as 1.855 x 10 5 M -1 s -1 This same plot was also used to determine the corresponding dissociation rate constant for this interaction, which was 3.175×10 -2 s -1 From these rate constants, K D is k off / k on The result is summarized in Table 3 below. off and k on The individual errors of the are very low and the overall fit to the data is well within the R maxThe observed binding rate was approximately 1% of the maximum response, which was satisfactory. Taken together, this suggests that the use of a single-site binding model to fit the data was appropriate. The evaluation indicates that binding is not mass-transfer limited, and the measured binding rate constants are considered valid.

[0300] [Table 3]

[0301] 10.2 Binding to ILT7-expressing cell lines The binding of underfucosylated ILT70137 to ILT7 was determined using cell lines stably expressing human or cynomolgus ILT7. The mean fluorescence intensity of cell-bound antibodies was assessed by flow cytometry. Cells were incubated with increasing concentrations of test antibodies ranging from 0.004 to 333.3 nM for 30 minutes at 4°C. After incubation, cells were washed with cold PBS and incubated with anti-human Alexa Fluor 647 antibody for 30 minutes at 4°C. Fluorescence intensity was then determined by FACS, and EC 50 Values ​​were calculated using a nonlinear fit equation in GraphPad Prism 6 software.

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

[0303] 10.3 ADCC activity against ILT7-expressing cell lines The potential of de-fucosylated ILT70137 to induce ADCC was measured by fluorescence-activated cell sorting (FACS) assay in target cells expressing human or cynomolgus ILT7. Target cells were incubated with increasing concentrations (0–6.66 × 10) of de-fucosylated ILT70137 or isotype control at a 1:5 ratio with the effector NK cell line KC1333. -9 For evaluation of target cell viability by flow cytometry, KC1333 cells were gated out using CD56, and dead cells were gated out using 7-amino-actinomycin D (7-AAD) viability staining. Viable target cells were defined as CD56-negative, 7-AAD-negative. The percentage of cytotoxicity was calculated using the following formula: % cytotoxicity = 100 - (percentage of viable target cells / percentage of viable targets in no-antibody control) x 100. The half-maximal effective concentration (EC 50 ) Values ​​were calculated using a nonlinear fit equation in GraphPad Prism 6 software. x-axis: antibody concentration.

[0304] The results are shown in Figure 19. De-fucosylated ILT70137 induced ADCC in cells expressing ILT7 in a dose-dependent manner and showed EC 50 was 4.19 pM 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 predominantly by plasmacytoid dendritic cells (pDCs) in peripheral blood mononuclear cell (PBMC) preparations. Therefore, the ability of hypofucosylated ILT70137 to induce ADCC in primary pDCs was indirectly measured by assessing its ability to block IFNα secretion in PBMCs. In these assays, purified PBMCs were plated 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 hypofucosylated ILT70137 and control antibodies 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 is expressed as pg / mL of supernatant in Figure 20. IC of ADCC 50 was calculated using a nonlinear fit equation in GraphPad Prism v5.01 software.

[0306] De-fucosylated ILT70137 dose-dependently reduced TLR9-mediated secretion of IFNα in PBMCs, reaching half-maximal inhibitory concentrations (IC 50 ) was 0.048 nM. These results demonstrate that underfucosylated ILT70137 effectively depletes naturally occurring primary human pDCs in PBMCs.

[0307] 10.5 Binding to Primary Plasmacytoid Dendritic Cells The specificity of de-fucosylated ILT70137 for human primary plasmacytoid dendritic cells (pDCs) was assessed by FACS in peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from human donors. To properly identify this dendritic cell subset, the markers CD123 (expressed on pDCs and basophils) and CD304 (unique to pDCs) were first utilized. pDCs are CD123+CD304+ double positive, and CD304 staining was sufficient to identify pDCs. See Figure 21 (top panel). De-fucosylated ILT70137 bound exclusively to CD304-positive cells, indicating that it uniquely binds to pDCs. See Figure 21 (bottom right panel). No significant binding to this population was observed with the human IgG1 isotype de-fucosylated control antibody R3-47. See Figure 21 (bottom left panel).

[0308] [Example 11] In vivo activity of ILT7 antibodies Three anti-ILT7 antibodies, namely, de-fucosylated 7C7, de-fucosylated ILT70080.6, and de-fucosylated IgG1 ILT70137, were administered to male cynomolgus monkeys, and all three antibodies were active in depleting plasmacytoid dendritic cells (pDCs).

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

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

[0311] However, the pathology of animals treated with reduced-fucosylated 7C7 and reduced-fucosylated ILT70137 differed. Increased spleen weight was observed in some animals treated with reduced-fucosylated 7C7. Microscopic findings were also observed in some animals treated with reduced-fucosylated 7C7. In particular, red pulp and macrophage hypertrophy / hyperplasia was observed in the spleen. Kupffer cell hypertrophy / hyperplasia was observed in the liver. Furthermore, immunohistochemistry demonstrated human IgG / 7C7- and monkey IgG-containing granule deposits associated with enlarged / hyperplastic Kupffer cells in the liver and red pulp macrophages in the spleen. These findings are consistent with excessive physiological clearance of immune complexes containing the drug (7C7) and anti-drug antibodies (ADAs). In contrast, no changes in organ weight or macroscopic or microscopic findings were observed with reduced-fucosylated ILT70137.

[0312] Furthermore, for two monkeys treated with the de-fucosylated 7C7 antibody, neutrophil counts fell below 1 E3 / μl, whereas no significant changes in neutrophil counts were observed in monkeys treated with control or de-fucosylated ILT70137.

[0313] Thus, although all three antibodies depleted pDCs in vivo, the excellent safety profile and lack of anti-drug antibodies following administration of underfucosylated ILT70137 is a surprising advantage.

[0314] [Example 12] Epitope mapping To determine the epitope bound by the ILT7 antibody, 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 is not recognized by the ILT7 monoclonal antibody. Therefore, we selected it for constructing chimeric variants. The chimeric polypeptides were generated by exchanging the extracellular Ig domain of ILT7 with its ILT1 counterpart. All of these constructs contained an N-terminal Flag tag. 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 specific embodiments fully reveals the general nature of the present invention, so that others, by applying the knowledge of those skilled in the art, can readily modify and / or adapt such specific embodiments without undue experimentation and without departing from the general concept of the present invention. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is understood that the words or terms used herein are for purposes of description and not of limitation, and that the words or terms used herein will be interpreted by one of ordinary skill in the art in light of the teaching and guidance.

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

[0317] [Sequence table] SEQUENCE LISTING <110> VIELA BIO, INC. <120> ILT7 BINDING MOLECULES AND METHODS OF USING THE SAME <130> PA25-266 <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 tcctgtgcag cctctggatt cacctttagc atctacccca tcgagtgggt gcgacaggct 120 cctggacagg gcctggaatg gatcggcaac ttccacccct acaacgacga caccaagtac 180 aacgagaagt tcaagggcag agtcaccatg accacagaca catccacgag cacagtgtac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtac gagaggcgac 300 gactacggcc tggactattg gggccagggc accctcgtga ccgtgtcctc t 351 <210> 12 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 7C7 VH <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 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 <h2 style=";text-align:left;direction:ltr">attacctgtg acgccagcca gaatgttcgt accgcagttg catggtatca gcagaaaccg 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggtaaagcac cgaaacgtct gatttatctg gcaagtaatc gtcataccgg tgttccgagc 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cgttttagcg gtagcggttc tggcaccgat tttaccctga ccattagcag cctgcagagc 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaagattttg ccacctatta ttgtctgcag cattggaatt atccgtttac ctttggtccg 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggtacaaaac tggaaattaa a 321<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 17<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 107<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Artificial Sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> 7C7 VL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 17<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Asn Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Asp Arg Val Thr Ile Thr Cys Asp Ala Ser Gln Asn Val Arg Thr Ala<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> 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 acctgacca 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 <h2 style=";text-align:left;direction:ltr"><213> Artificial Sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> ILT70080.4 VL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 66<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tcctatgagc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaagatt 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tcctgtgggg gagacagcgt tggcagtaca agtgtccact ggtaccagca gaagccggggc 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> caggcccctg tgatggtcat gttttataac agtgaccggc cctcagggat ccctgagcga 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttctctggct ccatctctgg gaacacggcc accctgacca tcagcaggggt cgaagccgggg 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gatgaggccg actattactg tcaggtgtgg gatactagta gtgatcatcc ggagttcggc 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggagggacca agctgaccgt ccta 324<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 67<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 108<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Artificial Sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> ILT70080.4 VL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 67<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 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 tcctgcagg catctggaga cagcttcagg aactagctg tcagttgggt gcgacaggcc 120 ccaggacaag gtcttgagtg gatgggagcg atgatgccta gttttggac aagggagcac 180 tcacagcagt tccaggggcag agtcaccctt accgcggacg atccacgag cacagcctac 240 atggaactga gcagtctaag atctgaggac acggccgtct attactgtgc gggtagtcgg 300 gactacaatg cttaccattt tggactggt ccccctgact tctggggccg aggaccctg 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 <h2 style=";text-align:left;direction:ltr">tcctatgtgc tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaggatt 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acctgtgggg gagacagcgt tggcagtaca agtgtccact ggtaccagca gaagccggggc 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> caggcccctg tgatggtcat gttttataac agtgaccggc cctcagggat ccctgagcga 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttctctggct ccaactctgg gaacacggcc accctgacca tcagcaggggt cgaagccggg 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gatgaggccg actattactg tcaggtgtgg gatactagta gtgatcatcc ggagttcggc 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggagggacca agctgaccgt ccta 324<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 77<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 108<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Artificial Sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> ILT70080.5 VL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 77<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Thr Ala Arg Ile Thr Cys Gly Gly Asp Ser Val Gly Ser Thr Ser Val<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 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 Assn 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 gttttggac aagggagcac 180 tcacagcagt tccagggcag agtcaccctt accgcggacg atccacgag cacagcctac 240 atggaactga gcagtctaag atctgaggac acggccgtct attactgtgc gggtagtcgg 300 gactacaatg cttaccattt tggactggt ccccctgact tctggggccg aggaccctg 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 <h2 style=";text-align:left;direction:ltr">acctgtgggg gagacagcgt tggcagtaca agtgtccact ggtaccagca gaagccggggc 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> caggcccctg tgctggtcat ttattataac agtgaccggc cctcagggat ccctgagcga 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttctctggct ccaactctgg gaacacggcc accctgacca tcagcaggggt cgaagccggg 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gatgaggccg actattactg tcaggtgtgg gatactagta gtgatcatcc ggagttcggc 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggagggacca agctgaccgt ccta 324<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 87<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 108<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Artificial Sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> ILT70080.6 VL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 87<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Thr Ala Arg Ile Thr Cys Gly Gly Asp Ser Val Gly Ser Thr Ser Val<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 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 acctgacca tcagcagggt cgaagccggg 240 gatgaggccg actattactg tcaggtgtgg gatactagta gtgatcatcc ggagttcggc 300 ggagggacca agctgaccgt cct 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>...

Claims

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.

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.

3. An isolated ILT7 binding protein comprising complementarity determining regions (CDRs) HCDR1, HDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the sequences of SEQ ID NOs: 203, 204, 205, 208, 209, and 210, respectively.

4. 4. The isolated ILT7 binding protein of any one of claims 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.

5. 5. The isolated ILT7 binding protein of claim 4, comprising a VH comprising SEQ ID NO: 202 and a VL comprising SEQ ID NO:

207.

6. 1. An isolated ILT7 binding protein comprising a VH comprising SEQ ID NO:

202.

7. 1. An isolated ILT7 binding protein comprising a VL comprising SEQ ID NO:

207.

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; 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 a VH and VL selected from the group consisting of:

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; 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 to ILT7 of an antibody comprising a VH and a VL selected from the group consisting of:

10. SEQ ID NOs: 13, 14, 15, 18, 19, and 20, respectively; SEQ ID NOs: 23, 24, 25, 28, 29, and 30, respectively; SEQ ID NOs: 33, 34, 35, 38, 39, and 40, respectively; SEQ ID NOs: 103, 104, 105, 108, 109, and 110, respectively; SEQ ID NOs: 213, 214, 215, 218, 219, and 220, respectively; SEQ ID NOs: 223, 224, 225, 228, 229, and 230, respectively; SEQ ID NOs: 233, 234, 235, 238, 239, and 240, respectively; and SEQ ID NOs: 243, 244, 245, 248, 249, and 250, respectively 1. An isolated ILT7 binding molecule comprising CDRs: HCDR1, HDR2, HCDR3, LCDR1, LCDR2, and LCDR3 selected from the group consisting of:

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 11. The isolated ILT7 binding protein of any one of claims 8 to 10, comprising a VH and VL that are at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to

12. The 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 SEQ ID NO: 242 and SEQ ID NO: 247, respectively 12. The ILT7 binding molecule of claim 11, comprising:

13. 12. 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.

14. 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 217, 227, 237, or 247.

15. 15. The isolated ILT7-binding molecule of any one of claims 1 to 14, which comprises an antibody or antigen-binding fragment thereof.

16. 16. The isolated ILT7-binding molecule of claim 15, wherein the antibody or antigen-binding fragment thereof is hypofucosylated.

17. The isolated ILT7-binding molecule of any one of claims 8 to 16, which binds to the Ig1 region of ILT7.

18. The isolated ILT7-binding molecule of any one of claims 8 to 16, which binds to the Ig2 region of ILT7.

19. 19. The isolated ILT7-binding molecule of any one of claims 1 to 18, which binds to human and cynomolgus monkey ILT7.

20. 20. The isolated ILT7-binding molecule of any one of claims 1 to 19, which inhibits interferon (IFN) alpha release from peripheral blood mononuclear cells (PBMC).

21. 21. The isolated ILT7-binding molecule of any one of claims 1 to 20, which has ADCC activity against plasmacytoid dendritic cells (pDCs) in PBMCs.

22. 22. The isolated ILT7 binding molecule of any one of claims 1 to 21, comprising a murine, human, chimeric, humanized, or resurfaced antibody or antigen-binding fragment thereof.

23. 23. The isolated ILT7-binding molecule of any one of claims 1 to 22, comprising an antibody, Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

24. 24. The isolated ILT7-binding molecule of any one of claims 1 to 23, which comprises a monoclonal antibody or an antigen-binding fragment thereof.

25. (a) IgA constant domain, (b) IgD constant domain, (c) IgE constant domain, (d) IgG1 constant domain; (e) IgG2 constant domain, (f) IgG3 constant domain; (g) an IgG4 constant domain, and (h) IgM constant domain 25. The isolated ILT7-binding molecule of any one of claims 1 to 24, comprising a heavy chain immunoglobulin constant domain selected from the group consisting of:

26. (a) an Ig kappa constant domain, and (b) Ig lambda constant domain 26. The isolated ILT7-binding molecule of any one of claims 1 to 25, comprising a light chain immunoglobulin constant domain selected from the group consisting of:

27. 27. The isolated ILT7 binding molecule of any one of claims 1 to 26, comprising a human IgG1 constant domain and a human lambda constant domain.

28. An isolated host cell producing a binding molecule according to any one of claims 1 to 27.

29. 1. 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 a 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.

30. 30. The polynucleotide of claim 29, comprising a sequence 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.

31. 1. 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 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.

32. 32. The polynucleotide of claim 31, comprising a sequence 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.

33. The polynucleotide of any one of claims 29 to 32, wherein the nucleic acid is operably linked to a regulatory sequence.

34. The polynucleotide of any one of claims 29 to 33, wherein an antibody or antigen-binding fragment thereof comprising said VH or said VL is capable of specifically binding to ILT7.

35. A polynucleotide encoding an ILT7 binding molecule according to any one of claims 1 to 27.

36. A vector comprising the polynucleotide according to any one of claims 29 to 35.

37. A polypeptide encoded by the polynucleotide according to any one of claims 29 to 35.

38. A host cell transformed with a polynucleotide according to claim 29 or 30 and a polynucleotide according to claim 31 or 32.

39. A host cell comprising the polynucleotide according to any one of claims 29 to 35, the vector according to claim 36, or the polypeptide according to claim 37.

40. 40. The host cell of claim 38 or 39, which is a mammalian host cell.

41. 41. The mammalian host cell of claim 40, which is an NS0 mouse myeloma cell, a PER.C6® human cell, or a Chinese hamster ovary (CHO) cell.

42. 42. The host cell of any one of claims 38 to 41, which lacks the enzyme alpha-1,6-fucosyltransferase.

43. 43. A method for producing an anti-ILT7 binding molecule, the method comprising culturing a host cell according to any one of claims 38 to 42 and recovering said binding molecule.

44. 44. An anti-ILT7 binding molecule produced by the method of claim 43.

45. 19. A method for detecting ILT7 expression in a sample, the method comprising: (a) contacting the sample with an ILT7 binding molecule of any one of claims 1 to 27 or 44; and (b) detecting binding of the binding molecule in the sample.

46. 134. A method for detecting plasmacytoid dendritic cells, the method comprising: (a) contacting a sample containing the cells with an ILT7-binding molecule of any one of claims 1 to 27 or 44; and (b) detecting binding of the binding molecule in the sample.

47. 1. A pharmaceutical composition comprising (a) an ILT7 binding molecule according to any one of claims 1 to 27 or 44, a polynucleotide according to any one of claims 29 to 35, a vector according to claim 36, a polypeptide according to claim 37, or a host cell according to any one of claims 28 or 38 to 42, and (b) a carrier.

48. 10. A method of reducing IFN-alpha release from plasmacytoid dendritic cells, the method comprising contacting plasmacytoid dendritic cells with a binding molecule of any one of claims 1 to 27 or 44, a polynucleotide of any one of claims 29 to 35, a vector of claim 36, a polypeptide of claim 37, a host cell of any one of claims 28 or 38 to 42, or a composition of claim 47.

49. 10. A method of treating a human subject having an autoimmune disease, comprising administering to the subject an effective amount of the binding molecule of any one of claims 1 to 27 or 44, the polynucleotide of any one of claims 29 to 35, the vector of claim 36, the polypeptide of claim 37, the host cell of any one of claims 28 or 38 to 42, or the composition of claim 47.

50. 10. A method of preventing an autoimmune disease in a human subject, comprising administering to the subject an effective amount of the binding molecule of any one of claims 1 to 27 or 44, the polynucleotide of any one of claims 29 to 35, the vector of claim 36, the polypeptide of claim 37, the host cell of claim 28 or any one of claims 38 to 42, or the composition of claim 47.

51. 51. The method of claim 49 or 50, wherein the autoimmune disease is systemic lupus erythematosus.

52. 51. The method of claim 49 or 50, wherein the autoimmune disease is rheumatoid arthritis.

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