Antibodies targeting ILT7 and uses thereof

Abstract

Disclosed herein are anti-ILT7 antibodies and antigen binding fragments, polynucleotides encoding the antibodies and antigen binding fragments, and pharmaceutical compositions comprising the antibodies and antigen binding fragments. The use of the anti-ILT7 antibodies and antigen binding fragments described herein in the treatment of conditions and disorders associated with complement activation is also disclosed.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to International Patent Application No. PCT / CN2023 / 082006, filed on March 16, 2023, the entire disclosure of which is incorporated herein by reference for all purposes.

[0003] References to sequence lists submitted electronically

[0004] This application incorporates, in the form of an XML file, a sequence list created on March 15, 2024, entitled “110961-1435499-018-001-02PCT”, with a size of 65,204 bytes. Technical Field

[0005] This invention relates to molecular biology, cell biology, and immunology. The invention includes anti-ILT7 antibodies and their use in the treatment of plasmacytoid dendritic cell (pDC) or type I interferon (type I IFN)-related immune disorders. Background Technology

[0006] Plasma cell-like dendritic cells (pDCs), responsible for producing type I interferon (IFN) and pro-inflammatory cytokines, are drivers of both innate and adaptive immune responses. Both pDCs and type I IFN are involved in a variety of immune disorders. ILT7, a member of the immunoglobulin-like transcript (ILT) or leukocyte immunoglobulin-like receptor (LIR) gene family, is selectively expressed in pDCs. Therefore, there is a need for ILT7 targets that can inhibit pDC-associated type I IFN release, for example, for the treatment and prevention of autoimmune diseases. However, limited success has been achieved in developing such ILT7 targets. The compositions and methods presented herein address these needs and offer relative advantages. Summary of the Invention

[0007] As used in this document, the terms “invention,” “the invention,” “this invention,” and “the present invention” are intended to refer broadly to all subject matter of this patent application and the claims below. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the patent claims below. The embodiments covered by this invention are defined by the claims, not by the content of this invention. The content of this invention is a high-level overview of various aspects of the invention and introduces some concepts among those described and illustrated in this document and the accompanying drawings. The content of this invention is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. This subject matter should be understood by referring to appropriate portions of the specification throughout, any or all figures, and each claim. Some exemplary embodiments of the invention are discussed below.

[0008] This document provides antibodies or antigen-binding fragments thereof that specifically bind to human ILT7, the antibodies or antigen-binding fragments comprising: (1) as defined by Kabat, (a) a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO: 11, 12, and 13, respectively; or variants thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs; and / or (b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 14, 15, and 16, respectively; or variants thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs; or (2) as defined by Chothia, (a) a VL comprising VL CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 11, 12, and 13, respectively. CDR1, VL CDR2, and VL CDR3; or variants thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDR; and / or (b) VH, the VH comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO: 17, 18, and 16, respectively; or variants thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VLCDR.

[0009] In some embodiments, the antibody or antigen-binding fragments provided herein comprise VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VHCDR3, respectively, having the amino acid sequences SEQ ID NO: 11, 12, 13, 14, 15, and 16, as defined by Kabat. In some embodiments, the antibody or antigen-binding fragments provided herein comprise VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3, respectively, having the amino acid sequences SEQ ID NO: 11, 12, 13, 17, 18, and 16, as defined by Chothia.

[0010] This document also provides antibodies or antigen-binding fragments thereof that specifically bind to human ILT7, said antibodies or antigen-binding fragments comprising: (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:9; and / or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:10. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise VL and VH having the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10, respectively.

[0011] This document also provides antibodies or antigen-binding fragments thereof that specifically bind to human ILT7, wherein the antigen or antigen-binding fragment comprises: (a) a VL comprising VL CDR1, VL CDR2, and VL CDR3 of a VL having the amino acid sequence of SEQ ID NO:9; and / or (b) a VH comprising VH CDR1, VH CDR2, and VH CDR3 of a VH having the amino acid sequence of SEQ ID NO:10.

[0012] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment.

[0013] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein is a humanized antibody or antigen-binding fragment. In some embodiments, the humanized anti-ILT7 antibody or antigen-binding fragment comprises: (a) VL, wherein the VL has at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:19-22; and / or (b) VH, wherein the VH has at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:23-28.

[0014] In some embodiments, the humanized anti-ILT7 antibody or antigen-binding fragment comprises VL and VH having the following amino acid sequences respectively: (1) SEQ ID NO: 19 and 23; (2) SEQ ID NO: 19 and 24; (3) SEQ ID NO: 19 and 25; (4) SEQ ID NO: 19 and 26; (5) SEQ ID NO: 19 and 27; (6) SEQ ID NO: 19 and 28; (7) SEQ ID NO: 20 and 23; (8) SEQ ID NO: 20 and 24; (9) SEQ ID NO: 20 and 25; (10) SEQ ID NO: 20 and 26; (11) SEQ ID NO: 20 and 27; (12) SEQ ID NO: 20 and 28; (13) SEQ ID NO: 21 and 23; (14) SEQ ID NO: 21 and 24; (15) SEQ ID NO: 21 and 25; (16) SEQ ID NO: 21 and 25; (17) SEQ ID NO:21 and 27; (18) SEQ ID NO:21 and 28; (19) SEQ ID NO:22 and 23; (20) SEQ ID NO:22 and 24; (21) SEQ ID NO:22 and 25; (22) SEQ ID NO:22 and 26; (23) SEQ ID NO:22 and 27; or (24) SEQ ID NO:22 and 28.

[0015] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein is selected from the group consisting of: Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-domain antibody (sdAb), and heavy chain antibody (HCAb). In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein may be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0016] In some embodiments, the anti-ILT7 antibody provided herein is an IgG1 antibody. In some embodiments, the anti-ILT7 IgG1 antibody provided herein comprises a light chain constant region (CL) having at least 85% sequence identity with κCL (Cκ; SEQ ID NO: 29). In some embodiments, the anti-ILT7 IgG1 antibody provided herein comprises a light chain constant region (CL) having at least 85% sequence identity with λCL (Cλ; SEQ ID NO: 30).

[0017] In some embodiments, the anti-ILT7 IgG1 antibody provided herein comprises a heavy chain constant region (CH) having at least 85% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:31 and 40-44.

[0018] In some embodiments of the anti-ILT7 IgG1 antibody provided herein, the heavy chain constant region (CH) comprises wild-type IgG1 CH, or comprises at least one amino acid mutation that enhances the antibody's ADCC (antibody-dependent cytotoxicity) or ADCP (antibody-dependent phagocytosis). In some embodiments, the CH region of the IgG1 antibody provided herein has amino acid substitutions at L234, L235, G236, S239, F243, H268, D270, R292, S298, Y300, V305, K326, A330, I332, E333, K334, P396, or any combination thereof, according to EU index numbers. In some embodiments, the CH region of the IgG1 antibody provided herein has amino acid substitutions, said amino acid substitutions being L234Y, L235Q, L235V, G236A, G236W, S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330M, A330L, I332E, E333A, K334A, K334E, or P396L or any combination thereof, according to EU index numbers. In the anti-ILT7 antibody provided herein... In some embodiments of the IgG1 antibody, the CH region is modified with amino acid substitutions selected from the group consisting of the following according to EU index numbers: (i) S298A, E333A, and K334A; (ii) S239D and I332E; (iii) S239D, A330L, and I332E; (iv) G236A; (v) G236A, S239D, and I332E; (vi) G236A, A330L, and I332E; (v ii) G236A, S239D, A330L, and I332E; (viii) F243L, R292P, Y300L, V305I, and P396L; (ix) L235V, F243L, R292P, Y300L, and P396L; (x) L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A; and (xi) D270E, K326D, A330M, and K334E. In some embodiments, the CH region has an amino acid sequence selected from the group consisting of SEQ ID NO:45-64.

[0019] In some embodiments of the anti-ILT7 IgG1 antibody provided herein, the Fc is unfucosylated.

[0020] This article also provides antibody or antigen-binding fragments that compete with the antibody or antigen-binding fragments described herein for binding to human ILT7.

[0021] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein is a bispecific antibody or a multispecific antibody.

[0022] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein is a monoclonal antibody or its antigen-binding fragment.

[0023] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment (1) provided herein is at a Kc of 500 nM or less as determined by SPR. D (1) Binds to human ILT7; (2) Does not specifically bind to LILR family members LILRA1, LILRA2 / ILT1, LILRA3 / ILT6, LILRA5 / ILT11, LILRA6 / ILT8, LILRB1 / ILT2, LILRB2 / ILT4, LILRB3 / ILT5, LILRB4 / ILT3, or LILB5; (3) Inhibits the release of interferon α (IFNα) via peripheral blood mononuclear cells (PBMCs); (4) Selectively binds to plasmacytoid dendritic cells (pDCs) in human PBMCs; (5) Exhibits natural killer cell (NK)-dependent ADCC activity against ILT7-expressing cells; (6) Exhibits neutrophil-dependent ADCC activity against ILT7-expressing cells; or (7) Exhibits macrophage-dependent ADCP activity against ILT7-expressing cells; or any combination of (1)-(7).

[0024] This article also provides an anti-ILT7 antibody or its antigen-binding fragment thereof, said anti-ILT7 antibody or its antigen-binding fragment (1) at a Kc of 500 nM or less as determined by SPR. D (1) Binds to human ILT7; (2) Does not specifically bind to LILR family members LILRA1, LILRA2 / ILT1, LILRA3 / ILT6, LILRA5 / ILT11, LILRA6 / ILT8, LILRB1 / ILT2, LILRB2 / ILT4, LILRB3 / ILT5, LILRB4 / ILT3, or LILB5; (3) Inhibits IFNα release via PBMCs; (4) Selectively binds to pDCs in human PBMCs; (5) Exhibits NK-dependent ADCC activity against ILT7-expressing cells; (6) Exhibits neutrophil-dependent ADCC activity against ILT7-expressing cells; or (7) Exhibits macrophage-dependent ADCP activity against ILT7-expressing cells; or any combination of (1)-(7).

[0025] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein (1) inhibits IFNα release from CpG-stimulated PBMCs in vitro with an EC50 of 1 nM or less; (2) exhibits NK-dependent ADCC activity against ILT7-expressing cells with an EC50 of 0.01 nM or less; (3) exhibits neutrophil-dependent ADCC activity against ILT7-expressing cells with an EC50 of 100 nM or less; (4) exhibits macrophage-dependent ADCP activity against ILT7-expressing cells with an EC50 of 10 nM or less; or (5) exhibits macrophage-dependent ADCP activity against ILT7-expressing cells with a maximum phagocytic index of 20% or higher; or any combination of (1)-(5).

[0026] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein (1) inhibits IFNα release via PBMCs with an EC50 ranging from 0.01 nM to 0.1 nM; (2) exhibits NK-dependent ADCC activity against ILT7-expressing cells with an EC50 ranging from 0.001 nM to 0.01 nM; (3) exhibits neutrophil-dependent ADCC activity against ILT7-expressing cells with an EC50 ranging from 1 nM to 50 nM; (4) exhibits macrophage-dependent ADCP activity against ILT7-expressing cells with an EC50 ranging from 0.5 nM to 5 nM; or (5) exhibits macrophage-dependent ADCP activity against ILT7-expressing cells with a maximum phagocytic index ranging from 20% to 80%; or any combination of (1)-(5).

[0027] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein exhibits neutrophil-dependent ADCC activity.

[0028] This article also provides polynucleotides encoding polypeptides of the anti-ILT7 antibody or antigen-binding fragments described herein. Vectors containing the polynucleotides described herein are also provided.

[0029] This document also provides host cells containing the polynucleotides or vectors described herein. In some embodiments, the host cells described herein (1) overexpress N-acetylglucosamine transferase III (GnTIII); (2) lack α-1,6-fucosyltransferase (FUT8); or (3) have low fucose content; or any combination of (1)-(3).

[0030] This document also provides methods for preparing the anti-ILT7 antibody or antigen-binding fragment described herein, the methods comprising culturing the host cells described herein under conditions that allow expression of the antibody or antibody fragment. In some embodiments, the methods provided herein comprise isolating the antibody from the culture.

[0031] This article also provides pharmaceutical compositions comprising a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment described herein and a pharmaceutically acceptable carrier.

[0032] This document also provides a method for reducing type I interferon (IFN) in subjects in need, the method comprising administering to the subject a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, the type I interferon is IFNα.

[0033] This article also provides a method for inhibiting or depleting pDC in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment described herein.

[0034] This article also provides a method for reducing autoimmunity in subjects in need, the method comprising administering to the subject an effective amount of the anti-ILT7 antibody or antigen-binding fragment described herein.

[0035] In some embodiments of the methods provided herein, the subject suffers from an autoimmune disease.

[0036] This article also provides a method for treating subjects with autoimmune diseases associated with type I IFN or pDC, the method comprising administering to the subject a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment described herein.

[0037] In some embodiments, the autoimmune disease is systemic lupus erythematosus (SLE).

[0038] In some embodiments, the method provided herein further includes administering additional therapy to the subject.

[0039] In some embodiments of the methods provided herein, the subject is a human being.

[0040] In some embodiments, this document provides for the use of the anti-ILT7 antibody or antigen-binding fragment described herein for reducing type I IFN. This document also provides for the use of the anti-ILT7 antibody or antigen-binding fragment described herein for preparing a medicament for reducing type I IFN. In some embodiments of the uses provided herein, the type I IFN is IFNα.

[0041] In some embodiments, this document provides for the use of the anti-ILT7 antibody or antigen-binding fragment described herein for inhibiting or depleting pDC. This document also provides for the use of the anti-ILT7 antibody or antigen-binding fragment described herein for preparing a medicament for inhibiting or depleting pDC.

[0042] In some embodiments, this document provides for the use of the anti-ILT7 antibody or antigen-binding fragment described herein for reducing autoimmunity. In some embodiments, this document provides for the use of the anti-ILT7 antibody or antigen-binding fragment described herein for the preparation of a medicament for reducing autoimmunity.

[0043] In some embodiments, this document provides for the use of the anti-ILT7 antibody or antigen-binding fragment described herein for the treatment of autoimmune diseases associated with type I IFN or pDC. In some embodiments, this document provides for the use of the anti-ILT7 antibody or antigen-binding fragment described herein for the preparation of a medicament for the treatment of autoimmune diseases associated with type I IFN or pDC. In some embodiments, the autoimmune disease is SLE. Attached Figure Description

[0044] Figure 1 ELISA results showing the binding of the ILT7 chimeric antibody, reference antibody (Tab1), and negative control antibody (hIgG1) to the human ILT7 protein are provided.

[0045] Figure 2A-2B Provided are illustrations of the ILT7 chimeric antibody, reference antibody (Tab1), and negative control antibody (hIgG1) in relation to 293F-human ILT7 cells ( Figure 2A ) and CHOK1-cynomolgus monkey ILT7 cells ( Figure 2B The results of flow cytometry analysis of the combination of )

[0046] Figure 3 Representative results from IFNα release assays performed on CpG-stimulated PBMCs are provided. The inhibitory activity of the ILT7 chimeric antibody against IFNα release was measured; results from the reference antibody (Tab1) and the negative control antibody (hIgG1) are also shown.

[0047] Figures 4A-4B Provided are humanized antibodies (Hu12) and reference antibodies (Tab1) against 293F-human ILT7 cells (Hu12 and Tab1). Figure 4A ) and CHOK1-cynomolgus monkey ILT7 cells ( Figure 4B The results of flow cytometry analysis of the combination of )

[0048] Figures 5A-5BRepresentative results are provided for the assay of IFNα release from CpG-stimulated PBMCs. The inhibitory activity of four humanized antibodies against cmAb12 against IFNα release was measured, along with the inhibitory activity of the reference antibody (Tab1) and the negative control antibody (hIgG1), as with cmAb12.

[0049] Figure 6 Representative results are provided for ELISA measurements of binding affinity of human ILT7 protein from hu-cmAb12 antibody, reference antibody (Tab1), and negative control antibody (hIgG1).

[0050] Figures 7A-7B The images show hu-cmAb12, reference antibody (Tab1), and negative control antibody (isotype) versus 293F-human ILT7 cells (…). Figure 7A ) and CHOK1-cynomolgus monkey ILT7 cells ( Figure 7B Representative flow cytometry results of the combination of )

[0051] Figure 8 Representative flow cytometry results are provided, showing the binding of hu-cmAb12, reference antibody (Tab1), and negative control antibody (isotype) to cell surface ILT7 on pDCs and other immune cells in human PBMCs.

[0052] Figures 9A-9B Representative results from IFNα release assays performed on CpG-stimulated PBMCs (from four donors) are provided (figures and summary tables, respectively). The inhibitory activity of hu-cmAb12 and the reference antibody (Tab1) on IFNα release was measured.

[0053] Figure 10 Representative results are provided for a cytotoxic activity assay showing the NK cell-dependent ADCC activity of hu-cmAb12, reference antibody (Tab1), and negative control antibody (isotype).

[0054] Figure 11 Representative results are provided for a cytotoxic activity assay showing neutrophil-dependent ADCC activity of hu-cmAb12, reference antibody (Tab1), and negative control antibody (isotype).

[0055] Figure 12A-12B Representative results of phagocytosis assays showing macrophage-dependent ADCP activity in three donors (figures and summary tables, respectively) are provided.

[0056] Figures 13A-13B Provided in humanized mice ( Figure 13A ) and crab-eating macaques ( Figure 13B Representative results of hematological cell changes after administration of hu-cmAb12, negative control antibody (isotype), or PBS infusion to peripheral blood. Detailed Implementation

[0057] This disclosure provides novel antibodies comprising antigen-binding fragments that specifically bind to ILT7 (e.g., human ILT7). Pharmaceutical compositions comprising therapeutically effective amounts of such antibodies or antigen-binding fragments are also disclosed herein. Furthermore, the use of such pharmaceutical compositions for the treatment of autoimmune diseases associated with plasmacytoid dendritic cells (pDCs) and / or type I interferon (IFN) is also disclosed herein.

[0058] dendritic cells (pDCs) are a subset of dendritic cells (DCs) in peripheral blood and secondary lymphoid organs. Although they constitute only about 0.1% to 0.8% of peripheral blood monocytes (PBMCs), these cells are driver phonemes for both innate and adaptive immune responses. pDCs enhance innate immune responses by inducing chemokine and myeloid cell recruitment, promoting monocyte recruitment and their differentiation into antigen-presenting cells (APCs), inducing dendritic cell maturation and activation, and supporting the recruitment, activation, and cytotoxicity of natural killer (NK) cells. pDCs also promote adaptive immune responses by promoting antigen presentation, supporting the activation and expansion of antigen-specific CD4+ Th cells, driving CD4+ T cell differentiation into Th2 and Treg cells, promoting CD8+ T cell survival and activity, and improving B cell survival, maturation, differentiation, and autoantibody production.

[0059] Importantly, pDCs are a major source of type I IFN (α / β), which promotes the function of NK cells, B cells, T cells, and myeloid dendritic cells. Both pDCs and type I IFN are known to be involved in the development of immune disorders such as autoimmune diseases. For example, *Annu. Rev. Pathol. Mech. Dis.* 2019, 14:369-93; *J. Immunol* 2020, 205:2941-2950; *Clinic Rev. Allerg. Immunol* 59, 248–272 (2020); *Front. Immunol.* 12:713779; *Int. J. Mol. Sci.* 2021, 22, 4190; *Rheumatology* 2017; 56:16621675.

[0060] Also known as leukocyte immunoglobulin-like receptor A4 (LIRA4 or LILRA4) or CD85g, immunoglobulin-like transcript-7 (ILT7) is a member of the immunoglobulin-like transcript (ILT) or leukocyte immunoglobulin-like receptor (LIR) gene family. ILT7 contains four immunoglobulin-like extracellular domains and a transmembrane domain. The extracellular portion is crucial for interaction with the ILT7 ligand bone marrow stromal cell antigen 2 (BST2), and the transmembrane domain of ILT7 contains positively charged residues that bind to FcεRIγ and inhibit pDC function via the ITAM-mediated signaling pathway.

[0061] Full-length human ILT7 is a 499-amino acid protein (Uniprot accession number P59901, SEQ ID NO:1) containing a signal peptide (amino acids 1-23; removed in mature protein), extracellular domains (amino acids 24-446), transmembrane domains (amino acids 447-467), and cytoplasmic domains (amino acids 468-499). The extracellular domains include four immunoglobulin-like C2 domains (amino acids 24-118, 123-213, 224-313, and 324-413).

[0062] MTLILTSLLFFGLSLLGPRTRVQAENLPKPILWAEPGPVITWHNPVTIWCQGTLEAQGYRLDKEGNSMSRHILKTLESENKVKLSIPSMMWEHAGRYHCYYQSPAGWSEPSDPLELVVTAYSRPT LSALPSPVVTSGVNVTLRCASRLGLGRFTLIEEGDHRLSWTLNSHQHNHGKFQALFPMGPLTFSNRGTFRCYGYENNTPYVWSEPSDPLQLLVSGVSRKPSLLTLQGPVVTPGENLTLQCGSDVG YIRYTLYKEGADGLPQRPGRQPQAGLSQANFTLSPVSRSYGGQYRCYGAHNVSSEWSAPSDPLDILIAGQISDRPSLSVQPGPTVTSGEKVTLLCQSWDPMFTFLLTKEGAAHPPLRLRSMYGAH KYQAEFPMSPVTSAHAGTYRCYGSRSSNPYLLSHPSEPLELVVSGATETLNPAQKKSDSKTAPHLQDYTVENLIRMGVAGLVLLFLGILLFEAQHSQRSPPRCSQEANSRKDNAPFRVVEPWEQI

[0063] (SEQ ID NO:1)

[0064] More information about human ILT7 can be found in public databases with the following IDs: HGNC: 15503; NCBI Entrez Gene: 23547; Ensembl: ENSG00000239961; 607517; UniProtKB / Swiss-Prot: Q8IZF0. Describes two isoform alternatively spliced ​​transcript variants of the human ILT7 gene (Uniprot IDs: P59901-1, P59901-2).

[0065] The sequence of ILT7 from the cynomolgus monkey is provided below:

[0066] MTPILTTLLCFGLSLGPRTCLQAENLLKPILWAEPGPVIIWKKPVTIWCQGTLEAQEYRLDKEGNSMLRHMLKTLESENKAKFSIPSMMWEHAGRYHCYYQSPAGWSEPSDPLELVVTAYSRPS LSALPSPVVTSGVNVTLRCASRLGLGRFTLIEEGDHRLSWTLDSHQHNHGKFQALFPVGPLTFSNRGTFRCYGYENNTPYVWSEPSDPLQLLVSGVSRKPSLLTLQGPVVAPGDNLTLQCGSDVG YIRYALYKEGGDGLPQRPGQQSQAGLSQASFTLNPVRGSHGGQYRCYGAHNVSSKWSAPSDPLDILIAGQIPDRPSLSVQLGPTVASGEKVTLLCQSWGPMFTFLLAKEGAAHPPLRLRSTYRAQ QYQAEFPMSPVTSAHAGTYRCYGSRSSDPYLLSHSSEPLELVVSEATETLNPAQNKSDSKTAPHLQDYTVENLIRMGIAGLVLVFLGILLFEAQQSQRSPTRCSQEVNSREDNAPFRVVEPWEQI

[0067] (SEQ ID NO:2)

[0068] ILT7 is selectively expressed on the surface of human pDCs, but not on myeloid DCs or other peripheral blood leukocytes. ILT7 transcripts are rarely detected in most human tissues, but are moderately enriched in lymphoid organs where pDCs reside. Without being bound by theory, the anti-ILT7 antibodies or antigen-binding fragments presented herein can be used to reduce pDC activity via NK / neutrophil-mediated ADCC (antibody-dependent cytotoxicity) and / or macrophage-mediated ADCP (antibody-dependent phagocytosis), and therefore could be used, for example, for the treatment and prevention of autoimmune diseases.

[0069] Before further describing this disclosure, it should be understood that this disclosure is not limited to the specific embodiments set forth herein, and it should also be understood that the terminology used herein is for the purpose of describing specific embodiments and is not intended to be limiting.

[0070] A. Definition

[0071] Unless otherwise defined herein, the scientific and technical terms used in this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the nomenclature and techniques used in conjunction with those described herein in cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry, and hybridization are well-known and commonly used nomenclature and techniques in the art.

[0072] The term “a” or “an” refers to one or more of the entities mentioned; for example, “antibody” is understood to mean one or more antibodies.

[0073] As used herein, the term “and / or” will be considered a specific disclosure of each of two particular features or components having or not having the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0074] As used herein, the term "about" is used to indicate a value that includes inherent error variation in the apparatus, the method used to determine the value, or variation present among study subjects. The term "about" encompasses the exact figures listed. In some embodiments, "about" means within 10% of a given value or range. In some embodiments, "about" means a variation of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the value indicated by "about". In some embodiments, "about" means a variation of ±1%, ±0.5%, ±0.2%, or ±0.1% of the value indicated by "about".

[0075] As used herein, the term "antibody" and its grammatical equivalents refer to an immunoglobulin molecule that recognizes and specifically binds to a target through at least one antigen-binding site, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or any combination thereof, wherein the antigen-binding site is typically located within a variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-domain antibodies (sdAbs; e.g., camel antibodies, alpaca antibodies), single-chain Fv (scFv) antibodies, heavy-chain antibodies (HCAbs), light-chain antibodies (LCAbs), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecule containing an antigen-binding site (e.g., a bivariate immunoglobulin molecule), provided that the antibody exhibits the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and human antibodies. Based on the identity of the heavy chain constant domains (referred to as α, δ, ε, γ, and μ, respectively), antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Unless otherwise explicitly stated, the term "antibody" as used herein includes the "antigen-binding fragment" of a complete antibody. As used herein, the term "antigen-binding fragment" refers to a portion or fragment of a complete antibody that is the antigen-determining variable region of the complete antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibody molecules (e.g., scFv), heavy chain antibodies (HCAb), light chain antibodies (LCAb), disulfide-linked scFv (dsscFv), bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, microantibodies, bivariate domain antibodies (DVD), single variable domain antibodies (sdAb; e.g., camel antibodies, alpaca antibodies), and single variable domain (VHH) of heavy chain antibodies, as well as bispecific or multispecific antibodies formed from antibody fragments. A “bispecific” antibody is an artificial hybrid antibody with two different antigen-binding sites that recognizes two different targets and specifically binds to said targets. Bispecific antibodies can be produced by a variety of methods, including hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., Journal of Immunology 148, 1547-1553 (1992).

[0076] As used herein, the term "humanized antibody" refers to a form of non-human (e.g., mouse) antibody, which is a specific immunoglobulin chain, a chimeric immunoglobulin, or a fragment thereof containing a minimum of non-human sequences. Typically, humanized antibodies are human immunoglobulins. In some cases, Fv frame region residues of human immunoglobulins are replaced by corresponding residues from antibodies derived from non-human species. In some cases, CDR residues are replaced by residues from CDRs derived from non-human species (e.g., mice, rats, hamsters, camels) that have the desired specificity, affinity, and / or binding capacity. Humanized antibodies can be further modified by substituting additional residues within the Fv frame region and / or within the substituted non-human residues to refine and optimize antibody specificity, affinity, and / or binding capacity. As used herein, the term "human antibody" refers to an antibody produced by humans, or an antibody prepared using any technique known in the art having an amino acid sequence corresponding to that of a human-produced antibody.

[0077] When used as a reference antibody, the term "heavy chain" refers to a polypeptide chain of approximately 50-70 kDa, wherein the amino-terminal portion comprises a variable region of approximately 120 to 130 or more amino acids and a carboxyl-terminal portion comprising a constant region. Based on the amino acid sequence of the heavy chain's constant region, the constant region can be one of five different types referred to as α ("alpha"), δ ("delta"), ε ("epsilon"), γ ("gamma"), and μ ("mu"). The different heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these different types of heavy chains respectively produce five well-known antibody classes: IgA, IgD, IgE, IgG, and IgM, including four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4. The heavy chain can be a human heavy chain.

[0078] When used as a reference antibody, the term "light chain" refers to a polypeptide chain of approximately 25 kDa, wherein the amino-terminal portion comprises a variable region of approximately 100 to approximately 110 or more amino acids and a carboxyl-terminal portion comprising a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two distinct types, referred to as kappa (κ) or lambda (λ). The amino acid sequences of light chains are well known in the art. Light chains can be human light chains.

[0079] The term "variable domain" or "variable region" refers to a portion of the light or heavy chain of an antibody, typically located at the amino terminus of the light or heavy chain. The heavy chain is approximately 120 to 130 amino acids long, and the light chain is approximately 100 to 110 amino acids long. This portion is responsible for the binding and specificity of each specific antibody to its specific antigen. Variable domains vary considerably in sequence between different antibodies. Sequence variability is concentrated in the CDR (constructive domain), while the less variable portion of the variable domain is called the framework region (FR). The CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions. The amino acid position numbering used in this article is based on the EU index, such as Kabat et al. (1991), Sequences of proteins of immunological interest (US Department of Health and Human Services, Washington, DC), 5th edition. Variable regions can be human variable regions.

[0080] CDR refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the VHβ-sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the VLβ-sheet framework of an antibody. Therefore, a CDR is a variable region sequence scattered within a frame region sequence. CDR regions are well known to those skilled in the art and have been defined using various methods / systems. These systems and / or definitions have been developed and refined over many years and include Kabat, Chothia, IMGT, AbM, and Contact. For example, Kabat defined the most hypervariable region within the variable (V) domain of an antibody (Kabat et al., *Journal of Biochemistry* 252:6609-6616 (1977); Kabat, *Advances in Protein Chemistry* 32:1-75 (1978)). The Chothia definition is based on the location of a structural loop region, which defines the CDR region sequence as those residues that do not belong to the conserved β-sheet framework and are therefore capable of adapting to different conformations (Chothia and Lesk, J.Mol.Biol. 196:901-917 (1987)). Both terms are well-established in the art. Alternatively, the IMGT system is based on the sequence variability and location within the variable region. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on the analysis of available antibody crystal structures. Software programs (e.g., abYsis) for antibody sequence analysis and CDR determination are available and known to those skilled in the art. The location of CDRs within the variable domains of typical antibodies has been determined by comparing a large number of structures (Al-Lazikani et al., J.Mol.Biol. 273:927-948 (1997); Morea et al., Methods 20:267-279 (2000)). Because the number of residues in the hypervariable region varies in different antibodies, in the typical variable domain numbering scheme (Al-Lazikani et al., ibid. (1997)), the additional residues relative to the typical position are conventionally numbered a, b, c, etc. next to the residue number. This type of nomenclature is similarly well known to those skilled in the art.

[0081] For example, the CDRs defined according to Kabat (high-level) or Chothia (structure) are shown in the table below.

[0082] <![CDATA[Kabat 1 ]]> <![CDATA[Chothia 2 ]]> Ring position VHCDR1 31-35 26-32 Connecting chains B and C VHCDR2 50-65 53-55 Chain connecting C' and C” VHCDR3 95-102 96-101 Connecting F and G chains VLCDR1 24-34 26-32 Connecting chains B and C VLCDR2 50-56 50-52 Chain connecting C' and C” VLCDR3 89-97 91-96 Connecting F and G chains

[0083] 1 Residue numbering follows the nomenclature of Kabat et al., as above.

[0084] 2 Residue numbering follows the nomenclature of Chothia et al., as above.

[0085] One or more CDRs can be covalently or non-covalently incorporated into a molecule to make it an immunoadhesin. Immunoadhesins can incorporate CDRs as part of a larger polypeptide chain, covalently link CDRs to another polypeptide chain, or non-covalently incorporate CDRs. CDRs allow immunoadhesins to bind to specific antigens of interest. CDR regions can be analyzed, for example, through the abysis website (abysis.org).

[0086] The terms “epitope” and “antigenic determinant” are used interchangeably herein and refer to a site on the surface of a target molecule that binds to an antibody or antigen-binding fragment, such as a localized region on the surface of an antigen. Target molecules may comprise proteins, peptides, nucleic acids, carbohydrates, or lipids. An epitope with immunogenic activity is a portion of a target molecule that elicits an immune response in an animal. An epitope of an antigenically active target molecule is a portion of a target molecule that binds to an antibody, as determined by any method well known in the art, including, for example, by an immunoassay. Antigenic epitopes are not necessarily immunogenic. Epitopes typically consist of chemically active surface groups of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics and specific charge characteristics. The term “epitope” includes linear epitopes and conformational epitopes. A region of a target molecule (e.g., a polypeptide) that contributes to an epitope may be a continuous amino acid of the polypeptide, or the epitope may originate together from two or more discontinuous regions of the target molecule. An epitope may or may not be a three-dimensional surface feature of the target molecule. Epitopes formed from consecutive amino acids (also known as linear epitopes) are typically retained after protein denaturation, while epitopes formed through tertiary folding (also known as conformational epitopes) are typically lost after protein denaturation. In a unique spatial conformation, an epitope typically comprises at least 3, and more commonly at least 5, 6, 7, or 8-10 amino acids.

[0087] As used herein, the term "specific binding" means that the interaction of a polypeptide or molecule with an epitope, protein, or target molecule is more frequent, faster, longer-lasting, more affinity, or a combination thereof than with an alternative substance, including both related and unrelated proteins. The binding moiety (e.g., antibody) that specifically binds to a target molecule (e.g., an antigen) can be identified by, for example, immunoassays, ELISA, biolayer interferometry (“BLI”), SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, the specific reaction will be at least twice the background signal or noise, and can be more than ten times the background. For a discussion of antibody specificity, see, for example, Paul (ed.), 1989. Fundamental Immunology Immunology, Second Edition Raven Press, New York, pp. 332-336. A binding moiety that specifically binds to a target molecule can bind to the target molecule with an affinity higher than its affinity for dissimilar molecules. In some embodiments, the binding moiety that specifically binds to a target molecule can bind to the target molecule with an affinity at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 times that of its affinity for dissimilar molecules. In some embodiments, the binding moiety that specifically binds to a particular target molecule binds to dissimilar molecules with such low affinity that the binding cannot be detected using the assays described herein or other assays known in the art. In some embodiments, “specific binding” means, for example, the binding moiety binding to a K+ level of about 0.1 mM or less. D Binding to a molecular target. In some embodiments, "specific binding" means that the peptide or molecule binds at a concentration of about 10 μM or less, or about 1 μM or less. D Binding to the target. In some embodiments, "specific binding" means that the peptide or molecule binds to a target at a concentration of about 0.1 μM or less, about 0.01 μM or less, or about 1 nM or less. DTarget binding. Due to sequence identity between homologous proteins in different species, specific binding can include peptides or molecules that recognize proteins or targets in more than one species. Similarly, due to homology within certain regions of the polypeptide sequences of different proteins, specific binding can include peptides or molecules that recognize more than one protein or target. It should be understood that in some embodiments, the binding portion (e.g., antibody) that specifically binds to a first target may or may not specifically bind to a second target. Therefore, “specific binding” does not necessarily require (although it may include) exclusive binding (i.e., binding to a single target). Thus, in some embodiments, the binding portion (e.g., antibody) specifically binds to more than one target. For example, in some cases, the antibody may contain two identical antigen-binding sites, each of which specifically binds to the same epitope on two or more proteins. In some alternative embodiments, the antibody may be bispecific and contain at least two antigen-binding sites with different specificities.

[0088] As used herein, the term "binding affinity" generally refers to the strength of the sum of non-covalent interactions between the binding moiety and the target molecule (e.g., antigen). Binding between the binding moiety and the target molecule is a reversible process, and the affinity of binding is typically reported as the equilibrium dissociation constant (K0). D K D It is the dissociation rate (k off or k d ) and association rate (k on or k a The ratio of ) to K. D The lower the value, the higher the affinity. Various methods for measuring affinity are known in the art, any of which can be used for the purposes of this disclosure. Specific illustrative embodiments include the following examples. In some embodiments, “K D "or "K D The value can be measured by means of determinations known in the art, such as by combining determinations. K D It can be measured in radiolabeled antigen binding assays (RIA) (Chen et al., (1999) Journal of Molecular Biology 293:865-881). K D or K D The value can also be measured using biological layer interferometry (BLI), for example using the Gator system (Probe Life) or the Octet-96 system (Sartorius AG). D or K DThe value can also be measured using Biacore's surface plasmon resonance (SPR) assay, such as the BIAcore™-2000 or BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). Binding affinity can also be measured using EC. 50 To quantify, the EC 50 It refers to the concentration of ligands present in a bound state for half of the target in the assay.

[0089] The terms “polypeptide,” “peptide,” “protein,” and their grammatical equivalents, which may be used interchangeably herein, refer to polymers of amino acids of any length, which may be linear or branched. They may include non-natural or modified amino acids or be interrupted by non-amino acid components. Polypeptides, peptides, or proteins may also be modified by, for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification.

[0090] As used herein, the term "variant" ("reference protein" or "reference peptide") in connection with a protein or peptide having specific sequence characteristics refers to a different protein or peptide that, compared to a reference protein or peptide, has one or more (e.g., about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and / or additions. Changes in amino acid sequence can be amino acid substitutions. Changes in amino acid sequence can be conserved amino acid substitutions. Changes in amino acid sequence can be amino acid deletions. A variant can be a fragment of a reference protein or peptide. Functional variants of a protein or peptide maintain the basic structural and functional properties of the reference protein or peptide.

[0091] The terms “polynucleotide,” “nucleic acid,” and their grammatical equivalents, which are used interchangeably herein, refer to polymers or oligomers of nucleotides of any length. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases (e.g., methylation, hydroxymethylation, or glycosylation), non-natural nucleotides, non-nucleotide building blocks exhibiting similar structure and / or function to natural nucleotides (i.e., “nucleotide analogs”), and / or any substrate that can be incorporated into the polymer by DNA or RNA polymerases. Nucleic acids or polynucleotides can be heterogeneous or homogeneous in composition, can be isolated from naturally occurring sources, or can be produced artificially or synthetically. Furthermore, nucleic acids can be DNA or RNA or mixtures thereof, and can exist permanently or transitionally in single-stranded or double-stranded forms, including homoduplex, heteroduplex, and hybrid states. Nucleic acid structures also include, for example, DNA / RNA helices, peptide nucleic acids (PNAs), morpholinonucleotides (see, for example, Braasch and Corey, Biochemistry, 4(14):4503-4510 (2002) and U.S. Patent No. 5,034,506), locked nucleic acids (LNAs; see Wahllestedt et al., Proceedings of the National Academy of Sciences, 97:5633-5638 (2000)), cyclohexenylnucleotides (see Wang, Journal of the American Chemical Society, 122:8595-8602 (2000)) and / or ribozymes.

[0092] As used herein in the context of two or more polynucleotides or peptides, the terms “identical,” “percentage of identity,” and their syntactic equivalents mean that, when compared and aligned against maximum correspondence (with gaps introduced if necessary), two or more identical sequences or subsequences are identical or have a specified percentage of identical nucleotide or amino acid residues, regardless of any conserved amino acid substitutions as part of sequence identity. The percentage of identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well known in the art. These algorithms and software include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, the two polynucleotides or peptides provided herein are substantially identical when compared and aligned against maximum correspondence, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide or amino acid residue identity, as measured using sequence comparison algorithms or by visual inspection. In some embodiments, identity exists on regions of amino acid sequences of at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or any integer value therebetween. In some embodiments, identity exists on longer regions exceeding 60-80 residues, such as at least about 80-100 residues, and in some embodiments, the sequences are substantially identical across the full length of the compared sequences, such as the coding region of a target protein or antibody. In some embodiments, identity exists on regions of nucleotide sequences of at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases, or any integer value therebetween. In some embodiments, identity exists on longer regions exceeding 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments, the sequences are substantially identical across the full length of the compared sequences, such as the nucleotide sequence encoding the protein of interest.

[0093] As used herein, the term "vector" and its grammatical equivalents refer to a medium for carrying genetic material (e.g., a polynucleotide sequence) that can be introduced into a host cell, where it can be replicated and / or expressed. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers operable for stable integration into the host cell chromosome. Additionally, a vector may include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes may include, for example, providing resistance to antibiotics or toxins, supplementing nutritional deficiencies, or supplying critical nutrients not present in the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., well-known in the art. When two or more polynucleotides are co-expressed, the two polynucleotides may be inserted into, for example, a single expression vector or a vector expressing the polynucleotide alone. For single-vector expression, the encoding polynucleotide may be operatively linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of the polynucleotide into the host cell can be confirmed using methods known in the art. Those skilled in the art will understand that polynucleotides are expressed in an amount sufficient to produce the desired product (e.g., an anti-ILT7 antibody or an antigen-binding fragment as described herein), and will further understand that expression levels can be optimized using methods well known in the art to obtain sufficient expression.

[0094] As used herein, the term "encoding" and its syntactic equivalents refer to the inherent property of a specific sequence of nucleotides in a polynucleotide or nucleic acid (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes, which have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological properties. Thus, if the transcription and translation of mRNA corresponding to a gene produces a protein, then the gene encodes that protein. Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes degenerate forms of each other and all nucleotide sequences encoding the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0095] "Isolated" polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions are polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions in a form not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions include polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions that have been purified to the point that they no longer exist in a form found in nature. In some embodiments, the isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions are substantially pure.

[0096] When used in this article in conjunction with a disease or symptom or a subject suffering from a disease or symptom, the term "treatment" and its grammatical equivalents refer to actions that suppress, eliminate, alleviate, and / or improve symptoms, the severity of symptoms, and / or the frequency of symptoms associated with the disease or symptom being treated.

[0097] As used herein, the term "application" and its grammatical equivalents refer to the act of delivering a therapeutic agent or pharmaceutical composition into a subject or causing said therapeutic agent or pharmaceutical composition to be delivered into a subject by the methods described herein or other methods known in the art. Therapeutic agents can be compounds, peptides, antibodies, cells, or populations of cells. Administering a therapeutic agent or pharmaceutical composition includes prescribing a therapeutic agent or pharmaceutical composition to be delivered into a subject. Exemplary forms of administration include oral dosage forms such as tablets, capsules, syrups, and suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP); transdermal dosage forms, including creams, jellies, powders, or patches; oral dosage forms; and inhaled powders, sprays, suspensions, and rectal suppositories.

[0098] As used herein, the terms "effective amount," "therapeutic effective amount," and their grammatical equivalents refer to the administration of a pharmaceutical agent, alone or as part of a pharmaceutical composition, and in a single dose or as part of a series of doses, to a subject in an amount that, when administered to the subject, produces any detectable, positive effect on any symptom, aspect, or characteristic of a disease, condition, or ailment. Therapeutic effective amounts can be determined by measuring the relevant physiological effects. The precise amount required varies from subject to subject, depending on the subject's age, weight and general condition, the severity of the ailment being treated, the clinician's judgment, etc. Those skilled in the art can determine the appropriate "effective amount" in any individual case using routine laboratory methods.

[0099] The terms “pharmaceuticalally acceptable carrier” or “pharmaceuticalally acceptable excipient” refer to materials suitable for administration to an individual with an active agent without causing undesirable biological effects or interacting with any other component of the pharmaceutical composition in an adverse manner.

[0100] As used herein, the term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, canines, felines, rodents, etc., that will become a recipient of a particular treatment. A subject may be a human. A subject may suffer from a particular disease or condition.

[0101] Scope: Throughout this disclosure, various aspects of the invention can be presented in a scope format. It should be understood that the use of a scope format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, the description of a scope should be considered as having specifically disclosed all possible sub-scopes and individual numerical values ​​within said scopes. For example, a description of a scope such as 1 to 6 should be considered as having specifically disclosed sub-scopes such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope.

[0102] Exemplary genes and peptides are described herein with reference to their GenBank numbers, GI numbers, and / or SEQ ID NOs. It should be understood that those skilled in the art can readily identify homologous sequences by referring to sequence sources, including but not limited to GenBank (ncbi.nlm.nih.gov / genbank / ) and EMBL (embl.org / ).

[0103] B. Anti-ILT7 antibody and antigen-binding fragment

[0104] This document provides antibodies or antigen-binding fragments thereof that specifically bind to ILT7 (e.g., human ILT7). In some embodiments, this document provides anti-ILT7 antibodies. In some embodiments, the antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody provided herein may be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0105] In some embodiments, this document provides antigen-binding fragments of anti-ILT7 antibodies. In some embodiments, the antigen-binding fragments provided herein may be single-domain antibodies (sdAbs), heavy-chain antibodies (HCAbs), Fab, Fab', F(ab')2, Fv, single-chain variable fragments (scFvs), or (scFv)2. In some embodiments, the antigen-binding fragment of an anti-ILT7 antibody is a single-domain antibody (sdAb). In some embodiments, the antigen-binding fragment of an anti-ILT7 antibody is a heavy-chain antibody (HCAb). In some embodiments, the antigen-binding fragment of an anti-ILT7 antibody is Fab. In some embodiments, the antigen-binding fragment of an anti-ILT7 antibody is Fab'. In some embodiments, the antigen-binding fragment of an anti-ILT7 antibody is F(ab')2. In some embodiments, the antigen-binding fragment of an anti-ILT7 antibody is Fv. In some embodiments, the antigen-binding fragment of an anti-ILT7 antibody is scFv. In some embodiments, the antigen-binding fragment of an anti-ILT7 antibody is a disulfide-linked scFv[(scFv)2]. In some embodiments, the antigen-binding fragment of an anti-ILT7 antibody is a bifunctional antibody (dAb).

[0106] In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise recombinant antibodies or antigen-binding fragments. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise monoclonal antibodies or antigen-binding fragments. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise polyclonal antibodies or antigen-binding fragments. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise camel-like (e.g., camel, dromedary camel, and llama) antibodies or antigen-binding fragments. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise chimeric antibodies or antigen-binding fragments. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise humanized antibodies or antigen-binding fragments. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise human antibodies or antigen-binding fragments. In some embodiments, the present invention provides anti-ILT7 human scFv.

[0107] In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein are isolated. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein are substantially pure.

[0108] In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise multispecific antibodies or antigen-binding fragments. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise bispecific antibodies or antigen-binding fragments. In some embodiments, the bispecific antibody or antigen-binding fragment comprises the anti-ILT7 antibody or antigen-binding fragment provided herein. In some embodiments, the bispecific antibody or antigen-binding fragment comprises the anti-ILT7 scFv provided herein.

[0109] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment provided herein includes a monovalent antigen-binding site. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment includes a monospecific binding site. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment includes a bivalent binding site.

[0110] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment. Monoclonal antibodies can be prepared by any method known to those skilled in the art. One exemplary method is to screen protein expression libraries, such as phage or ribosome display libraries. Phage display is described, for example, in the following literature: Ladner et al., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317; and WO 92 / 18619. In some embodiments, recombinant monoclonal antibodies are isolated from phage display libraries expressing the variable region or CDR of the desired species. Screening of phage libraries can be performed using various techniques known in the art.

[0111] In some embodiments, monoclonal antibodies are modified using recombinant DNA technology to generate alternative antibodies. In some embodiments, constant domains of the light and heavy chains of a mouse monoclonal antibody are replaced with constant regions of a human antibody to generate a chimeric antibody. In some embodiments, the constant regions are truncated or removed to generate desired antibody fragments of the monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable regions is used to optimize the specificity and / or affinity of the monoclonal antibody.

[0112] In some embodiments, this document provides an anti-ILT7 antibody clone, Ab12. Its sequence characteristics are described below. Specific CDR sequences defined herein are generally based on the Kabat or Chothia definitions. However, it should be understood that general references to one or more heavy chain CDRs and / or one or more light chain CDRs for a particular antibody cover all CDR definitions known to those skilled in the art.

[0113] Table 1. Amino acid sequence of the light chain variable region CDR (VL CDR) of Ab12

[0114]

[0115] Table 2 shows the amino acid sequence of the heavy chain variable region CDR (VH CDR) of Ab12.

[0116]

[0117] In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise one, two, three, four, five, and / or six CDRs of any of the antibodies described herein. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise a light chain variable region (VL) comprising one, two, and / or three light chain CDRs (VL CDRs) from Table 1. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise a heavy chain variable region (VH) comprising one, two, and / or three heavy chain CDRs (VH CDRs) from Table 2. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments provided herein comprise one, two, and / or three VL CDRs from Table 1 and one, two, and / or three VH CDRs from Table 2.

[0118] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising: VL, the VL comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 11; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 12; and / or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 13; or variants thereof having at most about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR; and / or VH, the VH comprising: (1) a VH CDR1 having the amino acid sequence selected from the group consisting of SEQ ID NO: 14 or 17; (2) a VH CDR2 having the amino acid sequence of SEQ ID NO: 15 or 18; and / or (3) a VH CDR3 having the amino acid sequence of SEQ ID NO: 16. CDR3; or variants thereof, said variants having up to about 3, about 5, about 8, about 10, about 12 or about 15 amino acid substitutions, additions and / or deletions in said VH CDR.

[0119] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising: a VL, the VL comprising: (1) a VL CDR1 having the amino acid sequence of SEQ ID NO: 11; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 12; or (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 13; or variants thereof having at most about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variant has at most about 5 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising: a VL, the VL comprising: (1) a VLCDR1 having the amino acid sequence of SEQ ID NO: 11; (2) a VL CDR2 having the amino acid sequence of SEQ ID NO: 12; and (3) a VL CDR3 having the amino acid sequence of SEQ ID NO: 13; or variants thereof having at most about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDR. In some embodiments, the variants have at most about 5 amino acid substitutions, additions, and / or deletions in the VLCDR.

[0120] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment having: a VL, wherein the VL comprises VL CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NO: 11, 12, and 13, respectively, as defined by Kabat; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDRs. In some embodiments, the variants have up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment having: a VL, wherein the VL comprises VL CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NO: 11, 12, and 13, as defined by Chothia; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDRs. In some embodiments, the variants have up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs.

[0121] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising: a VH, the VH comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 14 or 17; (2) a VH CDR2 having an amino acid sequence selected from SEQ ID NO: 15 or 18; or (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 16; or variants thereof having at most about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has at most about 5 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising: a VH, the VH comprising: (1) a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 14 or 17; (2) a VH CDR2 having an amino acid sequence selected from SEQ ID NO: 15 or 18; and (3) a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 16; or variants thereof having at most about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDR. In some embodiments, the variant has at most about 5 amino acid substitutions, additions, and / or deletions in the VHCDR.

[0122] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment having: a VH, wherein the VH comprises VH CDR1, CDR2, and CDR3 having amino acid sequences of SEQ ID NO: 14, 15, and 16, as defined by Kabat; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDRs. In some embodiments, the variants have up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment having: a VH, wherein the VH comprises VH CDR1, CDR2, and CDR3 having the amino acid sequences of SEQ ID NO: 17, 18, and 16, as defined by Chothia; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDRs. In some embodiments, the variants have up to about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs.

[0123] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, as defined by Kabat, comprising: (a) a VL comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO: 11, 12, and 13, respectively; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDRs; and / or (b) a VH comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 14, 15, and 16, respectively; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDRs.

[0124] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising: VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences SEQ ID NO: 11, 12, 13, 14, 15, and 16, as defined by Kabat; or variants thereof having at most about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDRs.

[0125] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, as defined by Chothia, comprising: (a) a VL comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO: 11, 12, and 13, respectively; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDRs; and / or (b) a VH comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of NO: 17, 18, and 16, respectively; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VH CDRs.

[0126] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising: VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences SEQ ID NO: 11, 12, 13, 17, 18, and 16, as defined by Chothia; or variants thereof having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions in the VL CDRs.

[0127] Table 3. Amino acid sequences of the light chain variable region (VL) and heavy chain variable region (VH) of the chimeric antibody Ab12 (cmAb12), as well as the humanized VL (hu-VL) and VH (hu-VH).

[0128]

[0129] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:9. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:10.

[0130] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising: (a) VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 9; and (b) VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising VL and VH, wherein the VL and VH have the amino acid sequences of SEQ ID NO:9 and 10, respectively.

[0131] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:9. In some embodiments, an anti-ILT7 antibody or antigen-binding fragment thereof has a VL having at least 85% sequence identity with SEQ ID NO:9. In some embodiments, an anti-ILT7 antibody or antigen-binding fragment thereof has a VL having at least 90% sequence identity with SEQ ID NO:9. In some embodiments, an anti-ILT7 antibody or antigen-binding fragment thereof has a VL having at least 95% sequence identity with SEQ ID NO:9. In some embodiments, an anti-ILT7 antibody or antigen-binding fragment thereof has a VL having at least 98% sequence identity with SEQ ID NO:9. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment thereof comprising a VL having the amino acid sequence of SEQ ID NO:9.

[0132] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:10. In some embodiments, an anti-ILT7 antibody or antigen-binding fragment thereof has a VH having at least 85% sequence identity with SEQ ID NO:10. In some embodiments, an anti-ILT7 antibody or antigen-binding fragment thereof has a VH having at least 90% sequence identity with SEQ ID NO:10. In some embodiments, an anti-ILT7 antibody or antigen-binding fragment thereof has a VH having at least 95% sequence identity with SEQ ID NO:10. In some embodiments, an anti-ILT7 antibody or an antigen-binding fragment thereof has a VH having at least 98% sequence identity with SEQ ID NO:10. In some embodiments, this document provides an antibody or an antigen-binding fragment thereof that specifically binds to ILT7, the antibody or the antigen-binding fragment thereof comprising a VH having the amino acid sequence of SEQ ID NO:10.

[0133] In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment comprises a humanized antibody or antigen-binding fragment. In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment comprises VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 from the antibody or antigen-binding fragment described herein. In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment comprises a variant of the anti-ILT7 antibody or antigen-binding fragment described herein. A variant of the anti-ILT7 antibody or antigen-binding fragment may comprise one to 30 amino acid substitutions, additions, and / or deletions of the anti-ILT7 antibody or antigen-binding fragment. A variant of the anti-ILT7 antibody or antigen-binding fragment may comprise one to 25 amino acid substitutions, additions, and / or deletions of the anti-ILT7 antibody or antigen-binding fragment. In some embodiments, a variant of the anti-ILT7 antibody or antigen-binding fragment comprises one to 20 substitutions, additions, and / or deletions of the anti-ILT7 antibody or antigen-binding fragment. In some embodiments, variants of the anti-ILT7 antibody or antigen-binding fragment contain one to 15 substitutions, additions, and / or deletions of the anti-ILT7 antibody or antigen-binding fragment. In some embodiments, variants of the anti-ILT7 antibody or antigen-binding fragment contain one to 10 substitutions, additions, and / or deletions of the anti-ILT7 antibody or antigen-binding fragment. In some embodiments, variants of the anti-ILT7 antibody or antigen-binding fragment contain one to five conserved amino acid substitutions, additions, and / or deletions of the anti-ILT7 antibody or antigen-binding fragment. In some embodiments, variants of the anti-ILT7 antibody or antigen-binding fragment contain one to three amino acid substitutions, additions, and / or deletions of the anti-ILT7 antibody or antigen-binding fragment. In some embodiments, the amino acid substitutions, additions, and / or deletions are conserved amino acid substitutions. In some embodiments, the conserved amino acid substitutions are in the CDR of the antibody or antigen-binding fragment. In some embodiments, the conserved amino acid substitutions are not in the CDR of the antibody or antigen-binding fragment. In some embodiments, the conserved amino acid substitutions are in the frame region of the antibody or antigen-binding fragment.

[0134] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising: (a) VL, the VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from SEQ ID NO:19-22; and / or (b) VH, the VH having an amino acid sequence identity selected from SEQ ID NO:19-22; The amino acid sequences of the group consisting of NO:23-28 have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the humanized antibody or its antigen-binding fragment comprises: VL, the VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:19; and VH, the VH having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:26.

[0135] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising: (a) a VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 19-22, as defined by Kabat or Chothia, the VL further comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO: 11, 12, and 13, respectively; and / or (b) a VH having an amino acid sequence selected from SEQ ID NO: 19-22, the VL having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 19-22; and / or (b) a VH having ... (c) a VL having at least 85%, at least 86%, at least 87%, at least 98%, at least 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: The amino acid sequences of the group consisting of NO:23-28 have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and the VH further has: (1) VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO:14, 15, and 16, respectively, as defined by Kabat; or (2) VHCDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO:17, 18, and 16, respectively, as defined by Chothia.In some embodiments, VL has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:19, as defined by Kabat or Chothia, and the VL further comprises VLCDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO:11, 12, and 13, respectively; and VH is identical to SEQ ID NO:19. The amino acid sequence of NO:26 has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and the VH further has: (1) VH CDR1, VH CDR2, and VHCDR3 having the amino acid sequences of SEQ ID NO:14, 15, and 16, respectively, as defined by Kabat; or (2) VHCDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO:17, 18, and 16, respectively, as defined by Chothia.

[0136] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 19 and 23, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 19 and 24, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 19 and 25, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 19 and 26, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 19 and 27, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 19 and 28, respectively. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 20 and 23, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 20 and 24, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 20 and 25, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 20 and 26, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 20 and 27, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 20 and 28, respectively. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 21 and 23, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 21 and 24, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 21 and 25, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 21 and 26, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 21 and 27, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO: 21 and 28, respectively. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising VL and VH, wherein VL and VH have the amino acid sequences of SEQ ID NO: 22 and 23, respectively.In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO:22 and 24, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO:22 and 25, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO:22 and 26, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO:22 and 27, respectively. In some embodiments, VL and VH have the amino acid sequences of SEQ ID NO:22 and 28, respectively.

[0137] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:19. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VL having at least 85% sequence identity with SEQ ID NO:19. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VL having at least 90% sequence identity with SEQ ID NO:19. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VL having at least 95% sequence identity with SEQ ID NO:19. Humanized anti-ILT7 antibodies or antigen-binding fragments thereof may have a VL having at least 98% sequence identity with SEQ ID NO:19. In some embodiments, this document provides humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, the humanized antibodies or antigen-binding fragments thereof comprising a VL having the amino acid sequence of SEQ ID NO:19.

[0138] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:20. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VL having at least 85% sequence identity with SEQ ID NO:20. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VL having at least 90% sequence identity with SEQ ID NO:20. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VL having at least 95% sequence identity with SEQ ID NO:20. Humanized anti-ILT7 antibodies or antigen-binding fragments thereof may have a VL having at least 98% sequence identity with SEQ ID NO:20. In some embodiments, this document provides humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, the humanized antibodies or antigen-binding fragments thereof comprising a VL having the amino acid sequence of SEQ ID NO:20.

[0139] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:21. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VL having at least 85% sequence identity with SEQ ID NO:21. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VL having at least 90% sequence identity with SEQ ID NO:21. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VL having at least 95% sequence identity with SEQ ID NO:21. Humanized anti-ILT7 antibodies or antigen-binding fragments thereof may have a VL having at least 98% sequence identity with SEQ ID NO:21. In some embodiments, this document provides humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, the humanized antibodies or antigen-binding fragments thereof comprising a VL having the amino acid sequence of SEQ ID NO:21.

[0140] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising a VL, wherein the VL has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:22. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VL having at least 85% sequence identity with SEQ ID NO:22. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VL having at least 90% sequence identity with SEQ ID NO:22. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VL having at least 95% sequence identity with SEQ ID NO:22. Humanized anti-ILT7 antibodies or antigen-binding fragments thereof may have a VL having at least 98% sequence identity with SEQ ID NO:22. In some embodiments, this document provides humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, the humanized antibodies or antigen-binding fragments thereof comprising a VL having the amino acid sequence of SEQ ID NO:22.

[0141] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:23. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 85% sequence identity with SEQ ID NO:23. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 90% sequence identity with SEQ ID NO:23. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 95% sequence identity with SEQ ID NO:23. Humanized anti-ILT7 antibodies or antigen-binding fragments thereof may have a VH having at least 98% sequence identity with SEQ ID NO:23. In some embodiments, this document provides humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, wherein the humanized antibody or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:23.

[0142] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:24. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 85% sequence identity with SEQ ID NO:24. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 90% sequence identity with SEQ ID NO:24. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 95% sequence identity with SEQ ID NO:24. Humanized anti-ILT7 antibodies or antigen-binding fragments thereof may have a VH having at least 98% sequence identity with SEQ ID NO:24. In some embodiments, this document provides humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, wherein the humanized antibody or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:24.

[0143] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:25. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 85% sequence identity with SEQ ID NO:25. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 90% sequence identity with SEQ ID NO:25. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 95% sequence identity with SEQ ID NO:25. Humanized anti-ILT7 antibodies or antigen-binding fragments thereof may have a VH having at least 98% sequence identity with SEQ ID NO:25. In some embodiments, this document provides humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, wherein the humanized antibody or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:25.

[0144] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:26. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 85% sequence identity with SEQ ID NO:26. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 90% sequence identity with SEQ ID NO:26. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 95% sequence identity with SEQ ID NO:26. Humanized anti-ILT7 antibodies or antigen-binding fragments thereof may have a VH having at least 98% sequence identity with SEQ ID NO:26. In some embodiments, this document provides humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, the humanized antibodies or antigen-binding fragments thereof comprising a VH having the amino acid sequence of SEQ ID NO:26.

[0145] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:27. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 85% sequence identity with SEQ ID NO:27. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 90% sequence identity with SEQ ID NO:27. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 95% sequence identity with SEQ ID NO:27. Humanized anti-ILT7 antibodies or antigen-binding fragments thereof may have a VH having at least 98% sequence identity with SEQ ID NO:27. In some embodiments, this document provides humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, wherein the humanized antibody or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:27.

[0146] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising a VH, wherein the VH has at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:28. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 85% sequence identity with SEQ ID NO:28. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 90% sequence identity with SEQ ID NO:28. A humanized anti-ILT7 antibody or antigen-binding fragment thereof may have a VH having at least 95% sequence identity with SEQ ID NO:28. Humanized anti-ILT7 antibodies or antigen-binding fragments thereof may have a VH having at least 98% sequence identity with SEQ ID NO:28. In some embodiments, this document provides humanized antibodies or antigen-binding fragments thereof that specifically bind to ILT7, the humanized antibodies or antigen-binding fragments thereof comprising a VH having the amino acid sequence of SEQ ID NO:28.

[0147] Anti-ILT7 antibodies or their antigen-binding fragments may comprise a combination of any VL and any VH disclosed herein. In some embodiments, the VL and VH are linked by a adapter. The adapter may be a flexible adapter or a rigid adapter. In some embodiments, the adapter has an amino acid sequence of (GGGGS)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 35). In some embodiments, the adapter has an amino acid sequence of (EAAAK)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 36). In some embodiments, the adapter has an amino acid sequence of (PA)nP, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 37).

[0148] In some embodiments, this document provides anti-ILT7 antibodies or antigen-binding fragments thereof comprising VL CDRs (SEQ ID NO: 9, 19, 20, 21, or 22) from the VL described herein, and / or VH CDRs (SEQ ID NO: 10, 23, 24, 25, 26, 27, or 28) from the VH described herein. Methods for identifying CDRs are well known in the art. For example, those skilled in the art are familiar with publicly available software programs (abYsis) for analyzing antibody sequences and determining CDRs.

[0149] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising: (a) a VL comprising VL CDRs 1, 2, and 3 of a VL having the amino acid sequence of SEQ ID NO: 9; and / or (b) a VH comprising VH CDRs 1, 2, and 3 of a VH having the amino acid sequence of SEQ ID NO: 10.

[0150] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising: (a) a VL comprising VL CDRs 1, 2, and 3 of a VL having an amino acid sequence selected from the group consisting of SEQ ID NO: 19-22; and / or (b) a VH comprising VH CDRs 1, 2, and 3 of a VH having an amino acid sequence selected from the group consisting of SEQ ID NO: 23-28.

[0151] In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VL, wherein the VL comprises VL CDRs 1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 9. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VL, wherein the VL comprises VL CDRs 1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 19. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VL, wherein the VL comprises VL CDRs 1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 20. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VL, wherein the VL comprises VL CDRs 1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 21. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VL, wherein the VL comprises VLCDRs 1, 2, and 3 from a VL having the amino acid sequence of SEQ ID NO: 22.

[0152] In some embodiments, this document provides a humanized antibody or antigen-binding fragment thereof that specifically binds to ILT7, the humanized antibody or antigen-binding fragment comprising a VH, wherein the VH comprises VH CDRs 1, 2, and 3 from the VH having the amino acid sequence of SEQ ID NO: 10. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VH, wherein the VH comprises VH CDRs 1, 2, and 3 from the VH having the amino acid sequence of SEQ ID NO: 23. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VH, wherein the VH comprises VH CDRs 1, 2, and 3 from the VH having the amino acid sequence of SEQ ID NO: 24. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VH, wherein the VH comprises VH CDRs 1, 2, and 3 from the VH having the amino acid sequence of SEQ ID NO:25. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VH, wherein the VH comprises VH CDRs 1, 2, and 3 from the VH having the amino acid sequence of SEQ ID NO:26. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment comprising a VH, wherein the VH comprises VH CDRs 1, 2, and 3 from the VH having the amino acid sequence of SEQ ID NO:27. In some embodiments, this document provides an antibody or antigen-binding fragment thereof that specifically binds to ILT7, the antibody or antigen-binding fragment thereof comprising VH, wherein the VH comprises VHCDR 1, 2 and 3 from the amino acid sequence of VH having SEQ ID NO:28.

[0153] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof provided herein is an antibody designated as cmAb12 (chimeric Ab12). In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof provided herein has a VL from cmAb12 (SEQ ID NO: 9). In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof provided herein has a VH from cmAb12 (SEQ ID NO: 10). The anti-ILT7 antibody or antigen-binding fragment thereof provided herein may have both a VL and a VH from cmAb12. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof provided herein has a VL comprising VL CDRs 1, 2, and 3 from the VL from cmAb12 (SEQ ID NO: 9). In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof provided herein has a VH comprising VL CDRs 1, 2, and 3 from the VH from cmAb12 (SEQ ID NO: 10). The anti-ILT7 antibody or its antigen-binding fragment provided herein may have VL and VH from cmAb12: VL containing VL CDRs 1, 2, and 3; and VH containing VH CDRs 1, 2, and 3. In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment provided herein is a variant of cmAb12. The cmAb12 variant may have a VL, which is a variant of the VL of cmAb12, having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions as shown in SEQ ID NO:9. The cmAb12 variant may have a VL, which is a variant of the VL of cmAb12, having up to about 5 amino acid substitutions, additions, and / or deletions as shown in SEQ ID NO:9. The cmAb12 variant may have a VH, which is a variant of the VH of cmAb12 having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions as shown in SEQ ID NO:10. The cmAb12 variant may have a VH, which is a variant of the VH of cmAb12 having up to about 5 amino acid substitutions, additions, and / or deletions as shown in SEQ ID NO:10. Amino acid substitutions, additions, and / or deletions may be in a VH CDR or a VL CDR. In some embodiments, amino acid substitutions, additions, and / or deletions are not in a CDR. In some embodiments, the cmAb12 variant has up to about 5 conserved amino acid substitutions. In some embodiments, the cmAb12 variant has up to 3 conserved amino acid substitutions. In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment provided herein is a humanized antibody or antigen-binding fragment derived from cmAb12.In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment provided herein is a human antibody or antigen-binding fragment derived from cmAb12.

[0154] In some embodiments, this document provides humanized antibodies against Ab12 (e.g., humanized Ab12, hu-cmAb12, hu-Ab12, or hu-12). In some embodiments, the humanized anti-ILT7 antibody or antigen-binding fragment thereof provided herein comprises a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:19-22. In some embodiments, the humanized anti-ILT7 antibody or antigen-binding fragment thereof provided herein comprises a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:23-28. In some embodiments, the humanized anti-ILT7 antibody or antigen-binding fragment thereof provided herein comprises a VL having an amino acid sequence selected from the group consisting of SEQ ID NO:19-22, and a VH having an amino acid sequence selected from the group consisting of SEQ ID NO:23-28. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof provided herein is a variant of the humanized Ab12 provided herein. Variant may have a VL, which is a variant of humanized Ab12 with a VL having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions selected from the group consisting of SEQ ID NO:19-22. Variant may have a VL, which is a variant of humanized Ab12 with a VL having up to about 5 amino acid substitutions, additions, and / or deletions selected from the group consisting of SEQ ID NO:19-22. Variant may have a VH, which is a variant of humanized Ab12 with a VH having up to about 3, about 5, about 8, about 10, about 12, or about 15 amino acid substitutions, additions, and / or deletions selected from the group consisting of SEQ ID NO:23-28. Variant may have a VH, which is a variant of the VH of humanized Ab12, having up to about 5 amino acid substitutions, additions, and / or deletions from the amino acid sequence selected from the group consisting of SEQ ID NO:23-28. In some embodiments, variants of humanized Ab12 have up to about 5 conserved amino acid substitutions. In some embodiments, the humanized antibody is the antibody “hu-cmAb12”, i.e., a humanized antibody comprising a VL having the amino acid sequence of SEQ ID NO:19 and a VH having the amino acid sequence of SEQ ID NO:26. Selected properties of the humanized antibody hu-cmAB12 are illustrated in Examples 8-17.

[0155] In some embodiments, the anti-ILT7 antibody provided herein is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, the antibody is an IgA antibody. In some embodiments, the antibody is an IgD antibody. In some embodiments, the antibody is an IgE antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgM antibody. In some embodiments, the antibody provided herein may be an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0156] In some embodiments, the anti-ILT7 antibody provided herein comprises a light chain and a heavy chain. The light chain may comprise a light chain constant domain (CL) and a light chain variable domain (VL). The heavy chain may comprise a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The VL / VH may be any VL / VH disclosed herein. In some embodiments, the light chain constant domain (CL) is κCL (Cκ; SEQ ID NO: 29). In some embodiments, the light chain constant domain (CL) is λCL (Cλ; SEQ ID NO: 30). In some embodiments, the heavy chain comprises a heavy chain constant domain (CH) from human IgA. In some embodiments, the heavy chain comprises a heavy chain constant domain (CH) from human IgD. In some embodiments, the heavy chain comprises a heavy chain constant domain (CH) from human IgE. In some embodiments, the heavy chain comprises a heavy chain constant domain (CH) from human IgG. In some embodiments, the heavy chain comprises a heavy chain constant domain (CH) from human IgM. In some embodiments, the heavy chain comprises a heavy chain constant domain (CH) from human IgG1 (e.g., SEQ ID NO: 31). In some embodiments, the heavy chain comprises a heavy chain constant domain (CH) from human IgG2 (e.g., SEQ ID NO:32). In some embodiments, the heavy chain comprises a heavy chain constant domain (CH) from human IgG3 (e.g., SEQ ID NO:33). In some embodiments, the heavy chain comprises a heavy chain constant domain (CH) from human IgG4 (e.g., SEQ ID NO:34). Any and all combinations of the VL / VH pairs disclosed herein that specifically bind to ILT7 (e.g., human ILT7) and the CL / CH disclosed herein or other CL / CH known in the art are explicitly contemplated herein.

[0157]

[0158]

[0159] In some embodiments, the antibody provided herein has a light chain constant region (CL) having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:29. In some embodiments, the antibody provided herein has a CL having the amino acid sequence of SEQ ID NO:29. In some embodiments, the antibody provided herein has a light chain constant region (CL) having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:30. In some embodiments, the antibody provided herein has a CL having the amino acid sequence of SEQ ID NO:30. In some embodiments, the antibody provided herein has a heavy chain constant region (CH) having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:31. In some embodiments, the antibody provided herein has a CH having the amino acid sequence of SEQ ID NO:31. In some embodiments, the antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:32. In some embodiments, the antibody provided herein has a CH having the amino acid sequence of SEQ ID NO:32. In some embodiments, the antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:33. In some embodiments, the antibody provided herein has a CH having the amino acid sequence of SEQ ID NO:33. In some embodiments, the antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:34. In some embodiments, the antibody provided herein has a CH having the amino acid sequence of SEQ ID NO:34.

[0160] In some embodiments, this document also provides antibody or antigen-binding fragments that compete with the antibody or antigen-binding fragments provided above for binding to ILT7 (e.g., human ILT7). An antibody that “competes with another antibody for binding to a target” refers to an antibody that inhibits (partially or completely) the binding of another antibody to a target. Known competition assays are used, such as... Surface plasmon resonance (SPR) analysis can determine whether two antibodies compete for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to the target. In some embodiments, an anti-ILT7 antibody or antigen-binding fragment competes with another antibody or antigen-binding fragment for binding to ILT7 and inhibits said binding by at least 50%, 60%, 70%, 80%, 90%, or 100%. The competition assay can be performed as described, for example, in the following literature: Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:l0.H0l / pdb.prot4277; or Chapter 11, “Using Antibodies,” edited by Harlow and David Lane, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, USA, 1999.

[0161] In some embodiments, this document provides antibody or antigen-binding fragments that competitively bind to ILT7 (e.g., human ILT7) with an anti-ILT7 antibody or antigen-binding fragment disclosed herein. In some embodiments, this document provides antibody or antigen-binding fragments that competitively bind to ILT7 (e.g., human ILT7) with a chimeric Ab12. In some embodiments, this document provides antibody or antigen-binding fragments that competitively bind to ILT7 (e.g., human ILT7) with a humanized Ab12 disclosed herein.

[0162] Epitope mapping is a method for identifying binding sites, regions, or epitopes on target proteins that bind to antibodies. Various methods for mapping epitopes on target proteins are known in the art. These methods include mutagenesis, including but not limited to shotgun mutagenesis, site-directed mutagenesis, and alanine scanning; domain or fragment scanning; peptide scanning (e.g., Pepscan technology); display methods (e.g., phage display, microbial display, and ribosome / mRNA display); methods involving proteolysis and mass spectrometry; and structure determination (e.g., X-ray crystallography and NMR). In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is characterized by methods including but not limited to N-terminal sequencing, amino acid analysis, HPLC, mass spectrometry, ion-exchange chromatography, and papain digestion.

[0163] As further detailed in the experimental section below, Ab12 does not compete with the baseline antibody daxdilimab for binding to human ILT7. In some embodiments, the anti-ILT7 antibody and antigen-binding fragments provided herein do not compete with daxdilimab for binding to human ILT7.

[0164] The anti-ILT7 antibodies or antigen-binding fragments disclosed herein can be analyzed for their physical, chemical, and / or biological properties using various methods known in the art. In some embodiments, the ability of the anti-ILT7 antibody to bind to ILT7 (e.g., human ILT7) is tested. In some embodiments, the ability of the anti-ILT7 antibody to bind to FcγR is tested. In some embodiments, the ability of the anti-ILT7 antibody to bind to FcγRIIA / CD32A is tested. In some embodiments, the ability of the anti-ILT7 antibody to bind to FcγRIIIA / CD16A is tested. Binding assays include, but are not limited to, BLI, SPR (e.g., Biacore), ELISA, and FACS. Additionally, the antibody's solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency can be evaluated.

[0165] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used with high affinity, for example, at 10... -6 M or smaller, 5×10 -7 M or smaller, 10 -7 M or smaller, 5×10 -8 M or smaller, 10 -8 M or smaller, 5×10 -9 M or smaller, 10 -9 M or smaller, 5×10 -10 M or smaller or 10 -10 M or smaller K D Binds to human ILT7. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is delivered at 5 × 10⁻⁶. -7 M or smaller K D Binds to human ILT7. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein binds with high affinity, for example, at about 10. -6 M, approximately 5×10 -7 M, approximately 10 - 7 M, approximately 5×10 -8 M, approximately 10 -8 M, approximately 5×10 -9 M, approximately 10 -9 M, approximately 5×10 -10 M or approximately 10 -10 M of K DBinds to human ILT7. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is at approximately 10 -7 M of K D Binds to human ILT7. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is in the range of 10. -10 M to 10 -6 M, 10 -9 M to 10 -6 M, 10 -8 M 10 -6 M, 10 -7 M to 10 -6 M, 10 -10 M to 5×10 -7 M, 10 -9 M to 5×10 -7 M, 10 -8 M to 5×10 -7 M or 10 - 7 M to 5×10 -7 M of K D Binds to human ILT7. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein binds with high affinity, for example at 10... -7 M to 10 -6 M of K D In conjunction with human ILT7. In some embodiments, K D Determined via BLI. In some embodiments, K D Determined by SPR. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein has high affinity, for example, at 10 as determined by SPR. -6 M or smaller, 5×10 -7 M or smaller, 10 -7 M or smaller, 5×10 - 8 M or smaller, 10 -8 M or smaller, 5×10 -9 M or smaller, 10 -9 M or smaller, 5×10 -10 M or smaller or 10 -10 M or smaller; or a range of 10 -10 M to 10 -6 M, 10 -9 M to 10 -6 M, 10 -8 M 10 -6 M, 10 -7 M to 10 -6 M, 10 -10 M to 5×10 -7M, 10 -9 M to 5×10 -7 M, 10 -8 M to 5×10 -7 M or 10 -7 M to 5×10 -7 M of K D It combines with human ILT7.

[0166] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein binds to both human ILT7 and cynomolgus monkey ILT7.

[0167] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein does not bind to other LILR family members. In some embodiments, the affinity of the anti-ILT7 antibody or antigen-binding fragment described herein for other LILR family member proteins is comparable to the affinity of an allotype antibody (e.g., hIgG1) for said family member proteins. Other LILR family member proteins include, but are not limited to: LILRA1, LILRA2 / ILT1, LILRA3 / ILT6, LILRA5 / ILT11, LILRA6 / ILT8, LILRB1 / ILT2, LILRB2 / ILT4, LILRB3 / ILT5, LILRB4 / ILT3, and LILB5. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein does not bind to one or more of the LILR family member proteins selected from the group consisting of: LILRA1, LILRA2 / ILT1, LILRA3 / ILT6, LILRA5 / ILT11, LILRA6 / ILT8, LILRB1 / ILT2, LILRB2 / ILT4, LILRB3 / ILT5, LILRB4 / ILT3, and LILB5. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein does not bind to any of the LILR family member proteins: LILRA1, LILRA2 / ILT1, LILRA3 / ILT6, LILRA5 / ILT11, LILRA6 / ILT8, LILRB1 / ILT2, LILRB2 / ILT4, LILRB3 / ILT5, LILRB4 / ILT3, and LILB5.

[0168] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein inhibits IFNα release via PBMCs. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein inhibits IFNα release via CpG-stimulated PBMCs in vitro. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein inhibits IFNα release via PBMCs in vivo. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein reduces IFNα levels in vivo. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is administered at EC50 concentrations of 10 nM or less, 5 nM or less, 1 nM or less, 0.5 nM or less, 0.1 nM or less, 0.08 nM or less, 0.05 nM or less, or 0.01 nM or less. 50 In vitro inhibition of IFNα release from PBMCs stimulated by CpG. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is administered at 0.1 nM or less EC50. 50 In vitro inhibition of IFNα release from PBMCs stimulated by CpG. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is administered at EC50 concentrations of about 10 nM, about 5 nM, about 1 nM, about 0.5 nM, about 0.1 nM, about 0.08 nM, about 0.05 nM, or about 0.01 nM. 50 In vitro inhibition of IFNα release from PBMCs stimulated by CpG. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is administered at approximately 0.05 nM EC. 50 In vitro inhibition of IFNα release from PBMCs stimulated by CpG. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is administered at EC50 concentrations ranging from 0.01 nM to 10 nM, 0.01 nM to 5 nM, 0.01 nM to 1 nM, 0.01 nM to 0.5 nM, 0.01 nM to 0.1 nM, or 0.01 nM to 0.05 nM. 50 In vitro inhibition of IFNα release from PBMCs stimulated by CpG. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is administered at 0.01 nM to 0.1 nM EC. 50 In vitro inhibition of IFNα release from PBMCs stimulated by CpG. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is administered at 0.01 nM to 1 nM EC. 50 In vitro inhibition of IFNα release from PBMCs stimulated by CpG. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used as a reference antibody, dastilimab, in EC... 5060% or less, 50% or less, 40% or less, or 30% or less of EC 50 Inhibits IFNα release from PBMCs stimulated by CpG. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used as a reference antibody, dastilimab, in EC... 50 50% or less of EC 50 Inhibits IFNα release from PBMCs stimulated by CpG. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used as a reference antibody, dastilimab, in EC... 50 EC 10-60%, 10-50%, 10-40%, 20-60%, 20-50%, or 20-40% 50 Inhibits IFNα release from PBMCs stimulated by CpG. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used as a reference antibody, dastilimab, in EC... 50 10-50% of EC 50 Inhibits IFNα release from PBMCs stimulated by CpG.

[0169] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein selectively binds to pDCs in human PBMCs. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein does not bind to T cells, B cells, NK cells, NKT cells, or monocytes in PBMCs.

[0170] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein exhibits ADCC and ADCP activities against ILT7-expressing cells such as pDCs. ADCC activity may be NK-dependent ADCC. ADCC activity may be neutrophil-dependent ADCC. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein depletes pDCs in vivo.

[0171] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is in EC50 of 0.05 nM or less, 0.02 nM or less, 0.01 nM or less, 0.008 nM or less, 0.005 nM or less, 0.002 nM or less, or 0.001 nM or less. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at 0.01 nM or less EC50. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at 0.008 nM or less EC50. 50It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at 0.005 nM or less EC50. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is in EC50 at approximately 0.05 nM, approximately 0.02 nM, approximately 0.01 nM, approximately 0.008 nM, approximately 0.005 nM, approximately 0.002 nM, or approximately 0.001 nM. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at approximately 0.008 nM EC50. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at approximately 0.005 nM EC. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is in the range of 0.001 nM to 0.05 nM, 0.001 nM to 0.02 nM, 0.001 nM to 0.01 nM, 0.001 nM to 0.05 nM, 0.005 nM to 0.05 nM, 0.005 nM to 0.02 nM, 0.005 nM to 0.01 nM, or 0.001 nM to 0.05 nM. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at an EC50 concentration ranging from 0.001 nM to 0.01 nM. 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used as a reference antibody for the EC of dastardilimab. 50 EC 10-60%, 10-50%, 10-40%, 20-60%, 20-50%, or 20-40% 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used as a reference antibody for the EC of dastardilimab. 50 10-50% of EC 50 It exhibits NK-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is used as a reference antibody for the EC of dastardilimab. 50 Approximately 40% of EC 50 It exhibits NK-dependent ADCC activity.

[0172] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein exhibits neutrophil-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at EC50 concentrations of 500 nM or less, 200 nM or less, 100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less. 50 It exhibits neutrophil-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at 100 nM or less EC50. 50 It exhibits neutrophil-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at 50 nM or less EC50. 50 It exhibits neutrophil-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is in the range of 1 nM to 500 nM, 1 nM to 200 nM, 1 nM to 100 nM, 1 nM to 80 nM, 1 nM to 50 nM, 1 nM to 20 nM, 5 nM to 500 nM, 5 nM to 200 nM, 5 nM to 100 nM, 5 nM to 80 nM, 5 nM to 50 nM, or 5 nM to 20 nM EC. 50 It exhibits neutrophil-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at EC50 in the range of 1 nM to 50 nM. 50 It exhibits neutrophil-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is in EC50 at approximately 500 nM, approximately 200 nM, approximately 100 nM, approximately 80 nM, approximately 50 nM, approximately 20 nM, approximately 10 nM, approximately 5 nM, or approximately 1 nM. 50 It exhibits neutrophil-dependent ADCC activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at approximately 10 nM EC50. 50 It exhibits neutrophil-dependent ADCC activity.

[0173] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at EC50 concentrations of 10 nM or less, 8 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, 0.5 nM or less, 0.2 nM or less, or 0.1 nM or less. 50It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at 10 nM or less EC50. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at 8 nM or less EC50. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is in EC50 at approximately 10 nM, approximately 8 nM, approximately 5 nM, approximately 2 nM, approximately 1 nM, approximately 0.5 nM, approximately 0.2 nM, or approximately 0.1 nM. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at approximately 1 nM EC. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is in the range of 0.01 nM to 100 nM, 0.01 nM to 50 nM, 0.01 nM to 10 nM, 0.1 nM to 100 nM, 0.1 nM to 50 nM, 0.1 nM to 10 nM, or 0.1 nM to 5 nM EC. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed in EC50 at a concentration ranging from 0.1 nM to 10 nM. 50 It exhibits macrophage-dependent ADCP activity. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed at EC50 in the range of 0.5 nM to 5 nM. 50 It exhibits macrophage-dependent ADCP activity.

[0174] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein exhibits a maximum phagocytic index of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein exhibits a maximum phagocytic index of about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein exhibits a maximum phagocytic index ranging from 20% to 80%, 20% to 70%, 20% to 60%, 30% to 80%, 30% to 70%, or 30% to 60%. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein exhibits a maximum phagocytic index ranging from 20% to 80%. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein exhibits a maximum phagocytic index ranging from 30% to 60%.

[0175] C. Variants and conjugates

[0176] This disclosure further considers additional variants and equivalents that are substantially homologous to the recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies or antibody fragments thereof described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector function, glycosylation, immunogenicity, and / or solubility. Those skilled in the art will understand that amino acid changes can alter the post-translational processes of the antibody, such as changing the number or location of glycosylation sites, or altering membrane anchoring properties.

[0177] The variation can be a substitution, deletion, or insertion of one or more nucleotides encoding an antibody or polypeptide, resulting in a change in the amino acid sequence relative to the native antibody or polypeptide sequence. In some embodiments, an amino acid substitution is the result of replacing an amino acid with another amino acid having similar structure and / or chemical properties, such as replacing leucine with serine, for example, a conserved amino acid substitution. Insertions or deletions can range from about 1 to 5 amino acids. In some embodiments, substitutions, deletions, or insertions relative to the parent molecule include substitutions of fewer than 25 amino acids, fewer than 20 amino acids, fewer than 15 amino acids, fewer than 10 amino acids, fewer than 5 amino acids, fewer than 4 amino acids, fewer than 3 amino acids, or fewer than 2 amino acids. In some embodiments, variations in the amino acid sequence that are biologically useful and / or relevant can be determined by systematically performing insertions, deletions, or substitutions in the sequence and testing the activity of the resulting variant protein compared to the parent protein.

[0178] In some embodiments, variants of the anti-ILT7 antibody or antigen-binding fragment described herein are provided. In some embodiments, variants of the anti-ILT7 antibody clone Ab12 (cmAb12 or hu-Ab12) are provided herein. In some embodiments, the variant comprises one to 30 amino acid substitutions, additions, and / or deletions of the parent antibody or antigen-binding fragment. In some embodiments, the variant comprises one to 25 amino acid substitutions, additions, and / or deletions of the parent antibody or antigen-binding fragment. In some embodiments, the variant comprises one to 20 substitutions, additions, and / or deletions of the parent antibody or antigen-binding fragment. In some embodiments, the variant comprises one to 15 substitutions, additions, and / or deletions of the parent antibody or antigen-binding fragment. In some embodiments, the variant comprises one to 10 substitutions, additions, and / or deletions of the parent antibody or antigen-binding fragment. In some embodiments, the variant comprises one to five amino acid substitutions, additions, and / or deletions of the parent antibody or antigen-binding fragment. In some embodiments, the variant comprises one to three amino acid substitutions, additions, and / or deletions of the parent antibody or antigen-binding fragment. In some embodiments, the amino acid substitution is in the CDR of the antibody or antigen-binding fragment. In some embodiments, the amino acid substitution is not in the CDR of the antibody or antigen-binding fragment. In some embodiments, amino acid substitutions are performed within the framework region of the antibody or antigen-binding fragment. In some embodiments, amino acid substitutions, additions, and / or deletions are conserved amino acid substitutions.

[0179] It is known in the art that constant regions of antibodies mediate several effector functions, and these effector functions can vary depending on the antibody isotype. For example, the binding of the C1 component of complement to the Fc region (which binds to the antigen) of an IgG or IgM antibody activates the complement system. Activation of complement is important in opsonization and lysis of cellular pathogens. Activation of complement also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity reactions. Additionally, the Fc region of an antibody can bind to cells expressing Fc receptors (FcRs). Many Fc receptors are specific to different classes of antibodies, including IgG (γ receptor), IgE (ε receptor), IgA (α receptor), and IgM (μ receptor). Binding of antibodies to Fc receptors on cell surfaces triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, killing cells that lyse antibody-coated target cells (a phenomenon known as antibody-dependent cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and control of immunoglobulin production.

[0180] As is known in the art, allotypes are polymorphic markers of IgG subclasses, corresponding to amino acid variations and detected serologically using antibody reagents. The human heavy γ chain allotype of IgG is designated as Gm (“γ marker”). Allotypes G1m, G2m, and G3m are carried by constant regions of the γ1, γ2, and γ3 chains, respectively, and are encoded by the IGHG1, IGHG2, and IGHG3 genes. The γ1 chain can express G1m alleles (combinations of G1m allotypes): G1m3; G1m3,1; G1m17,1; G1m17,1,2; G1m17,1,27; Gm17,1,28; and Gm17,1,27,28. The C-regions of the G1m3,1; G1m17,1; and G1m17,1,2 chains differ from the C-region of the G1m3 chain by two, three, and four amino acids, respectively. The correspondence between the G1m allele and the IGHG1 allele is known in the field, for example, Lefranc, Chapter 26- Immunoglobulin reservoir analysis and antibody humanization, *B-cell Molecular Biology (M... OLECULAR B IOLOGY OF BC ELLS (Second Edition), Academic Press, 2015, pp. 481-514 (Table 7). In IGHG1 CH1, the lysine at position 120 (K120) in chain G corresponds to the G1m17 isotype. Isoleucine I103 (chain F) is specific to the γ1 chain isotype. If arginine is expressed at position 120 (R120), the simultaneous presence of R120 and I103 corresponds to the expression of the G1m3 isotype. For the γ3 and γ4 isotypes (which also have R120, but T in 103), R120 corresponds only to the expression of the nG1m17 isoallotype (detected by an antibody reagent that identifies the marker as an isotype in one IgG subclass and isotypes in other subclasses). In IGHG1 CH3, aspartic acid D12 and leucine L14 (chain A) correspond to G1m1, while glutamic acid E12 and methionine M14 correspond to the nG1m1 allotypes and isoforms. Glycine at position 110 corresponds to G1m2, while alanine does not correspond to any allotype (G1m2-negative chain).

[0181] See below for exemplary allotypes of the human IgG1 heavy chain constant region (IgG1 CH). In some embodiments, this document provides an IgG1 antibody having a heavy chain constant region (CH) having at least 85% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:31 and 40-44. In some embodiments, the IgG1 antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:31. In some embodiments, the IgG1 antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:40. In some embodiments, the IgG1 antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:41. In some embodiments, the IgG1 antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:42. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:43. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:44.

[0182]

[0183]

[0184] The γ2 chain can express the G2m allele. Position 45.1 (first position on the transverse CD chain) corresponds to the presence or absence (G2m..) of the uniquely identified G2m allotype (G2m23). Valine V45.1 corresponds to G2m.., while methionine corresponds to G2m23.

[0185] The γ3 chain can express the G3m allele (a combination of G3m allotypes). G3m16 (W83), G3m21 (L82), and nG3m21 (P82) are located on CH2. Other G3m allotypes form two chimeras on CH3. G3m26 (R115), G3m5 (R115, F116), G3m28 (R115, Y116), nG3m5 (H115, Y116), G3m14 (M84, R115, F116), and G3m15 (M39, H115, Y116) form the first chimera. G3m11(S44), nG3m11(N44), G3m10(S44, I101), G3m24(S44, V101), G3m27(I101), G3m6(S44, E98), and G3m13(S44, Q98) form a second chimera.

[0186] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgA antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgD antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgE antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgG antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgM antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgG1 antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgG2 antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgG3 antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgG4 antibody. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgG1 antibody, wherein the IgG1 antibody may belong to any allotype known in the art. In some embodiments, the IgG1 antibody belongs to allotype G1m3; G1m3,1; G1m17,1; G1m17,1,2; G1m17,1,27; Gm17,1,28; or Gm17,1,27,28. In some embodiments, the IgG1 antibody belongs to allotype G1m3. In some embodiments, the IgG1 antibody belongs to allotype G1m3,1. In some embodiments, the IgG1 antibody belongs to allotype G1m17,1. In some embodiments, the IgG1 antibody belongs to allotype G1m17,1,2. In some embodiments, the IgG1 antibody belongs to allotype Gm17,1,28. In some embodiments, the IgG1 antibody belongs to allotype Gm17,1,27,28. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgG2 antibody, wherein the IgG2 antibody may belong to any allotype known in the art. In some embodiments, the IgG2 antibody belongs to allotype G2m23. In some embodiments, the IgG2 antibody belongs to the G2m allotype. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of a human IgG3 antibody, wherein the IgG3 antibody may belong to any allotype known in the art. In some embodiments, the IgG3 antibody belongs to allotypes G3m16, G3m21, G3m26, G3m5, G3m28, G3m14, G3m15, G3m11, G3m10, G3m24, G3m27, G3m6, or G3m13.In some embodiments, the IgG3 antibody belongs to allotype G3m16. In some embodiments, the IgG3 antibody belongs to allotype G3m21. In some embodiments, the IgG3 antibody belongs to allotype G3m26. In some embodiments, the IgG3 antibody belongs to allotype G3m5. In some embodiments, the IgG3 antibody belongs to allotype G3m28. In some embodiments, the IgG3 antibody belongs to allotype G3m14. In some embodiments, the IgG3 antibody belongs to allotype G3m15. In some embodiments, the IgG3 antibody belongs to allotype G3m11. In some embodiments, the IgG3 antibody belongs to allotype G3m10. In some embodiments, the IgG3 antibody belongs to allotype G3m24. In some embodiments, the IgG3 antibody belongs to allotype G3m27. In some embodiments, the IgG3 antibody belongs to allotype G3m6. In some embodiments, the IgG3 antibody belongs to allotype G3m13.

[0187] In some embodiments, at least one or more constant regions in the anti-ILT7 antibody or antigen-binding fragment described herein have been modified or deleted. In some embodiments, the antibody comprises modifications to one or more heavy chain constant regions (CH1, CH2, or CH3) and / or light chain constant regions (CL).

[0188] In some embodiments, the heavy chain constant region of the modified antibody includes at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibody includes more than one human constant region. In some embodiments, modification of the constant region includes the addition, deletion, or substitution of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or completely deleted from the constant region of the modified antibody. In some embodiments, the entire CH2 domain has been removed from the antibody (ΔCH2 construct). In some embodiments, the deleted constant region is replaced by a short amino acid spacer that provides some of the molecular flexibility typically conferred by the absence of a constant region. In some embodiments, the modified antibody includes a CH3 domain fused directly to the hinge region of the antibody. In some embodiments, the modified antibody includes a peptide spacer inserted between the hinge region and the modified CH2 and / or CH3 domains.

[0189] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment includes an Fc region. In some embodiments, the Fc region is fused by a hinge. The hinge may be an IgG1 hinge, an IgG2 hinge, or an IgG3 hinge. The amino acid sequences of the Fc regions of human IgG1, IgG2, IgG3, and IgG4 are known to those skilled in the art. In some cases, Fc regions with amino acid variations have been identified in natural antibodies. In some embodiments, the modified antibody (e.g., the modified Fc region) provides altered effector functions that, in turn, affect the antibody's biological properties. For example, in some embodiments, the deletion or inactivation of the constant region (through point mutation or other means) reduces the binding of the modified antibody to the Fc receptor during circulation. In some embodiments, constant region modification reduces the immunogenicity of the antibody. In some embodiments, constant region modification increases the serum half-life of the antibody. In some embodiments, constant region modification shortens the serum half-life of the antibody. In some embodiments, constant region modification enhances the antibody's ADCC and / or complement-dependent cytotoxicity (CDC). In some embodiments, constant region modification enhances the antibody-dependent phagocytosis (ADCP). In some embodiments, constant region modification reduces or removes the antibody's ADCC and / or CDC. In some embodiments, replacing specific amino acids in the human IgG1 Fc region with corresponding IgG2 or IgG4 residues reduces effector function (e.g., ADCC and CDC) in the modified antibody. In some embodiments, the antibody does not have one or more effector functions (e.g., "effectless" antibody). In some embodiments, the antibody does not bind to Fc receptors and / or complement factors. In some embodiments, the antibody does not have effector function. In some embodiments, constant region modification increases or enhances the antibody's ADCC and / or ADCP. In some embodiments, the constant region is modified to eliminate disulfide bonds or oligosaccharide moieties. In some embodiments, the constant region is modified to add / replace one or more amino acids to provide one or more cytotoxic, oligosaccharide, or carbohydrate attachment sites. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment includes a variant Fc region engineered with substitutions at specific amino acid positions compared to the native Fc region.

[0190] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises an IgG1 heavy chain constant region comprising one or more amino acid substitutions selected from the group consisting of the following according to EU index numbers: L234, L235, G236, S239, F243, H268, D270, R292, S298, Y300, V305, A330, I332, K326, E333, K334, and P396.

[0191] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises an IgG1 heavy chain constant region containing at least one amino acid substitution. The IgG1 heavy chain constant region may contain an L234 substitution. An L234 substitution may be, for example, L234Y. The IgG1 heavy chain constant region may contain an L235 substitution. An L235 substitution may be, for example, L235Q or L235V. The IgG1 heavy chain constant region may contain a G236 substitution. A G236 substitution may be, for example, G236A or G236W. The IgG1 heavy chain constant region may contain an S239 substitution. An S239 substitution may be, for example, S239D or S239M. The IgG1 heavy chain constant region may contain an F243 substitution. An F243 substitution may be, for example, F243L. The IgG1 heavy chain constant region may contain an H268 substitution. An H268 substitution may be, for example, H268D. The IgG1 heavy chain constant region may contain a D270 substitution. The D270 substitution can be, for example, D270E. The IgG1 heavy chain constant region can contain the R292 substitution. The R292 substitution can be, for example, R292P. The IgG1 heavy chain constant region can contain the S298 substitution. The S298 substitution can be, for example, S298A. The IgG1 heavy chain constant region can contain the Y300 substitution. The Y300 substitution can be, for example, Y300L. The IgG1 heavy chain constant region can contain the V305 substitution. The V305 substitution can be, for example, V305I. The IgG1 heavy chain constant region can contain the K326 substitution. The K326 substitution can be, for example, K326D. The IgG1 heavy chain constant region can contain the A330 substitution. The A330 substitution can be, for example, A330M or A330L. The IgG1 heavy chain constant region can contain the I332 substitution. The I332 substitution can be, for example, I332E. The IgG1 heavy chain constant region can contain the E333 substitution. The E333 substitution can be, for example, E333A. The constant region of the IgG1 heavy chain may contain a K334 substitution. The K334 substitution may be, for example, K334A or K334E. The constant region of the IgG1 heavy chain may contain a P396 substitution. The P396 substitution may be, for example, P396L.

[0192] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein comprises a constant region of the IgG1 heavy chain, the constant region of which comprises one or more amino acid substitutions selected from the group consisting of the following according to EU index numbers: L234Y, L235Q, L235V, G236A, G236W, S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330M, A330L, I332E, E333A, K334A, K334E, and P396L. In some embodiments, the IgG1 heavy chain constant region comprises one or more amino acid substitutions selected from the group consisting of the following according to EU index numbers: K214R, L234A, L235E, G237A, A330S, P331S, D356E, and L358M. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein comprises variants of the human IgG1 heavy chain constant region modified with amino acid substitutions of S298A, E333A, and K334A. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein comprises variants of the human IgG1 heavy chain constant region modified with amino acid substitutions of S239D and I332E. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein comprises variants of the human IgG1 heavy chain constant region modified with amino acid substitutions of S239D, A330L, and I332E. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein comprises variants of the human IgG1 heavy chain constant region modified with amino acid substitution of G236A. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein comprises variants of the human IgG1 heavy chain constant region modified by amino acid substitutions of G236A, S239D, and I332E. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein comprises variants of the human IgG1 heavy chain constant region modified by amino acid substitutions of G236A, S239D, A330L, and I332E. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein comprises variants of the human IgG1 heavy chain constant region modified by amino acid substitutions of F243L, R292P, Y300L, V305I, and P396L. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein comprises variants of the human IgG1 heavy chain constant region modified by amino acid substitutions of L235V, F243L, R292P, Y300L, and P396L.In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein comprises variants of the human IgG1 heavy chain constant region modified by amino acid substitutions of L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A. In some embodiments, the anti-ILT7 antibody and antigen-binding fragment described herein comprises variants of the human IgG1 heavy chain constant region modified by amino acid substitutions of D270E, K326D, A330M, and K334E. All amino acid substitutions are based on EU index numbers. Exemplary IgG1 allotypes of heavy chain constant regions (CH) with different mutations are provided below, which increase or enhance the ADCC and / or ADCP of the antibody. It is explicitly contemplated that the heavy chain constant region (CH) of any immunoglobulin (e.g., human IgG1) or other immunoglobulins known in the art is included in the antibodies disclosed herein, as well as any combination of mutations disclosed herein for increasing or enhancing the ADCC and / or ADCP of the antibody or other mutations known in the art.

[0193]

[0194]

[0195]

[0196]

[0197]

[0198] In some embodiments, this document provides an IgG1 antibody having a heavy chain constant region (CH) having at least 85% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:45-64. In some embodiments, the IgG1 antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:45. In some embodiments, the IgG1 antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:46. In some embodiments, the IgG1 antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:47. In some embodiments, the IgG1 antibody provided herein has a CH having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:48. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:49. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:50. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:51. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:52. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:53. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:54. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:55. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:56.In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:57. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:58. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:59. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:60. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:61. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:62. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:63. In some embodiments, the IgG1 antibody provided herein has a CH that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:64.

[0199] In some embodiments, the provided antibody or antigen-binding fragment comprises CH having the amino acid sequence of SEQ ID NO:55. In some embodiments (e.g., where the antibody or antigen-binding fragment is a humanized antibody hu-cmAb12), the antibody or antigen-binding fragment comprises: CH having the amino acid sequence of SEQ ID NO:55; VH having the amino acid sequence of SEQ ID NO:26; and VL having the amino acid sequence of SEQ ID NO:19. In some embodiments, the antibody or antigen-binding fragment comprises: CH having the amino acid sequence of SEQ ID NO:55; CL having the amino acid sequence of SEQ ID NO:30; VH having the amino acid sequence of SEQ ID NO:26; and VL having the amino acid sequence of SEQ ID NO:19. Selected properties of the humanized antibody hu-cmAB12 are illustrated in Examples 8-17.

[0200] In some embodiments, variants may include the addition of amino acid residues at the amino and / or carboxyl ends of an antibody or peptide. The length of the additional amino acid residues may range from one residue to one hundred or more residues. In some embodiments, variants contain an N-terminal methionyl residue. In some embodiments, variants contain additional peptides / proteins (e.g., an Fc region) to produce a fusion protein. In some embodiments, variants are engineered to be detectable and may contain a detectable marker and / or protein (e.g., a fluorescent tag or enzyme).

[0201] The variant antibodies or antigen-binding fragments described herein can be generated using methods known in the art, including but not limited to site-directed mutagenesis, alanine scan mutagenesis, and PCR mutagenesis. Methods for mutagenesis and nucleotide sequence alteration are well known in the art. See, for example, Walker and Gaastra eds. (1983), *Technologies for Molecular Biology* (T...). ECHNIQUES IN M OLECULAR B IOLOGY (MacMillan Publishing Company, New York); Kunkel, Proceedings of the National Academy of Sciences 82:488-492 (1985); Kunkel et al., Methods Enzymology 54:367-382 (1987); Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (M OLECULAR C LONING :AL ABORATORY M ANUAL(Cold Spring Harbor, NY); U.S. Patent No. 4,873,192; and references cited herein; all of which are incorporated herein by reference. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the polypeptide of interest can be found in the model of Dayhoff et al. (1978), *Atlas of Protein Sequence and Structure* (Natl. Biomed. Res. Found., Washington, DC), pp. 345-352, which is incorporated herein by reference in its entirety. Dayhoff et al.'s model uses a point-acceptance mutation (PAM) amino acid similarity matrix (PAM 250 matrix) to determine appropriate conserved amino acid substitutions. Conserved substitutions, such as exchanging one amino acid with another that has similar properties, can be beneficial. Examples of conserved amino acid substitutions taught by the PAM 250 matrix of Dayhoff et al.'s model include, but are not limited to, Gly→Ala, Val→Ile→Leu, Asp→Glu, Lys→Arg, Asn→Gln, and Phe→Trp→Tyr.

[0202] When constructing variants of anti-ILT7 binding molecules, such as antibodies or their antigen-binding fragments, variants, or derivatives, modifications are made such that the variant retains the desired properties, such as the ability to specifically bind to ILT7 and, in some embodiments, the ability to inhibit IFNα release and / or deplete pDC in vivo. Clearly, any mutations occurring in the DNA encoding the variant polypeptide cannot place the sequence outside the reading frame. In some embodiments, mutations occurring in the DNA do not produce complementary regions that could generate secondary mRNA structures.

[0203] In some embodiments, variants of the anti-ILT7 antibody or antigen-binding fragment disclosed herein may retain an ILT7 binding capacity similar to, the same as, or greater than that of the parent antibody or antigen-binding fragment. In some embodiments, the variant may be at least 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more identical in amino acid sequence to the parent antibody or antigen-binding fragment. In some embodiments, variants of the anti-ILT7 antibody or antigen-binding fragment comprise the amino acid sequence of the parent anti-ILT7 antibody or antigen-binding fragment having one or more conserved amino acid substitutions. Conserved amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties.

[0204] In some embodiments, variants of the anti-ILT7 antibody or antigen-binding fragment comprise the amino acid sequence of the parent antibody or antigen-binding fragment having one or more non-conservative amino acid substitutions. In some embodiments, variants of the anti-ILT7 antibody or antigen-binding fragment comprise the amino acid sequence of the parent binding antibody or antigen-binding fragment having one or more non-conservative amino acid substitutions, wherein the one or more non-conservative amino acid substitutions do not interfere with or inhibit one or more biological activities of the variant (e.g., ILT7 binding). In some embodiments, the one or more conserved amino acid substitutions and / or the one or more non-conservative amino acid substitutions can enhance the biological activity of the variant, such that the biological activity of the functional variant is increased compared to the parent antibody or antigen-binding fragment.

[0205] In some embodiments, the variant has one, two, three, four, or five amino acid substitutions in the CDRs of the binding portion (e.g., VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3).

[0206] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is chemically modified, either naturally or through intervention. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment has been chemically modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and / or linking to cellular ligands or other proteins. Any of these chemical modifications can be performed using known techniques. The anti-ILT7 antibody or antigen-binding fragment may contain one or more amino acid analogs (including, for example, non-natural amino acids), as well as other modifications known in the art.

[0207] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment disclosed herein is conjugated to at least one pharmaceutical agent to form an antibody conjugate. The conjugate may be, for example, an antibody conjugated to another protein, carbohydrate, lipid, steroid, immunosuppressant, or a mixture of molecules. Such antibody conjugates include, but are not limited to, modifications involving the conjugation of the antibody to one or more polymers. For example, the antibody or antigen-binding fragment may be conjugated to one or more water-soluble polymers. Conjugation to a water-soluble polymer reduces the likelihood of precipitation of the antibody or antigen-binding fragment in an aqueous environment, such as a physiological environment. Those skilled in the art can select a suitable water-soluble polymer based on considerations including, but not limited to, whether the polymer / antibody conjugate will be used to treat a patient, and if so, the pharmacological properties of the antibody (e.g., half-life, dosage, activity, antigenicity, and / or other factors).

[0208] To enhance the efficacy of antibody molecules as diagnostic or therapeutic agents, they are typically linked, covalently bound, or complexed with at least one desired molecule or moiety. Such molecules or moiety can be, but are not limited to, at least one effector or reporter molecule. Effector molecules contain molecules with desired activities, such as cytotoxic activity. Non-limiting examples of effector molecules already conjugated to antibodies include toxins, antitumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligonucleotides or polynucleotides. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules already conjugated to antibodies include enzymes, radiolabeled substances, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photosynthetic molecules, colored particles or ligands, enzymes (e.g., catalyzing colorimetric or fluorescent or bioluminescent reactions), substrates, and solid matrices such as biotin. Antibodies can contain one, two, or more of these labels.

[0209] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is chemically modified, either naturally or through intervention. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment has been chemically modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and / or linking to cellular ligands or other proteins. Any of these chemical modifications can be performed using known techniques. The anti-ILT7 antibody or antigen-binding fragment may contain one or more amino acid analogs (including, for example, non-natural amino acids), as well as other modifications known in the art.

[0210] Antibody conjugates can be used to deliver cytotoxic agents to target cells. This type of cytotoxic agent can improve antibody-mediated cytotoxicity and includes cytokines that directly or indirectly stimulate cell death, radioactive isotopes, chemotherapeutic agents (including prodrugs), bacterial toxins (e.g., pseudomonas exotoxin, diphtheria toxin, etc.), plant toxins (e.g., ricin, gelonin, etc.), chemical conjugates (e.g., maytansinoid toxin, calicheamicin, etc.), radioactive conjugates, enzyme conjugates (e.g., RNase conjugates, granzyme antibody-guided enzyme / prodrug therapy), etc.

[0211] Antibody conjugates are also used as diagnostic agents. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is conjugated to a detectable substance or molecule, making the agent suitable for diagnosis and / or detection. Detectable substances may include, but are not limited to, enzymes; prosthetic groups (e.g., biotin and flavin); fluorescent materials; bioluminescent materials such as luciferase; radioactive substances; positron-emitting metals; and magnetic metal ions.

[0212] Antibody diagnostics are generally divided into two categories: those used for in vitro diagnostics, such as various immunoassays, and those used for in vivo diagnostic protocols, often referred to as "antibody-guided imaging." Many suitable imaging agents are known in the art, as are methods for attaching them to antibodies (see, for example, U.S. Patents 5,021,236, 4,938,948, and 4,472,509). The imaging components used can be paramagnetic ions, radioisotopes, fluorescent dyes, NMR-detectable substances, MR hyperpolarized molecules, targeted sonication bubbles, and X-ray imaging agents.

[0213] Paramagnetic ions considered for use as conjugates include chromium (III), manganese (II), iron (III), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), and / or erbium (III), with gadolinium being particularly preferred. Ions that can be used in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum (III), gold (III), lead (II), and bismuth (III). Alternative useful isotopes are those used in hyperpolarized MRI, such as carbon-13 and silicon-29.

[0214] Radioisotopes used as conjugates or covalent dopants in imaging and radiotherapy include astatine-211, actinium-225, carbon-14, bismuth-212, chromium-51, chlorine-36, cobalt-57, cobalt-58, copper-64, copper-67, europium-152, fluorine-18, gallium-68, gallium-67, gold-198, hydrogen-3, iodine-123, iodine-125, and iodine-13. 1. Indium-111, iron-52, iron-59, lead-212, lutetium-177, phosphorus-32, rhenium-186, rhenium-188, rubidium-82, rhodium-99, selenium-75, sulfur-35, samarium-153, strontium-92, strontium-89, thallium-201, thorium-227, technetium-94m, technetium-99m, yttrium-86, yttrium-90, zirconium-86 and / or zirconium-89. F-18, Zr-89 and Cu-64 are generally preferred for PET imaging. Lu-177, At-211 and Yt-90 are generally preferred for radiotherapy. The radiolabeled monoclonal antibodies and antibody fragments of this disclosure can be generated according to methods well known in the art. For example, monoclonal antibodies can be iodinated by contact with sodium iodide and / or potassium iodide, as well as chemical oxidants such as sodium hypochlorite or enzymatic oxidants such as lactoperoxidase. Monoclonal antibodies according to this disclosure can be labeled with technetium-99m via a ligand exchange process, for example, by reducing pertechnetium with a stannous solution, chelating the reduced technetium onto a dextran gel column (Sephadex column), and then coating the antibody onto the column. Alternatively, direct labeling techniques can be used, for example, by incubating pertechnetium, a reducing agent such as SNCl2, a buffer solution such as sodium potassium phthalate solution, and the antibody. The intermediate functional group commonly used in introducing chelating agents to bind radioactive isotopes, which are present as metal ions, to antibodies is diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), monomeric or dendritic 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), deferoxamine (DFO), or 1-hydroxy-2(1H)-pyridone derivatives (e.g., 3,4,3-LI(1,2-HOPO) or HOPO).

[0215] Fluorescent labels considered for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Dansyl chloride, Dichlorotriazine fluorescein, FITC, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phycoerythrin, REG, Rhodamine Green, Rhodamine Red, Renografin, ROX, TAMRA, TET, Rhodamine Tetramethylisothiocyanate (TRITC), Texas Red, and / or Umbelliferone.

[0216] Another type of antibody considered in this disclosure is an antibody conjugate intended primarily for in vitro use, wherein the antibody is linked to a secondary binding ligand and / or an enzyme (enzyme tag) that produces a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include β-galactosidase, acetylcholinesterase, urease, alkaline phosphatase, (horseradish) catalase, or glucose oxidase. Preferred secondary binding ligands are biotin and avidin, as well as streptavidin compounds.

[0217] Several methods for attaching or conjugating antibodies to their conjugate portions are known in the art. Some attachment methods involve the use of metal chelates, such as organic chelating agents like diethylenetriaminepentaacetic anhydride (DTPA); ethylenediaminetetraacetic acid; monomeric or dendritic 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA); DFO; HOPO; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3a-6a-diphenylglyuron-3 (US Patents 4,472,509 and 4,938,948) attached to the antibody. Monoclonal antibodies can also be reacted with enzymes in the presence of conjugating agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these conjugating agents or by reaction with isothiocyanates. In U.S. Patent 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies, and the detectable imaging portion uses linkers such as methylparaben or N-succinimide-3-(4-hydroxyphenyl)propionate to bind to the antibody.

[0218] Another known method for site-specific attachment of molecules to antibodies involves the reaction of antibodies with hapten-based affinity tags. Essentially, the hapten-based affinity tag reacts with an amino acid at the antigen-binding site, thereby disrupting the site and blocking the specific antigen reaction.

[0219] Molecules containing azido groups can also be used to form covalent bonds with proteins via reactive daene intermediates generated by low-intensity ultraviolet light. Specifically, 2- and 8-azido analogs of purine nucleotides have been used as site-directed photoprobes to identify nucleotide-binding proteins in crude cell extracts. 2- and 8-azidonucleotides have also been used to map the nucleotide-binding domains of purified proteins and can be used as antibody binders.

[0220] The derivatization of immunoglobulins by selectively introducing thiol groups into the Fc region of the immunoglobulin using reaction conditions that do not alter the antibody binding site has also been considered. Antibody conjugates produced according to this method are disclosed to exhibit improved lifetime, specificity, and sensitivity (U.S. Patent 5,196,066, which is incorporated herein by reference). Site-specific attachment of effector or reporter molecules has also been disclosed in the literature, wherein the reporter or effector molecule is conjugated to carbohydrate residues in the Fc region. This method has been reported to produce antibodies with diagnostic and therapeutic potential that are currently under clinical evaluation.

[0221] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is conjugated with a steroid or immunosuppressant. In some embodiments, the antibody or antigen-binding fragment is conjugated with a steroid or immunosuppressant to form an ADC (antibody-drug conjugate). In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is conjugated with a steroid, which may be a corticosteroid. Corticosteroids may be, for example, dexamethasone, hydrocortisone, methylprednisolone, and prednisone. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is conjugated with an immunosuppressant, which may be an antimalarial drug (such as hydroxychloroquine, chloroquine), antimetabolite (such as methotrexate, azathioprine, mercaptopurine), calcineurin inhibitor (such as cyclosporin, tacrolimus), mycophenolic acid, mycophenolate mofetil, thalidomide, or acitretin.

[0222] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is conjugated to or partially conjugated to a cytotoxic agent. In some embodiments, the antibody or antigen-binding fragment is conjugated to a cytotoxic agent to form an ADC (antibody-drug conjugate). In some embodiments, antibody-drug conjugates or ADCs are a class of highly effective biopharmaceutical drugs designed as targeted therapies. ADCs consist of an antibody (a complete mAb or antibody fragment, such as scFv) linked to a bioactive cytotoxic / antiviral payload or drug via a stable chemical linker with unstable bonds. Antibody-drug conjugates are examples of biological conjugates and immunoconjugates. By combining the unique targeting capabilities of monoclonal antibodies with cytotoxic drugs, ADCs allow for sensitive differentiation between healthy and diseased tissues. This means that, compared to traditional systemic approaches, ADCs target and attack diseased cells, thus minimizing the impact on healthy cells.

[0223] In the development of ADC-based antitumor therapies, a warhead (e.g., a cell toxin or cytotoxin) is conjugated to an antibody that specifically targets a cellular marker (ideally, a protein found only in or on diseased cells). The antibody targets these proteins in vivo and attaches itself to the surface of diseased cells. A biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the targeted cell, which then absorbs or internalizes the antibody and cytotoxin. Once internalized, the ADC releases the cytotoxic drug and kills the cell or impairs cell replication. In other cases, the linker may cleave on the surface of the target cell or early endosomes, and therefore complete internalization is not required. Due to this targeting, ideally, the drug has fewer side effects and a wider therapeutic window compared to other agents.

[0224] In some embodiments, the cytotoxic portion of an ADC having the anti-ILT7 antibody or antigen-binding fragment described herein is a chemotherapeutic agent, including but not limited to methotrexate, adriamycin / doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepine (PBD), or other intercalating agents. In some embodiments, the cytotoxic portion is a microtubule inhibitor, including but not limited to auristatin, maytansine (e.g., DM1 and DM4), and tubulysin. In some embodiments, the cytotoxic portion is an enzymatically active toxin or fragment thereof of bacterial, fungal, plant, or animal origin, including but not limited to diphtheria A chain, non-bound active fragment of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, carnation protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, jatropha toxin curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecene. In some embodiments, the antibody or antigen-binding fragment is conjugated to one or more small molecule toxins such as chachiin, maytansin, trichothecene, and CC1065.

[0225] A stable link between an antibody and a cytotoxic agent is a key aspect of an ADC. Linkers are based on chemical motifs, including disulfides, hydrazones, or peptides (cleavable) or thioethers (non-cleavable), and control the distribution and delivery of the cytotoxic agent to target cells. Both cleavable and non-cleavable linker types have been shown to be safe in preclinical and clinical trials. The availability of better and more stable linkers alters the function of the chemical bonds. The type of cleavable or non-cleavable linker endows cytotoxic (e.g., anticancer) drugs with specific properties. For example, a non-cleavable linker holds the drug inside the cell. As a result, the entire antibody, linker, and cytotoxic agent enter the targeted cell, where the antibody is degraded to the amino acid level. The resulting complex—amino acid, linker, and cytotoxic agent—now becomes the active drug. Conversely, cleavable linkers are catalyzed by enzymes in or on the host cell, thereby releasing the cytotoxic agent. Common linker cleavage mechanisms are protease sensitivity, pH sensitivity, and glutathione sensitivity. Another type of cleavable linker adds an extra molecule between the cytotoxic drug and the cleavage site. This linker technology allows researchers to create more flexible ADCs without altering the cleavage kinetics. A novel method for peptide cleavage based on Edman degradation has also been developed. Future directions for ADC development also include the development of site-specific conjugates (TDCs) to further improve stability and therapeutic index, as well as α-emission immunoconjugates and antibody-conjugated nanoparticles.

[0226] The anti-ILT7 antibody or antigen-binding fragments described herein can be attached to a solid carrier. Such solid carriers include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the immobilized anti-ILT7 antibody or antigen-binding fragment is used for immunoassay. In some embodiments, the immobilized anti-ILT7 antibody or antigen-binding fragment is used to purify a target antigen (e.g., human ILT7).

[0227] D. Polynucleotides and carriers

[0228] This document also provides polynucleotides encoding the polypeptides described herein (e.g., anti-ILT7 antibodies or antigen-binding fragments). The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only the coding sequence of the polypeptide, as well as polynucleotides that include additional coding and / or non-coding sequences. The polynucleotides disclosed herein may be in the form of RNA or DNA. The DNA may be cDNA, genomic DNA, or synthetic DNA, and may be double-stranded or single-stranded. Single-stranded DNA may be a coding strand or a non-coding (antisense) strand. The polynucleotides disclosed herein may be mRNA.

[0229] This article explicitly considers polynucleotides encoding any anti-ILT7 antibody or antigen-binding fragment disclosed herein. For illustrative purposes, in some embodiments, the polynucleotides provided herein encode anti-ILT7 antibodies or antigen-binding fragments comprising (1) as defined by Kabat, (a) a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO: 11, 12, and 13, respectively; or variants thereof having at most about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs; and / or (b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO: 14, 15, and 16, respectively; or variants thereof having at most about 5 amino acid substitutions, additions, and / or deletions in the VH CDRs; or (2) as defined by Chothia, (a) a VL comprising, respectively having ..., SEQ ID NO: 14, 15, and 16, respectively; or (3) a heavy chain variable region (VH) comprising, respectively having, SEQ ID NO: 14, 15, and 16, respectively; or (4) a heavy chain variable region (VH) comprising, respectively having, SEQ ID NO: 14, 15, and 16, respectively; or (5) a heavy chain variable region (VH) comprising, respectively having, SEQ ID NO: 14, 15, and 16, respectively; or (6) a heavy chain variable region (VH) comprising, respectively having, SEQ ID NO: 14, 15, and 16, respectively; or (7) a heavy chain variable region (VH) comprising, respectively having, SEQ ID NO: 14 VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO: 11, 12, and 13; or variants thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs; and / or (b) VH comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO: 17, 18, and 16, respectively; or variants thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs.

[0230] In some embodiments, the polynucleotides provided herein encode an anti-ILT7 antibody or antigen-binding fragment comprising (a) a VL having at least 85%, 90%, 95%, 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:9; and / or (b) a VH having at least 85%, 90%, 95%, 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:10. The polynucleotide may be in the form of DNA. The polynucleotide may be in the form of mRNA.

[0231] In some embodiments, the polynucleotides provided herein encode the anti-ILT7 antibody or antigen-binding fragments disclosed herein, the anti-ILT7 antibody or antigen-binding fragments comprising: VL and VH, wherein the VL comprises VL CDR1, CDR2, and CDR3, and the VH comprises VH CDR1, CDR2, and CDR3, and wherein the VL CDR1, VL CDR2, VL CDR3, VHCDR1, VH CDR2, and VH CDR3 each have the following amino acid sequences: (1) SEQ ID NO: 11, 12, 13, 14, 15, and 16; or (2) SEQ ID NO: 11, 12, 13, 17, 18, and 16; or variants thereof, the variants having up to about 5 amino acid substitutions, additions, and / or deletions in the CDRs. The polynucleotides may be in the form of DNA. The polynucleotides may be in the form of mRNA.

[0232] In some embodiments, the polynucleotides provided herein encode the anti-ILT7 antibody or antigen-binding fragment disclosed herein, the anti-ILT7 antibody or antigen-binding fragment comprising VL and VH, wherein the VL and VH each have the following amino acid sequences: (1) SEQ ID NO: 9 and 10; (2) SEQ ID NO: 19 and 23; (3) SEQ ID NO: 19 and 24; (4) SEQ ID NO: 19 and 25; (5) SEQ ID NO: 19 and 26; (6) SEQ ID NO: 19 and 27; (7) SEQ ID NO: 19 and 28; (8) SEQ ID NO: 20 and 23; (9) SEQ ID NO: 20 and 24; (10) SEQ ID NO: 20 and 25; (11) SEQ ID NO: 20 and 26; (12) SEQ ID NO: 20 and 27; (13) SEQ ID NO: 20 and 28; (14) SEQ ID NO: 21 and 23; (15) SEQ ID NO: 21 and 23; SEQ ID NO:21 and 24; (16) SEQ ID NO:21 and 25; (17) SEQ ID NO:21 and 26; (18) SEQ ID NO:21 and 27; (19) SEQ ID NO:21 and 28; (20) SEQ ID NO:22 and 23; (21) SEQ ID NO:22 and 24; (22) SEQ ID NO:22 and 25; (23) SEQ ID NO:22 and 26; (24) SEQ ID NO:22 and 27; or (25) SEQ ID NO:22 and 28. Polynucleotides can be in the form of DNA. Polynucleotides can be in the form of mRNA.

[0233] In some embodiments, VL and VH are connected by a connector. The connector may be a flexible connector or a rigid connector. In some embodiments, the connector has an amino acid sequence of (GGGGS)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 35). In some embodiments, the connector has an amino acid sequence of (EAAAK)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 36). In some embodiments, the connector has an amino acid sequence of (PA)nP, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 37).

[0234] This disclosure also provides variants of the polynucleotides described herein, wherein the variants encode fragments, analogs, and / or derivatives of, for example, the anti-ILT7 antibody or antigen-binding fragments disclosed herein. In some embodiments, this disclosure provides a polynucleotide having a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the polynucleotide sequence encoding the anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, this disclosure provides a polynucleotide having a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the polynucleotide sequence encoding the anti-ILT7 antibody or antigen-binding fragment described herein.

[0235] As used herein, the phrase "a polynucleotide having at least about 95% identical nucleotide sequence to a reference sequence" means that the polynucleotide has the same nucleotide sequence as the reference sequence, except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference sequence. In other words, to obtain a polynucleotide having at least 95% identical nucleotide sequence to a reference sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' ends of the reference nucleotide sequence or at any position between these ends, said positions being scattered between nucleotides in the reference sequence or scattered within one or more consecutive groups in the reference sequence.

[0236] Polynucleotide variants may contain alterations to coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions but do not change the properties or activity of the encoded polypeptide. In some embodiments, polynucleotide variants contain silent substitutions that result in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can arise for a variety of reasons, such as optimizing codon expression in a particular host (e.g., changing codons in human mRNA to codons preferred by bacterial hosts such as E. coli). In some embodiments, polynucleotide variants contain at least one silent mutation in a non-coding or coding region of the sequence.

[0237] In some embodiments, polynucleotide variants are generated to modulate or alter the expression (or expression level) of the encoded peptide. In some embodiments, polynucleotide variants are generated to increase the expression of the encoded peptide. In some embodiments, polynucleotide variants are generated to decrease the expression of the encoded peptide. In some embodiments, the polynucleotide variant has increased expression of the encoded peptide compared to the parental polynucleotide sequence. In some embodiments, the polynucleotide variant has decreased expression of the encoded peptide compared to the parental polynucleotide sequence.

[0238] In some embodiments, the polynucleotide contains the coding sequence of a polypeptide (e.g., an antibody) fused to the polynucleotide in the same reading frame, the polynucleotide facilitating the expression and secretion of the polypeptide from the host cell (e.g., serving as a leader sequence for controlling the transport of the polypeptide). The polypeptide may have a leader sequence that is cleaved by the host cell to form the "mature" form of the polypeptide.

[0239] In some embodiments, the polynucleotide comprises the coding sequence of a polypeptide (e.g., an antibody) fused with a marker or tag sequence within the same reading frame. For example, in some embodiments, the marker sequence is a hexahistidine tag (HIS tag) (SEQ ID NO:65), which allows for efficient purification of the polypeptide fused with the marker. In some embodiments, when using a mammalian host (e.g., COS-7 cells), the marker sequence is a hemagglutinin (HA) tag derived from influenza hemagglutinin protein. In some embodiments, the marker sequence is a FLAG. TM Labels. In some embodiments, the logo is used in combination with other logos or labels.

[0240] In some embodiments, the polynucleotides are isolated. In some embodiments, the polynucleotides are substantially pure.

[0241] Vectors and cells comprising the polynucleotides described herein are also provided. In some embodiments, vectors comprising the polynucleotides provided herein are provided. The vector may be an expression vector. In some embodiments, the vectors provided herein comprise a polynucleotide encoding an anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, the vectors provided herein comprise a polynucleotide encoding a polypeptide that is part of an anti-ILT7 antibody or antigen-binding fragment described herein.

[0242] In some embodiments, this document provides recombinant expression vectors that can be used to amplify and express polynucleotides encoding anti-ILT7 antibodies or antigen-binding fragments described herein. For example, the recombinant expression vector can be a reproducible DNA construct comprising a synthetic or cDNA-derived DNA fragment encoding a polypeptide chain of an anti-ILT7 antibody, operatively linked to a suitable transcriptional and / or translational regulatory element derived from a mammalian, microbial, viral, or insect gene. In some embodiments, a viral vector is used. DNA regions are “operatively linked” when they are functionally related to each other. For example, if a promoter controls transcription of a sequence, the promoter is operatively linked to the coding sequence; or if a ribosome binding site is localized to allow translation, the ribosome binding site is operatively linked to the coding sequence. In some embodiments, structural elements intended for use in certain expression systems include a leader sequence for the extracellular secretion of a protein that enables translation by a host cell. In some embodiments, where a recombinant protein is expressed in the absence of a leader or transport sequence, the polypeptide may include an N-terminal methionine residue.

[0243] A variety of expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as those from *Escherichia coli*, including pCR1, pBR322, pMB9, and their derivatives, as well as broader host-scope plasmids, such as M13 and other filamentous single-stranded DNA bacteriophages. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is expressed by one or more vectors.

[0244] This document provides host cells comprising the vectors described herein. In some embodiments, the host cells are used for recombinant expression of the anti-ILT7 antibody described herein. Host cells may include prokaryotic, yeast, insect, or higher eukaryotic cells under the control of a suitable promoter. Suitable cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts, as well as protein production methods, including antibody production, are well known in the art.

[0245] Examples of suitable mammalian host cells include, but are not limited to, COS-7 (monkey kidney-derived), L-929 (mouse fibroblast-derived), C127 (mouse mammary tumor-derived), 3T3 (mouse fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human fetal kidney-derived) cell lines and their variants. Mammalian expression vectors may contain non-transcriptional elements such as origin of replication, appropriate promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanking non-transcriptional and 5' or 3' non-translational sequences, such as essential ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, and transcription termination sequences. Expression of recombinant proteins in insect cell culture systems (e.g., baculoviruses) also provides a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art.

[0246] This disclosure also provides host cells comprising the polypeptides described herein, polynucleotides encoding the polypeptides described herein, or vectors comprising such polynucleotides. In some embodiments, a host cell comprising a vector containing the polynucleotides disclosed herein is provided. In some embodiments, a host cell provided herein comprises a vector containing a polynucleotide encoding an anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, a host cell provided herein comprises a vector containing a polynucleotide encoding a polypeptide that is part of an anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, a host cell provided herein contains a polynucleotide encoding an anti-ILT7 antibody or antigen-binding fragment described herein. In some embodiments, a cell produces an anti-ILT7 antibody or antigen-binding fragment described herein.

[0247] E. Manufacturing method

[0248] This article also provides a method for preparing anti-ILT7 antibodies and their antigen-binding fragments, wherein the anti-ILT7 antibodies and their antigen-binding fragments include, but are not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies and their antigen-binding fragments.

[0249] Methods for antibody production are well known in the art. See, for example, Harlow et al., *Antibodies: A Laboratory Manual* (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., *Monoclonal Antibodies and T-Cell Hybridomas*, 563 681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. In some embodiments, monoclonal antibodies are prepared using hybridoma methods known to those skilled in the art. For example, hybridoma methods are used to immunize mice, rats, rabbits, hamsters, or other suitable host animals as described above. In some embodiments, lymphocytes are used for immunization in vitro. In some embodiments, the immunoantigen is a human protein or a fragment thereof.

[0250] Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. Hybridoma cells are selected using specialized culture media known in the art, and unfused lymphocytes and myeloma cells do not survive the selection process. Hybridomas producing monoclonal antibodies against selected antigens can be identified by a variety of methods, including but not limited to immunoprecipitation, Western blotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay). After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution or other techniques. Hybridomas can be propagated in in vitro cultures using standard methods or in vivo as ascites tumors. Monoclonal antibodies can be purified from culture media or ascites fluid according to standard methods in the art, including but not limited to affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.

[0251] In some embodiments, monoclonal antibodies are prepared using recombinant DNA techniques known to those skilled in the art. For example, polynucleotides encoding antibodies are isolated from mature B cells or hybridoma cells using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody via RT-PCR, and their sequences are determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector, which produces monoclonal antibodies when transfected into host cells that do not additionally produce immunoglobulins, such as *Escherichia coli*, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells.

[0252] The polynucleotides of antibody or antigen-binding fragments provided herein can be prepared, manipulated, and / or expressed using any well-established techniques known and available in the art. In some embodiments, the polynucleotides of antibody or antigen-binding fragments provided herein can be prepared recombinantly. Many vectors can be used. Examples of vectors are plasmids, autonomously replicating sequences, and transposon elements. Exemplary transposon systems such as Sleeping Beauty and PiggyBac can be used, which can be stably integrated into the genome (e.g., Ivics et al., Cell, 91(4):501-510(1997)). et al., (2007) Nucleic Acids Research. 35(12):e87. Other exemplary vectors include, but are not limited to: plasmids; phage particles; granules; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Examples of animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (e.g., SV40). Examples of expression vectors are the pCl-neo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST for lentivirus-mediated gene transfer and expression in mammalian cells. TM pLenti6 / V5-DEST TM And pLenti6.2 / V5-GW / lacZ (Invitrogen).

[0253] In some embodiments, the vector is a free vector or a vector maintained outside the chromosome. As used herein, the term "free" means a vector capable of replicating without integrating into the host's chromosomal DNA and not gradually lost from dividing host cells; this also means that the vector replicates outside the chromosome or in a free manner. The vector is engineered to contain a sequence encoding the origin of DNA replication or "ori" from lymphotropic herpesvirus or gamma herpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, particularly the origin of replication of lymphotropic herpesvirus or gamma herpesvirus corresponding to oriP of EBV. In some embodiments, the lymphotropic herpesvirus is Epstein Barrvirus (EBV), Kaposi's sarcoma herpesvirus (KSHV), Herpes virus saimiri (HS), or Marek's disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gamma herpesviruses. Typically, host cells contain viral replication transactivator proteins that activate replication.

[0254] The “expression control sequences,” “control elements,” or “regulatory sequences” present in expression vectors are those untranslated regions of the vector—the origin of replication, selection cassettes, promoters, enhancers, translation initiation signal (Shine Dalgarno or Kozak sequence) introns, polyadenylated sequences, and 5' and 3' untranslated regions—that interact with host cell proteins to carry out transcription and translation. The strength and specificity of these elements can vary. Depending on the vector system and the host used, any number of suitable transcriptional and translational elements can be used, including ubiquitous promoters and inducible promoters.

[0255] Illustrative and ubiquitous expression control sequences that can be used in this disclosure include, but are not limited to: cytomegalovirus (CMV) immediate early promoter, viral simian virus 40 (SV40) promoter (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Raul's sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, p7.5 and p11 promoters from vaccinia virus, elongation factor 1-α (EF1a) promoter, early growth promoter, etc. Response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa β, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinin (β-KIN), human ROSA26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, and β-actin promoter.

[0256] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoids or estrogen receptors (which can be induced by treatment with the corresponding hormone), metallothionein promoters (which can be induced by treatment with various heavy metals), MX-1 promoters (which can be induced by interferon), the mifepristone-regulated "gene switch" system (Sirin et al., 2003, *Gene*, 323:67), copper dimethylaminodithiocarbamate-inducible gene switches (WO 2002 / 088346), tetracycline-dependent regulatory systems, etc. The anti-ILT7 antibodies or antigen-binding fragments described herein can be generated by any method known in the art, including chemical synthesis and recombinant expression techniques. Unless otherwise stated, the practice of this invention employs conventional techniques from molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the scope of this art.

[0257] The polypeptides described herein can be prepared using a variety of techniques known in the art, including hybridoma and recombinant techniques or combinations thereof. In some embodiments, a recombinant expression vector is used to express a polynucleotide encoding the polypeptide described herein. For example, the recombinant expression vector may be a reproducible DNA construct comprising a synthetic or cDNA-derived DNA fragment encoding the polypeptide, operatively linked to a suitable transcriptional and / or translational regulatory element derived from a mammalian, microbial, viral, or insect gene. In some embodiments, the coding sequence of the polypeptide disclosed herein may be linked to such an expression vector for its expression in mammalian cells. In some embodiments, a viral vector is used. The DNA regions are “operatively linked” when they are functionally related to each other. For example, if a promoter controls transcription of a sequence, the promoter is operatively linked to the coding sequence; or if a ribosome binding site is positioned to allow translation, the ribosome binding site is operatively linked to the coding sequence. In some embodiments, structural elements intended for use in a yeast expression system include a leader sequence for the extracellular secretion of a protein that enables translation by a host cell. In some embodiments, where a recombinant protein is expressed in the absence of a leader or transport sequence, the polypeptide may include an N-terminal methionine residue.

[0258] A variety of expression host / vector combinations can be used. Suitable host cells for expression include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Appropriate cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts, as well as protein production methods, including antibody production, are well known in the art. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as those from *E. coli*, including pCR1, pBR322, pMB9, and their derivatives, as well as broader host-range plasmids, such as M13 and other filamentous single-stranded DNA bacteriophages.

[0259] Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney), L-929 (mouse fibroblast), C127 (mouse mammary tumor), 3T3 (mouse fibroblast), CHO (Chinese hamster ovary), HeLa (human cervical cancer), BHK (hamster kidney fibroblast), HEK-293 (human fetal kidney), and their variants. Mammalian expression vectors may contain non-transcriptional elements such as origin of replication, appropriate promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanked non-transcriptional and 5' or 3' non-translational sequences, such as essential ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, and transcription termination sequences. Expressing recombinant proteins in insect cell culture systems (e.g., baculoviruses) also provides a robust method for producing properly folded and biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art.

[0260] To prepare glycosylated anti-ILT7 antibodies and antigen-binding fragments, the following host cells can be used: (1) overexpressing N-acetylglucosamine transferase III (GnTIII); (2) lacking α-1,6-fucosyltransferase (FUT8); or (3) having low fucose content; or any combination of (1)-(3). In some embodiments, host cells overexpressing N-acetylglucosamine transferase III (GnTIII) are used herein. In some embodiments, host cells lacking α-1,6-fucosyltransferase (FUT8) are used herein. In some embodiments, host cells having low fucose content are used herein. In some embodiments, CHO host cells are used.

[0261] Peptides can be synthesized, in whole or in part, using chemical methods (see, for example, Caruthers (1980), *Nucleic Acids Res. Symp. Ser.*, 215; Horn (1980); and Banga, AK, *Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (T...)*). HERAPEUTIC P EPTIDES AND P ROTEINS ,F ORMULATION ,P ROCESSING AND D ELIVERY S YSTEMS(Technomic Publishing Co., Lancaster, PA, 1995). Peptide synthesis can be performed using a variety of solid-phase techniques (see, for example, Roberge, Science 269:202 (1995); Merrifield, Methods of Enzymology 289:3 (1997)), and automated synthesis can be performed, for example, using an ABI 431A peptide synthesizer (PerkinElmer) according to the manufacturer's instructions. Combinatorial methods can also be used to synthesize peptides. The synthesized residues and polypeptides can be synthesized using a variety of procedures and methods known in the art (see, for example, Organic Synthesis Collective Volume (O...). RGANIC S YNTHESES C OLLECTIVE V OLUMES (Journal of John Wiley & Sons, Inc., NY) Modified peptides can be produced by chemical modification methods (see, for example, Belousov, Nucleic Acid Research 25:3440 (1997); Frenkel, Free Radical Biology and Medicine 19:373 (1995); and Blommers, Biochemistry 33:7886 (1994)). Peptide sequence variations, derivatives, substitutions, and modifications can also be performed using methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR-based mutagenesis. Site-directed mutagenesis (Carter et al., Nucleic Acids Res., 13:4331 (1986); Zoller et al., Nucleic Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Genes, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philosophical Transactions of the Royal Society of London, Series A, 317:415 (1986)) and other techniques can be performed on cloned DNA to produce inventive peptide sequences, variants, fusions and chimeras, as well as their variants, derivatives, substitutions and modifications.

[0262] For in vivo use of antibodies in humans, human antibodies may be preferred. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences, including improvements to these techniques. See also U.S. Patents 4,444,887 and 4,716,111; and PCT disclosures WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741; each of these documents is incorporated herein by reference in its entirety. Human antibodies can also be antibodies in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources.

[0263] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment is a human antibody or antigen-binding fragment. Human antibodies can be prepared using various techniques known in the art. In some embodiments, human antibodies are produced from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are produced from lymphocytes isolated from an immunized individual. Cells that produce antibodies against a target antigen can be generated and isolated under any circumstances. In some embodiments, human antibodies are selected from phage libraries that express human antibodies. Alternatively, phage display technology can be used to generate human antibodies and antibody fragments in vitro from a reservoir of immunoglobulin variable regions from an unimmunized donor. Techniques for generating and using antibody phage libraries are well known in the art. After antibody identification, affinity maturation strategies known in the art, including but not limited to chain shuffling and site-directed mutagenesis, can be employed to generate human antibodies with higher affinity. In some embodiments, human antibodies are generated in transgenic mice containing human immunoglobulin loci. Following immunization, these mice can generate a complete reservoir of human antibodies without producing endogenous immunoglobulins.

[0264] Human antibodies can also be generated using transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy chain and light chain immunoglobulin gene complexes can be randomly introduced or introduced via homologous recombination into mouse embryonic stem cells. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. When introducing mouse heavy chain and light chain immunoglobulin genes into human immunoglobulin loci via homologous recombination, the mouse heavy chain and light chain immunoglobulin genes can be rendered nonfunctional, individually or simultaneously. For example, homozygous loss of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice has been described as resulting in complete suppression of endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. Chimeric mice are then fed to generate homozygous offspring expressing human antibodies. Transgenic mice are immunized in a normal manner with a selected antigen, such as all or part of the polypeptide of the present invention. For example, anti-ILT7 antibodies against the human ILT7 antigen can be obtained from immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgenes carried by these transgenic mice undergo rearrangement during B-cell differentiation and subsequently class switching and somatic mutations. Therefore, using such techniques, it is possible to generate therapeutically useful IgG, IgA, IgM, and IgE antibodies, including but not limited to IgG1 (γ1) and IgG3. For an overview of this technique for generating human antibodies, see Lonberg and Huszar (International Review of Immunology, 13:65-93 (1995)). For a detailed discussion of this technique for generating human antibodies and human monoclonal antibodies, as well as the human monoclonal antibodies and protocols for generating such antibodies, see, for example, PCT Publications WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patents 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598; each of these documents is incorporated herein by reference in its entirety. In addition, companies such as Amgen (Abgenix, Inc.) (Freemont, California) and Genpharm (San Jose, California) could participate in providing human antibodies against selected antigens using technologies similar to those described above.For a detailed discussion of the transfer of human germline immunoglobulin gene arrays in germline mutant mice that will lead to the production of human antibodies after antigen stimulation, see, for example, Jakobovits et al., Proceedings of the National Academy of Sciences, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993); and Duchosal et al., Nature, 355:258 (1992).

[0265] Human antibodies can also be derived from phage display libraries (Hoogenboom et al., Journal of Molecular Biology, 227:381 (1991); Marks et al., Journal of Molecular Biology, 222:581-597 (1991); Vaughan et al., Nature Biotech, 14:309 (1996)). Phage display technology (McCafferty et al., Nature, 348:552-553 (1990)) can be used to generate human antibodies and antibody fragments in vitro from gene libraries of immunoglobulin variable (V) domains from unimmunized donors. According to this technology, genes for major or minor coat proteins of filamentous phages, such as M13 or fd, are cloned within the antibody V domain gene frame and displayed as functional antibody fragments on the surface of phage particles. Because filamentous particles contain single-stranded DNA copies of the phage genome, selection based on the functional properties of the antibody also leads to the selection of genes encoding antibodies that express those properties. Therefore, phages mimic some of the properties of B cells. Phage display can be performed in various forms; for a review of these, see, for example, Johnson and Chiswell, Current Opinion in Structural Biology 3:564-571 (1993). V gene segments from several sources can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated various anti-oxazolone antibodies from a small, randomly combined library of the V gene derived from the spleen of immunized mice. A V gene bank derived from unimmunized human donors can be constructed, and antibodies against a wide variety of antigens, including autoantigens, can be substantially isolated using techniques described in the following literature: Mark et al., *Journal of Molecular Biology*, 222:581-597 (1991); or Griffith et al., *European Organization for Molecular Biology Journal*, 12:725-734 (1993). See also U.S. Patents 5,565,332 and 5,573,905, each of which is incorporated herein by reference in its entirety.

[0266] Human antibodies can also be generated from activated B cells in vitro (see U.S. Patents 5,567,610 and 5,229,275, each of which is incorporated herein by reference in its entirety). Human antibodies can also be generated in vitro using hybridoma techniques, such as, but not limited to, those described by Roder et al. (Enzymological Methods, 121:140-167 (1986)).

[0267] Alternatively, in some embodiments, the nonhuman antibody is humanized, wherein specific sequences or regions of the antibody are modified to increase similarity to naturally occurring human antibodies. In some embodiments, the antigen-binding domain is partially humanized. Various methods for generating humanized antibodies are known in the art. Methods for achieving high-affinity binding with humanized antibodies are known in the art. Non-limiting examples of such methods are hypermutation of the variable region and selection (affinity maturation) of cells expressing such high-affinity antibodies. In addition to using a display library, specific antigens (e.g., recombinant ILT7 or its epitopes) can be used to immunize nonhuman animals, such as rodents. In some embodiments, rodent antigen-binding fragments (e.g., mouse antigen-binding fragments) can be generated and isolated using methods known in the art and / or disclosed herein. In some embodiments, mice can be immunized with an antigen (e.g., recombinant ILT7 or its epitopes).

[0268] Humanized antibodies can be generated using techniques including, but not limited to, the following: CDR transplantation (see, for example, European Patent EP 239,400; International Publication WO 91 / 09967; and U.S. Patents 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein by reference in its entirety), inlaying, or surface remodeling (see, for example, European Patents EP 592,106 and EP 519,596; Padlan, 1991, *Molecular Immunology*, 28(4 / 5):489-498; Studnicka et al., 1994, *Protein Engineering*). Engineering), 7(6):805-814; and Roguska et al., 1994, Proceedings of the National Academy of Sciences (PNAS), 91:969-973, each of which is incorporated herein by reference in its entirety), chain truncation (see, for example, U.S. Patent No. 5,565,332, which is incorporated herein by reference in its entirety), and techniques disclosed, for example, in: U.S. Patent Application Publication No. US2005 / 0042664; U.S. Patent Application Publication No. US2005 / 0048617; U.S. Patent No. 6,407,213; U.S. Patent No. 5,766,886; International Publication No. WO 9317105; Tan et al., Journal of Immunology, 169:1119-25 (2002); Caldas et al., Protein Engineering, 13(5):353-60 (2000); Morea et al., Methods, 20(3):267-79 (2000); Baca et al., Journal of Biochemistry, 272(16):10678-84 (1997); Roguska et al., Protein Engineering, 9(10):895-904 (1996); Couto et al., Cancer Research, 55(23 Supplement):5973s-5977s (1995); Couto et al., Cancer Research, 55(8):1717-22 (1995); Sandhu JS, Genes, 150(2):409-10 (1994); and Pedersen et al., Journal of Molecular Biology, 235(3):959-73 (1994), each of which is incorporated herein by reference in its entirety. Typically, framework residues in the framework region can be altered by substituting corresponding residues from CDR donor antibodies, preferably to improve antigen binding.These framework substitutions were identified using methods well-known in the art, such as modeling the interaction between CDRs and framework residues to identify framework residues important for antigen binding, and identifying aberrant framework residues at specific locations through sequence comparison. (See, for example, Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entirety.)

[0269] Humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as “import” residues, which are typically derived from an “import” variable domain. Thus, humanized antibodies contain one or more CDRs from non-human immunoglobulin molecules and a framework region from a human. Humanization of antibodies is well known in the art and can be performed essentially as Winter and collaborators (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 replacing the rodent CDR or CDR sequence with the corresponding sequence of the human antibody (i.e., CDR transplantation (EP 239,400; PCT Publication No. WO)). Humanization is performed using documents such as 91 / 09967; and U.S. Patents 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; and 6,548,640, the contents of which are incorporated herein by reference in their entirety. In such humanized chimeric antibodies, substantially less than the complete human variable domain has been replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues have been replaced by residues from similar sites in rodent antibodies. Antibody humanization can also be achieved through matte finishing or surface remodeling (EP 592,106; EPEP). 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., Proceedings of the National Academy of Sciences, 91:969-973 (1994)) or chain truncation (US Patent No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

[0270] The selection of human variable domains (both light and heavy chains) for the preparation of humanized antibodies is intended to reduce antigenicity. Following a so-called "best-fit" approach, sequences of variable domains for rodent antibodies are screened against an entire library of known human variable domain sequences. The human sequence closest to the rodent is then accepted as the human frame (FR) for the humanized antibody (Sims et al., *Journal of Immunology*, 151:2296 (1993); Chothia et al., *Journal of Molecular Biology*, 196:901 (1987), the contents of which are incorporated herein by reference in their entirety). Another approach uses specific frames derived from common sequences of all human antibodies with specific light or heavy chain subgroups. The same frames can be used for several different humanized antibodies (Carter et al., *Proceedings of the National Academy of Sciences*, 89:4285 (1992); Presta et al., *Journal of Immunology*, 151:2623 (1993), the contents of which are incorporated herein by reference in their entirety).

[0271] Antibodies can be humanized while retaining high affinity for the target antigen and other favorable biological properties. For example, humanized antibodies can be prepared through a process that analyzes the parental sequence and various conceptual humanized products using a three-dimensional model of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs illustrating and demonstrating the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. These displays are examined to allow analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., analyzing residues that affect the ability of the candidate immunoglobulin to bind to the target antigen. In this way, FR residues can be selected and combined from the recipient and input sequences to achieve desired antibody properties, such as increased affinity for the target antigen. Typically, CDR residues are directly and most substantially involved in influencing antigen binding.

[0272] Humanized antibodies retain similar antigen specificity to the original antibodies, such as the ability to bind to the human ILT7 antigen. However, using certain humanization methods, “directed evolution” approaches can be used to increase the affinity and / or specificity of antibodies binding to specific antigens, as described by Wu et al., Journal of Molecular Biology, 294:151 (1999), the contents of which are incorporated herein by reference in their entirety.

[0273] The anti-ILT7 antibody or antigen-binding fragment described herein can be used to assess binding to human ILT7 via, for example, a standard ELISA assay. Briefly, a microtiter plate is coated with purified ILT7 and then blocked with bovine serum albumin. Antibody dilution (e.g., plasma dilution from ILT7-immunized mice) is added to each well and incubated. The plate is washed and incubated with a secondary reagent conjugated with horseradish peroxidase (HRP) (e.g., for human antibodies, goat anti-human IgG Fc-specific polyclonal reagent). After washing, the plate can be developed and analyzed using a spectrophotometer. Binding to cell lines expressing human ILT7, rather than to control cell lines not expressing ILT7, can then be further screened by flow cytometry using serum from immunized mice. In short, binding to anti-ILT7 antibodies can be assessed by incubating ILT7-expressing CHO cells with the anti-ILT7 antibody. Cells can be washed and binding detected using anti-human IgG Ab. Flow cytometry analysis can be performed using the FACScan flow cytometry instrument (Becton Dickinson, San Jose, CA). Mice producing the highest titers can be used for fusion.

[0274] The ELISA assay described above can be used to screen antibodies, and thus hybridomas that produce antibodies that react positively with the ILT7 immunogen. Hybridomas producing antibodies that bind to ILT7 with high affinity can then be subcloned and further characterized. A clone from each hybridoma, retaining the reactivity of the parental cells (by ELISA), can then be selected for preparing a cell bank and for antibody purification.

[0275] To purify anti-ILT7 antibodies, selected hybridomas can be cultured for monoclonal antibody purification. The supernatant can be filtered and concentrated, followed by affinity chromatography. The eluted IgG can be examined by gel electrophoresis and high-performance liquid chromatography to ensure purity. Buffer solutions can be exchanged, and concentrations can be determined. The monoclonal antibody can be aliquoted and stored.

[0276] To determine whether the selected anti-ILT7 monoclonal antibody binds to the unique epitope, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Binding of the biotinylated MAb can be detected using a streptavidin-labeled probe. Comparative studies using unlabeled and biotinylated monoclonal antibodies can be performed using ILT7-coated ELISA plates as described above.

[0277] To determine the isotype of purified antibodies, isotype ELISA can be performed using reagents specific to a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of a microtiter plate can be coated overnight at 4°C with 1 pg / mL anti-human immunoglobulin. After blocking with 1% BSA, the plate is reacted with the test monoclonal antibody or a purified isotype control antibody at ambient temperature for one to two hours. The wells can then be reacted with probes conjugated to human IgG1 or human IgM specific alkaline phosphatase. The plate is then developed and analyzed as described above.

[0278] To test the binding of monoclonal antibodies to live cells expressing ILT7, flow cytometry can be used, as described in the examples. Briefly, cell lines expressing membrane-bound ILT7 (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4°C for 1 hour. After washing, the cells are reacted with fluorescein-labeled anti-IgG antibodies under the same conditions as primary antibody staining. Samples can be analyzed using a FACScan instrument, which uses light and side-scattering properties to gate single cells and determine the binding of the labeled antibody. Alternative assays using fluorescence microscopy (in addition to or instead of flow cytometry) can be used. Cells can be precisely stained as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but sensitivity may decrease depending on the antigen density.

[0279] The reactivity of anti-ILT7 antibodies or antigen-binding fragments with the ILT7 antigen can be further tested using Western blotting. Briefly, cell extracts from ILT7-expressing cells can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigen is transferred to a nitrocellulose membrane, blocked with 20% mouse serum, and detected with the monoclonal antibody to be tested. IgG binding can be detected using anti-IgG alkaline phosphatase and visualized with BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

[0280] Methods for analyzing the binding affinity, cross-reactivity, and binding kinetics of various anti-ILT7 antibodies include standard assays known in the art, such as biolayer interferometry (BLI) using systems like the Gator system (Probe Lifetime Corporation) or the Octet-96 system (Sartorius Corporation), or BIACORE. TM 2000SPR Instruments (Biacore AB, Uppsala, Sweden) BIACORE TMSurface plasmon resonance (SPR) analysis.

[0281] F. Pharmaceutical Composition

[0282] This document also provides pharmaceutical compositions comprising an anti-ILT7 antibody or antigen-binding fragment disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can be used to suppress autoimmunity associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, the pharmaceutical composition can be used to treat diseases or conditions associated with type I IFN (e.g., IFNα) or pDC.

[0283] In some embodiments, the pharmaceutical compositions provided herein comprise the anti-ILT7 antibody or antigen-binding fragment provided herein. The anti-ILT7 antibody or antigen-binding fragment may be present in various concentrations. In some embodiments, the pharmaceutical compositions provided herein comprise 1-1000 mg / mL of the soluble anti-ILT7 antibody or antigen-binding fragment provided herein. The dosage can be readily adjusted by those skilled in the art; for example, a decrease in purity may necessitate an increase in dosage.

[0284] This document also provides kits for preparing pharmaceutical compositions having the anti-ILT7 antibody or antigen-binding fragment disclosed herein. In some embodiments, the kit comprises the anti-ILT7 antibody or antigen-binding fragment disclosed herein and a pharmaceutically acceptable carrier in one or more containers. In another embodiment, the kit may comprise the anti-ILT7 antibody or antigen-binding fragment disclosed herein for administration to a subject. In specific embodiments, the kit includes instructions for the preparation and / or administration of the anti-ILT7 antibody or antigen-binding fragment.

[0285] In some embodiments, pharmaceutical compositions are provided herein comprising an anti-ILT7 antibody or antigen-binding fragment or cell provided herein, wherein the compositions are suitable for topical application.

[0286] Pharmaceutically acceptable carriers that can be used in the compositions provided herein include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient (i.e., an anti-ILT7 antibody or antigen-binding fragment) may be coated in the material to protect the active ingredient from acids and other natural conditions that may inactivate it.

[0287] This document also provides pharmaceutical compositions or formulations that improve the stability of anti-ILT7 antibodies or antigen-binding fragments to allow for long-term storage. In some embodiments, the pharmaceutical compositions or formulations disclosed herein comprise: (a) the anti-ILT7 antibody or antigen-binding fragment disclosed herein; (b) a buffer; (c) a stabilizer; (d) a salt; (e) a compatibilizer; and / or (f) a surfactant. In some embodiments, the pharmaceutical compositions or formulations are stable for at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years, at least 5 years, or longer. In some embodiments, the pharmaceutical compositions or formulations are stable when stored at 4°C, 25°C, or 40°C.

[0288] Buffers used in the pharmaceutical compositions or formulations disclosed herein may be weak acids or weak bases, used to maintain the acidity (pH) of the solution near a selected value after the addition of another acid or base. Suitable buffers can maximize the stability of the pharmaceutical formulation by maintaining pH control of the formulation. Suitable buffers can also ensure physiological compatibility or optimize solubility. Rheological, viscosity, and other properties also depend on the pH of the formulation. Common buffers include, but are not limited to, histidine, citrate, succinate, acetate, and phosphate. In some embodiments, the buffer comprises histidine (e.g., L-histidine) and an isotonic agent, and the pH may be adjusted with an acid or base known in the art. In some embodiments, the buffer is L-histidine. In some embodiments, the pH of the formulation is maintained between about 2 and about 10, or between about 4 and about 8.

[0289] Stabilizers are added to pharmaceutical products to stabilize them. These agents can stabilize proteins in various ways. Common stabilizers include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine, or threonine; carbohydrates such as glucose, sucrose, trehalose, raffinose, or maltose; polyols such as glycerol, mannitol, or sorbitol; cyclodextrins or dextran of any type and molecular weight; or PEG. In some embodiments, stabilizers are selected to maximize the stability of the peptide in the lyophilized formulation. In some embodiments, the stabilizers are sucrose and / or arginine.

[0290] Compatibilizers can be added to pharmaceutical compositions or formulations to increase the volume and mass of the product, thereby facilitating accurate metering and handling. Common compatibilizers include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0291] Surfactants are amphiphilic substances having both hydrophilic and hydrophobic groups. Surfactants can be anionic, cationic, zwitterionic, or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxylates, nonylphenol ethoxylates, amine ethoxylates, polyethylene oxide, polypropylene oxide, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocoamide MEA, cocoamide DEA, polysorbate, or dodecyl dimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0292] The pharmaceutical compositions disclosed herein may further comprise one or more of the following: buffer systems, preservatives, tonifying agents, chelating agents, stabilizers, and / or surfactants, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. References may be made to... Remington: Pharmacy Remington: The Science and Practice of Pharmacy , 19th edition, 1995.

[0293] In some embodiments, the pharmaceutical composition is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multiphase materials. The term "aqueous formulation" is defined as a formulation containing at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension containing at least 50% w / w water.

[0294] In some embodiments, the pharmaceutical compositions disclosed herein are lyophilized, to which a physician or patient adds solvents and / or diluents prior to use.

[0295] The pharmaceutical compositions disclosed herein may also include pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbate palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0296] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions or formulations described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate flowability can be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0297] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. The sterilization process described above, along with the inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenolic sorbic acid, etc.), ensures the prevention of microbial presence. It may also be desirable to include exfoliating agents such as sugars and sodium chloride in the composition. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents with delayed absorption (e.g., aluminum monostearate and gelatin).

[0298] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in the pharmaceutical compositions described herein is considered, except that any conventional media or agents are incompatible with the active compound. Pharmaceutical compositions or formulations may contain preservatives or may not contain preservatives. Additional active compounds may be incorporated into the composition.

[0299] Pharmaceutical compositions or formulations must generally be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. In many cases, the composition may include isotonic agents such as sugars, polyols such as mannitol, sorbitol, or sodium chloride. Extended absorption of injectable compositions can be achieved by including agents that delay absorption, such as monostearate and gelatin.

[0300] As needed, sterile injectable solutions can be prepared by incorporating the active compound in the desired amount into a suitable solvent having one or a combination of the components listed above, followed by sterile microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and any other desired components from the components listed above. In the case of sterile powders used to prepare sterile injectable solutions, some preparation methods include vacuum drying and freeze-drying (lyophilization), which produce a powder containing the active ingredient plus any other desired components from its previously sterile filtered solution.

[0301] The amount of active ingredient that can be combined with a carrier material in the pharmaceutical compositions or formulations disclosed herein can vary. In some embodiments, the amount of active ingredient that can be combined with a carrier material is the amount that produces a therapeutic effect. Typically, in 100%, this amount will range from about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0302] The pharmaceutical compositions disclosed herein can be prepared using a carrier that protects the active ingredient from rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example... Slow-release and controlled-release drug delivery systems Systems) Edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0303] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment described herein is formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the active ingredient described herein crosses the BBB, the active ingredient may be formulated in, for example, liposomes. For methods of manufacturing liposomes, see, for example, U.S. Patents 4,522,811; 5,374,548; and 5,399,331. These liposomes may contain one or more portions that are selectively transported to specific cells or organs, thereby enhancing targeted drug delivery (see, for example, VVRanade (1989) *J. Clin. Pharmacol.* 29:685). Exemplary targeting components include folic acid or biotin (see, for example, U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem.Biophys.Res.Commun. 153:1038); antibodies (PGBloeman et al., (1995) FEBS Lett. 357:140; M. Owais et al., (1995) Antimicrobial Agents and Chemotherapy). Chemother.) 39:180); Surfactant protein A receptor (Briscoe et al. (1995) American Journal of Physiology 1233:134); and p120 (Schreier et al. (1994) Journal of Biochemistry 269:9090); see also K. Keinanen; ML Laukkanen (1994) European Federation of Biochemical Societies Letters 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0304] G. Methods and Applications

[0305] The antibody or antigen-binding fragments, compositions, and methods described herein have various in vitro and in vivo uses, relating to, for example, reducing type I IFN release via cells expressing ILT7, such as pDCs. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments described herein are humanized antibodies or antigen-binding fragments. For example, the anti-ILT7 antibody or antigen-binding fragments described herein can be administered in vitro or ex vivo to cells in cultures or to human subjects, such as in vivo, to selectively inhibit type I IFN release or inhibit pDC activity in various diseases. Therefore, this document provides a method for reducing autoimmunity in a subject, the method comprising administering the anti-ILT7 antibody or antigen-binding portion described herein to the subject such that the subject's autoimmunity associated with type I IFN or pDCs is reduced.

[0306] This disclosure also provides methods for reducing the release of cytokines (e.g., type I IFN) through cells expressing ILT7, such as pDCs, or for treating diseases or conditions associated with type I IFN-related or pDC-related diseases, such as autoimmune diseases.

[0307] In some embodiments, this document provides a method for reducing type I IFN (e.g., IFNα) in a subject of need, the method comprising administering to the subject a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment disclosed herein. In some embodiments, this document provides the use of the anti-ILT7 antibody or antigen-binding fragment disclosed herein for reducing type I IFN (e.g., IFNα). In some embodiments, this document provides the use of the anti-ILT7 antibody or antigen-binding fragment provided herein for preparing a medicament for reducing type I IFN (e.g., IFNα). In some embodiments, this document provides a method for reducing type I IFN (e.g., IFNα) in a subject of need, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, this document provides the use of the pharmaceutical composition disclosed herein for reducing type I IFN (e.g., IFNα). In some embodiments, this document provides the use of the pharmaceutical composition provided herein for preparing a medicament for reducing type I IFN (e.g., IFNα).

[0308] In some embodiments, this document provides a method for inhibiting or depleting pDC in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment disclosed herein. In some embodiments, this document provides the use of the anti-ILT7 antibody or antigen-binding fragment disclosed herein for inhibiting or depleting pDC. In some embodiments, this document provides the use of the anti-ILT7 antibody or antigen-binding fragment disclosed herein for preparing a medicament for inhibiting or depleting pDC. In some embodiments, this document provides a method for inhibiting or depleting pDC in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, this document provides the use of the pharmaceutical composition disclosed herein for inhibiting or depleting pDC. In some embodiments, this document provides the use of the pharmaceutical composition disclosed herein for preparing a medicament for inhibiting or depleting pDC. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment disclosed herein depletes pDC in a subject in need.

[0309] In some embodiments, this document provides methods for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC in a subject of need, the methods comprising administering to the subject a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment disclosed herein. In some embodiments, this document provides the use of the anti-ILT7 antibody or antigen-binding fragment disclosed herein for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, this document provides the use of the anti-ILT7 antibody or antigen-binding fragment disclosed herein for preparing a medicament for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, this document provides methods for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC in a subject of need, the methods comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, this document provides the use of the pharmaceutical composition disclosed herein for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, this document provides for the use of the pharmaceutical compositions provided herein in the preparation of a medicament for reducing autoimmunity associated with type I IFN (e.g., IFNα) or pDC.

[0310] In some embodiments, this document provides a method for treating an autoimmune disease associated with type I IFN (e.g., IFNα) or pDC in a subject of need, the method comprising administering to the subject a therapeutically effective amount of the anti-ILT7 antibody or antigen-binding fragment disclosed herein. In some embodiments, this document provides the use of the anti-ILT7 antibody or antigen-binding fragment disclosed herein for treating an autoimmune disease associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, this document provides the use of the anti-ILT7 antibody or antigen-binding fragment disclosed herein for preparing a medicament for treating an autoimmune disease associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, this document provides a method for treating an autoimmune disease associated with type I IFN (e.g., IFNα) or pDC in a subject of need, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition disclosed herein. In some embodiments, this document provides the use of the pharmaceutical composition disclosed herein for treating an autoimmune disease associated with type I IFN (e.g., IFNα) or pDC. In some embodiments, this document provides for the use of the pharmaceutical compositions provided herein in the preparation of a medicament for treating autoimmune diseases associated with type I IFN (e.g., IFNα) or pDC.

[0311] As is known in the art, dysregulation or overactivation of pDCs or the release of type I IFNs (e.g., IFNα) leads to unnecessary activation of the immune system, which is associated with the pathogenesis of various diseases such as autoimmune diseases. In some embodiments, diseases or conditions associated with pDCs or IFNα that can be treated with the anti-ILT7 antibodies or antigen-binding fragments or pharmaceutical compositions provided herein include lupus; lupus erythematosus such as systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), and discoid lupus erythematosus (DLE); and / or lupus nephritis (LN). In some embodiments, the disease or condition that can be treated with the anti-ILT7 antibodies or antigen-binding fragments or pharmaceutical compositions provided herein is lupus. In some embodiments, the disease or condition that can be treated with the anti-ILT7 antibodies or antigen-binding fragments or pharmaceutical compositions provided herein is SLE. In some embodiments, the disease or condition that can be treated with the anti-ILT7 antibodies or antigen-binding fragments or pharmaceutical compositions provided herein is CLE. In some embodiments, the disease or condition that can be treated with the anti-ILT7 antibodies or antigen-binding fragments or pharmaceutical compositions provided herein is DLE. In some embodiments, the disease or condition that can be treated with the anti-ILT7 antibody or antigen-binding fragment or pharmaceutical composition provided herein is LN.

[0312] In some embodiments, the methods provided herein facilitate a beneficial therapeutic response to an autoimmune response. In some embodiments, the methods provided herein result in improvement of disease-related symptoms, such as a decrease in IFNα levels, a decrease in the number or activity of pDCs, or a reduction in one or more other disease-related symptoms. Thus, for example, improvement in the disease can be characterized as a complete response. In some embodiments, screening techniques such as magnetic resonance imaging (MRI), X-ray imaging, computed tomography (CT), flow cytometry or fluorescence activated cell sorting (FACS) analysis, histology, gross pathology, and blood chemistry can be used to assess clinical response, including but not limited to techniques that measure changes detectable by ELISA, RIA, chromatography, etc.

[0313] The actual dose level of the active ingredient (i.e., anti-ILT7 antibody or antigen-binding fragment) in the pharmaceutical compositions described herein can be varied to obtain an amount of active ingredient effective in achieving the desired therapeutic response for a particular patient, composition, and administration mode without toxicity to the patient. The selected dose level will depend on various pharmacokinetic factors, including: the activity of the specific peptide described herein; route of administration; time of administration; excretion rate; duration of treatment; other drugs, compounds, and / or materials used in combination with the specific compound employed; the patient's age, sex, weight, condition, general health status, and prior medical history; and similar factors well known in the medical field.

[0314] Anti-ILT7 antibodies or antigen-binding fragments can be administered as sustained-release formulations, in which case a less frequent administration is required. Dosage and frequency vary depending on the half-life of the anti-ILT7 antibody or antigen-binding fragment in the patient's body. In therapeutic applications, relatively high doses at relatively short intervals are sometimes required until disease progression decreases or ceases and until the patient shows partial or complete improvement in their symptoms.

[0315] The anti-ILT7 antibody or antigen-binding fragment or pharmaceutical composition provided herein may be administered to a subject by any method known in the art, including but not limited to pleural administration, intravenous administration, subcutaneous administration, intralymphatic administration, intramuscular administration, intradermal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, spinal administration, or other parenteral administration routes, such as by injection or infusion, or direct administration to the thymus. As used herein, the phrase “parenteral administration” means a mode of administration typically administered by injection, other than enteral and local administration, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraocular, intraorbital, intracardiac, intracardiac, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. In some embodiments, subcutaneous administration is used. In some embodiments, intravenous administration is used. In some embodiments, oral administration is used. In some embodiments, the antibody or antigen-binding fragment provided herein is delivered locally. In another embodiment, the antibody or antigen-binding fragment provided herein is administered systemically.

[0316] In the methods disclosed herein, a therapeutically effective amount of the disclosed anti-ILT7 antibody or antigen-binding fragment or pharmaceutical composition is administered to a subject who may benefit from a reduction in IFNα levels. The subject may have unwanted, unregulated, or excessive pDC activation. The subject may be a mammal. In some embodiments, the subject is a human.

[0317] The anti-ILT7 antibodies, antigen-binding fragments, or pharmaceutical compositions provided herein can be administered using medical devices known in the art. For example, in some embodiments, needle-free subcutaneous injection devices, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556, can be used. Examples of well-known implants and modules used in this document include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses a permeable drug delivery system with multiple chambers; and U.S. Patent No. 4,475,196, which discloses a permeable drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0318] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment or pharmaceutical composition provided herein is administered in combination with additional therapies. These additional therapies may be administered before, simultaneously with, or after administration of the anti-ILT7 antibody or antigen-binding fragment, cells, or pharmaceutical composition described herein. Combination administration may include co-administration with a single pharmaceutical formulation or using individual formulations, or in any order but typically sequentially over a period of time, such that all active agents can exert their biological activity simultaneously. Those skilled in the art can readily determine appropriate regimens for administering the pharmaceutical compositions and combinations described herein, including the timing and dosage of additional agents for the combination therapy, based on the needs of the treated subject.

[0319] The antibody or antigen-binding fragments provided herein can also be used to detect ILT7. Methods for detecting the presence of human ILT7 antigen in a sample or measuring the amount of human ILT7 antigen are also covered, the methods comprising contacting a sample and a control sample with a monoclonal antibody, such as a humanized monoclonal antibody or its antigen-binding moiety, that specifically binds to human ILT7, under conditions allowing the formation of a complex between the antibody or antigen-binding fragment and human ILT7. The formation of the complex is then detected, wherein differential complex formation between the sample and the control sample indicates the presence of human ILT7 antigen in the sample. Furthermore, the anti-ILT7 antibody or antigen-binding fragments described herein can be used to purify human ILT7 by immunoaffinity purification. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments described herein are used to detect ILT7-expressing cells such as pDCs. In some embodiments, the anti-ILT7 antibody or antigen-binding fragments described herein are used to quantify ILT7 antigen or ILT7-expressing cells.

[0320] H. Illustrated Examples

[0321] Example 1: An antibody or antigen-binding fragment thereof specifically binds to human ILT7, the antibody or antigen-binding fragment comprising: (1) as defined by Kabat, (a) a light chain variable region (VL) comprising VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences of SEQ ID NO: 11, 12 and 13 respectively; or variants thereof having at most about 5 amino acid substitutions, additions and / or deletions in the VL CDRs; and / or (b) a heavy chain variable region (VH) comprising VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 14, 15 and 16 respectively; or variants thereof having at most about 5 amino acid substitutions, additions and / or deletions in the VH CDRs; or (2) as defined by Chothia, (a) a VL comprising ... at most 5 amino acid sequences of SEQ ID NO: 14, 15 and 16 respectively; and (c) a heavy chain variable region (VH) comprising VL CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 14, 15 and 16 respectively; and (c) a heavy chain variable region (VH) comprising VL CDR1, VH CDR2 and VH CDR3 having amino acid sequences of SEQ ID NO: 14, 15 and 16 respectively; and (c) a heavy chain variable region (VH) comprising VL CDR1 VL CDR1, VL CDR2, and VL CDR3 having amino acid sequences of SEQ ID NO: 11, 12, and 13; or variants thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs; and / or (b) VH comprising VH CDR1, VH CDR2, and VH CDR3 having amino acid sequences of SEQ ID NO: 17, 18, and 16, respectively; or variants thereof having up to about 5 amino acid substitutions, additions, and / or deletions in the VL CDRs.

[0322] Example 2: The antibody or antigen-binding fragment according to Example 1 comprises VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VHCDR3 having amino acid sequences of SEQ ID NO: 11, 12, 13, 14, 15 and 16, respectively, as defined by Kabat.

[0323] Example 3: The antibody or antigen-binding fragment according to Example 1 comprises VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VHCDR3 having amino acid sequences of SEQ ID NO: 11, 12, 13, 17, 18 and 16, respectively, as defined by Chothia.

[0324] Example 4: An antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment specifically binding to human ILT7, said antigen or antigen-binding fragment comprising: (a) VL, said VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:9; and / or (b) VH, said VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:10.

[0325] Example 5: The antibody or antigen-binding fragment according to Example 4 comprises VL and VH having amino acid sequences of SEQ ID NO:9 and 10, respectively.

[0326] Example 6: An antibody or antigen-binding fragment thereof specifically binds to human ILT7, wherein the antigen or antigen-binding fragment comprises: (a) VL, wherein the VL comprises VL CDR1, VL CDR2 and VL CDR3 from the amino acid sequence of VL having SEQ ID NO:9; and / or (b) VH, wherein the VH comprises VH CDR1, VH CDR2 and VH CDR3 from the amino acid sequence of VH having SEQ ID NO:10.

[0327] Example 7: An antibody or antigen-binding fragment according to any one of Examples 1 to 6, wherein the antibody or antigen-binding fragment is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment.

[0328] Example 8: An antibody or antigen-binding fragment according to Example 7, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment.

[0329] Example 9: An antibody or antigen-binding fragment according to Example 8, comprising: (a) VL, wherein the VL has at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of any of SEQ ID NO:19-22; and / or (b) VH, wherein the VH has at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of any of SEQ ID NO:23-28.

[0330] Example 10: An antibody or antigen-binding fragment according to Example 9, comprising: (a) VL, wherein the VL has at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:19; and (b) VH, wherein the VH has at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:26.

[0331] Example 11: An antibody or antigen-binding fragment according to Example 9, comprising: (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:19, as defined by Kabat or Chothia, the VL comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO:11, 12, and 13, respectively; and (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:26, the VH comprising: (1) VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO:14, 15, and 16, respectively, as defined by Kabat; or (2) VH CDR3 having the amino acid sequences of SEQ ID NO:14, 15, and 16, respectively, as defined by Chothia; The amino acid sequences of NO:17, 18 and 16 are VH CDR1, VH CDR2 and VH CDR3.

[0332] Example 12: The antibody or antigen-binding fragment according to Example 9 comprises VL and VH having the following amino acid sequences respectively: (1) SEQ ID NO: 19 and 23; (2) SEQ ID NO: 19 and 24; (3) SEQ ID NO: 19 and 25; (4) SEQ ID NO: 19 and 26; (5) SEQ ID NO: 19 and 27; (6) SEQ ID NO: 19 and 28; (7) SEQ ID NO: 20 and 23; (8) SEQ ID NO: 20 and 24; (9) SEQ ID NO: 20 and 25; (10) SEQ ID NO: 20 and 26; (11) SEQ ID NO: 20 and 27; (12) SEQ ID NO: 20 and 28; (13) SEQ ID NO: 21 and 23; (14) SEQ ID NO: 21 and 24; (15) SEQ ID NO: 21 and 25; (16) SEQ ID NO: 21 and 25; (17) SEQ ID NO:21 and 27; (18) SEQ ID NO:21 and 28; (19) SEQ ID NO:22 and 23; (20) SEQ ID NO:22 and 24; (21) SEQ ID NO:22 and 25; (22) SEQ ID NO:22 and 26; (23) SEQ ID NO:22 and 27; or (24) SEQ ID NO:22 and 28.

[0333] Example 13: An antibody or antigen-binding fragment according to Example 12, comprising: VL having the amino acid sequence of SEQ ID NO:19; and VH having the amino acid sequence of SEQ ID NO:26.

[0334] Example 14: An antibody or antigen-binding fragment according to any one of Examples 1 to 13, wherein the antibody or antigen-binding fragment is Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single-domain antibody (sdAb) or a heavy-chain antibody (HCAb).

[0335] Example 15: An antibody or antigen-binding fragment according to any one of Examples 1 to 13, wherein the antibody or antigen-binding fragment is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0336] Example 16: An antibody or antigen-binding fragment according to Example 15, wherein the antibody is an IgG1 antibody.

[0337] Example 17: An antibody or antigen-binding fragment according to Example 16, comprising a light chain constant region (CL) having at least 85% sequence identity with κCL (Cκ; SEQ ID NO:29).

[0338] Example 18: An antibody or antigen-binding fragment according to Example 16, comprising a light chain constant region (CL) having at least 85% sequence identity with λCL (Cλ; SEQ ID NO: 30).

[0339] Example 19: An antibody or antigen-binding fragment according to Example 16, comprising a heavy chain constant region (CH) having at least 85% sequence identity with the amino acid sequence of any of SEQ ID NO:31 and 40-44.

[0340] Example 20: An antibody or antigen-binding fragment according to any one of Examples 16 to 19, wherein the heavy chain constant region (CH) of the IgG1 antibody comprises wild-type IgG1 CH, or comprises at least one amino acid mutation that enhances the antibody’s ADCC (antibody-dependent cytotoxicity) or ADCP (antibody-dependent phagocytosis).

[0341] Example 21: An antibody or antigen-binding fragment according to Example 20, wherein the CH region of the IgG1 antibody has an amino acid substitution at L234, L235, G236, S239, F243, H268, D270, R292, S298, Y300, V305, K326, A330, I332, E333, K334, P396 or any combination thereof according to EU index numbers.

[0342] Example 22: An antibody or antigen-binding fragment according to Example 20, wherein the CH region of the IgG1 antibody has an amino acid substitution, wherein the amino acid substitution is L234Y, L235Q, L235V, G236A, G236W, S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330M, A330L, I332E, E333A, K334A, K334E or P396L or any combination thereof, according to EU index numbers.

[0343] Example 23: An antibody or antigen-binding fragment according to Example 20, wherein the CH region of the IgG1 antibody is modified with amino acid substitutions, the amino acid substitutions being the following according to EU index numbers: (i) S298A, E333A, and K334A; (ii) S239D and I332E; (iii) S239D, A330L, and I332E; (iv) G236A; (v) G236A, S239D, and I332E; (vi) G236A, A330L, and I332E; (vii) G236A, S239D, A330L and I332E; (viii) F243L, R292P, Y300L, V305I and P396L; (ix) L235V, F243L, R292P, Y300L and P396L; (x) L234Y, L235Q, G236W, S239M, H268D, D270E and S298A; or (xi) D270E, K326D, A330M and K334E.

[0344] Example 24: An antibody or antigen-binding fragment according to Example 23, wherein the CH region has an amino acid sequence selected from the group consisting of SEQ ID NO:45-64.

[0345] Example 25: An antibody or antigen-binding fragment according to Example 1, comprising: VL having the amino acid sequence of SEQ ID NO:19; VH having the amino acid sequence of SEQ ID NO:26; and CH having the amino acid sequence of SEQ ID NO:55.

[0346] Example 26: An antibody or antigen-binding fragment according to Example 1, comprising: VL having the amino acid sequence of SEQ ID NO:19; VH having the amino acid sequence of SEQ ID NO:26; CL having the amino acid sequence of SEQ ID NO:30; and CH having the amino acid sequence of SEQ ID NO:55.

[0347] Example 27: An antibody or antigen-binding fragment according to any one of Examples 16 to 26, wherein the Fc region of the IgG1 antibody is defucosylated.

[0348] Example 28: An antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment competing with an antibody or antigen-binding fragment according to any one of Examples 1 to 27 for binding to human ILT7.

[0349] Example 29: An antibody or antigen-binding fragment according to any one of Examples 1 to 28, wherein the antibody or antigen-binding fragment is a bispecific antibody or a multispecific antibody.

[0350] Example 30: An antibody or antigen-binding fragment according to any one of Examples 1 to 29, wherein the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment.

[0351] Example 31: An antibody or antigen-binding fragment according to any one of Examples 1 to 30, wherein the antibody or antigen-binding fragment: (1) binds to human ILT7 at a KD of 500 nM or less as determined by SPR; (2) does not bind to LILR family members LILRA1, LILRA2 / ILT1, LILRA3 / ILT6, LILRA5 / ILT11, LILRA6 / ILT8, LILLRB1 / ILT2, LILLRB2 / ILT4, LILLRB3 / ILT5, LILLRB4 / ILT3 or LILLRB4. (3) LB5-specific binding; (4) inhibition of interferon-α (IFNα) release via peripheral blood mononuclear cells (PBMCs); (5) selective binding to plasmacytoid dendritic cells (pDCs) in human PBMCs; (6) exhibiting natural killer cell (NK)-dependent ADCC activity against ILT7-expressing cells; (7) exhibiting neutrophil-dependent ADCC activity against ILT7-expressing cells; or (8) exhibiting macrophage-dependent ADCP activity against ILT7-expressing cells; or any combination of (1)-(7).

[0352] Example 32: An antibody or antigen-binding fragment thereof specifically binds to the protease domain of human ILT7, wherein the antibody or antigen-binding fragment: (1) binds to human ILT7 at a KD of 500 nM or less as determined by SPR; (2) does not bind to LILR family members LILRA1, LILRA2 / ILT1, LILRA3 / ILT6, LILRA5 / ILT11, LILRA6 / ILT8, LILRB1 / ILT2, LILRB2 / ILT4, ... (3) Specifically binds to LILRB3 / ILT5, LILRB4 / ILT3 or LILB5; (4) Inhibits IFNα release via PBMCs; (5) Selectively binds to pDCs in human PBMCs; (6) Exhibits NK-dependent ADCC activity against ILT7-expressing cells; (7) Exhibits neutrophil-dependent ADCC activity against ILT7-expressing cells; or (8) Exhibits macrophage-dependent ADCP activity against ILT7-expressing cells; or any combination of (1)-(7).

[0353] Example 33: An antibody or antigen-binding fragment according to Example 31 or 32, wherein the antibody or antigen-binding fragment (1) inhibits IFNα release from CpG-stimulated PBMCs in vitro with an EC50 of 1 nM or less; (2) exhibits NK-dependent ADCC activity against ILT7-expressing cells with an EC50 of 0.01 nM or less; (3) exhibits neutrophil-dependent ADCC activity against ILT7-expressing cells with an EC50 of 100 nM or less; (4) exhibits macrophage-dependent ADCP activity against ILT7-expressing cells with an EC50 of 10 nM or less; or (5) exhibits macrophage-dependent ADCP activity against ILT7-expressing cells with a maximum phagocytic index of 20% or higher; or any combination of (1)-(5).

[0354] Example 34: An antibody or antigen-binding fragment according to Example 33, wherein the antibody or antigen-binding fragment (1) inhibits IFNα release via PBMCs with an EC50 ranging from 0.01 nM to 0.1 nM; (2) exhibits NK-dependent ADCC activity against ILT7-expressing cells with an EC50 ranging from 0.001 nM to 0.01 nM; (3) exhibits neutrophil-dependent ADCC activity against ILT7-expressing cells with an EC50 ranging from 1 nM to 50 nM; (4) exhibits macrophage-dependent ADCP activity against ILT7-expressing cells with an EC50 ranging from 0.5 nM to 5 nM; or (5) exhibits macrophage-dependent ADCP activity against ILT7-expressing cells with a maximum phagocytic index ranging from 20% to 80%; or any combination of (1)-(5).

[0355] Example 35: An antibody or antigen-binding fragment according to any one of Examples 31 to 34, wherein the antigen or antigen-binding fragment exhibits neutrophil-dependent ADCC activity.

[0356] Example 36: A polynucleotide encoding a polypeptide of an antibody or antigen-binding fragment according to any one of Examples 1 to 35.

[0357] Example 37: A vector comprising the polynucleotide as described in Example 36.

[0358] Example 38: A host cell comprising the polynucleotide described in Example 36 or the vector described in Example 37.

[0359] Example 39: The host cell according to Example 38, wherein the host cell (1) overexpresses N-acetylglucosamine transferase III (GnTIII); (2) lacks α-1,6-fucosyltransferase (FUT8); or (3) has a low fucose content; or any combination of (1)-(3).

[0360] Example 40: A method for preparing an antibody or antigen-binding fragment thereof that specifically binds to human ILT7, the method comprising culturing host cells as described in Example 38 or 39 in a culture under conditions that allow expression of the antibody or antibody fragment.

[0361] Example 41: The method according to Example 40 further comprises isolating the antibody from the culture.

[0362] Example 42: A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of Examples 1 to 35, and a pharmaceutically acceptable carrier.

[0363] Example 43: A method for reducing type I interferon (IFN) in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of Examples 1 to 35.

[0364] Example 44: The method according to Example 43, wherein the type I interferon is IFNα.

[0365] Example 45: A method for inhibiting or depleting pDC in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of Examples 1 to 35.

[0366] Example 46: A method for reducing autoimmunity in a subject in need, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment according to any one of Examples 1 to 35.

[0367] Example 47: The method according to any one of Examples 43 to 46, wherein the subject suffers from an autoimmune disease.

[0368] Example 48: A method for treating an autoimmune disease associated with type I IFN or pDC in a subject of need, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of Examples 1 to 35.

[0369] Example 49: The method according to Example 47 or 48, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

[0370] Example 50: The method according to any one of Examples 43 to 49, further comprising administering additional therapy to the subject.

[0371] Example 51: The method according to any one of Examples 43 to 50, wherein the subject is a human.

[0372] Example 52: Use of an antibody or antigen-binding fragment according to any one of Examples 1 to 35 for reducing type I IFN.

[0373] Example 53: Use of an antibody or antigen-binding fragment according to any one of Examples 1 to 35 for the preparation of a medicament for reducing type I IFN.

[0374] Example 54: The use according to Example 52 or 53, wherein the type I IFN is IFNα.

[0375] Example 55: Use of an antibody or antigen-binding fragment according to any one of Examples 1 to 35 for inhibiting or depleting pDC.

[0376] Example 56: Use of an antibody or antigen-binding fragment according to any one of Examples 1 to 35 for the preparation of a medicament for inhibiting or depleting pDC.

[0377] Example 57: Use of an antibody or antigen-binding fragment according to any one of Examples 1 to 35 for reducing autoimmunity.

[0378] Example 58: Use of an antibody or antigen-binding fragment according to any one of Examples 1 to 35 for the preparation of a medicament for reducing autoimmunity.

[0379] Example 59: Use of an antibody or antigen-binding fragment according to any one of Examples 1 to 35 for the treatment of autoimmune diseases associated with type I IFN or pDC.

[0380] Example 60: Use of an antibody or antigen-binding fragment according to any one of Examples 1 to 35 for the preparation of a medicament for the treatment of autoimmune diseases associated with type I IFN or pDC.

[0381] Example 61: The use according to Example 59 or 60, wherein the autoimmune disease is SLE.

[0382] Unless otherwise indicated, the practice of this invention employs conventional techniques in molecular biology, cell biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields of expertise. These techniques are described in the references cited herein and are fully explained therein. See, for example, Maniatis et al. (1982), *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001), *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., *Contemporary Guide to Molecular Biology Experiments* (C URRENT P ROTOCOLS IN M OLECULAR B IOLOGY ), John Wiley & Sons (1987 and annual updates); *The Laboratory Manual of Contemporary Immunology* (C) URRENT P ROTOCOLSIN I MMUNOLOGY ), John Willie & Son Publishing Company (1987 and annual updates) Gait (ed.) (1984) "Oligonucleotide Synthesis: Practical Methods" LIGONUCLEOTIDE S YNTHESIS :AP RACTICAL A PPROACH )》, IRL Press; Eckstein (ed.) (1991) "Oligonucleotides and Analogs: Practical Methods" LIGONUCLEOTIDES AND A NALOGUES :AP RACTICAL A PPROACH ) , IRL Publishing; Birren et al. (editors) (1999) "Genome Analysis: A Laboratory Manual (G ENOME A NALYSIS :AL ABORATORY M ANUAL ), Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995) "Antibody Engineering (A)" NTIBODY E NGINEERING ), 2nd edition, Oxford University Press; Lo (ed.) (2006) Antibody Engineering: Methods and Protocols (Molecular Biology Approaches) (ANTIBODY E NGINEERING :M ETHODS AND P ROTOCOLS (M ETHODS IN M OLECULAR B IOLOGY Volume 248, Humana Press, Inc.; Each of the references cited herein is incorporated herein by reference in its entirety.

[0383] Example

[0384] The examples provided below are for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the invention should in no way be construed as limited to the examples below, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0385] In summary, the results from the study described below demonstrate that cmAb12 and its humanized version bind to human ILT7 with high affinity and are more effective than the benchmark antibody dastardilimab (“Tab1”; heavy chain: SEQ ID NO:38; and light chain: SEQ ID NO:39) in blocking IFNα release and inhibiting pDC.

[0386] Example 1: Immunization and Antibody Screening

[0387] method To generate anti-ILT7 antibodies, Balb / c and SJL mice were immunized using three different strategies. First, human ILT7-his protein was generated by fusing a his6 tag to the N-terminus of the extracellular domain of human ILT7 (amino acids 24-446 of SEQ ID NO:1). Second, a vector containing a sequence encoding full-length human ILT7 (SEQ ID NO:5) linked to a sequence encoding hFcεRiγ (SEQ ID NO:7) was generated and used for gene immunization and lentiviral packaging. Third, HEK-293F cells were infected with lentivirus to generate stable clones of full-length human ILT7, thus obtaining 293F-human ILT7 cells. All materials mentioned above were used as immunogens. Specifically, the human ILT7-his protein was administered via hock injection. Serum titers from the first round of screening were examined, and antibodies from mice with a strong immune response to ILT7 were selected for hybridoma generation. The binding affinity of the selected monoclonal antibodies to ILT7 was then rescreened using FACS and ELISA, and their activity in reducing IFNα release was further screened using an IFNα release assay.

[0388] Example 2: Generation and Characterization of Chimeric Antibodies

[0389] method Twenty-five antibodies were expressed and purified from hybridoma screening. Vectors expressing the heavy and light chains of chimeric antibodies were constructed. The heavy chain expression vector contained the coding sequence of the variable domain of the heavy chain of the chimeric antibody, which was linked to the coding sequence of the constant region of the human IgG1 heavy chain. Similarly, the light chain expression vector contained the coding sequence of the variable domain of the light chain of the chimeric antibody and the coding sequence of the constant region of the κ light chain.

[0390] Example 3: Binding of cmAb12 and ILT7 as measured by ELISA

[0391] method The binding of chimeric anti-ILT7 antibodies (including cmAb12) identified from screening to human ILT7-his protein was measured by ELISA, along with the isotype antibody hIgG1 as a negative control and the reference anti-ILT7 antibody Tab1. A 96-well ELISA plate was coated overnight at 4°C with PBS (2 μg / ml) containing human ILT7-his. The chimeric antibody was added to the wells and incubated with the coated protein at 37°C for 1 hour, followed by the addition of secondary HRP-labeled antibody. After incubation at 37°C for 1 hour, TMB substrate was added to the wells and incubated at room temperature for 15 minutes. The reaction was terminated by adding 1N HCl. The absorbance at 450 nm was measured using a microplate reader.

[0392] Results and Conclusions :like Figure 1 As shown, cmAb12 has a high affinity (0.07513 nM EC50) for EC50. 50 It binds to the human ILT7 protein.

[0393] Example 4: Binding of cmAb12 to ILT7-expressing cells as measured by flow cytometry

[0394] method The binding affinity of chimeric anti-ILT7 antibodies (including cmAb12) identified from screening to human ILT7 or cynomolgus monkey ILT7 expressed on the cell membrane was measured by flow cytometry. Vectors containing the coding sequences of full-length cynomolgus monkey ILT7 (SEQ ID NO:6) and hFcεRiγ (SEQ ID NO:7) were generated. The construction of the CHOK1-cynomolgus monkey ILT7 stable cell line was similar to that of 293F-human ILT7 cells. Both cell lines were seeded, resuspended, and incubated with the chimeric antibody at 4°C for 1 hour. Cells were then washed with cold FACS buffer (PBS + 2% FBS), resuspended, and incubated with the secondary antibody at 4°C for 1 hour. hIgG1 and Tab1 were used as controls.

[0395] Results and conclusions :like Figure 2A-2BThe concentration-MFI ("median fluorescence intensity") curves show that cmAb12 interacts with 293F-human ILT7 cells in a dose-dependent manner. Figure 2A ) and CHOK1-cynomolgus monkey ILT7 cells ( Figure 2B () binding. cmAb12 on EC50 cells of 293F-human ILT7 cells. 50 The concentration was 0.3121 nM, and it was effective against ECGs in CHOK1-cynomolgus monkey ILT7 cells. 50 It is 0.9385 nM.

[0396] Example 5: cmAb12 lacks cross-reactivity with other LILR family members.

[0397] method The binding specificity of chimeric antibodies (including cmAb12) to ILT7, one of the 10 associated LILR superfamily members, was assessed by ELISA. Plates coated with 1 μg / ml LILR superfamily proteins were incubated with the chimeric antibody at 37°C for 1 hour, followed by incubation with the secondary antibody. Detailed OD450 measurement results are listed in Table 4.

[0398] Results and Conclusions As shown in Table 4, cmAb12 does not specifically bind to other LILR family members (its affinity is comparable to that of the IgG negative control), indicating that it has high specificity for ILT7.

[0399] Table 4. Cross-reactivity among LILR family members

[0400]

[0401] Example 6: Blocking CpG-induced IFNα production in human PBMCs by cmAb12

[0402] method The activity of the anti-ILT7 chimeric antibody (including cmAb12) disclosed in this paper in reducing IFNα release was tested by an IFNα release assay. Briefly, PBMC cells (Allcells, frozen) were seeded in assay medium (1640 medium + 10% FBS) containing 200 ng / ml IL-2 and pre-incubated with the antibody at 4°C and 5% CO2 for 6 hours as instructed. ODN2216 solution (DNA containing CpG) was then added, and the cells were further incubated for 18 hours. The cell culture supernatant was then harvested, and IFNα levels were measured using the human IFNα pan ELISA development kit (HRP).

[0403] Results and Conclusions :like Figure 3As shown, cmAb12 inhibits IFNα release from PBMCs stimulated by CpG in a dose-dependent manner.

[0404] Example 7: Tabletop Sub-bins

[0405] method Epitope binning of the anti-ILT7 antibodies (including cmAb12) disclosed in this paper was performed using competitive FACS. Specifically, CHOK1 cells expressing human ILT7 were incubated together with an equal volume of reference antibody (Tab1-Alexa488) and the antibody to be tested at 4°C for 1 hour. The allotype antibody hIgG1 was used as a negative control. Cells were washed with PBS, and the relative MFI of different fluorescence signals was analyzed by flow cytometry.

[0406] The relative inhibition rate of each tested antibody against Tab1 was calculated as follows: [F(hIgG) - F(tested antibody)] * 100% / F(hIgG). F(hIgG) refers to the signal intensity of Tab1-Alexa488 in the presence of hIgG; F(tested antibody) refers to the signal intensity of Tab1-Alexa488 in the presence of the tested antibody. Therefore, the positive inhibition rate indicates that the tested antibody and Tab1 competitively bind to the same or overlapping epitopes on ILT7, which are grouped into bins. The results of the epitope binning analysis are shown in Table 5.

[0407] Results and Conclusions As shown in Table 5, the binding of Tab1-Alexa488 does not affect the binding of cmAb12, indicating that cmAb12 and Tab1 bind to different epitopes on human ILT7.

[0408] Table 5. Pallet bins measured by competitive FACS

[0409]

[0410]

[0411] Example 8: Humanization of cmAb12

[0412] methodFor the humanization of cmAb12, IgBLAST from NCBI was used to select the most appropriate human frame for transplanting rodent CDRs. Variable regions with high amino acid sequence identity (homology match or best fit) with rodent variable regions were used. cmAb12 was humanized by transplanting three VL CDRs into human VLs that were as homologous as possible to mouse VLs. Similarly, three VH CDRs were transplanted into human VHs that were as homologous as possible to mouse VHs. Kabat and Chothia numbering was then performed. Furthermore, the sequences of exemplary humanized antibodies are listed in Table 3. The binding and function of the humanized antibodies from cmAb12 were evaluated using the same experimental procedures described above.

[0413] Results and Conclusions :like Figures 4A-4B As shown, all humanized cmAb12 antibodies against 293F-human ILT7 cells ( Figure 4A ) and CHOK1-cynomolgus monkey ILT7 cells ( Figure 4B Maintain high binding affinity.

[0414] Example 9: CpG-induced IFNα production by a humanized antibody blocking cmAb12 in human PBMCs.

[0415] method The function of four humanized cmAb12 antibodies, cmAb12, positive control antibody (Tab1), and negative control antibody (hIgG1) in blocking CpG-induced IFNα secretion was evaluated under the same experimental procedures as described in Example 6.

[0416] Results and Conclusions :like Figure 5A and 5B As shown, four humanized antibodies against cmAb12 dose-dependently inhibited IFNα secretion in CpG-stimulated PBMCs.

[0417] Example 10: Binding of hu-cmAb12 to ILT7 as measured by Biacore and ELISA.

[0418] methodThe binding affinity of the humanized cmAb12 antibody hu-cmAb12 was further measured using surface plasmon resonance (SPR) technology with a Biacore 8K. As used herein, “hu-cmAb12” refers to a specific antibody clone variant of the Hu12-04 antibody of Examples 8 and 9. The hu-cmAb12 antibody comprises: VL, having the amino acid sequence of SEQ ID NO:19; VH, having the amino acid sequence of SEQ ID NO:26; and CH, having the amino acid sequence of SEQ ID NO:55. Hu-cmAb12 or Tab1 was immobilized on a CM-5 chip. Assays were performed at 25°C with a run buffer of 1×HEPES (10 mM HEPES, 150 mM NaCl, 3 mM EDTA) containing 0.005% Tween-20, pH 7.4. The diluted antibody was captured on the sensor chip by an Fc capture method. Human ILT7-his was used as the analyte, and the run buffer was used as the dissociation phase. Similarly, the ELISA was performed as described above. An isotype control antibody and a reference anti-ILT7 antibody, Tab1, were used as controls.

[0419] Results and Conclusions The KD (data not shown) of the hu-cmAb12 antibody binding to human ILT7, as measured by Biacore, was 111 nM, and the EC50 was 0.07911 μg / ml, as measured by ELISA. 50 ( Figure 6 Both are equivalent to Tab1.

[0420] Example 11: Binding of hu-cmAb12 to ILT7-expressing cells as measured by flow cytometry

[0421] method CHOK1-cynomolgus monkey ILT7 cells and 293F-human ILT7 cells were seeded, resuspended in a solution containing hu-cmAb12 antibody, and incubated at 4°C for 1 hour. Cells were washed with cold FACS buffer (PBS + 2% FBS) by centrifugation, then secondary antibody was added, and incubated at 4°C for 1 hour. Allotype control antibody and reference anti-ILT7 antibody Tab1 were used as negative and positive controls, respectively. The FACS concentration-MFI curve was then determined.

[0422] Results and Conclusions Representative results in Figures 7A-7B The figure shows the results of interaction with 293F-human ILT7 cells (…). Figure 7A ) and CHOK1-cynomolgus monkey ILT7 cells ( Figure 7B EC50 of 0.3434 nM and 1.649 nM of the bound hu-cmAb12 antibody 50 Equivalent to Tab1.

[0423] Example 12: Binding of hu-cmAb12 with human PBMCs

[0424] method The hu-cmAb12 antibody was conjugated with the fluorescent dye Alexa488, and PBMC cells (Auscelles, frozen) were seeded in assay medium (1640 medium + 10% FBS). All cells were first stained with live / dead dyes, and then the specific binding of hu-cmAb12 was detected in T cells, B cells, natural killer cells, natural killer T cells, monocytes, and plasmacytoid dendritic cells. Specifically, plasmacytoid dendritic cells were identified as having low CD11c, HLA-DR positivity, and CD123 positivity.

[0425] Results and Conclusions Representative results in Figure 8 Provided in [the text]. As shown in the figure, similar to Tab1, the hu-cmAb12 antibody specifically binds to pDCs in human PBMCs, but does not bind to T cells, B cells, NK cells, ...

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human ILT7, the antibody or antigen-binding fragment comprising: (1) as defined by Kabat, (a) a light chain variable region (VL) comprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions and / or deletions in the VL CDRs; and / or (b) a heavy chain variable region (VH) comprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions and / or deletions in the VH CDRs; or (2) as defined by Chothia, (a) a VL comprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions and / or deletions in the VL CDRs; and / or (b) a VH comprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 17, 18, and 16, respectively; or a variant thereof having up to about 5 amino acid substitutions, additions and / or deletions in the VL CDRs.

2. The antibody or antigen-binding fragment of claim 1, comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 11, 12, 13, 14, 15, and 16, respectively, as defined by Kabat.

3. The antibody or antigen-binding fragment of claim 1, comprising a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 11, 12, 13, 17, 18, and 16, respectively, as defined by Chothia.

4. An antibody or antigen-binding fragment thereof that specifically binds to human ILT7, the antibody or antigen-binding fragment comprising: (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9; and / or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:

10.

5. The antibody or antigen-binding fragment of claim 4, which comprises a VL and a VH having the amino acid sequences of SEQ ID NOs: 9 and 10, respectively.

6. An antibody or antigen-binding fragment thereof that specifically binds to human ILT7, comprising (a) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 from a VL having the amino acid sequence of SEQ ID NO: 9; and / or (b) a VH comprising a VH CDR1, a VH CDR2, and a VH CDR3 from a VH having the amino acid sequence of SEQ ID NO:

10.

7. The antibody or antigen-binding fragment of any one of claims 1-6, wherein the antibody or antigen-binding fragment is a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a human antibody or antigen-binding fragment.

8. The antibody or antigen-binding fragment of claim 7, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment.

9. The antibody or antigen-binding fragment of claim 8, comprising: (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 19-22; and / or (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23-28.

10. The antibody or antigen-binding fragment of claim 9, comprising: (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19; and (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:

26.

11. The antibody or antigen-binding fragment of claim 9, comprising: (a) a VL having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19, which comprises a VL CDR1, a VL CDR2, and a VL CDR3 having the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively, as defined by Kabat or Chothia; and (b) a VH having at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26, which comprises: (1) a VH CDR1, a VH CDR2, and a VH CDR3 having the amino acid sequences of SEQ ID NOs: 27, 28, and 29, respectively, as defined by Kabat, VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively; or (2) VH CDR1, VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 17, 18, and 16, respectively, as defined by Chothia.

12. The antibody or antigen-binding fragment of claim 9, which comprises a VL and a VH having the amino acid sequences of: (1) SEQ ID NOs: 19 and 23; (2) SEQ ID NOs: 19 and 24; (3) SEQ ID NOs: 19 and 25; (4) SEQ ID NOs: 19 and 26; (5) SEQ ID NOs: 19 and 27; (6) SEQ ID NOs: 19 and 28; (7) SEQ ID NOs: 20 and 23; (8) SEQ ID NOs: 20 and 24; (9) SEQ ID NOs: 20 and 25; (10) SEQ ID NOs: 20 and 26; (11) SEQ ID NOs: 20 and 27; (12) SEQ ID NOs: 20 and 28; (13) SEQ ID NOs: 21 and 23; (14) SEQ ID NOs: 21 and 24; (15) SEQ ID NOs: 21 and 25; (16) SEQ ID NOs: 21 and 26; (17) SEQ ID NOs: 21 and 27; (18) SEQ ID NOs: 21 and 28; (19) SEQ ID NOs: 22 and 23; (20) SEQ ID NOs: 22 and 24; (21) SEQ ID NOs: 22 and 25; (22) SEQ ID NOs: 22 and 26; (23) SEQ ID NOs: 22 and 27; or (24) SEQ ID NOs: 22 and 28, respectively.

13. The antibody or antigen-binding fragment of claim 12, which comprises a VL and a VH having the amino acid sequences of SEQ ID NOs: 19 and 26, respectively.

14. The antibody or antigen-binding fragment of any one of claims 1 to 13, wherein the antibody or antigen-binding fragment is a Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, single-domain antibody (sdAb), or heavy-chain antibody (HCAb).

15. The antibody or antigen-binding fragment of any one of claims 1 to 13, wherein the antibody or antigen-binding fragment is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

16. The antibody or antigen-binding fragment of claim 15, wherein the antibody is an IgG1 antibody.

17. The antibody or antigen-binding fragment of claim 16, which comprises a light chain constant region (CL) having at least 85% sequence identity to kappa CL (CK; SEQ ID NO: 29).

18. The antibody or antigen-binding fragment of claim 16, comprising a light chain constant region (CL) having at least 85% sequence identity to lambda CL (C lambda; SEQ ID NO: 30).

19. The antibody or antigen-binding fragment of claim 16, comprising a heavy chain constant region (CH) having at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 31 and 40-44.

20. The antibody or antigen-binding fragment of any one of claims 16-19, wherein the heavy chain constant region (CH) of the IgGl antibody comprises a wild-type IgGl CH, or comprises at least one amino acid mutation that enhances ADCC (antibody-dependent cellular cytotoxicity) or ADCP (antibody-dependent cellular phagocytosis) of the antibody.

21. The antibody or antigen-binding fragment of claim 20, wherein the CH region of the IgGl antibody has an amino acid substitution at L234, L235, G236, S239, F243, H268, D270, R292, S298, Y300, V305, K326, A330, I332, E333, K334, P396, or any combination thereof, numbered according to the EU index.

22. The antibody or antigen-binding fragment of claim 20, wherein the CH region of the IgGl antibody has an amino acid substitution that is L234Y, L235Q, L235V, G236A, G236W, S239D, S239M, F243L, H268D, D270E, R292P, S298A, Y300L, V305I, K326D, A330M, A330L, I332E, E333A, K334A, K334E, or P396L, or any combination thereof, numbered according to the EU index.

23. The antibody or antigen-binding fragment of claim 20, wherein the CH region of the IgGl antibody is modified by an amino acid substitution that is (i) S298A, E333A, and K334A; (ii) S239D and I332E; (iii) S239D, A330L, and I332E; (iv) G236A; (v) G236A, S239D, and I332E; (vi) G236A, A330L, and I332E; (vii) G236A, S239D, A330L, and I332E; (viii) F243L, R292P, Y300L, V305I, and P396L; (ix) L235V, F243L, R292P, Y300L, and P396L; (x) L234Y, L235Q, G236W, S239M, H268D, D270E, and S298A; or (xi) D270E, K326D, A330M, and K334E, numbered according to the EU index.

24. The antibody or antigen-binding fragment according to claim 23, wherein the CH region has the amino acid sequence of any one of SEQ ID NO:45-64.

25. The antibody or antigen-binding fragment according to claim 1, comprising: VL, wherein the VL has the amino acid sequence of SEQ ID NO:19; VH, wherein the VH has the amino acid sequence of SEQ ID NO:26; and CH, wherein CH has the amino acid sequence of SEQ ID NO:

55.

26. The antibody or antigen-binding fragment according to claim 1, comprising: VL, wherein the VL has the amino acid sequence of SEQ ID NO:19; VH, wherein the VH has the amino acid sequence of SEQ ID NO:26; CL, wherein the CL has the amino acid sequence of SEQ ID NO:30; and CH, wherein CH has the amino acid sequence of SEQ ID NO:

55.

27. The antibody or antigen-binding fragment according to any one of claims 16 to 26, wherein the Fc region of the IgG1 antibody is defucosylated.

28. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment competing with an antibody or antigen-binding fragment according to any one of claims 1 to 27 for binding to human ILT7.

29. The antibody or antigen-binding fragment according to any one of claims 1 to 28, wherein the antibody or antigen-binding fragment is a bispecific antibody or a multispecific antibody.

30. The antibody or antigen-binding fragment according to any one of claims 1 to 29, wherein the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment.

31. The antibody or antigen-binding fragment according to any one of claims 1 to 30, wherein the antibody or antigen-binding fragment: (1) bind to human ILT7 with a Kd of 500 nM or less as measured by SPR D binding to human ILT7; (2) It does not specifically bind to LILR family members LILRA1, LILRA2 / ILT1, LILRA3 / ILT6, LILRA5 / ILT11, LILRA6 / ILT8, LILRB1 / ILT2, LILRB2 / ILT4, LILRB3 / ILT5, LILRB4 / ILT3 or LILB5; (3) Inhibits the release of interferon-α (IFNα) via peripheral blood mononuclear cells (PBMCs); (4) It selectively binds to plasma-like dendritic cells (pDCs) in human PBMCs; (5) It exhibits natural killer (NK)-dependent ADCC activity against cells expressing ILT7; (6) Exhibits neutrophil-dependent ADCC activity against ILT7-expressing cells; or (7) Exhibits macrophage-dependent ADCP activity against cells expressing ILT7; or any combination of (1)-(7).

32. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment specifically binding to the protease domain of human ILT7, wherein said antibody or antigen-binding fragment: (1) binds to human ILT7 with a Kd of 500 nM or less as measured by SPR D human ILT7; (2) It does not specifically bind to LILR family members LILRA1, LILRA2 / ILT1, LILRA3 / ILT6, LILRA5 / ILT11, LILRA6 / ILT8, LILRB1 / ILT2, LILRB2 / ILT4, LILRB3 / ILT5, LILRB4 / ILT3 or LILB5; (3) Inhibits IFNα release via PBMC; (4) It selectively binds to pDCs in human PBMCs; (5) It exhibits NK-dependent ADCC activity against ILT7-expressing cells; (6) Exhibits neutrophil-dependent ADCC activity against ILT7-expressing cells; or (7) Exhibits macrophage-dependent ADCP activity against cells expressing ILT7; or any combination of (1)-(7).

33. The antibody or antigen-binding fragment according to claim 31 or 32, wherein the antibody or antigen-binding fragment: (1) In vitro inhibition of IFNα release from CpG-stimulated PBMCs with EC50 of 1 nM or less; (2) NK-dependent ADCC activity against ILT7-expressing cells was observed at EC50 of 0.01 nM or less; (3) It exhibits neutrophil-dependent ADCC activity against ILT7-expressing cells at EC50 of 100 nM or less. (4) Demonstrates macrophage-dependent ADCP activity against ILT7-expressing cells at EC50 levels of 10 nM or less; or (5) Demonstrates macrophage-dependent ADCP activity against ILT7-expressing cells with a maximum phagocytic index of 20% or higher; or any combination of (1)-(5).

34. The antibody or antigen-binding fragment of claim 33, wherein the antibody or antigen-binding fragment: (1) Inhibit IFNα release via PBMC with EC50 in the range of 0.01 nM to 0.1 nM; (2) EC50 in the range of 0.001 nM to 0.01 nM showed NK-dependent ADCC activity against ILT7-expressing cells; (3) EC50 in the range of 1 nM to 50 nM showed neutrophil-dependent ADCC activity against ILT7-expressing cells. (4) EC50 in the range of 0.5 nM to 5 nM exhibited macrophage-dependent ADCP activity against ILT7-expressing cells; or (5) Demonstrates macrophage-dependent ADCP activity against ILT7-expressing cells with a maximum phagocytic index ranging from 20% to 80%; or any combination of (1)-(5).

35. The antibody or antigen-binding fragment according to any one of claims 31 to 34, wherein the antibody or antigen-binding fragment exhibits neutrophil-dependent ADCC activity.

36. A polynucleotide encoding a polypeptide of an antibody or antigen-binding fragment according to any one of claims 1 to 35.

37. A vector comprising the polynucleotide according to claim 36.

38. A host cell comprising the polynucleotide of claim 36 or the vector of claim 37.

39. The host cell of claim 38, wherein the host cell: (1) Overexpression of N-acetylglucosamine transferase III (GnTIII); (2) Lack of α-1,6-fucosyltransferase (FUT8); or (3) Having a low fucose content; or any combination of (1)-(3).

40. A method for preparing an antibody or antigen-binding fragment thereof that specifically binds to human ILT7, the method comprising culturing host cells according to claim 38 or 39 in a culture under conditions that allow expression of said antibody or antibody fragment.

41. The method of claim 40, further comprising isolating the antibody from the culture.

42. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 35, and a pharmaceutically acceptable carrier.

43. A method for reducing type I interferon (IFN) in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 35.

44. The method of claim 43, wherein the type I interferon is IFNα.

45. A method for inhibiting or depleting the pDC of a subject in need, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 35.

46. ​​A method for reducing the autoimmunity of a subject in need, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 35.

47. The method according to any one of claims 43 to 46, wherein the subject suffers from an autoimmune disease.

48. A method for treating an autoimmune disease associated with type I IFN or pDC in a subject of need, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 35.

49. The method according to claim 47 or 48, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

50. The method according to any one of claims 43 to 49, further comprising administering an additional therapy to the subject.

51. The method according to any one of claims 43 to 50, wherein the subject is a human.

52. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 35 for reducing type I IFN.

53. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 35 for the preparation of a medicament for reducing type I IFN.

54. The use according to claim 52 or 53, wherein the type I IFN is IFNα.

55. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 35 for inhibiting or depleting pDC.

56. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 35 for the preparation of a medicament for inhibiting or depleting pDC.

57. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 35 for reducing autoimmunity.

58. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 35 for the preparation of a medicament for reducing autoimmunity.

59. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 35 for the treatment of autoimmune diseases associated with type I IFN or pDC.

60. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 35 for the preparation of a medicament for treating autoimmune diseases associated with type I IFN or pDC.

61. The use according to claim 59 or 60, wherein the autoimmune disease is SLE.

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